The NAC transcription factors OsNAC20 and OsNAC26 influence starch and storage protein accumulation by directly regulating the expression of starch and storage

Rice (Oryza sativa) endosperm is mainly occupied by homogeneous polygonal starch from inside to outside. However, morphologically different (heterogeneous) starches have been identified in some rice mutants. How these heterogeneous starches form remains unknown. A high-amylose rice line (TRS) generated through the antisense inhibition of starch branching synthase I (SBEI) and SBEIIb contains four heterogeneous starches: polygonal, aggregate, elongated, and hollow starch; these starches are regionally distributed in the endosperm from inside to outside. Here, we investigated the relationship between SBE dosage and the morphological architecture of heterogeneous starches in TRS endosperm from the view of the molecular structure of starch. The results indicated that their molecular structures underwent regular changes, including gradually increasing true amylose content but decreasing amylopectin content and gradually increasing the ratio of amylopectin long chain but decreasing the ratio of amylopectin short chain. Granule-bound starch synthase I (GBSSI) amounts in the four heterogeneous starches were not significantly different from each other, but SBEI, SBEIIa, and SBEIIb showed a gradually decreasing trend. Further immunostaining analysis revealed that the gradually decreasing SBEs acting on the formation of the four heterogeneous granules were mainly due to the spatial distribution of the three SBEs in the endosperm. It was suggested that the decreased amylopectin in starch might remove steric hindrance and provide extra space for abundant amylose accumulation when the GBSSI amount was not elevated. Furthermore, extra amylose coupled with altered amylopectin structure possibly led to morphological changes in heterogeneous granules.

The dry root tuber of Apios fortunei contained about 75% starch, indicating that it is an important starch resource. Starch displayed spherical, polygonal, and ellipsoidal granules with central hila. Granule sizes ranged from 3 to 30 μm with a 9.6 μm volume-weighted mean diameter. The starch had 35% apparent amylose content and exhibited C_A-type crystalline structure with 25.9% relative crystallinity. The short-range ordered degree in the granule external region was approximately 0.65, and the lamellar thickness was approximately 9.6 nm. The swelling power and water solubility began to increase from 70 °C and reached 28.7 g/g and 10.8% at 95 °C. Starch had typical bimodal thermal curve in water with gelatinization temperatures from 61.8 to 83.9 °C. The 7% (w/w) starch-water slurry had peak, hot, breakdown, final, and setback viscosities of 1689, 1420, 269, 2103, and 683 mPa s, respectively. Rapidly digestible starch, slowly digestible starch, and resistant starch were 6.04%, 10.96%, and 83.00% in native starch; 83.16%, 15.23%, and 1.61% in gelatinized starch; and 78.13%, 17.88%, and 3.99% in retrograded starch, respectively. The above physicochemical properties of A. fortunei starch were compared with those of maize A-type starch, potato B-type starch, and pea C-type starch. The hierarchical cluster analysis based on starch structural and functional property parameters showed that A. fortunei and pea starches had similar physicochemical properties and were more related to maize starch than potato starch.

Several starch synthesis regulators have been identified, but these regulators are situated in the terminus of the regulatory network. Their upstream regulators and the complex regulatory network formed between these regulators remain largely unknown. A previous study demonstrated that NAM, ATAF, and CUC (NAC) transcription factors, OsNAC20 and OsNAC26 (OsNAC20/26), redundantly and positively regulate the accumulation of storage material in rice (Oryza sativa) endosperm. In this study, we detected OsNAC25 as an upstream regulator and interacting protein of OsNAC20/26. Both OsNAC25 mutation and OE resulted in a chalky seed phenotype, decreased starch content, and reduced expression of starch synthesis–related genes, but the mechanisms were different. In the osnac25 mutant, decreased expression of OsNAC20/26 resulted in reduced starch synthesis; however, in OsNAC25-overexpressing plants, the OsNAC25–OsNAC20/26 complex inhibited OsNAC20/26 binding to the promoter of starch synthesis–related genes. In addition, OsNAC20/26 positively regulated OsNAC25. Therefore, the mutual regulation between OsNAC25 and OsNAC20/26 forms a positive regulatory loop to stimulate the expression of starch synthesis–related genes and meet the great demand for starch accumulation in the grain filling stage. Simultaneously, a negative regulatory loop forms among the 3 proteins to avoid the excessive expression of starch synthesis–related genes. Collectively, our findings demonstrate that both promotion and inhibition mechanisms between OsNAC25 and OsNAC20/26 are essential for maintaining stable expression of starch synthesis–related genes and normal starch accumulation.

Cereal crops with inhibition expression of starch branching enzyme (SBE) contain highly resistant starch in the endosperm, and have potential health benefits for human. However, seed plumpness is significantly different, with different inhibition effects of SBE expression, resulting in differently shrunken seeds. In this study, a transgenic resistant starch rice line, which has highly resistant starch and is developed through inhibition expression of SBEs, had non-, slightly, and moderately shrunken seeds with plumpness from high to low. The differently shrunken seeds had significantly different seed weight and starch content. Different morphological starch granules were detected in the endosperm and had similar spatial distribution pattern among the non-, slightly, and moderately shrunken seeds. Starches from differently shrunken seeds had similar amylose content and amylopectin structure. The protein amount of amylose/amylopectin synthesis key enzymes in endosperm was no different between differently shrunken seeds. The primary branch of the panicle had a higher percentage of non-shrunken seeds than did the secondary branch at the same part of the panicle. From the upper part to the base of the panicle, non-shrunken seeds gradually decreased but slightly and moderately shrunken seeds gradually increased. The above results indicated that the differently shrunken seeds in transgenic rice line had the same SBE dosage, and the starch morphology and structure had no relationship with seed plumpness. The grain position on the panicle influenced seed plumpness, but had no effect on starch properties.