Physiological changes in stomata and regulation of genes involved in CAM metabolism in polyploid Agave L. 

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Benjamín Abraham Ayil Gutierrez, Felipe Lorenzo Sanchez Teyer, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4284238/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In recent years there has been growing interest in increasing plant water use efficiency (WUE) through the introduction of crassulacean acid metabolism (CAM) into C3 crops. However, this task has been hampered because the scaling of CAM to other C3 plants requires knowledge of the enzymatic and regulatory pathways underpinning this temporal CO 2 pump. Agave presents CAM metabolism, to date research aimed at knowing the physiological and morphological adaptations related to CAM metabolism, only includes a small group of species analyzed. With the aim of knowing basic aspects related to the physiological response of polyploid (2 n =2 x =60 to 2 n =6 x =180) Agave accessions, we carried out genetic and physiological studies in A. tequilana Weber, A. fourcroydes Lem., and A. angustifolia Haw. Using AFLP markers, differences in genetic variability between the polyploid accessions and their diploid counterparts were found. Analysis of expression by real-time PCR showed that the regulation of CAM in Agave is accompanied by the transcription of RbcL, PEPC and PEPCK. Monitoring of the stomatal opening during the night showed differences according to the level of ploidy of the accessions. The genetic and physiological data obtained suggest that agave species present adaptations and flexibility in the transcriptional regulation of genes related to CAM metabolism, suggesting that some polyploid accessions of Agave L. could be more tolerant to drought and heat, adapting their CO2 exchange mechanisms according to the metabolic needs of each species of agave. Agave CAM polyploidy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 1. Introduction The introduce crassulacean acid metabolism (CAM) into C3 crops, with aim the increase plant water-use efficiency (WUE), has become relevant in recent years (Shen et al., 2022; Sonmez et al. 2022; Caferri and Bassi 2022; Zotz et al. 2023). It is important to carry out studies to understand how genetic, biochemical, and physiological factors behave in CAM plants under stressfull environmental factors. this could explain the differences between CAM and C3 photosynthesis and improve to engineer CAM into C3 plants (Rebecca and Hirasawa 2022; Yamaga-Hatakeyama et al. 2022). To perform this type of studies, Agave L. is postulated as a good model, because this species can perform CAM under stressful environmental conditions (Gentry 1982; Liu et al. 2018) and the Agave genus harbors polyploid species that have been documented to be able to better tolerate and adapt to global climate change (Palomino et al. 2012, 2015; Tamayo-Ordoñez et al. 2016b, 2018a). Agave spp. performs CAM photosynthesis under drought and CO 2 enrichment. Gentry (1982), studied Agave species from North America and to date research aimed at knowing the physiological and morphological adaptations related to CAM metabolism, only includes a small group of species analyzed (Nobel and Hartsock, 1978; Pimienta‐Barrios et al. 2001, 2005; Campos et al. 2014; Tamayo-Ordoñez et al. 2018a). For example, in Agave it has been described that CO 2 exchange is affected by drought and CO 2 enrichment. For example; A. americana, A. tequilana , A. asperrima Jacobi, A. cupreata Trel. et. A. Berger, A. durangensis Gentry, and A. salmiana Otto ex Salm-Dyck have been shown to affect their growth rate, biomass distribution, leaf thickness, and proline content at different water potential (Ψ). Species not adapted to dry, biomass production was inhibited (−3.5 MPa), but in species adapted to dry regions such as A. tequilana , A. durangensis , A. lechuguilla Torr., and A. salmiana , the number of leaves and plant coverage were maintained (Ramírez-Tobias et al. 2014). Also, A. Americana and A. angustifolia , they have shown that during drought periods 98% and 85% of CO 2 is fixed during the first eight night hours, similar to that reported in plants that perform strong CAM (Holtum and Winter 2014; Winter et al. 2014; Males and Griffiths 2017). Such physiological behavior is stimulated by CO 2 enrichment (800 ppm) during the night. At CO 2 concentrations of 800 ppm, fixation in A. angustifolia increases (62%) relative to low values of CO 2 fixed (200 and 400 ppm) (Winter et al. 2014). This suggests that Agave species can be better explored to understand why CAM plants increase nocturnal CO 2 assimilation under higher CO 2 concentrations and future to be able to propose them for introducing new traits aimed at creating crop varieties with enhanced CO2 capture and water- and light-use efficiency. On the other hand, Agave spp., has shown present ample tolerance to different stressful conditions, emphasizing the importance of carrying out studies for the selection of elite individuals with physiological and morphological differences that allow them to adapt even better to abiotic stresses. Agave can tolerate drought and high temperature through of control of water loss through leaf surfaces and CO 2 exchange (Pimienta-Barrios et al. 2001; Holtum and Winter 2014; Winter et al. 2014; Tamayo-Ordoñez et al. 2018b). Added to this, polyploidy has proved important role in the evolution and speciation of the genus (Wolfe 2001; Tamayo-Ordoñez et al. 2015, 2018a). In Agave spp., the occurrence of polyploidy has caused DNA regions to be subjected to genetic events, including recombination, amplification, duplication, transposition, and gene loss, which could have resulted in the high variability and diversity of these genomic regions and favoring the formation of new genes that may be fixed in the course of evolution according to the climatic condition and geographical distribution of the species (Tamayo-Ordoñez et al., 2018a). This genus has been shown to inhabit geographical areas where there are high temperatures and water scarcity, this has contributed to the development of morphological (thorns, leaf succulence, stomatal density) and physiological (CAM Metabolism) characters, related to withstanding drought and high temperatures. In addition, in the polyploid species of Agave L., due to the increase in the genome, it is possible that there are more isoforms of protein-coding genes involved in CAM metabolism that could be functionally specialized. As a result of these genetic, morphological, and physiological changes, it is possible that the polyploid species of Agave L. adapt in a better way to global warming and drought (Sattler et al. 2016; Tamayo-Ordoñez et al. 2018b). Few studies have been done about the tolerance to environmental stress of Agave plants with different ploidy levels to find out if polyploids are more stress tolerant or have favorable morphological and physiological features relative to lower ploidy level plants. In that context, Tamayo-Ordoñez et al. (2016b) focused in knowing if polyploid species of Agave are more sensitive to abiotic and biotic stress, and in analyzing the morphological, physiological, and molecular factors involved in their improved responses. These authors described that the size of stomata and the suprastomatal cavity increases according to higher levels of ploidy in the accessions analyzed (Tamayo-Ordoñez et al. 2016b) and the stomatal density is lower as the level of ploidy increases in Agave L. Suggesting that polyploid species of Agave spp. could perform CO2 fixation more efficiently compared to their counterparts with lower ploidy levels. In addition, the differences found in the stomatal density could result in a different physiological response to drought among the analyzed accessions, allowing certain accessions of Agave respond differentially during prolonged periods of drought. In Betula papyrifera Marsh. and A. thaliana Heynh in Holl& Heynh have been shown to have fewer stomata per unit area and smaller stomatal indices than their diploid counterparts (Li et al.1996, 2012). Stomatal indices according to ploidy numbers have also been related to adaptation to stress and previous studies described a direct relation between water deficit, stomatal dimensions, and ploidy level affecting the plant’suptake of water (Li et al. 1996, 2012; Balao et al. 2011). In Agave L., adaptations preventing physiological damages due to draught include nocturnal assimilation of CO2, thick cuticles, low stomata density, and succulent leaves. The latter two adaptations enable water stored in the leaf parenchyma to continuously move to the chlorenchyma during dry periods (Pimienta-Barrios et al. 2005), thus conferring the plants the capacity for with standing up to seven years of draught (Stewart 2015). In this research, the goals was to relate the ploidy levels of Agave spp., with differences in CAM induction, for it determine if there are differences in genetic variability in polyploid species, Know if there is a differential physiological mechanism in the fixation of CO 2 (related to the stomatal opening and closure), it could be related to adaptation to stress by drought, and know if there is a differential genetic regulation of genes coding for enzymes involved in CAM metabolism. The obtained results indicate that polyploid species of Agave reflect physiological adaptations and genetic changes that could help them to better respond against global warming. 2. Material and methods 2.1 Plant material For all the studies reported in this investigation, three representative individuals of each varieties analyzed belonging to three species ( Agave tequilana Weber, Agave fourcroydes Lem. and Agave angustifolia Haw) were included in this research (Table S1). The selected Agave accessions included varieties and variants (not registrated yet) with different ploidy levels (Table S1). All these accessions were directly collected from the field and adapted to two ecoregions: the Regional Roger Orellana-CICY Botanical Garden (RO-CICY) in the city of Merida, and the Germplasm Bank of the Scientific-Technological Park of Yucatán (GB-PCTY), located in the Sierra Papacal, 20 km northwest of Merida (21° 07' 20" N, 89° 43' 41" W). The accessions from the GB-PCTY belong to the Agave spp. collection described by Pulido-Salas and GarcíaMarín (2003) and their ploidy level was characterized as a part of this research. Herbarium specimens from leaves of each accession were made and deposited in the CICY herbarium (Table S1). 2.2 Variability of Agave L. by AFLP The AFLP method was performed following Tamayo-Ordóñez et al. (2012). For the selection of the best four sets of primers, we initially evaluated 35 combinations of primers (Table S2). AFLP reactions with selective primers were performed with a touchdown PCR program for most primer sets. The amplification conditions were carried out according to Tamayo-Ordoñez et al. (2012). PCR products were electrophoresed in a CEQ 8800 sequencer (Perkin–Elmer Inc., Foster City, CA). The obtained electropherograms were analyzed using the software GeneMarker v.1.75 (Perkin-Elmer, Inc., Boston, MA) All calculations were performed using the NTSYS-pc 2.1 (Exeter Software Co., New York) software (Rohlf 2000). Only strong, reproducible, and clearly distinguished bands were used in a binary matrix. In order to find out if the AFLP markers could discriminate between polyploid accessions, the unweighted pair group was analyzed in all the studied accessions with UPGMA method, and genetic distances among accessions were calculated according to Nei and Li (1979). The reliability and robustness of the dendrograms were tested by bootstrap analysis with 1000 replications to assess branch support using FreeTree software (Pavlíek et al. 1999). Analyses of percentages of polymorphism, index of similarity, and marker bands were made with the Free Tree (Pavlíek et al. 1999) software. 2.3 Determination of stomatal aperture and closure In the first instance, in order to know if there are differences in the opening and closing of stomata during the nocturnal period (19:00h at 7:00h) of CAM metabolism in polyploid species compared to their counterparts with lower level of ploidia, a determination of stomatal aperture and closure was carried out, including two varieties with different ploidy level representative of the species A. angustifolia Haw. and A. fourcroydes Lem. Samples of leaves (1 cm 3 ) from the adaxial and abaxial epidermis were collected at 19:00 h, 23:00 h, 3:00 h and 7:00 h in two ecoregions (RO-CICY and GB-PCTY). The preparation of the samples is made according Tamayo-Ordoñez et al. (2016b). The samples were then mounted on metallic stubs with carbon conductive adhesive tape (Electron Microscopy Science) and sputter coated with a 150 Å gold layer (Denton Vaccum Desk II). Length of the guard cells and suprastomatic cavity area, stomatal size and density were calculated for the abaxial and adaxial epidermis at a magnification of 100x (0.1213 mm 2 ). Counts and measurements were made in ten fields of each leaf for each accession. Sample analysis and image recording were made using a scanning electron microscope (Jeol, JSM-6360LV). The gathered data were subjected to statistical analysis by Tukey tests evaluated at P>0.05. 2.4 Phylogenetic analysis of NADH, RbcL, PEPC, and PEPCK genes NADH gene (Nad4 subunit dehydrogenase 4) amplification was conducted using the primers previously reported by Tamayo-Ordoñez et al. (2012). PEPC, PEPCK, and RbcL primers were designed from Ananas comosus (AJ312631.1), Arabidopsis (NM_119948.4) and Agave tequilana (GW667494.1), respectively (Table S3). PCR reactions were carried out in a volume of 50 μL containing 25 ng of genomic DNA, 130 μM dNTPs, 15 μM of each primer, 2.5 units of Taq polymerase, and 1X PCR reaction buffer (Life Technologies, Rockville, MD, U.S.A.) with 1.5 mM MgCl 2 . PCR conditions included one cycle of 3 min at 94 ºC for initial denaturation, followed by 35 cycles of 1 min at 94 ºC, 1 min at alignment temperature (tm) according to each set of primers used (Table S3), 1 min at 72 ºC, and finally, 7 min at 72 ºC. PCR products were separated by electrophoresis in 1.2% agarose gels. Purification, cloning and sequencing of PCR products was carried out according to Tamayo-Ordoñez et al. (2016b). The nucleotide sequences were aligned (BLASTX) and compared with those in the GenBank database. DNAMAN version 4.0 was used to translate these sequences and to identify the open reading frame. Predicted amino acid (aa) sequences relative to the NADH, RbcL, and PEPC nucleotide sequences were used in combination with related sequences retrieved from GenBank to build a phylogenetic aligning. Conserved aligned regions (>90%) were selected in all sequences, phylogenetic analysis was performed in software MEGA version 6.0 (Tamura et al. 2013). Domains of interest for both proteins were identified with the CDD (Derbyshire et al. 2015). 2.5 Tertiary structure (3D) of NADH, RbcL, PEPC, and PEPCK enzymes Tertiary structure analysis of NADH, RbcL, PEPC, and PEPCK was performed with SWISS-MODEL (Biasini et al. 2014). Values of sequence identity (%), Global Model Quality Estimation and Qualitative Model Energy Analysis, were considered. As references, sequences corresponding to NADH- quinone-oxidoreductase subunit L from Escherichia coli (Efremov and Sazanov 2011), ribose bisphosphate carboxylase small chain from Oryza sativa (Matsumura et al. 2012) , phosphoenolpyruvate carboxylase from Arabidopsis thaliana Heynh in Holl and Heynh (unpublished) and phosphoenolpyruvate carboxykinase from Escherichia coli (Sudom et al. 2003) were included. 2.6 Determination of relative expression of the NADH, RbcL, PEPC, and PEPCK RNA isolation and cDNA synthesis were conducted according to Tamayo-Ordóñez et al. (2015). The PEPC and PEPCK primers used for relative expression analysis were those reported by Aragón et al. (2013). For phylogenetic analyses, a RbcL primer was designed from Agave tequilana (GW667494.1), a NADH primer was designed from the isolated NADH sequence, and, as reference genes, the 18S rDNA genes were used according to Tamayo-Ordoñez et al. 2015 (Table S3). Amplifications of the NADH, PEPC, PEPCK, and RbcL genes were carried out as described above and with the same PCR amplification conditions used for the phylogenetic analysis. The melt curve analysis and negative controls for the reference and target genes were always included in the experiments in order to eliminate DNA contamination. The relative expression of each gene was determined by the ∆∆Cq method between the target (NADH, PEPC, PEPCK and RbcL) and reference (18S rDNA) genes (Tamayo-Ordoñez et al., 2015), by the following equation: Relative expression = (E ref ) Ctref /(E target ) Ctarget (Pfaffl 2001). All analyses included 3 biological replicates, each with three technical replicates. 3. Results 3.1 Polyploidy and the genomic consequences in Agave Many authors have described that the increase of genome size in polyploid species is accompanied by genetic changes, resulting in greater variability and genetic diversity in polyploid plants (Chen 2010; Wendel 2000; Moghe and Shiu 2014; Tamayo-Ordoñez et al. 2016a). The dendrogram obtained from the AFLP marker showed two clusters, cluster I having two subgroups denominated as A and B (Fig. 1). The subgroup A showed the clustering of hexaploid and diploid accessions of Agave angustifolia (AAM1-RO, AAM2-RO, AAM3-RO, AAC1-RO, AAC2-RO, AAC3-RO, CHA1-PCTY, CHA5-PCTY and CHA6-PCTY), and the subgroup B included the diploid accessions of A. tequilana (AT1-RO, AT2-RO, AT3-RO, 1444-2-PCTY, 1444-3-PCTY, and 0345b-PCTY; Fig. 1). Cluster II grouped the triploid and pentaploid accessions of A. fourcroydes . According to the global variability, the AFLP marker indicated a closer genetic proximity between A. angustifolia and A. tequilana . The analysis of similarity indexes and percentages of polymorphism in accessions belonging to the same species (considering ploidy level as an important factor) indicated that Agave accessions containing polyploid varieties had low similarity indexes (0.76) compared to the results from the analysis including only diploid accessions like A. tequilana (2 n = 2 x = 60) and A. angustifolia (2 n = 2 x = 60) (0.83). The analysis of A. angustifolia (2 n = 2 x = 60) and A. angustifolia (2 n = 6 x = 180) showed percentages of polymorphism of 75 and a similarity index of 0.76, while accessions of A. fourcroydes (2 n = 3 x = 90) and A. fourcroydes (2 n = 5 x = 150) showed percentages of polymorphism of 79 and a similarity index of 0.73 (Table 1). Apparently, polyploidy does have an effect on genome size and genetic variability in Agave . The analysis of correlation of alleles indicated that, the accessions of A. tequilana cultivated in the RO-CICY share a greater proportion of alleles, similar in the same proportion as A. angustifolia cultivated in both microclimates (RO-CICY and GB-PCTY). Likewise, the varieties of A. fourcroydes apparently contain a narrow genetic germplasm, reflected in the conservation of genetic material (Fig. 2). Table 1. Polymorphism and index of genetic variation of Agave L. Especie Total number of individuals Accessions (number of individuals evaluated by each accession)* Total numbers of loci Total numbers of polymorphic loci Total numbers of common loci Polymorphic percentage Ɨ Index of similarity Agave spp. 33 ATA-RO (3), AAM-RO (3), AAC-RO(3), AFK-RO(3), AFS-RO (3), AFY-RO (3), 1444-PCTY (2), 345b-PCTY (1), CHA-PCTY(3), KK-PCTY (3), SK-PCTY(3) and YK-PCTY (3) 201 182 19 90.54 0.66 Agave tequilana Weber. (2 n = 2 x = 60) and Agave angustifolia Haw. (2 n = 2 x = 60) 9 ATA-RO (3), AAM-RO (3), 1444-PCTY (2), and 345b-PCTY (1) 73 33 40 45.20 0.83 Agave tequilana Weber. (2 n = 2 x = 60) 6 ATA-RO (3), 1444-PCTY (2) and 345b-PCTY (1) 44 20 28 45.45 0.85 Agave angustifolia Haw. (2 n = 2 x = 60) and Agave angustifolia Haw. (2 n = 6 x = 180) 9 AAM-RO (3), AAC-RO(3) and CHA-PCTY(3) 82 62 20 75.60 0.76 Agave fourcroydes Lem. (2 n = 3 x = 90) and Agave fourcroydes Lem. (2 n = 5 x = 150) 12 AFK-RO(3), AFS-RO (3), AFY-RO (3), KK-PCTY (3), SK-PCTY(3) and YK-PCTY (3) 142 113 29 79.57 0.73 Agave tequilana Weber. ‘Azul’ (2 n = 2 x = 60) 6 ATA-RO (3), 1444-PCTY (2) and 345b-PCTY (1) 48 20 28 41.66 0.85 Agave angustifolia Haw ‘Marginata’ (2 n = 2 x = 60) 3 AAM-RO (3) 68 33 35 48.52 0.81 Agave angustifolia Haw. ‘Chelem ki’ (2 n = 6 x = 180) 6 AAC-RO(3) and CHA-PCTY(3) 63 38 25 60.31 0.77 Agave fourcroydes Lem. ‘Kitam ki’ (2 n = 3 x = 90) 6 AFK-RO(3) and KK-PCTY (3) 103 51 52 49.51 0.81 Agave fourcroydes Lem. ‘Sacki ki’ (2 n = 5 x = 150) 6 AFS-RO (3) and SK-PCTY(3) 139 82 49 58.99 0.74 Agave fourcroydes Lem. ‘Yaax ki’ (2 n = 5 x = 150) 6 AFY-RO (3) and YK-PCTY (3) 101 57 44 56.43 0.77 *The specifications of the collections are described in the table S1. Ɨ Polymorphic percentage= (Total numbers of polymorphic loci/ Total numbers of loci)*100 Polyploidy is a very common phenomenon in species of angiosperms and vascular plants (Moghe and Shiu 2014; Wendel 2000). Nowadays, the ancestors of many polyploid plant species, like those in Agave , remain to be unknown. The genus Agave includes triploid (e.g., A. fourcroydes ‘Kitam ki’ 2 n =3 x =90) to octoploid species (e.g., A. datylio 2 n =8 x =240; Castorena-Sánchez, 1990), however, despite having species with a wide range of ploidy level, most studies –conducted in few species of the genus– have focused on cytogenetic characterization. In addition, it is suggested that due to the presence of bimodal karyotype with a basic chromosome number of n =30 (5 long acrocentric chromosomes and 25 small metacentric or submetacentric chromosomes; Moreno-Salazar et al. 2007; McKain et al. 2012; Palomino et al. 2015) the genus is possibly of allopolyploid origin (McKain et al. 2012). Allopolyploidy can be associated with an increased number of gene copies and, therefore, it implies the generation of redundant genes. These genes, mainly generated by duplication, are involved in epigenetic regulation, become specialized to perform a complementary function or may eventually be lost. The functional partitioning and differences in expression patterns of these redundant genes are associated to changes (physiological and phenotypic) (Tamayo-Ordoñez et al. 2016) that confer advantages in allopolyploid species. Otherwise, the genus Agave is of recent origin (6-8 Mya and 1.5-3 Mya), has a high index of species diversity (0.32 to 0.56 species per million years) relative to angiosperms in general (0.089-0.07 species per million years), and according to the model for evolution of rDNA regions proposed by Tamayo-Ordoñez et al. (2018), it is possible that certain genomic regions are undergoing an evolutionary stage of fixation of certain genes copies. This evolutionary process suggests that genes are subjected to genetic events including recombination, amplification, duplication, transposition, and gene loss that could have resulted in the high variability and diversity observed in the Agave genome. The use in Agave of ISSR (Vargas-Ponce et al. 2009; Aguirre-Dugua and Eguiarte 2013), AFLP (Sánchez-Teyer et al. 2009) and SSAP (Bousios et al. 2007) molecular markers has indicated variability and genetic diversity between wild and cultivated populations of A. angustifolia , A. tequilana , and A. fourcroydes , among other species. The Agave accessions we analyzed showed high genetic variability. The accessions with the highest level of ploidy –like A. angustifolia ‘Chelem ki’ (2 n = 6 x = 180), A. fourcroydes ‘Sack ki’ (2n = 5x= 150), and A. fourcroydes ‘Yaax ki’ (2 n = 5 x = 150)– showed the lowest similarity indexes (<78) in comparison to the other analyzed accessions. Apparently, polyploidy has an effect on genome size and genetic variability in Agave , and the presence of these polyploid species within the genus could be a factor that contributes to the high species diversity index (0.32 to 0.56 species per million years) of the genus. Also, according to Good-Avila et al. (2006), the process of speciation in Agave has been coincident with increasing aridity in central Mexico, which suggests that high retrotransposition activity in response to water deficit may have had an important role in speciation. Tamayo-Ordoñez et al. (2016b) found that in some species of Agave there is differential regulation of genes associated with biotic and abiotic stress factors depending on their habitat and proposed that stressing environmental factors could have contributed to gene diversity that expresses as speciation and species’ adaptation. Cultivation of some species of Agave since pre-Columbian time (Casas et al. 2016) and the involved pressure from anthropic domestication processes, artificial selection, and intensive cultivation has resulted in its species having different degrees of domestication (Colunga-GarcíaMarín et al. 2004). But, unfortunately, until now only a handful of species have been studied with the goal of obtaining evidence of their domestication and management processes. Studies made of the rDNA, NBS-LRR, and LEA genes in cultivated polyploid species of Agave have revealed that A. tequilana ‘Azul’ (2n = 2x = 60) –used for production of tequila– and A. fourcroydes ‘Kitam ki’ (2n = 3x = 90) –used for fiber production– have variants of these genes that could be involved in gene evolution, rRNA functionality, and activity of proteins participating in defense responses. This strongly suggests that anthropic pressure might have had negative effects on their variability, genetic diversity, and response to abiotic and biotic stress. Consequently, it must be emphasized that morphological, molecular, and physiological studies be made before attempting to use polyploid Agave plants in order to achieve their sustainable use and to avoid negative consequences on the variability and genetic diversity of the genus. Also, based on the possible allopolyploid origin of Agave , its genome may contain valuable information that could be analyzed and used for genetic improvement of economical important crops exposed to biotic and abiotic stresses induced by global climate change (Tamayo-Ordoñez et al., 2018). 3.2 Implications of the Conservation of NADH, RbcL, PEPC and PEPCK enzymes in Agave In CAM and C4 plants, the activity of key enzymes such as PEPC, PPDK, NAD (P) –ME, and PEPCK is much higher than in C3 plants. Even so, the cellular compartmentalization of said enzymes differs between CAM and C4 plants. CAM plants have evolved specific diurnal and nocturnal patterns of expression and regulation to accommodate the flux of carbon through gluconeogenesis and glycolysis necessary to meet the nocturnal demand for PEP and diurnal decarboxylation of four-carbon organic acids. The characterization of certain specific CAM genes (PEPC and PEPCK) that are related to the cyclic electron transport and chlororespiration (NADH), and to enzymes related to carbon fixation (RbcL) and their expression patterns in CAM plants, could clarify the molecular mechanisms subjected to evolution and expression of said enzymes. In Agave , the basic enzymes and metabolites necessary for the optional functioning of CAM are not yet known, so in this work we isolated partial regions of the enzymes and studied their phylogenetic relationships with members of the classes Liliopsida and Magnoliopsida. Analysis of aa substitutions found in the sequence of the enzymes NADH, rbcL, PEPC, and PEPCK and its impact in the conformation of the 3D structures were included. Phylogenetic analyses indicated that the NADH enzyme sequences from Agave spp. were more closely related with the families Asparagaceae and Agavoidaceae. The genus Allium , representative of the Amaryllidaceae family, formed a separate group (Fig. 3A). In the phylogenetic analysis of the RbcL, PEPC, and PEPCK enzymes we included accessions belonging to the class Liliopsida and Magnoliopsida. Sequences close to the family Agavaceae were not included due to the scarce information available. RbcL of Agave grouped with species in genera belonging to the class Liliopsida. Agave accessions were more closely related with accessions from the genus Oryza (Fig. 3B). The genera belonging to the class Magnoliopsida, formed a separate group. PEPC of Agave grouped with members of the order Poales ( Oryza , Aegilops , Tillandsia , and Ananas ). Some plant accessions belonging to genera in the class Liliopsida ( Asparagus , Oryza , Setaria , Aegilops , Hordeum , and Triticum ) grouped with members of genera in the class Magnoliopsida ( Herrania , Theobroma , Gossypium , Jatropha , Manihot , Ricinus , Populus , Curcubita , and Arabidopsis ; Fig. 4A). Presence of isoforms of the PEPC enzyme in C3 and C4 plants has been widely described in plants (Paulus et al. 2013), and it is possible that the formation of two different groups including aa sequences of PEPC from Oryza and Aegilops is a consequence of the presence of PEPC isoforms in these genus. For this part, PEPCK sequences of Agave that grouped with members of genera from the class Liliopsida ( Elaeis , Phoenix , Musa , Panicum , Sorghum and Asparagus ), were more closely related with the genus Asparagus . Members of genera in the class Magnoliopsida formed a separate group (Fig. 4B). The chloroplast NADH dehydrogenase-like complex (NDH) is comprised of many subunits. The plastid genomes of flowering plants also have 11 genes (ndhA–ndhK; Ifuku et al., 2011); a subunit specific to photosynthetic NDH is NdhL (dehydrogenase subunit L). In this study we identified a partial region of dehydrogenase subunit L in Agave that showed high conservation when compared with other members of the Asparagaceae family. Analysis of aa substitutions found in NADH enzyme from the Agavaceae and Amaryllidaceae families, indicated that Agavaceae presents 2% of substitutions from V (117) to L, 12% from V (298) to A, and 12% from A (301) to V (Table S4). In the family Amaryllidaceae we found 33% of substitution from V (117) to I, 33% from V (298) to I, and 33% from A (301) to E (Table S4). Analysis of the effect of aa substitutions on 3D structures indicated that these changes do not affect the tertiary structure of the NADH enzyme. Our three-dimensional (3D) models of NADH dehydrogenase of the families Asparagaceae and Amaryllidaceae had a sequence identity of >40% with NADH- quinone-oxidoreductase subunit L (Fig. 5 and Table S5). 3D models of NdhL (320aa) from the families Asparagaceae ( Agave ) and Amaryllidaceae ( Allium ) showed structures similar with the NADH-quinone-oxidoreductase subunit L described in E. coli (Efremov and Sazanov 2011) (Fig. 5). In both families, it was possible to identify the formation of nine alpha helices (Fig. 5B-D and Table S5). This results, suggesting that in the analyzed plant accessions of Agave with eight million years of evolution (Tamayo-Ordoñez et al. 2018) the dehydrogenase subunit L enzyme presents structural conservation. The importance of conservation in this enzyme, lies in that the chloroplast NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport and chlororespiration, which in angiosperms further associates with photosystem I (PSI) to form a super-complex (Yamori et al. 2015). For this part, in our analysis of point mutations of PEPC enzyme, total aa substitutions (100% change) were detected in 22 aa residues from Liliopsida and in 28 aa residues from Magnoliopsida. This result indicated that 18% of the PEPC enzyme sequence analyzed differed between classes (Table S7). These differences found in the aa substitutions affect the grouping support between the aa sequences of the PEPC enzyme in the classes Liliopsida and Magnoliopsida, which was previously reflected in the phylogenetic tree of this enzyme (Fig. 4A). In Agave , The conservation of A and R in the substrate binding ((PWIF(A/S)WTQR) and inhibitory site ((DLLEGDPYLKQ(R/G)IRLRDSYIT)) of PEPC enzyme, was 100%, which suggests according to Paulus et al. (2013), possibly, it is of the C3-type in Agave and other members of to the classes Magnoliopsida and Liliopsida. Also, 3D models of PEPC from Liliopsida (genera Agave and Ananas ) and Magnoliopsida (genera Arabidopsis and Gossypium ) showed similar structures (identity >85% with the phosphoenolpyruvate carboxylase (PEPC) of A. thaliana model) (Fig. 6 and Table S5); in both classes it was possible to identify the formation of 11 alpha helices (Fig. 6B-D and Table S5). 3D models indicated that the PEPCK from Agave had a sequence identity >45% with the phosphoenolpyruvate carboxykinase (PEPCK) described for the E. coli model (Fig. 7A and Table S5). 3D models of PEPCK from the classes Liliopsida (genera Agave and Asparagus ) and Magnoliopsida (genera Arabidopsis , Brassica , and Arachis ) showed similar structures between them, and it was possible to identify the conservation of aa residues that allow the union of Mg + , Ca 2+ , and pyruvic acid –important for the function of the enzyme (Sudom et al. 2013; Fig. 7B-F and Table S5). The ATP binding site could not be identified in the plant accessions that we analyzed. C4 photosynthesis has evolved independently more than 62 times, including 7500 species in 19 families, or 3% of the flowering plant species (Deng et al. 2016). Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) has shown plays a key role in the carbon metabolism of C4 and CAM plants, and markedly improves photosynthetic efficiency and water use efficiency (Driever and Kromdijk 2013). Deng et al. (2016) analyzed 60 available published plant genomes and delimited that the PEPC family consists of three distinct subfamilies (PPC-1, PPC-2, and PPC-3). The monocot CAM –or C4-related PEPC– originated from the PPC-1M1 clade and WGD may increase the number of copies of the PEPC gene, suggesting the formation of more isoforms of the PEPC in Plants CAM (Fan et al., 2013; O’Leary et al., 2011). It is possible that the broad amino acid changes (18%) found between the classes Magnoliopsida and Liliopsida, reported in this study, may be due to the presence of isoforms in the accessions representative of each class. D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39; Rubisco) catalyzes the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation (Bracher et al. 2017). Land plants, have hexadecameric type I rubisco with an approximate molecular mass of 550 kDa composed of eight large (L;55 kDa) and eight small (S;15 kDa) subunits (L8S8). A. thaliana produces four distinct small-subunit isoforms (RbcS1A, RbcS1B, RbcS2B and RbcS3B) (Valegård et al. 2018). In the analysis of RbcL enzyme, it was possible to identify unique substitutions between members of the classes Liliopsida and Magnoliopsida. Total aa substitutions (100% change) were detected in aa residues such as such as S/Q (1), R/K(22), I/V (37), Q/E (68), and S/M (Table S6). Our 3D models indicated that the RbcL from Agave had a sequence identity of 99% with the ribose bisphosphate carboxylase small chain (Fig. 8A and Table S5) described in the O. sativa model (Matsumura et al. 2012). 3D models of RbcL from genera in the classes Liliopsida ( Oryza , Agave , and Aegilops ) and Magnoliopsida ( Tragopogon and Arachis ) showed similar structures. In both classes, we were able to identify the formation of four alpha helices and two beta folds (Fig. 8B-D and Table S5). In Aegilops and Tragopogon , we observed that the length of the first alpha helix was lower compared to the reference model (Matsumura et al. 2012). The differences found between these tertiary structures can be derived from the presence of different isoforms that code for the small subunit of RbcL, as it has been described A. thaliana (Valegård et al. 2018). Because Rubisco catalyzes the rate-limiting step of C3-photosynthesis, many studies have been carried out for future improvement of Rubisco activity by genetic modification to increase the productivity of crop plants (Borland et al. 2011; 2014). The regulation of rubisco activity goes hand in hand with a negative coordination of PEPC regulation, suggesting a complex coregulation of both carboxylases compete for CO 2 during the early morning hours. According to the results obtained, it seems that the enzymes rbcL, PEPCK, PEPC and NADH are conserved between the classes Liliopsid and Magnoliopsida, however the presence of amino acid changes (>18%) in the enzymes PEPC and rbcL, could indicate the presence of isoforms, and genes that could be functionally specialized to better perform their catalytic activity. The participation of these two enzymes is related to photosynthetic efficiency and water use efficiency, suggesting that they are in constant evolutionary change according to the conditions of water limitation, high temperatures, increase of CO2 as a result of global warming, which we are currently experiencing. It is important to mention that the grouping of Agave with monocots in the order Poales, suggests that the genus possibly has genetic material that allows it to tolerate environments where abiotic stresses are extreme, as described for that order (Linder and Rudall 2005). Bouchenak-Khelladi et al. (2014) described that CO 2 -concentrating mechanisms counteract the effects of low atmospheric CO 2 and reduce phototranspiration. It is believed that the parallel evolution of C4 and CAM photosynthesis in Poaceae, Cyperaceae, and Bromeliaceae is an adaptation to changes in atmospheric CO 2 concentrations. Combinations of extrinsic and intrinsic factors might have played a role in shifts in diversification rates and may explain the variation in species richness in Poales. In the genus Agave the richness in diversity is not yet fully known, so the physiological exploration of a larger number of species could help us to know if the changes in atmospheric CO 2 concentrations during the evolution of the genus allowed the diversification of the C3, C4, and CAM responses. 3.3 Differences in stomata and transcriptional regulation of CAM-related genes in Agave L. With the aim of assessing the physiological changes that could be observed in stomata and that could impact the dynamics of CO 2 exchange and transpiration, we focused on determining the opening and closing of stomata by scanning electron microscopy in the two Agave species with different levels of ploidy ( A. angustifolia and A. fourcroydes ) at 19:00 h, 23:00 h, 3:00 h and 7:00 h. Differences in the opening and closing of stomata on the abaxial and adaxial leaf surfaces were observed (Fig. 9 and Table 2). Table 2. Features of stomata in Agave L. Characteristics* A. angustifolia Haw. ‘Marginata’ (2 n =2 x =60) Ɨ▲ A. angustifolia Haw. ‘Chelem ki’ (2 n =6 x =180) Ɨ▲ A. fourcroydes Lem. ‘Kitam ki’ (2 n =3 x =90) Ɨ▲ A. fourcroydes Lem. ‘Sac ki’ (2 n =5 x =150) Ɨ▲ Leaf section Adaxial/Abaxial Adaxial/Abaxial Adaxial/Abaxial Adaxial/Abaxial Stomata area (µM 2 ) 997±56 a /1161±32 a 2080±240 b /2059±167 b 1106±66 a /1149±63 a 1621±73 b /1619±54 b Stomata density (number mm -2 )* 58±3 a /47±2 a 32±2 b /34±2 b 45±1 a /44±1 a 36±2 b /38±1 b Guard cells area (µM 2 ) 19:00 PM: 853±6 a /850±4 a 23:00 PM: 859±38 a /889±40 a 3:00 AM: 1,161±50 b /792±62 a 7:00 AM: 954 ±18 a /1,000±52 a 19:00 PM: 1001±3 a /1135±61 b 23:00 PM: 1475±9 d /1470±23 d 3:00 AM: 1310±61 d /1380±83 d 7:00 AM: 1448±35 d /1516±60 d 19:00 PM: 915±33 a /919 a ±44 c 23:00 PM:769±3 c /756±13 c 3:00 AM: 873±18 a /889±33 a 7:00 AM: 887±30 a /1002±30 a 19:00 PM: 1071±1 b /1254±46 b 23:00 PM: 1140±64 b /1179±39 b 3:00 AM: 1342±2 d /1152±18 b 7:00 AM: 1051±26 a /998±47 a Suprastomatic cavity area (µM 2 ) 19:00 PM: 77±4 a /330±17 c 23:00 PM: 50±0 a /317±33 c 3:00 AM: 231±28 c /306±7 c 7:00 AM: 159 ±9 b / 216±20 c 19:00 PM: 383±59 c /510±17 c 23:00 PM: 865±37 d /1683±19 e 3:00 AM: 450±9 c /740±1 d 7:00 AM: 630 ±23 d /955±2 c 19:00 PM: 101±3 a /225±29 c 23:00 PM: 291±15 c /294±36 c 3:00 AM: 364±8 c /326±12 c 7:00 AM: 181±3 b /398±2 c 19:00 PM: 555±43 d /319±17 c 23:00 PM: 390±9 c /377±8 c 3:00 AM: 350±28 c /576±67 d 7:00 AM: 170±4 b* /153±4 b* Ɨ Lowercase letters (a, b, c, d or e) indicate significantly different values (Student’s t test; p<0.05). ▲ Values are averages of triplicates obtained from analysis of 30 stomata in 3 leaves of three plants of each accession ± standard error *Values of suprastomatic cavity area (µM 2 ) at 7:00AM were obtained from stomata that remained open and represent only 25% of the total stomata observed Comparing different ploidy level accessions from the same species of Agave , those with higher ploidy level ( A. angustifolia ‘Chelem ki’ and A. fourcroydes ‘Sac ki’) have a lower stomatal density (32±2 and 52±3) and their stomata are 1.5 to 2 times larger in comparison to their lower ploidy level counterparts ( A. angustifolia ‘Marginata’ and A. fourcroydes ‘Kitam ki’) (Fig. 10 and Table 2). Analyses of guard cell area (μM 2 ) showed larger size in polyploid plants of the species A. angustifolia 'Chelem ki' and A. fourcroydes 'Sac ki,' coinciding with stomata size (Fig. 9 and Table 2). Stomatal indices according to ploidy numbers have also been related to adaptation to stress (Balao et al. 2011; Jordan et al. 2015; Males and Griffiths 2017). Analysis of suprastomatic cavity area (µM 2 ) showed important changes between the polyploid species. A. angustifolia ‘Chelem ki’ showed a greater suprastomatic cavity area (1683±19 µM 2 ) compared to A. angustifolia ‘Marginata’ (317±33 µM 2 ) at 23:00 h (Fig. 11 and Table 2). Lower values of suprastomatic cavity area in A. angustifolia ‘Marginata’ were observed at 19:00 h and 23:00 h. Suprastomatic cavity area in the abaxial section of the leaf did not show significant differences along the temporal course, however, for A. angustifolia ‘Marginata,’ the suprastomatic cavity area was higher on the abaxial section of the leaf in comparison to the stomata located on the adaxial part of the leaf (Fig. 11). In the hexaploid species A. angustifolia 'Chelem ki', high values of suprastomatic cavity area were observed at 23:00 h in both sections of the leaves (adaxial: 865±37 µM 2 and abaxial: 1683±19 µM 2 ; Table 2). The lowest values of suprastomatic cavity on the adaxial and abaxial surfaces were observed at 19:00 h. Similar to what we found in A. angustifolia 'Marginata,' the suprastomatic opening on the abaxial surface was always greater compared to the same on the adaxial surface. Behavior of stomata opening in A. fourcroydes showed patterns different to those observed in A. angustifolia . The pentaploid species A. fourcroydes 'Sack ki' showed the highest values of suprastomatic cavity area at 3:00 h (576±67 μM2) and 19:00 h (555±43 μM 2 ), both on the abaxial and on the adaxial surfaces. Suprastomatic cavity area was not as large as that reported in the hexaploid species A. angustifolia 'Chelem ki' (abaxial: 1683±19 μM 2 ). In addition, in the adaxial leaf section, 75% of the stomata analyzed proved to be closed and the aperture of the remaining 25% indicated low values of suprastomatic cavity area (adaxial: 170±4 μM 2 and abaxial: 153±4 μM 2 ) (Table 2). Triploid A. fourcroydes ‘Kitam ki’ showed few significant differences along time, indicating that in this species the opening of the stomata is maintained longer; which is different to what was observed in A. angustifolia Haw 'Chelem ki', in which immediately after the stomatal opening at 23:00 h., suprastomatic cavity area decreases drastically (Fig. 9 and Table 2) Relative expression of the PEPC gene showed significant differences, raising its expression at 3h and 23h (Fig. 12). The highest values in PEPC expression were identified at 23 h in the accessions cultivated in the BGR-RO (Fig. 12A), coinciding with the highest values of suprastomatic cavity area. Similar trends were found in the expression of the PEPCK enzyme, which also indicated higher values at 3h and 23h, in plants belonging to both microclimates (Fig. 13). Highest values of RbcL expression were observed at 15h for both plantations (BGR-RO and GB-PCTY; Fig. 14). Regarding NADH, there were no significant differences during the evaluated period (data not shown). 4. Discussion CAM photosynthesis is a flexible phenomenon in plants. It has been proposed that there is a continuous photosynthetic expression from C3 to CAM and, in this progression, several types of CAM photosynthesis can be found ranging from a weak to a strong degree of expression (Males and Griffiths 2017; Liu et al. 2018). Differences found in stomatal size and density of Agave depending on their ploidy level could be an indicator of differences in water uptake and tolerance to drought, which could be a product of their adaptation to CO 2 concentration changes (Winter et al. 2014; Driever and Kromdijk 2013; Tamayo-Ordoñez et al . 2018b). There is evidence that totally compacted cells, or stomata without air spaces, and the development of cuticular waxes, diminish the hydric state when the ambient temperature increases. Also, research conducted in the Proteaceae family has indicated that the ancient changes in genome size clearly influenced stomatal size, but adaptation to habitat strongly modified the genome-stomatal size relationship (Jordan et al. 2015). This suggests that an increase in CO 2 concentrations could impact on stomatal size an frequency, so it is possible that the polyploid species ( A. angustifolia 'Chelem ki' and A. fourcroydes ‘Sac ki’) showing larger stomata, lower stomatal density, and higher wax content may be better able to tolerate stress in climates where water is a limiting factor, and that this increased tolerance resulted from genetic, physiological and morphological changes during the polyploidization process and the increased CO 2 concentration that are currently experienced (Tamayo-Ordoñez et al. 2016b; Tamayo-Ordoñez et al. 2018b). In addition, although plants such as Agave can control the loss of water by transpiration –for example, A. americana and A. deserti (Ehrler 1969; Nobel and Hartsock 1978)– through their ability to open stomata at night, avoiding desiccation, and regulating its temperature through perspiration, there is evidence suggesting that agave plants with frequent irrigation can alter their metabolism causing the opening of stomata during light hours (Geydan and Melgarejo 2005). In Agave –with the exception of A. fourcroydes 'Kitam ki', the opening patterns between 19:00 h and 7:00 h when sunlight is already present, can be influenced by the frequent irrigation of plantations, and conditions of culture can result in metabolic and physiological differences of CAM plants. In Opuntia elatior , demonstrate that C 3 photosynthesis, drought-stress-related facultative CAM, and developmentally controlled constitutive CAM can all contribute to the early growth of O. elatior (Winter et al. 2011), it is possible that under non-stressful conditions and frequent watering members of the class Magnoliopsida ( Agave and Opuntia ), showing a C3 metabolic behavior. The coincidence in an increase in the expression of the PEPCK and PEPC gene during the evaluated nocturnal hours (23h and 3h; Fig. 12 and 13), open the possibility of coordinated regulation between both enzymes. Also, regulation of rubisco activity goes hand in hand with a negative coordination of PEPC regulation, suggesting a complex co-regulation of both carboxylases compete for CO 2 during the early morning hours (Bailey et al. 2007; O’Leary et al. 2011; Deng et al. 2016). The highest values in RbcL expression obtained in this work, coincided at 11:00 h and 15:00 h, times in which PEPC expression was lower. In addition, the differences in 3D structure of RbcL in some accessions of the analyzed plants suggest the possible presence of isoforms that code for the RbcL small subunit. Also, the wide diversity in aminoacid substitutions found in PEPC and RbcL of the analyzed plants could reflect the presence of isoforms of this enzyme, which could result from the evolution of CAM metabolism in plants (Borland et al. 2014; Heckmann et al. 2016; Valegard et al. 2018). For the evolution of C3 to C4 to be carried out it is necessary that less mutational changes are present in the original C3 genes (Williams et al. 2012; Christin et al. 2013, 2014, 2015; Heckmann et al. 2016) and the isoforms take on greater importance (O’Leary et al. 2011; Deng et al. 2016; Bracher et al. 2017). Polyploidy is a primary source that can lead to the neofunctionalization of genes, and according to physiological Agave data may present C3-CAM metabolism that would indicate the possibility of still conserving original C3 genes, which according to environmental conditions could favor the expression of C4 syndrome, responding better under conditions of heat, aridity, salinity and high light (Sage et al. 2012, 2014) Heyduk et al. (2016), resolving the phylogeny of the subfamily Agavoideae, ancestral state reconstruction shows three independent origins of CAM in the group. These origins are associated with a shift in climate space toward warmer, drier habitats. The large genera of Agave and Yucca have a center of diversity in the southwestern deserts of North America, however a number of species have distributions outside of the iconic desert range. These derived species may suggest that ancestrally the Agavoideae was composed of non-desert dwellers and established lineages migrated into more arid regions after an early radiation within the group. A movement into arid regions would require that those desert regions were in place already, and that species that moved there had an ability to grow in arguably some of the harshest conditions on the planet. In addition, morphological data (3D venation and large cells) indicated that the last common ancestor of Yucca and Agave was C3 with CAM-like leaf anatomy and Sage (2002) suggested that leaf and cell succulence may arise first in a C3 ancestor, followed by evolution of PEPC function to recapture respired CO 2 , eventually leading to circadian control and full-fledged CAM function (Sage 2004; Sage et al. 2012). The physiological and genetic data found in this work suggest that derived from the great diversity of the genus, it is possible to find species of Agave L., C3-CAM intermediate range. Further work on whether diversification rates vary between C3 and CAM lineages will give insight into how the evolution of CAM might be promoting biodiversity in the Agavoideae. Also, future studies of water stress in these Agave accessions could be a near goal that could help to sustain whether polyploid agave plants could tolerate water stress (Tamayo-Ordoñez et al. 2016b; Tamayo-Ordoñez et al. 2018a). According to the obtained physiological and genetic results, apparently Agave can behave like C3-CAM (Optional CAM). It seems that the nocturnal intake of CO 2 achieved by opening its stomata can be made at night or during the day, depending on the conditions of its cultivation. It is possible that these facultative CAM plants maintain a positive balance and present an improvement in WUE and reduced photorespiration (Andrade et al. 2007) positioning Agave as a promising model for different biotechnological applications in the face of global climate change (Tamayo-Ordoñez et al. 2018b). Conclusion CAM plants, such as Agave L., could be used for sustainable production on irrigated lands using up to 80% less water to produce similar amounts of biomass compared with C3 species. Thus, research emphasizes that according to the genetic and physiological differences found in Agave L., it is possible that the genus harbors species with C3-CAM metabolism, suggesting the possibility of still conserving ancestral C3 genes, which according to environmental conditions could favor the expression of C4 syndrome, which could result in C4 evolution, under abiotic stress (heat, aridity, salinity and high light. This opens up the possibility of expanding the agricultural uses of these CAM specie, in the face of this future climate change, with high priority to ensure that food, feed, and fiber needs are in future warmer climates with diminishing arable land and water resources. Additionally, polyploidy agaves showed physiological and genetic changes, which could help them to respond differently according to the growing environment, facilitating its adaptation in environments where water is a limiting factor. Declarations Acknowledgements The authors wish to express their gratitude to the staff in charge of Germplasm Bank of the Scientific-Technological Park of Yucatán (GB-PCTY) and Regional Roger Orellana-CICY Botanical Garden (RG-CICY) for the facilities granted for the collection of plant material. Funding. This work was supported by the National Council of Science and Technology of Mexico (CONACYT) with the Science Projects, CB-50268 and CB-155356 Conflict of Interest: The authors declare that they have no conflict of interest. Author contributions: Conceptualization, Y.J.T.-O., and M.C.T.O.; methodology, B.A.A.-G., F.L.S.T., F.A.T.O.; formal analysis, L.C.R.Z., and F.A.B.P. investigation, Y.J.T.-O., and M.C.T.-O.; writing—original draft preparation, Y.J.T.-O., B.A.A.G, and F.A.T.O; writing—review and editing, V.H.R.G, L.C.R.Z, and F.L.S.T. All authors have read and agreed to the published version of the manuscript. References Aguirre-Dugua X, Eguiarte, LE (2013) Genetic diversity, conservation and sustainable use of wild Agave cupreata and Agave potatorum extracted for mezcal production in Mexico. J Arid Environ 90:36–44. https://doi.org/10.1016/j.jaridenv.2012.10.018 Andrade JL, de la Barrera E, Reyes-García C et al. (2007). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4284238","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":297106984,"identity":"ef2f0b5e-821e-4ea3-8780-7652d71a09d4","order_by":0,"name":"Benjamín Abraham Ayil Gutierrez","email":"","orcid":"","institution":"Instituto Politécnico Nacional","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Benjamín","middleName":"Abraham Ayil","lastName":"Gutierrez","suffix":""},{"id":297106985,"identity":"12d9c3dc-a1bd-4e53-944c-0e3b6fd35cbd","order_by":1,"name":"Felipe Lorenzo Sanchez Teyer","email":"","orcid":"","institution":"Centro de Investigacion Cientifica de Yucatan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"Lorenzo Sanchez","lastName":"Teyer","suffix":""},{"id":297106986,"identity":"cb6ac683-7250-42ab-b988-5aded030443e","order_by":2,"name":"Luis Carlos Rodriguez Zapata","email":"","orcid":"","institution":"Centro de Investigacion Cientifica de Yucatan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luis","middleName":"Carlos Rodriguez","lastName":"Zapata","suffix":""},{"id":297106988,"identity":"41330ae8-cd86-4af1-804a-0fd5f3d7f079","order_by":3,"name":"Felipe Barredo Pool","email":"","orcid":"","institution":"Centro de Investigacion Cientifica de Yucatan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"Barredo","lastName":"Pool","suffix":""},{"id":297106991,"identity":"67dc939c-e482-4233-a133-406cd4fc3a4e","order_by":4,"name":"Victor Hugo Ramos Garcia","email":"","orcid":"","institution":"Autonomous University of Tamaulipas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Victor","middleName":"Hugo Ramos","lastName":"Garcia","suffix":""},{"id":297106995,"identity":"dafdb47b-0093-4e3a-b5fe-4b2c7cd13e31","order_by":5,"name":"Francisco Alberto Tamayo Ordoñez","email":"","orcid":"","institution":"Autonomous University of Carmen","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"Alberto Tamayo","lastName":"Ordoñez","suffix":""},{"id":297106999,"identity":"49068d8d-fb80-46a7-ba03-c466e1ccfd7b","order_by":6,"name":"Yahaira de Jesus Tamayo Ordoñez","email":"","orcid":"","institution":"Instituto Politécnico Nacional","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yahaira","middleName":"de Jesus Tamayo","lastName":"Ordoñez","suffix":""},{"id":297107003,"identity":"0e45a4dd-42a8-4855-9639-49d061407ce4","order_by":7,"name":"Maria Concepcion Tamayo Ordoñez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBADOSA2ADEYGwiqZYNQxqRrSWwgWovu/OanG3622aRvOH5444MfDDayGw5wp0ng02J2jM3sZm9bWu6GM2nFhj0MacYbDvBuNsCvhcHsBm/b4dwNB3LMpBkYDicCtWx8gF8L+7ebf9sOpxucf2P+m4HhP0jLhgP4tfCY3QbakmBwI8eMmYHhADG25JTdljmXZjjzxrNiyR6DZOOZhwn55fDxbTfflNnI851P3vjhR4WdbN/x3m14QwwMGNlgLJDxzATVg8AfolSNglEwCkbBSAUATk1SiI5OAXAAAAAASUVORK5CYII=","orcid":"","institution":"Autonomous University of Coahuila","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Concepcion Tamayo","lastName":"Ordoñez","suffix":""}],"badges":[],"createdAt":"2024-04-17 23:44:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4284238/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4284238/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55793646,"identity":"ef04b90a-eb03-4d80-8f79-219aa4ed539c","added_by":"auto","created_at":"2024-05-03 10:10:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":213561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of genetic variability of polyploid species of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAgave\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eL. by AFLP. \u003c/strong\u003eFor the evaluation of the genetic distance, the coefficient NEI72 was used. In the dendrogram, the accessions were labeled according to Table 1.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/3dafe6e4b592ea5a403a63a8.png"},{"id":55793648,"identity":"5af20d4f-b556-41bd-be03-3f18705eca4b","added_by":"auto","created_at":"2024-05-03 10:10:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":145170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic relationship of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAgave\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e L. species\u003c/strong\u003e. For the correlation of the \u003cem\u003eAgave \u003c/em\u003eaccessions, the CORR coefficient of the statistical program NTSys-PC (version 2.1) was used. In the allele correlation analysis, \u003cem\u003eAgave\u003c/em\u003e accessions were labeled according to Table S1.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/2da2763761f6886b913b6591.png"},{"id":55794156,"identity":"452db4ca-97ad-417c-9159-570d06fcd993","added_by":"auto","created_at":"2024-05-03 10:18:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1366941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic trees based on aminoacid partial sequences of NADH and rbcL enzymes in plants.\u003c/strong\u003e A) Analysis of NADH enzyme (960 bp ~320 aa) were included 40 and 3 accessions belonging to the order Asparagales and to the Amaryllidaceae family. B) Analysis of rbcL enzyme (348 bp ~116 aa), were included 32 and 14 accessions belonging to Liliopsida and Magnoliopsida classes. Phylogeny was reconstructed by maximum likehood and cluster confidence was tested by 1000 bootstrap iterations. The amino acid sequences were aligned with Aligment Explorer/CLUSTALW program and the software Genetic and Molecular Evolution Analyses (MEGA versión 6.0). The sequences of amino acids corresponding to the different varieties of \u003cem\u003eAgave\u003c/em\u003e L. are indicated by circles of blue color.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/c0b64a600725287c579b5b1c.png"},{"id":55793647,"identity":"94223a9b-a689-41f7-b125-83aa5e287415","added_by":"auto","created_at":"2024-05-03 10:10:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1354627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic trees based on aminoacid partial sequences of PEPC and PEPCK enzymes in plants.\u003c/strong\u003e A) Analysis of PEPC enzyme (846bp ~275 aa) were included 17 and 18 accessions belonging to Liliopsida and Magnoliosida classes. B) Analysis of PEPCK enzyme (870 bp ~290 aa) were included 14 and 31 accessions belonging to Liliopsida and Magnoliopsida classes. Phylogeny was reconstructed by maximum likehood and cluster confidence was tested by 1000 bootstrap iterations. The amino acid sequences were aligned with Aligment Explorer/CLUSTALW program and the software Genetic and Molecular Evolution Analyses (MEGA versión 6.0). The sequences of amino acids corresponding to the different varieties of \u003cem\u003eAgave\u003c/em\u003e L. are indicated by circles of blue color.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/fec87ce0e6e8419246d2129c.png"},{"id":55793658,"identity":"2802be60-e8f6-4f4b-b588-a643c66742bb","added_by":"auto","created_at":"2024-05-03 10:10:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":420777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein structures of NADH enzyme in members belonging to the Asparagaceae and Amaryllidaceae families. \u003c/strong\u003eA) Complete structure of NADH-Quinone-Oxidoreductase from \u003cem\u003eEscherichia coli\u003c/em\u003e. Modeling of three-dimensional structure of the partial enzyme NADH-Quinone-Oxidoreductase subunit L from B) \u003cem\u003eEscherichia coli\u003c/em\u003e), C) \u003cem\u003eAgave tequilana\u003c/em\u003e Weber ‘Azul’ (submitted) and D) \u003cem\u003eAllium thunbergii \u003c/em\u003eG. Don (AGL72522.1) they are illustrated.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/889bb9e7c5f331919d72d3c9.png"},{"id":55793649,"identity":"588b9392-589f-4f71-872d-8b83bebf4aa6","added_by":"auto","created_at":"2024-05-03 10:10:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":451465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein structures of phosphoenolpyruvate carboxylase (PEPC) enzyme in members belonging to the Liliopsida and Magnoliopsida classes. \u003c/strong\u003eA) Complete structure of PEPC from \u003cem\u003eArabidopsis thaliana \u003c/em\u003eL. Modeling of three-dimensional structure of the partial enzyme PEPC from B) \u003cem\u003eArabidopsis thaliana \u003c/em\u003eL. (AEE75592.1), C) \u003cem\u003eGossypium arboreum\u003c/em\u003eL. (XP_017611070.1), D) \u003cem\u003eAnanas comosus \u003c/em\u003eL. (CAC84929.1) and E) \u003cem\u003eAgave tequilana\u003c/em\u003eWeber ‘Azul’ (submitted) they are illustrated.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/89025f1870d073be01f7f7aa.png"},{"id":55793659,"identity":"cfb4b8c8-bb5c-4c24-90a5-07f70165fdfa","added_by":"auto","created_at":"2024-05-03 10:10:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":740864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein structures of phosphoenolpyruvate carboxykinase (PEPCK) enzyme in members belonging to the Liliopsida and Magnoliopsida classes. \u003c/strong\u003eModeling of three-dimensional structure of PEPCK enzyme from A)\u003cem\u003e Escherichia coli, \u003c/em\u003eB) \u003cem\u003eAsparagus officinalis \u003c/em\u003eL. (XP_020259720.1), C) \u003cem\u003eAgave tequilana\u003c/em\u003eWeber ‘Azul’ (submitted), D)\u003cem\u003e Brassica rapa \u003c/em\u003eL. (XP_009132424.1), E) \u003cem\u003eArachis duranensis \u003c/em\u003e(XP_015963313.1)and\u003cem\u003e \u003c/em\u003eF)\u003cem\u003e Arabidopsis thaliana L. \u003c/em\u003e(NP_195500.1).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/45662b041b772a41cd96670d.png"},{"id":55794155,"identity":"23ae8e54-c95e-4541-8153-7bb79547269d","added_by":"auto","created_at":"2024-05-03 10:18:24","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":412555,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein structures of RuBisCo enzyme in members belonging to the Liliopsida and Magnoliopsida classes. \u003c/strong\u003eA) Complete structure of RuBisCo from \u003cem\u003eOryza sativa \u003c/em\u003eL. Modeling of three-dimensional structure of the partial Ribose bisphosphate carboxylase small chain from B) \u003cem\u003eOryza sativa \u003c/em\u003eL (XP_015620270.1). C) \u003cem\u003eAgave tequilana \u003c/em\u003eWeber ‘Azul’ (submitted), D) \u003cem\u003eAegilops longissima \u003c/em\u003eSchweinf. \u0026amp; Muschl (BAA35153.1), E) \u003cem\u003eTragopogon pratensis \u003c/em\u003eL.\u003cem\u003e \u003c/em\u003e(ALJ30132.1) and F) \u003cem\u003eArachis duranensis\u003c/em\u003e Krapov \u0026amp; W.C. Greg. (XP_015962947.1), they are illustrated.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/7e84c48e691de22d6e379c55.png"},{"id":55793663,"identity":"a58c1064-7ead-4627-9572-05eb1931860d","added_by":"auto","created_at":"2024-05-03 10:10:24","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2110633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScanning electron microscopy of stomata during temporary course in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAgave \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eL. \u003c/strong\u003eI-VIII) \u003cem\u003eA. angustifolia\u003c/em\u003e Haw. ‘Marginata’ (2n = 2x = 60) for the adaxial (I-IV) and abaxial (V-VIII) epidermis analyzed at 19:00 h (I and V), 23:00 h (II and VI), 3:00 h (III and VII) and 7:00 h (IV and VIII). XI-XVI) \u003cem\u003eA. angustifolia\u003c/em\u003e Haw. ‘Chelem ki’ (2n = 6x = 180) for the adaxial (IX-XII) and abaxial (XIII-XVI) epidermis analyzed at 19:00 h (IX and XIII), 23:00 h (X and XIV), 3:00 h (XI and XV) and 7:00 h (XII and XVI). XVII-XXIV) \u003cem\u003eA. fourcroydes\u003c/em\u003e Lem. ‘Sac ki’ (2n = 5x = 150) for the adaxial (XVII-XXIV) and abaxial (XVII-XX) epidermis analyzed at 19:00 h (XVII and XXI), 23:00 h (XVIII and XXII), 3:00 h (XIX and XXIII) and 7:00 h (XX and XXIV). XXV-XXXII) \u003cem\u003eA. angustifolia\u003c/em\u003e Haw. ‘Chelem ki’ (2n = 6x = 180) for the adaxial (XXV-XXXVIII) and abaxial (XXIX-XXXII) epidermis analyzed at 19:00 h (XXV and XXIX), 23:00 h (XXVI and XXX), 3:00 h (XXVII and XXXI) and 7:00 h (XXVIII and XXXII). Bars measure 100 µM, respectively.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/eb9f9b3c16f36710786102d6.png"},{"id":55793655,"identity":"b33882f9-dac9-41af-bfb9-e85205e0ee3a","added_by":"auto","created_at":"2024-05-03 10:10:24","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":125236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStomata area (µM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e) of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. fourcroydes\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Lem. and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. angustifolia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Haw.\u003c/strong\u003e \u003cstrong\u003eduring temporary course. \u003c/strong\u003eThe analysis was carried out at times of 19:00 h, 23:00 h, 3:00 h, 7:00 h. Lowercase letters (a and b) indicate significantly different values (Student’s t-test; p \u0026lt; 0.05). Values are averages of triplicates obtained from analysis of 30 stomata in 3 leaves of three plants of each accession ± standard error.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/cc2b317929493cbb7afdaee9.png"},{"id":55793651,"identity":"88e54c09-7c33-431c-883e-e5bf0054bdb6","added_by":"auto","created_at":"2024-05-03 10:10:24","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":316760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\n\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuprastomatic cavity area (µM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e) of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. fourcroydes\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Lem. and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. angustifolia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Haw.\u003c/strong\u003e \u003cstrong\u003eduring temporary course. \u003c/strong\u003eThe analysis was carried out at times of 19:00 h, 23:00 h, 3:00 h, 7:00 h.\u003cstrong\u003e \u003c/strong\u003eLowercase letters (a, b, c, d or e) indicate significantly different values (Student’s t-test; p \u0026lt; 0.05). Values are averages of triplicates obtained from 3 leaves of five plants of each accession ± standard error.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/3209d3f7ffd608171cacc098.png"},{"id":55794157,"identity":"c91fc96a-309f-41cc-976c-f3cb6c38ed0a","added_by":"auto","created_at":"2024-05-03 10:18:24","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":25188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression relative of the phosphoenolpyruvate carboxylase (PEPC) gene evaluated by RT-qPCR in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAgave\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e L.\u003c/strong\u003e The analysis was carried out on plants adapted in the BGR-RO (A) and PCTY (B) during a temporary course during daytime hours (11:00 h and 15:00 h) and at night (23:00 h and 3:00 h). All values are averages of three replicates ± SE and lowercase letters (a, b, c, d or e) indicate significantly different values (Student’s t-test; p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/3bb630f8639296c7725d1b94.png"},{"id":55793656,"identity":"939c78a4-58e8-4d13-b970-ae593408a49f","added_by":"auto","created_at":"2024-05-03 10:10:24","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":33427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression relative of the phosphoenolpyruvate carboxykinase (PEPCK) gene evaluated by RT-qPCR in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAgave\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e L.\u003c/strong\u003e The analysis was carried out on plants adapted in the BGR-RO (A) and PCTY (B) during a temporary course during daytime hours (11:00 h and 15:00 h) and at night (23:00 h and 3:00 h). All values are averages of three replicates ± SE and lowercase letters (a, b, or c) indicate significantly different values (Student’s t-test; p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure13.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/eddbe6809578caca4f3061b3.png"},{"id":55793657,"identity":"47b2dc66-7d38-4556-a4ba-bc08b121e6fd","added_by":"auto","created_at":"2024-05-03 10:10:24","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":31558,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression relative of the rbcL gene evaluated by RT-qPCR in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAgave\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e L.\u003c/strong\u003e The analysis was carried out on plants adapted in the BGR-RO (A) and PCTY (B) during a temporary course during daytime hours (11:00 h and 15:00 h) and at night (23:00 h and 3:00 h). All values are averages of three replicates ± SE and lowercase letters (a, b, or c) indicate significantly different values (Student’s t-test; p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure14.png","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/0a4be4af0235afadffd456e6.png"},{"id":71342337,"identity":"864c2fde-b01b-42ce-9cdb-42d098e11fd9","added_by":"auto","created_at":"2024-12-13 13:32:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9143380,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4284238/v1/07aa1594-ffa4-4cbe-ba09-26a5f5a3c738.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Physiological changes in stomata and regulation of genes involved in CAM metabolism in polyploid Agave L. ","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe introduce crassulacean acid metabolism (CAM) into C3 crops, with aim the increase plant water-use efficiency (WUE), has become relevant in recent years (Shen et al., 2022; Sonmez et al. 2022; Caferri and Bassi 2022;\u0026nbsp;Zotz et al. 2023). It is important to carry out studies to understand how genetic, biochemical, and physiological factors behave in CAM plants under stressfull environmental factors. this could explain the differences between CAM and C3 photosynthesis and improve to engineer CAM into C3 plants (Rebecca and Hirasawa 2022; Yamaga-Hatakeyama et al. 2022).\u0026nbsp;To perform this type of studies, \u003cem\u003eAgave\u003c/em\u003e L. is postulated as a good model, because this species can perform CAM under stressful environmental conditions (Gentry 1982; Liu et al. 2018) and the Agave genus harbors polyploid species that have been documented to be able to better tolerate and adapt to global climate change (Palomino et al. 2012, 2015; Tamayo-Ordo\u0026ntilde;ez et al. 2016b, 2018a).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAgave\u003c/em\u003e spp. performs CAM photosynthesis under drought and CO\u003csub\u003e2\u003c/sub\u003e enrichment. Gentry (1982), studied \u003cem\u003eAgave\u003c/em\u003e species from North America and to date research aimed at knowing the physiological and morphological adaptations related to CAM metabolism, only includes a small group of species analyzed (Nobel and Hartsock, 1978; Pimienta‐Barrios et al. 2001, 2005; Campos et al. 2014; Tamayo-Ordo\u0026ntilde;ez et al. 2018a). For example, in \u003cem\u003eAgave\u003c/em\u003e it has been described\u0026nbsp;that CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eexchange is affected by drought and CO\u003csub\u003e2\u003c/sub\u003e enrichment. For example; \u003cem\u003eA. americana,\u003c/em\u003e \u003cem\u003eA. tequilana\u003c/em\u003e, \u003cem\u003eA. asperrima\u003c/em\u003e Jacobi, \u003cem\u003eA. cupreata\u0026nbsp;\u003c/em\u003eTrel. et. A. Berger, \u003cem\u003eA. durangensis\u003c/em\u003e Gentry, and \u003cem\u003eA. salmiana\u003c/em\u003e Otto ex Salm-Dyck have been shown to affect their growth rate, biomass distribution, leaf thickness, and proline content at different water potential (\u0026Psi;). Species not adapted to dry, biomass production was inhibited (\u0026minus;3.5 MPa), but in species adapted to dry regions such as \u003cem\u003eA. tequilana\u003c/em\u003e, \u003cem\u003eA. durangensis\u003c/em\u003e, \u003cem\u003eA. lechuguilla\u003c/em\u003e Torr., and \u003cem\u003eA. salmiana\u003c/em\u003e, the number of leaves and plant coverage were maintained (Ram\u0026iacute;rez-Tobias et al. 2014). Also, \u003cem\u003eA. Americana\u003c/em\u003e and \u003cem\u003eA. angustifolia\u003c/em\u003e, they have shown that during drought periods 98% and 85% of CO\u003csub\u003e2\u003c/sub\u003e is fixed during the first eight night hours, similar to that reported in plants that perform strong CAM (Holtum and Winter 2014; Winter et al. 2014;\u0026nbsp;Males and Griffiths 2017). Such physiological behavior is stimulated by CO\u003csub\u003e2\u003c/sub\u003e enrichment (800 ppm) during the night. At CO\u003csub\u003e2\u003c/sub\u003e concentrations of 800 ppm, fixation in \u003cem\u003eA. angustifolia\u003c/em\u003e increases (62%) relative to low values of CO\u003csub\u003e2\u003c/sub\u003e fixed (200 and 400 ppm) (Winter et al. 2014). This suggests that \u003cem\u003eAgave\u003c/em\u003e species can be better explored to understand why CAM plants increase nocturnal CO\u003csub\u003e2\u003c/sub\u003e assimilation under higher CO\u003csub\u003e2\u003c/sub\u003e concentrations and future to be able to propose them for\u0026nbsp;introducing new traits aimed at creating crop varieties with enhanced CO2 capture and water- and light-use efficiency.\u003c/p\u003e\n\u003cp\u003eOn the other hand, \u003cem\u003eAgave\u003c/em\u003e spp., has shown present ample tolerance to different stressful conditions,\u0026nbsp;emphasizing the importance of carrying out studies for the selection of elite individuals with physiological and morphological differences that allow them to adapt even better to abiotic stresses.\u0026nbsp;\u003cem\u003eAgave\u003c/em\u003e can tolerate drought and high temperature through of control of water loss through leaf surfaces and CO\u003csub\u003e2\u003c/sub\u003e exchange (Pimienta-Barrios et al. 2001; Holtum and Winter 2014; Winter et al. 2014; Tamayo-Ordo\u0026ntilde;ez et al. 2018b). Added to this, polyploidy has proved important role in the evolution and speciation of the genus (Wolfe 2001; Tamayo-Ordo\u0026ntilde;ez et al. 2015, 2018a).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn Agave spp., the occurrence of polyploidy has caused DNA regions to be subjected to genetic events, including recombination, amplification, duplication, transposition, and gene loss, which could have resulted in the high variability and diversity of these genomic regions and favoring the formation of new genes that may be fixed in the course of evolution according to the climatic condition and geographical distribution of the species \u0026nbsp;(Tamayo-Ordo\u0026ntilde;ez et al., 2018a). This genus has been shown to inhabit geographical areas where there are high temperatures and water scarcity, this has contributed to the development of morphological (thorns, leaf succulence, stomatal density) and physiological (CAM Metabolism) characters, related to withstanding drought and high temperatures. In addition, in the polyploid species of \u003cem\u003eAgave\u0026nbsp;\u003c/em\u003eL., due to the increase in the genome, it is possible that there are more isoforms of protein-coding genes involved in CAM metabolism that could be functionally specialized. As a result of these genetic, morphological, and physiological changes,\u0026nbsp;it is possible that the polyploid species of \u003cem\u003eAgave\u003c/em\u003e L. adapt in a better way to global warming and drought (Sattler et al. 2016;\u0026nbsp;Tamayo-Ordo\u0026ntilde;ez et al. 2018b).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFew studies have been done about the tolerance to environmental stress of \u003cem\u003eAgave\u003c/em\u003e plants with different ploidy levels to find out if polyploids are more stress tolerant or have favorable morphological and physiological features relative to lower ploidy level plants. In that context, Tamayo-Ordo\u0026ntilde;ez et al. (2016b) focused in knowing if polyploid species of \u003cem\u003eAgave\u0026nbsp;\u003c/em\u003eare more sensitive to abiotic and biotic stress, and in analyzing the morphological, physiological, and molecular factors involved in their improved responses. These authors described that the size of stomata and the suprastomatal cavity increases according to higher levels of ploidy in the accessions analyzed (Tamayo-Ordo\u0026ntilde;ez et al. 2016b) and the stomatal density is lower as the level of ploidy increases in \u003cem\u003eAgave\u003c/em\u003e L. Suggesting that polyploid species of \u003cem\u003eAgave\u003c/em\u003e spp. could perform CO2 fixation more efficiently compared to their counterparts with lower ploidy levels. In addition, the differences found in the stomatal density could result in a different physiological response to drought among the analyzed accessions, allowing certain accessions of \u003cem\u003eAgave\u003c/em\u003e respond differentially during prolonged periods of drought. \u0026nbsp;In \u003cem\u003eBetula papyrifera\u003c/em\u003e Marsh. and \u003cem\u003eA. thaliana\u003c/em\u003e Heynh in Holl\u0026amp; Heynh have been shown to have fewer stomata per unit area and smaller stomatal indices than their diploid counterparts (Li et al.1996, 2012). Stomatal indices according to ploidy numbers have also been related to adaptation to stress and previous studies described a direct relation between water deficit, stomatal dimensions, and ploidy level affecting the plant\u0026rsquo;suptake of water (Li et al. 1996, 2012; Balao et al. 2011). In \u003cem\u003eAgave\u003c/em\u003e L., adaptations preventing physiological damages due to draught include nocturnal assimilation of CO2, thick cuticles, low stomata density, and succulent leaves. The latter two adaptations enable water stored in the leaf parenchyma to continuously move to the chlorenchyma during dry periods (Pimienta-Barrios et al. 2005), thus conferring the plants the capacity for with standing up to seven years of draught (Stewart 2015).\u003c/p\u003e\n\u003cp\u003eIn this research, the goals was to relate the ploidy levels of \u003cem\u003eAgave\u003c/em\u003e spp., with differences in CAM induction, for it determine if there are differences in genetic variability in polyploid species, Know if there is a differential physiological mechanism in the fixation of CO\u003csub\u003e2\u003c/sub\u003e (related to the stomatal opening and closure), it could be related to adaptation to stress by drought, and know if there is a differential genetic regulation of genes coding for enzymes involved in CAM metabolism. The obtained results indicate that polyploid species of \u003cem\u003eAgave\u003c/em\u003e reflect physiological adaptations and genetic changes that could help them to better respond against global warming.\u003c/p\u003e"},{"header":"2.\tMaterial and methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.1 Plant material\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor all the studies reported in this investigation, three representative individuals of each varieties analyzed belonging to three species (\u003cem\u003eAgave tequilana\u003c/em\u003e Weber, \u003cem\u003eAgave fourcroydes\u003c/em\u003e Lem. and \u003cem\u003eAgave angustifolia\u0026nbsp;\u003c/em\u003eHaw) were included in this research (Table S1). The selected \u003cem\u003eAgave\u003c/em\u003e accessions included varieties and variants (not registrated yet) with different ploidy levels (Table S1). \u0026nbsp;All these accessions were directly collected from the field and adapted to two ecoregions: the Regional Roger Orellana-CICY Botanical Garden (RO-CICY) in the city of Merida, and the Germplasm Bank of the Scientific-Technological Park of Yucat\u0026aacute;n (GB-PCTY),\u0026nbsp;located in the Sierra Papacal,\u0026nbsp;20 km northwest of Merida (21\u0026deg; 07\u0026apos; 20\u0026quot; N, 89\u0026deg; 43\u0026apos; 41\u0026quot; W).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe accessions from the GB-PCTY belong to the \u003cem\u003eAgave\u003c/em\u003e spp. collection described by Pulido-Salas and Garc\u0026iacute;aMar\u0026iacute;n (2003) and their ploidy level was characterized as a part of this research. Herbarium specimens from leaves of each accession were made and deposited in the CICY herbarium (Table S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.2 Variability of Agave L. by AFLP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe AFLP method was performed following Tamayo-Ord\u0026oacute;\u0026ntilde;ez et al. (2012). For the selection of the best four sets of primers, we initially evaluated 35 combinations of primers (Table S2). AFLP reactions with selective primers were performed with a touchdown PCR program for most primer sets. The amplification conditions were carried out according to Tamayo-Ordo\u0026ntilde;ez et al. (2012). PCR products were electrophoresed in a CEQ 8800 sequencer (Perkin\u0026ndash;Elmer Inc., Foster City, CA). The obtained electropherograms were analyzed using the software GeneMarker v.1.75 (Perkin-Elmer, Inc., Boston, MA)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll calculations were performed using the NTSYS-pc 2.1 (Exeter Software Co., New York) software (Rohlf 2000). Only strong, reproducible, and clearly distinguished bands were used in a binary matrix. In order to find out if the AFLP markers could discriminate between polyploid accessions, the unweighted pair group was analyzed in all the studied accessions with UPGMA method, and genetic distances among accessions were calculated according to Nei and Li (1979). The reliability and robustness of the dendrograms were tested by bootstrap analysis with 1000 replications to assess branch support using FreeTree software (Pavl\u0026iacute;ek et al. 1999). Analyses of percentages of polymorphism, index of similarity, and marker bands were made with the Free Tree (Pavl\u0026iacute;ek\u003cem\u003e\u0026nbsp;\u003c/em\u003eet al. 1999) software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eDetermination of stomatal aperture and closure\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the first instance, in order to know if there are differences in the opening and closing of stomata during the nocturnal period (19:00h at 7:00h) of CAM metabolism in polyploid species compared to their counterparts with lower level of ploidia, a determination of stomatal aperture and closure was carried out, including two varieties with different ploidy level representative of the species \u003cem\u003eA. angustifolia\u003c/em\u003e Haw. and \u003cem\u003eA. fourcroydes\u003c/em\u003e Lem.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSamples of leaves (1 cm\u003csup\u003e3\u003c/sup\u003e) from the adaxial and abaxial epidermis were collected at 19:00 h, 23:00 h, 3:00 h and 7:00 h in two ecoregions (RO-CICY and GB-PCTY). The preparation of the samples is made according Tamayo-Ordo\u0026ntilde;ez et al. (2016b). The samples were then mounted on metallic stubs with carbon conductive adhesive tape (Electron Microscopy Science) and sputter coated with a 150 \u0026Aring; gold layer (Denton Vaccum Desk II). Length of the guard cells and suprastomatic cavity area, stomatal size and density were calculated for the abaxial and adaxial epidermis at a magnification of 100x (0.1213 mm\u003csup\u003e2\u003c/sup\u003e). Counts and measurements were made in ten fields of each leaf for each accession. Sample analysis and image recording were made using a scanning electron microscope (Jeol, JSM-6360LV). The gathered data were subjected to statistical analysis by Tukey tests evaluated at P\u0026gt;0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ePhylogenetic analysis of NADH, RbcL, PEPC, and PEPCK genes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNADH gene (Nad4 subunit dehydrogenase 4) amplification was conducted using the primers previously reported by Tamayo-Ordo\u0026ntilde;ez et al. (2012). PEPC, PEPCK, and RbcL primers were designed from \u003cem\u003eAnanas comosus\u003c/em\u003e (AJ312631.1), \u003cem\u003eArabidopsis\u003c/em\u003e (NM_119948.4) and \u003cem\u003eAgave tequilana\u003c/em\u003e (GW667494.1), respectively (Table S3). PCR reactions were carried out in a volume of 50 \u0026mu;L containing 25 ng of genomic DNA, 130 \u0026mu;M dNTPs, 15 \u0026mu;M of each primer, 2.5 units of \u003cem\u003eTaq\u0026nbsp;\u003c/em\u003epolymerase, and 1X PCR reaction buffer (Life Technologies, Rockville, MD, U.S.A.) with 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e. PCR conditions included one cycle of 3 min at 94 \u0026ordm;C for initial denaturation, followed by 35 cycles of 1 min at 94 \u0026ordm;C, 1 min at alignment temperature (tm) according to each set of primers used (Table S3), 1 min at 72 \u0026ordm;C, and finally, 7 min at 72 \u0026ordm;C. PCR products were separated by electrophoresis in 1.2% agarose gels. Purification, cloning and sequencing of PCR products was carried out according to Tamayo-Ordo\u0026ntilde;ez et al. (2016b).\u003c/p\u003e\n\u003cp\u003eThe nucleotide sequences were aligned (BLASTX) and compared with those in the GenBank database.\u0026nbsp;DNAMAN version 4.0\u0026nbsp;was used to translate these sequences and to identify the open reading frame. Predicted amino acid (aa) sequences relative to the\u0026nbsp;NADH, RbcL, and PEPC nucleotide sequences were used in combination with related sequences retrieved from GenBank to build a phylogenetic aligning. Conserved aligned regions (\u0026gt;90%) were selected in all sequences,\u0026nbsp;phylogenetic analysis was performed in software MEGA version 6.0 (Tamura et al. 2013). Domains of interest for both proteins were identified with the CDD (Derbyshire et al. 2015).\u003c/p\u003e\n\u003cp\u003e2.5\u0026nbsp;\u003cstrong\u003e\u003cem\u003eTertiary structure (3D) of NADH,\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eRbcL, PEPC, and PEPCK\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;enzymes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTertiary structure analysis of NADH, RbcL, PEPC, and PEPCK was performed with SWISS-MODEL (Biasini et al. 2014). Values of sequence identity (%), Global Model Quality Estimation and Qualitative Model Energy Analysis, were considered. As references, sequences corresponding to NADH- quinone-oxidoreductase subunit L from \u003cem\u003eEscherichia coli\u003c/em\u003e (Efremov\u0026nbsp;and Sazanov\u0026nbsp;2011),\u0026nbsp;ribose bisphosphate carboxylase small chain from\u0026nbsp;\u003cem\u003eOryza sativa\u003c/em\u003e (Matsumura et al. 2012)\u003cem\u003e,\u0026nbsp;\u003c/em\u003ephosphoenolpyruvate carboxylase from\u003cem\u003e\u0026nbsp;Arabidopsis thaliana\u0026nbsp;\u003c/em\u003eHeynh in Holl and Heynh\u0026nbsp;(unpublished)\u003cem\u003e\u0026nbsp;\u003c/em\u003eand phosphoenolpyruvate carboxykinase from\u0026nbsp;\u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003e(Sudom et al. 2003)\u0026nbsp;were included.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.6 Determination of relative expression of the NADH, RbcL, PEPC, and PEPCK\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA isolation and cDNA synthesis were conducted according to Tamayo-Ord\u0026oacute;\u0026ntilde;ez et al. (2015). The PEPC and PEPCK primers used for relative expression analysis were those reported by\u0026nbsp;Arag\u0026oacute;n\u0026nbsp;et al. (2013). For phylogenetic analyses, a RbcL primer was designed from \u003cem\u003eAgave tequilana\u003c/em\u003e (GW667494.1), a NADH primer was designed from the isolated NADH sequence, and, as reference genes, the\u0026nbsp;18S rDNA genes were used according to Tamayo-Ordo\u0026ntilde;ez et al. 2015 (Table S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmplifications of the NADH, PEPC, PEPCK, and RbcL genes were carried out as described above and with the same PCR amplification conditions used for the phylogenetic analysis. The melt curve analysis and negative controls for the reference and target genes were always included in the experiments in order to eliminate DNA contamination. The relative expression of each gene was determined by the ∆∆Cq method between the target (NADH, PEPC, PEPCK and RbcL) and reference (18S rDNA) genes\u0026nbsp;(Tamayo-Ordo\u0026ntilde;ez et al., 2015), by the following equation: Relative expression = (E\u003csub\u003eref\u003c/sub\u003e)\u003csup\u003eCtref\u003c/sup\u003e/(E\u003csub\u003etarget\u003c/sub\u003e)\u003csup\u003eCtarget\u003c/sup\u003e (Pfaffl 2001). All analyses included 3 biological replicates, each with three technical replicates. \u003c/p\u003e"},{"header":"3.\tResults ","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.1 Polyploidy and the genomic consequences in Agave\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMany authors have described that the increase of genome size in polyploid species is accompanied by genetic changes, resulting in greater variability and genetic diversity in polyploid plants (Chen 2010; Wendel 2000; Moghe and Shiu 2014; Tamayo-Ordo\u0026ntilde;ez et al. 2016a). The dendrogram obtained from the AFLP marker showed two clusters, cluster I having two subgroups denominated as A and B (Fig. 1). The subgroup A showed the clustering of hexaploid and diploid accessions of \u003cem\u003eAgave angustifolia\u003c/em\u003e (AAM1-RO, AAM2-RO, AAM3-RO, AAC1-RO, AAC2-RO, AAC3-RO, CHA1-PCTY, CHA5-PCTY and CHA6-PCTY), and the subgroup B included the diploid accessions of \u003cem\u003eA. tequilana\u003c/em\u003e (AT1-RO, AT2-RO, AT3-RO, 1444-2-PCTY, 1444-3-PCTY, and 0345b-PCTY; Fig. 1). Cluster II grouped the triploid and pentaploid accessions of \u003cem\u003eA. fourcroydes\u003c/em\u003e. According to the global variability, the AFLP marker indicated a closer genetic proximity between \u003cem\u003eA. angustifolia\u003c/em\u003e and \u003cem\u003eA. tequilana\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe analysis of similarity indexes and percentages of polymorphism in accessions belonging to the same species (considering ploidy level as an important factor) indicated that \u003cem\u003eAgave\u003c/em\u003e accessions containing polyploid varieties had low similarity indexes (0.76) compared to the results from the analysis including only diploid accessions like \u003cem\u003eA. tequilana\u0026nbsp;\u003c/em\u003e(2\u003cem\u003en\u003c/em\u003e = 2\u003cem\u003ex\u0026nbsp;\u003c/em\u003e= 60) and\u003cem\u003e\u0026nbsp;A. angustifolia\u0026nbsp;\u003c/em\u003e(2\u003cem\u003en\u003c/em\u003e = 2\u003cem\u003ex\u003c/em\u003e = 60) (0.83). The analysis of \u003cem\u003eA. angustifolia\u003c/em\u003e (2\u003cem\u003en\u003c/em\u003e = 2\u003cem\u003ex\u0026nbsp;\u003c/em\u003e= 60)\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eA. angustifolia\u0026nbsp;\u003c/em\u003e(2\u003cem\u003en\u003c/em\u003e = 6\u003cem\u003ex\u0026nbsp;\u003c/em\u003e= 180) showed percentages of polymorphism of 75 and a similarity index of 0.76, while accessions of \u003cem\u003eA. fourcroydes\u0026nbsp;\u003c/em\u003e(2\u003cem\u003en\u003c/em\u003e = 3\u003cem\u003ex\u0026nbsp;\u003c/em\u003e= 90) and \u003cem\u003eA. fourcroydes\u003c/em\u003e (2\u003cem\u003en\u003c/em\u003e = 5\u003cem\u003ex\u0026nbsp;\u003c/em\u003e= 150) showed percentages of polymorphism of 79 and a similarity index of 0.73 (Table 1). Apparently, polyploidy does have an effect on genome size and genetic variability in \u003cem\u003eAgave\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe analysis of correlation of alleles indicated that, the accessions of \u003cem\u003eA. tequilana\u003c/em\u003e cultivated in the RO-CICY share a greater proportion of alleles, similar in the same proportion as \u003cem\u003eA. angustifolia\u003c/em\u003e cultivated in both microclimates (RO-CICY and GB-PCTY). Likewise, the varieties of \u003cem\u003eA. fourcroydes\u003c/em\u003e apparently contain a narrow genetic germplasm, reflected in the conservation of genetic material (Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"8\" valign=\"top\"\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePolymorphism and index of genetic variation of \u003cem\u003eAgave\u003c/em\u003e L.\u0026nbsp;\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cstrong\u003eEspecie\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\" valign=\"top\"\u003e\u003cstrong\u003eTotal number of individuals\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\" valign=\"top\"\u003e\u003cstrong\u003eAccessions \u0026nbsp; \u0026nbsp; (number of individuals evaluated by each accession)*\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\" valign=\"top\"\u003e\u003cstrong\u003eTotal numbers of loci\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\" valign=\"top\"\u003e\u003cstrong\u003eTotal numbers of polymorphic loci\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\" valign=\"top\"\u003e\u003cstrong\u003eTotal numbers of common loci\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\" valign=\"top\"\u003e\u003cstrong\u003ePolymorphic percentage\u003csup\u003eƗ\u003c/sup\u003e\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\" valign=\"top\"\u003e\u003cstrong\u003eIndex of similarity\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cem\u003eAgave\u003c/em\u003e spp.\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e33\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\"\u003eATA-RO (3), AAM-RO (3), AAC-RO(3), AFK-RO(3), AFS-RO (3), AFY-RO (3),\u0026nbsp;1444-PCTY (2), 345b-PCTY (1), CHA-PCTY(3), KK-PCTY (3), SK-PCTY(3) and YK-PCTY (3)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\"\u003e201\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e182\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\"\u003e19\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\"\u003e90.54\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\"\u003e0.66\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cem\u003eAgave tequilana\u0026nbsp;\u003c/em\u003eWeber.\u003cem\u003e\u0026nbsp;\u0026nbsp;\u003c/em\u003e(2\u003cem\u003en\u003c/em\u003e = 2\u003cem\u003ex\u0026nbsp;\u003c/em\u003e= 60) and\u003cem\u003e\u0026nbsp;Agave angustifolia\u0026nbsp;\u003c/em\u003eHaw.\u003cem\u003e\u0026nbsp;\u003c/em\u003e(2\u003cem\u003en\u003c/em\u003e = 2\u003cem\u003ex\u003c/em\u003e = 60)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e9\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\"\u003eATA-RO (3), AAM-RO (3), 1444-PCTY (2), and 345b-PCTY (1)\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\"\u003e73\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e33\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\"\u003e40\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\"\u003e45.20\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\"\u003e0.83\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cem\u003eAgave tequilana\u0026nbsp;\u003c/em\u003eWeber.\u003cem\u003e\u0026nbsp;\u0026nbsp;\u003c/em\u003e(2\u003cem\u003en\u003c/em\u003e = 2\u003cem\u003ex\u003c/em\u003e= 60)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e6\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\"\u003eATA-RO (3), 1444-PCTY (2) and \u0026nbsp;345b-PCTY (1)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\"\u003e44\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e20\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\"\u003e28\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\"\u003e45.45\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\"\u003e0.85\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cem\u003eAgave angustifolia\u003c/em\u003e Haw.\u003cem\u003e\u0026nbsp;\u003c/em\u003e(2\u003cem\u003en\u003c/em\u003e = 2\u003cem\u003ex\u003c/em\u003e= 60)\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAgave angustifolia\u003c/em\u003e Haw.\u003cem\u003e\u0026nbsp;\u003c/em\u003e(2\u003cem\u003en\u003c/em\u003e = 6\u003cem\u003ex\u003c/em\u003e= 180)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e9\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\"\u003eAAM-RO (3), AAC-RO(3) and\u0026nbsp;CHA-PCTY(3)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\"\u003e82\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e62\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\"\u003e20\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\"\u003e75.60\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\"\u003e0.76\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cem\u003eAgave fourcroydes\u003c/em\u003e Lem.\u003cem\u003e\u0026nbsp;\u003c/em\u003e(2\u003cem\u003en\u003c/em\u003e = 3\u003cem\u003ex\u003c/em\u003e= 90) and \u003cem\u003eAgave fourcroydes\u003c/em\u003e Lem. (2\u003cem\u003en\u003c/em\u003e = 5\u003cem\u003ex\u003c/em\u003e= 150)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e12\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\"\u003eAFK-RO(3), AFS-RO (3), AFY-RO (3),\u0026nbsp;KK-PCTY (3), SK-PCTY(3) and YK-PCTY (3)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\"\u003e142\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e113\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\"\u003e29\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\"\u003e79.57\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\"\u003e0.73\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cem\u003eAgave tequilana\u003c/em\u003e Weber. \u0026lsquo;Azul\u0026rsquo; (2\u003cem\u003en\u003c/em\u003e = 2\u003cem\u003ex\u0026nbsp;\u003c/em\u003e= 60)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e6\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\"\u003eATA-RO (3),\u0026nbsp;1444-PCTY (2) and 345b-PCTY (1)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\"\u003e48\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e20\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\"\u003e28\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\"\u003e41.66\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\"\u003e0.85\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cem\u003eAgave angustifolia\u003c/em\u003e Haw \u0026lsquo;Marginata\u0026rsquo;\u003cem\u003e\u0026nbsp;\u003c/em\u003e(2\u003cem\u003en\u003c/em\u003e = 2\u003cem\u003ex\u003c/em\u003e = 60)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e3\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\"\u003eAAM-RO (3)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\"\u003e68\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e33\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\"\u003e35\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\"\u003e48.52\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\"\u003e0.81\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cem\u003eAgave angustifolia\u003c/em\u003e Haw. \u0026lsquo;Chelem ki\u0026rsquo; (2\u003cem\u003en\u003c/em\u003e = 6\u003cem\u003ex\u003c/em\u003e = 180)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e6\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\"\u003eAAC-RO(3)\u0026nbsp;and CHA-PCTY(3)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\"\u003e63\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e38\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\"\u003e25\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\"\u003e60.31\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\"\u003e0.77\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cem\u003eAgave fourcroydes\u003c/em\u003e Lem. \u0026lsquo;Kitam ki\u0026rsquo; (2\u003cem\u003en\u003c/em\u003e = 3\u003cem\u003ex\u003c/em\u003e= 90)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e6\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\"\u003eAFK-RO(3) and\u0026nbsp;KK-PCTY (3)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\"\u003e103\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e51\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\"\u003e52\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\"\u003e49.51\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\"\u003e0.81\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cem\u003eAgave fourcroydes\u003c/em\u003e Lem. \u0026lsquo;Sacki ki\u0026rsquo; (2\u003cem\u003en\u003c/em\u003e = 5\u003cem\u003ex\u003c/em\u003e= 150)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e6\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\"\u003eAFS-RO (3) and\u0026nbsp;SK-PCTY(3)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\"\u003e139\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e82\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\"\u003e49\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\"\u003e58.99\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\"\u003e0.74\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.28263103802672%\" valign=\"top\"\u003e\u003cem\u003eAgave fourcroydes\u003c/em\u003e Lem. \u0026lsquo;Yaax ki\u0026rsquo; (2\u003cem\u003en\u003c/em\u003e = 5\u003cem\u003ex\u003c/em\u003e= 150)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e6\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"29.08530318602261%\"\u003eAFY-RO (3) and\u0026nbsp;YK-PCTY (3)\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.858170606372045%\"\u003e101\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.735868448098664%\"\u003e57\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.708119218910586%\"\u003e44\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.810894141829394%\"\u003e56.43\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.7831449126413155%\"\u003e0.77\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e*The specifications of the collections are described in the table S1. \u003csup\u003eƗ\u003c/sup\u003ePolymorphic percentage= (Total numbers of polymorphic loci/ Total numbers of loci)*100\u003c/p\u003e\n\u003cp\u003ePolyploidy is a very common phenomenon in species of angiosperms and vascular plants (Moghe and Shiu 2014; Wendel 2000). Nowadays, the ancestors of many polyploid plant species, like those in \u003cem\u003eAgave\u003c/em\u003e, remain to be unknown. The genus \u003cem\u003eAgave\u003c/em\u003e includes triploid (e.g., \u003cem\u003eA. fourcroydes\u003c/em\u003e \u0026lsquo;Kitam ki\u0026rsquo; 2\u003cem\u003en\u003c/em\u003e=3\u003cem\u003ex\u003c/em\u003e=90) to octoploid species (e.g., \u003cem\u003eA. datylio\u0026nbsp;\u003c/em\u003e2\u003cem\u003en\u003c/em\u003e=8\u003cem\u003ex\u003c/em\u003e=240; Castorena-S\u0026aacute;nchez, 1990), however, despite having species with a wide range of ploidy level, most studies \u0026ndash;conducted in few species of the genus\u0026ndash; have focused on cytogenetic characterization. In addition, it is suggested that due to the presence of bimodal karyotype with a basic chromosome number of \u003cem\u003en\u003c/em\u003e=30 (5 long acrocentric chromosomes and 25 small metacentric or submetacentric chromosomes; Moreno-Salazar et al. 2007; McKain et al. 2012; Palomino et al. 2015) the genus is possibly of allopolyploid origin (McKain et al. 2012).\u003c/p\u003e\n\u003cp\u003eAllopolyploidy can be associated with an increased number of gene copies and, therefore, it implies the generation of redundant genes. These genes, mainly generated by duplication, are involved in epigenetic regulation, become specialized to perform a complementary function or may eventually be lost. The functional partitioning and differences in expression patterns of these redundant genes are associated to changes (physiological and phenotypic) (Tamayo-Ordo\u0026ntilde;ez et al. 2016) that confer advantages in allopolyploid species.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOtherwise, the genus \u003cem\u003eAgave\u003c/em\u003e is of recent origin (6-8 Mya and 1.5-3 Mya), has a high index of species diversity (0.32 to 0.56 species per million years) relative to angiosperms in general (0.089-0.07 species per million years), and according to the model for evolution of rDNA regions proposed by Tamayo-Ordo\u0026ntilde;ez et al. (2018), it is possible that certain genomic regions are undergoing an evolutionary stage of fixation of certain genes copies. This evolutionary process suggests that genes are subjected to genetic events including recombination, amplification, duplication, transposition, and gene loss that could have resulted in the high variability and diversity observed in the \u003cem\u003eAgave\u003c/em\u003e genome.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe use in \u003cem\u003eAgave\u003c/em\u003e of ISSR (Vargas-Ponce et al. 2009; Aguirre-Dugua and Eguiarte 2013), AFLP (S\u0026aacute;nchez-Teyer et al. 2009) and SSAP (Bousios et al. 2007) molecular markers has indicated variability and genetic diversity between wild and cultivated populations of \u003cem\u003eA. angustifolia\u003c/em\u003e, \u003cem\u003eA. tequilana\u003c/em\u003e, and \u003cem\u003eA. fourcroydes\u003c/em\u003e, among other species. The \u003cem\u003eAgave\u003c/em\u003e accessions we analyzed showed high genetic variability. The accessions with the highest level of ploidy \u0026ndash;like \u003cem\u003eA. angustifolia\u003c/em\u003e \u0026lsquo;Chelem ki\u0026rsquo; (2\u003cem\u003en\u003c/em\u003e = 6\u003cem\u003ex\u003c/em\u003e = 180), \u003cem\u003eA. fourcroydes\u003c/em\u003e \u0026lsquo;Sack ki\u0026rsquo; (2n = 5x= 150), and \u003cem\u003eA. fourcroydes\u003c/em\u003e \u0026lsquo;Yaax ki\u0026rsquo; (2\u003cem\u003en\u003c/em\u003e = 5\u003cem\u003ex\u003c/em\u003e= 150)\u0026ndash; showed the lowest similarity indexes (\u0026lt;78) in comparison to the other analyzed accessions. Apparently, polyploidy has an effect on genome size and genetic variability in \u003cem\u003eAgave\u003c/em\u003e, and the presence of these polyploid species within the genus could be a factor that contributes to the high species diversity index (0.32 to 0.56 species per million years) of the genus. Also, according to Good-Avila et al. (2006), the process of speciation in \u003cem\u003eAgave\u003c/em\u003e has been coincident with increasing aridity in central Mexico, which suggests that high retrotransposition activity in response to water deficit may have had an important role in speciation. Tamayo-Ordo\u0026ntilde;ez et al. (2016b) found that in some species of \u003cem\u003eAgave\u0026nbsp;\u003c/em\u003ethere is differential regulation of genes associated with biotic and abiotic stress factors depending on their habitat and proposed that stressing environmental factors could have contributed to gene diversity that expresses as speciation and species\u0026rsquo; adaptation.\u003c/p\u003e\n\u003cp\u003eCultivation of some species of \u003cem\u003eAgave\u003c/em\u003e since pre-Columbian time (Casas et al. 2016) and the involved pressure from anthropic domestication processes, artificial selection, and intensive cultivation has resulted in its species having different degrees of domestication (Colunga-Garc\u0026iacute;aMar\u0026iacute;n et al. 2004). But, unfortunately, until now only a handful of species have been studied with the goal of obtaining evidence of their domestication and management processes. Studies made of the rDNA, NBS-LRR, and LEA genes in cultivated polyploid species of \u003cem\u003eAgave\u003c/em\u003e have revealed that \u003cem\u003eA. tequilana\u003c/em\u003e \u0026lsquo;Azul\u0026rsquo; (2n = 2x = 60) \u0026ndash;used for production of tequila\u0026ndash; and \u003cem\u003eA. fourcroydes\u003c/em\u003e \u0026lsquo;Kitam ki\u0026rsquo; (2n = 3x = 90) \u0026ndash;used for fiber production\u0026ndash; have variants of these genes that could be involved in gene evolution, rRNA functionality, and activity of proteins participating in defense responses. This strongly suggests that anthropic pressure might have had negative effects on their variability, genetic diversity, and response to abiotic and biotic stress.\u003c/p\u003e\n\u003cp\u003eConsequently, it must be emphasized that morphological, molecular, and physiological studies be made before attempting to use polyploid \u003cem\u003eAgave\u0026nbsp;\u003c/em\u003eplants in order to achieve their sustainable use and to avoid negative consequences on the variability and genetic diversity of the genus. Also, based on the possible allopolyploid\u003cem\u003e\u0026nbsp;\u003c/em\u003eorigin of \u003cem\u003eAgave\u003c/em\u003e, its genome may contain valuable information that could be analyzed and used for genetic improvement of economical important crops exposed to biotic and abiotic stresses induced by global climate change (Tamayo-Ordo\u0026ntilde;ez et al., 2018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.2 Implications of the Conservation of NADH, RbcL, PEPC and PEPCK enzymes in Agave\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn CAM and C4 plants, the activity of key enzymes such as PEPC, PPDK, NAD (P) \u0026ndash;ME, and PEPCK is much higher than in C3 plants. Even so, the cellular compartmentalization of said enzymes differs between CAM and C4 plants. CAM plants have evolved specific diurnal and nocturnal patterns of expression and regulation to accommodate the flux of carbon through gluconeogenesis and glycolysis necessary to meet the nocturnal demand for PEP and diurnal decarboxylation of four-carbon organic acids. The characterization of certain specific CAM genes (PEPC and PEPCK) that are related to the cyclic electron transport and chlororespiration (NADH), and to enzymes related to carbon fixation (RbcL) and their expression patterns in CAM plants, could clarify the molecular mechanisms subjected to evolution and expression of said enzymes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eAgave\u003c/em\u003e, the basic enzymes and metabolites necessary for the optional functioning of CAM are not yet known, so in this work we isolated partial regions of the enzymes and studied their phylogenetic relationships with members of the classes Liliopsida and Magnoliopsida. Analysis of aa substitutions found in the sequence of the enzymes NADH, rbcL, PEPC, and PEPCK and its impact in the conformation of the 3D structures were included.\u003c/p\u003e\n\u003cp\u003ePhylogenetic analyses indicated that the NADH enzyme sequences from \u003cem\u003eAgave\u003c/em\u003e spp. were more closely related with the families Asparagaceae and Agavoidaceae. The genus \u003cem\u003eAllium\u003c/em\u003e, representative of the Amaryllidaceae family, formed a separate group (Fig. 3A). In the phylogenetic analysis of the RbcL, PEPC, and PEPCK enzymes we included accessions belonging to the class Liliopsida and Magnoliopsida. Sequences close to the family Agavaceae were not included due to the scarce information available. RbcL of \u003cem\u003eAgave\u003c/em\u003e grouped with species in genera belonging to the class Liliopsida. \u003cem\u003eAgave\u003c/em\u003e accessions were more closely related with accessions from the genus \u003cem\u003eOryza\u003c/em\u003e (Fig. 3B). The genera belonging to the class Magnoliopsida, formed a separate group.\u003c/p\u003e\n\u003cp\u003ePEPC of \u003cem\u003eAgave\u003c/em\u003e grouped with members of the order Poales (\u003cem\u003eOryza\u003c/em\u003e, \u003cem\u003eAegilops\u003c/em\u003e, \u003cem\u003eTillandsia\u003c/em\u003e, and \u003cem\u003eAnanas\u003c/em\u003e). Some plant accessions belonging to genera in the class Liliopsida (\u003cem\u003eAsparagus\u003c/em\u003e, \u003cem\u003eOryza\u003c/em\u003e, \u003cem\u003eSetaria\u003c/em\u003e, \u003cem\u003eAegilops\u003c/em\u003e, \u003cem\u003eHordeum\u003c/em\u003e, and \u003cem\u003eTriticum\u003c/em\u003e) grouped with members of genera in the class Magnoliopsida (\u003cem\u003eHerrania\u003c/em\u003e, \u003cem\u003eTheobroma\u003c/em\u003e, \u003cem\u003eGossypium\u003c/em\u003e, \u003cem\u003eJatropha\u003c/em\u003e, \u003cem\u003eManihot\u003c/em\u003e, \u003cem\u003eRicinus\u003c/em\u003e, \u003cem\u003ePopulus\u003c/em\u003e, \u003cem\u003eCurcubita\u003c/em\u003e, and \u003cem\u003eArabidopsis\u003c/em\u003e; Fig. 4A). Presence of isoforms of the PEPC enzyme in C3 and C4 plants has been widely described in plants (Paulus et al. 2013), and it is possible that the formation of two different groups including aa sequences of PEPC from \u003cem\u003eOryza\u003c/em\u003e and \u003cem\u003eAegilops\u003c/em\u003e is a consequence of the presence of PEPC isoforms in these genus. For this part, PEPCK sequences of \u003cem\u003eAgave\u003c/em\u003e that grouped with members of genera from the class Liliopsida (\u003cem\u003eElaeis\u003c/em\u003e, \u003cem\u003ePhoenix\u003c/em\u003e, \u003cem\u003eMusa\u003c/em\u003e, \u003cem\u003ePanicum\u003c/em\u003e, \u003cem\u003eSorghum\u003c/em\u003e and \u003cem\u003eAsparagus\u003c/em\u003e), were more closely related with the genus \u003cem\u003eAsparagus\u003c/em\u003e. Members of genera in the class Magnoliopsida formed a separate group (Fig. 4B).\u003c/p\u003e\n\u003cp\u003eThe chloroplast NADH dehydrogenase-like complex (NDH) is comprised of many subunits. The plastid genomes of flowering plants also have 11 genes (ndhA\u0026ndash;ndhK; Ifuku et al., 2011); a subunit specific to photosynthetic NDH is NdhL (dehydrogenase subunit L). In this study we identified a partial region of dehydrogenase subunit L in \u003cem\u003eAgave\u003c/em\u003e that showed high conservation when compared with other members of the Asparagaceae family. Analysis of aa substitutions found in NADH enzyme from the Agavaceae and Amaryllidaceae families, indicated that Agavaceae presents 2% of substitutions from V (117) to L, 12% from V (298) to A, and 12% from A (301) to V (Table S4). In the family Amaryllidaceae we found 33% of substitution from V (117) to I, 33% from V (298) to I, and 33% from A (301) to E (Table S4). Analysis of the effect of aa substitutions on 3D structures indicated that these changes do not affect the tertiary structure of the NADH enzyme. Our three-dimensional (3D) models of NADH dehydrogenase of the families Asparagaceae and Amaryllidaceae had a sequence identity of \u0026gt;40% with NADH- quinone-oxidoreductase subunit L (Fig. 5 and Table S5). 3D models of NdhL (320aa) from the families Asparagaceae (\u003cem\u003eAgave\u003c/em\u003e) and Amaryllidaceae (\u003cem\u003eAllium\u003c/em\u003e) showed structures similar with the NADH-quinone-oxidoreductase subunit L described in \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003e(Efremov and Sazanov 2011) (Fig. 5). In both families, it was possible to identify the formation of nine alpha helices (Fig. 5B-D and Table S5). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis results, suggesting that in the analyzed plant accessions of \u003cem\u003eAgave\u003c/em\u003e with eight million years of evolution (Tamayo-Ordo\u0026ntilde;ez et al. 2018) the dehydrogenase subunit L enzyme presents structural conservation. The importance of conservation in this enzyme, lies in that the chloroplast NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport and chlororespiration, which in angiosperms further associates with photosystem I (PSI) to form a super-complex (Yamori et al. 2015).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor this part, in our analysis of point mutations of PEPC enzyme, total aa substitutions (100% change) were detected in 22 aa residues from Liliopsida and in 28 aa residues from Magnoliopsida. This result indicated that 18% of the PEPC enzyme sequence analyzed differed between classes (Table S7). These differences found in the aa substitutions affect the grouping support between the aa sequences of the PEPC enzyme in the classes Liliopsida and Magnoliopsida, which was previously reflected in the phylogenetic tree of this enzyme (Fig. 4A). In \u003cem\u003eAgave\u003c/em\u003e, The conservation of A and R in the substrate binding ((PWIF(A/S)WTQR) \u0026nbsp;and inhibitory site ((DLLEGDPYLKQ(R/G)IRLRDSYIT)) of PEPC enzyme, was 100%, which suggests according to Paulus et al. (2013), possibly, it is of the C3-type in \u003cem\u003eAgave\u003c/em\u003e and other members of to the classes Magnoliopsida and Liliopsida. Also, 3D models of PEPC from Liliopsida (genera \u003cem\u003eAgave\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Ananas\u003c/em\u003e) and Magnoliopsida (genera \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003eGossypium\u003c/em\u003e) showed similar structures (identity \u0026gt;85% with the phosphoenolpyruvate carboxylase (PEPC)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eof \u003cem\u003eA. thaliana\u003c/em\u003e model) (Fig. 6 and Table S5); in both classes it was possible to identify the formation of 11 alpha helices (Fig. 6B-D and Table S5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3D models indicated that the PEPCK from \u003cem\u003eAgave\u003c/em\u003e had a sequence identity \u0026gt;45% with the phosphoenolpyruvate carboxykinase (PEPCK) described for the \u003cem\u003eE. coli\u003c/em\u003e model (Fig. 7A and Table S5). 3D models of PEPCK from the classes Liliopsida (genera \u003cem\u003eAgave\u003c/em\u003e and \u003cem\u003eAsparagus\u003c/em\u003e) and Magnoliopsida (genera \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eBrassica\u003c/em\u003e, and \u003cem\u003eArachis\u003c/em\u003e) showed similar structures between them, and it was possible to identify the conservation of aa residues that allow the union of Mg\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, and pyruvic acid \u0026ndash;important for the function of the enzyme (Sudom et al. 2013; Fig. 7B-F and Table S5). The ATP binding site could not be identified in the plant accessions that we analyzed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC4 photosynthesis has evolved independently more than 62 times, including 7500 species in 19 families, or 3% of the flowering plant species (Deng et al. 2016). Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) has shown plays a key role in the carbon metabolism of C4 and CAM plants, and markedly improves photosynthetic efficiency and water use efficiency (Driever and Kromdijk 2013). Deng et al. (2016) analyzed 60 available published plant genomes and delimited that the PEPC family consists of three distinct subfamilies (PPC-1, PPC-2, and PPC-3). The monocot CAM \u0026ndash;or C4-related PEPC\u0026ndash; originated from the PPC-1M1 clade and WGD may increase the number of copies of the PEPC gene, suggesting the formation of more isoforms of the PEPC in Plants CAM (Fan et al., 2013; O\u0026rsquo;Leary et al., 2011). It is possible that the broad amino acid changes (18%) found between the classes Magnoliopsida and Liliopsida, reported in this study, may be due to the presence of isoforms in the accessions representative of each class.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD-Ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39; Rubisco) catalyzes the initial steps of photosynthetic carbon reduction and photorespiratory carbon oxidation (Bracher et al. 2017). Land plants, have hexadecameric type I rubisco with an approximate molecular mass of 550 kDa composed of eight large (L;55 kDa) and eight small (S;15 kDa) subunits (L8S8). \u003cem\u003eA. thaliana\u003c/em\u003e produces four distinct small-subunit isoforms (RbcS1A, RbcS1B, RbcS2B and RbcS3B) (Valeg\u0026aring;rd et al. 2018). In the analysis of RbcL enzyme, it was possible to identify unique substitutions between members of the classes Liliopsida and Magnoliopsida. Total aa substitutions (100% change) were detected in aa residues such as such as S/Q (1), R/K(22), I/V (37), Q/E (68), and S/M (Table S6). Our 3D models indicated that the RbcL from \u003cem\u003eAgave\u003c/em\u003e had a sequence identity of 99% with the ribose bisphosphate carboxylase small chain (Fig. 8A and Table S5) described in the \u003cem\u003eO. sativa\u003c/em\u003e model (Matsumura et al. 2012). 3D models of RbcL from genera in the classes Liliopsida (\u003cem\u003eOryza\u003c/em\u003e, \u003cem\u003eAgave\u003c/em\u003e, and \u003cem\u003eAegilops\u003c/em\u003e) and Magnoliopsida (\u003cem\u003eTragopogon\u003c/em\u003e and \u003cem\u003eArachis\u003c/em\u003e) showed similar structures. In both classes, we were able to identify the formation of four alpha helices and two beta folds (Fig. 8B-D and Table S5). In \u003cem\u003eAegilops\u003c/em\u003e and \u003cem\u003eTragopogon\u003c/em\u003e, we observed that the length of the first alpha helix was lower compared to the reference model (Matsumura et al. 2012). The differences found between these tertiary structures can be derived from the presence of different isoforms that code for the small subunit of RbcL, as it has been described \u003cem\u003eA. thaliana\u003c/em\u003e (Valeg\u0026aring;rd et al. 2018).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause Rubisco catalyzes the rate-limiting step of C3-photosynthesis, many studies have been carried out for future improvement of Rubisco activity by genetic modification to increase the productivity of crop plants (Borland et al. 2011; 2014). The regulation of rubisco activity goes hand in hand with a negative coordination of PEPC regulation, suggesting a complex coregulation of both carboxylases compete for CO\u003csub\u003e2\u003c/sub\u003e during the early morning hours. According to the results obtained, it seems that the enzymes rbcL, PEPCK, PEPC and NADH are conserved between the classes Liliopsid and Magnoliopsida, however the presence of amino acid changes (\u0026gt;18%) in the enzymes PEPC and rbcL, could indicate the presence of isoforms, and genes that could be functionally specialized to better perform their catalytic activity. The participation of these two enzymes is related to photosynthetic efficiency and water use efficiency, suggesting that they are in constant evolutionary change according to the conditions of water limitation, high temperatures, increase of CO2 as a result of global warming, which we are currently experiencing.\u003c/p\u003e\n\u003cp\u003eIt is important to mention that the grouping of \u003cem\u003eAgave\u003c/em\u003e with monocots in the order Poales, suggests that the genus possibly has genetic material that allows it to tolerate environments where abiotic stresses are extreme, as described for that order (Linder and Rudall 2005). Bouchenak-Khelladi et al. (2014) described that CO\u003csub\u003e2\u003c/sub\u003e-concentrating mechanisms counteract the effects of low atmospheric CO\u003csub\u003e2\u003c/sub\u003e and reduce phototranspiration. It is believed that the parallel evolution of C4 and CAM photosynthesis in Poaceae, Cyperaceae, and Bromeliaceae is an adaptation to changes in atmospheric CO\u003csub\u003e2\u003c/sub\u003e concentrations. Combinations of extrinsic and intrinsic factors might have played a role in shifts in diversification rates and may explain the variation in species richness in Poales. In the genus \u003cem\u003eAgave\u003c/em\u003e the richness in diversity is not yet fully known, so the physiological exploration of a larger number of species could help us to know if the changes in atmospheric CO\u003csub\u003e2\u003c/sub\u003e concentrations during the evolution of the genus allowed the diversification of the C3, C4, and CAM responses.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003e3.3 Differences in stomata and transcriptional regulation of CAM-related genes in Agave L.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWith the aim of assessing the physiological changes that could be observed in stomata and that could impact the dynamics of CO\u003csub\u003e2\u003c/sub\u003e exchange and transpiration, we focused on determining the opening and closing of stomata by scanning electron microscopy in the two \u003cem\u003eAgave\u003c/em\u003e species with different levels of ploidy (\u003cem\u003eA. angustifolia\u003c/em\u003e and \u003cem\u003eA. fourcroydes\u003c/em\u003e) at 19:00 h, 23:00 h, 3:00 h and 7:00 h. Differences in the opening and closing of stomata on the abaxial and adaxial leaf surfaces were observed (Fig. 9 and Table 2).\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"954\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"5\"\u003e\u003cstrong\u003eTable 2. \u0026nbsp;Features of stomata in \u003cem\u003eAgave\u003c/em\u003e L.\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.780104712041886%\"\u003e\u003cstrong\u003eCharacteristics*\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e\u003cem\u003eA. angustifolia\u003c/em\u003e Haw.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026lsquo;Marginata\u0026rsquo; (2\u003cem\u003en\u003c/em\u003e=2\u003cem\u003ex\u003c/em\u003e=60)\u003csup\u003eƗ▲\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e\u003cem\u003eA. angustifolia\u003c/em\u003e Haw.\u0026nbsp;\u003cbr\u003e\u0026lsquo;Chelem ki\u0026rsquo; (2\u003cem\u003en\u003c/em\u003e=6\u003cem\u003ex\u003c/em\u003e=180)\u003csup\u003eƗ▲\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e\u003cem\u003eA. fourcroydes\u003c/em\u003e Lem.\u003cbr\u003e\u0026nbsp;\u0026lsquo;Kitam ki\u0026rsquo; (2\u003cem\u003en\u003c/em\u003e=3\u003cem\u003ex\u003c/em\u003e=90)\u003csup\u003eƗ▲\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e\u003cem\u003eA. fourcroydes\u0026nbsp;\u003c/em\u003eLem.\u0026nbsp;\u003cbr\u003e\u0026lsquo;Sac ki\u0026rsquo; (2\u003cem\u003en\u003c/em\u003e=5\u003cem\u003ex\u003c/em\u003e=150)\u003csup\u003eƗ▲\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.780104712041886%\"\u003e\u003cstrong\u003eLeaf section\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003eAdaxial/Abaxial\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003eAdaxial/Abaxial\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.848167539267017%\"\u003eAdaxial/Abaxial\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003eAdaxial/Abaxial\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.780104712041886%\"\u003e\u003cstrong\u003eStomata area (\u0026micro;M\u003csup\u003e2\u003c/sup\u003e)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e\u0026nbsp;\u003cbr\u003e997\u0026plusmn;56\u003csup\u003ea\u003c/sup\u003e/1161\u0026plusmn;32\u003csup\u003ea\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e2080\u0026plusmn;240\u003csup\u003eb\u003c/sup\u003e/2059\u0026plusmn;167\u003csup\u003eb\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e\u0026nbsp;\u003cbr\u003e1106\u0026plusmn;66\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e/1149\u0026plusmn;63\u003csup\u003ea\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e\u0026nbsp;\u003cbr\u003e1621\u0026plusmn;73\u003csup\u003eb\u003c/sup\u003e/1619\u0026plusmn;54\u003csup\u003eb\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.780104712041886%\"\u003e\u003cstrong\u003eStomata density (number mm\u003csup\u003e-2\u003c/sup\u003e)*\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e\u0026nbsp;\u003cbr\u003e58\u0026plusmn;3\u003csup\u003ea\u003c/sup\u003e/47\u0026plusmn;2\u003csup\u003ea\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e32\u0026plusmn;2\u003csup\u003eb\u003c/sup\u003e/34\u0026plusmn;2\u003csup\u003eb\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e45\u0026plusmn;1\u003csup\u003ea\u003c/sup\u003e/44\u0026plusmn;1\u003csup\u003ea\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e36\u0026plusmn;2\u003csup\u003eb\u003c/sup\u003e/38\u0026plusmn;1\u003csup\u003eb\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.780104712041886%\"\u003e\u003cstrong\u003eGuard cells area (\u0026micro;M\u003csup\u003e2\u003c/sup\u003e)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e19:00 PM: 853\u0026plusmn;6\u003csup\u003ea\u003c/sup\u003e/850\u0026plusmn;4\u003csup\u003ea\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e23:00 PM: 859\u0026plusmn;38\u003csup\u003ea\u003c/sup\u003e/889\u0026plusmn;40\u003csup\u003ea\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e3:00 AM: 1,161\u0026plusmn;50\u003csup\u003eb\u003c/sup\u003e/792\u0026plusmn;62\u003csup\u003ea\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e7:00 AM: 954 \u0026plusmn;18\u003csup\u003ea\u003c/sup\u003e/1,000\u0026plusmn;52\u003csup\u003ea\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e19:00 PM: 1001\u0026plusmn;3\u003csup\u003ea\u003c/sup\u003e/1135\u0026plusmn;61\u003csup\u003eb\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e23:00 PM: 1475\u0026plusmn;9\u003csup\u003ed\u003c/sup\u003e/1470\u0026plusmn;23\u003csup\u003ed\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e3:00 AM: 1310\u0026plusmn;61\u003csup\u003ed\u003c/sup\u003e/1380\u0026plusmn;83\u003csup\u003ed\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e7:00 AM: 1448\u0026plusmn;35\u003csup\u003ed\u003c/sup\u003e/1516\u0026plusmn;60\u003csup\u003ed\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e19:00 PM: 915\u0026plusmn;33\u003csup\u003ea\u003c/sup\u003e/919\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;44\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e23:00 PM:769\u0026plusmn;3\u003csup\u003ec\u003c/sup\u003e/756\u0026plusmn;13\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e3:00 AM: 873\u0026plusmn;18\u003csup\u003ea\u003c/sup\u003e/889\u0026plusmn;33\u003csup\u003ea\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e7:00 AM: 887\u0026plusmn;30\u003csup\u003ea\u003c/sup\u003e/1002\u0026plusmn;30\u003csup\u003ea\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e19:00 PM: 1071\u0026plusmn;1\u003csup\u003eb\u003c/sup\u003e/1254\u0026plusmn;46\u003csup\u003eb\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e23:00 PM: 1140\u0026plusmn;64\u003csup\u003eb\u003c/sup\u003e/1179\u0026plusmn;39\u003csup\u003eb\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e3:00 AM: 1342\u0026plusmn;2\u003csup\u003ed\u003c/sup\u003e/1152\u0026plusmn;18\u003csup\u003eb\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e7:00 AM: 1051\u0026plusmn;26\u003csup\u003ea\u003c/sup\u003e/998\u0026plusmn;47\u003csup\u003ea\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.780104712041886%\"\u003e\u003cstrong\u003eSuprastomatic cavity area (\u0026micro;M\u003csup\u003e2\u003c/sup\u003e)\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e19:00 PM: 77\u0026plusmn;4\u003csup\u003ea\u003c/sup\u003e/330\u0026plusmn;17\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e23:00 PM: 50\u0026plusmn;0\u003csup\u003ea\u003c/sup\u003e/317\u0026plusmn;33\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e3:00 AM: 231\u0026plusmn;28\u003csup\u003ec\u003c/sup\u003e/306\u0026plusmn;7\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e7:00 AM: 159 \u0026plusmn;9\u003csup\u003eb\u003c/sup\u003e/\u003csup\u003e\u0026nbsp;\u003c/sup\u003e216\u0026plusmn;20\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e19:00 PM: 383\u0026plusmn;59\u003csup\u003ec\u003c/sup\u003e/510\u0026plusmn;17\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e23:00 PM: 865\u0026plusmn;37\u003csup\u003ed\u003c/sup\u003e/1683\u0026plusmn;19\u003csup\u003ee\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e3:00 AM: 450\u0026plusmn;9\u003csup\u003ec\u003c/sup\u003e/740\u0026plusmn;1\u003csup\u003ed\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e7:00 AM: 630 \u0026plusmn;23\u003csup\u003ed\u003c/sup\u003e/955\u0026plusmn;2\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"18.848167539267017%\"\u003e19:00 PM: 101\u0026plusmn;3\u003csup\u003ea\u003c/sup\u003e/225\u0026plusmn;29\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e23:00 PM: 291\u0026plusmn;15\u003csup\u003ec\u003c/sup\u003e/294\u0026plusmn;36\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e3:00 AM: 364\u0026plusmn;8\u003csup\u003ec\u003c/sup\u003e/326\u0026plusmn;12\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e7:00 AM: 181\u0026plusmn;3\u003csup\u003eb\u003c/sup\u003e/398\u0026plusmn;2\u003csup\u003ec\u003c/sup\u003e\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.790575916230367%\"\u003e19:00 PM: 555\u0026plusmn;43\u003csup\u003ed\u003c/sup\u003e/319\u0026plusmn;17\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e23:00 PM: 390\u0026plusmn;9\u003csup\u003ec\u003c/sup\u003e/377\u0026plusmn;8\u003csup\u003ec\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e3:00 AM: 350\u0026plusmn;28\u003csup\u003ec\u003c/sup\u003e/576\u0026plusmn;67\u003csup\u003ed\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003cbr\u003e7:00 AM: 170\u0026plusmn;4\u003csup\u003eb*\u003c/sup\u003e/153\u0026plusmn;4\u003csup\u003eb*\u003c/sup\u003e\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003eƗ\u003c/sup\u003eLowercase letters (a, b, c, d or e) indicate significantly different values (Student\u0026rsquo;s t test; p\u0026lt;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003e▲\u003c/sup\u003e\u003c/em\u003eValues are averages of triplicates obtained from analysis of 30 stomata in 3 leaves of three plants of each accession \u0026plusmn; standard error\u003c/p\u003e\n\u003cp\u003e*Values of suprastomatic cavity area (\u0026micro;M\u003csup\u003e2\u003c/sup\u003e) at 7:00AM were obtained from stomata that remained open and represent only 25% of the total stomata observed\u003c/p\u003e\n\u003cp\u003eComparing different ploidy level accessions from the same species of \u003cem\u003eAgave\u003c/em\u003e, those with higher ploidy level (\u003cem\u003eA. angustifolia\u003c/em\u003e \u0026lsquo;Chelem ki\u0026rsquo; and \u003cem\u003eA. fourcroydes\u003c/em\u003e \u0026lsquo;Sac ki\u0026rsquo;) have a lower stomatal density (32\u0026plusmn;2 and 52\u0026plusmn;3) and their stomata are 1.5 to 2 times larger in comparison to their lower ploidy level counterparts (\u003cem\u003eA. angustifolia\u003c/em\u003e \u0026lsquo;Marginata\u0026rsquo; and \u003cem\u003eA. fourcroydes\u003c/em\u003e \u0026lsquo;Kitam ki\u0026rsquo;) (Fig. 10 and Table 2). Analyses of guard cell area (\u0026mu;M\u003csup\u003e2\u003c/sup\u003e) showed larger size in polyploid plants of the species \u003cem\u003eA. angustifolia\u003c/em\u003e \u0026apos;Chelem ki\u0026apos; and \u003cem\u003eA. fourcroydes\u003c/em\u003e \u0026apos;Sac ki,\u0026apos; coinciding with stomata size (Fig. 9 and Table 2). Stomatal indices according to ploidy numbers have also been related to adaptation to stress (Balao et al. 2011; Jordan et al. 2015; Males and Griffiths 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnalysis of suprastomatic cavity area (\u0026micro;M\u003csup\u003e2\u003c/sup\u003e) showed important changes between the polyploid species. \u003cem\u003eA. angustifolia\u003c/em\u003e \u0026lsquo;Chelem ki\u0026rsquo; showed a greater suprastomatic cavity area (1683\u0026plusmn;19 \u0026micro;M\u003csup\u003e2\u003c/sup\u003e) compared to \u003cem\u003eA. angustifolia\u003c/em\u003e \u0026lsquo;Marginata\u0026rsquo; (317\u0026plusmn;33 \u0026micro;M\u003csup\u003e2\u003c/sup\u003e) at 23:00 h (Fig. 11 and Table 2). Lower values of suprastomatic cavity area in \u003cem\u003eA. angustifolia\u003c/em\u003e \u0026lsquo;Marginata\u0026rsquo; were observed at 19:00 h and 23:00 h. Suprastomatic cavity area in the abaxial section of the leaf did not show significant differences along the temporal course, however, for \u003cem\u003eA. angustifolia\u003c/em\u003e \u0026lsquo;Marginata,\u0026rsquo; the suprastomatic cavity area was higher on the abaxial section of the leaf in comparison to the stomata located on the adaxial part of the leaf (Fig. 11).\u003c/p\u003e\n\u003cp\u003eIn the hexaploid species \u003cem\u003eA. angustifolia\u003c/em\u003e \u0026apos;Chelem ki\u0026apos;, high values of suprastomatic cavity area were observed at 23:00 h in both sections of the leaves (adaxial: 865\u0026plusmn;37 \u0026micro;M\u003csup\u003e2\u003c/sup\u003e and abaxial: 1683\u0026plusmn;19 \u0026micro;M\u003csup\u003e2\u003c/sup\u003e; Table 2). The lowest values of suprastomatic cavity on the adaxial and abaxial surfaces were observed at 19:00 h. Similar to what we found in \u003cem\u003eA. angustifolia\u003c/em\u003e \u0026apos;Marginata,\u0026apos; the suprastomatic opening on the abaxial surface was always greater compared to the same on the adaxial surface.\u003c/p\u003e\n\u003cp\u003eBehavior of stomata opening in \u003cem\u003eA. fourcroydes\u003c/em\u003e showed patterns different to those observed in \u003cem\u003eA. angustifolia\u003c/em\u003e. The pentaploid species \u003cem\u003eA. fourcroydes\u003c/em\u003e \u0026apos;Sack ki\u0026apos; showed the highest values of suprastomatic cavity area at 3:00 h (576\u0026plusmn;67 \u0026mu;M2) and 19:00 h (555\u0026plusmn;43 \u0026mu;M\u003csup\u003e2\u003c/sup\u003e), both on the abaxial and on the adaxial surfaces. Suprastomatic cavity area was not as large as that reported in the hexaploid species \u003cem\u003eA. angustifolia\u003c/em\u003e \u0026apos;Chelem ki\u0026apos; (abaxial: 1683\u0026plusmn;19 \u0026mu;M\u003csup\u003e2\u003c/sup\u003e). In addition, in the adaxial leaf section, 75% of the stomata analyzed proved to be closed and the aperture of the remaining 25% indicated low values of suprastomatic cavity area (adaxial:\u0026nbsp;170\u0026plusmn;4\u0026nbsp;\u0026mu;M\u003csup\u003e2\u003c/sup\u003e and abaxial: 153\u0026plusmn;4 \u0026mu;M\u003csup\u003e2\u003c/sup\u003e) (Table 2). Triploid \u003cem\u003eA. fourcroydes\u003c/em\u003e \u0026lsquo;Kitam ki\u0026rsquo; showed few significant differences along time, indicating that in this species the opening of the stomata is maintained longer; which is different to what was observed in \u003cem\u003eA. angustifolia\u003c/em\u003e Haw \u0026apos;Chelem ki\u0026apos;, in which immediately after the stomatal opening at 23:00 h., suprastomatic cavity area decreases drastically (Fig. 9 and Table 2)\u003c/p\u003e\n\u003cp\u003eRelative expression of the PEPC gene showed significant differences, raising its expression at 3h and 23h (Fig. 12). The highest values in PEPC expression were identified at 23 h in the accessions cultivated in the BGR-RO (Fig. 12A), coinciding with the highest values of suprastomatic cavity area. Similar trends were found in the expression of the PEPCK enzyme, which also indicated higher values at 3h and 23h, in plants belonging to both microclimates (Fig. 13). Highest values of RbcL expression were observed at 15h for both plantations (BGR-RO and GB-PCTY; Fig. 14). Regarding NADH, there were no significant differences during the evaluated period (data not shown).\u003c/p\u003e"},{"header":"4.\tDiscussion","content":"\u003cp\u003eCAM photosynthesis is a flexible phenomenon in plants. It has been proposed that there is a continuous photosynthetic expression from C3 to CAM and, in this progression, several types of CAM photosynthesis can be found ranging from a weak to a strong degree of expression (Males and Griffiths 2017; Liu et al. 2018).\u003c/p\u003e\n\u003cp\u003eDifferences found in stomatal size and density of \u003cem\u003eAgave\u003c/em\u003e depending on their ploidy level could be an indicator of differences in water uptake and tolerance to drought, which could be a product of their adaptation to CO\u003csub\u003e2\u003c/sub\u003e concentration changes (Winter et al. 2014; Driever and Kromdijk 2013; Tamayo-Ordo\u0026ntilde;ez et al\u003cem\u003e.\u003c/em\u003e 2018b). There is evidence that totally compacted cells, or stomata without air spaces, and the development of cuticular waxes, diminish the hydric state when the ambient temperature increases. Also, research conducted in the Proteaceae family has indicated that the ancient changes in genome size clearly influenced stomatal size, but adaptation to habitat strongly modified the genome-stomatal size relationship (Jordan et al. 2015). This suggests that an increase in CO\u003csub\u003e2\u003c/sub\u003e concentrations could impact on stomatal size an frequency, so it is possible that the polyploid species (\u003cem\u003eA. angustifolia\u003c/em\u003e \u0026apos;Chelem ki\u0026apos; and \u003cem\u003eA. fourcroydes\u003c/em\u003e \u0026lsquo;Sac ki\u0026rsquo;) showing larger stomata, lower stomatal density, and higher wax content may be better able to tolerate stress in climates where water is a limiting factor, and that this increased tolerance resulted from genetic, physiological and morphological changes during the polyploidization process and the increased CO\u003csub\u003e2\u003c/sub\u003e concentration that are currently experienced (Tamayo-Ordo\u0026ntilde;ez et al. 2016b; Tamayo-Ordo\u0026ntilde;ez et al. 2018b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, although plants such as \u003cem\u003eAgave\u003c/em\u003e can control the loss of water by transpiration \u0026ndash;for example, \u003cem\u003eA. americana\u003c/em\u003e and \u003cem\u003eA. deserti\u003c/em\u003e (Ehrler 1969; Nobel and Hartsock 1978)\u0026ndash; through their ability to open stomata at night, avoiding desiccation, and regulating its temperature through perspiration, there is evidence suggesting that agave plants with frequent irrigation can alter their metabolism causing the opening of stomata during light hours (Geydan and Melgarejo 2005). In \u003cem\u003eAgave\u003c/em\u003e \u0026ndash;with the exception of \u003cem\u003eA. fourcroydes\u003c/em\u003e \u0026apos;Kitam ki\u0026apos;, the opening patterns between 19:00 h and 7:00 h when sunlight is already present, can be influenced by the frequent irrigation of plantations, and conditions of culture can result in metabolic and physiological differences of CAM plants. In \u003cem\u003eOpuntia elatior\u003c/em\u003e, demonstrate that C\u003csub\u003e3\u003c/sub\u003e photosynthesis, drought-stress-related facultative CAM, and developmentally controlled constitutive CAM can all contribute to the early growth of \u003cem\u003eO. elatior\u003c/em\u003e (Winter et al. 2011), it is possible that under non-stressful conditions and frequent watering members of the class Magnoliopsida (\u003cem\u003eAgave\u003c/em\u003e and \u003cem\u003eOpuntia\u003c/em\u003e), showing a C3 metabolic behavior.\u003c/p\u003e\n\u003cp\u003eThe coincidence in an increase in the expression of the PEPCK and PEPC gene during the evaluated nocturnal hours (23h and 3h; Fig. 12 and 13), open the possibility of coordinated regulation between both enzymes. Also, regulation of rubisco activity goes hand in hand with a negative coordination of PEPC regulation, suggesting a complex co-regulation of both carboxylases compete for CO\u003csub\u003e2\u003c/sub\u003e during the early morning hours (Bailey et al. 2007; O\u0026rsquo;Leary et al. 2011; Deng et al. 2016). The highest values in RbcL expression obtained in this work, coincided at 11:00 h and 15:00 h, times in which PEPC expression was lower. In addition, the differences in 3D structure of RbcL in some accessions of the analyzed plants suggest the possible presence of isoforms that code for the RbcL small subunit. Also, the wide diversity in aminoacid substitutions found in PEPC and RbcL of the analyzed plants could reflect the presence of isoforms of this enzyme, which could result from the evolution of CAM metabolism in plants (Borland et al. 2014; Heckmann et al. 2016; Valegard et al. 2018).\u003c/p\u003e\n\u003cp\u003eFor the evolution of C3 to C4 to be carried out it is necessary that less mutational changes are present in the original C3 genes (Williams et al. 2012; Christin et al. 2013, 2014, 2015; Heckmann et al. 2016) and the isoforms take on greater importance (O\u0026rsquo;Leary et al. 2011; Deng et al. 2016; Bracher et al. 2017). Polyploidy is a primary source that can lead to the neofunctionalization of genes, and according to physiological \u003cem\u003eAgave\u003c/em\u003e data may present C3-CAM metabolism that would indicate the possibility of still conserving original C3 genes, which according to environmental conditions could favor the expression of C4 syndrome, responding better under conditions of heat, aridity, salinity and high light (Sage et al. 2012, 2014)\u003c/p\u003e\n\u003cp\u003eHeyduk et al. (2016), resolving the phylogeny of the subfamily Agavoideae, ancestral state reconstruction shows three independent origins of CAM in the group. These origins are associated with a shift in climate space toward warmer, drier habitats. The large genera of \u003cem\u003eAgave\u003c/em\u003e and \u003cem\u003eYucca\u003c/em\u003e have a center of diversity in the southwestern deserts of North America, however a number of species have distributions outside of the iconic desert range. These derived species may suggest that ancestrally the Agavoideae was composed of non-desert dwellers and established lineages migrated into more arid regions after an early radiation within the group. A movement into arid regions would require that those desert regions were in place already, and that species that moved there had an ability to grow in arguably some of the harshest conditions on the planet. In addition, morphological data (3D venation and large cells) indicated that the last common ancestor of \u003cem\u003eYucca\u003c/em\u003e and \u003cem\u003eAgave\u003c/em\u003e was C3 with CAM-like leaf anatomy and Sage (2002) suggested\u0026nbsp;that leaf and cell succulence may arise first in a C3 ancestor, followed by evolution of PEPC function to recapture respired CO\u003csub\u003e2\u003c/sub\u003e, eventually leading to circadian control and full-fledged CAM function (Sage 2004; Sage et al. 2012).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe physiological and genetic data found in this work suggest that derived from the great diversity of the genus, it is possible to find species of \u003cem\u003eAgave\u0026nbsp;\u003c/em\u003eL., C3-CAM intermediate range. Further work on whether diversification rates vary between C3 and CAM lineages will give insight into how the evolution of CAM might be promoting biodiversity in the Agavoideae. Also, future studies of water stress in these \u003cem\u003eAgave\u003c/em\u003e accessions could be a near goal that could help to sustain whether polyploid agave plants could tolerate water stress (Tamayo-Ordo\u0026ntilde;ez et al. 2016b; Tamayo-Ordo\u0026ntilde;ez et al. 2018a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to the obtained physiological and genetic results, apparently \u003cem\u003eAgave\u003c/em\u003e can behave like C3-CAM (Optional CAM). It seems that the nocturnal intake of CO\u003csub\u003e2\u003c/sub\u003e achieved by opening its stomata can be made at night or during the day, depending on the conditions of its cultivation. It is possible that these facultative CAM plants maintain a positive balance and present an improvement in WUE and reduced photorespiration (Andrade et al. 2007) positioning \u003cem\u003eAgave\u003c/em\u003e as a promising model for different biotechnological applications in the face of global climate change (Tamayo-Ordo\u0026ntilde;ez et al. 2018b).\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCAM plants, such as \u003cem\u003eAgave\u003c/em\u003e L., could be used for sustainable production on irrigated lands using up to 80% less water to produce similar amounts of biomass compared with C3 species. Thus, research emphasizes that according to the genetic and physiological differences found in \u003cem\u003eAgave\u003c/em\u003e L., it is possible that the genus harbors species with C3-CAM metabolism, suggesting the possibility of still conserving ancestral C3 genes, which according to environmental conditions could favor the expression of C4 syndrome, which could result in C4 evolution, under abiotic stress (heat, aridity, salinity and high light. This opens up the possibility of expanding the agricultural uses of these CAM specie, in the face of this future climate change, with high priority to ensure that food, feed, and fiber needs are in future warmer climates with diminishing arable land and water resources. Additionally, polyploidy agaves showed physiological and genetic changes, which could help them to respond differently according to the growing environment, facilitating its adaptation in environments where water is a limiting factor.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to express their gratitude to the staff in charge of Germplasm Bank of the Scientific-Technological Park of Yucat\u0026aacute;n (GB-PCTY) and Regional Roger Orellana-CICY Botanical Garden (RG-CICY) for the facilities granted for the collection of plant material.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u003c/strong\u003e This work was supported by the National Council of Science and Technology of Mexico (CONACYT) with the Science Projects, CB-50268 and CB-155356\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e The authors declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e Conceptualization, Y.J.T.-O., and M.C.T.O.; methodology, B.A.A.-G., F.L.S.T., F.A.T.O.; formal analysis, L.C.R.Z., and F.A.B.P. investigation, Y.J.T.-O., and M.C.T.-O.; writing\u0026mdash;original draft preparation, Y.J.T.-O., B.A.A.G, and F.A.T.O; writing\u0026mdash;review and editing, V.H.R.G, L.C.R.Z, \u0026nbsp; and F.L.S.T. 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Ann Bot 132(4):685\u0026ndash;698, https://doi.org/10.1093/aob/mcac158\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Agave, CAM, polyploidy","lastPublishedDoi":"10.21203/rs.3.rs-4284238/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4284238/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn recent years there has been growing interest in increasing plant water use efficiency (WUE) through the introduction of crassulacean acid metabolism (CAM) into C3 crops. However, this task has been hampered because the scaling of CAM to other C3 plants requires knowledge of the enzymatic and regulatory pathways underpinning this temporal CO\u003csub\u003e2\u003c/sub\u003e pump.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAgave\u003c/em\u003e presents CAM metabolism, to date research aimed at knowing the physiological and morphological adaptations related to CAM metabolism, only includes a small group of species analyzed. With the aim of knowing basic aspects related to the physiological response of polyploid (2\u003cem\u003en\u003c/em\u003e=2\u003cem\u003ex\u003c/em\u003e=60 to 2\u003cem\u003en\u003c/em\u003e=6\u003cem\u003ex\u003c/em\u003e=180) \u003cem\u003eAgave\u003c/em\u003e accessions, we carried out genetic and physiological studies in \u003cem\u003eA. tequilana\u003c/em\u003e Weber, \u003cem\u003eA. fourcroydes\u003c/em\u003e Lem., and \u003cem\u003eA. angustifolia\u003c/em\u003e Haw. Using AFLP markers, differences in genetic variability between the polyploid accessions and their diploid counterparts were found. Analysis of expression by real-time PCR showed that the regulation of CAM in \u003cem\u003eAgave\u003c/em\u003e is accompanied by the transcription of RbcL, PEPC and PEPCK. Monitoring of the stomatal opening during the night showed differences according to the level of ploidy of the accessions.\u003c/p\u003e\n\u003cp\u003eThe genetic and physiological data obtained suggest that agave species present adaptations and flexibility in the transcriptional regulation of genes related to CAM metabolism, suggesting that some polyploid accessions of \u003cem\u003eAgave\u003c/em\u003e L. could be more tolerant to drought and heat, adapting their CO2 exchange mechanisms according to the metabolic needs of each species of agave.\u003c/p\u003e","manuscriptTitle":"Physiological changes in stomata and regulation of genes involved in CAM metabolism in polyploid Agave L. 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