Effect of gibberellin on crown root development in the mutant of the rice plasmodesmal Germin-like protein OsGER4.

Rice is an essential but highly stress-susceptible crop, whose root system plays an important role in plant development and stress adaptation. The rice root system architecture is controlled by gene regulatory networks involving different phytohormones including auxin, jasmonate, and gibberellin. Gibberellin is generally known as a molecular clock that interacts with different pathways to regulate root meristem development. The exogenous treatment of rice plantlets with Gibberellin reduced the number of crown roots, whilst the exogenous jasmonic acid treatment enhanced them by involving a Germin-like protein OsGER4. Due to those opposite effects, this study aims to investigate the effect of Gibberellin on crown root development in the rice mutant of the plasmodesmal Germin-like protein OsGER4. Under exogenous gibberellin treatment, the number of crown roots significantly increased in osger4 mutant lines and decreased in the OsGER4 overexpressed lines. GUS staining showed that OsGER4 was strongly expressed in rice root systems, particularly crown and lateral roots under GA3 application. Specifically, OsGER4 was strongly expressed from the exodermis, epidermis, sclerenchyma to the endodermis layers of the crown root, along the vascular bundle and throughout LR primordia. The plasmodesmal protein OsGER4 is suggested to be involved in crown root development by maintaining hormone homeostasis, including Gibberillin.

The Germin-like protein gene OsGER4 is involved in heat stress response in rice root development.

Rice (Oryza sativa L.) is one of the most important dietary carbohydrate sources for half of the world's population. However, it is not well adapted to environmental stress conditions, necessitating to create new and improved varieties to help ensure sufficient rice production in the face of rising populations and shrinking arable land. Recently, the development of the CRISPR/Cas9 gene editing system has allowed researchers to study functional genomics and engineer new rice varieties with great efficiency compared to conventional methods. In this study, we investigate the involvement of OsGER4, a germin-like protein identified by a genome-wide association study that is associated with rice root development under a stress hormone jasmonic acids treatment. Analysis of the OsGER4 promoter region revealed a series of regulatory elements that connect this gene to ABA signaling and water stress response. Under heat stress, osger4 mutant lines produce a significantly lower crown root than wild-type Kitaake rice. The loss of OsGER4 also led to the reduction of lateral root development. Using the GUS promoter line, OsGER4 expression was detected in the epidermis of the crown root primordial, in the stele of the crown root, and subsequently in the primordial of the lateral root. Taken together, these results illustrated the involvement of OsGER4 in root development under heat stress by regulating auxin transport through plasmodesmata, under control by both ABA and auxin signaling.

Genome-wide association study reveals useful QTL and genes controlling the fatty acid composition in rice bran oil using Vietnamese rice landraces.

In rice (Oryza sativa L.), rice bran contains valuable nutritional constituents, such as high unsaturated fat content, tocotrienols, inositol, γ-oryzanol, and phytosterols, all of which are of nutritional and pharmaceuticals interest. There is now a rising market demand for rice bran oil, which makes research into their content and fatty acid profile an area of interest. As it is evident that lipid content has a substantial impact on the eating, cooking, and storage quality of rice, an understanding of the genetic mechanisms that determine oil content in rice is of great importance, equal to that of rice quality. Therefore, in this study, we performed a genome-wide association study on the composition and oil concentration of 161 Vietnamese rice varieties. Five categories of fatty acids in rice bran were discovered and the bran oil concentration profile in different rice accessions was identified. We also identified 229 important markers related to the fatty acid composition of bran oil, distributed mainly on chromosomes 1 and 7. Seven quantitative trait loci and five potential genes related to unsaturated fatty acid content were detected, including OsKASI, OsFAD, OsARF, OsGAPDH, and OsMADS29. These results provide insights into the genetic basis of rice bran oil composition, which is pivotal to the metabolic engineering of rice plants with desirable bran oil content through candidate genes selection.

The Germin-like protein OsGER4 is involved in promoting crown root development under exogenous jasmonic acid treatment in rice.

In rice (Oryza sativa L.), crown roots (CRs) have many important roles in processes such as root system expansion, water and mineral uptake, and adaptation to environmental stresses. Phytohormones such as auxin, cytokinin, and ethylene are known to control CR initiation and development in rice. However, the role of jasmonic acid (JA) in CR development remained elusive. Here, we report that JA promotes CR development by regulating OsGER4, a rice Germin-like protein. Root phenotyping analysis revealed that exogenous JA treatment induced an increase in CR number in a concentration-dependent manner. A subsequent genome-wide association study and gene expression analyses pinpointed a strong association between the Germin-like protein OsGER4 and the increase in CR number under exogenous JA treatment. The ProGER4::GUS reporter line showed that OsGER4 is a hormone-responsive gene involved in various stress responses, mainly confined to epidermal and vascular tissues during CR primordia development and to vascular bundles of mature crown and lateral roots. Notable changes in OsGER4 expression patterns caused by the polar auxin transport inhibitor NPA support its connection to auxin signaling. Phenotyping experiments with OsGER4 knockout mutants confirmed that this gene is required for CR development under exogenous JA treatment. Overall, our results provide important insights into JA-mediated regulation of CR development in rice.

CROWN ROOTLESS1 binds DNA with a relaxed specificity and activates OsROP and OsbHLH044 genes involved in crown root formation in rice.

In cereals, the root system is mainly composed of post-embryonic shoot-borne roots, named crown roots. The CROWN ROOTLESS1 (CRL1) transcription factor, belonging to the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) family, is a key regulator of crown root initiation in rice (Oryza sativa). Here, we show that CRL1 can bind, both in vitro and in vivo, not only the LBD-box, a DNA sequence recognized by several ASL/LBD transcription factors, but also another not previously identified DNA motif that was named CRL1-box. Using rice protoplast transient transactivation assays and a set of previously identified CRL1-regulated genes, we confirm that CRL1 transactivates these genes if they possess at least a CRL1-box or an LBD-box in their promoters. In planta, ChIP-qPCR experiments targeting two of these genes that include both a CRL1- and an LBD-box in their promoter show that CRL1 binds preferentially to the LBD-box in these promoter contexts. CRISPR/Cas9-targeted mutation of these two CRL1-regulated genes, which encode a plant Rho GTPase (OsROP) and a basic helix-loop-helix transcription factor (OsbHLH044), show that both promote crown root development. Finally, we show that OsbHLH044 represses a regulatory module, uncovering how CRL1 regulates specific processes during crown root formation.

Discovery of new genetic determinants of morphological plasticity in rice roots and shoots under phosphate starvation using GWAS.

Phosphorus is an essential nutrient for plants that is often in short supply. In rice (Oryza sativa L.), inorganic phosphate (Pi) deficiency leads to various physiological disorders that consequently affect plant productivity. In this study, a large-scale phenotyping experiment using 160 Vietnamese rice landraces was performed under greenhouse conditions, by employing an alpha lattice design with three replicates, to identify quantitative trait loci (QTLs) associated with plant growth inhibition caused by Pi deficiency. Rice plantlets were grown for six weeks in the PVC sand column (16 cm diameter × 80 cm height) supplied with Pi-deficient medium (10 µM P) or full-Pi Yoshida medium (320 µM P). The effects of Pi deficiency on the number of crown roots, root length, shoot length, root weight, shoot weight and total weight were studied. From 36 significant markers identified using a genome-wide association study, 21 QTLs associated with plant growth inhibition under Pi starvation were defined. In total, 158 candidate genes co-located with the defined QTLs were identified. Interestingly, one QTL (qRST9.14) was associated with all three weight-traits. The co-located gene GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE 13 was found to be potentially involved in Pi transport. Understanding the molecular mechanisms of Pi-starvation responses, and identifying the potential QTLs responsible for low-Pi stress tolerance, will provide valuable information for developing new varieties tolerant of low-Pi conditions.

A genome-wide association study reveals the quantitative trait locus and candidate genes that regulate phosphate efficiency in a Vietnamese rice collection.

The crucial role of phosphate (Pi) for plant alongside the expected depletion of non-renewable phosphate rock have created an urgent need for phosphate-efficient rice varieties. In this study, 157 greenhouse-grown Vietnamese rice landraces were treated under Pi-deficient conditions to discover the genotypic variation among biochemical traits, including relative efficiency of phosphorus use (REP), relative root to shoot weight ratio (RRSR), relative physiological phosphate use efficiency (RPPUE), and relative phosphate uptake efficiency (RPUpE). Plants were grown in Yoshida nutrient media with either a full (320 µM) or a low Pi supply (10 µM) over six weeks. This genome-wide association study led to the discovery of 31 significant single nucleotide polymorphisms, 18 quantitative trait loci (QTLs), and 85 candidate genes. A common QTL named qRPUUE9.16 was found among the three investigated traits. Some interesting candidate genes, such as PLASMA MEMBRANE PROTEIN1 (OsPM1), CALMODULIN-RELATED CALCIUM SENSOR PROTEIN 15 (OsCML15), phosphatases 2C (PP2C), STRESS-ACTIVATED PROTEIN KINASE (OsSAPK2), and GLYCEROPHOSPHORYL DIESTER PHOSPHODIESTERASES (GDPD13), were found strongly correlated to the Pi starvation. RNA sequencing transcriptomes revealed that 45 out of 85 candidate genes were significantly regulated under Pi starvation. Furthermore, nearly two-thirds of genotypes did not possess the OsPsTOL1 gene; however, no significant difference was observed in response to Pi deficiency between genotypes with or without this gene, suggesting that other QTLs in rice may resist Pi starvation. These results provide new information on the genetics of nutrient use efficiency in rice and may potentially assist with developing more phosphate-efficient rice plants.

Unraveling the Genetic Elements Involved in Shoot and Root Growth Regulation by Jasmonate in Rice Using a Genome-Wide Association Study.

BACKGROUND: Due to their sessile life style, plant survival is dependent on the ability to build up fast and highly adapted responses to environmental stresses by modulating defense response and organ growth. The phytohormone jasmonate plays an essential role in regulating these plant responses to stress. RESULTS: To assess variation of plant growth responses and identify genetic determinants associated to JA treatment, we conducted a genome-wide association study (GWAS) using an original panel of Vietnamese rice accessions. The phenotyping results showed a high natural genetic variability of the 155 tested rice accessions in response to JA for shoot and root growth. The level of growth inhibition by JA is different according to the rice varieties tested. We conducted genome-wide association study and identified 28 significant associations for root length (RTL), shoot length (SHL), root weight (RTW), shoot weight (SHW) and total weight (TTW) in response to JA treatment. Three common QTLs were found for RTL, RTW and SHL. Among a list of 560 candidate genes found to co-locate with the QTLs, a transcriptome analysis from public database for the JA response allows us to identify 232 regulated genes including several JA-responsive transcription factors known to play a role in stress response. CONCLUSION: Our genome-wide association study shows that common and specific genetic elements are associated with inhibition of shoot and root growth under JA treatment suggesting the involvement of a complex JA-dependent genetic control of rice growth inhibition at the whole plant level. Besides, numerous candidate genes associated to stress and JA response are co-located with the association loci, providing useful information for future studies on genetics and breeding to optimize the growth-defense trade-off in rice.