Background Helictotrichon virescens is a perennial grass that is primarily distributed in high altitude areas of 2000 ~ 4500 m. It is widely cultivated in the Qinghai-Tibet Plateau of China, strongly resistant to cold, and an essential part of the wild herbs in this region. However, the molecular mechanism of the response of H. virescens to low temperature stress and the key regulatory genes for specific biological processes are poorly understood. Results Physiological and transcriptome analyses were used to study the cold stress response mechanism in H virescens. During the low temperature stress period, the content of chlorophyll a and b decreased more and more with the delay of the treatment time. Among them, the difference between the controls was not significant, and the difference between the control and the treatment was significant. At the same time, the expression of related differential genes was up-regulated during low temperature treatment. In addition, the plant circadian pathway is crucial for their response to cold stress. The expression of differentially expressed genes that encode LHY and HY5 were strongly up-regulated during cold stress. Conclusions This study should help to fully understand how H. virescens responds to low temperatures. It answers pertinent questions in the response of perennial herbs to cold stress, i.e., how light and low temperature signals integrate to regulate plant circadian rhythms and Decrease of content of chlorophylls (which can be also accompanied with decrease of total quantity of reaction centers) leads to an increase in photosynthetic damage.

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Helictotrichon virescens is a perennial herbaceous plant with a life expectancy of about 10 years. It has high cold and heat resistance and can successfully survive over winter in the habitats with a temperature range of −25 to 25°C. Therefore, this study aimed to identify the key genes regulating low-temperature stress responses in H. virescens and analyze cold tolerant at molecular level. This study used RNA sequencing (RNA-Seq) and weighted gene co-expression network analysis (WGCNA) to identify the hub genes associated with cold tolerance in H. virescens. RT-PCR was conducted, homologous genes were identified, and related bioinformatics were analyzed to verify the identified hub genes. Moreover, WGCNA analysis showed that only the brown module had the highest correlation with the active-oxygen scavenging enzymes [peroxide (POD), superoxide dismutase (SOD), and catalase (CAT)]. The expression levels of three hub genes in the brown module (Cluster-37118.47362, cluster-37118.47713, and cluster-37118.66740) were significantly higher under low-temperature stress than those under control conditions. Furthermore, gene ontology (GO) and KEGG annotations showed that the three hub genes were mainly enriched in the metabolism pathways of sphingolipids, selenocompounds, glyoxylate, and dicarboxylate, carotenoids biosynthesis, and other biological pathways. The results of this study also showed that the subcellular localization prediction results showed that the cold tolerance hub genes were all localized to the plasma membrane. By constructing a protein interaction network, it was found that the hub gene Cluster-37118.66740 interacted with Sb09g003460.1 and Sb04g020180.1 proteins in Sorghum bicolor. By constructing phylogenetic trees of the four species of H. virescens, Sorghum bicolo, Oryza sativa Japonica, and Arabidopsis thaliana, the results showed that, the hub gene Cluster 37118.66740 (of H. virescens) and Os03g0340500 (of Oryza sativa Japonica) belonged to the same ancestral branch and were in the same subfamily. Thus, this study provides methodology and guidance to identify the cold tolerance genes for other herbage and their cold tolerant molecular mechanisms at molecular level.

Summary Low-temperature chilling injury in early spring or late spring severely restricts maize production. Therefore, improving its temperature tolerance is one of the important goals of maize breeding. The wild maize relatives such as Z. perennis and T. dactyloides have strong cold tolerance and thus can be used to improve the cold tolerance of cultivated maize. In the previous study, the allohexaploid MTP ( Zea . Z. perennis and T. dactyloides ) was created as a genetic bridge material and the introgressed cross of the backbone maize inbred line to obtain the MTP-maize introgression line with strong cold tolerance. In this study, F 2:3 populations was developed of the cold-tolerant maize introgression line IB030 and the cold-sensitive maize introgression line IB021. A high-density genetic linkage map was constructed using SSR genetic markers and performed QTL mapping for related traits at 2°C, RNA-seq was performed using cold-tolerant introgression line IB030 and recurrent parent B73 (cold-sensitive) after low temperature stress at seedling stage. The key genes controlling REC, SDW and SFW of maize introgression line seedlings were located by forward genetics combined with WGCNA analysis strategy. Key genes function verification results showed that, Zm00001d037590 REC and Zm00001d012321 REC have the highest expression levels in introgression line IB030, followed by the wild type B73, and finally the EMS mutant after low temperature stress, these results initially concluded that key genes positively regulate the cold tolerance of maize, and also laid a theoretical and practical foundation for improvement of cultivated maize against low-temperature stress.