A wild rice CSSL population facilitated identification of salt tolerance genes and rice germplasm innovation.

Salt stress is one of the major factors that limits rice production. Therefore, identification of salt-tolerant alleles from wild rice is important for rice breeding. In this study, we constructed a set of chromosome segment substitution lines (CSSLs) using wild rice as the donor parent and cultivated rice Nipponbare (Nip) as the recurrent parent. Salt tolerance germinability (STG) was evaluated, and its association with genotypes was determined using this CSSL population. We identified 17 QTLs related to STG. By integrating the transcriptome and genome data, four candidate genes were identified, including the previously reported AGO2 and WRKY53. Compared with Nip, wild rice AGO2 has a structure variation in its promoter region and the expression levels were upregulated under salt treatments; wild rice WRKY53 also has natural variation in its promoter region, and the expression levels were downregulated under salt treatments. Wild rice AGO2 and WRKY53 alleles have combined effects for improving salt tolerance at the germination stage. One CSSL line, CSSL118 that harbors these two alleles was selected. Compared with the background parent Nip, CSSL118 showed comprehensive salt tolerance and higher yield, with improved transcript levels of reactive oxygen species scavenging genes. Our results provided promising genes and germplasm resources for future rice salt tolerance breeding.

The GW5-WRKY53-SGW5 module regulates grain size variation in rice.

Grain size is a crucial agronomic trait that affects stable yield, appearance, milling quality, and domestication in rice. However, the molecular and genetic relationships among QTL genes (QTGs) underlying natural variation for grain size remain elusive. Here, we identified a novel QTG SGW5 (suppressor of gw5) by map-based cloning using an F2 segregation population by fixing same genotype of the master QTG GW5. SGW5 positively regulates grain width by influencing cell division and cell size in spikelet hulls. Two nearly isogenic lines exhibited a significant differential expression of SGW5 and a 12.2% increase in grain yield. Introducing the higher expression allele into the genetic background containing the lower expression allele resulted in increased grain width, while its knockout resulted in shorter grain hulls and dwarf plants. Moreover, a cis-element variation in the SGW5 promoter influenced its differential binding affinity for the WRKY53 transcription factor, causing the differential SGW5 expression, which ultimately leads to grain size variation. GW5 physically and genetically interacts with WRKY53 to suppress the expression of SGW5. These findings elucidated a new pathway for grain size regulation by the GW5-WRKY53-SGW5 module and provided a novel case for generally uncovering QTG interactions underlying the genetic diversity of an important trait in crops.

C4 rice engineering, beyond installing a C4 cycle.

C4 photosynthesis in higher plants is carried out by two distinct cell types: mesophyll cells and bundle sheath cells, as a result highly concentrated carbon dioxide is released surrounding RuBisCo in chloroplasts of bundle sheath cells and the photosynthetic efficiency is significantly higher than that of C3 plants. The evolution of the dual-cell C4 cycle involved complex modifications to leaf anatomy and cell ultra-structures. These include an increase in leaf venation, the formation of Kranz anatomy, changes in chloroplast morphology in bundle sheath cells, and increases in the density of plasmodesmata at interfaces between the bundle sheath and mesophyll cells. It is predicted that cereals will be in severe worldwide shortage at the mid-term of this century. Rice is a staple food that feeds more than half of the world's population. If rice can be engineered to perform C4 photosynthesis, it is estimated that rice yield will be increased by at least 50% due to enhanced photosynthesis. Thus, the Second Green Revolution has been launched on this principle by genetically installing C4 photosynthesis into C3 crops. The studies on molecular mechanisms underlying the changes in leaf morphoanatomy involved in C4 photosynthesis have made great progress in recent years. As there are plenty of reviews discussing the installment of the C4 cycle, we focus on the current progress and challenges posed to the research regarding leaf anatomy and cell ultra-structure modifications made towards the development of C4 rice.

Different contributions of PROG1 and TAC1 to the angular kinematics of the main culm and tillers of wild rice (Oryza rufipogon).

MAIN CONCLUSION: PROG1 is necessary but insufficient for the main culm inclination while TAC1 partially takes part in it, and both genes promote tiller inclination in Asian wild rice. Asian wild rice (Oryza rufipogon), the ancestor of cultivated rice (O. sativa), has a prostrate architecture, with tillers branching from near the ground. The main culm of each plant grows upward and then tilts during the vegetative stage. Genes controlling tiller angle have been reported; however, their genetic contributions to the culm movement have not been quantified. Here, we quantified their genetic contributions to angular kinematics in the main culm and tillers. For the main culm inclination, one major QTL surrounding the PROG1 region was found. In cultivated rice, tillers firstly inclined and lately rose, while it kept inclining in wild rice. It was suggested that PROG1 affected the tiller elevation angle in the later kinematics, whereas TAC1 was weakly associated with the tiller angle in the whole vegetative stage. Micro-computed tomography (micro-CT) suggested that these angular changes are produced by the bending of culm bases. Because near-isogenic lines (NILs) of wild rice-type Prog1 and Tac1 alleles in the genetic background of cultivated rice did not show the prostrate architecture, the involvement of another gene(s) for inclination of the main culm was suggested. Our findings will not only contribute to the understanding of the morphological transition during domestication but also be used in plant breeding to precisely reproduce the ideal plant architecture by combining the effects of multiple genes.

Genetic potential of grain-related traits in rice landraces: phenomics and multi-locus association analyses.

BACKGROUND: Grain length, width, weight, and the number of grains per panicle are crucial determinants contributing to yield in cereal crops. Understanding the genetic basis of grain-related traits has been the main research object in crop science. METHODS AND RESULTS: Kerala has a collection of different rice landraces. Characterization of these valuable genetic resources for 39 distinct agro-morphological traits was carried out in two seasons from 2017 to 2019 directly in farmers field. Most characteristics were polymorphic except ligule shape, leaf angle, and panicle axis. The results of principal component analysis implied that leaf length, plant height, culm length, flag leaf length, and grain-related traits were the principal discriminatory characteristics of rice landraces. For identifying the genetic basis of key grain traits of rice, three multi locus GWAS models were performed based on 1,47,994 SNPs in 73 rice accessions. As a result, 48 quantitative trait nucleotides (QTNs) were identified to be associated with these traits. After characterization of their function and expression, 15 significant candidate genes involved in regulating grain width, number of grains per panicle, and yield were identified. CONCLUSIONS: The detected QTNs and candidate genes in this study could be further used for marker-assisted high-quality breeding of rice.

Thickness-stiffness trade-off improves lodging resistance in rice.

Lodging of cereal crops significantly reduces grain yield and quality, making lodging resistance a prime target for breeding programs. However, lodging resistance among different rice (Oryza sativa L.) cultivars in the field remains largely unknown, as is the relationship between the major properties of culms such as their morphological and mechanical properties. Here, we investigated the morphological and mechanical properties of 12 rice cultivars by considering different internodes within culms. We detected variation in these two traits among cultivars: one set of cultivars had thicker but softer culms (thickness-type), while the other set of cultivars showed stiffer but thinner culms (stiffness-type). We designate this variation as a thickness-stiffness trade-off. We then constructed a mechanical model to dissect the mechanical and/or morphological constraints of rice culms subjected to their own weight (self-weight load). Through modeling, we discovered that ear weight and the morphology of the highest internode were important for reducing deflection, which may be important factors to achieve higher lodging resistance. The mechanical theory devised in this study could be used to predict the deflection of rice culms and may open new avenues for novel mechanics-based breeding techniques.

Advances in Rice Seed Shattering.

Seed shattering is an important trait that wild rice uses to adapt to the natural environment and maintain population reproduction, and weedy rice also uses it to compete with the rice crop. The loss of shattering is a key event in rice domestication. The degree of shattering is not only one of the main reasons for rice yield reduction but also affects its adaptability to modern mechanical harvesting methods. Therefore, it is important to cultivate rice varieties with a moderate shattering degree. In this paper, the research progress on rice seed shattering in recent years is reviewed, including the physiological basis, morphological and anatomical characteristics of rice seed shattering, inheritance and QTL/gene mapping of rice seed shattering, the molecular mechanism regulating rice seed shattering, the application of seed-shattering genes, and the relationship between seed-shattering genes and domestication.

CRISPR-Cas9 Mediated Mutation in OsPUB43 Improves Grain Length and Weight in Rice by Promoting Cell Proliferation in Spikelet Hull.

Grain weight, a crucial trait that determines the grain yield in rice, is influenced by grain size. Although a series of regulators that control grain size have been identified in rice, the mechanisms underlying grain development are not yet well understood. In this study, we identified OsPUB43, a U-box E3 ubiquitin ligase, as an important negative regulator determining the gain size and grain weight in rice. Phenotypes of large grain are observed in ospub43 mutants, whereas overexpression of OsPUB43 results in short grains. Scanning electron microscopy analysis reveals that OsPUB43 modulates the grain size mainly by inhibiting cell proliferation in the spikelet hull. The OsPUB43 protein is localized in the cytoplasm and nucleus. The ospub43 mutants display high sensitivity to exogenous BR, while OsPUB43-OE lines are hyposensitive to BR. Furthermore, the transient transcriptional activity assay shows that OsBZR1 can activate the expression of OsPUB43. Collectively, our results indicate that OsPUB43 negatively controls the gain size by modulating the expression of BR-responsive genes as well as MADS-box genes that are required for lemma/palea specification, suggesting that OsPUB43 has a potential valuable application in the enlargement of grain size in rice.

A New RING Finger Protein, PLANT ARCHITECTURE and GRAIN NUMBER 1, Affects Plant Architecture and Grain Yield in Rice.

Developing methods for increasing the biomass and improving the plant architecture is important for crop improvement. We herein describe a gene belonging to the RING_Ubox (RING (Really Interesting New Gene) finger domain and U-box domain) superfamily, PLANT ARCHITECTURE and GRAIN NUMBER 1 (PAGN1), which regulates the number of grains per panicle, the plant height, and the number of tillers. We used the CRISPR/Cas9 system to introduce loss-of-function mutations to OsPAGN1. Compared with the control plants, the resulting pagn1 mutant plants had a higher grain yield because of increases in the plant height and in the number of tillers and grains per panicle. Thus, OsPAGN1 may be useful for the genetic improvement of plant architecture and yield. An examination of evolutionary relationships revealed that OsPAGN1 is highly conserved in rice. We demonstrated that OsPAGN1 can interact directly with OsCNR10 (CELL NUMBER REGULATOR10), which negatively regulates the number of rice grains per panicle. A transcriptome analysis indicated that silencing OsPAGN1 affects the levels of active cytokinins in rice. Therefore, our findings have clarified the OsPAGN1 functions related to rice growth and grain development.

QTL mapping and candidate gene mining of flag leaf size traits in Japonica rice based on linkage mapping and genome-wide association study.

BACKGROUND: As one of the most important factors of the japonica rice plant, leaf shape affects the photosynthesis and carbohydrate accumulation directly. Mining and using new leaf shape related genes/QTLs can further enrich the theory of molecular breeding and accelerate the breeding process of japonica rice. METHODS: In the present study, 2 RILs and a natural population with 295 japonica rice varieties were used to map QTLs for flag leaf length (FL), flag leaf width (FW) and flag leaf area (FLA) by linkage analysis and genome-wide association study (GWAS) throughout 2 years. RESULTS: A total of 64 QTLs were detected by 2 ways, and pleiotropic QTLs qFL2 (Chr2_33,332,579) and qFL10 (Chr10_10,107,835; Chr10_10,230,100) consisted of overlapping QTLs mapped by linkage analysis and GWAS throughout the 2 years were identified. CONCLUSIONS: The candidate genes LOC_Os02g54254, LOC_Os02g54550, LOC_Os10g20160, LOC_Os10g20240, LOC_Os10g20260 were obtained, filtered by linkage disequilibrium (LD), and haplotype analysis. LOC_Os10g20160 (SD-RLK-45) showed outstanding characteristics in quantitative real-time PCR (qRT-PCR) analysis in leaf development period, belongs to S-domain receptor-like protein kinases gene and probably to be a main gene regulating flag leaf width of japonica rice. The results of this study provide valuable resources for mining the main genes/QTLs of japonica rice leaf development and molecular breeding of japonica rice ideal leaf shape.

SYL3-k increases style length and yield of F1 seeds via enhancement of endogenous GA4 content in Oryza sativa L. pistils.

KEY MESSAGE: SYL3-k allele increases the outcrossing rate of male sterile line and the yield of hybrid F1 seeds via enhancement of endogenous GA4 content in Oryza sativa L. pistils. The change in style length might be an adaptation of rice cultivation from south to north in the northern hemisphere. The style length (SYL) in rice is one of the major factors influencing the stigma exertion, which affects the outcross rate of male sterile line and the yield of hybrid F1 seeds. However, the biological mechanisms underlying SYL elongation remain elusive. Here, we report a map-based cloning and characterisation of the allele qSYL3-k. The qSYL3-k allele encodes a MADS-box family transcription factor, and it is expressed in various rice organs. The qSYL3-k allele increases SYL via the elongation of cell length in the style, which is associated with a higher GA4 content in the pistil. The expression level of OsGA3ox2 in pistils with qSYL3-k alleles is significantly higher than that in pistils with qSYL3-n allele on the same genome background of Nipponbare. The yield of F1 seeds harvested from plants with 7001SSYL3-k alleles was 16% higher than that from plants with 7001SSYL3-n allele. The sequence data at the qSYL3 locus in 136 accessions showed that alleles containing the haplotypes qSYL3AA, qSYL3AG, and qSYL3GA increased SYL, whereas those containing the haplotype qSYL3GG decreased it. The frequency of the haplotype qSYL3GG increases gradually from the south to north in the northern hemisphere. These findings will facilitate improvement in SYL and yield of F1 seeds henceforward.

Pinpointing genomic regions associated with root system architecture in rice through an integrative meta-analysis approach.

KEY MESSAGE: Applying an integrated meta-analysis approach led to identification of meta-QTLs/ candidate genes associated with rice root system architecture, which can be used in MQTL-assisted breeding/ genetic engineering of root traits. Root system architecture (RSA) is an important factor for facilitating water and nutrient uptake from deep soils and adaptation to drought stress conditions. In the present research, an integrated meta-analysis approach was employed to find candidate genes and genomic regions involved in rice RSA traits. A whole-genome meta-analysis was performed for 425 initial QTLs reported in 34 independent experiments controlling RSA traits under control and drought stress conditions in the previous twenty years. Sixty-four consensus meta-QTLs (MQTLs) were detected, unevenly distributed on twelve rice chromosomes. The confidence interval (CI) of the identified MQTLs was obtained as 0.11-14.23 cM with an average of 3.79 cM, which was 3.88 times narrower than the mean CI of the original QTLs. Interestingly, 52 MQTLs were co-located with SNP peak positions reported in rice genome-wide association studies (GWAS) for root morphological traits. The genes located in these RSA-related MQTLs were detected and explored to find the drought-responsive genes in the rice root based on the RNA-seq and microarray data. Multiple RSA and drought tolerance-associated genes were found in the MQTLs including the genes involved in auxin biosynthesis or signaling (e.g. YUCCA, WOX, AUX/IAA, ARF), root angle (DRO1-related genes), lateral root development (e.g. DSR, WRKY), root diameter (e.g. OsNAC5), plant cell wall (e.g. EXPA), and lignification (e.g. C4H, PAL, PRX and CAD). The genes located within both the SNP peak positions and the QTL-overview peaks for RSA are suggested as novel candidate genes for further functional analysis. The promising candidate genes and MQTLs can be used as basis for genetic engineering and MQTL-assisted breeding of root phenotypes to improve yield potential, stability and performance in a water-stressed environment.

Closer vein spacing by ectopic expression of nucleotide-binding and leucine-rich repeat proteins in rice leaves.

KEY MESSAGE: Elevated expression of nucleotide-binding and leucine-rich repeat proteins led to closer vein spacing and higher vein density in rice leaves. To feed the growing global population and mitigate the negative effects of climate change, there is a need to improve the photosynthetic capacity and efficiency of major crops such as rice to enhance grain yield potential. Alterations in internal leaf morphology and cellular architecture are needed to underpin some of these improvements. One of the targets is to generate a "Kranz-like" anatomy in leaves that includes decreased interveinal spacing close to that in C4 plant species. As C4 photosynthesis has evolved from C3 photosynthesis independently in multiple lineages, the genes required to facilitate C4 may already be present in the rice genome. The Taiwan Rice Insertional Mutants (TRIM) population offers the advantage of gain-of-function phenotype trapping, which accelerates the identification of rice gene function. In the present study, we screened the TRIM population to determine the extent to which genetic plasticity can alter vein density (VD) in rice. Close vein spacing mutant 1 (CVS1), identified from a VD screening of approximately 17,000 TRIM lines, conferred heritable high leaf VD. Increased vein number in CVS1 was confirmed to be associated with activated expression of two nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. Overexpression of the two NB-LRR genes individually in rice recapitulates the high VD phenotype, due mainly to reduced interveinal mesophyll cell (M cell) number, length, bulliform cell size and thus interveinal distance. Our studies demonstrate that the trait of high VD in rice can be achieved by elevated expression of NB-LRR proteins limited to no yield penalty.

Molecular basis underlying rice tiller angle: Current progress and future perspectives.

Crop plant architecture is an important agronomic trait that contributes greatly to crop yield. Tiller angle is one of the most critical components that determine crop plant architecture, which in turn substantially affects grain yield mainly owing to its large influence on plant density. Gravity is a fundamental physical force that acts on all organisms on earth. Plant organs sense gravity to control their growth orientation, including tiller angle in rice (Oryza sativa). This review summarizes recent research advances made using rice tiller angle as a research model, providing insights into domestication of rice tiller angle, genetic regulation of rice tiller angle, and shoot gravitropism. Finally, we propose that current discoveries in rice can shed light on shoot gravitropism and improvement of plant tiller/branch angle in other species, thereby contributing to agricultural production in the future.

Xuezhikang alleviates lipid accumulation via AMPK activation in hepatocellular steatosis model

Abstract Xuezhikang (XZK) is an extract of Chinese red yeast rice. It has multiple protective effects in cardiovascular systems. However, the underlying mechanism by which XZK affects free fatty acid (FFA)-induced lipogenesis in hepatocellular steatosis model is still unknown. Herein, we investigated this mechanism in HepG2 cells. The HepG2 cells were treated with palmitate acid (PA) to induce lipogenesis. Then the PA-induced HepG2 cells were subsequently treated with XZK. After 24 h of treatment, we determined the intracellular triglyceride (TG) contents and average areas of lipid droplets. To study the involvement of AMPK signaling pathway, we pre-treated the PA-induced HepG2 cells with Compound C, an AMPK inhibitor, before XZK treatment. Expressions of p-AMPK and AMPK were determined by Western blot. The results showed that XZK decreased TG content and lipid accumulation in hepatocellular steatosis model. Compound C abolished the effects of XZK. These results demonstrated for the first time that XZK protects hepatocytes against lipid accumulation induced by free fatty acids. Its effects may be mediated by the activation of AMPK pathway.

Identification of Genomic Regions Controlling Chalkiness and Grain Characteristics in a Recombinant Inbred Line Rice Population Based on High-Throughput SNP Markers.

Rice (Oryza sativa L.) is the primary food for half of the global population. Recently, there has been increasing concern in the rice industry regarding the eating and milling quality of rice. This study was conducted to identify genetic information for grain characteristics using a recombinant inbred line (RIL) population from a japonica/indica cross based on high-throughput SNP markers and to provide a strategy for improving rice quality. The RIL population used was derived from a cross of "Kaybonnet (KBNT lpa)" and "ZHE733" named the K/Z RIL population, consisting of 198 lines. A total of 4133 SNP markers were used to identify quantitative trait loci (QTLs) with higher resolution and to identify more accurate candidate genes. The characteristics measured included grain length (GL), grain width (GW), grain length to width ratio (RGLW), hundred grain weight (HGW), and percent chalkiness (PC). QTL analysis was performed using QTL IciMapping software. Continuous distributions and transgressive segregations of all the traits were observed, suggesting that the traits were quantitatively inherited. A total of twenty-eight QTLs and ninety-two candidate genes related to rice grain characteristics were identified. This genetic information is important to develop rice varieties of high quality.

A Short Review: Bioactivity of Fermented Rice Bran.

Rice bran is a by-product of the rice milling process, which refers to the processing of brown rice into polished rice. It contains a considerable amount of functional bioactive compounds. However, the utilization of these compounds is limited and calls for an effort to ferment rice bran. One of the methods that can significantly increase the added value of rice bran as well as its bioactivity is the solid-state fermentation. It can also be one of the strategies that help in the production of rice bran as a functional ingredient with higher bioactivity for health promotion.

Enhancement of drought tolerance in rice by silencing of the OsSYT-5 gene.

Improvement of drought tolerance of crops is a great challenge in conditions of increasing climate change. This report describes that the silencing of the synaptotagmin-5 (OsSYT-5) gene encoding the rice Ca2+ sensing protein with a C2 domain led to a significant improvement of rice tolerance to water deficit stress. Transgenic lines with suppressed expression of the OsSYT-5 gene exhibited an enhanced photosynthetic rate but reduced stomatal conductance and transpiration during water deficit stress. The abscisic acid (ABA) content under both normal and drought conditions was elevated in the leaves of the transgenic rice as compared to the wild type. The silencing of the OsSYT-5 gene affected the expression of several genes associated with ABA-related stress signaling in the transgenic rice plants. In the water deficit experiment, the transgenic lines with a silenced OsSYT-5 gene exhibited symptoms of drought stress seven days later than the wild type. Transgenic lines with suppressed OsSYT-5 gene expression exhibited higher pollen viability and produced more grains compared to the wild type at both normal and drought stress conditions.

Association mapping of drought tolerance and agronomic traits in rice (Oryza sativa L.) landraces.

BACKGROUND: Asian cultivars were predominantly represented in global rice panel selected for sequencing and to identify novel alleles for drought tolerance. Diverse genetic resources adapted to Indian subcontinent were not represented much in spite harboring useful alleles that could improve agronomic traits, stress resilience and productivity. These rice accessions are valuable genetic resource in developing rice varieties suited to different rice ecosystem that experiences varying drought stress level, and at different crop stages. A core collection of rice germplasm adapted to Southwestern Indian peninsular genotyped using SSR markers and characterized by contrasting water regimes to associate genomic regions for physiological, root traits and yield related traits. Genotyping-By-Sequencing of selected accessions within the diverse panel revealed haplotype variation in genic content within genomic regions mapped for physiological, morphological and root traits. RESULTS: Diverse rice panel (99 accessions) were evaluated in field and measurements on plant physiological, root traits and yield related traits were made over five different seasons experiencing varying drought stress intensity at different crop stages. Traits like chlorophyll stability index, leaf rolling, days to 50% flowering, chlorophyll content, root volume and root biomass were identified as best predictors of grain yield under stress. Association mapping revealed genetic variation among accessions and revealed 14 genomic targets associated with different physiological, root and plant production traits. Certain accessions were found to have beneficial allele to improve traits, plant height, root length and spikelet fertility, that contribute to the grain yield under stress. Genomic characterization of eleven accessions revealed haplotype variation within key genomic targets on chromosomes 1, 4, 6 and 11 for potential use as molecular markers to combine drought avoidance and tolerance traits. Genes mined within the genomic QTL intervals identified were prioritized based on tissue specific expression level in publicly available rice transcriptome data. CONCLUSION: The genetic and genomic resources identified will enable combining traits with agronomic value to optimize yield under stress and hasten trait introgression into elite cultivars. Alleles associated with plant height, specific leaf area, root length from PTB8 and spikelet fertility and grain weight from PTB26 can be harnessed in future rice breeding program.

Microstructure investigation of plant architecture with X-ray microscopy.

In recent years, the plant morphology has been well studied by multiple approaches at cellular and subcellular levels. Two-dimensional (2D) microscopy techniques offer imaging of plant structures on a wide range of magnifications for researchers. However, subcellular imaging is still challenging in plant tissues like roots and seeds. Here we use a three-dimensional (3D) imaging technology based on the X-ray microscope (XRM) and analyze several plant tissues from different plant species. The XRM provides new insights into plant structures using non-destructive imaging at high-resolution and high contrast. We also utilized a workflow aiming to acquire accurate and high-quality images in the context of the whole specimen. Multiple plant samples including rice, tobacco, Arabidopsis and maize were used to display the differences of phenotypes. Our work indicates that the XRM is a powerful tool to investigate plant microstructure in high-resolution scale. Our work also provides evidence that evaluate and quantify tissue specific differences for a range of plant species. We also characterize novel plant tissue phenotypes by the XRM, such as seeds in Arabidopsis, and utilize them for novel observation measurement. Our work represents an evaluated spatial and temporal resolution solution on seed observation and screening.