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.

Global Analysis of Gene Expression Profiles in Brassica napus Developing Seeds Reveals a Conserved Lipid Metabolism Regulation with Arabidopsis thaliana

In order to study Brassica napus fatty acid （FA） metabolism and relevant regulatory networks, a systematic identification of fatty acid （FA） biosynthesis-related genes was conducted. Following gene identification, gene expression profiles during B. napus seed development and FA metabolism were performed by cDNA chip hybridization （〉8000 EST clones from seed）. The results showed that FA biosynthesis and regulation, and carbon flux, were conserved between B. napus and Arabidopsis. However, a more critical role of starch metabolism was detected for B. napus seed FA metabolism and storage-component accumulation when compared with Arabidopsis. In addition, a crucial stage for the transition of seed-to-sink tissue was 17-21 d after flowering （DAF）, whereas FA biosynthesis-related genes were highly expressed pri- marily at 21 DAF. Hormone （auxin and jasmonate） signaling is found to be important for FA metabolism. This study helps to reveal the global regulatory network of FA metabolism in developing B. napus seeds.

Phenolic Composition Analysis and Gene Expression in Developing Seeds of Yellow-and Black-seeded Brassica napus

Breeders have focused on yellow-seeded Brassica napus （rapeseed） for its better quality com- pared with the black-seeded variety. Moreover, flavonoids have been associated with this kind of rapeseed. In this study, we applied lipid chromatography-electrospray ionization mass spec- trometry （LC-ESI-MSn ） to compare flavonoids in developing seeds of natural black-seeded B. na- pus and yellow-seeded introgression lines selected from progenies of B. napus–Sinapis alba so- matic hybrids. Aside from the most abundant phenolic compounds （sinapine and sinapic acid） and 1, 2-disinapoylglucose, 16 different flavonoids were identified and quantified, including （-）- epicatechin, five monocharged oligomers of （-）-epicatechin （[DP 2]- , [DP 3]- , [DP 4] [DP 2]- B2 and [DP 2]- B5）, quercetin, kaempferol, isorhamnetin-dihexoside, kaempferol-sinapoyl-trihexoside, isorhamnetin- sinapoyl-trihexoside, isorhamnetin-hexoside-sulfate, and isorhamnetin-3-O-glucoside. Most of the flavonoids accumulated with seed development, whereas some rapidly decreased during maturation. The content of these flavonoids was lower in the yellow-seeded materials than in the black seeds. In addition, variations of insoluble procyanidin oligomers and soluble phenolic acids were observed among both rapeseed varieties. Transcriptome changes of genes participating in the flavonoid pathway were discovered by quantitative reverse transcription polymerase chain reaction analysis. Consistent with flavonoid changes identified by high performance liquid chromatography analysis, the expression of most genes in the flavonoid biosynthetic pathway was also downregulated.

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.

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.

Dynamic changes of nutrient content in herbaceous peony seed

Herbaceous peony is a promising oilseed crop.‘Hangshao’and‘Fenghuang Niepan’are two cultivars with high yield and oil content In this study,seed reserved nutrient of these two genotypes were investigated during seed development process.Results showed that‘Hangshao’and‘Fenghuang Niepan’matured approximately 85 d after flowering(DAF).The development p rocess could be divided into four stages:seed formation and enlargement stage(S1),seed inclusion and enrichment stage(S2),crude fat rapid accumulation stage(S3)and dehydration and color transition stage(S4).During this process,100-seed fresh weight(HFW)and 100-seed dry weight(HDW)increased,whereas water content(WC)decreased.HFW,HDW and WC for‘Hangshao’seed at 85 DAF were28.56 g,20.78 g and 37.50%respectively,whereas those for‘Fenghuang Niepan’seed were 27.2 g,19.78 g,and30.85%respectively.In terms of the primary metabolites measuring,soluble protein content was consistently low(only 2.24%-3.15%),while starch content was relatively high(5.60%-24.81%).They displayed a trend of gradual increasing in early stage,followed by rapid decline.Crude fat tended to accumulate continuously with a linear upward trend starting at 40 DAF,and its maximum level was reached at 85 DAF(27.57%for‘Hangshao’and29.57%for‘Fenghuang Niepan’).For the secondary metabolites determination,total phenols content was relatively steady,maintaining at 107.7-129.9 mg/g for both cultivars,while total flavonoids content varied within25.33-65.33 mg/g for‘Hangshao’and 24.33-52.37 mg/g for Tenghuang Niepan’.The maximum level for both cultivars occurred at 55 DAF.Furthermore,crude fat content was found to be highly correlated with WC by correlation analysis and linear regression model definition(P≤0.01 and r=0.954).Thus,WC was recommended for evaluating crude fat in peony seed.These findings could provide scientific reference for high-yielding production practice for oleaginous peony plant.

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.

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.

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.

Several Strategic Issues in the Development of China's Seed Industry

Chinese agricultural industry and rural economy are currently undergoing a strategic restructuring. The Seed Act has been thoroughly enforced. The Development of West China program has also got started in an all-round way. Not long ago China entered the World Trade Organization. All these not only present great opportunities but also severe challenges to China.

Planting the Seeds of Development

ON a farm in Marirangwe in suburban Harare, an octogenarian dairy farmer watched Chinese agricultural expert Liu Xiaohui spellbound as he operated a mobile milking machine.

Cone and Ovule Development in Platycladus orientalis (Cupressaceae)

Seed cone in Platycladus orientalis (L.) France consists of four or five pairs of decussate bracts. Usually, two pairs of the fertile bracts in the middle of the cone subtend six ovules, which initiate in an acropetal manner. Only one ovule presents on each of the upper fertile bract, while two ovules initiate from a common primordium in the axil of lower bracts. In Beijing, most female cones initiated in July. All parts of the cone formed before dormancy, which occurred during November to the next January. After pollination in March, bract morphology changed dramatically; intercalary growth of the bract base formed a conspicuous protuberance, in which inverted vascular system developed. Furthermore, ovules on different pairs of bracts initiated in an acropetal manner and two ovules in each lower fertile bract initiated from a common primordium, which was different from the basipetal initiation of ovules and independently formed single ovule as reported by Takaso in Calltris

Synthesis and Degradation of the Major Allergens in Developing and Germinating Soybean Seed

Gly m Bd 28K, Gly m Bd 30K and Gly m Bd 60K are the major soybean （Glycine max （L.） Merr.） allergens limiting the consumption of a good protein source for sensitive individuals. However, little is known about their temporal-spatial expression during seed development and upon germination. The present data shows that soy allergens accumulated in both the embryonic axes and cotyledon, but expression patterns differed depending on the specific allergen. Allergens accumulated sooner and to a greater level in cotyledons than in embryonic axes. Gly m Bd 28 began at 14 d after flowering, 7 to 14 d earlier than Gly m Bd 30K and Gly m Bd 60K. Comparatively, their degradation was faster and more profound in embryonic axes than in cotyledons. Gly m Bd 60K began to decline at 36 h after imbibition and remained detectable up to 108 h in cotyledons. In contrast, the Glym Bd 60K protein was reduced at 24 h, and eventually disappeared at 96 h. In cotyledons Gly m Bd 28K first declined at 24 h, then increased from 36 h to 48 h, followed by its large reduction at 72 h after seed germination. These findings provide useful information on soy allergen biosynthesis and will help move forward towards developing a hypoallergenic soybean for safer food.

High-throughput sequencing-based genome-wide identification of micro RNAs expressed in developing cotton seeds

Micro RNAs(mi RNAs) have been shown to play critical regulatory roles in gene expression in cotton. Although a large number of mi RNAs have been identified in cotton fibers, the functions of mi RNAs in seed development remain unexplored. In this study, a small RNA library was constructed from cotton seeds sampled at 15 days post-anthesis(DPA) and was subjected to high-throughput sequencing. A total of 95 known mi RNAs were detected to be expressed in cotton seeds. The expression pattern of these identified mi RNAs was profiled and 48 known mi RNAs were differentially expressed between cotton seeds and fibers at 15 DPA. In addition, 23 novel mi RNA candidates were identified in 15-DPA seeds. Putative targets for 21 novel and 87 known mi RNAs were successfully predicted and 900 expressed sequence tag(EST) sequences were proposed to be candidate target genes, which are involved in various metabolic and biological processes, suggesting a complex regulatory network in developing cotton seeds. Furthermore, mi RNA-mediated cleavage of three important transcripts in vivo was validated by RLM-5′ RACE. This study is the first to show the regulatory network of mi RNAs that are involved in developing cotton seeds and provides a foundation for future studies on the specific functions of these mi RNAs in seed development.

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.

Studies on the Programmed Cell Death in Rice During Starchy Endosperm Development

Morphological variations of the nucleus in starchy endosperm cell were observed by theelectron-transmisson microscope during endosperm development in rice. Along with thedevelopment of the starchy endosperm, the nuclei of the cells showed chromatin condensation,the typical feature of programmed cell death (PCD). The nuclei also showed nucleusdeformation, disruption of nuclear envelope, nucleoplasm leaking into the cytoplasm andnucleus disintegration resulting in nuclear residue formation. From the nucleus deformationto the nucleus disintegration, the morphological changes of the nucleus were orderlyprogressive. This indicated that the cell death of starchy endosperm in rice wasprogrammed cell death. Evans Blue staining observation showed that the cell death wasinitially detected in the central part of starchy endosperm in rice, then expandedoutward. The activities of superoxide dismutase (SOD) and catalase (CAT) in rice starchyendosperm both descended continuously as development progressed. The analysis of DNA ofrice starchy endosperm did not show the presence of DNA laddering. The above resultsshowed that the cell death of starchy endosperm in rice was a special form of PCD.

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.