The parent-of-origin lncRNA MISSEN regulates rice endosperm development

The cereal endosperm is a major factor determining seed size and shape. However, the molecular mechanisms of endosperm development are not fully understood. Long noncoding RNAs (lncRNAs) function in various biological processes. Here we show a lncRNA, MISSEN, that plays an essential role in early endosperm development in rice (Oryza sativa). MISSEN is a parent-of-origin lncRNA expressed in endosperm, and negatively regulates endosperm development, leading to a prominent dent and bulge in the seed. Mechanistically, MISSEN functions through hijacking a helicase family protein (HeFP) to regulate tubulin function during endosperm nucleus division and endosperm cellularization, resulting in abnormal cytoskeletal polymerization. Finally, we revealed that the expression of MISSEN is inhibited by histone H3 lysine 27 trimethylation (H3K27me3) modification after pollination. Therefore, MISSEN is the first lncRNA identified as a regulator in endosperm development, highlighting the potential applications in rice breeding.

Comparative Phosphoproteomic Analysis Reveals the Response of Starch Metabolism to High-Temperature Stress in Rice Endosperm

High-temperature stress severely affects rice grain quality. While extensive research has been conducted at the physiological, transcriptional, and protein levels, it is still unknown how protein phosphorylation regulates seed development in high-temperature environments. Here, we explore the impact of high-temperature stress on the phosphoproteome of developing grains from two indica rice varieties, 9311 and Guangluai4 (GLA4), with different starch qualities. A total of 9994 phosphosites from 3216 phosphoproteins were identified in all endosperm samples. We identified several consensus phosphorylation motifs ([sP], [LxRxxs], [Rxxs], [tP]) induced by high-temperature treatment and revealed a core set of protein kinases, splicing factors, and regulatory factors in response to high-temperature stress, especially those involved in starch metabolism. A detailed phosphorylation scenario in the regulation of starch biosynthesis (AGPase, GBSSI, SSIIa, SSIIIa, BEI, BEIIb, ISA1, PUL, PHO1, PTST) in rice endosperm was proposed. Furthermore, the dynamic changes in phosphorylated enzymes related to starch synthesis (SSIIIa-Ser94, BEI-Ser562, BEI-Ser620, BEI-Ser821, BEIIb-Ser685, BEIIb-Ser715) were confirmed by Western blot analysis, which revealed that phosphorylation might play specific roles in amylopectin biosynthesis in response to high-temperature stress. The link between phosphorylation-mediated regulation and starch metabolism will provide new insights into the mechanism underlying grain quality development in response to high-temperature stress.

Temporal Changes in Transcripts of MITE Transposable Elements during Rice Endosperm Development

The repression of transcription from transposable elements (TEs) by DNA methylation is necessary to maintain genome integrity and prevent harmful mutations. However, under certain circumstances, TEs are thought to escape from the host defense system and reactivate their transcription. In Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), DNA demethylases target the sequences derived from TEs in the central cell, the progenitor cell for the endosperm in the female gametophyte. This genome-wide DNA demethylation is also observed in the endosperm after fertilization. In this study, we used a custom microarray to survey the transcripts generated from TEs during the rice endosperm development and at selected timepoints in the embryo as a control. The expression patterns of TE transcripts are dynamically up- and downregulated during endosperm development, especially for miniature inverted-repeat transposable elements (MITEs). Surprisingly, some TE transcripts were directionally controlled, while the other DNA transposons and retrotransposons were not. We also discovered the NF-Y binding motif, CCAAT, in the region near the 5′ terminal inverted repeat of Youren, one of the transcribed MITEs in the endosperm. Our results uncover dynamic changes in TE activity during endosperm development in rice.

Biochemical and Molecular Assessment of Cooking Quality and Nutritional Value of Pigmented and Non-pigmented Whole Grain Rice

To date, most people prefer softer and stickier rice with high glycaemic index, which has led to the study of the association between the dietary fibre profile and the textural properties to balance between the eating quality, and health benefits. A slight variation was observed in the dietary fibre composition among rice varieties with different amylose content. The percentage of insoluble dietary fibre (IDF) in whole grain rice varied from 1.65–4.32 while the percentage of soluble dietary fibre (SDF) ranged from 0.26–1.37. We demonstrated that the SDF/IDF ratio was higher in soft texture rice with low amylose and it shared a negative correlation with hardness and chewiness of cooked whole grain rice determined by a texture analyser, which suggested that the distribution of SDF throughout the rice endosperm influences the reduction of the hardness of cooked rice. According to the importance of dietary fibre, we proposed a simple linear regression method to estimate the amount of IDF and total dietary fibre with r = 0.97 and 0.92, respectively, of whole grain rice based on its bran layer, determined by the alternative alkaline method. Furthermore, low amylose rice has higher content of -glucan and pectin, which are classified as soluble dietary fibre, than high amylose rice. The percentage of -glucan and pectin in whole grain rice ranged from 0.03 and 0.07 respectively, for high amylose rice to 0.14 and 0.27 respectively for low amylose rice,

Laser Microdissection Transcriptome Data Derived Gene Regulatory Networks of Developing Rice Endosperm Revealed Tissue- and Stage-specific Regulators Modulating Starch Metabolism

Rice (Oryza sativa L.) filial seed tissues are heterozygous in its function, which accumulate distinct storage compounds spatially in starchy endosperm and aleurone. In this study, we identified the 18 tissue- and stage-specific gene co-regulons in the developing endosperm by isolating four fine tissues dorsal aleurone layer (AL), central starchy endosperm (CSE), dorsal starchy endosperm (DSE), and lateral starchy endosperm (LSE) at two developmental stages (7 days after flowering, DAF and 12DAF) using laser microdissection (LM) coupled with gene expression analysis of a 44K microarray. The derived co-expression regulatory networks depict that distinct set of starch biosynthesis genes expressed preferentially at first in CSE at 7 DAF and extend its spatial expression to LSE and DSE by 12 DAF. Interestingly, along with the peak of starch metabolism we noticed accumulation of transcripts related to phospholipid and glycolipid metabolism in CSE during 12 DAF. The spatial distribution of starch accumulation in distinct zones of starchy endosperm contains specific transcriptional factors and hormonal-regulated genes. Genes related to programmed cell death (PCD) were specifically expressed in CSE at 12DAF, when starch accumulation was already completed in that tissue. The aleurone layer present in the outermost endosperm accumulates transcripts of lipid, tricarboxylic acid metabolism, several transporters, while starch metabolism and PCD is not pronounced. These regulatory cascades are likely to play a critical role in determining the positional fate of cells and offer novel insights into the molecular physiological mechanisms of endosperm development from early to middle storage phase.

Grain Quality and Starch Physicochemical Properties of Chalky Rice Mutant

Rice mutants with altered starch components and properties are important genetic resources for grain quality and starch structure analysis. Accordingly, in the present study, two mutants of the transcription factor OsbZIP09 were generated (osbzip09a and osbzip09b), and the rice grain quality and physicochemical starch properties of the mutant and wild-type lines were compared. The OsbZIP09 mutants exhibit a chalky grain owing to loosely packed, small, spherical starch granules in the ventral region of the endosperm. Furthermore, grain-quality profile analysis showed that OsbZIP09 deficiency leads to increased apparent amylose content but decreased gel consistency. Structural analysis of the mutant starches revealed that the mutant rice lines contain more amylopectin short chains and fewer intermediate chains, leading to lower crystallinity and lower gelatinization properties than those of the wild-type rice. Moreover, the OsbZIP09 mutants rice presented a significantly higher pasting curve and corresponding parameters than the wild-type rice. The results from this work strongly indicate that the transcription factor OsbZIP09 plays an important role in rice grain quality and starch fine structure modification, and extend our understanding of starch biosynthesis in rice endosperm.

CHARACTERISATION OF HORDEUM VULGARE CELLULOSE SYNTHASE-LIKE F6 PROMOTER VIA TRANSGENE EXPRESSION IN RICE

Beta-glucan in cereal crops is known as a functional food, which can reduce cardiovascular diseases by lowering blood cholesterol levels. However, beta-glucan content is relatively low in rice grains, despite being relatively abundant in barley and oat grains. Taking advantage of rice as the staple food for Asians, increasing beta-glucan content in rice for their consumption may help to reduce cardiovascular-related diseases among them. Previous attempts in increasing beta-glucan content in rice via transgene expression of beta-glucan synthase genes from barley into rice were unsuccessful due to the use of non-tissue specific as well as constitutively expressing promoter. The current transgenic expression study was performed to characterise the promoter of beta-glucan synthase gene in barley using beta-glucuronidase (GUS) reporter gene. Two fragments of HvCslF6 promoter (2771 bp and 1257 bp) were successfully fused with GUS reporter gene and integrated into rice plants, demonstrated that the promoter was functional in the heterologous plant system. The presence of blue GUS staining was observed on the leaf, root, stem, and grain of the transgenic rice regardless of the promoter length used and stayed functional up to the next generation. GUS qualitative analysis confirmed that the shorter promoter length generated a stronger GUS activity in comparison to the longer one. This indicated that the presence of repressor elements in between the -2771 bp and -1257 bp regions. The preliminary results shed light on the strong promoter activity in the rice endosperm tissue. It can become an alternative to the collection of plant promoters that can be used for grain quality improvement and biofortification.