Regulatory loops between rice transcription factors OsNAC25 and OsNAC20/26 balance starch synthesis.

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 overexpression 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 three 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.

RGA1 regulates grain size, rice quality and seed germination in the small and round grain mutant srg5.

BACKGROUND: Generating elite rice varieties with high yield and superior quality is the main goal of rice breeding programs. Key agronomic traits, including grain size and seed germination characteristics, affect the final yield and quality of rice. The RGA1 gene, which encodes the α-subunit of rice G-protein, plays an important role in regulating rice architecture, seed size and abiotic stress responses. However, whether RGA1 is involved in the regulation of rice quality and seed germination traits is still unclear. RESULTS: In this study, a rice mutant small and round grain 5 (srg5), was identified in an EMS-induced rice mutant library. Systematic analysis of its major agronomic traits revealed that the srg5 mutant exhibited a semi-dwarf plant height with small and round grain and reduced panicle length. Analysis of the physicochemical properties of rice showed that the difference in rice eating and cooking quality (ECQ) between the srg5 mutant and its wild-type control was small, but the appearance quality was significantly improved. Interestingly, a significant suppression of rice seed germination and shoot growth was observed in the srg5 mutant, which was mainly related to the regulation of ABA metabolism. RGA1 was identified as the candidate gene for the srg5 mutant by BSA analysis. A SNP at the splice site of the first intron disrupted the normal splicing of the RGA1 transcript precursor, resulting in a premature stop codon. Additional linkage analysis confirmed that the target gene causing the srg5 mutant phenotype was RGA1. Finally, the introduction of the RGA1 mutant allele into two indica rice varieties also resulted in small and round rice grains with less chalkiness. CONCLUSIONS: These results indicate that RGA1 is not only involved in the control of rice architecture and grain size, but also in the regulation of rice quality and seed germination. This study sheds new light on the biological functions of RGA1, thereby providing valuable information for future systematic analysis of the G-protein pathway and its potential application in rice breeding programs.

Development of introgression lines and mapping of qGW2, a novel QTL that confers grain width, in rice (Oryza sativa L.).

Rice grain size is a key determinant of both grain yield and quality. Identification of favorable alleles for use in rice breeding may help to meet the demand for increased yield. In this study, we developed a set of 210 introgression lines (ILs) by using indica variety Huanghuazhan as the donor parent and erect-panicle japonica rice variety Wuyujing3R as the recurrent parent. A total of 133 ILs were selected for high-throughput sequencing. Using specific-locus amplified fragment (SLAF) sequencing technology, 10,103 high-quality SLAF labels evenly distributed on 12 chromosomes were obtained and selected for subsequent analysis. Using a high-density map, quantitative trait locus (QTL) mapping of grain size-related traits was performed, and a total of 38 QTLs were obtained in two environments. Furthermore, qGW2, a novel QTL that controls grain width on chromosome 2, was validated and delimited to a region of 309 kb via substitution mapping. These findings provide new genetic material and a basis for future fine mapping and cloning of favorable QTLs. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01453-0.

Lysine-rich rice partially enhanced the growth and development of skeletal system with better skeletal microarchitecture in young rats.

Rice is the primary staple food for half of the world's population but is low in lysine content. Previously, we developed transgenic rice with enhanced free lysine content in rice seeds (lysine-rich rice), which was shown safe for consumption and improved the growth in rats. However, the effects of lysine-rich rice on skeletal growth and development remained unknown. In this study, we hypothesized that lysine-rich rice improved skeletal growth and development in weaning rats. Male weaning Sprague-Dawley rats received lysine-rich rice (HFL) diet, wild-type rice (WT) diet, or wild-type rice with various contents of lysine supplementation diet for 70 days. Bone microarchitectures were examined by microcomputed tomography, bone strength was investigated by mechanical test, and dynamics of bone growth were examined by histomorphometric analysis. In addition, we explored the molecular mechanism of lysine and skeletal growth through biochemical testing of growth hormone, bone turnover marker, and amino acid content of rat serum analysis, as well as in a cell culture system. Results indicated that the HFL diet improved rats' bone growth, strength, and microarchitecture compared with the WT diet group. In addition, the HFL diet increased the serum essential amino acids, growth hormone (insulin-like growth factor-1), and bone formation marker concentrations. The cell culture model showed that lysine deficiency reduced insulin-like growth factor-1 and Osterix expression, Akt/mammalian target of rapamycin signaling, and matrix mineralization, and inhibited osteoblast differentiation associated with bone growth. Our findings showed that lysine-rich rice improved skeletal growth and development in weaning rats. A further increase of rice lysine content is highly desirable to fully optimize bone growth and development.

Two ABCI family transporters, OsABCI15 and OsABCI16, are involved in grain-filling in rice.

Seed development is critical for plant reproduction and crop yield, with panicle seed-setting rate, grain-filling, and grain weight being key seed characteristics for yield improvement. However, few genes are known to regulate grain filling. Here, we identify two adenosine triphosphate (ATP)-binding cassette (ABC)I-type transporter genes, OsABCI15 and OsABCI16, involved in rice grain-filling. Both genes are highly expressed in developing seeds, and their proteins are localized to the plasma membrane and cytosol. Interestingly, knockout of OsABCI15 and OsABCI16 results in a significant reduction in seed-setting rate, caused predominantly by the severe empty pericarp phenotype, which differs from the previously reported low seed-setting phenotype resulting from failed pollination. Further analysis indicates that OsABCI15 and OsABCI16 participate in ion homeostasis and likely export ions between filial tissues and maternal tissues during grain filling. Importantly, overexpression of OsABCI15 and OsABCI16 enhances seed-setting rate and grain yield in transgenic plants and decreases ion accumulation in brown rice. Moreover, the OsABCI15/16 orthologues in maize exhibit a similar role in kernel development, as demonstrated by their disruption in transgenic maize. Therefore, our findings reveal the important roles of two ABC transporters in cereal grain filling, highlighting their value in crop yield improvement.

Effect of melting combined with ice recrystallization on porous starch preparation: Pore-forming properties, granular morphology, functionality, and multi-scale structures.

In this work, critical melting (CM) combined with freeze-thawing treatment (FT, freezing at -20 â„ƒ and -80 â„ƒ, respectively) was used to prepare porous starch. The results showed that CM combined with the slow freezing rate (-20 â„ƒ) can prepare porous starch with characteristics of grooves and cavities, while combined with the rapid freezing rate (-80 â„ƒ) can prepare with holes and channels, especially after repeating FT cycles. Compared with the native counterpart, the specific surface area, pore volume, and average diameter of CMFT-prepared porous starch were significantly increased to 4.07 m2/g, 7.29 cm3/g × 10-3, and 3.57 nm, respectively. CMFT significantly increased the thermal stability of starch, in which the To, Tp, and Tc significantly increased from 63.32, 69.62, and 72.90 (native) to ∼69, 72, and 76 °C, respectively. CMFT significantly increased water and oil absorption of porous starch from 91.20 % and 72.00 % (native) up to ∼163 % and 94 %, respectively. Moreover, CMFT-prepared porous starch had a more ordered double-helical structure, which showed in the significantly increased relative crystallinity, semi-crystalline lamellae structure, and the proportion of the double helix structure of starch. The synergistic effect of melting combined with ice recrystallization can be used as an effective way to prepare structure-stabilized porous starch.

Mapping of Rf20(t), a minor fertility restorer gene for rice wild abortive cytoplasmic male sterility in the maintainer line 'Zhenshan97B'.

Three-line hybrid rice has primarily been developed on wild abortive (WA)-type cytoplasmic male sterility (CMS) and has helped increase the yield of rice globally. The development of WA-type CMS lines and hybrids was expedited through the identification and mapping of the fertility restorer gene (Rf) in maintainers. This study observed fertile plants in WA-TianfengA/Zhenshan97B//TianfengB population, indicating that the maintainer line 'Zhenshan97B' should carry Rfs for WA-type CMS. Several advanced backcross populations were generated with the genetic background of the 'WA-TianfengA,' and the pollen fertility levels of the backcrossed individuals in BC3F1, BC4F1 and BC4F2 populations are governed by a new gene, Rf20(t), from 'Zhenshan97B.' Employing bulk segregant analysis of fertile and sterile pools from the BC4F1 population, Rf20(t) was genetically mapped to a candidate region on chromosome 10. Subsequently, Rf20(t) was located between RM24883 and RM24919 through recombination analysis of molecular markers using the BC4F2 population. Implementing a substitution mapping strategy, Rf20(t) was ultimately mapped to a 245-kb region between the molecular markers STS10-122 and STS10-126 and obtained the most likely candidate gene LOC_Os10g02650, which is predicted to encode pentatricopeptide repeat-containing (PPR) protein. These results enhance our understanding of the fertility restoration of WA-type CMS lines, facilitating the development of high-quality pairs of WA-type CMS and maintainer lines.

Revelation of rice molecular design breeding: the blend of tradition and modernity.

As one of the major staple crops, rice feeds more than one half of the world population. Due to increasing population and dramatic climate change, the rice varieties with higher yield performance and excellent overall agronomic performance should be developed. The raise of molecular design breeding concept provides opportunity to get new breakthrough for variety development, and it is important to clarify the efficient gene combination during actual breeding. In this review, we summarize the recent advances about rice variety improvement either by marker assisted selection (MAS) breeding or popular gene editing technique, which will be beneficial to understand different aspects of the molecular design breeding. We provide genetic views for the classical MAS application, including the genetic effect of key genes and their combinations, the recurrent genome recovery rate at different backcross generations, linkage drag and recombination selection. Moreover, we compare the breeding value of recently-developed molecular techniques, including the advantage of high-throughput genotyping and the way and effect of gene editing in creating useful traits. Considering the current status and actual demands of rice breeding, we raise the strategy to take advantages of both traditional breeding resources and popular molecular techniques, which might pave the way to optimize the process of molecular design breeding in future.

Gene editing of non-coding regulatory DNA and its application in crop improvement.

The development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system has provided precise and efficient strategies to edit target genes and generate transgene-free crops. Significant progress has been made in the editing of protein-coding genes; however, studies on the editing of non-coding DNA with regulatory roles lags far behind. Non-coding regulatory DNAs, including those which can be transcribed into long non-coding RNAs (lncRNAs), and miRNAs, together with cis-regulatory elements (CREs), play crucial roles in regulating plant growth and development. Therefore, the combination of CRISPR/Cas technology and non-coding regulatory DNA has great potential to generate novel alleles that affect various agronomic traits of crops, thus providing valuable genetic resources for crop breeding. Herein, we review recent advances in the roles of non-coding regulatory DNA, attempts to edit non-coding regulatory DNA for crop improvement, and potential application of novel editing tools in modulating non-coding regulatory DNA. Finally, the existing problems, possible solutions, and future applications of gene editing of non-coding regulatory DNA in modern crop breeding practice are also discussed.

The OsNAC24-OsNAP protein complex activates OsGBSSI and OsSBEI expression to fine-tune starch biosynthesis in rice endosperm.

Starch accounts for up to 90% of the dry weight of rice endosperm and is a key determinant of grain quality. Although starch biosynthesis enzymes have been comprehensively studied, transcriptional regulation of starch-synthesis enzyme-coding genes (SECGs) is largely unknown. In this study, we explored the role of a NAC transcription factor, OsNAC24, in regulating starch biosynthesis in rice. OsNAC24 is highly expressed in developing endosperm. The endosperm of osnac24 mutants is normal in appearance as is starch granule morphology, while total starch content, amylose content, chain length distribution of amylopectin and the physicochemical properties of the starch are changed. In addition, the expression of several SECGs was altered in osnac24 mutant plants. OsNAC24 is a transcriptional activator that targets the promoters of six SECGs; OsGBSSI, OsSBEI, OsAGPS2, OsSSI, OsSSIIIa and OsSSIVb. Since both the mRNA and protein abundances of OsGBSSI and OsSBEI were decreased in the mutants, OsNAC24 functions to regulate starch synthesis mainly through OsGBSSI and OsSBEI. Furthermore, OsNAC24 binds to the newly identified motifs TTGACAA, AGAAGA and ACAAGA as well as the core NAC-binding motif CACG. Another NAC family member, OsNAP, interacts with OsNAC24 and coactivates target gene expression. Loss-of-function of OsNAP led to altered expression in all tested SECGs and reduced the starch content. These results demonstrate that the OsNAC24-OsNAP complex plays key roles in fine-tuning starch synthesis in rice endosperm and further suggest that manipulating the OsNAC24-OsNAP complex regulatory network could be a potential strategy for breeding rice cultivars with improved cooking and eating quality.

Highly sensitive and selective Sb2WO6 microspheres in detecting VOC biomarkers in cooked rice: Experimental and density functional theory study.

The palatability of cooked rice is susceptible to the flavor and effective detection of volatile organic compounds (VOCs) can avoid deterioration and improve the taste quality. Herein, hierarchical antimony tungstate (Sb2WO6) microspheres are synthesized through a solvothermal process and the effect of solvothermal temperature on the room temperature gas-sensing properties of gas sensors is investigated. Outstanding sensitivity towards VOC biomarkers (nonanal, 1-octanol, geranyl acetone and 2-pentylfuran) in cooked rice is achieved and the sensors exhibit remarkable stability and reproducibility, which are contributed to the formation of the hierarchical microsphere structure, larger specific surface area, narrower band gap and increased oxygen vacancy content. The kinetic parameters combined with principal component analysis (PCA) effectively distinguish the four VOCs while the enhanced sensing mechanism was substantiated through density functional theory (DFT) calculation. This work provides a strategy for fabricating high performance Sb2WO6 gas sensors which can be practically applied to food industry.

Effects of enhanced starch-xanthan gum synergism on their physicochemical properties, functionalities, structural characteristics, and digestibility.

The limited and unstable interactions between potato starch (PS) and xanthan gum (XG) by simple mixing (SM) lead it difficult to induce substantial changes in starchy products. Structural unwinding and rearrangement of PS and XG by critical melting and freeze-thawing (CMFT) were used to promote PS/XG synergism, and the physicochemical, functionalities, and structural properties were investigated. Compared to "Native" and SM, CMFT promoted the formation of large clusters with a rough granular surface and wrapped by a matrix composed of released soluble starches and XG (SEM), thus making the composite more compact to thermal processes, such as the significantly decreased WSI and SP, and increased the melting temperatures. The enhanced synergism of PS/XG after CMFT effectively decreased the breakdown viscosity from ~3600 (Native) to ~300 mPa·s and increased the final viscosity from ~2800 (Native) to ~4800. CMFT significantly increased the functional properties of PS/XG composite, including water/oil absorptions and resistant starch content. CMFT caused the partial melting and loss of large packaged structures in starch (XRD, FTIR, and NMR), and the melting and the loss of crystalline structure controlled at approximately 20 % and 30 %, respectively, are the most effective for promoting PS/XG interaction.

Combined effects of SSII-2RNAi and different Wx alleles on rice grain transparency and physicochemical properties.

Near-isogenic lines Nip(Wxb/SSII-2), Nip(Wxb/ss2-2), Nip(Wxmw/SSII-2), Nip(Wxmw/ss2-2), Nip(Wxmp/SSII-2) and Nip(Wxmp/ss2-2) in the Nipponbare (Nip) background containing the SSII-2RNAi cassette combined with different Waxy (Wx) alleles were investigated in terms of rice grain transparency and quality profiles. Rice lines carrying the SSII-2RNAi cassette displayed downregulation of SSII-2, SSII-3 and Wx genes. Introduction of the SSII-2RNAi cassette decreased apparent amylose content (AAC) in all transgenic lines, but grain transparency differed between low AAC rice lines. Grains from Nip(Wxb/SSII-2) and Nip(Wxb/ss2-2) were transparent, while those of rice were increasingly translucent with decreasing moisture due to cavities within starch granules. Rice grain transparency was positively correlated with grain moisture and AAC, but negatively correlated with cavity area within starch granules. Starch fine structure analysis revealed a marked increase in short amylopectin chains with DP 6-12, but a decrease in intermediate chains with DP 13-24, resulting in decreased gelatinisation temperature. Starch crystalline structure analysis showed that the transgenic rice starches have lower crystallinity and lamellar repeat distance than controls due to differences in starch fine structure. The results highlight the molecular basis underpinning rice grain transparency, and provide strategies for improving rice grain transparency.

Co-Overexpression of Two Key Source Genes, OsBMY4 and OsISA3, Improves Multiple Key Traits of Rice Seeds.

Optimized source-sink interactions are determinants of both rice yield and quality. However, most source genes have not been well studied in rice, a major grain crop. In this study, OsBMY4 and OsISA3, the key ß-amylase and debranching enzymes that control transient starch degradation in rice leaves, were co-overexpressed in rice in order to accelerate starch degradation efficiency and increase the sugar supply for sink organs. Systematic analyses of the transgenic rice indicated that co-overexpression of OsBMY4 and OsISA3 not only promoted rice yield and quality, but also improved seed germination and stress tolerance. Moreover, since the OsBMY4 gene has not been characterized, we generated osbmy4 mutants using CRIPSR/Cas9 gene editing, which helped to reveal the roles of ß-amylase in rice yield and quality. This study demonstrated that specific modulation of the expression of some key source genes improves the source-sink balance and leads to improvements in multiple key traits of rice seeds.

Genetic architecture of seed glycerolipids in Asian cultivated rice.

Glycerolipids are essential for rice development and grain quality but its genetic regulation remains unknown. Here we report its genetic base using metabolite-based genome-wide association study and metabolite-based quantitative traits locus (QTL) analyses based on lipidomic profiles of seeds from 587 Asian cultivated rice accessions and 103 chromosomal segment substitution lines, respectively. We found that two genes encoding phosphatidylcholine (PC):diacylglycerol cholinephosphotransferase (OsLP1) and granule-bound starch synthase I (Waxy) contribute to variations in saturated triacylglycerol (TAG) and lyso-PC contents, respectively. We demonstrated that allelic variation in OsLP1 sequence between indica and japonica results in different enzymatic preference for substrate PC-16:0/16:0 and different saturated TAG levels. Further evidence demonstrated that OsLP1 also affects heading date, and that co-selection of OsLP1 and a flooding-tolerant QTL in Aus results in the abundance of saturated TAGs associated with flooding tolerance. Moreover, we revealed that the sequence polymorphisms in Waxy has pleiotropic effects on lyso-PC and amylose content. We proposed that rice seed glycerolipids have been unintentionally shaped during natural and artificial selection for adaptive or import seed quality traits. Collectively, our findings provide valuable genetic resources for rice improvement and evolutionary insights into seed glycerolipid variations in rice.

The Effects of Rf4 and the Genetic Mechanism Behind Fertility Restoration of Wild Abortive Cytoplasmic Male Sterility (WA-CMS) in Japonica Rice (Oryza sativa ssp. Japonica).

Wild abortive-type cytoplasmic male sterility (WA-type CMS) has been exclusively used in hybrid seed production in indica rice cultivars, and fertility restoration in WA-type CMS is controlled by two major restorer genes, Rf3 and Rf4, through a sporophytic mechanism. However, the genetic mechanism underlying fertility restoration in WA-type CMS in japonica cultivars is poorly understood. In the present study, C418, a leading Chinsurah Boro II- (BT)-type japonica restorer line, showed partial restoration ability in WA-type japonica CMS lines. The 1:1 segregation ratio of partially fertile to sterile plants in a three-cross F1 population indicated that fertility restoration is controlled by one dominant gene. Gene mapping and sequencing results revealed that the target gene should be Rf4. The Rf4 gene restores fertility through a sporophytic mechanism, but the Rf4 pollen grains show a preferential fertilization in the testcross F1 plants. Furthermore, Rf4 was confirmed to have only a minor effect on fertility restoration in WA-type japonica CMS lines, and Rf gene dosage effects influenced the fertility restoration of WA-type CMS in japonica rice. The results of our study not only provide valuable insights into the complex genetic mechanisms underlying fertility restoration of WA-type CMS but will also facilitate the efficient utilization of WA-type CMS in japonica rice lines.