Physiological and lipidomic response of exogenous choline chloride alleviating salt stress injury in Kentucky bluegrass (Poa pratensis).

Introduction: Choline participates in plant stress tolerance through glycine betaine (GB) and phospholipid metabolism. As a salt-sensitive turfgrass species, Kentucky bluegrass (Poa pratensis) is the main turfgrass species in cool-season areas. Methods: To improve salinity tolerance and investigate the effects of choline on the physiological and lipidomic responses of turfgrass plants under salinity stress conditions, exogenous choline chloride was applied to Kentucky bluegrass exposed to salt stress. Results: From physiological indicators, exogenous choline chloride could alleviate salt stress injury in Kentucky bluegrass. Lipid analysis showed that exogenous choline chloride under salt-stress conditions remodeled the content of phospholipids, glycolipids, and lysophospholipids. Monogalactosyl diacylglycerol, digalactosyl diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophosphatidylcholine content were increased and phosphatidic acid content were decreased in plants after exogenous choline chloride under salt treatment. Plant leaf choline content increased, but GB was not detected in exogenous choline chloride treatment plants under nonstress or salt-stress conditions. Discussion: GB synthesis pathway related genes showed no clear change to choline chloride treatment, whereas cytidyldiphosphate-choline (CDP-choline) pathway genes were upregulated by choline chloride treatment. These results reveal that lipid remodeling through choline metabolism plays an important role in the salt tolerance mechanism of Kentucky bluegrass. Furthermore, the lipids selected in this study could serve as biomarkers for further improvement of salt-sensitive grass species.

Basic Helix-Loop-Helix Transcription Factors: Regulators for Plant Growth Development and Abiotic Stress Responses.

Plant basic helix-loop-helix (bHLH) transcription factors are involved in many physiological processes, and they play important roles in the abiotic stress responses. The literature related to genome sequences has increased, with genome-wide studies on the bHLH transcription factors in plants. Researchers have detailed the functionally characterized bHLH transcription factors from different aspects in the model plant Arabidopsis thaliana, such as iron homeostasis and abiotic stresses; however, other important economic crops, such as rice, have not been summarized and highlighted. The bHLH members in the same subfamily have similar functions; therefore, unraveling their regulatory mechanisms will help us to identify and understand the roles of some of the unknown bHLH transcription factors in the same subfamily. In this review, we summarize the available knowledge on functionally characterized bHLH transcription factors according to four categories: plant growth and development; metabolism synthesis; plant signaling, and abiotic stress responses. We also highlight the roles of the bHLH transcription factors in some economic crops, especially in rice, and discuss future research directions for possible genetic applications in crop breeding.

MicroRNA398: A Master Regulator of Plant Development and Stress Responses.

MicroRNAs (miRNAs) play crucial roles in plant development and stress responses, and a growing number of studies suggest that miRNAs are promising targets for crop improvement because they participate in the regulation of diverse, important agronomic traits. MicroRNA398 (miR398) is a conserved miRNA in plants and has been shown to control multiple stress responses and plant growth in a variety of species. There are many studies on the stress response and developmental regulation of miR398. To systematically understand its function, it is necessary to summarize the evolution and functional roles of miR398 and its target genes. In this review, we analyze the evolution of miR398 in plants and outline its involvement in abiotic and biotic stress responses, in growth and development and in model and non-model plants. We summarize recent functional analyses, highlighting the role of miR398 as a master regulator that coordinates growth and diverse responses to environmental factors. We also discuss the potential for fine-tuning miR398 to achieve the goal of simultaneously improving plant growth and stress tolerance.

Identification of bHLH genes through genome-wide association study and antisense expression of ZjbHLH076/ZjICE1 influence tolerance to low temperature and salinity in Zoysia japonica.

Abiotic stress greatly affects plant growth and developmental processes, resulting in poor productivity. A variety of basic helix-loop-helix (bHLH) transcription factors (TFs) that play important roles in plant abiotic stress response pathways have been identified. However, bHLH proteins of Zoysia japonica, one of the warm-season turfgrasses, have not been widely studied. In this study, 141 bHLH genes (ZjbHLHs) were identified and classified into 22 subfamilies. The ZjbHLHs were mapped on 19 chromosomes except for Chr17 and one pair of the tandemly arrayed genes was identified on Chr06. Also, the co-linearity of ZjbHLHs was found to have been driven mostly by segmental duplication events. The subfamily IIIb genes of our present interest, possessed various stress responsive cis-elements in their promoters. ZjbHLH076/ZjICE1, a MYC-type bHLH TF in subfamily IIIb was analyzed by overexpression and its loss-of-function via overexpressing a short ZjbHLH076/ZjICE1 fragment in the antisense direction. The overexpression of ZjbHLH076/ZjICE1 enhanced the tolerance to cold and salinity stress in the transgenic Z. japonica plants. However, the anti-sense expression of ZjbHLH076/ZjICE1 showed sensitive to these abiotic stresses. These results suggest that ZjbHLH076/ZjICE1 would be a promising candidate for the molecular breeding program to improve the abiotic stress tolerance of Z. japonica.

Small RNAs: The Essential Regulators in Plant Thermotolerance.

Small RNAs (sRNAs) are a class of non-coding RNAs that consist of 21-24 nucleotides. They have been extensively investigated as critical regulators in a variety of biological processes in plants. sRNAs include two major classes: microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis and functional pathways. Due to global warming, high-temperature stress has become one of the primary causes for crop loss worldwide. Recent studies have shown that sRNAs are involved in heat stress responses in plants and play essential roles in high-temperature acclimation. Genome-wide studies for heat-responsive sRNAs have been conducted in many plant species using high-throughput sequencing. The roles for these sRNAs in heat stress response were also unraveled subsequently in model plants and crops. Exploring how sRNAs regulate gene expression and their regulatory mechanisms will broaden our understanding of sRNAs in thermal stress responses of plant. Here, we highlight the roles of currently known miRNAs and siRNAs in heat stress responses and acclimation of plants. We also discuss the regulatory mechanisms of sRNAs and their targets that are responsive to heat stress, which will provide powerful molecular biological resources for engineering crops with improved thermotolerance.

A novel basic helix-loop-helix transcription factor, ZjICE2 from Zoysia japonica confers abiotic stress tolerance to transgenic plants via activating the DREB/CBF regulon and enhancing ROS scavenging.

KEY MESSAGE: ZjICE2 works as a positive regulator in abiotic stress responses and ZjICE2 is a valuable genetic resource to improve abiotic stress tolerance in the molecular breeding program of Zoysia japonica. The basic helix-loop-helix (bHLH) family transcription factors (TFs) play an important role in response to biotic or abiotic stresses in plants. However, the functions of bHLH TFs in Zoysia japonica, one of the warm-season turfgrasses, remain poorly understood. Here, we identified ZjICE2 from Z. japonica, a novel MYC-type bHLH transcription factor that was closely related to ICE homologs in the phylogenetic tree, and its expression was regulated by various abiotic stresses. Transient expression of ZjICE2-GFP in onion epidermal cells revealed that ZjICE2 was a nuclear-localized protein. Also, ZjICE2 bound the MYC cis-element in the promoter of dehydration responsive element binding 1 of Z. japonica (ZjDREB1) using yeast one-hybrid assay. A phenotypic analysis showed that overexpression of the ZjICE2 in Arabidopsis enhanced tolerance to cold, drought, and salt stresses. The transgenic Arabidopsis and Z. japonica accumulated more transcripts of cold-responsive DREB/CBFs and their downstream genes than the wild type (WT) after cold treatment. Furthermore, the transgenic plants exhibited an enhanced Reactive oxygen species (ROS) scavenging ability, which resulted in an efficient maintenance of oxidant-antioxidant homeostasis. In addition, overexpression of the ZjICE2 in Z. japonica displayed intensive cold tolerance with increases in chlorophyll contents and photosynthetic efficiency. Our study suggests that ZjICE2 works as a positive regulator in abiotic stress responses and the ICE-DREB/CBFs response pathway involved in cold stress tolerance is also conserved in Z. japonica. These results provide a valuable genetic resource for the molecular breeding program especially for warm-season grasses as well as other leaf crop plants.

Zoysia japonica MYC type transcription factor ZjICE1 regulates cold tolerance in transgenic Arabidopsis.

ICE1 (Inducer of CBF Expression 1) is a regulator of cold-induced transcriptome, which plays an important role in plant cold response pathway. To enhance the cold tolerance of Zoysia japonica, one of the warm-season turfgrasses, it is helpful to understand the cold response mechanism in Zoysia japonica. We identified stress-responsive ZjICE1 from Zoysia japonica and characterized its function in cold stress. Our results showed that ZjICE1 shared the typical feature of ICE homolog proteins belonging to a nucleic protein. Transactivation activity assay revealed that ZjICE1 bound to the MYC cis-element in the ZjDREB1's promotor. The ZjICE1 overexpressed transgenic Arabidopsis showed enhanced tolerance to cold stress with an increases in SOD, POD, and free proline content and reduction in MDA content. They also induced the transcripts abundance of cold-responsive genes (CBF1, CBF2, CBF3, COR47A, KIN1, and RD29A) after cold treatment. These results suggest that ZjICE1 is a positive regulator in Zoysia japonica plant during cold stress and can be a useful gene for the molecular breeding program to develop the cold tolerant zoysiagrass. Furthermore, the ZjICE1 also conferred resistance to salt and drought stresses, providing the better understanding of the basic helix-loop-helix (bHLH) gene family in abiotic stress responses.