Genome-wide analysis of nuclear factor Y genes and functional investigation of watermelon ClNF-YB9 during seed development

The nuclear factor Y(NF-Y) gene family is a class of transcription factors that are widely distributed in eukaryotes and are involved in various biological processes. However, the NF-Y gene family members in watermelon, a valued and nutritious fruit, remain largely unknown and their functions have not been characterized. In the present study, 22 ClNF-Y genes in watermelon, 29 CsNF-Y genes in cucumber, and 24CmNF-Y genes in melon were identified based on the whole-genome investigation and their protein properties, gene location, gene structure, motif composition, conserved domain, and evolutionary relationship were investigated. ClNF-YB9 from watermelon and its homologs in cucumber and melon were expressed specifically in seeds. Its expression remained low in the early stages of watermelon seed development,increased at 20 days after pollination(DAP), and peaked at 45–50 DAP. Moreover, the knockout mutant Clnf-yb9 exhibited abnormal leafy cotyledon phenotype, implying its critical role during seed formation.Finally, protein interaction assays showed that ClNF-YB9 interacts with all ClNF-YCs and the ClNF-YB9-YC4 heterodimer was able to recruit a ClNF-YA7 subunit to assemble a complete NF-Y complex, which may function in seed development. This study revealed the structure and evolutionary relationships of the NF-Y gene family in Cucurbitaceae and the novel function of ClNF-YB9 in regulating seed development in watermelon.

The pentatricopeptide repeat protein EMP601 functions in maize seed development by affecting RNA editing of mitochondrial transcript ccmC

Although several pentatricopeptide repeat(PPR) proteins are involved in post-transcriptional processing of mitochondrial RNA, it is unclear which specific protein is involved in the RNA editing of ccmC in maize(Zea mays). Here we report the identification of the maize empty pericarp 601(emp601) mutant and the map-based cloning of the Emp601 gene, which encodes an E2-type PPR protein that is targeted to mitochondria. A single-nucleotide deletion in the emp601 mutant caused a frameshift and introduced a premature stop codon into the predicted EMP601. This mutation was associated with reduced accumulation of mitochondrial complex Ⅲ as well as with inhibition of growth and differentiation of basal endosperm transfer layer cells, leading to final degeneration of the embryo and endosperm. We determine that loss of EMP601 function prevents the C-to-U RNA editing of the mitochondrial transcript ccmC at position 358.EMP601 binds to the ccmC transcript and directly interacts with Multiple organellar RNA editing factor 8and may be a component of the plant mitochondrial editosome. We conclude that EMP601 functions in RNA editing of mitochondrial ccmC transcripts and influences mitochondrial function and seed development.

A Pleiotropic Drug Resistance Family Protein Gene Is Required for Rice Growth, Seed Development and Zinc Homeostasis

Zinc(Zn) is an essential mineral element for plant growth and development. Zn deficiency in crops frequently occurs in many types of soils. It is therefore crucial to identify genetic resources linking Zn acquisition traits and development of crops with improved Zn-use efficiency for sustainable crop production. In this study, we functionally identified a rice uncharacterized ABCG(ATP-binding cassette G-subfamily) gene encoding a PDR20(pleiotropic drug resistance 20) metal transporter for mediation of rice growth, seed development and Zn accumulation. OsPDR20 was localized to the plasma membrane, but it was not transcriptionally induced under Zn deficiency, rather was sufficiently up-regulated under high level of Zn stress. Yeast(Saccharomyces cerevisiae) transformed with OsPDR20 displayed a relatively lower Zn accumulation with attenuated cellular growth, suggesting that OsPDR20 had an activity for Zn transport. Knocking-down OsPDR20 by RNA interference(RNAi) compromised rice growth with shorter plant height and decreased biomass in rice plantlets grown under hydroponic media. Zn concentration in the roots of OsPDR20 knocked-down rice lines declined under Zn deficiency, while they remained unchanged compared with the wild type under normal Zn supply. A rice lifelong field trial demonstrated that OsPDR20 mutation impaired the capacity of seed development, with shortened panicle and seed length, compromised spikelet fertility, and reduced grain number per plant or grain weight per unit area. Interestingly, OsPDR20 mutation elevated the accumulation of Zn in husk and brown rice over the wild type. Overall, this study pointed out that OsPDR20 is fundamentally required for rice growth and seed development through Zn transport and homeostasis.

Roles of ABA Signal Transduction during Higher Plant Seed Development and Germination

ABA is one of the 5 phytohormones in higher plants, which is also the most important hormone that regulates higher plants in response to environmental stress, by ABA signal transduction. Understanding ABA signal transduction at the molecular level is crucial to biology and ecology, and rational breeding complied with corresponding eco-environmental changes. Great advancements have taken place over the past 10 years by application of the Arabidopsis experimental system. Many components involved in ABA signal transduction have been isolated and identified and a clear overall picture of gene expression and control for this transduction has become an accepted fact. On the basis of the work in our laboratory, in conjunction with the data available at the moment, the authors have attempted to integrate ABA signal transduction pathways into a common one and give some insights into the relationship between ABA signal transduction and other hormone signal transduction pathways, with an emphasis upon the ABA signal transduction during higher plant seed development. A future challenge in this field is that different experimental systems are applied and various receptors and genes need to be characterized through the utilization of microarray chips.

Differential Gene Expression Between Cross-Fertilized and Self-Fertilized Kernels During the Early Stages of Seed Development in Wheat

In order to understand molecular basis of cross-fertilized kernel advantage and heterosis, improved differential display of mRNA was used in this study to analyze alterations in gene expression between cross-fertilized and self-fertilized kernels at 2, 4, 6, 8, 10 and 12 days after pollination (DAP) by using 3 wheat hybrids with different level of heterosis. Four patterns of differential expression were observed: (i) bands observed in cross-fertilized kernels but not in self-fertilized kernels (BCnS); (ii) bands occurring in only self-fertilized kernels but not in cross-fertilized kernels (BSnC); (iii) cDNA over-expressed in cross-fertilized kernels compared to self-fertilized kernels (OEC); (iv) cDNA under-expressed in cross-fertilized kernels compared to self-fertilized kernels (UEC). Further analysis showed that BCnS is positively correlated with heterosis, but BSnC is negatively correlated with heterosis. Four differentially expressed cDNA fragments were verified by reverse-northern blot and sequence homology search in GenBank showed that one of them was new sequences; the other exhibited higher similarity to NBS-LRR type resistance protein, 1,6-bisphosphatase and photosystem Ⅱ chlorophyll a-binding protein psbB, respectively, which indicated diverse pathways may be involved in heterosis formation.

Genome-wide analysis of the B3 transcription factors reveals that RcABI3/VP1 subfamily plays important roles in seed development and oil storage in castor bean(Ricinus communis)

The B3 transcription factors(TFs)in plants play vital roles in numerous biological processes.Although B3 genes have been broadly identified in many plants,little is known about their potential functions in mediating seed development and material accumulation.Castor bean(Ricinus communis)is a non-edible oilseed crop considered an ideal model system for seed biology research.Here,we identified a total of 61 B3 genes in the castor bean genome,which can be classified into five subfamilies,including ABI3/VP1,HSI,ARF,RAV and REM.The expression profiles revealed that RcABI3/VP1 subfamily genes are significantly up-regulated in the middle and later stages of seed development,indicating that these genes may be associated with the accumulation of storage oils.Furthermore,through yeast one-hybrid and tobacco transient expression assays,we detected that ABI3/VP1 subfamily member RcLEC2 directly regulates the transcription of RcOleosin2,which encodes an oil-body structural protein.This finding suggests that RcLEC2,as a seed-specific TF,may be involved in the regulation of storage materials accumulation.This study provides novel insights into the potential roles and molecular basis of B3 family proteins in seed development and material accumulation.

Integration of embryo–endosperm interaction into a holistic and dynamic picture of seed development using a rice mutant with notched-belly kernels

Interaction between the embryo and endosperm affects seed development,an essential process in yield formation in crops such as rice.Signals that mediate communication between embryo and endosperm are largely unknown.We used the notched-belly(NB)mutant with impaired communication between embryo and endosperm to investigate the effect of the embryo on developmental staging of the endosperm and signaling pathways in the embryo that regulate endosperm development.Hierachical clustering of m RNA datasets from embryo and endosperm samples collected during development in NB and a wild type showed a delaying effect of the embryo on the developmental transition of the endosperm by extension of the middle stage.K-means clustering further identified coexpression modules of gene sets specific to embryo and endosperm development.Combined gene expression and biochemical analysis showed that T6 P–Sn RK1,gibberellin and auxin signaling by the embryo regulate endosperm developmental transition.We propose a new seed developmental staging system for rice and identify the most detailed signature of rice grain formation to date.These will direct genetic strategies for rice yield improvement.

UDP-glucosyltransferase 71C4 controls the flux of phenylpropanoid metabolism to shape cotton seed development

Seeds play a crucial role in plant reproduction,making it essential to identify genes that affect seed development.In this study,we focused on UDP-glucosyltransferase 71C4(UGT71C4)in cotton,a member of the glycosyltransferase family that shapes seed width and length,thereby influencing seed index and seed cotton yield.Overexpression of UGT71C4 results in seed enlargement owing to its glycosyltransferase activity on flavonoids,which redirects metabolic flux from lignin to flavonoid metabolism.This shift promotes cell proliferation in the ovule via accumulation of flavonoid glycosides,significantly enhancing seed cotton yield and increasing the seed index from 10.66 g to 11.91 g.By contrast,knockout of UGT71C4 leads to smaller seeds through activation of the lignin metabolism pathway and redirection of metabolic flux back to lignin synthesis.This redirection leads to increased ectopic lignin deposition in the ovule,inhibiting ovule growth and development,and alters yield components,increasing the lint percentage from 41.42%to 43.40%and reducing the seed index from 10.66 g to 8.60 g.Our research sheds new light on seed size development and reveals potential pathways for enhancing seed yield.

The DEAD-box RNA helicase ZmRH48 is required for the splicing of multiple mitochondrial introns,mitochondrial complex biosynthesis,and seed development in maize

RNA helicases participate in nearly all aspects of RNA metabolism by rearranging RNAs or RNA–protein complexes in an adenosine triphosphatedependent manner.Due to the large RNA helicase families in plants,the precise roles of many RNA helicases in plant physiology and development remain to be clarified.Here,we show that mutations in maize(Zea mays)DEAD-box RNA helicase48(Zm RH48)impair the splicing of mitochondrial introns,mitochondrial complex biosynthesis,and seed development.Loss of Zm RH48 function severely arrested embryogenesis and endosperm development,leading to defective kernel formation.Zm RH48 is targeted to mitochondria,where its deficiency dramatically reduced the splicing efficiency of five cis-introns(nad5 intron 1;nad7 introns 1,2,and 3;and ccm Fc intron 1)and one trans-intron(nad2 intron 2),leading to lower levels of mitochondrial complexes I andⅢ.Zm RH48 interacts with two unique pentatricopeptide repeat(PPR)proteins,PPR-SMR1 and SPR2,which are required for the splicing of over half of all mitochondrial introns.PPR-SMR1 interacts with SPR2,and both proteins interact with P-type PPR proteins and Zm-m CSF1 to facilitate intron splicing.These results suggest that Zm RH48 is likely a component of a splicing complex and is critical for mitochondrial complex biosynthesis and seed development.

Arabidopsis Transcription Factor Genes NF-YA 1, 5, 6, and 9 Play Redundant Roles in Male Gametogenesis, Embryogenesis, and Seed Development

Nuclear factor Y （NF-Y） is a highly conserved transcription factor presented in all eukaryotic organisms, and is a heterotrimer consisting of three subunits： NF-YA, NF-YB, and NF-YC. In Arabidopsis, these three subunits are encoded by multigene families. The best-studied member of the NF-Y transcription factors is LEAFY COTYLEDON1 （LEC1）, a NF-YB family member, which plays a critical role in embryogenesis and seed maturation. However, the function of most NF-Y genes remains elusive. Here, we report the characterization of four genes in the NF-YA family. We found that a gain- of-function mutant of NF-YA1 showed defects in male gametogenesis and embryogenesis. Consistently, overexpression of NF-YA1, 5, 6, and 9 affects male gametogenesis, embryogenesis, seed morphology, and seed germination, with a stronger phenotype when overexpressing NF-YA1 and NF-YA9. Moreover, overexpression of these NF-YA genes also causes hypersensitivity to abscisic acid （ABA） during seed germination, retarded seedling growth, and late flowering at different degrees. Intriguingly, overexpression of NF-YA1, 5, 6, and 9 is sufficient to induce the formation of somatic embryos from the vegetative tissues. However, single or double mutants of these NF-YA genes do not have detectable phenotype. Collectively, these results provide evidence that NF-YA1, 5, 6, and 9 play redundant roles in male gameto- phyte development, embryogenesis, seed development, and post-germinative growth.

Expression Patterns of ABA and GA Metabolism Genes and Hormone Levels during Rice Seed Development and Imbibition: A Comparison of Dormant and Non-Dormant Rice Cultivars

Seed dormancy is an important agronomic trait in cereals. Using deep dormant （N22）, medium dormant （ZH11）, and non-dormant （G46B） rice cultivars, we correlated seed dormancy phenotypes with abscisic acid （ABA） and gibberellin （GA） metabolism gene expression profiles and phytohormone levels during seed development and imbibition. A time course analysis of ABA and GA content during seed development showed that N22 had a high ABA level at early and middle seed developmental stages, while at late developmental stage it declined to the level of ZHll; however, its ABA/GA ratio maintained at a high level throughout seed development. By contrast, G46B had the lowest ABA content during seed development though at early developmental stage its ABA level was close to that of ZH11, and its ABA/GA ratio peaked at late developmental stage that was at the same level of ZHll. Compared with N22 and G46B, ZH11 had an even and medium ABA level during seed development and its ABA/GA ratio peaked at the middle developmental stage. Moreover, the seed development time-point having high ABA/GA ratio also had relatively high transcript levels for key genes in ABA and GA metabolism pathways across three cultivars. These indicated that the embryo-imposed dormancy has been induced before the late developmental stage and is determined by ABA/GA ratio. A similar analysis during seed imbibition showed that ABA was synthesized in different degrees for the three cultivars. In addition, water uptake assay for intact mature seeds suggested that water could permeate through husk barrier into seed embryo for all three cultivars; however, all three cultivars showed distinct colors by vanillin-staining indicative of the existence of flavans in their husks, which are dormancy inhibition compounds responsible for the husk-imposed dormancy.

Boosting Seed Development as a New Strategy to Increase Cotton Fiber Yield and Quality

Cotton （Gossypium spp.） is the most important textile crop worldwide due to its cellulosic mature fibers, which are single-celled hairs initiated from the cotton ovule epidermis at anthesis. Research to improve cotton fiber yield and quality in recent years has been largely focused on identifying genes regulating fiber cell initiation, elonga- tion and cellulose synthesis. However, manipulating some of those candidate genes has yielded no effect or only a marginally positive effect on fiber yield or quality. On the other hand, evolutionary comparison and transgenic studies have clearly shown that cotton fiber growth is intimately controlled by seed development. Therefore,I propose that enhancing seed development could be a more effective and achievable strategy to increase fiber yield and quality.

Sugar-Hormone Cross-Talk in Seed Development： Two Redundant Pathways of IAA Biosynthesis Are Regulated Differentially in the Invertase- Deficient miniature 1 （mn 1） Seed Mutant in Maize

The miniature1 （mn1） seed phenotype is a loss-of-function mutation at the Mnl locus that encodes a cell wall invertase; its deficiency leads to pleiotropic changes including altered sugar levels and decreased levels of IAA throughout seed development. To understand the molecular details of such a sugar-hormone relationship, we have initiated studies on IAA biosynthesis genes in developing seeds of maize. Two tryptophan-dependent pathways of IAA biosynthesis, tryptamine （TAM） and indole-3-pyruvic acid （IPA）, are of particular interest. We report on molecular isolation and characterization of an endosperm-specific ZmTARelatedl （ZmTarl） gene of the IPA branch; we have also reported recently on ZmYucl gene in the TAM branch. Comparative gene expression analyses here have shown that （1） the ZmTarl transcripts were approximately 10-fold higher levels than the ZmYucl; （2） although both genes showed the highest level of expression at 8-12 d after pollination （DAP） coincident with an early peak in IAA levels, the two showed highly divergent （antagonistic） response at 12 and 16 DAP but similar patterns at 20 and 28 DAP in the Mnl and ran1 endosperm. The Western blot analyses for the ZmTAR1 protein, however, displayed disconcordant protein/transcript expression patterns. Overall, these data report novel observations on redundant trp-dependent pathways of auxin biosynthesis in developing seeds of maize, and suggest that homeostatic control of IAA in this important sink is highly complex and may be regulated by both sucrose metabolism and developmental signals.

Defective Kernel 39 encodes a PPR protein required for seed development in maize

RNA editing is a posttranscriptional process that is important in mitochondria and plastids of higher plants. All RNA editing-specific trans-factors reported so far belong to PLS-class of pentatricopeptide repeat(PPR)proteins. Here, we report the map-based cloning and molecular characterization of a defective kernel mutant dek39 in maize. Loss of Dek39 function leads to delayed embryogenesis and endosperm development, reduced kernel size, and seedling lethality. Dek39 encodes an E subclass PPR protein that targets to both mitochondria and chloroplasts, and is involved in RNA editing in mitochondrial NADH dehydrogenase3(nad3) at nad3-247 and nad3-275. C-to-U editing of nad3-275 is not conserved and even lost in Arabidopsis, consistent with the idea that no close DEK39 homologs are present in Arabidopsis. However, the amino acids generated by editing nad3-247 and nad3-275 are highly conserved in many other plant species, and the reductions of editing at these two sites decrease the activity of mitochondria NADH dehydrogenase complex I,indicating that the alteration of amino acid sequence is necessary for Nad3 function. Our results indicate that Dek39 encodes an E sub-class PPR protein that is involved in RNA editing of multiple sites and is necessary for seed development of maize.

Arabidopsis RAN 1 Mediates Seed Development through Its Parental .Ratio by Affecting the Onset of Endosperm Cellularization

Although previous studies have demonstrated that endosperm development is influenced by its parental genome constitution, the genetic basis and molecular mechanisms that control parent-of-origin effects require further elucidation. Here we show that the Ras-related nuclear protein 1 （RAN1） regulates endosperm development in Arabidopsis thaliana. Reciprocal crosses between wild-type （WT） and transgenic lines misexpressing RAN1 （msRAN1） gave rise to small F1 seeds when RAN1 down-regulated/up-regulated individuals were used as a male/female parent; in contrast, F1 seeds were aborted when RAN1 down-regulated/up-regulated plants were used as a female/male parent, suggesting that seed development is affected by the parental genome ratio of RAN1. Whereas RAN1 expression in wild-type plants is reduced before the onset of endosperm cellularization, F1 seeds from reciprocal crosses between WT and msRAN1 showed abnormal endosperm cellularization and ectopic expression of RAN1. The expression of MINISEED3 （MINI3）-a gene that also controls endosperm cellularization-was also affected in these reciprocal crosses, and the misregulation of MINI3 activity rescued F1 seeds when msRAN1 plants were used in reciprocal crosses. Taken together, our results suggest that the parental ratio of RAN1 regulates the onset of endosperm cellularization through its genetic interaction with MINI3.

Arabinan Metabolism during Seed Development and Germination in Arabidopsis

Arabinans are found in the pectic network of many cell walls, where, along with galactan, they are present as side chains of Rhamnogalacturonan I. Whilst arabinans have been reported to be abundant polymers in the cell walls of seeds from a range of plant species, their proposed role as a storage reserve has not been thoroughly investigated. In the cell walls of Arabidopsis seeds, arabinose accounts for approximately 40% of the monosaccharide composition of non- cellulosic polysaccharides of embryos. Arabinose levels decline to -15% during seedling establishment, indicating that cell wall arabinans may be mobilized during germination. Immunolocalization of arabinan in embryos, seeds, and seedlings reveals that arabinans accumulate in developing and mature embryos, but disappear during germination and seedling establishment. Experiments using 14C-arabinose show that it is readily incorporated and metabolized in growing seedlings, indicating an active catabolic pathway for this sugar. We found that depleting arabinans in seeds using a fungal arabinanase causes delayed seedling growth, lending support to the hypothesis that these polymers may help fuel early seedling growth.

An MCIA-like complex is required for mitochondrial complex Ⅰ assembly and seed development in maize

During adaptive radiation,mitochondria have co-evolved with their hosts,leading to gain or loss of subunits and assembly factors of respiratory complexes.Plant mitochondrial complex Ⅰ harbors40 nuclearand 9 mitochondrial-encoded subunits,and is formed by stepwise assembly during which different intermediates are integrated via various assembly factors.In mammals,the mitochondrial complex Ⅰ intermediate assembly(MCIA)complex is required for building the membrane arm module.However,plants have lost almost all of the MCIA complex components,giving rise to the hypothesis that plants follow an ancestral pathway to assemble the membrane arm subunits.Here,we characterize a maize crumpled seed mutant,crk1,and reveal by map-based cloning that CRK1 encodes an ortholog of human complex Ⅰ assembly factor 1,zNDUFAF1,the only evolutionarily conserved MCIA subunit in plants.zNDUFAF1 is localized in the mitochondria and accumulates in two intermediate complexes that contain complex Ⅰ membrane arm subunits.Disruption of zNDUFAF1 results in severe defects in complex Ⅰ assembly and activity,a cellular bioenergetic shift to aerobic glycolysis,and mitochondrial vacuolation.Moreover,we found that zNDUFAF1,the putative mitochondrial import inner membrane translocase ZmTIM17-1,and the isovaleryl-coenzyme A dehydrogenase ZmIVD1 interact each other,and could be co-precipitated from the mitochondria and co-migrate in the same assembly intermediates.Knockout of either ZmTIM17-1 or ZmIVD1 could lead to the significantly reduced complex Ⅰ stability and activity as well as defective seeds.These results suggest that zNDUFAF1,ZmTIM17-1 and ZmIVD1 probably form an MCIA-like complex that is essential for the biogenesis of mitochondrial complex Ⅰ and seed development in maize.Our findings also imply that plants and mammals recruit MCIA subunits independently for mitochondrial complex Ⅰ assembly,highlighting the importance of parallel evolution in mitochondria adaptation to their hosts.

Ectopic expression of OsNF-YA8,an endosperm-specific nuclear factor Y transcription-factor gene,causes vegetative and reproductive development defects in rice

Nuclear factor Y(NF-Y),a group of conserved transcription-factor complexes that consist of NF-YA,B,and C subunits,is essential for developmental regulation and for responses to environmental changes in eukaryotes.We previously found that some NF-Y genes,such as OsNF-YA8,were expressed specifically in the endosperm of rice.In the present study,overexpression of OsNF-YA8 in rice resulted in reduced plant height due to suppressed cell elongation in internodes.Gibberellin(GA)biosynthetic genes,including OsCPS1,OsGA20ox1,and OsGA20ox2,were down-regulated.OsNF-YA8 bound to the promoters of these genes to repress their expression.Endogenous GA content was decreased in OsNF-YA8 overexpressors,whose dwarf phenotype could be partially rescued by exogenous GA treatment.The findings suggested that ectopic expression of OsNF-YA8 causes defective GA biosynthesis in vegetative stage.Heading date in OsNF-YA8 overexpressors was delayed,especially under short-day conditions.OsNFYA8 bound to the promoter of Heading Date 3a(Hd3a),the florigen gene in rice,to negatively regulate flowering.Either ectopic activation or knockout of OsNF-YA8 impaired seed development,as indicated by reduced seed size and increased grain chalkiness.These results suggest that ectopic expression of the endosperm-specific OsNF-YA8 in rice disrupts both vegetative and reproductive development.

Post-Fertilization Development of Seed in French Bean Genotypes： Changes in Seed Quality

An experiment was conducted to evaluate physiological maturity of French bean genotypes at District Seed Farm ＂D＂ Block, Bidhan Chandra Krishi Viswavidyalaya, Kalyani, Nadia, West Bengal during winter season of both 2012-2013 and 2013-2014. All the five genotypes were grown with sufficient population in three replications following randomized block design （RBD） for the purpose of developmental studies. Developing pods were harvested at different stages, starting from 7 days after anthesis （7 DAA） to 49 DAA with an interval of 7 d, including the field maturity stage, thus leading to study on maximum seven different stages. Harvested pods and seeds were utilized for this development programme through dry seed weight, germination and vigour index. Enhancement in dry matter accumulation in seed continued till 42 DAA for Selection 9 and Victoria in both the years; it was 49 and 42 DAA for Sonali in respective years; it was 49 DAA for Abhay in both the years; and for Deepali, it was 42 DAA along with statistically similar magnitude at 49 DAA. Significant enhancement in germination potential of developing seeds continued till 42 DAA for Selection 9 and Deepali, while it was 49 DAA for the other three genotypes. Enhancement in vigour index continued till 49 DAA for all the genotypes except for Selection 9, for which it continued till 42 DAA and then declined.

The molecular mechanism of WRINKLED1 transcription factor regulating oil accumulation in developing seeds of castor bean

The transcription factor WRINKLED1(WRI1),a member of AP2 gene family that contain typical AP2 domains,has been considered as a master regulator regulating oil biosynthesis in oilseeds.However,the regulatory mechanism of RcWRI1 in regulating oil accumulation during seed development has not been clearly addressed.Castor bean(Ricinus communis)is one of the most important non-edible oil crops and its seed oils are rich in hydroxy fatty acids,widely applied in industry.In this study,based on castor bean reference genome,three RcWRIs genes(RcWRI1,RcWRI2 and RcWRI3)were identified and the expressed association of RcWRI1 with oil accumulation were determined.Heterologous transformation of RcWRI1 significantly increased oil content in tobacco leaf,confirming that RcWRI1 activate lipid biosynthesis pathway.Using DNA Affinity Purification sequencing(DAP-seq)technology,we confirmed RcWRI1 binding with Transcription Start Site of genes and identified 7961 WRI1-binding candidate genes.Functionally,these identified genes were mainly involved in diverse metabolism pathways(including lipid biosynthesis).Three cis-elements AW-box([CnTnG](n)7[CG])and AW-boxes like([GnAnC](n)6[GC]/[GnAnC](n)7[G])bound with RcWRI1 were identified.Co-expression network analysis of RcWRI1 further found that RcWRI1 might be widely involved in biosynthesis of storage materials during seed development.In particular,yeast one hybrid experiments found that both AP2 domains within RcWRI1 were required in binding targeted genes.These results not only provide new evidence to understand the regulatory mechanism of RcWRI1 in regulation of oil accumulation during castor bean seed development,but also give candidate gene resource for subsequent genetic improvement toward increasing oil content in oilseed crops.