Identification of seed development regulatory genes is the key for the genetic improvement in rice grain quality. NF-Ys are the important transcription factors, but their roles in rice grain quality control and the underlying molecular mechanism remain largely unknown. Here, we report the functional characterization a rice NF-Y heterotrimer complex NF-YB1-YC12-bHLH144, which is formed by the binding of NF-YB1 to NF-YC12 and then bHLH144 in a sequential order. Knock-out of each of the complex genes resulted in alteration of grain qualities in all the mutants as well as reduced grain size in crnf-yb1 and crnf-yc12. RNA-seq analysis identified 1496 genes that were commonly regulated by NF-YB1 and NF-YC12, including the key granule-bound starch synthase gene Wx. NF-YC12 and bHLH144 maintain NF-YB1 stability from the degradation mediated by ubiquitin/26S proteasome, while NF-YB1 directly binds to the 'G-box' domain of Wx promoter and activates Wx transcription, hence to regulate rice grain quality. Finally, we revealed a novel grain quality regulatory pathway controlled by NF-YB1-YC12-bHLH144 complex, which has great potential for rice genetic improvement.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.13048

NF-YB1-YC12-bHLH144 complex directly activates Wx to regulate grain quality in rice (Oryza sativa L.).

Starch is a vital energy source for living organisms and is a key raw material and additive in the food and non-food industries. Starch has received continuous attention in multiple research fields. The endosperm of cereals (e.g., rice, corn, wheat, and barley) is the most important site for the synthesis of storage starch. Around 2010, several excellent reviews summarized key progress in various fields of starch research, serving as important references for subsequent research. In the past 10 years, many achievements have been made in the study of starch synthesis and regulation in cereals. The present review provides an update on research progress in starch synthesis of cereal endosperms over the past decade, focusing on new enzymes and non-enzymatic proteins involved in starch synthesis, regulatory networks of starch synthesis, and the use of elite alleles of starch synthesis-related genes in cereal breeding programs. We also provide perspectives on future research directions that will further our understanding of cereal starch biosynthesis and regulation to support the rational design of ideal quality grain.

Starch biosynthesis in cereal endosperms: An updated review over the last decade.

The LAFL (i.e. LEC1, ABI3, FUS3, and LEC2) master transcriptional regulators interact to form different complexes that induce embryo development and maturation, and inhibit seed germination and vegetative growth in Arabidopsis. Orthologous genes involved in similar regulatory processes have been described in various angiosperms including important crop species. Consistent with a prominent role of the LAFL regulators in triggering and maintaining embryonic cell fate, their expression appears finely tuned in different tissues during seed development and tightly repressed in vegetative tissues by a surprisingly high number of genetic and epigenetic factors. Partial functional redundancies and intricate feedback regulations of the LAFL have hampered the elucidation of the underpinning molecular mechanisms. Nevertheless, genetic, genomic, cellular, molecular, and biochemical analyses implemented during the last years have greatly improved our knowledge of the LALF network. Here we summarize and discuss recent progress, together with current issues required to gain a comprehensive insight into the network, including the emerging function of LEC1 and possibly LEC2 as pioneer transcription factors.

/pdf/molecular-and-epigenetic-regulations-and-functions-of-the-7f9c4wvd06.pdf

Molecular and epigenetic regulations and functions of the LAFL transcriptional regulators that control seed development.

Seed development is a complex period of the flowering plant life cycle. After fertilization, the three main regions of the seed, embryo, endosperm and seed coat, undergo a series of developmental processes that result in the production of a mature seed that is developmentally arrested, desiccated, and metabolically quiescent. These processes are highly coordinated, both temporally and spatially, to ensure the proper growth and development of the seed. The transcription factor, LEAFY COTYLEDON1 (LEC1), is a central regulator that controls several aspects of embryo and endosperm development, including embryo morphogenesis, photosynthesis, and storage reserve accumulation. Thus, LEC1 regulates distinct sets of genes at different stages of seed development. Despite its critical importance for seed development, an understanding of the mechanisms underlying LEC1's multifunctionality is only beginning to be obtained. Recent studies describe the roles of specific transcription factors and the hormones, gibberellic acid and abscisic acid, in controlling the activity and transcriptional specificity of LEC1 across seed development. Moreover, studies indicate that LEC1 acts as a pioneer transcription factor to promote epigenetic reprogramming during embryogenesis. In this review, we discuss the mechanisms that enable LEC1 to serve as a central regulator of seed development.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/jipb.12806

Central role of the LEAFY COTYLEDON1 transcription factor in seed development.

Starch and storage proteins, the primary storage substances of cereal endosperm, are a major source of food for humans. However, the transcriptional regulatory networks of the synthesis and accumulation of storage substances remain largely unknown. Here, we identified a rice endosperm-specific gene, NF-YC12, that encodes a putative nuclear factor-Y transcription factor subunit C. NF-YC12 is expressed in the aleurone layer and starchy endosperm during grain development. Knockout of NF-YC12 significantly decreased grain weight as well as altering starch and protein accumulation and starch granule formation. RNA-sequencing analysis revealed that in the nf-yc12 mutant genes related to starch biosynthesis and the metabolism of energy reserves were enriched in the down-regulated category. In addition, starch and protein contents in seeds differed between NF-YC12-overexpression lines and the wild-type. NF-YC12 was found to interact with NF-YB1. ChIP-qPCR and yeast one-hybrid assays showed that NF-YC12 regulated the rice sucrose transporter OsSUT1 in coordination with NF-YB1 in the aleurone layer. In addition, NF-YC12 was directly bound to the promoters of FLO6 (FLOURY ENDOSPERM6) and OsGS1;3 (glutamine synthetase1) in developing endosperm. This study demonstrates a transcriptional regulatory network involving NF-YC12, which coordinates multiple pathways to regulate endosperm development and the accumulation of storage substances in rice seeds.

NF-YC12 is a key multi-functional regulator of accumulation of seed storage substances in rice.

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor that consists of three subunits, NF-YA, NF-YB, and NF-YC. Gene functions of NF-Ys during endosperm development are not well understood. In this study, we identified eight rice NF-Y-encoding genes, namely OsNF-YA8, OsNF-YB1,9, and OsNF-YC8,9,10,11,12, that are predominantly expressed in the endosperm. Interestingly, the close homologs of these OsNF-Ys are present only in monocot species and are also preferentially expressed in the endosperm, suggesting that they have roles in the regulation of endosperm development. A systemic analysis of interactions between rice endosperm-preferential NF-Ys in yeast revealed that OsNF-YBs and OsNF-YCs could interact with each other. We also found that the endosperm-preferential OsNF-YBs and OsNF-YCs could interact with some ethylene response factors (ERFs) of rice. Unlike OsNF-YC8,9,10, the members of OsNF-YB1,9 or OsNF-YC 11,12 showed no transcriptional activation when present alone. However, they displayed functional activity while in dimer form. In addition, OsNF-YB1-knockout lines showed significant changes in seed morphology, further confirming its role in endosperm development. Our findings provide evidence that a group of phylogenetically conserved NF-Ys is probably differentiated in monocots to regulate endosperm development.

A group of nuclear factor Y transcription factors are sub-functionalized during endosperm development in monocots.

Following double fertilization, plant endosperm nuclei undergo syncytial divisions, followed by synchronous cellularization. Cellularization is a key event during endosperm development, but our understanding of its regulation is limited to Arabidopsis. In this study we show that OsbZIP76 regulates cellularization in rice (Oryza sativa). Activation of OsbZIP76 coincided with the initiation of cellularization, and its knockdown or knockout mutants exhibited precocious cellularization. Genes involved in endosperm development or starch biosynthesis were prematurely activated in the osbzip76 caryopsis. As a putative transcription factor, OsbZIP76 alone lacked transcriptional activation activity; however, it interacted with the nuclear factor Y (NF-Y) family transcription factors OsNF-YB9 and OsNF-YB1 in yeast and in planta. OsbZIP76 and OsNF-YB9 were predominantly expressed in the endosperm and the proteins colocalized. Seeds of osnf-yb1 and osbzip76 mutants showed reduced size and reduced apparent amylose content. The parent-of-origin-dependent expression of OsbZIP76 is variable in different rice accessions. In summary, OsbZIP76 is an endosperm-expressed imprinted gene that regulates endosperm development in rice.

OsbZIP76 interacts with OsNF‐YBs and regulates endosperm cellularization in rice (Oryza sativa)

Nuclear factor Y (NF-Y), also known as Heme Activator Protein or CCAAT-binding factor, is a heterotrimeric transcription factor that consists of three subunits, NF-YA, NF-YB, and NF-YC. Although NF-Ys play multiple roles in plant development, their functions during endosperm development are not well understood. In this study, we identified eight rice (Oryza sativa) NF-Y encoding genes, including OsNF-YA8, OsNF-YB1,9, and OsNF-YC8,9,10,11,12, which predominantly express in the endosperm. Interestingly, their closest homologs could only be found in monocot species and these genes also showed an endosperm-preferential expression pattern, which suggest that the phylogenetically related group of NF-Ys may be involved in the regulation of endosperm development. A systemic analysis of the interactions between rice endosperm-preferential NF-Ys in yeast revealed that NF-YBs and NF-YCs could interact with each other in all the combinations that we tested. OsNF-YA8 is a recently evolved NF-YA in rice. NF-YA does not usually interact with NF-YB monomers in plants; however, OsNF-YA8 could interact with OsNF-YB9. Our results also indicated that the endosperm-preferential OsNF-YBs, as well as the OsNF-YCs, could interact with some endosperm-specific ERFs of rice. Unlike the OsNF-YC8,9,10, OsNF-YB1,9 or OsNF-YC11,12 alone lacks transcriptional activation activity. However, the dimers they formed displayed transcriptional activation activities. Considering that mutated OsNF-YB1 can severely impair endosperm development in rice, our findings strongly suggest that there is a group of phylogenetically conserved NF-Ys that have differentiated in monocots to regulate endosperm development.

https://www.biorxiv.org/content/biorxiv/early/2017/06/12/149047.full.pdf

A group of nuclear factor Y transcription factors are likely sub-functionalized in the endosperm development of rice and other monocots

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor that consists of three subunits, NF-YA, NF-YB, and NF-YC. Although NF-Ys play multiple roles in plant development, their functions during endosperm development are not well understood. In this study, we identified eight NF-Y encoding genes, including OsNF-YA8, OsNF-YB1,9, and OsNF-YC8,9,10,11,12, which predominantly express in the rice endosperm. Interestingly, the closest homologs of these OsNF-Ys are present only in the monocot species. All the genes are preferentially expressed in the endosperm, suggesting their roles in the regulation of endosperm development. A systemic analysis of the interactions between rice endosperm-preferential NF-Ys in yeast revealed that NF-YBs and NF-YCs could interact with each other. OsNF-YA8 is a recently evolved NF-YA in rice. Generally, NF-YA does not interact with NF-YB monomers in plants; however, in the present study, we found that OsNY-YA8 interacts with OsNF-YB9. Our results also indicated that the endosperm-preferential OsNF-YBs and OsNF-YCs could interact with some ethylene response factors (ERFs) of rice. Unlike the OsNF-YC8,9,10, the members of OsNF-YB1, 9 or OsNF-YC 11 and 12 showed lack of transcriptional activation when present alone. However, they displayed functional activity while in dimer form. In addition, OsNF-YB1 knockout lines showed significant changes in the seed morphology, further confirms its role in endosperm development. Our findings have provided strong evidences that the group of phylogenetically conserved NF-Ys are differentiated in monocots to regulate the endosperm development.

https://www.biorxiv.org/content/biorxiv/early/2017/06/19/149047.full.pdf

Hui Deng

Papers

A group of nuclear factor Y transcription factors are sub-functionalized during endosperm development in monocots.

OsbZIP76 interacts with OsNF‐YBs and regulates endosperm cellularization in rice (Oryza sativa)

A group of nuclear factor Y transcription factors are likely sub-functionalized in the endosperm development of rice and other monocots

A group of nuclear factor Y transcription factors are likely sub-functionalized in the endosperm development of monocot