Heterologous Expressions of foxtail millet (Seteria italica) mitogen-activated protein kinase kinase (SiMKK) group A genes regulate root development under salt stress in Arabidopsis thaliana

Heterologous Expressions of foxtail millet (Seteria italica) mitogen-activated protein kinase kinase (SiMKK) group A genes regulate root development under salt stress in Arabidopsis thaliana | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Heterologous Expressions of foxtail millet (Seteria italica) mitogen-activated protein kinase kinase (SiMKK) group A genes regulate root development under salt stress in Arabidopsis thaliana Yaqiong Li, Kai Huang, Huazhuan He, Yuhuan Yang, Xiaoxia Meng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3887368/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Aug, 2024 Read the published version in Plant Growth Regulation → Version 1 posted 6 You are reading this latest preprint version Abstract The MAPK cascade is evolutionarily conserved in eukaryotes and involved in the regulation of plant growth, development, resistance to biotic and abiotic stress. However, the gene function of MAPK cascade in foxtail millet ( Seteria italica ) is rarely studied. In this study, RNA sequencing was performed and MAPK cascade was the main enrichment pathway in foxtail millet after salt treatment. Meanwhile, fourteen genes encoded and mitogen-activated protein kinase kinases (SiMKKs) were identified which could be divided into 4 subfamilies. Under salt treatment, the expression levels of 11 SiMKKs were upregulated and the expression level of SiMKK6-2 in group A had the biggest increase. SiMKK1 and SiMKK6-1 , which were the other two member of in the same subfamily, also significantly upregulated under salt stress. Overexpression of these three genes in Arabidopsis thaliana reduced the sensitivity of roots to salt stress. Transgenic plants had more lateral roots. The decrease of primary root length of transgenic plants under salt stress was significantly lower than that of wild type plants. These three genes are involved in regulating the development of primary and lateral roots of plants, which can maintain better root development to improve plant tolerance to salt stress. MAPK cascade foxtail millet salt stress transcriptome root development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction As sessile organisms, plants have to survive under environmental stresses that inhibit their growth and development (Fang and Xiong 2015 ). One of the major threats to plant growth and agricultural production is the loss of water as global weather trends shift toward drier and the aggravation of soil salinization (Gong et al. 2020 ). When subjected to abiotic stress, plant cells activate various response pathways to restore cellular and organism-level homeostasis. Among them, a number of necessary signaling pathways function under multiple stresses such as Ca 2+ signaling, the production of reactive oxygen species and phosphoprotein cascades (Zhu 2016 ). In eukaryotes, mitogen-activated protein kinase (MAPK) cascades are highly conserved modules downstream of signal receptors which play important roles in transducing extracellular stimuli into intracellular responses (Meng and Zhang 2013 ; Chen et al. 2021b ). A classical MAPK module consisting of three classes of protein kinases named MAPKs, MAPKKs (MAPK kinases) and MAPKKKs (MAPKK kinases). When stimulated, cell surface–localized receptors recognize signal molecules, and then activate MAPK signaling cascade through phosphorylation to efficiently amplify the signal (Liang and Zhou 2018 ; Xu and Zhang 2015 ). The Arabidopsis thaliana genome contains approximately 60 MAPKKKs, 10 MAPKKs and 20 MAPKs (Komis et al. 2018 ; Ichimura et al. 2002 ). The similar gene families encoding proteins involved in MAPK cascades have also been identified in other plants species, such as rice, maize, and cucumber (Kong et al. 2013 ; Wang et al. 2015 ; Chen et al. 2021a ; Hamel et al. 2006 ). The number of MAPKKs is less than that of MAPKKs and MAPKs, which indicates that the same MAPKK can integrate the signals received by different MAPKKs and transmit them to multiple MAPKs. According to sequence similarity, MAPKKs can be divided into four groups (A-D). Group A includes AtMKK1/2/6, group B includes AtMKK3, group C includes AtMKK4/5, and group D includes AtMKK7/8/9/10 (Meng and Zhang 2013 ; Hamel et al. 2006 ). Some early studies have revealed that several members of MAPK cascades are key components in plants responses to abiotic stresses. For example, The ABA-INSENSITIVE PROTEIN KINASE 1‐MKK5‐MPK6 cascade positively regulates the ABA‐mediated stomatal closure and root growth in Arabidopsis (Li et al. 2017 ). During osmotic stress, Raf-like MAPKKKs ARK1, ARK2 and ARK3 interact with and phosphorylate OST1 to positively regulate the stress responses (Katsuta et al. 2020 ). In maize, ZmMPK5 phosphorylates and enhances the stability and activity of a dehydrogenase ZmABA2 that contributes to ABA biosynthesis (Ma et al. 2016 ). Foxtail millet ( Setaria italica ), one of the representative cereals of ancient times in the world, gets a lot of attention in recent years owing to the unique composition of its seeds containing high content of health-promoting properties (Lu et al. 2009 ; Sachdev et al. 2021 ). Because of the high tolerance to drought and barren, foxtail millet is widely cultivated in semi-arid regions and is also thought to be an important crop to ensure food and nutritional security in the face of a quickly growing world population, particularly during abnormal situations (Muthamilarasan and Prasad 2021 ; Lu et al. 2009 ). On the other hand, foxtail millet has a small diploid genome of ~ 490 Mb and the its genome was fully sequenced. Recently, an Arabidopsis-like foxtail millet was constructed which provided a good plant model for plant gene functional studies (Yang et al. 2020 ; Peng and Zhang 2020 ). For the above reasons, it is effective to study the molecular mechanisms of abiotic stress tolerance in foxtail millet and some stress-tolerance genes were identified through sequence characteristics and expression patterns under stress. For example, it was found that the promotor of SiARDP contained ABA response element and the expression level of SiARDP was induced by ABA. Overexpression of SiARDP improved the drought resistance of plants (Li et al. 2014 ). However, few studies have uncovered the functions of conserved signaling pathways like MAPK cascade in foxtail millet. In this study, transcriptome analysis revealed that MAPK signaling pathway plays an important role in foxtail millet response to salt stress. We identified 14 MKK genes in foxtail millet genome and analyzed the functions of SiMKK1 SiMKK6-1 and SiMKK6-2 in salt stress responses. By this study, we may provide new insights into the role of MAPK cascade in foxtail millet. Materials and Methods Plant materials and growth conditions A foxtail millet variety (Yugu1/YG1), the Arabidopsis thaliana (Col-0 wild type and transgenic lines in Col-0 background) were used in this study. Arabidopsis thaliana grown in the growth room at 23°C with 16-h-light /8-h-dark. Foxtail millet seedlings were grown in a growth chamber under greenhouse conditions with a 16 h light (25℃)/8 h dark (23℃) cycle. Foxtail millet seedlings with three leaves were transferred to Hoagland’s solution and precultured for 24 h. For NaCl treatment, seedlings were cultured in Hoagland’s solution containing 250 mM NaCl. Leaves were collected un-der a time series treatment (0, 3, 6, 9, and 12 h for NaCl treatment) for RNA extraction. Transcriptome sequencing and analyses Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonucle-ase/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferentially 370 ~ 420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. We performed Gene Ontology (GO) enrichment analysis by the GOseq R packages which is a published method (Young et al. 2010 ). KEGG database ( http://www.genome.jp/kegg/ ) was used to discover the regulatory pathways of genes (Kanehisa et al. 2008 ). We used a software KOBAS (Mao et al. 2005 ) to investigate enrichment of DEGs in KEGG pathways. Phylogenetic analysis The amino acid sequence of Arabidopsis MKK was obtained from TAIR website ( https://www.arabidopsis.org/ ). The MKK genes of foxtail millet, maize and sorghum were retrieved from Phytozome database by using the amino acid sequence of Arabidopsis MKK as the submission sequence. The amino acid sequence of rice MKK (Hamel et al. 2006 ) was also obtained from Phytozome database. Multiple sequence alignment was performed by ClustalW, and the phylogenetic tree was constructed by maximum likelihood method with MEGA 6.0 software. Gene and protein properties analysis Chromosome positional information of SiMKKs was obtained from Phytozome database, and images were drawn by MapChart software. The cis-regulatory elements in the promoter region of the SiMKKs gene were identified by PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). The exon and intron structure diagrams of the SiMKKs gene were drawn on GSDS2.0 ( http://gsds.gao-lab.org/ ) website. The protein parameters were evaluated though Expasy ProtParam tool ( https://web.expasy.org/protparam/ ). Total RNA extraction and qRT-PCR RNA extraction was performed according to the instructions of RNApure Plant Kit (CWBIO CW0559S). The first-strand cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (RR047A; Takara). qRT-PCR was performed using the Bio-Rad CFX96TM Touch real-time PCR detection system with SYBR Premix Ex TaqTM II (RR820A; Takara). Transcript levels were normalized to the SiACTIN (Seita.5G464000). Vector construction and plants transformation The SiMKK1 , SiMKK6-1 and SiMKK6-2 coding regions were amplified with primers (Supplemental Table 1). Then the fragments were subcloned into pSuper1300 (GFP) vector under the control of the CaMV 35S promoter. The resulting constructs were introduced into the Agrobacterium tumefaciens strain GV3101. Transformation of Arabidopsis plants was performed using the floral dip method (Clough and Bent 1998 ). Phenotypic evaluation Arabidopsis seeds were grown on the 1/2 MS medium for 4 days and then seed-lings of wild type and transgenic lines were transferred to 1/2 MS medium with or without 100 mM NaCl and grown for additional 3 days for primary root length measurement. Arabidopsis seeds were grown 1/2 MS medium with or without 100 mM NaCl and grown for 2 weeks for lateral roots observation. The Statistical Analysis Statistical assays of three independent experiments were performed using online ANOVA tool ( http://vassarstats.net/anova1u.html ). The resulting ANOVA P -values were used to indicate significant differences between multiple groups. Results Transcriptome analysis of foxtail millet under salt stress To elucidate the molecular mechanism of stress-resistance in foxtail millet, we conducted the RNA sequencing. In three leaves and one-half leaf period, seedlings of YG1 were respectively treated with 300 mM NaCl (experiment group, YG1_Na) and distilled water (control group, YG1_CK) for 24h. Then, the total RNA was extracted from the top leave of each seedling (three biological replicates per group and six samples in total). Through RNA-sequencing, we obtained 144,677,546 clean reads with a total base number of 41.84 G and the GC contents were between 58.04% and 59.73%. Q20 and Q30 values of 6 samples were all above 90.66%. We compared the clean reads with the genome of YG1, and the mapping rate of each sample was over 95% indicating that transcriptome data were of high quality and could be further analyzed (Supplemental Table 1). The data showed there were 16477 and 16131 genes expressed in experiment group and control group respectively, 15507 of which were expressed in both groups (Fig. 1 A). Then, differentially expressed genes (DEGs) were identified by comparing the read counts of genes in the two groups. In total, 1740 genes displayed different expression pattern after salt treatment, among which 1349 genes were upregulated and 391 were downregulated (Fig. 1 B). Enrichment analysis of DEGs induced by salt treatment To further understand the biological function of DEGs, gene ontology (GO) enrichment analysis was performed and 30 significant enrichment GO terms are presented. As shown in Fig. 2 , the significantly enriched GO terms of Biological Process (BP) included the “stress responses” and “response to chemical”. The most enriched Cellular Component (CC) was “extracellular region” revealed the plant needed to modulate metabolism to cope with external salt stress. In addition, the significantly enriched GO terms of molecular function (MF) were “DNA-binding transcription factor activity” and “transcription regulator activity” suggested that transcriptional regulation were mainly activated by salt stimulus. Next, we performed enrichment analyses of DEGs based on Kyoto Encyclopedia of Genes and Genomes (KEGG) which revealed that the DEGs were mainly enriched in 96 pathways (Supplemental Table 3). Among them, DEGs were most significantly enriched in the MAPK signaling pathway (Fig. 3 ). Genome-wide characterization of the Mitogen-activated protein kinase kinases in Seteria italica (SiMKKs) The transcriptome analysis indicated that MAPK signaling pathway was essential for the responses to salt stress in foxtail millet. However, few investigations were focus on the MAPK signaling of foxtail millet and the components of MAPK cascades had not yet been identified. Therefore, we performed a comprehensive identification to confirm Mitogen-activated protein kinase kinases in Seteria italica (SiMKKs). In this assay, the amino acid sequences of ten Arabidopsis MKKs (TAIR: https://www.arabidopsis.org/ ) were used as queries for BLAST searches of the foxtail millet genome ( https://phytozome-next.jgi.doe.gov/ ). As results, a total number of fourteen SiMKKs genes were identified and the physicochemical characteristics of the corresponding proteins were predicted (Table 1). SiMKKs were distributed on seven chromosomes: three each on chromosome 1, chromosome 5 and chromosome 9; two on chromosome 4; and one each on chromosome 2, chromosome 3 and chromosome 6 (Supplemental Fig. 1). MAPK cascades are conserved in plants and MKK genes have been identified in other species. To study the phylogenetic relationship between foxtail millet and other species, we constructed a phylogenetic tree of MKK proteins from Seteria italica (Si), Arabidopsis thaliana (At), Oryza sativa (Os). As shown in Supplemental Fig. 2, the phylogenetic analysis suggested that MKKs from different species all could be divided into four groups (A, B, C and D). MKKs in foxtail millet (SiMKKs) were named based on the result of phylogenetic analysis. Gene structural and conserved domain analysis To further clarify the evolutionary relationships and explore the structural diversity of SiMKKs , the distribution of exon-intron structure was analyzed and displayed. As shown in Fig. 4 A, SiMKK1 , SiMKK3-1 and SiMKK3-2 contained eight introns; SiMKK3-3 contained nine introns; both SiMKK6-1 and SiMKK6-2 had seven introns; SiMKK10-4 had one intron and the other SiMKKs didn’t contain any introns. It was showed that members in same group had similar exon-intron structures. On the other hand, we performed promoter analysis of SiMKKs which predicted the cis-elements in the 2.0 kb promoter region upstream from the start codon (Fig. 4 B). All SiMKKs contains more than one MYB-binding sites and MYC-binding sites. The numbers of CGTCA-motif, TGACG-motif and G-box were significantly higher than other motifs. For phytohormone response element, the results showed there were more ABA response elements (ABRE) than auxin response elements (ARE) in the promoters of SiMKKs , which indicated SiMKKs might play critical roles in stress responses. Next, we predicted the conserved domains of SiMKKs with an online tool ( https://prosite.expasy.org/prosite.html ). As the result shown in Fig. 4 C, all SiMKKs contained protein kinase domain, ATP binding region and kinase activate sites. Meanwhile, members of group B (SiMKK3-1, SiMKK3-2 and SiMKK3-3) contained nuclear transport factor 2 domain at the C-terminal of these proteins. SiMKK genes expression patterns in response to salt stress To explore the response of SiMKK genes to salt stress, we treated seedlings of YG1 with 200 mM NaCl and detected the expression patterns of 14 SiMKKs genes within 12 hours after salt treatment. Except for SiMKK4-2 , SiMKK10-2 and SiMKK10-5 , the expression level of the other 11 SiMKKs up-regulated within 12 h of salt treatment. Among genes with up-regulation expression, the expression levels SiMKK3-1 , SiMKK10-1 and of all three genes in group A increased at least fivefold. In addition, the expression level of SiMKK6-2 increased by more than 20 times 3 hours after salt treatment which was the highest in SiMKK gene family. Root development of heterologous overexpression lines of SiMKK1 , SiMKK6-1 and SiMKK6-2 in Arabidopsis As the expression levels of the three genes in subgroup A were all significantly increased under salt treatment, and 6 − 2 was the largest increase among all family members. Next, we took a closer look at these three genes. Through the existing transcriptome data, we analyzed the expression patterns of 14 MKK genes in different tissues and different growth periods, and the data showed that the expression levels of the three genes in group A were high in multiple tissues such as root and leaf. In addition, we also predicted the interacting proteins of the three proteins, and the results showed that the predicted interacting proteins of 6 − 1 and 6 − 2 were the same. SiMKK1 shares many common interacting proteins with 6 − 1 and 6 − 2, with 8 of the 10 proteins with the highest likelihood of interacting being the same. Therefore, we hypothesized that the three proteins might have very similar functions. We cloned CDS of these three genes, constructed 35S promoter-driven expression vectors, and transformed them into Arabidopsis thaliana to obtain heterologous overexpression lines. Through phenotypic observation, we found that all overexpressed transgenic strains could have more developed roots under normal growth conditions, and the number of lateral roots was significantly higher than that of wild Arabidopsis. We planted the plants on a medium containing 100mM NaCl, and the results showed that under salt stress, the number of lateral roots of the transgenic plants was also significantly higher than that of the wild type. And two separate lines of the same gene showed similar phenotypes. In addition, we also tested whether these three genes were involved in the regulation of taproot length. We grew wild-type plants and overexpressed strains on MS medium and medium containing 100mM NaCl respectively, and then measured the root length. The results showed that taproot length had no significant difference when there was no salt stress. The root length of transgenic plants was significantly longer than that of the wild type. Through calculation of relative root length (the ratio of average root length under salt stress to average root length under normal growth), we found that overexpression of these three millet MKK genes could significantly reduce the shortening of taproots under salt stress. Discussion Saline soils are estimated to increase in ~ 50% of irrigated lands because of the chemical fertilizers application (Kumar et al. 2020 ). Therefore, it is of great significance to study the mechanism of plant salt resistance at the molecular level is critical for crop production. Foxtail millet is one of the representative cereals with high nutritional value and strong stress resistance which is ideal for food and nutritional security (Muthamilarasan and Prasad 2015 ). Owing to its short life cycle, small plant size, and availability of whole-genome sequencing data, foxtail millet is a suitable model for the discovery of stress response genes (Peng and Zhang 2020 ). SiDi19-3 encoded a transcription factor contains Cys2/His2-type zinc-finger motifs and SiDi19-3 overexpression increased the transcript levels of most Na+/H + antiporter (NHX), salt overly sensitive (SOS), and calcineurin B-like protein (CBL) genes and improved the salt tolerance of plants (Xiao et al. 2023 ). SiMYB16 and SiMYB19 were both upregulated by salt stress. Overexpression of SiMYB16 and SiMYB19 enhanced salt tolerance of plants by regulating the biosynthesis of lignin and ABA signal transduction respectively (Yu et al. 2023 ; Xu et al. 2022 ). Salt stress resulted in decreased biomass, decreased relative water content and membrane damage in foxtail millet (Rathinapriya et al. 2020 ). Soil salinity provokes water and nutrient imbalance which causes plant growth inhibition and plant roots perceive these stresses in the soil and adapt their architecture accordingly (Karlova et al. 2021 ). When challenged with high salt stress, the growth of primary and lateral roots is arrested. (Gandullo et al. 2021 ). Salt-resistant plant could cope with environmental changes through various growth regulation, including root development regulation. Excellent root development is an important guarantee for plants to maintain water and nutrient balance under salt stress. In this study, we provide new gene information for foxtail millet response to salt stress and revealed three genes that enhanced plant salt tolerance by modulating root development. The full utilization of these three genes in breeding is expected to enhance the stress resistance of millet by improving root development. MAPK cascades are ubiquitous signaling modules in eukaryotes which involved in almost all fundamental cellular processes (Ichimura et al. 2002 ; Xu and Zhang 2015 ; Pawson and Scott 2005 ). Hence, MAPK signaling pathway is also employed to activate the stress resistance of plants under salt stress. The mismatch of between the numbers of MKKs and MAPK substrates suggests that individual MKKs have the capacity to address more than one MAPK and MKKs might be core hubs in multiple signal pathways (Kong et al. 2013 ). In Arabidopsis, five out of ten MKKs (AtMKK1, AtMKK2, AtMKK5, AtMKK7 and AtMKK9) were involved in regulating plant responses to salt stress (Shen et al. 2019 ; Xing et al. 2015 ; Teige et al. 2004 ; Matsuoka et al. 2002 ). In this study, we also found the expression level of 11 SiMKKs were up-regulated under salt treatment, five of which were significantly up-regulated indicating the resistance of foxtail millet to salt stress were also dependent on the participation of multiple MKKs. MKKs in plant were involved in plant response to salt stress by regulating various pathways. For instance, AtMKK5 modulated the expression of iron superoxide dismutase gene and ZmMKK1 enhanced the expression of ROS scavenging enzyme- and ABA-related genes (Cai et al. 2014 ). SiMKK1, SiMKK6-1 and SiMKK6-2 could promote root development, increase the length of primary root and the number of lateral roots under stress conditions, which was conducive to ensure the absorption of water and nutrients in high salt soil. Similar results were found in rice. OsMKK1, and OsMKK6 regulated the transcript levels of the genes related to auxin content enhancement to regulate lateral root growth under salt stress (Yang et al. 2021 ; Kumar and Sinha 2013 ). These results indicate that the maintenance of root development is an important way for plants resistance to salt stress and MKKs is conservative in regulating root development because SiMKK1/6 in rice and foxtail millet showed high similarity in sequence and were close in evolution (see Supplemental Fig. 2). On the other hand, MPK3 and MPK6 are implicated in plant salinity stress in several different contexts, probably a result of the sharing of a regulatory component/event by multiple stress pathways (Zhang and Zhang 2022 ). MPK3/6 play critical roles in lateral root emergence (Zhu et al. 2019 ), however, there is not enough evidence provided MPK3 regulated root development under salt stress. Through prediction of protein interaction, we found that SiMKK1 might interact with MPK3 and MPK6 in foxtail millet (labeled as K4ABG1 and K3XXF6 in Supplemental Fig. 4). SiMKK6-1 and SiMKK6-2 might interact with MKK3 in foxtail millet. These results provide a probability for further study to investigate whether signals were transduced from members of group A of SiMKKs to MPK3/6 in foxtail millet to promote root development under salt stress. Conclusion In summary, we demonstrated the importance of MAPK signaling pathway in resistance of foxtail millet to salt stress through RNA sequencing and analysis, and demonstrated that SiMKK1, SiMKK6-1 and SiMKK6-2 could maintain the development of primary root and lateral roots under salt stress through expression patterns and transgenic technology. This study laid a foundation for the analysis of the stress resistance mechanism of millet, and provided important genetic resources for cultivating crops with high stress resistance. Declarations Funding This research was funded by the National Natural Science Foundation of China, 32101760 and 32101722; Natural Science Foundation of Shanxi Province, grant number, 20210302123420 and 20210302123362; The Distinguished and Excellent Young Scholar Cultivation Project of Shanxi Agricultural University, 2022YQPYGC02. Author Contributions Yaofei Zhao designed this study. Yaqiong Li, Kai Huang and Huazhuan He performed the experiments. Yuhuan Yang and Xiaoxia Meng participated in the data analysis. Yaofei Zhao and Yaqiong Li wrote the manuscript. Yaofei Zhao and Guiyun Yan reviewed and edited the manuscript. Conflict of interest The authors have no conflict of interest to declare. References Cai G, Wang G, Wang L, Liu Y, Pan J, Li D (2014) A maize mitogen-activated protein kinase kinase, ZmMKK1, positively regulated the salt and drought tolerance in transgenic Arabidopsis. J Plant Physiol 171(12):1003–1016. 10.1016/j.jplph.2014.02.012 Chen J, Wang L, Yuan M (2021a) Update on the Roles of Rice MAPK Cascades. Int J Mol Sci 22(4). 10.3390/ijms22041679 Chen X, Ding Y, Yang Y, Song C, Wang B, Yang S, Guo Y, Gong Z (2021b) Protein kinases in plant responses to drought, salt, and cold stress. J Integr Plant Biol 63(1):53–78. 10.1111/jipb.13061 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743. 10.1046/j.1365-313x.1998.00343.x Fang Y, Xiong L (2015) General mechanisms of drought response and their application in drought resistance improvement in plants. Cell Mol Life Sci 72(4):673–689. 10.1007/s00018-014-1767-0 Gandullo J, Ahmad S, Darwish E, Karlova R, Testerink C (2021) Phenotyping Tomato Root Developmental Plasticity in Response to Salinity in Soil Rhizotrons. Plant Phenomics 2021:2760532. 10.34133/2021/2760532 Gong Z, Xiong L, Shi H, Yang S, Herrera-Estrella LR, Xu G, Chao DY, Li J, Wang PY, Qin F, Li J, Ding Y, Shi Y, Wang Y, Yang Y, Guo Y, Zhu JK (2020) Plant abiotic stress response and nutrient use efficiency. Sci China Life Sci 63(5):635–674. 10.1007/s11427-020-1683-x Hamel LP, Nicole MC, Sritubtim S, Morency MJ, Ellis M, Ehlting J, Beaudoin N, Barbazuk B, Klessig D, Lee J, Martin G, Mundy J, Ohashi Y, Scheel D, Sheen J, Xing T, Zhang S, Seguin A, Ellis BE (2006) Ancient signals: comparative genomics of plant MAPK and MAPKK gene families. Trends Plant Sci 11(4):192–198. 10.1016/j.tplants.2006.02.007 Ichimura K, Shinozaki K, Tena G, Sheen J, Henry Y, Champion A, Kreis M, Zhang S, Hirt H, Wilson C, Heberle-Bors E, Ellis BE, Morris PC, Innes RW, Ecker JR, Scheel D, Klessig DF, Machida Y, Mundy J, Ohashi Y, Walker JC (2002) Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci 7(7):301–308. 10.1016/s1360-1385(02)02302-6 Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y (2008) KEGG for linking genomes to life and the environment. Nucleic Acids Res 36(Database issue):D480–484. 10.1093/nar/gkm882 Karlova R, Boer D, Hayes S, Testerink C (2021) Root plasticity under abiotic stress. Plant Physiol 187(3):1057–1070. 10.1093/plphys/kiab392 Katsuta S, Masuda G, Bak H, Shinozawa A, Kamiyama Y, Umezawa T, Takezawa D, Yotsui I, Taji T, Sakata Y (2020) Arabidopsis Raf-like kinases act as positive regulators of subclass III SnRK2 in osmostress signaling. Plant J 103(2):634–644. 10.1111/tpj.14756 Komis G, Šamajová O, Ovečka M, Šamaj J (2018) Cell and Developmental Biology of Plant Mitogen-Activated Protein Kinases. Annu Rev Plant Biol 69:237–265. 10.1146/annurev-arplant-042817-040314 Kong X, Pan J, Zhang D, Jiang S, Cai G, Wang L, Li D (2013) Identification of mitogen-activated protein kinase kinase gene family and MKK-MAPK interaction network in maize. Biochem Biophys Res Commun 441(4):964–969. 10.1016/j.bbrc.2013.11.008 Kumar A, Singh S, Gaurav AK, Srivastava S, Verma JP (2020) Plant Growth-Promoting Bacteria: Biological Tools for the Mitigation of Salinity Stress in Plants. Front Microbiol 11:1216. 10.3389/fmicb.2020.01216 Kumar K, Sinha AK (2013) Overexpression of constitutively active mitogen activated protein kinase kinase 6 enhances tolerance to salt stress in rice. Rice (New York NY) 6(1):25. 10.1186/1939-8433-6-25 Li C, Yue J, Wu X, Xu C, Yu J (2014) An ABA-responsive DRE-binding protein gene from Setaria italica, SiARDP, the target gene of SiAREB, plays a critical role under drought stress. J Exp Bot 65(18):5415–5427. 10.1093/jxb/eru302 Li K, Yang F, Zhang G, Song S, Li Y, Ren D, Miao Y, Song CP (2017) AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade. Plant Physiol 173(2):1391–1408. 10.1104/pp.16.01386 Liang X, Zhou JM (2018) Receptor-Like Cytoplasmic Kinases: Central Players in Plant Receptor Kinase-Mediated Signaling. Annu Rev Plant Biol 69:267–299. 10.1146/annurev-arplant-042817-040540 Lu H, Zhang J, Liu KB, Wu N, Li Y, Zhou K, Ye M, Zhang T, Zhang H, Yang X, Shen L, Xu D, Li Q (2009) Earliest domestication of common millet (Panicum miliaceum) in East Asia extended to 10,000 years ago. Proc Natl Acad Sci USA 106(18):7367–7372. 10.1073/pnas.0900158106 Ma F, Ni L, Liu L, Li X, Zhang H, Zhang A, Tan M, Jiang M (2016) ZmABA2, an interacting protein of ZmMPK5, is involved in abscisic acid biosynthesis and functions. Plant Biotechnol J 14(2):771–782. 10.1111/pbi.12427 Mao X, Cai T, Olyarchuk JG, Wei L (2005) Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinf (Oxford England) 21(19):3787–3793. 10.1093/bioinformatics/bti430 Matsuoka D, Nanmori T, Sato K, Fukami Y, Kikkawa U, Yasuda T (2002) Activation of AtMEK1, an Arabidopsis mitogen-activated protein kinase kinase, in vitro and in vivo: analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings. Plant J 29(5):637–647. 10.1046/j.0960-7412.2001.01246.x Meng X, Zhang S (2013) MAPK Cascades in Plant Disease Resistance Signaling. Annu Rev Phytopathol 51:245–266. 10.1146/annurev-phyto-082712-102314 Muthamilarasan M, Prasad M (2015) Advances in Setaria genomics for genetic improvement of cereals and bioenergy grasses. Theor Appl Genet 128(1):1–14. 10.1007/s00122-014-2399-3 Muthamilarasan M, Prasad M (2021) Small Millets for Enduring Food Security Amidst Pandemics. Trends Plant Sci 26(1):33–40. 10.1016/j.tplants.2020.08.008 Pawson T, Scott JD (2005) Protein phosphorylation in signaling – 50 years and counting. Trends Biochem Sci 30(6):286–290. https://doi.org/10.1016/j.tibs.2005.04.013 Peng R, Zhang B (2020) Foxtail Millet: A New Model for C4 Plants. Trends Plant Sci 26(3):199–201. 10.1016/j.tplants.2020.12.003 Rathinapriya P, Pandian S, Rakkammal K, Balasangeetha M, Alexpandi R, Satish L, Rameshkumar R, Ramesh M (2020) The protective effects of polyamines on salinity stress tolerance in foxtail millet (Setaria italica L.), an important C4 model crop. Physiol Mol Biol Plants 26(9):1815–1829. 10.1007/s12298-020-00869-0 Sachdev N, Goomer S, Singh LR (2021) Foxtail millet: a potential crop to meet future demand scenario for alternative sustainable protein. J Sci Food Agric 101(3):831–842. https://doi.org/10.1002/jsfa.10716 Shen L, Zhuang B, Wu Q, Zhang H, Nie J, Jing W, Yang L, Zhang W (2019) Phosphatidic acid promotes the activation and plasma membrane localization of MKK7 and MKK9 in response to salt stress. Plant Sci 287:110190. 10.1016/j.plantsci.2019.110190 Teige M, Scheikl E, Eulgem T, Dóczi R, Ichimura K, Shinozaki K, Dangl JL, Hirt H (2004) The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol Cell 15(1):141–152. 10.1016/j.molcel.2004.06.023 Wang J, Pan C, Wang Y, Ye L, Wu J, Chen L, Zou T, Lu G (2015) Genome-wide identification of MAPK, MAPKK, and MAPKKK gene families and transcriptional profiling analysis during development and stress response in cucumber. BMC Genomics 16(1):386. 10.1186/s12864-015-1621-2 Xiao S, Wan Y, Guo S, Fan J, Lin Q, Zheng C, Wu C (2023) Transcription Factor SiDi19-3 Enhances Salt Tolerance of Foxtail Millet and Arabidopsis. Int J Mol Sci 24(3). 10.3390/ijms24032592 Xing Y, Chen WH, Jia W, Zhang J (2015) Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress. J Exp Bot 66(19):5971–5981. 10.1093/jxb/erv305 Xu C, Luo M, Sun X, Yan J, Shi H, Yan H, Yan R, Wang S, Tang W, Zhou Y, Wang C, Xu Z, Chen J, Ma Y, Jiang Q, Chen M, Sun D (2022) SiMYB19 from Foxtail Millet (Setaria italica) Confers Transgenic Rice Tolerance to High Salt Stress in the Field. Int J Mol Sci 23(2). 10.3390/ijms23020756 Xu J, Zhang S (2015) Mitogen-activated protein kinase cascades in signaling plant growth and development. Trends Plant Sci 20(1):56–64. 10.1016/j.tplants.2014.10.001 Yang J, Ji L, Liu S, Jing P, Hu J, Jin D, Wang L, Xie G (2021) The CaM1-associated CCaMK-MKK1/6 cascade positively affects lateral root growth via auxin signaling under salt stress in rice. J Exp Bot 72(18):6611–6627. 10.1093/jxb/erab287 Yang Z, Zhang H, Li X, Shen H, Gao J, Hou S, Zhang B, Mayes S, Bennett M, Ma J, Wu C, Sui Y, Han Y, Wang X (2020) A mini foxtail millet with an Arabidopsis-like life cycle as a C4 model system. Nat Plants 6(9):1167–1178. 10.1038/s41477-020-0747-7 Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol 11(2):R14. 10.1186/gb-2010-11-2-r14 Yu Y, Guo DD, Min DH, Cao T, Ning L, Jiang QY, Sun XJ, Zhang H, Tang WS, Gao SQ, Zhou YB, Xu ZS, Chen J, Ma YZ, Chen M, Zhang XH (2023) Foxtail millet MYB-like transcription factor SiMYB16 confers salt tolerance in transgenic rice by regulating phenylpropane pathway. Plant Physiol Biochem 195:310–321. 10.1016/j.plaphy.2022.11.032 Zhang M, Zhang S (2022) Mitogen-activated protein kinase cascades in plant signaling. J Integr Plant Biol 64(2):301–341. 10.1111/jipb.13215 Zhu J-K (2016) Abiotic Stress Signaling and Responses in Plants. Cell 167(2):313–324. 10.1016/j.cell.2016.08.029 Zhu Q, Shao Y, Ge S, Zhang M, Zhang T, Hu X, Liu Y, Walker J, Zhang S, Xu J (2019) A MAPK cascade downstream of IDA-HAE/HSL2 ligand-receptor pair in lateral root emergence. Nat Plants 5(4):414–423. 10.1038/s41477-019-0396-x Supplementary Files Supplementalfile.rar Cite Share Download PDF Status: Published Journal Publication published 23 Aug, 2024 Read the published version in Plant Growth Regulation → Version 1 posted Editorial decision: Major revisions 06 Apr, 2024 Reviewers agreed at journal 20 Mar, 2024 Reviewers invited by journal 20 Mar, 2024 Editor invited by journal 02 Feb, 2024 Editor assigned by journal 23 Jan, 2024 First submitted to journal 21 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3887368","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":281696318,"identity":"2edec1a1-0bdc-470f-b8c1-918a6902a580","order_by":0,"name":"Yaqiong Li","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yaqiong","middleName":"","lastName":"Li","suffix":""},{"id":281696319,"identity":"01e61ccb-6be5-4730-9262-a9bfd92cd185","order_by":1,"name":"Kai Huang","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Huang","suffix":""},{"id":281696323,"identity":"2183c376-6cfc-40d0-b6c8-62b9192766f6","order_by":2,"name":"Huazhuan He","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Huazhuan","middleName":"","lastName":"He","suffix":""},{"id":281696325,"identity":"f712f5b5-bbe3-426f-b13a-0fd3c53d520a","order_by":3,"name":"Yuhuan Yang","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuhuan","middleName":"","lastName":"Yang","suffix":""},{"id":281696327,"identity":"83df687a-0e47-482c-a41e-5bc17839b39c","order_by":4,"name":"Xiaoxia Meng","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxia","middleName":"","lastName":"Meng","suffix":""},{"id":281696328,"identity":"36420a7e-689d-4a5d-9b88-34b85f17e70b","order_by":5,"name":"Guiyun Yan","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Guiyun","middleName":"","lastName":"Yan","suffix":""},{"id":281696329,"identity":"3362e621-7360-44c3-b65e-71873337b0a6","order_by":6,"name":"Yaofei Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYDAC5gNsQNIGymMjRgtbAkhZGulaDpOgxeAY+7MHPyrO2xscP2PA8KHsMAP/7AZCWhjSDXvO3E6c2ZNjwDjj3GEGiTsHCGi533BMgrftdgI/Q44BM2/bYQYDiQRCtjC2Sf5tO2fPxv/GgPkvcVqY2aR52w4w9ksAbWEkRovkMTY2aZkzyYkzZzwrONhzLp1H4gYBLXzAEJN8U2Fnb3A+eeODH2XWcvwzCGhROIDEAbF58KsHAvkGgkpGwSgYBaNgxAMAWHNBFDENlpMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2412-5073","institution":"Shanxi Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Yaofei","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2024-01-22 08:27:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3887368/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3887368/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10725-024-01180-8","type":"published","date":"2024-08-23T15:57:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53283468,"identity":"3d4412a3-fa29-43f8-8764-db53c180aafa","added_by":"auto","created_at":"2024-03-22 21:04:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":157287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of RNA sequencing data.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The Venn diagram showed the number of genes expressed in the experimental group (YG1_Na) and the control group (YG1_CK). (B) Number of upregulated and downregulated DEGs in the two groups.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887368/v1/b998d424eecd906425d5e516.jpg"},{"id":53283466,"identity":"cd48e8db-c5ac-4aa1-a77c-f5a5695cc412","added_by":"auto","created_at":"2024-03-22 21:04:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":278519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnriched GO terms of DEGs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe top ten terms of BP, CC and MF were displayed. The y-axis represents the significant level of enrichment.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887368/v1/f24c5ea3f0f557b59170c2bc.jpg"},{"id":53283470,"identity":"c558b9de-f80f-4828-bf72-6ff0d86ea055","added_by":"auto","created_at":"2024-03-22 21:04:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":245656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnriched KEGG pathways of the DEGs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe y-axis shows the description of the corresponding KEGG pathways. The top 20 KEGG pathways enriched in DEGs are shown.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887368/v1/0f1523148f8d055bb135d7b0.jpg"},{"id":53283606,"identity":"92ef9b60-13dc-4b51-a8c8-208dff3efa20","added_by":"auto","created_at":"2024-03-22 21:12:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":475516,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene structure analysis and protein domain prediction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Structure of \u003cem\u003eSiMKK\u003c/em\u003e genes with exon(s) in yellow, UTR regions in blue, and solid lines between exons indicating introns. (B) The cis-elements prediction in the 2.0 kb promoter region upstream from the start codon of \u003cem\u003eSiMKKs\u003c/em\u003e. (C) Structures of SiMKK proteins.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887368/v1/a89b1f541878b3245204d1fd.jpg"},{"id":53283605,"identity":"95222f04-bad5-477c-ac62-f731b55e9615","added_by":"auto","created_at":"2024-03-22 21:12:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":409983,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relative expression levels of SiMKKs were detected by quantitative real time PCR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Subgroup A; (B) Subgroup B; (C) Subgroup C; (D) Subgroup D. Three biological replicates of each sample were used for qRT-PCR analysis. SiACTIN (Seita 5G464000) was used as an endoge-nous control. Bars with different lowercase letters are significantly different at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (ANOVA).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887368/v1/289178c35173e42ef389f6f2.jpg"},{"id":53283472,"identity":"c16ea274-a1f8-4f90-8ac8-6896d6a500b0","added_by":"auto","created_at":"2024-03-22 21:04:24","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":328972,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic evaluation of lateral roots development of Arabidopsis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phenotype of wild type Arabidopsis and heterologous overexpression transgenic lines grown on 1/2 MS medium with or without 100 mM NaCl and for 2 weeks. Bar = 2 cm. (B) and (C) The number of lateral roots. 30 independent seedlings of each line were counted. Each point on the graph represents a biological duplicate, the same hereafter. Bars with different lowercase letters are significantly different at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887368/v1/74d221adc45ff272cd51ee9c.jpg"},{"id":53283607,"identity":"2e941ced-34ce-4478-bc1f-6d6c28e57c96","added_by":"auto","created_at":"2024-03-22 21:12:24","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":388999,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic evaluation of primary roots growth of Arabidopsis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phenotype of seed-lings grown on 1/2 MS medium with or without 100 mM NaCl. Bar = 1 cm. (B) Measurement of primary root length. 20 independent seedlings of each line were measured. (C) Relative root growth. The ratio of average root length grown on 1/2 MS medium with or without 100 mM NaCl. Bars with different lowercase letters are significantly different at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and ns means no statis-tically significant difference.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887368/v1/1bf5714f0e9074403da495da.jpg"},{"id":63300348,"identity":"2ff1e16f-9492-417b-b7f3-6feba62da682","added_by":"auto","created_at":"2024-08-26 16:13:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2927246,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3887368/v1/a64f10ee-2e43-49ad-a307-1e9b55f5d2e4.pdf"},{"id":53283474,"identity":"d3307055-4585-4514-8227-42f00d6bc300","added_by":"auto","created_at":"2024-03-22 21:04:25","extension":"rar","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":496497,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfile.rar","url":"https://assets-eu.researchsquare.com/files/rs-3887368/v1/051f58926b2d6bd10e79b01e.rar"}],"financialInterests":"","formattedTitle":"Heterologous Expressions of foxtail millet (Seteria italica) mitogen-activated protein kinase kinase (SiMKK) group A genes regulate root development under salt stress in Arabidopsis thaliana","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs sessile organisms, plants have to survive under environmental stresses that inhibit their growth and development (Fang and Xiong \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). One of the major threats to plant growth and agricultural production is the loss of water as global weather trends shift toward drier and the aggravation of soil salinization (Gong et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). When subjected to abiotic stress, plant cells activate various response pathways to restore cellular and organism-level homeostasis. Among them, a number of necessary signaling pathways function under multiple stresses such as Ca\u003csup\u003e2+\u003c/sup\u003e signaling, the production of reactive oxygen species and phosphoprotein cascades (Zhu \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn eukaryotes, mitogen-activated protein kinase (MAPK) cascades are highly conserved modules downstream of signal receptors which play important roles in transducing extracellular stimuli into intracellular responses (Meng and Zhang \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). A classical MAPK module consisting of three classes of protein kinases named MAPKs, MAPKKs (MAPK kinases) and MAPKKKs (MAPKK kinases). When stimulated, cell surface\u0026ndash;localized receptors recognize signal molecules, and then activate MAPK signaling cascade through phosphorylation to efficiently amplify the signal (Liang and Zhou \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu and Zhang \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The Arabidopsis thaliana genome contains approximately 60 MAPKKKs, 10 MAPKKs and 20 MAPKs (Komis et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ichimura et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The similar gene families encoding proteins involved in MAPK cascades have also been identified in other plants species, such as rice, maize, and cucumber (Kong et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e; Hamel et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The number of MAPKKs is less than that of MAPKKs and MAPKs, which indicates that the same MAPKK can integrate the signals received by different MAPKKs and transmit them to multiple MAPKs. According to sequence similarity, MAPKKs can be divided into four groups (A-D). Group A includes AtMKK1/2/6, group B includes AtMKK3, group C includes AtMKK4/5, and group D includes AtMKK7/8/9/10 (Meng and Zhang \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hamel et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Some early studies have revealed that several members of MAPK cascades are key components in plants responses to abiotic stresses. For example, The ABA-INSENSITIVE PROTEIN KINASE 1‐MKK5‐MPK6 cascade positively regulates the ABA‐mediated stomatal closure and root growth in Arabidopsis (Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). During osmotic stress, Raf-like MAPKKKs ARK1, ARK2 and ARK3 interact with and phosphorylate OST1 to positively regulate the stress responses (Katsuta et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In maize, ZmMPK5 phosphorylates and enhances the stability and activity of a dehydrogenase ZmABA2 that contributes to ABA biosynthesis (Ma et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFoxtail millet (\u003cem\u003eSetaria italica\u003c/em\u003e), one of the representative cereals of ancient times in the world, gets a lot of attention in recent years owing to the unique composition of its seeds containing high content of health-promoting properties (Lu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sachdev et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Because of the high tolerance to drought and barren, foxtail millet is widely cultivated in semi-arid regions and is also thought to be an important crop to ensure food and nutritional security in the face of a quickly growing world population, particularly during abnormal situations (Muthamilarasan and Prasad \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). On the other hand, foxtail millet has a small diploid genome of ~\u0026thinsp;490 Mb and the its genome was fully sequenced. Recently, an Arabidopsis-like foxtail millet was constructed which provided a good plant model for plant gene functional studies (Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Peng and Zhang \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For the above reasons, it is effective to study the molecular mechanisms of abiotic stress tolerance in foxtail millet and some stress-tolerance genes were identified through sequence characteristics and expression patterns under stress. For example, it was found that the promotor of \u003cem\u003eSiARDP\u003c/em\u003e contained ABA response element and the expression level of \u003cem\u003eSiARDP\u003c/em\u003e was induced by ABA. Overexpression of \u003cem\u003eSiARDP\u003c/em\u003e improved the drought resistance of plants (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, few studies have uncovered the functions of conserved signaling pathways like MAPK cascade in foxtail millet. In this study, transcriptome analysis revealed that MAPK signaling pathway plays an important role in foxtail millet response to salt stress. We identified 14 MKK genes in foxtail millet genome and analyzed the functions of \u003cem\u003eSiMKK1 SiMKK6-1\u003c/em\u003e and \u003cem\u003eSiMKK6-2\u003c/em\u003e in salt stress responses. By this study, we may provide new insights into the role of MAPK cascade in foxtail millet.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003eA foxtail millet variety (Yugu1/YG1), the Arabidopsis thaliana (Col-0 wild type and transgenic lines in Col-0 background) were used in this study. Arabidopsis thaliana grown in the growth room at 23\u0026deg;C with 16-h-light /8-h-dark. Foxtail millet seedlings were grown in a growth chamber under greenhouse conditions with a 16 h light (25℃)/8 h dark (23℃) cycle. Foxtail millet seedlings with three leaves were transferred to Hoagland\u0026rsquo;s solution and precultured for 24 h. For NaCl treatment, seedlings were cultured in Hoagland\u0026rsquo;s solution containing 250 mM NaCl. Leaves were collected un-der a time series treatment (0, 3, 6, 9, and 12 h for NaCl treatment) for RNA extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome sequencing and analyses\u003c/h2\u003e \u003cp\u003eTotal RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonucle-ase/polymerase activities. After adenylation of 3\u0026rsquo; ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferentially 370\u0026thinsp;~\u0026thinsp;420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.\u003c/p\u003e \u003cp\u003eWe performed Gene Ontology (GO) enrichment analysis by the GOseq R packages which is a published method (Young et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). KEGG database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to discover the regulatory pathways of genes (Kanehisa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). We used a software KOBAS (Mao et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) to investigate enrichment of DEGs in KEGG pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe amino acid sequence of Arabidopsis MKK was obtained from TAIR website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The \u003cem\u003eMKK\u003c/em\u003e genes of foxtail millet, maize and sorghum were retrieved from Phytozome database by using the amino acid sequence of Arabidopsis MKK as the submission sequence. The amino acid sequence of rice MKK (Hamel et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) was also obtained from Phytozome database. Multiple sequence alignment was performed by ClustalW, and the phylogenetic tree was constructed by maximum likelihood method with MEGA 6.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGene and protein properties analysis\u003c/h2\u003e \u003cp\u003eChromosome positional information of \u003cem\u003eSiMKKs\u003c/em\u003e was obtained from Phytozome database, and images were drawn by MapChart software. The cis-regulatory elements in the promoter region of the \u003cem\u003eSiMKKs\u003c/em\u003e gene were identified by PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The exon and intron structure diagrams of the \u003cem\u003eSiMKKs\u003c/em\u003e gene were drawn on GSDS2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.gao-lab.org/\u003c/span\u003e\u003cspan address=\"http://gsds.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) website. The protein parameters were evaluated though Expasy ProtParam tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA extraction and qRT-PCR\u003c/h2\u003e \u003cp\u003e RNA extraction was performed according to the instructions of RNApure Plant Kit (CWBIO CW0559S). The first-strand cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (RR047A; Takara). qRT-PCR was performed using the Bio-Rad CFX96TM Touch real-time PCR detection system with SYBR Premix Ex TaqTM II (RR820A; Takara). Transcript levels were normalized to the \u003cem\u003eSiACTIN\u003c/em\u003e (Seita.5G464000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVector construction and plants transformation\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eSiMKK1\u003c/em\u003e, \u003cem\u003eSiMKK6-1\u003c/em\u003e and \u003cem\u003eSiMKK6-2\u003c/em\u003e coding regions were amplified with primers (Supplemental Table\u0026nbsp;1). Then the fragments were subcloned into pSuper1300 (GFP) vector under the control of the CaMV 35S promoter. The resulting constructs were introduced into the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101. Transformation of \u003cem\u003eArabidopsis\u003c/em\u003e plants was performed using the floral dip method (Clough and Bent \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic evaluation\u003c/h2\u003e \u003cp\u003eArabidopsis seeds were grown on the 1/2 MS medium for 4 days and then seed-lings of wild type and transgenic lines were transferred to 1/2 MS medium with or without 100 mM NaCl and grown for additional 3 days for primary root length measurement. Arabidopsis seeds were grown 1/2 MS medium with or without 100 mM NaCl and grown for 2 weeks for lateral roots observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical assays of three independent experiments were performed using online ANOVA tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://vassarstats.net/anova1u.html\u003c/span\u003e\u003cspan address=\"http://vassarstats.net/anova1u.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The resulting ANOVA \u003cem\u003eP\u003c/em\u003e-values were used to indicate significant differences between multiple groups.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cdiv id=\"Sec11\" type=\"Results\" class=\"Section2\"\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eTranscriptome analysis of foxtail millet under salt stress\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanism of stress-resistance in foxtail millet, we conducted the RNA sequencing. In three leaves and one-half leaf period, seedlings of YG1 were respectively treated with 300 mM NaCl (experiment group, YG1_Na) and distilled water (control group, YG1_CK) for 24h. Then, the total RNA was extracted from the top leave of each seedling (three biological replicates per group and six samples in total). Through RNA-sequencing, we obtained 144,677,546 clean reads with a total base number of 41.84 G and the GC contents were between 58.04% and 59.73%. Q20 and Q30 values of 6 samples were all above 90.66%. We compared the clean reads with the genome of YG1, and the mapping rate of each sample was over 95% indicating that transcriptome data were of high quality and could be further analyzed (Supplemental Table\u0026nbsp;1). The data showed there were 16477 and 16131 genes expressed in experiment group and control group respectively, 15507 of which were expressed in both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Then, differentially expressed genes (DEGs) were identified by comparing the read counts of genes in the two groups. In total, 1740 genes displayed different expression pattern after salt treatment, among which 1349 genes were upregulated and 391 were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEnrichment analysis of DEGs induced by salt treatment\u003c/h2\u003e \u003cp\u003eTo further understand the biological function of DEGs, gene ontology (GO) enrichment analysis was performed and 30 significant enrichment GO terms are presented. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the significantly enriched GO terms of Biological Process (BP) included the \u0026ldquo;stress responses\u0026rdquo; and \u0026ldquo;response to chemical\u0026rdquo;. The most enriched Cellular Component (CC) was \u0026ldquo;extracellular region\u0026rdquo; revealed the plant needed to modulate metabolism to cope with external salt stress. In addition, the significantly enriched GO terms of molecular function (MF) were \u0026ldquo;DNA-binding transcription factor activity\u0026rdquo; and \u0026ldquo;transcription regulator activity\u0026rdquo; suggested that transcriptional regulation were mainly activated by salt stimulus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we performed enrichment analyses of DEGs based on Kyoto Encyclopedia of Genes and Genomes (KEGG) which revealed that the DEGs were mainly enriched in 96 pathways (Supplemental Table\u0026nbsp;3). Among them, DEGs were most significantly enriched in the MAPK signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGenome-wide characterization of the Mitogen-activated protein kinase kinases in\u003c/b\u003e \u003cb\u003eSeteria italica\u003c/b\u003e \u003cb\u003e(SiMKKs)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe transcriptome analysis indicated that MAPK signaling pathway was essential for the responses to salt stress in foxtail millet. However, few investigations were focus on the MAPK signaling of foxtail millet and the components of MAPK cascades had not yet been identified. Therefore, we performed a comprehensive identification to confirm Mitogen-activated protein kinase kinases in \u003cem\u003eSeteria italica\u003c/em\u003e (SiMKKs). In this assay, the amino acid sequences of ten Arabidopsis MKKs (TAIR: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used as queries for BLAST searches of the foxtail millet genome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). As results, a total number of fourteen \u003cem\u003eSiMKKs\u003c/em\u003e genes were identified and the physicochemical characteristics of the corresponding proteins were predicted (Table\u0026nbsp;1). \u003cem\u003eSiMKKs\u003c/em\u003e were distributed on seven chromosomes: three each on chromosome 1, chromosome 5 and chromosome 9; two on chromosome 4; and one each on chromosome 2, chromosome 3 and chromosome 6 (Supplemental Fig.\u0026nbsp;1). MAPK cascades are conserved in plants and MKK genes have been identified in other species. To study the phylogenetic relationship between foxtail millet and other species, we constructed a phylogenetic tree of MKK proteins from \u003cem\u003eSeteria italica\u003c/em\u003e (Si), \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (At), \u003cem\u003eOryza sativa\u003c/em\u003e (Os). As shown in Supplemental Fig.\u0026nbsp;2, the phylogenetic analysis suggested that MKKs from different species all could be divided into four groups (A, B, C and D). MKKs in foxtail millet (SiMKKs) were named based on the result of phylogenetic analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGene structural and conserved domain analysis\u003c/h2\u003e \u003cp\u003eTo further clarify the evolutionary relationships and explore the structural diversity of \u003cem\u003eSiMKKs\u003c/em\u003e, the distribution of exon-intron structure was analyzed and displayed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cem\u003eSiMKK1\u003c/em\u003e, \u003cem\u003eSiMKK3-1\u003c/em\u003e and \u003cem\u003eSiMKK3-2\u003c/em\u003e contained eight introns; \u003cem\u003eSiMKK3-3\u003c/em\u003e contained nine introns; both \u003cem\u003eSiMKK6-1\u003c/em\u003e and \u003cem\u003eSiMKK6-2\u003c/em\u003e had seven introns; \u003cem\u003eSiMKK10-4\u003c/em\u003e had one intron and the other \u003cem\u003eSiMKKs\u003c/em\u003e didn\u0026rsquo;t contain any introns. It was showed that members in same group had similar exon-intron structures. On the other hand, we performed promoter analysis of \u003cem\u003eSiMKKs\u003c/em\u003e which predicted the cis-elements in the 2.0 kb promoter region upstream from the start codon (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). All \u003cem\u003eSiMKKs\u003c/em\u003e contains more than one MYB-binding sites and MYC-binding sites. The numbers of CGTCA-motif, TGACG-motif and G-box were significantly higher than other motifs. For phytohormone response element, the results showed there were more ABA response elements (ABRE) than auxin response elements (ARE) in the promoters of \u003cem\u003eSiMKKs\u003c/em\u003e, which indicated \u003cem\u003eSiMKKs\u003c/em\u003e might play critical roles in stress responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we predicted the conserved domains of SiMKKs with an online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://prosite.expasy.org/prosite.html\u003c/span\u003e\u003cspan address=\"https://prosite.expasy.org/prosite.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). As the result shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, all SiMKKs contained protein kinase domain, ATP binding region and kinase activate sites. Meanwhile, members of group B (SiMKK3-1, SiMKK3-2 and SiMKK3-3) contained nuclear transport factor 2 domain at the C-terminal of these proteins.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSiMKK\u003c/b\u003e \u003cb\u003egenes expression patterns in response to salt stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the response of \u003cem\u003eSiMKK\u003c/em\u003e genes to salt stress, we treated seedlings of YG1 with 200 mM NaCl and detected the expression patterns of 14 \u003cem\u003eSiMKKs\u003c/em\u003e genes within 12 hours after salt treatment. Except for \u003cem\u003eSiMKK4-2\u003c/em\u003e, \u003cem\u003eSiMKK10-2\u003c/em\u003e and \u003cem\u003eSiMKK10-5\u003c/em\u003e, the expression level of the other 11 \u003cem\u003eSiMKKs\u003c/em\u003e up-regulated within 12 h of salt treatment. Among genes with up-regulation expression, the expression levels \u003cem\u003eSiMKK3-1\u003c/em\u003e, \u003cem\u003eSiMKK10-1\u003c/em\u003e and of all three genes in group A increased at least fivefold. In addition, the expression level of \u003cem\u003eSiMKK6-2\u003c/em\u003e increased by more than 20 times 3 hours after salt treatment which was the highest in \u003cem\u003eSiMKK\u003c/em\u003e gene family.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRoot development of heterologous overexpression lines of\u003c/b\u003e \u003cb\u003eSiMKK1\u003c/b\u003e, \u003cb\u003eSiMKK6-1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eSiMKK6-2\u003c/b\u003e \u003cb\u003ein Arabidopsis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs the expression levels of the three genes in subgroup A were all significantly increased under salt treatment, and 6\u0026thinsp;\u0026minus;\u0026thinsp;2 was the largest increase among all family members. Next, we took a closer look at these three genes. Through the existing transcriptome data, we analyzed the expression patterns of 14 MKK genes in different tissues and different growth periods, and the data showed that the expression levels of the three genes in group A were high in multiple tissues such as root and leaf. In addition, we also predicted the interacting proteins of the three proteins, and the results showed that the predicted interacting proteins of 6\u0026thinsp;\u0026minus;\u0026thinsp;1 and 6\u0026thinsp;\u0026minus;\u0026thinsp;2 were the same. SiMKK1 shares many common interacting proteins with 6\u0026thinsp;\u0026minus;\u0026thinsp;1 and 6\u0026thinsp;\u0026minus;\u0026thinsp;2, with 8 of the 10 proteins with the highest likelihood of interacting being the same. Therefore, we hypothesized that the three proteins might have very similar functions.\u003c/p\u003e \u003cp\u003eWe cloned CDS of these three genes, constructed 35S promoter-driven expression vectors, and transformed them into Arabidopsis thaliana to obtain heterologous overexpression lines. Through phenotypic observation, we found that all overexpressed transgenic strains could have more developed roots under normal growth conditions, and the number of lateral roots was significantly higher than that of wild Arabidopsis. We planted the plants on a medium containing 100mM NaCl, and the results showed that under salt stress, the number of lateral roots of the transgenic plants was also significantly higher than that of the wild type. And two separate lines of the same gene showed similar phenotypes.\u003c/p\u003e \u003cp\u003eIn addition, we also tested whether these three genes were involved in the regulation of taproot length. We grew wild-type plants and overexpressed strains on MS medium and medium containing 100mM NaCl respectively, and then measured the root length. The results showed that taproot length had no significant difference when there was no salt stress. The root length of transgenic plants was significantly longer than that of the wild type. Through calculation of relative root length (the ratio of average root length under salt stress to average root length under normal growth), we found that overexpression of these three millet MKK genes could significantly reduce the shortening of taproots under salt stress.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSaline soils are estimated to increase in ~\u0026thinsp;50% of irrigated lands because of the chemical fertilizers application (Kumar et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, it is of great significance to study the mechanism of plant salt resistance at the molecular level is critical for crop production. Foxtail millet is one of the representative cereals with high nutritional value and strong stress resistance which is ideal for food and nutritional security (Muthamilarasan and Prasad \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Owing to its short life cycle, small plant size, and availability of whole-genome sequencing data, foxtail millet is a suitable model for the discovery of stress response genes (Peng and Zhang \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eSiDi19-3\u003c/em\u003e encoded a transcription factor contains Cys2/His2-type zinc-finger motifs and \u003cem\u003eSiDi19-3\u003c/em\u003e overexpression increased the transcript levels of most Na+/H\u0026thinsp;+\u0026thinsp;antiporter (NHX), salt overly sensitive (SOS), and calcineurin B-like protein (CBL) genes and improved the salt tolerance of plants (Xiao et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eSiMYB16 and SiMYB19\u003c/em\u003e were both upregulated by salt stress. Overexpression of \u003cem\u003eSiMYB16\u003c/em\u003e and \u003cem\u003eSiMYB19\u003c/em\u003e enhanced salt tolerance of plants by regulating the biosynthesis of lignin and ABA signal transduction respectively (Yu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Salt stress resulted in decreased biomass, decreased relative water content and membrane damage in foxtail millet (Rathinapriya et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Soil salinity provokes water and nutrient imbalance which causes plant growth inhibition and plant roots perceive these stresses in the soil and adapt their architecture accordingly (Karlova et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). When challenged with high salt stress, the growth of primary and lateral roots is arrested. (Gandullo et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Salt-resistant plant could cope with environmental changes through various growth regulation, including root development regulation. Excellent root development is an important guarantee for plants to maintain water and nutrient balance under salt stress. In this study, we provide new gene information for foxtail millet response to salt stress and revealed three genes that enhanced plant salt tolerance by modulating root development. The full utilization of these three genes in breeding is expected to enhance the stress resistance of millet by improving root development.\u003c/p\u003e \u003cp\u003eMAPK cascades are ubiquitous signaling modules in eukaryotes which involved in almost all fundamental cellular processes (Ichimura et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Xu and Zhang \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Pawson and Scott \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Hence, MAPK signaling pathway is also employed to activate the stress resistance of plants under salt stress. The mismatch of between the numbers of MKKs and MAPK substrates suggests that individual MKKs have the capacity to address more than one MAPK and MKKs might be core hubs in multiple signal pathways (Kong et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In Arabidopsis, five out of ten MKKs (AtMKK1, AtMKK2, AtMKK5, AtMKK7 and AtMKK9) were involved in regulating plant responses to salt stress (Shen et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Xing et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Teige et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Matsuoka et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In this study, we also found the expression level of 11 \u003cem\u003eSiMKKs\u003c/em\u003e were up-regulated under salt treatment, five of which were significantly up-regulated indicating the resistance of foxtail millet to salt stress were also dependent on the participation of multiple MKKs.\u003c/p\u003e \u003cp\u003eMKKs in plant were involved in plant response to salt stress by regulating various pathways. For instance, AtMKK5 modulated the expression of iron superoxide dismutase gene and ZmMKK1 enhanced the expression of ROS scavenging enzyme- and ABA-related genes (Cai et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). SiMKK1, SiMKK6-1 and SiMKK6-2 could promote root development, increase the length of primary root and the number of lateral roots under stress conditions, which was conducive to ensure the absorption of water and nutrients in high salt soil. Similar results were found in rice. OsMKK1, and OsMKK6 regulated the transcript levels of the genes related to auxin content enhancement to regulate lateral root growth under salt stress (Yang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kumar and Sinha \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These results indicate that the maintenance of root development is an important way for plants resistance to salt stress and MKKs is conservative in regulating root development because SiMKK1/6 in rice and foxtail millet showed high similarity in sequence and were close in evolution (see Supplemental Fig.\u0026nbsp;2). On the other hand, MPK3 and MPK6 are implicated in plant salinity stress in several different contexts, probably a result of the sharing of a regulatory component/event by multiple stress pathways (Zhang and Zhang \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). MPK3/6 play critical roles in lateral root emergence (Zhu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), however, there is not enough evidence provided MPK3 regulated root development under salt stress. Through prediction of protein interaction, we found that SiMKK1 might interact with MPK3 and MPK6 in foxtail millet (labeled as K4ABG1 and K3XXF6 in Supplemental Fig.\u0026nbsp;4). SiMKK6-1 and SiMKK6-2 might interact with MKK3 in foxtail millet. These results provide a probability for further study to investigate whether signals were transduced from members of group A of SiMKKs to MPK3/6 in foxtail millet to promote root development under salt stress.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we demonstrated the importance of MAPK signaling pathway in resistance of foxtail millet to salt stress through RNA sequencing and analysis, and demonstrated that SiMKK1, SiMKK6-1 and SiMKK6-2 could maintain the development of primary root and lateral roots under salt stress through expression patterns and transgenic technology. This study laid a foundation for the analysis of the stress resistance mechanism of millet, and provided important genetic resources for cultivating crops with high stress resistance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China, 32101760 and 32101722; Natural Science Foundation of Shanxi Province, grant number, 20210302123420 and 20210302123362; The Distinguished and Excellent Young Scholar Cultivation Project of Shanxi Agricultural University, 2022YQPYGC02.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYaofei Zhao designed this study. Yaqiong Li, Kai Huang and Huazhuan He performed the experiments. Yuhuan Yang and Xiaoxia Meng participated in the data analysis. Yaofei Zhao and Yaqiong Li wrote the manuscript. Yaofei Zhao and Guiyun Yan reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCai G, Wang G, Wang L, Liu Y, Pan J, Li D (2014) A maize mitogen-activated protein kinase kinase, ZmMKK1, positively regulated the salt and drought tolerance in transgenic Arabidopsis. J Plant Physiol 171(12):1003\u0026ndash;1016. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jplph.2014.02.012\u003c/span\u003e\u003cspan address=\"10.1016/j.jplph.2014.02.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J, Wang L, Yuan M (2021a) Update on the Roles of Rice MAPK Cascades. Int J Mol Sci 22(4). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms22041679\u003c/span\u003e\u003cspan address=\"10.3390/ijms22041679\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Ding Y, Yang Y, Song C, Wang B, Yang S, Guo Y, Gong Z (2021b) Protein kinases in plant responses to drought, salt, and cold stress. J Integr Plant Biol 63(1):53\u0026ndash;78. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jipb.13061\u003c/span\u003e\u003cspan address=\"10.1111/jipb.13061\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735\u0026ndash;743. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1046/j.1365-313x.1998.00343.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-313x.1998.00343.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang Y, Xiong L (2015) General mechanisms of drought response and their application in drought resistance improvement in plants. Cell Mol Life Sci 72(4):673\u0026ndash;689. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00018-014-1767-0\u003c/span\u003e\u003cspan address=\"10.1007/s00018-014-1767-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGandullo J, Ahmad S, Darwish E, Karlova R, Testerink C (2021) Phenotyping Tomato Root Developmental Plasticity in Response to Salinity in Soil Rhizotrons. Plant Phenomics 2021:2760532. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.34133/2021/2760532\u003c/span\u003e\u003cspan address=\"10.34133/2021/2760532\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong Z, Xiong L, Shi H, Yang S, Herrera-Estrella LR, Xu G, Chao DY, Li J, Wang PY, Qin F, Li J, Ding Y, Shi Y, Wang Y, Yang Y, Guo Y, Zhu JK (2020) Plant abiotic stress response and nutrient use efficiency. Sci China Life Sci 63(5):635\u0026ndash;674. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11427-020-1683-x\u003c/span\u003e\u003cspan address=\"10.1007/s11427-020-1683-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamel LP, Nicole MC, Sritubtim S, Morency MJ, Ellis M, Ehlting J, Beaudoin N, Barbazuk B, Klessig D, Lee J, Martin G, Mundy J, Ohashi Y, Scheel D, Sheen J, Xing T, Zhang S, Seguin A, Ellis BE (2006) Ancient signals: comparative genomics of plant MAPK and MAPKK gene families. Trends Plant Sci 11(4):192\u0026ndash;198. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tplants.2006.02.007\u003c/span\u003e\u003cspan address=\"10.1016/j.tplants.2006.02.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIchimura K, Shinozaki K, Tena G, Sheen J, Henry Y, Champion A, Kreis M, Zhang S, Hirt H, Wilson C, Heberle-Bors E, Ellis BE, Morris PC, Innes RW, Ecker JR, Scheel D, Klessig DF, Machida Y, Mundy J, Ohashi Y, Walker JC (2002) Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci 7(7):301\u0026ndash;308. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s1360-1385(02)02302-6\u003c/span\u003e\u003cspan address=\"10.1016/s1360-1385(02)02302-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y (2008) KEGG for linking genomes to life and the environment. Nucleic Acids Res 36(Database issue):D480\u0026ndash;484. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkm882\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkm882\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarlova R, Boer D, Hayes S, Testerink C (2021) Root plasticity under abiotic stress. Plant Physiol 187(3):1057\u0026ndash;1070. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/plphys/kiab392\u003c/span\u003e\u003cspan address=\"10.1093/plphys/kiab392\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatsuta S, Masuda G, Bak H, Shinozawa A, Kamiyama Y, Umezawa T, Takezawa D, Yotsui I, Taji T, Sakata Y (2020) Arabidopsis Raf-like kinases act as positive regulators of subclass III SnRK2 in osmostress signaling. Plant J 103(2):634\u0026ndash;644. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/tpj.14756\u003c/span\u003e\u003cspan address=\"10.1111/tpj.14756\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKomis G, Šamajov\u0026aacute; O, Ovečka M, Šamaj J (2018) Cell and Developmental Biology of Plant Mitogen-Activated Protein Kinases. Annu Rev Plant Biol 69:237\u0026ndash;265. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-arplant-042817-040314\u003c/span\u003e\u003cspan address=\"10.1146/annurev-arplant-042817-040314\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong X, Pan J, Zhang D, Jiang S, Cai G, Wang L, Li D (2013) Identification of mitogen-activated protein kinase kinase gene family and MKK-MAPK interaction network in maize. Biochem Biophys Res Commun 441(4):964\u0026ndash;969. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbrc.2013.11.008\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2013.11.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar A, Singh S, Gaurav AK, Srivastava S, Verma JP (2020) Plant Growth-Promoting Bacteria: Biological Tools for the Mitigation of Salinity Stress in Plants. Front Microbiol 11:1216. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2020.01216\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2020.01216\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar K, Sinha AK (2013) Overexpression of constitutively active mitogen activated protein kinase kinase 6 enhances tolerance to salt stress in rice. Rice (New York NY) 6(1):25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1939-8433-6-25\u003c/span\u003e\u003cspan address=\"10.1186/1939-8433-6-25\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Yue J, Wu X, Xu C, Yu J (2014) An ABA-responsive DRE-binding protein gene from Setaria italica, SiARDP, the target gene of SiAREB, plays a critical role under drought stress. J Exp Bot 65(18):5415\u0026ndash;5427. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jxb/eru302\u003c/span\u003e\u003cspan address=\"10.1093/jxb/eru302\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi K, Yang F, Zhang G, Song S, Li Y, Ren D, Miao Y, Song CP (2017) AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade. Plant Physiol 173(2):1391\u0026ndash;1408. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1104/pp.16.01386\u003c/span\u003e\u003cspan address=\"10.1104/pp.16.01386\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang X, Zhou JM (2018) Receptor-Like Cytoplasmic Kinases: Central Players in Plant Receptor Kinase-Mediated Signaling. Annu Rev Plant Biol 69:267\u0026ndash;299. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-arplant-042817-040540\u003c/span\u003e\u003cspan address=\"10.1146/annurev-arplant-042817-040540\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu H, Zhang J, Liu KB, Wu N, Li Y, Zhou K, Ye M, Zhang T, Zhang H, Yang X, Shen L, Xu D, Li Q (2009) Earliest domestication of common millet (Panicum miliaceum) in East Asia extended to 10,000 years ago. Proc Natl Acad Sci USA 106(18):7367\u0026ndash;7372. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.0900158106\u003c/span\u003e\u003cspan address=\"10.1073/pnas.0900158106\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa F, Ni L, Liu L, Li X, Zhang H, Zhang A, Tan M, Jiang M (2016) ZmABA2, an interacting protein of ZmMPK5, is involved in abscisic acid biosynthesis and functions. Plant Biotechnol J 14(2):771\u0026ndash;782. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/pbi.12427\u003c/span\u003e\u003cspan address=\"10.1111/pbi.12427\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMao X, Cai T, Olyarchuk JG, Wei L (2005) Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinf (Oxford England) 21(19):3787\u0026ndash;3793. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/bioinformatics/bti430\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/bti430\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsuoka D, Nanmori T, Sato K, Fukami Y, Kikkawa U, Yasuda T (2002) Activation of AtMEK1, an Arabidopsis mitogen-activated protein kinase kinase, in vitro and in vivo: analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings. Plant J 29(5):637\u0026ndash;647. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1046/j.0960-7412.2001.01246.x\u003c/span\u003e\u003cspan address=\"10.1046/j.0960-7412.2001.01246.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng X, Zhang S (2013) MAPK Cascades in Plant Disease Resistance Signaling. Annu Rev Phytopathol 51:245\u0026ndash;266. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-phyto-082712-102314\u003c/span\u003e\u003cspan address=\"10.1146/annurev-phyto-082712-102314\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuthamilarasan M, Prasad M (2015) Advances in Setaria genomics for genetic improvement of cereals and bioenergy grasses. Theor Appl Genet 128(1):1\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00122-014-2399-3\u003c/span\u003e\u003cspan address=\"10.1007/s00122-014-2399-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuthamilarasan M, Prasad M (2021) Small Millets for Enduring Food Security Amidst Pandemics. Trends Plant Sci 26(1):33\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tplants.2020.08.008\u003c/span\u003e\u003cspan address=\"10.1016/j.tplants.2020.08.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePawson T, Scott JD (2005) Protein phosphorylation in signaling \u0026ndash; 50 years and counting. Trends Biochem Sci 30(6):286\u0026ndash;290. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tibs.2005.04.013\u003c/span\u003e\u003cspan address=\"10.1016/j.tibs.2005.04.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng R, Zhang B (2020) Foxtail Millet: A New Model for C4 Plants. Trends Plant Sci 26(3):199\u0026ndash;201. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tplants.2020.12.003\u003c/span\u003e\u003cspan address=\"10.1016/j.tplants.2020.12.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRathinapriya P, Pandian S, Rakkammal K, Balasangeetha M, Alexpandi R, Satish L, Rameshkumar R, Ramesh M (2020) The protective effects of polyamines on salinity stress tolerance in foxtail millet (Setaria italica L.), an important C4 model crop. Physiol Mol Biol Plants 26(9):1815\u0026ndash;1829. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12298-020-00869-0\u003c/span\u003e\u003cspan address=\"10.1007/s12298-020-00869-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSachdev N, Goomer S, Singh LR (2021) Foxtail millet: a potential crop to meet future demand scenario for alternative sustainable protein. J Sci Food Agric 101(3):831\u0026ndash;842. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jsfa.10716\u003c/span\u003e\u003cspan address=\"10.1002/jsfa.10716\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen L, Zhuang B, Wu Q, Zhang H, Nie J, Jing W, Yang L, Zhang W (2019) Phosphatidic acid promotes the activation and plasma membrane localization of MKK7 and MKK9 in response to salt stress. Plant Sci 287:110190. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plantsci.2019.110190\u003c/span\u003e\u003cspan address=\"10.1016/j.plantsci.2019.110190\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeige M, Scheikl E, Eulgem T, D\u0026oacute;czi R, Ichimura K, Shinozaki K, Dangl JL, Hirt H (2004) The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol Cell 15(1):141\u0026ndash;152. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molcel.2004.06.023\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2004.06.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Pan C, Wang Y, Ye L, Wu J, Chen L, Zou T, Lu G (2015) Genome-wide identification of MAPK, MAPKK, and MAPKKK gene families and transcriptional profiling analysis during development and stress response in cucumber. BMC Genomics 16(1):386. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12864-015-1621-2\u003c/span\u003e\u003cspan address=\"10.1186/s12864-015-1621-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao S, Wan Y, Guo S, Fan J, Lin Q, Zheng C, Wu C (2023) Transcription Factor SiDi19-3 Enhances Salt Tolerance of Foxtail Millet and Arabidopsis. Int J Mol Sci 24(3). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms24032592\u003c/span\u003e\u003cspan address=\"10.3390/ijms24032592\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXing Y, Chen WH, Jia W, Zhang J (2015) Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress. J Exp Bot 66(19):5971\u0026ndash;5981. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jxb/erv305\u003c/span\u003e\u003cspan address=\"10.1093/jxb/erv305\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu C, Luo M, Sun X, Yan J, Shi H, Yan H, Yan R, Wang S, Tang W, Zhou Y, Wang C, Xu Z, Chen J, Ma Y, Jiang Q, Chen M, Sun D (2022) SiMYB19 from Foxtail Millet (Setaria italica) Confers Transgenic Rice Tolerance to High Salt Stress in the Field. Int J Mol Sci 23(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms23020756\u003c/span\u003e\u003cspan address=\"10.3390/ijms23020756\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu J, Zhang S (2015) Mitogen-activated protein kinase cascades in signaling plant growth and development. Trends Plant Sci 20(1):56\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tplants.2014.10.001\u003c/span\u003e\u003cspan address=\"10.1016/j.tplants.2014.10.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Ji L, Liu S, Jing P, Hu J, Jin D, Wang L, Xie G (2021) The CaM1-associated CCaMK-MKK1/6 cascade positively affects lateral root growth via auxin signaling under salt stress in rice. J Exp Bot 72(18):6611\u0026ndash;6627. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jxb/erab287\u003c/span\u003e\u003cspan address=\"10.1093/jxb/erab287\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Z, Zhang H, Li X, Shen H, Gao J, Hou S, Zhang B, Mayes S, Bennett M, Ma J, Wu C, Sui Y, Han Y, Wang X (2020) A mini foxtail millet with an Arabidopsis-like life cycle as a C4 model system. Nat Plants 6(9):1167\u0026ndash;1178. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41477-020-0747-7\u003c/span\u003e\u003cspan address=\"10.1038/s41477-020-0747-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoung MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol 11(2):R14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/gb-2010-11-2-r14\u003c/span\u003e\u003cspan address=\"10.1186/gb-2010-11-2-r14\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu Y, Guo DD, Min DH, Cao T, Ning L, Jiang QY, Sun XJ, Zhang H, Tang WS, Gao SQ, Zhou YB, Xu ZS, Chen J, Ma YZ, Chen M, Zhang XH (2023) Foxtail millet MYB-like transcription factor SiMYB16 confers salt tolerance in transgenic rice by regulating phenylpropane pathway. Plant Physiol Biochem 195:310\u0026ndash;321. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plaphy.2022.11.032\u003c/span\u003e\u003cspan address=\"10.1016/j.plaphy.2022.11.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang M, Zhang S (2022) Mitogen-activated protein kinase cascades in plant signaling. J Integr Plant Biol 64(2):301\u0026ndash;341. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jipb.13215\u003c/span\u003e\u003cspan address=\"10.1111/jipb.13215\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu J-K (2016) Abiotic Stress Signaling and Responses in Plants. Cell 167(2):313\u0026ndash;324. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2016.08.029\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2016.08.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Q, Shao Y, Ge S, Zhang M, Zhang T, Hu X, Liu Y, Walker J, Zhang S, Xu J (2019) A MAPK cascade downstream of IDA-HAE/HSL2 ligand-receptor pair in lateral root emergence. Nat Plants 5(4):414\u0026ndash;423. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41477-019-0396-x\u003c/span\u003e\u003cspan address=\"10.1038/s41477-019-0396-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"MAPK cascade, foxtail millet, salt stress, transcriptome, root development","lastPublishedDoi":"10.21203/rs.3.rs-3887368/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3887368/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe MAPK cascade is evolutionarily conserved in eukaryotes and involved in the regulation of plant growth, development, resistance to biotic and abiotic stress. However, the gene function of MAPK cascade in foxtail millet (\u003cem\u003eSeteria italica\u003c/em\u003e) is rarely studied. In this study, RNA sequencing was performed and MAPK cascade was the main enrichment pathway in foxtail millet after salt treatment. Meanwhile, fourteen genes encoded and mitogen-activated protein kinase kinases (SiMKKs) were identified which could be divided into 4 subfamilies. Under salt treatment, the expression levels of 11 \u003cem\u003eSiMKKs\u003c/em\u003e were upregulated and the expression level of \u003cem\u003eSiMKK6-2\u003c/em\u003e in group A had the biggest increase. \u003cem\u003eSiMKK1\u003c/em\u003e and \u003cem\u003eSiMKK6-1\u003c/em\u003e, which were the other two member of in the same subfamily, also significantly upregulated under salt stress. Overexpression of these three genes in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e reduced the sensitivity of roots to salt stress. Transgenic plants had more lateral roots. The decrease of primary root length of transgenic plants under salt stress was significantly lower than that of wild type plants. These three genes are involved in regulating the development of primary and lateral roots of plants, which can maintain better root development to improve plant tolerance to salt stress.\u003c/p\u003e","manuscriptTitle":"Heterologous Expressions of foxtail millet (Seteria italica) mitogen-activated protein kinase kinase (SiMKK) group A genes regulate root development under salt stress in Arabidopsis thaliana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-22 21:04:19","doi":"10.21203/rs.3.rs-3887368/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-04-06T20:51:39+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-20T08:23:35+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-20T08:14:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Growth Regulation","date":"2024-02-02T06:42:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-23T05:41:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Growth Regulation","date":"2024-01-21T22:01:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fe284c2f-7c8d-45a2-af0b-ab8e43655109","owner":[],"postedDate":"March 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:04:43+00:00","versionOfRecord":{"articleIdentity":"rs-3887368","link":"https://doi.org/10.1007/s10725-024-01180-8","journal":{"identity":"plant-growth-regulation","isVorOnly":false,"title":"Plant Growth Regulation"},"publishedOn":"2024-08-23 15:57:47","publishedOnDateReadable":"August 23rd, 2024"},"versionCreatedAt":"2024-03-22 21:04:19","video":"","vorDoi":"10.1007/s10725-024-01180-8","vorDoiUrl":"https://doi.org/10.1007/s10725-024-01180-8","workflowStages":[]},"version":"v1","identity":"rs-3887368","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3887368","identity":"rs-3887368","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}