High temperature often leads to the failure of grain filling in rice (Oryza sativa) to cause yield loss, while the mechanism is not well elucidated yet. Here, we report that two seed-specific NAM/ATAF/CUC domain transcription factors, ONAC127 and ONAC129, are responsive to heat stress and involved in the grain filling process of rice. ONAC127 and ONAC129 are dominantly expressed in the pericarp and can form a heterodimer during rice grain filling. CRISPR/Cas9 induced mutants and overexpression lines were then generated to investigate the functions of these two transcription factors. Interestingly, both knock-out and overexpression plants showed incomplete grain filling and shrunken grains, which became more severe under heat stress. Transcriptome analysis revealed that ONAC127 and ONAC129 mainly regulate stimulus response and nutrient transport. ChIP-seq analysis identified that the direct targets of ONAC127 and ONAC129 in developing rice seeds include monosaccharide transporter OsMST6, sugar transporter OsSWEET4, calmodulin-like protein OsMSR2 and AP2/ERF factor OsEATB. These results suggest that ONAC127 and ONAC129 may regulate grain filling through affecting sugar transportation and abiotic stress responses. Overall, this study demonstrates a transcriptional regulatory network involving ONAC127 and ONAC129 and coordinating multiple pathways to modulate seed development and heat stress response at rice reproductive stage.

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.

22High temperature usually leads to the failure of grain filling during caryopsis 23 development, resulting in the loss of yield, however the mechanism is not yet well 24 elucidated. Here, we report that two rice caryopsis-specific NAM/ATAF/CUC domain 25 transcription factors, ONAC127 and ONAC129, respond to heat stress and are involved 26 in the caryopsis filling process. ONAC127 and ONAC129 are dominantly expressed in 27 pericarp during grain filling and can form a heterodimer. To investigate the functions 28 of these two ONACs, we obtained CRISPR/cas9 induced mutants and overexpression 29 lines of them. Interestingly, we found that both knock-out and overexpression plants 30showed incompletely filling and shrunken phenotype of caryopses, which became more 31 severe under heat stress. The shrunken caryopses of these transgenic lines are usually 32 with ectopic accumulation of starch in the pericarp. Transcriptome analyses revealed 33 that ONAC127 and ONAC129 mainly regulate stimulus response, cell wall construction 34 and nutrient transport etc. ChIP-seq analyses identified the direct targets of ONAC127 35 and ONAC129 in developing caryopses, including monosaccharide transporter 36OsMST6, sugar transporter OsSWEET4, calmodulin-like protein OsMSR2 and 37Ethylene-Response AP2/ERF Factor OsEATB. The result suggested that ONAC127 and 38 ONAC129 might regulate the caryopsis filling through sugar transportation and abiotic 39 stress responses. Overall, this study demonstrates the transcriptional regulatory 40 networks involving ONAC127 and ONAC129, which coordinates multiple pathways 41 to modulate caryopsis development and heat stress response at rice filling stage. 42 43 rice caryopsis is mainly composed of endosperm filled with starch grains, and the 48 biosynthesis of starch is closely related to carbohydrates transportation. Carbohydrates 49 are synthesized in leaves and delivered to caryopses via phloem (Patrick, 1997), then 50 unloaded to the pericarp as the energy and materials for starch biosynthesis (Zhang et 51 al., 2007). The dorsal vascular bundle passes through the pericarp is the main nutrient 52 transport tissue in caryopsis (Oparka and Gates, 1981). While the vascular bundles are 53 not contiguous with endosperm tissue (Hoshikawa, 1984), the apoplasmic pathway is 54 the only way for nutrient to reach the starchy endosperm. (Matsuda et al., 1979). 55Carbohydrates may enter the nucellar epidermis directly through plasmodesmata, 56 then be transported to the apoplasmic space with sugar transporter protein OsSWEETs 57 (sugar will eventually be exported transporters) and partially hydrolyzed into 58 monosaccharide by cell wall invertase OsCINs. The monosaccharide is transported into 59 the aleurone layer mainly by monosaccharide transporter OsMSTs while the rest un-60 hydrolyzed sucrose is directly transported into the aleurone layer via sucrose transporter 61OsSUTs (Yang et al., 2018). The nutrient transportation is also regulated by a range of 62 transcription factors such as OsNF-YB1 and OsNF-YC12, ...