Shuangcheng Ding

Cinnamoyl-CoA reductases (CCRs) and their homologs, CCR-like proteins, play key roles in plant secondary metabolism and defense against environmental stresses; however, their functions in heat stress responses remain unclear. In this study, phylogenetic and sequence alignment analyses revealed that OsCCR14 encodes a CCR-like protein. qRT-PCR assays showed that OsCCR14 is predominantly expressed in roots and is induced by heat stress. Notably, overexpression of OsCCR14 increased lignin content, and transgenic plants with enhanced OsCCR14 expression exhibited higher flavonoid accumulation in roots. Conversely, knockout of OsCCR14 reduced flavonoid content and impaired seedling heat tolerance. Furthermore, OsCCR14 overexpression improved heat tolerance, accompanied by increased root lignification and flavonoid accumulation. These results indicate that OsCCR14 acts as a critical regulator of lignin and flavonoid metabolism, thereby providing a potential target for enhancing crop heat tolerance.

Heat stress (HT) exerts significant negative impacts on plant growth, development, and productivity. In this study, we identified OsLAC12, a heat-induced laccase-encoding gene, as a key regulator of heat tolerance in rice. Functional validation confirmed that OsLAC12 encodes an active laccase, with supporting evidence showing that roots of OsLAC12 overexpression lines exhibited ~1.5-fold higher laccase activity and nearly three times the lignin content compared to the wild type (WT). Under HT, oslac12 mutants showed significantly lower seedling survival rates (~13–22%) than WT (~43%), whereas OE lines displayed higher survival rates (~68–74%). At the heading stage, OsLAC12 overexpression lines activated the antioxidant enzyme system in spikelets, regulated reactive oxygen species (ROS) levels in anthers, and mitigated HT-induced damage to pollen viability. In contrast, oslac12 mutants showed an average 10.8% reduction in pollen viability compared to WT, accompanied by significantly lower seed-setting rates. Collectively, our findings demonstrate that the laccase OsLAC12 positively regulates rice resistance to HT stress, providing critical insights for improving plant thermotolerance through genetic improvement of cell wall-associated pathways.

Glucose functions as both an essential energy source and a critical signaling molecule, playing pivotal roles in regulating plant growth, development, and stress responses. Here, we report that AtCHYR1, a previously characterized RING-type ubiquitin E3 ligase involved in drought tolerance, also participates in glucose signaling. Exposure to high glucose levels significantly inhibits AtCHYR1 expression, particularly in root tips, while low glucose conditions, such as osmotic stress, sugar starvation, and dark conditions, induce its expression. Importantly, analysis of chyr1 mutants and plants overexpressing AtCHYR1 revealed that AtCHYR1 positively regulated the high glucose-mediated inhibition of germination and root growth, as well as starvation-induced growth retardation, through enhanced reactive oxygen species (ROS) accumulation in root tips. Additionally, transcriptional levels of glucose-activated pathogenesis-related (PR) and defense-related genes were reduced, while hypoxia-associated and ROS-inducing genes were significantly upregulated in AtCHYR1-overexpressing plants. Collectively, our findings provide novel insights into the role of AtCHYR1 in plant responses to fluctuating sugar availability and its control of ROS homeostasis during seed germination and plant growth.

RING-type E3 ligases are critical regulators of diverse plant processes, yet their roles in maize remain poorly defined, particularly in drought responses. To address this knowledge gap, we conducted an integrative analysis of maize RING genes, combining evolutionary profiling, and candidate-gene association studies to identify drought-associated candidates. Key genes were further characterized via quantitative expression profiling, subcellular localization, in vitro ubiquitination assay, and functional validation in transgenic Arabidopsis. This study aimed to provide new insights into the maize RING gene family’s role in drought stress adaptation. We employed three distinct methods and identified a total of 590 proteins. Phylogenetic analysis revealed that these proteins could be grouped into 11 separate clusters. Our findings suggested that the expansion of the RING family in maize was likely due to gene duplication events. Notably, genetic variations in ZmRING-93 were significantly associated with drought tolerance, and its expression was up-regulated under various abiotic stress conditions and hormone treatments. We further discovered that ZmRING-93 was a functional ubiquitin E3 ligase that localized to the nucleus, cytoplasm, and parts of the endoplasmic reticulum. Transgenic Arabidopsis plants overexpressing ZmRING-93 exhibited enhanced drought tolerance, with a lower water loss rate, further supporting the importance of ZmRING-93 in drought tolerance. These findings revealed that ZmRING-93 contributed to drought tolerance in maize and provided a basis for further investigation of the role of RING domain-containing proteins.

The objective of this study is to identify the members of maize ZmC2s gene family, to analyze the association between their genetic variations and heat tolerance, and to lay a foundation for clarifying its function and molecular mechanism in maize heat tolerance. Using the C2 protein domain PF00168, hmmsearch was applied to search for the members of the ZmC2s gene family from maize B73 genome. The protein isoelectric point, molecular weight, phylogenetic evolution, and gene family replication were analyzed. Using the method of candidate gene association analysis, the association between the natural variations of ZmC2s and the heat tolerance of maize seedlings was conducted, and the important heat-tolerant candidate genes of maize ZmC2s gene family were found. The relative gene expression level of the heat-tolerant candidate gene under stress was identified by Real time fluorescent quantitative PCR (RT-qPCR). The subcellular expression sites of heat-tolerant candidate gene were identified by transforming maize protoplasts. A total of 95 maize ZmC2s genes were identified from the reference genome B73 in maize. According to the order of their physical coordinates, 95 maize ZmC2s genes were named from ZmC2-1 to ZmC2-95, respectively. The length of the 95 proteins was 130-2141, the isoelectric point was 4.1-10.8, and the molecular weight was 14.1-230.1. The evolution tree of C2 gene in maize, rice, and sorghum genomes was constructed. We found that C2 genes can be divided into three major cluster branches, and each cluster branch can be subdivided into two small cluster branches. Analyzing the whole genome collinearity data of maize, rice, and sorghum, 59 ZmC2s genes were detected to have corresponding replication genes in rice and sorghum genomes. A candidate-gene based on the association analysis of ZmC2s showed that ZmC2-15/60/91 were important candidate genes for heat tolerance in maize (P ≤ 0.001, MLM), among which ZmC2-15 was the most significantly associated to heat tolerance at seedling stage (P ≤ 0.000,01, MLM), and the relative expression level of ZmC2-15 was up-regulated under various stress treatments. Subcellular localization indicated that ZmC2-15 was localized in the cytoplasm, nuclear membrane, and endoplasmic reticulum. The overexpression of ZmC2-15 improved plant heat tolerance. ZmC2-15 can be used as an important candidate gene for regulating heat tolerance in maize.

Key message Cytoplasm-localized RING ubiquitin E3 ligase AtCHYR2 involved in plant glucose responses during germination and post-germinative growth. Abstract 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.

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.

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.

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.