Takeshi Kuroha

Rice leaf hydrophobicity and gas films are conferred by a wax synthesis gene (LGF1) and contribute to flood tolerance

Floods impede gas (O2 and CO2 ) exchange between plants and the environment. A mechanism to enhance plant gas exchange under water comprises gas films on hydrophobic leaves, but the genetic regulation of this mechanism is unknown. We used a rice mutant (dripping wet leaf 7, drp7) which does not retain gas films on leaves, and its wild-type (Kinmaze), in gene discovery for this trait. Gene complementation was tested in transgenic lines. Functional properties of leaves as related to gas film retention and underwater photosynthesis were evaluated. Leaf Gas Film 1 (LGF1) was identified as the gene determining leaf gas films. LGF1 regulates C30 primary alcohol synthesis, which is necessary for abundant epicuticular wax platelets, leaf hydrophobicity and gas films on submerged leaves. This trait enhanced underwater photosynthesis 8.2-fold and contributes to submergence tolerance. Gene function was verified by a complementation test of LGF1 expressed in the drp7 mutant background, which restored C30 primary alcohol synthesis, wax platelet abundance, leaf hydrophobicity, gas film retention, and underwater photosynthesis. The discovery of LGF1 provides an opportunity to better understand variation amongst rice genotypes for gas film retention ability and to target various alleles in breeding for improved submergence tolerance for yield stability in flood-prone areas.

Pectin RG-I rhamnosyltransferases represent a novel plant-specific glycosyltransferase family

Pectin is one of the three key cell wall polysaccharides in land plants and consists of three major structural domains: homogalacturonan, rhamnogalacturonan I (RG-I) and RG-II. Although the glycosyltransferase required for the synthesis of the homogalacturonan and RG-II backbone was identified a decade ago, those for the synthesis of the RG-I backbone, which consists of the repeating disaccharide unit [→2)-α-l-Rha-(1u2009→u20094)-α-d-GalUA-(1→], have remained unknown. Here, we report the identification and characterization of Arabidopsis RG-I:rhamnosyltransferases (RRTs), which transfer the rhamnose residue from UDP-β-l-rhamnose to RG-I oligosaccharides. RRT1, which is one of the four Arabidopsis RRTs, is a single-spanning transmembrane protein, localized to the Golgi apparatus. RRT1 was highly expressed during formation of the seed coat mucilage, which is a specialized cell wall with abundant RG-I. Loss-of-function mutation in RRT1 caused a reduction in the level of RG-I in the seed coat mucilage. The RRTs belong to a novel glycosyltransferase family, now designated GT106. This is a large plant-specific family, and glycosyltransferases in this family seem to have plant-specific roles, such as biosynthesis of plant cell wall polysaccharides.Pectin, the major gelling component of the plant cell wall, is rich in galacturonic acids that compose the backbones of pectic polysaccharides. Now, researchers have identified a new family of enzymes responsible for synthesizing the backbone of pectin, RG-I.

Arabidopsis Regenerating Protoplast: A Powerful Model System for Combining the Proteomics of Cell Wall Proteins and the Visualization of Cell Wall Dynamics

The development of a range of sub-proteomic approaches to the plant cell wall has identified many of the cell wall proteins. However, it remains difficult to elucidate the precise biological role of each protein and the cell wall dynamics driven by their actions. The plant protoplast provides an excellent means not only for characterizing cell wall proteins, but also for visualizing the dynamics of cell wall regeneration, during which cell wall proteins are secreted. It therefore offers a unique opportunity to investigate the de novo construction process of the cell wall. This review deals with sub-proteomic approaches to the plant cell wall through the use of protoplasts, a methodology that will provide the basis for further exploration of cell wall proteins and cell wall dynamics.

eQTLS regulating transcript variations associated with rapid internode elongation in deepwater rice

To avoid low oxygen, oxygen deficiency or oxygen deprivation, deepwater rice cultivated in flood planes can develop elongated internodes in response to submergence. Knowledge of the gene regulatory networks underlying rapid internode elongation is important for an understanding of the evolution and adaptation of major crops in response to flooding. To elucidate the genetic and molecular basis controlling their deepwater response we used microarrays and performed expression quantitative trait loci (eQTL) and phenotypic QTL (phQTL) analyses of internode samples of 85 recombinant inbred line (RIL) populations of non-deepwater (Taichung 65)- and deepwater rice (Bhadua). After evaluating the phenotypic response of the RILs exposed to submergence, confirming the genotypes of the populations, and generating 188 genetic markers, we identified 10,047 significant eQTLs comprised of 2,902 cis-eQTLs and 7,145 trans-eQTLs and three significant eQTL hotspots on chromosomes 1, 4, and 12 that affect the expression of many genes. The hotspots on chromosomes 1 and 4 located at different position from phQTLs detected in this study and other previous studies. We then regarded the eQTL hotspots as key regulatory points to infer causal regulatory networks of deepwater response including rapid internode elongation. Our results suggest that the downstream regulation of the eQTL hotspots on chromosomes 1 and 4 is independent, and that the target genes are partially regulated by SNORKEL1 and SNORKEL2 genes (SK1/2), key ethylene response factors. Subsequent bioinformatic analyses, including gene ontology-based annotation and functional enrichment analysis and promoter enrichment analysis, contribute to enhance our understanding of SK1/2-dependent and independent pathways. One remarkable observation is that the functional categories related to photosynthesis and light signaling are significantly over-represented in the candidate target genes of SK1/2. The combined results of these investigations together with genetical genomics approaches using structured populations with a deepwater response are also discussed in the context of current molecular models concerning the rapid internode elongation in deepwater rice. This study provides new insights into the underlying genetic architecture of gene expression regulating the response to flooding in deepwater rice and will be an important community resource for analyses on the genetic basis of deepwater responses.

Ethylene-gibberellin signaling underlies adaptation of rice to periodic flooding

How rice defeats the floodwaters Deepwater rice varieties grow taller when flooded, in a growth response driven by the plant hormones gibberellin and ethylene. This keeps the leaves above the water. Kuroha et al. identified the genes underlying this phenotype, which encode a component of the gibberellin biosynthetic pathway and its regulatory ethylene-responsive transcription factor. This genetic relay drives growth of the plant stem internodes in response to flooding. Modern cultivated deepwater rice, which has been domesticated for adaptation to the monsoon season of Bangladesh, emerged from the genetic variation found in wild rice strains over a broader geographic region. Science, this issue p. 181 Ethylene-inducible activation of gibberellin biosynthesis helps rice survive long periods of submersion in flooded plots. Most plants do poorly when flooded. Certain rice varieties, known as deepwater rice, survive periodic flooding and consequent oxygen deficiency by activating internode growth of stems to keep above the water. Here, we identify the gibberellin biosynthesis gene, SD1 (SEMIDWARF1), whose loss-of-function allele catapulted the rice Green Revolution, as being responsible for submergence-induced internode elongation. When submerged, plants carrying the deepwater rice–specific SD1 haplotype amplify a signaling relay in which the SD1 gene is transcriptionally activated by an ethylene-responsive transcription factor, OsEIL1a. The SD1 protein directs increased synthesis of gibberellins, largely GA4, which promote internode elongation. Evolutionary analysis shows that the deepwater rice–specific haplotype was derived from standing variation in wild rice and selected for deepwater rice cultivation in Bangladesh.

Time-course transcriptomics analysis reveals key responses of submerged deepwater rice to flooding

Comparative transcriptomics and targeted analyses provide insights into the roles of phytohormone synthesis, signaling, and turnover during submergence-induced internode elongation in deepwater rice. Water submergence is an environmental factor that limits plant growth and survival. Deepwater rice (Oryza sativa) adapts to submergence by rapidly elongating its internodes and thereby maintaining its leaves above the water surface. We performed a comparative RNA sequencing transcriptome analysis of the shoot base region, including basal nodes, internodes, and shoot apices of seedlings at two developmental stages from two varieties with contrasting deepwater growth responses. A transcriptomic comparison between deepwater rice cv C9285 and nondeepwater rice cv Taichung 65 revealed both similar and differential expression patterns between the two genotypes during submergence. The expression of genes related to gibberellin biosynthesis, trehalose biosynthesis, anaerobic fermentation, cell wall modification, and transcription factors that include ethylene-responsive factors was significantly different between the varieties. Interestingly, in both varieties, the jasmonic acid content at the shoot base decreased during submergence, while exogenous jasmonic acid inhibited submergence-induced internode elongation in cv C9285, suggesting that jasmonic acid plays a role in the submergence response of rice. Furthermore, a targeted de novo transcript assembly revealed transcripts that were specific to cv C9285, including submergence-induced biotic stress-related genes. Our multifaceted transcriptome approach using the rice shoot base region illustrates a differential response to submergence between deepwater and nondeepwater rice. Jasmonic acid metabolism appears to participate in the submergence-mediated internode elongation response of deepwater rice.

Quantitative confocal imaging method for analyzing cellulose dynamics during cell wall regeneration in Arabidopsis mesophyll protoplasts

Abstract The network structure of cellulose fibrils provides mechanical properties to the primary cell wall, thereby determining the shapes and growth patterns of plant cells. Despite intensive studies, the construction process of the network structure in muro remains largely unknown, mainly due to the lack of a robust, straightforward technique to evaluate network configuration. Here, we developed a quantitative confocal imaging method for general use in the study of cell wall dynamics in protoplasts derived from Arabidopsis leaf mesophyll cells. Confocal imaging of regenerating cell walls in protoplasts stained with Calcofluor allowed us to visualize the cellulose network, comprising strings of bundled cellulosic fibrils. Using image analysis techniques, we measured several metrics including total length, which is a measure of the spread of the cellulose network. The total length increased during cell wall regeneration. In a proof‐of‐concept experiment using microtubule‐modifying agents, oryzalin, an inhibitor of microtubule polymerization, inhibited the increase in total length and caused abnormal orientation of the network, as shown by the decrease in the average angle of the cellulose with respect to the cell long axis. Taxol, a microtubule stabilizer, stimulated the bundling of cellulose fibrils, as shown by the increase in skewness in the fluorescence intensity distribution of Calcofluor, and inhibited the increase in total length. These results demonstrate the validity of this method for quantitative imaging of the cellulose network, providing an opportunity to gain insight into the dynamic aspects of cell wall regeneration.