Laser microdissection-based gene expression analysis in the aleurone layer and starchy endosperm of developing rice caryopses in the early storage phase

Cai

et al. 2019

New Phytologist

Summary Endosperm, the major storage organ in cereal grains, determines grain yield and quality. Despite the fact that a role for P‐type pentatricopeptide repeat (PPR) proteins in the regulation of endosperm development has emerged, molecular functions of many P‐type PPR proteins remain obscure. Here, we report a rice endosperm defective mutant, floury endosperm10 (flo10), which developed smaller starch grains in starchy endosperm and abnormal cells in the aleurone layer. Map‐based cloning and rescued experiments showed that FLO10 encodes a P‐type PPR protein with 26 PPR motifs, which is localized to mitochondria. Loss of function of FLO10 affected the trans‐splicing of the mitochondrial nad1 intron 1, which was accompanied by the increased accumulation of the nad1 exon 1 and exons 2–5 precursors. The failed formation of mature nad1 led to a dramatically decreased assembly and activity of complex I, reduced ATP production, and changed mitochondrial morphology. In addition, loss of function of FLO10 significantly induced an alternative respiratory pathway involving alternative oxidase. These results reveal that FLO10 plays an important role in the maintenance of mitochondrial function and endosperm development through its effect on the trans‐splicing of the mitochondrial nad1 intron 1 in rice.

Section: Discussionmentioning

confidence: 97%

Rice FLOURY ENDOSPERM10 encodes a pentatricopeptide repeat protein that is essential for the trans‐splicing of mitochondrial nad1 intron 1 and endosperm development

Cai

et al. 2019

New Phytologist

“…A fully developed endosperm, which occupies most of the seed space, has accumulated large amounts of storage substances. The endosperm consists of an outer aleurone layer (AL) inside which is the starchy endosperm (SE) (Becraft, 2001; Krishnan and Dayanandan, 2003; Zuo and Li, 2014; Ishimaru et al , 2015; Wu et al , 2016). Proteins, lipids, and minerals are accumulated in the AL, whereas starch and small amounts of storage proteins are mainly stored in the SE (Olsen, 2004; Zheng and Wang, 2014).…”

Section: Introductionmentioning

confidence: 99%

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

Xiong

Journal of Experimental Botany

et al. 2019

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.

“…For validating the spatial and temporal expression patterns and further exploring the specific expression distribution of ONAC127 and ONAC129 in caryopsis, we mechanically isolated rice caryopses at 5-14 DAP to the mixture of pericarp and aleurone layer, starch endosperm and embryo for qRT-PCR analyses by the method described by Bai (Bai et al, 2016). The aleurone layer specifically expressed gene oleosin and the starch endosperm specifically expressed gene SDBE were used as markers (Ishimaru et al, 2015) (Supplemental Fig. S1).…”

Section: Resultsmentioning

confidence: 99%

A Heat Stress Responsive NAC Transcription Factor Heterodimer Plays Key Roles in Rice Grain Filling

Huang

Jiang

et al. 2020

Preprint

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, ...