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 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.

Glycosylphosphatidylinositol anchor lipid remodeling directs proteins to the plasma membrane and governs cell wall mechanics.

Glycosylphosphatidylinositol (GPI) anchoring is a common protein modification that targets proteins to the plasma membrane (PM). Knowledge about the GPI lipid tail, which guides the secretion of GPI-anchored proteins (GPI-APs), is limited in plants. Here, we report that rice (Oryza sativa) BRITTLE CULM16 (BC16), a membrane-bound O-acyltransferase (MBOAT) remodels GPI lipid tails and governs cell wall biomechanics. The bc16 mutant exhibits fragile internodes, resulting from reduced cell wall thickness and cellulose content. BC16 is the only MBOAT in rice and is located in the endoplasmic reticulum and Golgi apparatus. Yeast gup1Δ mutant restoring assay and GPI lipid composition analysis demonstrated BC16 as a GPI lipid remodelase. Loss of BC16 alters GPI lipid structure and disturbs the targeting of BC1, a GPI-AP for cellulose biosynthesis, to the PM lipid nanodomains. Atomic force microscopy revealed compromised deposition of cellulosic nanofibers in bc16, leading to an increased Young's modulus and abnormal mechanical properties. Therefore, BC16-mediated lipid remodeling directs the GPI-APs, such as BC1, to the cell surface to fulfill multiple functions, including cellulose organization. Our work unravels a mechanism by which GPI lipids are remodeled in plants and provides insights into the control of cell wall biomechanics, offering a tool for breeding elite crops with improved support strength.

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.

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.

Brassinosteroids regulate rice seed germination through the BZR1-RAmy3D transcriptional module.

Seed dormancy and germination, two physiological processes unique to seed-bearing plants, are critical for plant growth and crop production. The phytohormone brassinosteroid (BR) regulates many aspects of plant growth and development, including seed germination. The molecular mechanisms underlying BR control of rice (Oryza sativa) seed germination are mostly unknown. We investigated the molecular regulatory cascade of BR in promoting rice seed germination and post-germination growth. Physiological assays indicated that blocking BR signaling, including introducing defects into the BR-insensitive 1 (BRI1) receptor or overexpressing the glycogen synthase kinase 2 (GSK2) kinase delayed seed germination and suppressed embryo growth. Our results also indicated that brassinazole-resistant 1 (BZR1) is the key downstream transcription factor that mediates BR regulation of seed germination by binding to the alpha-Amylase 3D (RAmy3D) promoter, which affects α-amylase expression and activity and the degradation of starch in the endosperm. The BZR1-RAmy3D module functions independently from the established Gibberellin MYB-alpha-amylase 1A (RAmy1A) module of the gibberellin (GA) pathway. We demonstrate that the BZR1-RAmy3D module also functions in embryo-related tissues. Moreover, RNA-sequencing (RNA-seq) analysis identified more potential BZR1-responsive genes, including those involved in starch and sucrose metabolism. Our study successfully identified the role of the BZR1-RAmy3D transcriptional module in regulating rice seed germination.

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.

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.

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.

Suppressing chlorophyll degradation by silencing OsNYC3 improves rice resistance to Rhizoctonia solani, the causal agent of sheath blight.

Necrotrophic fungus Rhizoctonia solani Kühn (R. solani) causes serious diseases in many crops worldwide, including rice and maize sheath blight (ShB). Crop resistance to the fungus is a quantitative trait and resistance mechanism remains largely unknown, severely hindering the progress on developing resistant varieties. In this study, we found that resistant variety YSBR1 has apparently stronger ability to suppress the expansion of R. solani than susceptible Lemont in both field and growth chamber conditions. Comparison of transcriptomic profiles shows that the photosynthetic system including chlorophyll biosynthesis is highly suppressed by R. solani in Lemont but weakly in YSBR1. YSBR1 shows higher chlorophyll content than that of Lemont, and inducing chlorophyll degradation by dark treatment significantly reduces its resistance. Furthermore, three rice mutants and one maize mutant that carry impaired chlorophyll biosynthesis all display enhanced susceptibility to R. solani. Overexpression of OsNYC3, a chlorophyll degradation gene apparently induced expression by R. solani infection, significantly enhanced ShB susceptibility in a high-yield ShB-susceptible variety '9522'. However, silencing its transcription apparently improves ShB resistance without compromising agronomic traits or yield in field tests. Interestingly, altering chlorophyll content does not affect rice resistance to blight and blast diseases, caused by biotrophic and hemi-biotrophic pathogens, respectively. Our study reveals that chlorophyll plays an important role in ShB resistance and suppressing chlorophyll degradation induced by R. solani infection apparently improves rice ShB resistance. This discovery provides a novel target for developing resistant crop to necrotrophic fungus R. solani.

Lysine biofortification of crops to promote sustained human health in the 21st century.

Crop biofortification is pivotal in preventing malnutrition, with lysine considered the main limiting essential amino acid (EAA) required to maintain human health. Lysine deficiency is predominant in developing countries where cereal crops are the staple food, highlighting the need for efforts aimed at enriching the staple diet through lysine biofortification. Successful modification of aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS) feedback inhibition has been used to enrich lysine in transgenic rice plants without yield penalty, while increases in the lysine content of quality protein maize have been achieved via marker-assisted selection. Here, we reviewed the lysine metabolic pathway and proposed the use of metabolic engineering targets as the preferred option for fortification of lysine in crops. Use of gene editing technologies to translate the findings and engineer lysine catabolism is thus a pioneering step forward.

qFC6, a major gene for crude fat content and quality in rice.

KEY MESSAGE: qFC6, a major quantitative trait locus for rice crude fat content, was fine mapped to be identical with Wx. FC6 negatively regulates crude fat content and rice quality. Starch, protein and lipids are the three major components in rice endosperm. The lipids content in rice influences both storage and quality. In this study, we identified a quantitative trait locus (QTL), qFC6, for crude fat (free lipids) content through association analysis and linkage analysis. Gene-based association analysis revealed that LOC_Os06g04200, also known as Wx, was the candidate gene for qFC6. Complementation and knockout transgenic lines revealed that Wx negatively regulates crude fat content. Lipid composition and content analysis by gas chromatography and taste evaluation analysis showed that FC6 positively influenced bound lipids content and negatively affected both free lipids content and taste. Besides, higher free lipids content rice varieties exhibit more lustrous appearance after cooking and by adding extra oil during cooking could improve rice luster and taste score, indicating that higher free lipids content may make rice more lustrous and delicious. Together, we cloned a QTL coordinating rice crude fat content and eating quality and assisted in uncovering the genetic basis of rice lipid content and in the improvement of rice eating quality.

Characterization of qPL5: a novel quantitative trait locus (QTL) that controls panicle length in rice (Oryza sativa L.).

Panicle length (PL) is an important trait that determines panicle architecture and strongly affects grain yield and quality in rice. However, this trait has not been well characterized genetically, and its contribution to yield improvement is not well understood. Characterization of novel genes related to PL is of great significance for breeding high-yielding rice varieties. In our previous research, we identified qPL5, a quantitative trait locus for PL. In this study, we aimed to determine the exact position of qPL5 in the rice genome and identify the candidate gene. Through substitution mapping, we mapped qPL5 to a region of 21.86 kb flanked by the molecular marker loci STS5-99 and STS5-106 in which two candidate genes were predicted. By sequence analysis and relative expression analysis, LOC-Os05g41230, which putatively encodes a BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 precursor, was considered to be the most likely candidate gene for qPL5. In addition, we successfully developed a pair of near-isogenic lines (NILs) for qPL5 in different genetic backgrounds to evaluate the genetic effects of qPL5. Agronomic trait analysis of the NILs indicated that qPL5 positively contributes to plant height, grain number per panicle, panicle length, grain yield per plant, and flag leaf length, but it had no influence on heading date and grain-size-related traits. Therefore, qPL5 and the markers tightly linked to it should be available for molecular breeding of high-yielding varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01339-z.

Allelic Diversification of the Wx and ALK Loci in Indica Restorer Lines and Their Utilisation in Hybrid Rice Breeding in China over the Last 50 Years.

Hybrid rice technology has been used for more than 50 years, and eating and cooking quality (ECQ) has been a major focus throughout this period. Waxy (Wx) and alkaline denaturation (ALK) genes have received attention owing to their pivotal roles in determining rice characteristics. However, despite significant effort, the ECQ of restorer lines (RLs) has changed very little. By contrast, obvious changes have been seen in inbred rice varieties (IRVs), and the ECQ of IRVs is influenced by Wx, which reduces the proportion of Wxa and increases the proportion of Wxb, leading to a decrease in amylose content (AC) and an increase in ECQ. Meanwhile, ALK is not selected in the same way. We investigated Wx alleles and AC values of sterile lines of female parents with the main mating combinations in widely used areas. The results show that almost all sterile lines were Wxa-type with a high AC, which may explain the low ECQ of hybrid rice. Analysis of hybrid rice varieties and RLs in the last 5 years revealed serious homogenisation among hybrid rice varieties.

Improving rice eating and cooking quality by coordinated expression of the major starch synthesis-related genes, SSII and Wx, in endosperm.

KEY MESSAGE: Coordinated regulation of amylose and amylopectin synthesis via manipulation of SSII-2, SSII-3 and Wx expression in endosperm can improve rice eating and cooking quality. With increasing rice consumption worldwide, many researchers are working to increase the yield and improve grain quality, especially eating and cooking quality (ECQ). The rice ECQ is mainly controlled by the expression of starch synthesis-related genes (SSRGs) in endosperm. Although the Wx and SSII-3/SSIIa/ALK genes, two major SSRGs, have been manipulated to improve rice ECQ via various breeding approaches, new methods to further improve ECQ are desired. In our previous study, we enhanced rice ECQ by knocking down SSII-2 expression in the japonica Nipponbare cultivar (carrying the Wxb allele) via RNA interference. Herein, the SSII-2 RNAi was introduced into two Nipponbare-derived near-isogenic lines (NILs), Nip(Wxa) and Nip(wx), carrying Wxa and wx alleles respond for high and no amylose levels, respectively. Analysis of physicochemical properties revealed that the improved grain quality of SSII-2 RNAi transgenic lines was achieved by coordinated downregulating the expression of SSII-2, SSII-3 and Wx. To further confirm this conclusion, we generated ssii-2, ssii-3 and ssii-2ssii-3 mutants via CRISPR/Cas9 technique. The amylopectin structure of the resulting ssii-2sii-3 mutants was similar to that in SSII-2 RNAi transgenic lines, and the absence of SSII-2 decreased the amylose content, gelatinisation temperature and rapid visco-analyser profile, indicating essential roles for SSII-2 in the regulation of amylopectin biosynthesis and amylose content in rice endosperm. The effect of SSII-2 was seen only when the activity of SSII-3 was very low or lacking. Our study provides novel approaches and valuable germplasm resources for improving ECQ via plant breeding.

Lysine biofortification in rice by modulating feedback inhibition of aspartate kinase and dihydrodipicolinate synthase.

Lysine is the main limiting essential amino acid (EAA) in the rice seeds, which is a major energy and nutrition source for humans and livestock. In higher plants, the rate-limiting steps in lysine biosynthesis pathway are catalysed by two key enzymes, aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS), and both are extremely sensitive to feedback inhibition by lysine. In this study, two rice AK mutants (AK1 and AK2) and five DHDPS mutants (DHDPS1-DHDPS5), all single amino acid substitution, were constructed. Their protein sequences passed an allergic sequence-based homology alignment. Mutant proteins were recombinantly expressed in Escherichia coli, and all were insensitive to the lysine analog S-(2-aminoethyl)-l-cysteine (AEC) at concentrations up to 12 mm. The AK and DHDPS mutants were transformed into rice, and free lysine was elevated in mature seeds of transgenic plants, especially those expressing AK2 or DHDPS1, 6.6-fold and 21.7-fold higher than the wild-type (WT) rice, respectively. We then engineered 35A2D1L plants by simultaneously expressing modified AK2 and DHDPS1, and inhibiting rice LKR/SDH (lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase). Free lysine levels in two 35A2D1L transgenic lines were 58.5-fold and 39.2-fold higher than in WT and transgenic rice containing native AK and DHDPS, respectively. Total free amino acid and total protein content were also elevated in 35A2D1L transgenic rice. Additionally, agronomic performance analysis indicated that transgenic lines exhibited normal plant growth, development and seed appearance comparable to WT plants. Thus, AK and DHDPS mutants may be used to improve the nutritional quality of rice and other cereal grains.

Gibberellins orchestrate panicle architecture mediated by DELLA-KNOX signalling in rice.

Panicle architecture is a key determinant of grain yield in cereals, but the mechanisms governing panicle morphogenesis and organ development remain elusive. Here, we have identified a quantitative trait locus (qPA1) associated with panicle architecture using chromosome segment substitution lines from parents Nipponbare and 9311. The panicle length, branch number and grain number of Nipponbare were significantly higher than CSSL-9. Through map-based cloning and complementation tests, we confirmed that qPA1 was identical to SD1 (Semi Dwarf1), which encodes a gibberellin 20-oxidase enzyme participating in gibberellic acid (GA) biosynthesis. Transcript analysis revealed that SD1 was widely expressed during early panicle development. Analysis of sd1/osga20ox2 and gnp1/ osga20ox1 single and double mutants revealed that the two paralogous enzymes have non-redundant functions during panicle development, likely due to differences in spatiotemporal expression; GNP1 expression under control of the SD1 promoter could rescue the sd1 phenotype. The DELLA protein SLR1, a component of the GA signalling pathway, accumulated more highly in sd1 plants. We have demonstrated that SLR1 physically interacts with the meristem identity class I KNOTTED1-LIKE HOMEOBOX (KNOX) protein OSH1 to repress OSH1-mediated activation of downstream genes related to panicle development, providing a mechanistic link between gibberellin and panicle architecture morphogenesis.

Glucan, Water-Dikinase 1 (GWD1), an ideal biotechnological target for potential improving yield and quality in rice.

The source-sink relationship determines the overall agronomic performance of rice. Cloning and characterizing key genes involved in the regulation of source and sink dynamics is imperative for improving rice yield. However, few source genes with potential application in rice have been identified. Glucan, Water-Dikinase 1 (GWD1) is an essential enzyme that plays a pivotal role in the first step of transitory starch degradation in source tissues. In the present study, we successfully generated gwd1 weak mutants by promoter editing using CRISPR/Cas9 system, and also leaf-dominant overexpression lines of GWD1 driven by Osl2 promoter. Analysis of the gwd1 plants indicated that promoter editing mediated down-regulation of GWD1 caused no observable effects on rice growth and development, but only mildly modified its grain transparency and seed germination. However, the transgenic pOsl2::GWD1 overexpression lines showed improvements in multiple key traits, including rice yield, grain shape, rice quality, seed germination and stress tolerance. Therefore, our study shows that GWD1 is not only involved in transitory starch degradation in source tissues, but also plays key roles in the seeds, which is a sink tissue. In conclusion, we find that GWD1 is an ideal biotechnological target with promising potential for the breeding of elite rice cultivars via genetic engineering.

Identification of Structure-Controlling Rice Biosynthesis Enzymes.

The main enzymes controlling the chain-length distributions (CLDs) of starches are starch synthases (SSs), starch branching enzymes (SBEs), and debranching enzymes (DBEs), which have various isoforms, denoted as SSI, SSII-1, etc. Different isozymes dominate the CLD in different ranges of degrees of polymerization (DPs). Models have been developed for the CLDs in terms of the activities of isoforms of these enzymes, in terms of two parameters: ßi, which is the ratio of the activity of SBE to that of SS in set i, and hi, which is the relative activity of SS in that set. These provide good fits to data but without specifying which isozymes are in set i. Here, CLDs for amylopectin and amylose synthesis in rice endosperm are explored. Molecular weight distributions of the different chains formed in 87 rice varieties were obtained using size-exclusion chromatography following enzymatic debranching (converting a complex branched macromolecule to linear polymers), and fitted by the biosynthesis-based models. The mutants of each isoform among tested rice varieties were identified by amino-acid mutations in coding sequences based on the extraction and analysis of whole gene sequences. The significant differences between mutant groups of different isoforms indicate that SSI, SSII-3, SSIII-1, SSIII-2, and SBEI as well as GBSSI (an isozyme of granule-bound starch synthase) belong to the enzymes sets that control amylose biosynthesis. Further, GBSSI is in the enzyme sets that control amylopectin chains. This enables specification of all isozymes and the DP range, which they dominate, over the entire DP range. As the CLD controls many functional properties of rice, this can help breeders target and develop improved rice species.