Ubiquitin E3 ligase AtCHYR2 functions in glucose regulation of germination and post-germinative growth in Arabidopsis thaliana.

KEY MESSAGE: Cytoplasm-localized RING ubiquitin E3 ligase AtCHYR2 involved in plant glucose responses during germination and post-germinative growth. CHY ZINC FINGER AND RING PROTEIN (CHYR) containing both a CHY zinc finger and a C3H2C3-type RING domain plays important roles in plant drought tolerance and the abscisic acid (ABA) response; however, their functions in sugar signaling pathways are less studied. Here, we report a glucose (Glc) response gene AtCHYR2, a homolog of RZFP34/CHYR1, which is induced by various abiotic stresses, ABA, and sugar treatments. In vitro, we demonstrated that AtCHYR2 is a cytoplasm-localized RING ubiquitin E3 ligase. Overexpression of AtCHYR2 led to hypersensitivity to Glc and enhanced Glc-mediated inhibition of cotyledon greening and post-germinative growth. Contrastingly, AtCHYR2 loss-of-function plants were insensitive to Glc-regulated seed germination and primary root growth, suggesting that AtCHYR2 is a positively regulator of the plant glucose response. Additionally, physiological analyses showed that overexpression AtCHYR2 increased stomata aperture and photosynthesis under normal condition, and promoted accumulation of endogenous soluble sugar and starch in response to high Glc. Genome-wide RNA sequencing analysis showed that AtCHYR2 affects a major proportion of Glc-responsive genes. Particularly, sugar marker gene expression analysis suggested that AtCHYR2 enhances the Glc response via a signaling pathway dependent on glucose metabolism. Taken together, our findings show that a novel RING ubiquitin E3 ligase, AtCHYR2, plays an important role in glucose responses in Arabidopsis.

VisProDom: an interactive Shiny/R application for displaying protein domains with transcriptional features.

BACKGROUND: Both the protein domains and transcript structures influence protein functional variation. The genomic location of both protein domains and transcript structural features can be described using the genomic coordinates of their encoded sequences. However, the coordinates of protein domains and transcriptional features often differ greatly, and it is difficult to view them in combination at the genome-wide level. In this paper, we describe the development of a new tool that allows users to visualize domains and transcript features together, using either built-in or uploaded genome datasets, and export publication-ready figures.  RESULTS: We developed a user-friendly, independent R package and Shiny web application named "VisProDom". VisProDom consists of a genome-wide database containing entire annotated transcripts merged with annotated protein domains from the Pfam database. The built-in dataset includes 82 files, which merge genome general feature format (GFF) annotations with rpsblast tabular outputs from protein sequence searches in the Pfam database. Multiple genomes can be simultaneously screened for protein domains or transcript names. VisProDom includes step-by-step introductions and clickable elements for ease of use. CONCLUSION: VisProDom can display hundreds of transcripts alongside protein domains and export figures in a publication-ready format. This makes it a valuable tool for molecular evolution and comparative genomics.

Genome-Wide Analysis of TCP Family Genes in Zea mays L. Identified a Role for ZmTCP42 in Drought Tolerance.

The Teosinte-branched 1/Cycloidea/Proliferating (TCP) plant-specific transcription factors (TFs) have been demonstrated to play a fundamental role in plant development and organ patterning. However, it remains unknown whether or not the TCP gene family plays a role in conferring a tolerance to drought stress in maize, which is a major constraint to maize production. In this study, we identified 46 ZmTCP genes in the maize genome and systematically analyzed their phylogenetic relationships and synteny with rice, sorghum, and Arabidopsis TCP genes. Expression analysis of the 46 ZmTCP genes in different tissues and under drought conditions, suggests their involvement in maize response to drought stress. Importantly, genetic variations in ZmTCP32 and ZmTCP42 are significantly associated with drought tolerance at the seedling stage. RT-qPCR results suggest that ZmTCP32 and ZmTCP42 RNA levels are both induced by ABA, drought, and polyethylene glycol treatments. Based on the significant association between the genetic variation of ZmTCP42 and drought tolerance, and the inducible expression of ZmTCP42 by drought stress, we selected ZmTCP42, to investigate its function in drought response. We found that overexpression of ZmTCP42 in Arabidopsis led to a hypersensitivity to ABA in seed germination and enhanced drought tolerance, validating its function in drought tolerance. These results suggested that ZmTCP42 functions as an important TCP TF in maize, which plays a positive role in drought tolerance.

Arabidopsis RZFP34/CHYR1, a Ubiquitin E3 Ligase, Regulates Stomatal Movement and Drought Tolerance via SnRK2.6-Mediated Phosphorylation.

Abscisic acid (ABA) is a phytohormone that plays a fundamental role in plant development and stress response, especially in the regulation of stomatal closure in response to water deficit stress. The signal transduction that occurs in response to ABA and drought stress is mediated by protein phosphorylation and ubiquitination. This research identified Arabidopsis thaliana RING ZINC-FINGER PROTEIN34 (RZP34; renamed here as CHY ZINC-FINGER AND RING PROTEIN1 [CHYR1]) as an ubiquitin E3 ligase. CHYR1 expression was significantly induced by ABA and drought, and along with its corresponding protein, was expressed mainly in vascular tissues and stomata. Analysis of CHYR1 gain-of-function and loss-of-function plants revealed that CHYR1 promotes ABA-induced stomatal closure, reactive oxygen species production, and plant drought tolerance. Furthermore, CHYR1 interacted with SNF1-RELATED PROTEIN KINASE2 (SnRK2) kinases and could be phosphorylated by SnRK2.6 on the Thr-178 residue. Overexpression of CHYR1(T178A), a phosphorylation-deficient mutant, interfered with the proper function of CHYR1, whereas CHYR1(T178D) phenocopied the gain of function of CHYR1. Thus, this study identified a RING-type ubiquitin E3 ligase that functions positively in ABA and drought responses and detailed how its ubiquitin E3 ligase activity is regulated by SnRK2.6-mediated protein phosphorylation.

Micrometeorological monitoring reveals that canopy temperature is a reliable trait for the screening of heat tolerance in rice.

Micrometeorological monitoring is not just an effective method of determining the impact of heat stress on rice, but also a reliable way of understanding how to screen for heat tolerance in rice. The aim of this study was to use micrometeorological monitoring to determine varietal differences in rice plants grown under two weather scenarios-Long-term Heat Scenario (LHS) and Normal Weather Scenario (NWS)- so as to establish reliable methods for heat tolerance screening. Experiments were conducted with two heat susceptible varieties-Mianhui 101 and IR64-and two heat tolerant varieties, Quanliangyou 681 and SDWG005. We used staggered sowing method to ensure that all varieties flower at the same time. Our results showed that heat tolerant varieties maintained lower canopy temperature compared to heat susceptible varieties, not just during the crucial flowering period of 10 am to 12 pm, but throughout the entire day and night. The higher stomatal conductance rate observed in heat tolerant varieties possibly decreased their canopy temperatures through the process of evaporative cooling during transpiration. Conversely, we found that panicle temperature cannot be used to screen for heat tolerance at night, as we observed no significant difference in the panicle temperature of heat tolerant and heat susceptible varieties at night. However, we also reported that higher panicle temperature in heat susceptible varieties decreased spikelet fertility rate, while low panicle temperature in heat tolerant varieties increased spikelet fertility rate. In conclusion, the results of this study showed that canopy temperature is probably the most reliable trait to screen for heat tolerance in rice.

IntAssoPlot: An R Package for Integrated Visualization of Genome-Wide Association Study Results With Gene Structure and Linkage Disequilibrium Matrix.

Genome-wide association study (GWAS), exploring the historical and evolutionary recombinations at the population level, is a major method adopted to identify quantitative trait loci (QTL) for complex traits. However, to summarize GWAS results, gene structure, and linkage disequilibrium (LD) in a single view, multiple tools are required. It is tedious to generate these three results and manually put them together; moreover, it may eventually lead to inaccuracies. On the other hand, genotype markers are usually detected by DNA- and/or RNA-Seq. For GWAS analysis based on RNA-Seq, markers from DNA-Seq provide more genetic information when displaying LD. The currently released software package does not have this function for an integrated analysis of LD, using genotypic markers different from that in association analysis. Here, we present an R package, IntAssoPlot, which provides an integrated visual display of GWAS results, along with LD and gene structure information, in a publication-ready format. The main panel of an IntAssoPlot plot has a connecting line linking the genome-wide association P-values on the -log10 scale with the gene structure and LD matrix. Importantly, IntAssoPlot is designed to plot GWAS results with LD calculated from genotypes different from those in GWAS analysis. IntAssoPlot provides a powerful visualization tool to gain an integrated insight into GWAS results. The functions provided by IntAssoPlot increase the efficiency by revealing GWAS results in a publication-ready format. Inspection of the output image can provide important biological information, including the loci that passed the genome-wide significance threshold, genes located at or near the significant loci, and the extent of LD within the selected region.

Transcriptomic Analysis Reveals Important Roles of Lignin and Flavonoid Biosynthetic Pathways in Rice Thermotolerance During Reproductive Stage.

Rice is one of the major staple cereals in the world, but heat stress is increasingly threatening its yield. Analyzing the thermotolerance mechanism from new thermotolerant germplasms is very important for rice improvement. Here, physiological and transcriptome analyses were used to characterize the difference between two germplasms, heat-sensitive MH101 and heat-tolerant SDWG005. Two genotypes exhibited diverse heat responses in pollen viability, pollination characteristics, and antioxidant enzymatic activity in leaves and spikelets. Through cluster analysis, the global transcriptomic changes indicated that the ability of SDWG005 to maintain a steady-state balance of metabolic processes played an important role in thermotolerance. After analyses of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, we found that the thermotolerance mechanism in SDWG00 was associated with reprogramming the cellular activities, such as response to abiotic stress and metabolic reorganization. In contrast, the down-regulated genes in MH101 that appeared to be involved in DNA replication and DNA repair proofreading, could cause serious injury to reproductive development when exposed to high temperature during meiosis. Furthermore, we identified 77 and 11 differentially expressed genes (DEGs) involved in lignin and flavonoids biosynthetic pathways, respectively. Moreover, we found that more lignin deposition and flavonoids accumulation happened in SDWG005 than in MH101 under heat stress. The results indicated that lignin and flavonoid biosynthetic pathways might play important roles in rice heat resistance during meiosis.

Identification of Maize CC-Type Glutaredoxins That Are Associated with Response to Drought Stress.

Global maize cultivation is often adversely affected by drought stress. The CC-type glutaredoxin (GRX) genes form a plant-specific subfamily that regulate plant growth and respond to environmental stresses. However, how maize CC-type GRX (ZmGRXCC) genes respond to drought stress remains unclear. We performed a TBLASTN search to identify ZmGRXCCs in the maize genome and verified the identified sequences using the NCBI conservative domain database (CDD). We further established a phylogenetic tree using Mega7 and surveyed known cis-elements in the promoters of ZmGRXCCs using the PlantCARE database. We found twenty-one ZmGRXCCs in the maize genome by a genome-wide investigation and compared their phylogenetic relationships with rice, maize, and Arabidopsis. The analysis of their redox active sites showed that most of the 21 ZmGRXCCs share similar structures with their homologs. We assessed their expression at young seedlings and adult leaves under drought stress and their expression profiles in 15 tissues, and found that they were differentially expressed, indicating that different ZmGRXCC genes have different functions. Notably, ZmGRXCC14 is up-regulated at seedling, V12, V14, V16, and R1 stages. Importantly, significant associations between genetic variation in ZmGRXCC14 and drought tolerance are found at the seedling stage. These results will help to advance the study of the function of ZmGRXCCs genes under drought stress and understand the mechanism of drought resistance in maize.

Genome-wide analysis of maize OSCA family members and their involvement in drought stress.

BACKGROUND: Worldwide cultivation of maize is often impacted negatively by drought stress. Hyperosmolality-gated calcium-permeable channels (OSCA) have been characterized as osmosensors in Arabidopsis. However, the involvement of members of the maize OSCA (ZmOSCA) gene family in response to drought stress is unknown. It is furthermore unclear which ZmOSCA gene plays a major role in genetic improvement of drought tolerance in Maize. METHODS: We predicted the protein domain structure and transmembrane regions by using the NCBI Conserved Domain Database database and TMHMM server separately. The phylogeny tree was built by Mega7. We used the mixed linear model in TASSEL to perform the family-based association analysis. RESULTS: In this report, 12 ZmOSCA genes were uncovered in the maize genome by a genome-wide survey and analyzed systematically to reveal their synteny and phylogenetic relationship with the genomes of rice, maize, and sorghum. These analyses indicated a relatively conserved evolutionary history of the ZmOSCA gene family. Protein domain and transmembrane analysis indicated that most of the 12 ZmOSCAs shared similar structures with their homologs. The result of differential expression analysis under drought at various stages, as well as the expression profiles in 15 tissues, revealed a functional divergence of ZmOSCA genes. Notably, the expression level of ZmOSCA4.1 being up-regulated in both seedlings and adult leaves. Notably, the association analysis between genetic variations in these genes and drought tolerance was detected. Significant associations between genetic variation in ZmOSCA4.1 and drought tolerance were found at the seedling stage. Our report provides a detailed analysis of the ZmOSCAs in the maize genome. These findings will contribute to future studies on the functional characterization of ZmOSCA proteins in response to water deficit stress, as well as understanding the mechanism of genetic variation in drought tolerance in maize.

Bulked Segregant RNA-seq Reveals Differential Expression and SNPs of Candidate Genes Associated with Waterlogging Tolerance in Maize.

Waterlogging has increasingly become one of the major constraints to maize productivity in some maize production zones because it causes serious yield loss. Bulked segregant RNA-seq (BSR-seq) has been widely applied to profile candidate genes and map associated Single Nucleotide Polymorphism (SNP) markers in many species. In this study, 10 waterlogging sensitive and eight tolerant inbred lines were selected from 60 maize inbred lines with waterlogging response determined and preselected by the International Maize and Wheat Improvement Center (CIMMYT) from over 400 tropical maize inbred lines. BSR-seq was performed to identify differentially expressed genes and SNPs associated with waterlogging tolerance. Upon waterlogging stress, 354 and 1094 genes were differentially expressed in the tolerant and sensitive pools, respectively, compared to untreated controls. When tolerant and sensitive pools were compared, 593 genes were differentially expressed under untreated and 431 genes under waterlogged conditions, of which 122 genes overlapped. To validate the BSR-seq results, the expression levels of six genes were determined by qRT-PCR. The qRT-PCR results were consistent with BSR-seq results. Comparison of allelic polymorphism in mRNA sequences between tolerant and sensitive pools revealed 165 (normal condition) and 128 (waterlogged condition) high-probability SNPs. We found 18 overlapping SNPs with genomic positions mapped. Eighteen SNPs were contained in 18 genes, and eight and nine of 18 genes were responsive to waterlogging stress in tolerant and sensitive lines, respectively. Six alleles of the 18 originated from tolerant pool were significantly up-regulated under waterlogging, but not those from sensitive pool. Importantly, one allele (GRMZM2G055704) of the six genes was mapped between umc1619 and umc1948 on chromosome 1 where a QTL associated with waterlogging tolerance was identified in a previous research, strongly indicating that GRMZM2G055704 is a candidate gene responsive to waterlogging. Our research contributes to the knowledge of the molecular mechanism for waterlogging tolerance in maize.