Wheat E3 ubiquitin ligase TaGW2-6A degrades TaAGPS to affect seed size.

TaGW2 has been identified as a key determinant of the grain weight in wheat (Triticum aestivum L.). In our previous study, we found that the grain size differs in Chinese Spring (CS) and its TaGW2-6A allelic variant (NIL31). In addition, the expression of the key starch biosynthesis enzyme gene TaAGPS differs significantly in the two materials. However, the underlying molecular mechanism associated with the action of TaGW2-6A has not been reported. In the present study, we found that TaGW2-6A-CS interacted with TaAGPS, whereas TaGW2-6A-NIL31 did not interact with it in vitro and in vivo. Furthermore, we found that the C-terminal LXLX domain (376-424 aa) of TaGW2-6A recognized TaAGPS. However, the TaGW2-6A allelic variant lacked this key interaction region due to premature translation termination. We also found that TaGW2-6A-CS can ubiquitinate TaAGPS and degrade it via the 26 S proteasome pathway. In addition, our analysis of the activity of ADP-glucose pyrophosphorylase (AGPase) indicated that the AGPase level in the endosperm cells was lower in CS than NIL31. Cytological observations demonstrated that the average number of starch granules and the average area of starch granules in endosperm cells were lower in CS than NIL31. The overexpression of TaAGPS positively regulated the seed size in transgenic Arabidopsis. Our findings provide novel insights into the molecular mechanism that allows TaGW2-6A-TaAGPS to regulate seed size via the starch synthesis pathway.

Influence of TaGW2-6A on seed development in wheat by negatively regulating gibberellin synthesis.

Gibberellins (GA) are involved in seed development and E3 ubiquitin-ligases actively participate in GA perception and signal transduction. TaGW2-6A encodes a RING E3 ubiquitin-ligase that negatively regulates grain size. Therefore, Chinese Spring (CS) and its TaGW2-6A allelic variants (NIL31) were investigated to elucidate the relative contribution of GA to the regulation of seed development in wheat. The expression levels of GA biosynthesis and response genes were higher in NIL31 than CS, especially those of GA 3-oxidase and GASA4. The expression of TaGW2-6A exhibited the opposite pattern compared with those of the GA biosynthesis and response genes in CS and NIL31. The results showed that the GA content of NIL31 was significantly higher than that of CS. Thus, TaGW2-6A had a negative relationship on GA synthesis and response genes. Moreover, after GA treatment, CS and NIL31 exhibited the opposite phenotypes and GA contents. These results demonstrate that allelic variation in TaGW2-6A increases the seed size via the GA hormone pathway. Transcriptional analysis and cytological analysis showed that TaGW2-6A allelic variants regulated GA synthesis via GA 3-oxidases, thereby leading to the higher expression of GASA4 to control endosperm cell elongation and division during grain filling. Finally, germination experiments were performed to elucidate the relationships between TaGW2-6A and GA synthesis and response genes in wheat with full fertility. These results provide new insights into the effects of the ubiquitination system mediated by TaGW2-6A on the GA hormone signaling pathway, thereby improving our understanding of the role of TaGW2-6A in seed development in wheat.

TaGW2-6A allelic variation contributes to grain size possibly by regulating the expression of cytokinins and starch-related genes in wheat.

MAIN CONCLUSION: Functional allelic variants of TaGW2 - 6A produce large grains, possibly via changes in endosperm cells and dry matter by regulating the expression of cytokinins and starch-related genes via the ubiquitin-proteasome system. In wheat, TaGW2-6A coding region allelic variants are closely related to the grain width and weight, but how this region affects grain development has not been fully elucidated; thus, we explored its influence on grain development based mainly on histological and grain filling analyses. We found that the insertion type (NIL31) TaGW2-6A allelic variants exhibited increases in cell numbers and cell size, thereby resulting in a larger (wider) grain size with an accelerated grain milk filling rate, and increases in grain width and weight. We also found that cytokinin (CK) synthesis genes and key starch biosynthesis enzyme AGPase genes were significantly upregulated in the TaGW2-6A allelic variants, while CK degradation genes and starch biosynthesis-negative regulators were downregulated in the TaGW2-6A allelic variants, which was consistent with the changes in cells and grain filling. Thus, we speculate that TaGW2-6A allelic variants are linked with CK signaling, but they also influence the accumulation of starch by regulating the expression of related genes via the ubiquitin-proteasome system to control the grain size and grain weight.

Identification of Key Proteins and Networks Related to Grain Development in Wheat (Triticum aestivum L.) by Comparative Transcription and Proteomic Analysis of Allelic Variants in TaGW2-6A.

In wheat, coding region allelic variants of TaGW2-6A are closely associated with grain width and weight, but the genetic mechanisms involved remain unclear. Thus, to obtain insights into the key functions regulated by TaGW2-6A during wheat grain development, we performed transcriptional and proteomic analyses of TaGW2-6A allelic variants. The transcription results showed that the TaGW2-6A allelic variants differed significantly by several orders of magnitude. Each allelic variant of TaGW2-6A reached its first transcription peak at 6 days after anthesis (DAA), but the insertion type TaGW2-6A allelic variant reached its second peak earlier than the normal type, i.e., at 12 DAA rather than 20 DAA. In total, we identified 228 differentially accumulated protein spots representing 138 unique proteins by two-dimensional gel electrophoresis and tandem MALDI-TOF/TOF-MS in these three stages. Based on the results, we found some key proteins that are closely related to wheat grain development. The results of this analysis improve our understanding of the genetic mechanisms related to TaGW2-6A during wheat grain development as well as providing insights into the biological processes involved in seed formation.