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.

Starch branching enzyme IIb (BEIIb) and soluble starch synthase IIa (SSIIa) play important roles in starch biosynthesis in cereals. Deficiency in the BEIIb gene produces the amylose extender (ae) mutant rice strain with increased amylose content (AC) and changes in the amylopectin structure. The SSIIa gene is responsible for the genetic control of gelatinization temperature (GT). The combined effects of BEIIb and SSIIa alleles on the AC, fine structures, and physicochemical properties of starches from 12 rice accessions including 10 recombinant inbred lines (RIL) and their two parents were examined in this study. Under the active BEIIb background, starches with the SSIIa-GC allele showed a higher GT than those with the SSIIa-TT allele, resulting from a lower proportion of A chain and a larger proportion of B1 chains in the amylopectin of SSIIa-GC. However, starch with the BEIIb mutant allele (be2b) in combination with any SSIIa genotype displayed more amylose long chains, higher amylose content, B2 and B3 chains, and molecular order, but smaller relative crystallinity and proportion of amylopectin A and B1 chains than those with BEIIb, leading to a higher GT and lower paste viscosities. These results suggest that BEIIb is more important in determining the structural and physicochemical properties than SSIIa. These results provide additional insights into the structure-function relationship in indica rice rather than that in japonica rice and are useful for breeding rice with high amylose content and high resistant starch.

The Shanlan landrace rice in Hainan Province, China, is a unique upland rice germplasm that holds significant value as a genetic resource for rice breeding. However, its genetic diversity and its usefulness in rice breeding have not been fully explored. In this study, a total of eighty-four Shanlan rice, three typical japonica rice cultivars, and three typical indica rice cultivars were subjected to resequencing of their genomes. As a result, 11.2 million high-quality single nucleotide polymorphisms (SNPs) and 1.6 million insertion/deletions (InDels) were detected. Population structure analysis showed all the rice accessions could be divided into three main groups, i.e., Geng/japonica 1 (GJ1), GJ2, and Xian/indica (XI). However, the GJ1 group only had seven accessions including three typical japonica cultivars, indicating that most Shanlan landrace rice are different from the modern japonica rice. Principal component analysis (PCA) showed that the first three principal components explained 60.7% of the genetic variation. Wide genetic diversity in starch physicochemical parameters, such as apparent amylose content (AAC), pasting viscosity, texture properties, thermal properties, and retrogradation representing the cooking and eating quality was also revealed among all accessions. The genome-wide association study (GWAS) for these traits was conducted and identified 32 marker trait associations in the entire population. Notably, the well-known gene Waxy (Wx) was identified for AAC, breakdown viscosity, and gumminess of the gel texture, and SSIIa was identified for percentage of retrogradation and peak gelatinization temperature. Upon further analysis of nucleotide diversity in Wx, six different alleles, wx, Wx^a, Wx^b, Wxⁱⁿ, Wx^la/mw, and Wx^lv in Shanlan landrace rice were identified, indicating rich gene resources in Shanlan rice for quality rice breeding. These findings are expected to contribute to the development of new rice with premium quality.

The improvement of the cooking and eating quality (CEQ) of rice is one of the major objectives of current rice-breeding programs. A few major genes such as Waxy (Wx) and starch synthase IIa (SSIIa) have been successfully applied in molecular breeding. However, their interactive effects on CEQ have not been fully understood. In this study, a recombinant inbred line (RIL) population was constructed by crossing the white-core mutant GM645 with the transparent phenotype of the japonica rice variety Tainung 67 (TN67). GM645 and TN67 contain different alleles of FLOURY ENDOSPERM2 (FLO2), Wx, and SSIIa. The effects of different allele combinations of FLO2, Wx, and SSIIa on the CEQ of rice were investigated. The inbred lines with the mutation allele flo2 had a significantly lower apparent amylose content (AAC), viscosity characteristics except for setback (SB), and gel texture properties compared to those lines with the FLO2 allele. The allelic combination of FLO2 and Wx significantly affected the AAC, breakdown (BD), and gel textural properties, which could explain most of the variations in those rice quality traits that were correlated with AAC. The allelic combination of FLO2 and SSIIa significantly affected the hot paste viscosity (HPV) and pasting temperature (PT). The Wx × SSIIa interaction had a significant effect on the PT. The interaction of FLO2, Wx and SSIIa significantly affected the AAC, cold paste viscosity (CPV), PT, and consistency viscosity (CS). These results highlight the important roles of these quality-related genes in regulating the CEQ of rice and provide new clues for rice-quality improvement by marker-assisted selection.