Peanut LEAFY COTYLEDON1-type genes participate in regulating the embryo development and the accumulation of storage lipids

Peanut LEAFY COTYLEDON1-type genes participate in regulating the embryo development and the accumulation of storage lipids | 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 Peanut LEAFY COTYLEDON1-type genes participate in regulating the embryo development and the accumulation of storage lipids Guiying Tang, Pingli Xu, Chunyu Jiang, Guowei Li, Lei Shan, Shubo Wan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3913572/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Apr, 2024 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract LEAFY COTYLEDON1 (LEC1) isa member of the nuclear factor Y (NF-Y) family of transcription factors and has been identified as a key regulator of embryonic development. In the present study, two LEC1-type genes from Arachis hypogeae were identified and designated as AhNF-YB1 and AhNF-YB10 ; these genes belong to subgenome A and subgenome B, respectively. The functions of AhNF-YB1 and AhNF-YB10 were investigated by complementation analysis of their defective phenotypes of the Arabidopsis lec1-2 mutant and by ectopic expression in wild-type Arabidopsis. The results indicated that both AhNF-YB1 and AhNF-YB10 participate in regulating embryogenesis, embryo development, and reserve deposition in cotyledons and that they have partial functional redundancy. In contrast, AhNF-YB10 complemented almost all the defective phenotypes of lec1-2 in terms of embryonic morphology and hypocotyl length, while AhNF-YB1 had only a partial effect. In addition, 30%-40% of the seeds of the AhNF-YB1 transformants exhibited a decreasing germination ratio and longevity. Therefore, appropriate spatiotemporal expression of these genes is necessary for embryo morphogenesis at the early development stage and is responsible for seed maturation at the mid-late development stage. On the other hand, overexpression of AhNF-YB1 or AhNF-YB10 at the middle to late stages of Arabidopsis seed development improved the weight, oil content, and fatty acid composition of the transgenic seeds. Moreover, the expression levels of several genes associated with fatty acid synthesis and embryogenesis were significantly greater in developing AhNF-YB10 -overexpressing seeds than in control seeds. This study provides a theoretical basis for breeding oilseed crops with high yields and high oil content. LEAFY COTYLEDON-1 type gene Seed development Fatty acid (FA) synthesis Oil accumulation Yields Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cultivated peanut ( Arachis hypogeae L.) is one of the most important oil crops grown in tropical and subtropical regions worldwide. Its kernels are rich in oil, protein and other nutrients that are beneficial for human health (Akhtar et al. 2014 ). The oil content in peanut kernels is approximately 45%-56%, and two major unsaturated fatty acids (USFAs), oleic acid (36%-67%) and linoleic acid (15%-43%), constitute 80% of the total fatty acid (FA) content (Barkley et al. 2013 ). Diets with high levels of USFAs are nutritionally beneficial for lowering cholesterol levels and reducing systolic blood pressure (Terés et al. 2008 ; Akhtar et al. 2014 ). Therefore, given the growing need for a healthy diet, the demand for high-quality peanut oil is increasing. In turn, increasing the oil content and improving the FA composition are major goals for guiding scientific research currently and for the foreseeable future. Transcription factors (TFs) regulate the expression of downstream target genes by binding to specific motifs in promoters. Due to their regulatory effects on target genes or interactions with other TFs at multiple levels, some TFs can control the entire pathways of various biological processes. The transcription factor NF-YB is a subunit of the NF-Y (Nuclear Factor Y) heterotrimeric complex and plays an important role in the specific binding of the NF-Y trimer complex to DNA (Zemzoumi et al. 1999 ); it is involved in regulating the growth, development and stress tolerance of a wide array of plants (Lotan et al. 1998 ; Siriwardana et al. 2014 ; Su et al. 2018 ; Niu and He 2019 ). On the basis of the presence of 16 conserved amino acid residues in the NF-YB B domain, these proteins can be divided into two classes, namely, LEC1-type and non-LEC1-type, in Arabidopsis thaliana (Kwong et al. 2003 ). AtNF-YB9 (LEAFY COTYLEDON 1, AtLEC1) and AtNF-YB6 (AtLEC1-LIKE, AtL1L) in Arabidopsis are LEC1-type proteins, and the remaining 11 members are non-LEC1-type proteins. LEC1 has been demonstrated to be a key regulator of seed development and controls several crucial processes, including embryogenesis, endosperm development, and the deposition of seed reserves in plants (Jo et al. 2020 ). The LEC1 null mutant exhibits defects in normal embryos, acquisition of desiccation tolerance, reserve accumulation, and suppression of germination (Harada 2001 ; Santos-Mendoza et al. 2008 ). In the Arabidopsis LEC1 gain-of-function mutant, turnip ( tnp ), whose promoter has a longer fragment deletion 436 bp upstream from the start codon (ATG), exhibits ectopic accumulation of storage products, and undergoes partial de-etiolation when the seedlings are grown in the dark (Casson and Lindsey 2006 ). Ectopic expression of LEC1 was sufficient to induce somatic embryogenesis from vegetative cells (Lotan et al. 1998 ). In addition to analyses of the physiological function and primary molecular mechanism of LEC1 in Arabidopsis and in several other staple crop species, several studies have focused on the genetic improvement of reserve substance accumulation via the use of LEC1 . For example, overexpression of the corn ZmLEC1 gene under the embryo-specific weak EARLY EMBRYO PROTEIN 1 ( EAP1 ) promoter could significantly increase the oil content of transgenic maize (Shen et al. 2010 ). In Brassica napus , LEC1 regulates the expression of multiple genes related to promoting sucrose (Suc) and lipid accumulation in seeds, resulting in a significantly increased FA level in transgenic canola (Mu JY et al. 2008 ; Tan et al. 2011 ). Several researchers have also shown that LEC1 plays essential roles in starch synthesis in rice seeds (Yang et al. 2017 ; Das et al. 2019 ). To date, the biological function of LEC1 in peanut has not been elucidated. In the present study, 16 NF-YB genes were identified from the genome of cultivated peanut; among these genes, AhNF-YB1 and AhNF-YB10 are LEC1 -type genes, and the remaining 14 AhNF-YBs are non- LEC1 -type genes. The functions of the two LEC1 -type genes were investigated via complementation analysis in the Arabidopsis lec1-2 mutant and ectopic expression in wild-type Arabidopsis. The results indicated that most or some of the defective phenotypes of lec1-2 were complemented in the transgenic AhNF-YB1 and AhNF-YB10 mutant plants. Overexpression of AhNF-YB1 and AhNF-YB10 in Arabidopsis impacted seed weight, oil content, and FA content. Moreover, the normal function of these genes was found to be dependent on proper spatiotemporal expression. Materials and Methods Identification and phylogenetic analysis of NF-YB family members in the peanut cultivar and two probable diploid ancestors The sequences of the Arabidopsis thaliana NF-YB family members were downloaded from The Arabidopsis Information Resource (TAIR) ( https://www.arabidopsis.org/ ) and used as query sequences to search the sequences of Arachis hypogeae , Arachis duranensis and Arachis ipaensis via BLASTP in the Peanutbase ( https://www.peanutbase.org/ ) and NCBI (National Center for Biotechnology Information) databases. In addition, the putative NF-YBs of the peanut cultivar and the two diploid wild species were confirmed via InterProScan 56.0 ( http://www.Ebi.ac.uk/inerpro/ ). Their molecular weights (MWs) and isoelectric points (pIs) were calculated using ExPASy. Chromosome distribution information for AhNF-YBs was extracted from gff3 annotation files of Arachis hypogeae by TBtools (Chen et al. 2020 ). The NF-YB protein sequences of Arabidopsis thaliana and three Arachis species were aligned by the MUSCLE method (Kumar et al. 2016 ), and an unrooted phylogenetic tree was established using MEGA X by employing the neighbour-joining (NJ) method (Kumar et al. 2018 ). Plant material and growth conditions The peanut cultivar Fenghua No. 1 (FH1) was maintained and cultivated by our group. The roots, stems, leaves, flowers and seeds at different developmental stages were taken from peanut plants grown in the natural environment of Yinmaquan Farm in Jinan and kept in a -80°C freezer before isolation of total RNA. To ensure consistency of seed development, the pegs were labelled by tying with thin plastic threads. Two ecotypes of Arabidopsis thaliana (Wassilewskija, WS; and Columbia, COL) were maintained in our laboratory, and the lec1-2 (CS3867) mutant obtained from the Arabidopsis Biological Resource Center (ABRC) was used for transformation in this study. The embryos of homozygous lec1-2 mutants before desiccation were sown on 1/2 MS medium to rescue their desiccation-intolerant phenotype, and the defective seedlings were subsequently transplanted to pots with culture substrate after 10 days. The wild-type WS, COL, lec1-2 mutant and transgenic Arabidopsis plants were grown in a greenhouse at room temperature under a 16 h light/8 h dark photoperiod and 65% relative humidity. To measure the thousand-seed weight and oil content in the wild-type, transgenic Arabidopsis and lec1-2 mutant plants, the plants were always grown in the same chamber at the same time. Generation of plant expression vectors and transgenic Arabidopsis plants harbouring AhNF-YB1 and AhNF-YB10 The 1044-bp AtLEC1 promoter ( AtLEC1P ) upstream of ATG in Arabidopsis was cloned and identified by sequencing. With pCAMBIA-3301 as the initial vector, two vectors with 211-bp promoter of Napin A (211P) from Brassica napus (Tan et al. 2011 ) and two vectors driven by AtLEC1 P expressing AhNF-YB1 and AhNF-YB10 were constructed and were designated pC3301-211P: AhNF-YB1/10 (pC211P: B1/10 ) and pC3301- AtLEC1P : AhNF-YB1 /10 (pC AtLEC1 P: B1 / 10 ), respectively. Transgenic Arabidopsis plants were generated via the floral dip method. The seeds of transgenic Arabidopsis plants harbouring AhNF-YB1 or AhNF-YB10 were screened on basta-containing 1/2 MS medium. The transgenic lines with one copy of the exogenous gene were identified by the 3:1 segregation ratio of the T 2 plants with and without basta resistance. The Arabidopsis overexpression lines were also identified by amplifying the insertion fragment from the genomic DNA using primers located on the P211 promoter and ORF of AhNF-YB1/10 . Measurement of seed weight, germination rate and longevity Thousand-grain weight was measured to evaluate the size and the plumpness of the Arabidopsis seeds. The hundred-seed method was used for determination of the weight per thousand seeds following the process described by Wang (Wang et al. 2007 ). For seed germination and longevity assessment in Arabidopsis, the seeds were kept in 1.5 mL centrifuge tubes with allochroic silica gel and stored at room temperature. The germination ability of the plants was tested separately after harvesting for 1 week, 1 month, and 3 months. One hundred sterile seeds were sown on 1/2 MS medium, kept in the dark and cold (4 ℃) for 2 days, and subsequently transferred into a chamber for growth at 22 ℃± 2 ℃ with a 16/8 hr light/dark cycle for 7 days. Then, the number of seedlings with green opening cotyledons was counted. The viability of the seeds preserved for 3 months at room temperature was evaluated via 2,3,5-triphenyltetrazolium chloride (TTC) staining. Two hundred disinfected seeds in each assay were stained by incubating in 1% tetrazolium solution in darkness at 30°C for 48 hr. The viability of the seeds was estimated by the displayed colour of 2,3,5-triphenyl formazan after staining, wherein the darker the red colour was, the more viable the seeds were. The stained seeds were observed by stereomicroscopy (Waes and Debergh 1986 ; Salvi et al. 2016 ). All the experiments were carried out in triplicate. Quantitative RT‒PCR (qRT‒PCR) analysis of gene expression The total RNAs from the different peanut tissues and Arabidopsis seeds were isolated following the protocol for an RNA isolation kit provided by Huayueyang Biotech Company, Ltd., Beijing, China. First-strand cDNA was synthesized from 1 µg of total RNA using the Evo M-MLV RT-Kit with gDNA Clean for qPCR II. Each cDNA sample was diluted 10-20-fold in sterile water for qRT‒PCR. The expression level was normalized using peanut AhACTIN 7 or AtACTIN as internal controls. The primers used are listed in Supplemental Table 1. The qRT‒PCRs were performed in 8-tube strips, and the data were analysed according to methods described previously (Zhu et al. 2022 ). Analysis of oil content and fatty acid composition in Arabidopsis seeds The Soxhlet extraction method was used to analyse the seed oil content. Seeds (1.5 g) were baked at 80°C for 4 hr and then cooled to room temperature. The following steps, including grinding, extracting and weighing, were performed according to previously described methods (Tang et al. 2018 ). For each sample, triplicate analyses were performed, and the mean values of the triplicates were used to calculate the oil content. FA analysis was performed by gas chromatography/mass spectrometry (GC/MS) using an HP-6890 instrument equipped with a DB-23 column (60 m× 250 µm × 0.25 µm). Approximately 300 mg was needed per seed sample. The pretreatment of the seed samples was performed as described previously (Tang et al. 2018 ). The loading volume of the methyl esterified sample was 1 µL. The temperature was set to 180°C in the beginning, subsequently increased to 200°C at a 4°C/min, and then held for 15 min; finally, the temperature was increased to 230°C at 10°C/min and held for 10 min. The injection and detection port temperatures were 260℃ and 270℃, respectively. FAs were separated using N 2 as the carrier gas at a flow rate of 2.0 mL/min, and the split ratio was 30:1. The experiment was carried out in triplicate. Statistical analysis All the data are presented as the mean value and the corresponding standard error of at least three replicates. Statistical analyses were performed in Excel 2019 and IBM SPSS Statistics 26 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) with Tukey’s post hoc test and Student’s t - test were used to indicate significant differences. All the bar charts in the figures were prepared using GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego CA, USA) or Excel. Results Identification and phylogenetic analysis of the NF-YB gene family in Arachis hypogeae A total of 16 AhNF-YB genes in peanut were identified and named according to their physical location on the chromosome. The encoded proteins, AhNF-YB1~16, varied from 151 to 226 amino acids in length. The detailed information, including sequence ID, chromosomal location, number of amino acids, protein isoelectric point (pI) and protein molecular weight (MW), is listed in Supplemental Table 2. According to the 16 amino acid residues conserved in LEC1-type NF-YB, AhNF-YB1 on Chr 01 and AhNF-YB10 on Chr 11 are classified as LEC1-type proteins, and the remaining 14 AhNF-YBs are classified as non-LEC1-type proteins. To investigate the phylogenetic relationship of NF-YBs, 16 AhNF-YB members in cultivated peanut, 8 AdNF-YBs in Arachis duranensis , 8 AiNF-YBs in Arachis ipaensis , and 13 AtNF-YBs in Arabidopsis were used to construct a phylogenetic tree. As shown in Fig. 1, the NF-YBs were divided naturally into three clades, designated I, II, and III. Clade I included ten members, among those AhNF-YB1 and XP015940529.1 (from Arachis duranensis ), and AhNF-YB10 and XP016195843.1 (from Arachis ipaensis ) shared relatively high homology and clustered into a subclade with Arabidopsis AtNF-YB6 and AtNF-YB9, which belong to the LEC1 type. AhNF-YB1 and AhNF-YB10 had 63% and 62% sequence similarity with AtNF-YB6 respectively, and had 56% and 55% sequence similarity with AtNF-YB9 respectively; while another subclade of clade I consists of AhNF-YB6, AhNF-YB14 and their homologue from wild peanut of A or B genome. Except AhNF-YB6 and AhNF-YB14 from Clade I, the other non-LEC1-type members of the A subgenome and B subgenome in Arachis hypogeae were classified into clade II (4 members) and III (8 members), and those genes located on the corresponding chromosomes were clustered together with the relatives from the two diploid Arachis species. According to previous reports, LEC1-type NF-YBs play essential roles in seed developmental processes, including embryogenesis and deposition of storage reserves in seeds (West et al. 1994; Shen et al. 2010; Huang et al. 2015; Zhu et al. 2018). Owing to the focus on seed development and reserve accumulation in our research, two LEC1 -type genes in peanut, AhNF-YB1 and AhNF-YB10, were chosen for further functional identification and analysis. Expression patterns of AhNF-YB1 and AhNF-YB10 in peanut The expression patterns of AhNF-YB1 and AhNF-YB10 in various organs and in seeds at different developmental stages were investigated via qRT‒PCR. The results demonstrated that AhNF-YB1 is constitutively expressed at different levels in the roots, stems, leaves, flowers and seeds (order from high to low: seeds, roots, leaves, flowers, and stems). AhNF-YB10 was expressed specifically in seeds (Fig. 2a). During seed development, the two genes exhibited similar expression patterns; their expression started at the early stage of seed development (at 10 DAP), increased at the middle stage, and then decreased obviously at the late stage. In addition, AhNF-YB1 was significantly more highly expressed thanwas AhNF-YB10 at the 20 DAP, 30 DAP and 40 DAP, whereas the expression level of AhNF-YB10 was greater than that of AhNF-YB1 at early developmental stage (at 10 DAP) and later stages (at 50, 60, and 70 DAP) (Fig. 2b). Ectopic expression of two AhNF-YB genes in the Arabidopsis lec1-2 mutant To determine the function of the two AhNF-YB genes, homozygous lec1-2 mutant plants derived from heterozygous lec1-2 seeds purchased from the ABRC were first distinguished by their embryonic phenotype and subsequently rescued on 1/2 MS medium. Initially, pC211P: B1 and pC211P: B10 , which harbour the 211-bp promoter of the rape Nap A gene, were used as transformation vectors (Supplemental Fig. 1a). Three transformants of the T 1 generation containing the pC211P: B10 construct in the lec1-2 homozygous background were successfully obtained by selection on 1/2 MS medium supplemented with 10 mg/L Basta. Furthermore, the phenotypes of the T 2 transgenic plants were investigated. Unlike the lec1-2 homozygous seeds, which cannot germinate normally because of their desiccation intolerance (West et al. 1994), most of the seeds in the three transgenic lines possessed the ability to germinate. However, the morphologies of several of the transgenic seeds, including round unbent cotyledons, shorter embryonic axes, and roots with constricted ends, were also similar to those of the mutant lec1-2 . Unlike those on lec1-2, trichomes on the adaxial surfaces of the cotyledons of the transgenic seedlings were not found(Fig. 3 F-O). In the present study, transgenic progenies with the pC211P: B1 construct were not obtained. It is possible that AhNF-YB1 could not complement the phenotype of desiccation intolerance in lec1-2 seeds. The 211-bp promoter of NapA retains a seed-specific expression pattern similar to that of the full-length promoter, and its expression is greater at the later stage of seed development (Tan et al. 2011). To explore why most of the defects in lec1-2 had not been complemented in the pC211P: B1/10 transgenic seeds, the AhNF-YB1 and AhNF-YB10 constructs under the control ofthe AtLEC1 promoter were transferred into lec1-2 (Supplemental Fig. 1b). More than a dozen transformants of the two genes were successfully obtained. Phenotypic analysis of the transgenic plants revealed that the phenotypes of the transgenic plants harbouring the exogenous gene AhNF-YB10 were similar to those of the wild-type plants, in which the morphology of the embryos was normal and the seeds germinated normally (Fig. 3. A-E, P-T). However, in the transgenic plants harbouring AhNF-YB1 , only approximately 60%-70% of the transformants were similar to WS-type Arabidopsis plants; the seeds of these plants had elliptic cotyledons and bent axes, and the etiolated plants had longer hypocotyls. The remaining 30%-40% of the seeds were similar to those of the lec1-2 mutant in appearance; however, some of them could germinate, but the seedlings had shorter hypocotyls even though they were in darkness (Fig. 3. U-Y). Furthermore, the longevity and vigour of the seeds of plants ectopically expressing AhNF-YB1 and AhNF-YB10 were examined. The results showed that the germination rates of seeds of the AhNF-YB10 transgenic lines and the wild-type control were hardly affected by storage for three months after harvesting, while the germination rates of the seeds from the AhNF-YB1 transformants significantly decreased with increasing duration of preservation in comparison to those of the control seeds (Fig. 4a). The results of the TTC assay showed that all the lec1-2 seeds were not stained because of seed inviability (Fig. 4b. D), and the seeds of the AhNF-YB10 transgenic plants and WS control plants were stained red or dark red (Fig. 4b. A, C, F), while the most AhNF-YB1 transgenic seeds were pale red or had no dyeing (Fig. 4b. B, E). These results indicated that ageing treatment significantly decreased the longevity of the AhNF-YB1 -transformed seeds. To explore why the P211 and AtLEC1 promoters triggering AhNF-YB1/10 expression in the lec1-2 background caused different phenotypes in transgenic Arabidopsis, the expression profiles of these transgenic lines during seed development were checked. The results showed that AhNF-YB10 expression driven by P211 was hardly detectable at 4 DAF and markedly increased at 8 DAF and 13 DAF. In addition, driven by the AtLEC1 promoter, the expression levels were greatest at 4 DAF and decreased with seed development. AhNF-YB1 exhibited an expression pattern similar to that of AhNF-YB10 under the control of the AtLEC1 promoter but was expressed at lower levels (Supplemental Fig. 2). The degree to which AhNF-YB1 and AhNF-YB10 restored the defects in lec1-2 was related to the transcript levels of these genes. Moreover, the expression levels of key genes involved in seed development and FA synthesis were elevated in the transgenic plants (Supplemental Fig. 3). The oil contents and seed weights of the AhNF-YB1/10 -overexpressing Arabidopsis lines Considering the important role of plant LEC1-type NF-YBs in reserve deposition, we tried to improve the oil content by overexpressing AhNF-YB1/10 via the P211 promoter in Arabidopsis seeds. Multiple independent transgenic lines were obtained by screening on basta-containing medium and detection via PCR (Supplemental Fig. 4). No developmental abnormalities were observed throughout their life cycle (Supplemental Fig. 5). Analysis of the transgenic seeds revealed that the crude fat content in the transgenic seeds was elevated by 6.2%-29.1% compared to that in COL(Table 1). The content of the main FAs in the AhNF-YB1/10 transgenic seeds increased to different degrees, among which the improvements in the C18:1n9c, C18:2n6c, C18:3n3, C20:1 and C22:1n9 contents were more significant (Table 2). These findings suggested that the increase in seed oil content was due to the increase in several major FAs. Table 1 Content of crude fat in Arabidopsis seeds (% dry weight). Mature seeds were used for the analysis of the oil content via the Soxhlet method. Asterisks indicate statistically significant differences compared with the control.(Student’s t test: * P ＜0.05; ** P ＜0.01) Genotype Crude fat content (%) Percent increase (%) COL 24.46 (±0.43) - OE-AhNF-B1, #11 27.99 (±0.56)** 14.43 OE-AhNF-B1, #13 28.25 (±0.51)** 15.50 OE-AhNF-B1, #15 30.69 (±0.63)** 25.47 OE-AhNF-B10, #1 33.34 (±0.59)** 36.33 OE-AhNF-B10, #10 32.36 (±0.68)** 32.31 OE-AhNF-B10, #15 28.92 (±0.51)** 18.23 Table 2 Composition of the main fatty acids in Arabidopsis seeds (mg/g). Mature seeds were used for the GC analysis. Asterisks indicate statistically significant differences compared with the control (Student’s t test: * P ＜0.05; ** P ＜0.01) Fatty acid COL OE-AhNF-B1, #11 OE-AhNF-B1, #13 OE-AhNF-B1, #15 OE-AhNF-B10, #1 OE-AhNF-B10, #10 OE-AhNF-B10, #15 C16:0 23.91 ± 0.50 24.10±0.71 23.34±0.10 24.57±0.45 28.52±0.37** 27.98±0.64** 23.40±0.18 C18:0 8.30±0.24 8.16±0.16 7.73±0.06 8.87±0.16* 10.37±0.15** 10.11±0.32** 7.99±0.06 C18:1n9c 34.64±0.68 36.36±0.48** 30.47±0.44** 35.88±0.89 41.56±1.04** 47.49±1.75** 37.74±0.55** C18:2n6c 83.28±2.03 83.78±1.04 81.35±0.56 86.78±1.54 97.31±1.02** 97.35±2.02** 82.06±0.71 C18:3n3 52.01±0.46 52.56±0.34 54.27±0.76* 58.42±1.14** 72.62±0.61** 64.33±1.44** 53.95±0.75* C20:0 6.22±0.27 6.33±0.15 7.15±0.00** 7.66±0.19** 7.28±0.13** 7.06±0.17* 6.29±0.01 C20:1 53.50±1.85 55.21±0.92 58.61±0.55* 63.23±1.63** 73.09±1.24** 67.51±2.41** 55.72±0.45 C21:0 5.84±0.18 6.14±0.09 6.96±0.04** 6.79±0.16** 7.73±0.11** 6.85±0.14** 5.74±0.03 C22:1n9 6.27±0.35 6.71±0.19 9.03±0.05** 8.81±0.25** 7.62±0.17** 6.88±0.17 6.52±0.03 Compared with that of the nontransgenic COL plants, the thousand-seed weight increased by 3.31%-27.81% in the AhNF-YB1 -OE and AhNF-YB10 -OE transgenic Arabidopsis plants (Table 3), but the other traits related to plant growth and development did not change. Table 3 The 1000-seed weight of the transgenic Arabidopsis lines. Asterisks indicate statistically significant differences compared with the control (Student’s t test: * P ＜0.05; ** P ＜0.01) Genotype Thousand-seed weight (mg) % Increase over control OE-AhNF-B1, #11 20.13* 6.62 OE-AhNF-B1, #13 19.50 3.31 OE-AhNF-B1, #15 22.00** 16.56 OE-AhNF-B10, #1 24.13** 27.81 OE-AhNF-B10, #10 21.13** 11.92 OE-AhNF-B10, #15 19.63 3.97 COL 18.86 - Expression analysis of the genes associated with fatty acid synthesis and embryogenesis in developing AhNF-YB10 -OE seeds To elucidate the molecular basis of the improvements in FA content, oil content and thousand-seed weight in transgenic Arabidopsis plants overexpressing these genes, the genes involved in FA synthesis and embryogenesis were analysed at the transcriptional level. Endogenous AtLEC1 in AhNF-YB10 transgenic Arabidopsis and COL were expressed at higher levels at the early stage of seed development, and their expression decreased to a moderately low level at the mid-late stage (Supplemental Fig. 6a). In contrast, the expression level of exogenous AhNF-YB10 in the OE lines increased with seed development. At 8 and 13 DAF, increased expression of AhNF-YB10 was detected (Supplemental Fig. 6b). Thus, the seeds of Arabidopsis at 13 DAF were subjected to further gene expression analysis. As shown in Fig. 5, the expression level of several genes in the FA de novo synthetic pathway, including dihydrolipoamide dehydrogenase 1 ( LPD1 ), pyruvate dehydrogenase E1 α ( PDH-E1α ), acetyl-CoA carboxylase2 ( ACC2 ), biotin carboxyl carrier protein ( BCCP2 ) for preparation of acetyl coenzyme A which is the precursor of FA synthesis; acyl carrier protein ( ACP1 ), malonyl-CoA: ACP transacylase ( MCAT ), fatty acyl-ACP thioesterase A ( Fat A ), enoyl-ACP reductase ( EAR ), fatty-acid chain elongase ( FAE ) for fatty acyl transference, reduction, elongation; stearoyl-ACP desaturase ( FAB2 ), Δ-12 fatty acid desaturase2 ( FAD2 ), ω-3 fatty acid desaturase3 ( FAD3 ) for dehydrogenation at specific position of FAs; oleosin1 ( OLE1 ) for lipid accumulation and the glycolytic genes fructose 1,6 bisphosphate aldolase ( FPA1 ), phosphofructokinase-1 ( PFK1 ), adenosine diphosphoglucose pyrophosphprylase ( AGP ) were upregulated to varying degrees in AhNF-YB10 transgenic lines. Owing to the increase in 1000-seed weight in the transgenic plants, the transcript levels of several TF genes regulating seed development, including LEC2 , FUS3 , and WRI1, were determined. The expression of these genes increased in comparison to that in the untransformed control. Discussion AhNF-YB1 and AhNF-YB10 are subfunctionalized for embryo development The NF-YB TFs are classified into LEC1-type and non-LEC1-type proteins according to the 16 amino acid residues in the conserved region of the B domain (Lee et al. 2003 ). There are relatively few LEC1 -type genes in plants, ranging in number from 1 to 6, while more than 10 non- LEC1 -type genes are generally present (Cagliari et al. 2014 ). The functions of LEC1 -type genes were originally revealed in Arabidopsis. The lec1 mutant plants exhibited leafy cotyledons; hence, the gene was named thus (Meinke 1992 ; West et al. 1994 ). The other LEC1 -type gene in Arabidopsis, designated LEC1-LIKE (L1L), which shares 83% sequence identity with AtLEC1, is also required for normal embryo development (Kwong et al. 2003 ). The phylogenetic relationship of LEC1 -type members, including a vast number of taxa, indicated that LEC1 -type genes originated in vascular plants, and only one LEC1 -type gene was found in the ancient plants Selaginella, Picea abies and Pinus sylvestris , whose protein sequences are more similar to AtL1L than to AtLEC1 (Cagliari et al. 2014 ). In our study, there was only one LEC1 -type gene in two diploid ancestral species, Arachis duranensis and Arachis ipaensis , and two in the cultivar Arachis hypogeae . In addition, these four LEC1 -type genes were most closely related to AtL1L in terms of the sequence of their encoded proteins. It was speculated that these genes, as the sole LEC1 -type genes, can indispensably control embryo development in these two diploid species. However, how do the two LEC1 -type genes cooperate to function in Arachis hypogeae ? AhNF-YB1 and AhNF-YB10 exhibited different expression patterns. AhNF-YB1 was expressed in all the organs analysed, and its expression pattern was similar to that of AtL1L ; however, similar to that of AtLEC1 , AhNF-YB10 expression was restricted mainly to the embryo, and AhNF-YB1 was expressed at a greater level than AhNF-YB10 in mid-stage seeds. Our prior studies on the two promoters of AhNF-YBs revealed that AhLEC1A and AhLEC1B correspond to AhNF-YB10 and AhNF-YB1 , respectively, in the present study. Research also demonstrated that they have different activities and spatiotemporal expression characteristics when they drive GUS expression in transgenic Arabidopsis (Tang et al. 2015 ; Tang et al. 2021 ). In our study, the expression of AhNF-YB10 , which is expressed at a relatively high level in the kernel, almost completely complemented the defects observed in the lec1-2 mutant, as indicated by the embryo morphology and seed germination rate. Although more than half of the AhNF-YB1 transgenic plants in the lec1-2 background exhibited a restored phenotype compared with the wild-type plants, 30%-40% of the transgenic plants and seedlings partially or completely retained the mutant phenotype. Previous research indicated that AtLEC1 was downregulated in the lec1-4 mutant, resulting in a decreased seed germination rate, decreased seed vigour, and a defective seedling phenotype in the dark (Huang et al. 2015 ). To some extent, the phenotypic features of ectopic expression of AhNF-YB1 in some transgenic lec1-2 mutants were similar to those of the receptor line lec1-2 , which could be attributed to its lower expression in embryos. Thus, we suggest that AhNF-YB10 located on the B subgenome may play a primary role in embryogenesis and embryonic development and maturation and that AhNF-YB1 on the A subgenome has partial functional redundancy with AhNF-YB10. The normal function of AhNF-YB10 depends on proper spatiotemporal expression LEC1 is a central regulator of the whole process of embryo development. In this work, we explored the phenotypes of lines overexpressing AhNF-YB10 in the lec1-2 background under the control of two different promoters, the truncated NapA promoter P211 and the AtLEC1 promoter. The transgenic seeds harbouring pC211P: B10 acquired desiccation tolerance and could germinate after drying. However, the other defects of lec1-2 , such as round cotyledons, shorter hypocotyls, and constricted root apices, remained unchanged in the transgenic embryos. When the 1044 bp AtLEC1 promoter replaced the P211 promoter, the pC AtLEC1P : B10 transgenic plants exhibited almost complete complementation of all the defects in the lec1-2 mutant. AtLEC1 is expressed during the whole process of seed development and at higher levels during early embryo development than in late-maturing-stage embryos, and it is indispensable for the specification of cotyledon identity and the completion of embryo maturation (Lotan et al. 1998 ). Previous studies on the NapA promoter indicated that its expression was barely detectable at the globule to heart stages, increased significantly at 10 to 15 days after pollination (DAP), and peaked at 30 DAP; moreover, its sequences between positions − 152 and + 44 are essential for the seed-specific expression pattern (Stålberg et al. 1993 ; Ellerström et al. 1996 ). Additionally, the deletion promoter of the − 211 nt upstream sequence maintained the original expression pattern in seeds but only held 4% of the activity of the wild-type 1101 bp promoter (Tan et al. 2011 ). Consequently, the lack of expression of AhNF-YB10 in embryos before heart embryo development in association with pC211P: B10 might cause defects at early stages of development and further during seedling establishment in the dark. The expression pattern of AhNF-YB10 during embryo development is similar to that of Arabidopsis AtLEC1. Therefore, appropriate spatiotemporal expression of AhNF-YB10 is necessary for embryo morphogenesis at the early development stage and is responsible for seed maturation at the mid-late development stage. Overexpressing AhNF-YB1 or AhNF-B10 could increase the yields and oil content of seeds Given the increasing global demand for edible oils, improving oil content and production is a major objective for breeding oilseed crops. Yields and oil content are controlled by multiple biological pathways involving many genes. In view of its indispensable role in seed development, many efforts have been made to utilize the LEC1 gene to improve yields and nutrient composition. However, the ectopic expression of master transcription factors, including LEC1 , controlled by the 35S promoter often leads to adverse effects on growth or development (Shen et al. 2010 ; Pouvreau et al. 2011 ; Tan et al. 2011 ). Therefore, moderate expression of LEC1 in the tissues of interest is vital for successful molecular breeding. In this work, a truncated Nap A promoter was used to drive the expression of AhNF-YB1 or AhNF-YB10 in transgenic wild-type Arabidopsis plants. This promoter could mainly stimulate the transcription of downstream genes during the middle to late stages of seed development without adverse effects on vegetative organ development (Shen et al. 2010 ; Pouvreau et al. 2011 ; Tan et al. 2011 ). The overexpression of either of these genes in Arabidopsis seeds increased the thousand-grain weight and the seed oil content to different extents. Consistent with the findings of previous studies of LEC1 -type genes in other plants (Tan et al. 2011 ; Elahi et al. 2016 ; Deng et al. 2023 ), the expression levels of several genes involved in de novo FA synthesis and seed development were greater in the AhNF-YB1 or AhNF-YB10 overexpression line than in the control. Most of the analysed genes related to acetyl-CoA precursors, FA chain elongation, lipid accumulation and embryo development exhibited increased expression in the developing seeds of the transgenic plants. Analysis of the levels of major FAs demonstrated that the proportion of USFAs was elevated, and the expression of some corresponding genes encoding desaturase increased. These results demonstrated that the two LEC1 -type genes in peanut perform conserved functions as key regulators during embryo development and reserve deposition. These studies provide an approach for the genetic improvement of peanut and other oil crops. Conclusions In summary, we identified and studied the functions of two LEC1 -type genes in Arachis hypogeae . It was found that both AhNF-YB1 and AhNF-YB10 participate in regulating embryogenesis, embryo development, and reserve deposition in cotyledons and that they have partial functional redundancy. On the other hand, overexpression of AhNF-YB1 or AhNF-YB10 at the middle to late stages of Arabidopsis seed development improved the weight, oil content, FA composition of the transgenic seeds. This study provides a theoretical basis for breeding oilseed crops with high yields and high oil contents. Declarations Author contribution statement GT conducted the experiments and drafted the manuscript, PX and CJ conducted the experiments, GL analyzed the data. LS and SW designed the study and revised the manuscript. All authors read and approved the final version. Conflict of interest The authors declare that they have no competing interests. Funding This research was supported by grants from Natural Science Foundation of Shandong Province (ZR2021MC054), National Natural Science Foundation of China (30971546), Shandong Provincial Key Research and Development Program (2023LZGCQY019), Shandong Provincial Key Research and Development Program (2021LZGC025). Acknowledgements We thank Jianru Zuo (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for kindly providing us with the plasmid with 211-bp promoter of Napin A (211P) and the methods of vector construction. References Akhtar S，Khalid N，Ahmed I et al. 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Biotechnol Biofuels 11:46. https://doi.org/10.1186/s13068-018-1049-4 Supplementary Files Supplementdata20240129.docx Cite Share Download PDF Status: Published Journal Publication published 20 Apr, 2024 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Minor revisions 13 Mar, 2024 Reviewers agreed at journal 06 Feb, 2024 Reviewers invited by journal 05 Feb, 2024 Editor assigned by journal 05 Feb, 2024 First submitted to journal 01 Feb, 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3913572","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271190273,"identity":"576510ca-a105-4685-ba45-d7b173ed7f8d","order_by":0,"name":"Guiying Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYFACxgaGBCjrwYcfEnL8zMyHHxCjRQLIYjac2WNjLNnOlmZAjF0gLWzSPGxpiQbneRQk8CmVj0huk3i4o7aOf3b7ZcMZPIcTjA/zMBgw1NhE49JieCOxTSLxzHEJiTtnCh98sDicZ3aY98ADhmNpuQ24tMwAaWk7JsFwIycZZEux2WG+BAPGhsOEtcjfyEkD+uVw4uZmHgMJfFrkJcBaaiQMbqQfA3t/AzMBLQY8D5stEtsOSG68kQMJZInDwEBOwOMX+fb0hzd/ttXxy91IfwiJyv7Dhx98qLHBbcsBBhZgLBwGMnmQIjABh3KwLQ0MzB8YGOqATPYHeNSNglEwCkbBSAYAMM9jPw9yRe0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0001-4758-6673","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Guiying","middleName":"","lastName":"Tang","suffix":""},{"id":271190274,"identity":"fb1ac3ef-4f89-48f2-b743-47abf20f33a4","order_by":1,"name":"Pingli Xu","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Pingli","middleName":"","lastName":"Xu","suffix":""},{"id":271190275,"identity":"f9f41251-8dea-4dd5-875e-c61cff46d9b0","order_by":2,"name":"Chunyu Jiang","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chunyu","middleName":"","lastName":"Jiang","suffix":""},{"id":271190276,"identity":"80d3f31b-a2de-4174-90ff-d662fda83107","order_by":3,"name":"Guowei Li","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Guowei","middleName":"","lastName":"Li","suffix":""},{"id":271190277,"identity":"9691ce77-3603-4240-aeca-1108518bceb4","order_by":4,"name":"Lei Shan","email":"","orcid":"https://orcid.org/0000-0001-7250-8944","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Shan","suffix":""},{"id":271190278,"identity":"5cdf8ea1-dcb1-4092-bd92-7bd8d98e5358","order_by":5,"name":"Shubo Wan","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shubo","middleName":"","lastName":"Wan","suffix":""}],"badges":[],"createdAt":"2024-01-31 10:54:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3913572/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3913572/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00299-024-03209-8","type":"published","date":"2024-04-20T22:39:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50787697,"identity":"a6380405-b21a-495e-b4a1-55b08e2022ad","added_by":"auto","created_at":"2024-02-07 09:49:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":87689,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships of NF-YBs derived from \u003cem\u003eArachis hypogeae\u003c/em\u003e, \u003cem\u003eArachis\u003c/em\u003e \u003cem\u003eduranensis\u003c/em\u003e,\u003cem\u003eArachis ipaensis\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. The unrooted phylogenetic tree was constructed using MEGA-X via the neighbour-joining method with 1,000 bootstrap replicates. The I-III subgroups are represented by different colours\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3913572/v1/504194c8ad7b98fe5d99aa24.jpg"},{"id":50787699,"identity":"a73d8bb5-a2fd-4360-a247-077e56a930e7","added_by":"auto","created_at":"2024-02-07 09:49:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33403,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10 \u003c/em\u003ein peanut plants. (a) Relative expression levels in different organs, as determined by qRT‒PCR. Roots, stems and leaves were collected from the 2-week-old seedlings. Flowers were picked at the full-bloom stage. Seeds were collected at 30 DAP. (b) Relative expression levels in seeds at different developmental stages. The relative expression level was calculated by the 2\u003csup\u003e-ΔCt\u003c/sup\u003e method using \u003cem\u003eAhACTIN\u003c/em\u003e 7 as an internal control. The data presented here are the mean values of three replicates, with error bars indicating SEs. The asterisks represent significant differences determined by Student’s \u003cem\u003et\u003c/em\u003e test. *, \u003cem\u003eP\u003c/em\u003e＜0.05; **, \u003cem\u003eP\u003c/em\u003e＜0.01.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3913572/v1/ee60c13a00309b37329dfe3d.jpg"},{"id":50787702,"identity":"49374fdb-f2ea-448b-ba27-dbaaae8dd243","added_by":"auto","created_at":"2024-02-07 09:49:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111268,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic characteristics of transgenic \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10\u003c/em\u003e in the \u003cem\u003elec1-2\u003c/em\u003emutant background. Embryos and siliques were removed at 13-15 days after flowering. Seeds were dried at room temperature. The etiolated seedlings were further grown in the dark for 3 days after germination, and the seven-day-old green seedlings were subsequently grown in light.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3913572/v1/0bcc287998892cddf3b9b1cc.jpg"},{"id":50787700,"identity":"ad2270ab-4279-4693-b418-f37256184ed1","added_by":"auto","created_at":"2024-02-07 09:49:34","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68517,"visible":true,"origin":"","legend":"\u003cp\u003eThe viability and germination rate of transgenic \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10 lec1-2\u003c/em\u003e mutant seeds. (a) Germination rate of transgenic and WS Arabidopsis seeds stored for 1 week, 1 month, or 3 months at room temperature after harvest. (b) TTC staining of Arabidopsis seeds kept at room temperature for 3 months after harvest. A. The WS ecotype.\u003c/p\u003e\n\u003cp\u003eB.\u003cem\u003epCAtLEC1P\u003c/em\u003e: \u003cem\u003eB1\u003c/em\u003e, #3. C. \u003cem\u003epCAtLEC1P\u003c/em\u003e: \u003cem\u003eB10\u003c/em\u003e, #1. D. The \u003cem\u003elec1-2\u003c/em\u003emutant. E. \u003cem\u003epCAtLEC1P\u003c/em\u003e: \u003cem\u003eB1\u003c/em\u003e, #4. F.\u003cem\u003e pCAtLEC1P\u003c/em\u003e: B10, #3\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3913572/v1/dbcc66be2f5e141dc49e3e7c.jpg"},{"id":50787698,"identity":"108beee6-da76-4b7c-9f48-fe74375e5e33","added_by":"auto","created_at":"2024-02-07 09:49:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":116536,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of \u003cem\u003eAhNF-B10\u003c/em\u003e affects the expression levels of several genes related to lipid accumulation and seed development in developing Arabidopsis seeds. Total RNA was extracted from seeds collected at 13 DAF. The relative expression level was calculated by the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method using \u003cem\u003eAtActin\u003c/em\u003e as an internal control. Significant differences at the \u003csup\u003e*\u003c/sup\u003ep＜0.05 and \u003csup\u003e**\u003c/sup\u003ep＜0.01 levels were determined via Student’s \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e\n\u003cp\u003eNote: DAF, days after flowering; \u003cem\u003eB10, #1\u003c/em\u003e: OE-\u003cem\u003eAhNF-B10\u003c/em\u003e, \u003cem\u003e#1\u003c/em\u003e, \u003cem\u003eB10, #10\u003c/em\u003e: OE-\u003cem\u003eAhNF-B10\u003c/em\u003e, \u003cem\u003e#1\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3913572/v1/bb9d2b8575aad4f1f833b197.jpg"},{"id":55690366,"identity":"9e4b7048-dd8c-4493-9796-f837635bbfb7","added_by":"auto","created_at":"2024-05-01 22:39:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1125947,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3913572/v1/bbf3720d-c611-4cde-a2e5-29ef94103feb.pdf"},{"id":50787703,"identity":"075bfe37-596e-4d9e-884f-9ffda8750c4d","added_by":"auto","created_at":"2024-02-07 09:49:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3073924,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementdata20240129.docx","url":"https://assets-eu.researchsquare.com/files/rs-3913572/v1/62254ac5ee047d4e213d7573.docx"}],"financialInterests":"","formattedTitle":"Peanut LEAFY COTYLEDON1-type genes participate in regulating the embryo development and the accumulation of storage lipids","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCultivated peanut (\u003cem\u003eArachis hypogeae\u003c/em\u003e L.) is one of the most important oil crops grown in tropical and subtropical regions worldwide. Its kernels are rich in oil, protein and other nutrients that are beneficial for human health (Akhtar et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The oil content in peanut kernels is approximately 45%-56%, and two major unsaturated fatty acids (USFAs), oleic acid (36%-67%) and linoleic acid (15%-43%), constitute 80% of the total fatty acid (FA) content (Barkley et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Diets with high levels of USFAs are nutritionally beneficial for lowering cholesterol levels and reducing systolic blood pressure (Ter\u0026eacute;s et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Akhtar et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, given the growing need for a healthy diet, the demand for high-quality peanut oil is increasing. In turn, increasing the oil content and improving the FA composition are major goals for guiding scientific research currently and for the foreseeable future.\u003c/p\u003e \u003cp\u003eTranscription factors (TFs) regulate the expression of downstream target genes by binding to specific motifs in promoters. Due to their regulatory effects on target genes or interactions with other TFs at multiple levels, some TFs can control the entire pathways of various biological processes. The transcription factor NF-YB is a subunit of the NF-Y (Nuclear Factor Y) heterotrimeric complex and plays an important role in the specific binding of the NF-Y trimer complex to DNA (Zemzoumi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1999\u003c/span\u003e); it is involved in regulating the growth, development and stress tolerance of a wide array of plants (Lotan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Siriwardana et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Niu and He \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). On the basis of the presence of 16 conserved amino acid residues in the NF-YB B domain, these proteins can be divided into two classes, namely, LEC1-type and non-LEC1-type, in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Kwong et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). AtNF-YB9 (LEAFY COTYLEDON 1, AtLEC1) and AtNF-YB6 (AtLEC1-LIKE, AtL1L) in Arabidopsis are LEC1-type proteins, and the remaining 11 members are non-LEC1-type proteins.\u003c/p\u003e \u003cp\u003eLEC1 has been demonstrated to be a key regulator of seed development and controls several crucial processes, including embryogenesis, endosperm development, and the deposition of seed reserves in plants (Jo et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The \u003cem\u003eLEC1\u003c/em\u003e null mutant exhibits defects in normal embryos, acquisition of desiccation tolerance, reserve accumulation, and suppression of germination (Harada \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Santos-Mendoza et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In the Arabidopsis \u003cem\u003eLEC1\u003c/em\u003e gain-of-function mutant, \u003cem\u003eturnip\u003c/em\u003e (\u003cem\u003etnp\u003c/em\u003e), whose promoter has a longer fragment deletion 436 bp upstream from the start codon (ATG), exhibits ectopic accumulation of storage products, and undergoes partial de-etiolation when the seedlings are grown in the dark (Casson and Lindsey \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Ectopic expression of \u003cem\u003eLEC1\u003c/em\u003e was sufficient to induce somatic embryogenesis from vegetative cells (Lotan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In addition to analyses of the physiological function and primary molecular mechanism of \u003cem\u003eLEC1\u003c/em\u003e in Arabidopsis and in several other staple crop species, several studies have focused on the genetic improvement of reserve substance accumulation via the use of \u003cem\u003eLEC1\u003c/em\u003e. For example, overexpression of the corn \u003cem\u003eZmLEC1\u003c/em\u003e gene under the embryo-specific weak \u003cem\u003eEARLY EMBRYO PROTEIN 1\u003c/em\u003e (\u003cem\u003eEAP1\u003c/em\u003e) promoter could significantly increase the oil content of transgenic maize (Shen et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In \u003cem\u003eBrassica napus\u003c/em\u003e, LEC1 regulates the expression of multiple genes related to promoting sucrose (Suc) and lipid accumulation in seeds, resulting in a significantly increased FA level in transgenic canola (Mu JY et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Several researchers have also shown that LEC1 plays essential roles in starch synthesis in rice seeds (Yang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Das et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo date, the biological function of LEC1 in peanut has not been elucidated. In the present study, 16 \u003cem\u003eNF-YB\u003c/em\u003e genes were identified from the genome of cultivated peanut; among these genes, \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10\u003c/em\u003e are \u003cem\u003eLEC1\u003c/em\u003e-type genes, and the remaining 14 \u003cem\u003eAhNF-YBs\u003c/em\u003e are non-\u003cem\u003eLEC1\u003c/em\u003e-type genes. The functions of the two \u003cem\u003eLEC1\u003c/em\u003e-type genes were investigated via complementation analysis in the Arabidopsis \u003cem\u003elec1-2\u003c/em\u003e mutant and ectopic expression in wild-type Arabidopsis. The results indicated that most or some of the defective phenotypes of \u003cem\u003elec1-2\u003c/em\u003e were complemented in the transgenic \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10\u003c/em\u003e mutant plants. Overexpression of \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10\u003c/em\u003e in Arabidopsis impacted seed weight, oil content, and FA content. Moreover, the normal function of these genes was found to be dependent on proper spatiotemporal expression.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eIdentification and phylogenetic analysis of NF-YB family members in the peanut cultivar and two probable diploid ancestors\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe sequences of the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e NF-YB family members were downloaded from The Arabidopsis Information Resource (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) and used as query sequences to search the sequences of \u003cem\u003eArachis hypogeae\u003c/em\u003e, \u003cem\u003eArachis duranensis\u003c/em\u003e and \u003cem\u003eArachis ipaensis\u003c/em\u003e via BLASTP in the Peanutbase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.peanutbase.org/\u003c/span\u003e\u003cspan address=\"https://www.peanutbase.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and NCBI (National Center for Biotechnology Information) databases. In addition, the putative NF-YBs of the peanut cultivar and the two diploid wild species were confirmed via InterProScan 56.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.Ebi.ac.uk/inerpro/\u003c/span\u003e\u003cspan address=\"http://www.Ebi.ac.uk/inerpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Their molecular weights (MWs) and isoelectric points (pIs) were calculated using ExPASy. Chromosome distribution information for AhNF-YBs was extracted from gff3 annotation files of \u003cem\u003eArachis hypogeae\u003c/em\u003e by TBtools (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The NF-YB protein sequences of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and three \u003cem\u003eArachis\u003c/em\u003e species were aligned by the MUSCLE method (Kumar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and an unrooted phylogenetic tree was established using MEGA X by employing the neighbour-joining (NJ) method (Kumar et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and growth conditions\u003c/h2\u003e \u003cp\u003eThe peanut cultivar Fenghua No. 1 (FH1) was maintained and cultivated by our group. The roots, stems, leaves, flowers and seeds at different developmental stages were taken from peanut plants grown in the natural environment of Yinmaquan Farm in Jinan and kept in a -80\u0026deg;C freezer before isolation of total RNA. To ensure consistency of seed development, the pegs were labelled by tying with thin plastic threads.\u003c/p\u003e \u003cp\u003eTwo ecotypes of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Wassilewskija, WS; and Columbia, COL) were maintained in our laboratory, and the \u003cem\u003elec1-2\u003c/em\u003e (CS3867) mutant obtained from the Arabidopsis Biological Resource Center (ABRC) was used for transformation in this study. The embryos of homozygous \u003cem\u003elec1-2\u003c/em\u003e mutants before desiccation were sown on 1/2 MS medium to rescue their desiccation-intolerant phenotype, and the defective seedlings were subsequently transplanted to pots with culture substrate after 10 days. The wild-type WS, COL, \u003cem\u003elec1-2\u003c/em\u003e mutant and transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants were grown in a greenhouse at room temperature under a 16 h light/8 h dark photoperiod and 65% relative humidity. To measure the thousand-seed weight and oil content in the wild-type, transgenic Arabidopsis and \u003cem\u003elec1-2\u003c/em\u003e mutant plants, the plants were always grown in the same chamber at the same time.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of plant expression vectors and transgenic Arabidopsis plants harbouring\u003c/b\u003e \u003cb\u003eAhNF-YB1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eAhNF-YB10\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 1044-bp \u003cem\u003eAtLEC1\u003c/em\u003e promoter (\u003cem\u003eAtLEC1P\u003c/em\u003e) upstream of ATG in Arabidopsis was cloned and identified by sequencing. With pCAMBIA-3301 as the initial vector, two vectors with 211-bp promoter of \u003cem\u003eNapin A\u003c/em\u003e (211P) from \u003cem\u003eBrassica napus\u003c/em\u003e (Tan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and two vectors driven by \u003cem\u003eAtLEC1\u003c/em\u003eP expressing \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10\u003c/em\u003e were constructed and were designated pC3301-211P:\u003cem\u003eAhNF-YB1/10\u003c/em\u003e (pC211P:\u003cem\u003eB1/10\u003c/em\u003e) and pC3301-\u003cem\u003eAtLEC1P\u003c/em\u003e:\u003cem\u003eAhNF-YB1\u003c/em\u003e/10 (pC\u003cem\u003eAtLEC1\u003c/em\u003eP:\u003cem\u003eB1\u003c/em\u003e/\u003cem\u003e10\u003c/em\u003e), respectively. Transgenic Arabidopsis plants were generated via the floral dip method. The seeds of transgenic Arabidopsis plants harbouring \u003cem\u003eAhNF-YB1\u003c/em\u003e or \u003cem\u003eAhNF-YB10\u003c/em\u003e were screened on basta-containing 1/2 MS medium. The transgenic lines with one copy of the exogenous gene were identified by the 3:1 segregation ratio of the T\u003csub\u003e2\u003c/sub\u003e plants with and without basta resistance. The Arabidopsis overexpression lines were also identified by amplifying the insertion fragment from the genomic DNA using primers located on the P211 promoter and ORF of \u003cem\u003eAhNF-YB1/10\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of seed weight, germination rate and longevity\u003c/h2\u003e \u003cp\u003eThousand-grain weight was measured to evaluate the size and the plumpness of the Arabidopsis seeds. The hundred-seed method was used for determination of the weight per thousand seeds following the process described by Wang (Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor seed germination and longevity assessment in Arabidopsis, the seeds were kept in 1.5 mL centrifuge tubes with allochroic silica gel and stored at room temperature. The germination ability of the plants was tested separately after harvesting for 1 week, 1 month, and 3 months. One hundred sterile seeds were sown on 1/2 MS medium, kept in the dark and cold (4 ℃) for 2 days, and subsequently transferred into a chamber for growth at 22 ℃\u0026plusmn; 2 ℃ with a 16/8 hr light/dark cycle for 7 days. Then, the number of seedlings with green opening cotyledons was counted. The viability of the seeds preserved for 3 months at room temperature was evaluated via 2,3,5-triphenyltetrazolium chloride (TTC) staining. Two hundred disinfected seeds in each assay were stained by incubating in 1% tetrazolium solution in darkness at 30\u0026deg;C for 48 hr. The viability of the seeds was estimated by the displayed colour of 2,3,5-triphenyl formazan after staining, wherein the darker the red colour was, the more viable the seeds were. The stained seeds were observed by stereomicroscopy (Waes and Debergh \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Salvi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). All the experiments were carried out in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT‒PCR (qRT‒PCR) analysis of gene expression\u003c/h2\u003e \u003cp\u003eThe total RNAs from the different peanut tissues and Arabidopsis seeds were isolated following the protocol for an RNA isolation kit provided by Huayueyang Biotech Company, Ltd., Beijing, China. First-strand cDNA was synthesized from 1 \u0026micro;g of total RNA using the Evo M-MLV RT-Kit with gDNA Clean for qPCR II. Each cDNA sample was diluted 10-20-fold in sterile water for qRT‒PCR. The expression level was normalized using peanut \u003cem\u003eAhACTIN 7\u003c/em\u003e or \u003cem\u003eAtACTIN\u003c/em\u003e as internal controls. The primers used are listed in Supplemental Table\u0026nbsp;1. The qRT‒PCRs were performed in 8-tube strips, and the data were analysed according to methods described previously (Zhu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of oil content and fatty acid composition in Arabidopsis seeds\u003c/h2\u003e \u003cp\u003eThe Soxhlet extraction method was used to analyse the seed oil content. Seeds (1.5 g) were baked at 80\u0026deg;C for 4 hr and then cooled to room temperature. The following steps, including grinding, extracting and weighing, were performed according to previously described methods (Tang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For each sample, triplicate analyses were performed, and the mean values of the triplicates were used to calculate the oil content.\u003c/p\u003e \u003cp\u003eFA analysis was performed by gas chromatography/mass spectrometry (GC/MS) using an HP-6890 instrument equipped with a DB-23 column (60 m\u0026times; 250 \u0026micro;m \u0026times; 0.25 \u0026micro;m). Approximately 300 mg was needed per seed sample. The pretreatment of the seed samples was performed as described previously (Tang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The loading volume of the methyl esterified sample was 1 \u0026micro;L. The temperature was set to 180\u0026deg;C in the beginning, subsequently increased to 200\u0026deg;C at a 4\u0026deg;C/min, and then held for 15 min; finally, the temperature was increased to 230\u0026deg;C at 10\u0026deg;C/min and held for 10 min. The injection and detection port temperatures were 260℃ and 270℃, respectively. FAs were separated using N\u003csub\u003e2\u003c/sub\u003e as the carrier gas at a flow rate of 2.0 mL/min, and the split ratio was 30:1. The experiment was carried out in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the data are presented as the mean value and the corresponding standard error of at least three replicates. Statistical analyses were performed in Excel 2019 and IBM SPSS Statistics 26 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s post hoc test and Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e- test were used to indicate significant differences. All the bar charts in the figures were prepared using GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego CA, USA) or Excel.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification and phylogenetic analysis of the NF-YB gene family in \u003cem\u003eArachis hypogeae\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 16 \u003cem\u003eAhNF-YB\u003c/em\u003e genes in peanut were identified and named according to their physical location on the chromosome. The encoded proteins, AhNF-YB1~16, varied from 151 to 226 amino acids in length. The detailed information, including sequence ID, chromosomal location, number of amino acids, protein isoelectric point (pI) and protein molecular weight (MW), is listed in Supplemental Table 2. According to the 16 amino acid residues conserved in LEC1-type NF-YB, AhNF-YB1 on Chr 01 and AhNF-YB10 on Chr 11 are classified as LEC1-type proteins, and the remaining 14 AhNF-YBs are classified as non-LEC1-type proteins.\u003c/p\u003e\n\u003cp\u003eTo investigate the phylogenetic relationship of NF-YBs, 16 AhNF-YB members in cultivated peanut, 8 AdNF-YBs in \u003cem\u003eArachis duranensis\u003c/em\u003e, 8 AiNF-YBs in \u003cem\u003eArachis ipaensis\u003c/em\u003e, and 13 AtNF-YBs in Arabidopsis were used to construct a phylogenetic tree. As shown in Fig. 1, the NF-YBs were divided naturally into three clades, designated I, II, and III. Clade I included ten members, among those AhNF-YB1 and XP015940529.1 (from \u003cem\u003eArachis duranensis\u003c/em\u003e), and AhNF-YB10 and XP016195843.1 (from \u003cem\u003eArachis ipaensis\u003c/em\u003e) shared relatively high homology and clustered into a subclade with Arabidopsis AtNF-YB6 and AtNF-YB9, which belong to the LEC1 type. AhNF-YB1 and AhNF-YB10 had 63% and 62% sequence similarity with AtNF-YB6 respectively, and had 56% and 55% sequence similarity with AtNF-YB9 respectively; while another subclade of clade I consists of AhNF-YB6, AhNF-YB14 and their homologue from wild peanut of A or B genome. Except AhNF-YB6 and AhNF-YB14 from Clade I, the other non-LEC1-type members of the A subgenome and B subgenome in \u003cem\u003eArachis hypogeae\u0026nbsp;\u003c/em\u003ewere classified into clade II (4 members) and III (8 members), and those genes located on the corresponding chromosomes were clustered together with the relatives from the two diploid \u003cem\u003eArachis\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003eAccording to previous reports, LEC1-type NF-YBs play essential roles in seed developmental processes, including embryogenesis and deposition of storage reserves in seeds (West et al. 1994; Shen et al. 2010; Huang et al. 2015; Zhu et al. 2018). Owing to the focus on seed development and reserve accumulation in our research, two \u003cem\u003eLEC1\u003c/em\u003e-type genes in peanut, \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10,\u0026nbsp;\u003c/em\u003ewere chosen for further functional identification and analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression patterns of \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10\u003c/em\u003e in peanut\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression patterns of \u003cem\u003eAhNF-YB1\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAhNF-YB10\u0026nbsp;\u003c/em\u003ein various organs and in seeds at different developmental stages were investigated via qRT‒PCR. The results demonstrated that \u003cem\u003eAhNF-YB1\u003c/em\u003e is constitutively expressed at different levels in the roots, stems, leaves, flowers and seeds (order from high to low: seeds, roots, leaves, flowers, and stems). \u003cem\u003eAhNF-YB10\u003c/em\u003e was expressed specifically in seeds (Fig. 2a). During seed development, the two genes exhibited similar expression patterns; their expression started at the early stage of seed development (at 10 DAP), increased at the middle stage, and then decreased obviously at the late stage. In addition, \u003cem\u003eAhNF-YB1\u003c/em\u003e was significantly more highly expressed thanwas\u003cem\u003e\u0026nbsp;AhNF-YB10\u003c/em\u003e at the 20 DAP, 30 DAP and 40 DAP, whereas the expression level of \u003cem\u003eAhNF-YB10\u0026nbsp;\u003c/em\u003ewas greater than that of \u003cem\u003eAhNF-YB1\u003c/em\u003e at early developmental stage (at 10 DAP) and later stages (at 50, 60, and 70 DAP) (Fig. 2b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEctopic expression of two \u003cem\u003eAhNF-YB\u003c/em\u003e genes in the Arabidopsis \u003cem\u003elec1-2\u003c/em\u003e mutant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the function of the two \u003cem\u003eAhNF-YB\u003c/em\u003e genes, homozygous \u003cem\u003elec1-2\u003c/em\u003e mutant plants derived from heterozygous \u003cem\u003elec1-2\u003c/em\u003e seeds purchased from the ABRC were first distinguished by their embryonic phenotype and subsequently rescued on 1/2 MS medium. Initially, pC211P:\u003cem\u003eB1\u003c/em\u003e and pC211P:\u003cem\u003eB10\u003c/em\u003e, which harbour the 211-bp promoter of the rape \u003cem\u003eNap A\u0026nbsp;\u003c/em\u003egene, were used as transformation vectors (Supplemental Fig. 1a). Three transformants of the T\u003csub\u003e1\u003c/sub\u003e generation containing the pC211P:\u003cem\u003eB10\u0026nbsp;\u003c/em\u003econstruct in the \u003cem\u003elec1-2\u003c/em\u003e homozygous background were successfully obtained by selection on 1/2 MS medium supplemented with 10 mg/L Basta. Furthermore, the phenotypes of the T\u003csub\u003e2\u003c/sub\u003e transgenic plants were investigated. Unlike the \u003cem\u003elec1-2\u003c/em\u003e homozygous seeds, which cannot germinate normally because of their desiccation intolerance (West et al. 1994), most of the seeds in the three transgenic lines possessed the ability to germinate. However, the morphologies of several of the transgenic seeds, including round unbent cotyledons, shorter embryonic axes, and roots with constricted ends, were also similar to those of the mutant \u003cem\u003elec1-2\u003c/em\u003e. Unlike those on \u003cem\u003elec1-2,\u0026nbsp;\u003c/em\u003etrichomes on the adaxial surfaces of the cotyledons of the transgenic seedlings were not found(Fig. 3 F-O). In the present study, transgenic progenies with the pC211P:\u003cem\u003eB1\u003c/em\u003e construct were not obtained. It is possible that \u003cem\u003eAhNF-YB1\u0026nbsp;\u003c/em\u003ecould not complement the phenotype of desiccation intolerance in \u003cem\u003elec1-2\u0026nbsp;\u003c/em\u003eseeds.\u003c/p\u003e\n\u003cp\u003eThe 211-bp promoter of \u003cem\u003eNapA\u003c/em\u003e retains a seed-specific expression pattern similar to that of the full-length promoter, and its expression is greater at the later stage of seed development (Tan et al. 2011). To explore why most of the defects in \u003cem\u003elec1-2\u003c/em\u003e had not been complemented in the pC211P:\u003cem\u003eB1/10\u003c/em\u003e transgenic seeds, the \u003cem\u003eAhNF-YB1\u003c/em\u003e and\u003cem\u003e\u0026nbsp;AhNF-YB10\u003c/em\u003e constructs under the control ofthe\u003cem\u003e\u0026nbsp;AtLEC1\u0026nbsp;\u003c/em\u003epromoter were transferred into \u003cem\u003elec1-2\u0026nbsp;\u003c/em\u003e(Supplemental Fig. 1b). More than a dozen transformants of the two genes were successfully obtained. Phenotypic analysis of the transgenic plants revealed that the phenotypes of the transgenic plants harbouring the exogenous gene \u003cem\u003eAhNF-YB10\u0026nbsp;\u003c/em\u003ewere similar to those of the wild-type plants, in which the morphology of the embryos was normal and the seeds germinated normally (Fig. 3. A-E, P-T). However, in the transgenic plants harbouring \u003cem\u003eAhNF-YB1\u003c/em\u003e, only approximately 60%-70% of the transformants were similar to WS-type Arabidopsis plants; the seeds of these plants had elliptic cotyledons and bent axes, and the etiolated plants had longer hypocotyls. The remaining 30%-40% of the seeds were similar to those of the \u003cem\u003elec1-2\u0026nbsp;\u003c/em\u003emutant in appearance; however, some of them could germinate, but the seedlings had shorter hypocotyls even though they were in darkness (Fig. 3. U-Y). Furthermore, the longevity and vigour of the seeds of plants ectopically expressing \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10\u003c/em\u003e were examined. The results showed that the germination rates of seeds of the \u003cem\u003eAhNF-YB10\u003c/em\u003e transgenic lines and the wild-type control were hardly affected by storage for three months after harvesting, while the germination rates of the seeds from the \u003cem\u003eAhNF-YB1\u003c/em\u003e transformants significantly decreased with increasing duration of preservation in comparison to those of the control seeds (Fig. 4a). The results of the TTC assay showed that all the \u003cem\u003elec1-2\u003c/em\u003e seeds were not stained because of seed inviability (Fig. 4b. D), and the seeds of the \u003cem\u003eAhNF-YB10\u0026nbsp;\u003c/em\u003etransgenic plants and WS control plants were stained red or dark red (Fig. 4b. A, C, F), while the most \u003cem\u003eAhNF-YB1\u0026nbsp;\u003c/em\u003etransgenic seeds were pale red or had no dyeing (Fig. 4b. B, E). These results indicated that ageing treatment significantly decreased the longevity of the \u003cem\u003eAhNF-YB1\u003c/em\u003e-transformed seeds. To explore why the P211 and \u003cem\u003eAtLEC1\u003c/em\u003e promoters triggering \u003cem\u003eAhNF-YB1/10\u003c/em\u003e expression in the \u003cem\u003elec1-2\u003c/em\u003e background caused different phenotypes in transgenic Arabidopsis, the expression profiles of these transgenic lines during seed development were checked. The results showed that \u003cem\u003eAhNF-YB10\u003c/em\u003e expression driven by P211 was hardly detectable at 4 DAF and markedly increased at 8 DAF and 13 DAF. In addition, driven by the \u003cem\u003eAtLEC1\u003c/em\u003e promoter, the expression levels were greatest at 4 DAF and decreased with seed development. \u003cem\u003eAhNF-YB1\u003c/em\u003e exhibited an expression pattern similar to that of \u003cem\u003eAhNF-YB10\u003c/em\u003e under the control of the \u003cem\u003eAtLEC1\u003c/em\u003e promoter but was expressed at lower levels (Supplemental Fig. 2). The degree to which \u003cem\u003eAhNF-YB1\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAhNF-YB10\u0026nbsp;\u003c/em\u003erestored the defects in\u003cem\u003e\u0026nbsp;lec1-2\u003c/em\u003e was related to the transcript levels of these genes. Moreover, the expression levels of key genes involved in seed development and FA synthesis were elevated in the transgenic plants (Supplemental Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe oil contents and seed weights of the \u003cem\u003eAhNF-YB1/10\u003c/em\u003e-overexpressing Arabidopsis lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsidering the important role of plant LEC1-type NF-YBs in reserve deposition, we tried to improve the oil content by overexpressing \u003cem\u003eAhNF-YB1/10\u003c/em\u003e via the P211 promoter in Arabidopsis seeds. Multiple independent transgenic lines were obtained by screening on basta-containing medium and detection via PCR (Supplemental Fig. 4). No developmental abnormalities were observed throughout their life cycle (Supplemental Fig. 5). Analysis of the transgenic seeds revealed that the crude fat content in the transgenic seeds was elevated by 6.2%-29.1% compared to that in COL(Table 1). The content of the main FAs in the \u003cem\u003eAhNF-YB1/10\u0026nbsp;\u003c/em\u003etransgenic seeds increased to different degrees, among which the improvements in the C18:1n9c, C18:2n6c, C18:3n3, C20:1 and C22:1n9 contents were more significant (Table 2). These findings suggested that the increase in seed oil content was due to the increase in several major FAs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Content of crude fat in Arabidopsis seeds (% dry weight). Mature seeds were used for the analysis of the oil content via the Soxhlet method. Asterisks indicate statistically significant differences compared with the control.(Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test: *\u003cem\u003eP\u003c/em\u003e＜0.05; **\u003cem\u003eP\u003c/em\u003e＜0.01)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eGenotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eCrude fat content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003ePercent increase (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eCOL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e24.46 (\u0026plusmn;0.43)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B1, #11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e27.99 (\u0026plusmn;0.56)**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e14.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B1, #13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e28.25 (\u0026plusmn;0.51)**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e15.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B1, #15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e30.69 (\u0026plusmn;0.63)**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e25.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B10, #1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e33.34 (\u0026plusmn;0.59)**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e36.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B10, #10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e32.36 (\u0026plusmn;0.68)**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e32.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B10, #15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e28.92 (\u0026plusmn;0.51)**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e18.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Composition of the main fatty acids in Arabidopsis seeds (mg/g). Mature seeds were used for the GC analysis. Asterisks indicate statistically significant differences compared with the control (Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test: *\u003cem\u003eP\u003c/em\u003e＜0.05; **\u003cem\u003eP\u003c/em\u003e＜0.01)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eFatty acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003eCOL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.072434607645874%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B1, #11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B1, #13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B1, #15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B10, #1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B10, #10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B10, #15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eC16:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e23.91 \u0026plusmn; 0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.072434607645874%\" valign=\"top\"\u003e\n \u003cp\u003e24.10\u0026plusmn;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e23.34\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e24.57\u0026plusmn;0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e28.52\u0026plusmn;0.37**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e27.98\u0026plusmn;0.64**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e23.40\u0026plusmn;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eC18:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e8.30\u0026plusmn;0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.072434607645874%\" valign=\"top\"\u003e\n \u003cp\u003e8.16\u0026plusmn;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e7.73\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e8.87\u0026plusmn;0.16*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e10.37\u0026plusmn;0.15**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e10.11\u0026plusmn;0.32**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e7.99\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eC18:1n9c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e34.64\u0026plusmn;0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.072434607645874%\" valign=\"top\"\u003e\n \u003cp\u003e36.36\u0026plusmn;0.48**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e30.47\u0026plusmn;0.44**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e35.88\u0026plusmn;0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e41.56\u0026plusmn;1.04**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e47.49\u0026plusmn;1.75**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e37.74\u0026plusmn;0.55**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eC18:2n6c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e83.28\u0026plusmn;2.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.072434607645874%\" valign=\"top\"\u003e\n \u003cp\u003e83.78\u0026plusmn;1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e81.35\u0026plusmn;0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e86.78\u0026plusmn;1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e97.31\u0026plusmn;1.02**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e97.35\u0026plusmn;2.02**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e82.06\u0026plusmn;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eC18:3n3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e52.01\u0026plusmn;0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.072434607645874%\" valign=\"top\"\u003e\n \u003cp\u003e52.56\u0026plusmn;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e54.27\u0026plusmn;0.76*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e58.42\u0026plusmn;1.14**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e72.62\u0026plusmn;0.61**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e64.33\u0026plusmn;1.44**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e53.95\u0026plusmn;0.75*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eC20:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e6.22\u0026plusmn;0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.072434607645874%\" valign=\"top\"\u003e\n \u003cp\u003e6.33\u0026plusmn;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e7.15\u0026plusmn;0.00**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e7.66\u0026plusmn;0.19**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e7.28\u0026plusmn;0.13**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e7.06\u0026plusmn;0.17*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e6.29\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eC20:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e53.50\u0026plusmn;1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.072434607645874%\" valign=\"top\"\u003e\n \u003cp\u003e55.21\u0026plusmn;0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e58.61\u0026plusmn;0.55*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e63.23\u0026plusmn;1.63**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e73.09\u0026plusmn;1.24**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e67.51\u0026plusmn;2.41**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e55.72\u0026plusmn;0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eC21:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e5.84\u0026plusmn;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.072434607645874%\" valign=\"top\"\u003e\n \u003cp\u003e6.14\u0026plusmn;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e6.96\u0026plusmn;0.04**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e6.79\u0026plusmn;0.16**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e7.73\u0026plusmn;0.11**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e6.85\u0026plusmn;0.14**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e5.74\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003eC22:1n9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\" valign=\"top\"\u003e\n \u003cp\u003e6.27\u0026plusmn;0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.072434607645874%\" valign=\"top\"\u003e\n \u003cp\u003e6.71\u0026plusmn;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e9.03\u0026plusmn;0.05**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e8.81\u0026plusmn;0.25**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e7.62\u0026plusmn;0.17**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e6.88\u0026plusmn;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.273641851106639%\" valign=\"top\"\u003e\n \u003cp\u003e6.52\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eCompared with that of the nontransgenic COL plants, the thousand-seed weight increased by 3.31%-27.81% in the \u003cem\u003eAhNF-YB1\u003c/em\u003e-OE and \u003cem\u003eAhNF-YB10\u003c/em\u003e-OE transgenic Arabidopsis plants (Table 3), but the other traits related to plant growth and development did not change.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e The 1000-seed weight of the transgenic Arabidopsis lines. Asterisks indicate statistically significant differences compared with the control (Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test: *\u003cem\u003eP\u003c/em\u003e＜0.05; **\u003cem\u003eP\u003c/em\u003e＜0.01)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.31858407079646%\" valign=\"top\"\u003e\n \u003cp\u003eGenotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.283185840707965%\" valign=\"top\"\u003e\n \u003cp\u003eThousand-seed weight (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.39823008849557%\" valign=\"top\"\u003e\n \u003cp\u003e% Increase over control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.31858407079646%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B1, #11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.283185840707965%\" valign=\"top\"\u003e\n \u003cp\u003e20.13*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.39823008849557%\" valign=\"top\"\u003e\n \u003cp\u003e6.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.31858407079646%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B1, #13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.283185840707965%\" valign=\"top\"\u003e\n \u003cp\u003e19.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.39823008849557%\" valign=\"top\"\u003e\n \u003cp\u003e3.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.31858407079646%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B1, #15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.283185840707965%\" valign=\"top\"\u003e\n \u003cp\u003e22.00**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.39823008849557%\" valign=\"top\"\u003e\n \u003cp\u003e16.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.31858407079646%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B10, #1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.283185840707965%\" valign=\"top\"\u003e\n \u003cp\u003e24.13**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.39823008849557%\" valign=\"top\"\u003e\n \u003cp\u003e27.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.31858407079646%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B10, #10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.283185840707965%\" valign=\"top\"\u003e\n \u003cp\u003e21.13**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.39823008849557%\" valign=\"top\"\u003e\n \u003cp\u003e11.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.31858407079646%\" valign=\"top\"\u003e\n \u003cp\u003eOE-AhNF-B10, #15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.283185840707965%\" valign=\"top\"\u003e\n \u003cp\u003e19.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.39823008849557%\" valign=\"top\"\u003e\n \u003cp\u003e3.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.31858407079646%\" valign=\"top\"\u003e\n \u003cp\u003eCOL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.283185840707965%\" valign=\"top\"\u003e\n \u003cp\u003e18.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.39823008849557%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eExpression analysis of the genes associated with fatty acid synthesis and embryogenesis in developing \u003cem\u003eAhNF-YB10\u003c/em\u003e-OE seeds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the molecular basis of the improvements in FA content, oil content and thousand-seed weight in transgenic Arabidopsis plants overexpressing these genes, the genes involved in FA synthesis and embryogenesis were analysed at the transcriptional level. Endogenous \u003cem\u003eAtLEC1\u0026nbsp;\u003c/em\u003ein \u003cem\u003eAhNF-YB10\u0026nbsp;\u003c/em\u003etransgenic Arabidopsis and COL were expressed at higher levels at the early stage of seed development, and their expression decreased to a moderately low level at the mid-late stage (Supplemental Fig. 6a). In contrast, the expression level of exogenous \u003cem\u003eAhNF-YB10\u0026nbsp;\u003c/em\u003ein the OE lines increased with seed development. At 8 and 13 DAF, increased expression of \u003cem\u003eAhNF-YB10\u003c/em\u003e was detected (Supplemental Fig. 6b). Thus, the seeds of Arabidopsis at 13 DAF were subjected to further gene expression analysis. As shown in Fig. 5, the expression level of several genes in the FA de novo synthetic pathway, including \u003cem\u003edihydrolipoamide dehydrogenase 1\u0026nbsp;\u003c/em\u003e(\u003cem\u003eLPD1\u003c/em\u003e), \u003cem\u003epyruvate dehydrogenase E1 \u0026alpha;\u003c/em\u003e (\u003cem\u003ePDH-E1\u0026alpha;\u003c/em\u003e), \u003cem\u003eacetyl-CoA carboxylase2\u0026nbsp;\u003c/em\u003e(\u003cem\u003eACC2\u003c/em\u003e), \u003cem\u003ebiotin carboxyl carrier protein\u003c/em\u003e (\u003cem\u003eBCCP2\u003c/em\u003e) for preparation of acetyl coenzyme A which is the precursor of FA synthesis; \u003cem\u003eacyl carrier protein\u003c/em\u003e (\u003cem\u003eACP1\u003c/em\u003e), \u003cem\u003emalonyl-CoA: ACP transacylase\u003c/em\u003e (\u003cem\u003eMCAT\u003c/em\u003e), \u003cem\u003efatty acyl-ACP thioesterase\u003c/em\u003e A (\u003cem\u003eFat A\u003c/em\u003e), \u003cem\u003eenoyl-ACP reductase\u003c/em\u003e (\u003cem\u003eEAR\u003c/em\u003e), \u003cem\u003efatty-acid chain elongase\u003c/em\u003e (\u003cem\u003eFAE\u003c/em\u003e) for fatty acyl transference, reduction, elongation; \u003cem\u003estearoyl-ACP desaturase\u0026nbsp;\u003c/em\u003e(\u003cem\u003eFAB2\u003c/em\u003e),\u0026nbsp;\u0026Delta;-12 \u003cem\u003efatty acid desaturase2\u003c/em\u003e (\u003cem\u003eFAD2\u003c/em\u003e), \u003cem\u003e\u0026omega;-3\u003c/em\u003e \u003cem\u003efatty acid desaturase3\u003c/em\u003e (\u003cem\u003eFAD3\u003c/em\u003e) for dehydrogenation at specific position of FAs; \u003cem\u003eoleosin1\u003c/em\u003e (\u003cem\u003eOLE1\u003c/em\u003e) for lipid accumulation and the glycolytic genes \u003cem\u003efructose 1,6 bisphosphate aldolase\u003c/em\u003e (\u003cem\u003eFPA1\u003c/em\u003e), \u003cem\u003ephosphofructokinase-1\u003c/em\u003e (\u003cem\u003ePFK1\u003c/em\u003e), \u003cem\u003eadenosine diphosphoglucose pyrophosphprylase\u003c/em\u003e (\u003cem\u003eAGP\u003c/em\u003e) were upregulated to varying degrees in \u003cem\u003eAhNF-YB10\u003c/em\u003e transgenic lines. Owing to the increase in 1000-seed weight in the transgenic plants, the transcript levels of several TF genes regulating seed development, including \u003cem\u003eLEC2\u003c/em\u003e, \u003cem\u003eFUS3\u003c/em\u003e, and \u003cem\u003eWRI1,\u003c/em\u003e were determined. The expression of these genes increased in comparison to that in the untransformed control.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eAhNF-YB1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eAhNF-YB10\u003c/b\u003e \u003cb\u003eare subfunctionalized for embryo development\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe NF-YB TFs are classified into LEC1-type and non-LEC1-type proteins according to the 16 amino acid residues in the conserved region of the B domain (Lee et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). There are relatively few \u003cem\u003eLEC1\u003c/em\u003e-type genes in plants, ranging in number from 1 to 6, while more than 10 non-\u003cem\u003eLEC1\u003c/em\u003e-type genes are generally present (Cagliari et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The functions of \u003cem\u003eLEC1\u003c/em\u003e-type genes were originally revealed in Arabidopsis. The \u003cem\u003elec1\u003c/em\u003e mutant plants exhibited leafy cotyledons; hence, the gene was named thus (Meinke \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; West et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The other \u003cem\u003eLEC1\u003c/em\u003e-type gene in Arabidopsis, designated LEC1-LIKE (L1L), which shares 83% sequence identity with AtLEC1, is also required for normal embryo development (Kwong et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The phylogenetic relationship of \u003cem\u003eLEC1\u003c/em\u003e-type members, including a vast number of taxa, indicated that \u003cem\u003eLEC1\u003c/em\u003e-type genes originated in vascular plants, and only one \u003cem\u003eLEC1\u003c/em\u003e-type gene was found in the ancient plants \u003cem\u003eSelaginella, Picea abies\u003c/em\u003e and \u003cem\u003ePinus sylvestris\u003c/em\u003e, whose protein sequences are more similar to AtL1L than to AtLEC1 (Cagliari et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In our study, there was only one \u003cem\u003eLEC1\u003c/em\u003e-type gene in two diploid ancestral species, \u003cem\u003eArachis duranensis\u003c/em\u003e and \u003cem\u003eArachis ipaensis\u003c/em\u003e, and two in the cultivar \u003cem\u003eArachis hypogeae\u003c/em\u003e. In addition, these four \u003cem\u003eLEC1\u003c/em\u003e-type genes were most closely related to \u003cem\u003eAtL1L\u003c/em\u003e in terms of the sequence of their encoded proteins. It was speculated that these genes, as the sole \u003cem\u003eLEC1\u003c/em\u003e-type genes, can indispensably control embryo development in these two diploid species. However, how do the two \u003cem\u003eLEC1\u003c/em\u003e-type genes cooperate to function in \u003cem\u003eArachis hypogeae\u003c/em\u003e? \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10\u003c/em\u003e exhibited different expression patterns. \u003cem\u003eAhNF-YB1\u003c/em\u003e was expressed in all the organs analysed, and its expression pattern was similar to that of \u003cem\u003eAtL1L\u003c/em\u003e; however, similar to that of \u003cem\u003eAtLEC1\u003c/em\u003e, \u003cem\u003eAhNF-YB10\u003c/em\u003e expression was restricted mainly to the embryo, and \u003cem\u003eAhNF-YB1\u003c/em\u003e was expressed at a greater level than \u003cem\u003eAhNF-YB10\u003c/em\u003e in mid-stage seeds. Our prior studies on the two promoters of \u003cem\u003eAhNF-YBs\u003c/em\u003e revealed that \u003cem\u003eAhLEC1A\u003c/em\u003e and \u003cem\u003eAhLEC1B\u003c/em\u003e correspond to \u003cem\u003eAhNF-YB10\u003c/em\u003e and \u003cem\u003eAhNF-YB1\u003c/em\u003e, respectively, in the present study. Research also demonstrated that they have different activities and spatiotemporal expression characteristics when they drive GUS expression in transgenic Arabidopsis (Tang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In our study, the expression of \u003cem\u003eAhNF-YB10\u003c/em\u003e, which is expressed at a relatively high level in the kernel, almost completely complemented the defects observed in the \u003cem\u003elec1-2\u003c/em\u003e mutant, as indicated by the embryo morphology and seed germination rate. Although more than half of the \u003cem\u003eAhNF-YB1\u003c/em\u003e transgenic plants in the \u003cem\u003elec1-2\u003c/em\u003e background exhibited a restored phenotype compared with the wild-type plants, 30%-40% of the transgenic plants and seedlings partially or completely retained the mutant phenotype. Previous research indicated that \u003cem\u003eAtLEC1\u003c/em\u003e was downregulated in the \u003cem\u003elec1-4\u003c/em\u003e mutant, resulting in a decreased seed germination rate, decreased seed vigour, and a defective seedling phenotype in the dark (Huang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). To some extent, the phenotypic features of ectopic expression of \u003cem\u003eAhNF-YB1\u003c/em\u003e in some transgenic \u003cem\u003elec1-2\u003c/em\u003e mutants were similar to those of the receptor line \u003cem\u003elec1-2\u003c/em\u003e, which could be attributed to its lower expression in embryos. Thus, we suggest that \u003cem\u003eAhNF-YB10\u003c/em\u003e located on the B subgenome may play a primary role in embryogenesis and embryonic development and maturation and that \u003cem\u003eAhNF-YB1\u003c/em\u003e on the A subgenome has partial functional redundancy with \u003cem\u003eAhNF-YB10.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe normal function of\u003c/b\u003e \u003cb\u003eAhNF-YB10\u003c/b\u003e \u003cb\u003edepends on proper spatiotemporal expression\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLEC1 is a central regulator of the whole process of embryo development. In this work, we explored the phenotypes of lines overexpressing \u003cem\u003eAhNF-YB10\u003c/em\u003e in the \u003cem\u003elec1-2\u003c/em\u003e background under the control of two different promoters, the truncated \u003cem\u003eNapA\u003c/em\u003e promoter P211 and the \u003cem\u003eAtLEC1\u003c/em\u003e promoter. The transgenic seeds harbouring pC211P:\u003cem\u003eB10\u003c/em\u003e acquired desiccation tolerance and could germinate after drying. However, the other defects of \u003cem\u003elec1-2\u003c/em\u003e, such as round cotyledons, shorter hypocotyls, and constricted root apices, remained unchanged in the transgenic embryos. When the 1044 bp \u003cem\u003eAtLEC1\u003c/em\u003e promoter replaced the P211 promoter, the pC\u003cem\u003eAtLEC1P\u003c/em\u003e:\u003cem\u003eB10\u003c/em\u003e transgenic plants exhibited almost complete complementation of all the defects in the \u003cem\u003elec1-2\u003c/em\u003e mutant. \u003cem\u003eAtLEC1\u003c/em\u003e is expressed during the whole process of seed development and at higher levels during early embryo development than in late-maturing-stage embryos, and it is indispensable for the specification of cotyledon identity and the completion of embryo maturation (Lotan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Previous studies on the \u003cem\u003eNapA\u003c/em\u003e promoter indicated that its expression was barely detectable at the globule to heart stages, increased significantly at 10 to 15 days after pollination (DAP), and peaked at 30 DAP; moreover, its sequences between positions \u0026minus;\u0026thinsp;152 and +\u0026thinsp;44 are essential for the seed-specific expression pattern (St\u0026aring;lberg et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Ellerstr\u0026ouml;m et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Additionally, the deletion promoter of the \u0026minus;\u0026thinsp;211 nt upstream sequence maintained the original expression pattern in seeds but only held 4% of the activity of the wild-type 1101 bp promoter (Tan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Consequently, the lack of expression of \u003cem\u003eAhNF-YB10\u003c/em\u003e in embryos before heart embryo development in association with pC211P:\u003cem\u003eB10\u003c/em\u003e might cause defects at early stages of development and further during seedling establishment in the dark. The expression pattern of \u003cem\u003eAhNF-YB10\u003c/em\u003e during embryo development is similar to that of Arabidopsis \u003cem\u003eAtLEC1.\u003c/em\u003e Therefore, appropriate spatiotemporal expression of \u003cem\u003eAhNF-YB10\u003c/em\u003e is necessary for embryo morphogenesis at the early development stage and is responsible for seed maturation at the mid-late development stage.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpressing\u003c/b\u003e \u003cb\u003eAhNF-YB1\u003c/b\u003e \u003cb\u003eor\u003c/b\u003e \u003cb\u003eAhNF-B10\u003c/b\u003e \u003cb\u003ecould increase the yields and oil content of seeds\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven the increasing global demand for edible oils, improving oil content and production is a major objective for breeding oilseed crops. Yields and oil content are controlled by multiple biological pathways involving many genes. In view of its indispensable role in seed development, many efforts have been made to utilize the \u003cem\u003eLEC1\u003c/em\u003e gene to improve yields and nutrient composition. However, the ectopic expression of master transcription factors, including \u003cem\u003eLEC1\u003c/em\u003e, controlled by the 35S promoter often leads to adverse effects on growth or development (Shen et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Pouvreau et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Therefore, moderate expression of \u003cem\u003eLEC1\u003c/em\u003e in the tissues of interest is vital for successful molecular breeding.\u003c/p\u003e \u003cp\u003eIn this work, a truncated \u003cem\u003eNap\u003c/em\u003e A promoter was used to drive the expression of \u003cem\u003eAhNF-YB1\u003c/em\u003e or \u003cem\u003eAhNF-YB10\u003c/em\u003e in transgenic wild-type Arabidopsis plants. This promoter could mainly stimulate the transcription of downstream genes during the middle to late stages of seed development without adverse effects on vegetative organ development (Shen et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Pouvreau et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The overexpression of either of these genes in Arabidopsis seeds increased the thousand-grain weight and the seed oil content to different extents. Consistent with the findings of previous studies of \u003cem\u003eLEC1\u003c/em\u003e-type genes in other plants (Tan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Elahi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Deng et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the expression levels of several genes involved in de novo FA synthesis and seed development were greater in the \u003cem\u003eAhNF-YB1\u003c/em\u003e or \u003cem\u003eAhNF-YB10\u003c/em\u003e overexpression line than in the control. Most of the analysed genes related to acetyl-CoA precursors, FA chain elongation, lipid accumulation and embryo development exhibited increased expression in the developing seeds of the transgenic plants. Analysis of the levels of major FAs demonstrated that the proportion of USFAs was elevated, and the expression of some corresponding genes encoding desaturase increased. These results demonstrated that the two \u003cem\u003eLEC1\u003c/em\u003e-type genes in peanut perform conserved functions as key regulators during embryo development and reserve deposition. These studies provide an approach for the genetic improvement of peanut and other oil crops.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we identified and studied the functions of two \u003cem\u003eLEC1\u003c/em\u003e-type genes in \u003cem\u003eArachis hypogeae\u003c/em\u003e. It was found that both \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10\u003c/em\u003e participate in regulating embryogenesis, embryo development, and reserve deposition in cotyledons and that they have partial functional redundancy. On the other hand, overexpression of \u003cem\u003eAhNF-YB1\u003c/em\u003e or \u003cem\u003eAhNF-YB10\u003c/em\u003e at the middle to late stages of Arabidopsis seed development improved the weight, oil content, FA composition of the transgenic seeds. This study provides a theoretical basis for breeding oilseed crops with high yields and high oil contents.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAuthor contribution statement\u003c/h2\u003e \u003cp\u003eGT conducted the experiments and drafted the manuscript, PX and CJ conducted the experiments, GL analyzed the data. LS and SW designed the study and revised the manuscript. All authors read and approved the final version.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by grants from Natural Science Foundation of Shandong Province (ZR2021MC054), National Natural Science Foundation of China (30971546), Shandong Provincial Key Research and Development Program (2023LZGCQY019), Shandong Provincial Key Research and Development Program (2021LZGC025).\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank Jianru Zuo (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for kindly providing us with the plasmid with 211-bp promoter of \u003cem\u003eNapin A\u003c/em\u003e (211P) and the methods of vector construction.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkhtar S，Khalid N，Ahmed I et al. 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Biotechnol Biofuels 11:46. https://doi.org/10.1186/s13068-018-1049-4\u003c/li\u003e\n\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"LEAFY COTYLEDON-1 type gene, Seed development, Fatty acid (FA) synthesis, Oil accumulation, Yields","lastPublishedDoi":"10.21203/rs.3.rs-3913572/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3913572/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLEAFY COTYLEDON1 (LEC1) isa member of the nuclear factor Y (NF-Y) family of transcription factors and has been identified as a key regulator of embryonic development. In the present study, two\u003cem\u003e \u003c/em\u003eLEC1-type genes from \u003cem\u003eArachis hypogeae \u003c/em\u003ewere identified and designated as \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10\u003c/em\u003e; these genes belong to subgenome A and subgenome B, respectively. The functions of \u003cem\u003eAhNF-YB1\u003c/em\u003eand \u003cem\u003eAhNF-YB10\u003c/em\u003e were investigated by complementation analysis of their defective phenotypes of the Arabidopsis \u003cem\u003elec1-2 \u003c/em\u003emutant and by ectopic expression in wild-type Arabidopsis. The results indicated that both \u003cem\u003eAhNF-YB1\u003c/em\u003e and \u003cem\u003eAhNF-YB10 \u003c/em\u003eparticipate in regulating embryogenesis, embryo development, and reserve deposition in cotyledons and that they have partial functional redundancy. In contrast, \u003cem\u003eAhNF-YB10 \u003c/em\u003ecomplemented almost all the defective phenotypes of \u003cem\u003elec1-2 \u003c/em\u003ein terms of embryonic morphology and hypocotyl length, while \u003cem\u003eAhNF-YB1\u003c/em\u003e had only a partial effect. In addition, 30%-40% of the seeds of the \u003cem\u003eAhNF-YB1 \u003c/em\u003etransformants exhibited a decreasing germination ratio and longevity. Therefore, appropriate spatiotemporal expression of these genes is necessary for embryo morphogenesis at the early development stage and is responsible for seed maturation at the mid-late development stage. On the other hand, overexpression of \u003cem\u003eAhNF-YB1\u003c/em\u003eor \u003cem\u003eAhNF-YB10\u003c/em\u003e at the middle to late stages of Arabidopsis seed development improved the weight, oil content, and fatty acid composition of the transgenic seeds. Moreover, the expression levels of several genes associated with fatty acid synthesis and embryogenesis were significantly greater in developing \u003cem\u003eAhNF-YB10\u003c/em\u003e-overexpressing seeds than in control seeds. This study provides a theoretical basis for breeding oilseed crops with high yields and high oil content.\u003c/p\u003e","manuscriptTitle":"Peanut LEAFY COTYLEDON1-type genes participate in regulating the embryo development and the accumulation of storage lipids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-07 09:49:29","doi":"10.21203/rs.3.rs-3913572/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2024-03-14T03:55:07+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-02-07T02:34:03+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-05T14:16:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-05T06:12:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2024-02-02T01:35:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fb7c8e5c-ef12-4f4d-94b4-246b409fe1b9","owner":[],"postedDate":"February 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-01T22:39:10+00:00","versionOfRecord":{"articleIdentity":"rs-3913572","link":"https://doi.org/10.1007/s00299-024-03209-8","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2024-04-20 22:39:10","publishedOnDateReadable":"April 20th, 2024"},"versionCreatedAt":"2024-02-07 09:49:29","video":"","vorDoi":"10.1007/s00299-024-03209-8","vorDoiUrl":"https://doi.org/10.1007/s00299-024-03209-8","workflowStages":[]},"version":"v1","identity":"rs-3913572","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3913572","identity":"rs-3913572","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}