Transcriptome Analysis of Cold-Tolerant and Cold-Sensitive Strawberry Cultivars Reveals the Defense Mechanisms Against Cold Stress.

Cold stress, triggered by particularly low temperatures, is one of the most severe forms of abiotic stress in pepper plants and a major constraint to the global pepper industry, threatening crop production and food security. To acclimatize to extreme conditions, the plant undergoes numerous modifications, including genetic and metabolic modulations. A thorough study of both the genetic and metabolic alterations of plants in response to cold stress is vital to understanding and developing the cold stress resistance mechanism. This study implemented transcriptome and metabolome analyses to evaluate the cold stress response in cold-tolerant and cold-sensitive pepper species. The weighted gene co-expression network revealed three significant modules related to cold stress tolerance in Capsicum pubescens. We identified 17 commonly enriched genes among both species at different time points in 10 different comparisons, including the AP2 transcription factor, LRR receptor-like serine, hypersensitivity-related 4-like protein, and uncharacterized novel.295 and novel.6172 genes. A pathway enrichment analysis indicated that these DEGs were mainly associated with the MAPK signaling pathway, hormone signaling pathway, and primary and secondary metabolism. Additionally, 21 significantly differentially accumulated metabolites (DAMs) were identified in both species after 6 h of cold stress. A transcriptome and metabolome integrated analysis revealed that 54 genes correlated with metabolites enriched in five different pathways. Most genes and metabolites involved in carbohydrate metabolism, the TCA cycle, and flavonoid biosynthesis pathways were upregulated in cold-tolerant plants under cold stress. Together, the results of this study provide a comprehensive gene regulatory and metabolic network in response to cold stress and identified some key genes and metabolic pathways involved in pepper cold tolerance. This study lays a foundation for the functional characterization and development of pepper cultivars with improved cold tolerance.

Climate changes and environmental stresses become severe over the past few decades. In particular, different abiotic stresses reduce the yield and quality of crop, leading to the threaten of global food security. With the deciphering of rice genome and advancement of functional genomics technology, researchers were able to gradually reveal the mechanism of abiotic stress tolerance mechanisms in rice and to identify essential genes for breeding to improve stress tolerance. In this thesis, we used TNG67 (japonica) and TCN1 (indica) rice cultivars with contrastive tolerance to cold and salt stresses as studying materials. A custom designed oligonucleotide array, Rice OneArray® v1 microarray platform (Phalanx Biotech Group Inc.) was used for transcriptomic analysis of shoot and root tissues of these two cultivars under cold or salt treatment and subsequent recovery. The results showed that TNG67 which is tolerant to cold and salt stresses can enhance TCA (tricarboxylic acid) cycle and PCD (programmed cell death) pathways under cold stress while it shifts to fermentation pathway for energy production and enhances the efficiency of Calvin cycle under salt stress and recovery, respectively. In addition, activation of SOS pathway may partially contribute to salt tolerance of TNG67. Increase of genes expressions related to phytohormone biosynthesis and response of ABA, PA, JA, and auxin can help TNG67 in cold stress tolerance. Besides, maintaining the balance and crosstalk of different hormones through the induction of gene expressions related to ABA, ET, PA, auxin, JA and the decrease of gene expressions associated with GA and CK responses may also be quite important for salt tolerance of TNG67. The crosstalk of ET with CK and JA in rice may play a role in the restoration of cold and salt stress. Also, we investigated the possible transcription factors (TFs) which may be the candidate genes that control cold or salt stress tolerance in rice. The induction or repression of TFs under stresses includes NACs and WRKYs, and MYB and AP2/ERF. NACs and WRKYs were the major TFs that may participate in cold tolerance, and MYB and AP2/ERF may involve in salt stress tolerance. Taken together aforementioned results, the cold- and salt-tolerance exhibit distinct regulatory mechanisms in TNG67 vs. TCN1. Interestingly, comparing the DEGs in shoots or roots of both rice cultivars under stresses, the venn diagram analysis showed that TNG67 and TCN1 shared less differentially expressed genes (DEGs) between cold and salt treatment. Although cold and salt stress can cause similar phenotypes and physiological damages, the molecular basis of cellular regulation mechanism can be quite different. Understanding the difference of cold and salt tolerance mechanisms in details is important in the future for us to breed rice precisely to cope with various abiotic stresses.

Peanut (Arachis hypogaea L.), also called groundnut is an important oil and cash crop grown widely in the world. The annual global production of groundnuts has increased to approximately 50 million tons, which provides a rich source of vegetable oils and proteins for humans. Low temperature (non-freezing) is one of the major factors restricting peanut growth, yield, and geographic distribution. Since the complexity of cold-resistance trait, the molecular mechanism of cold tolerance and related gene networks were largely unknown in peanut. In this study, comparative transcriptomic analysis of two peanut cultivars (SLH vs. ZH12) with differential cold tolerance under low temperature (10°C) was performed using Oxford Nanopore Technology (ONT) platform. As a result, we identified 8,949 novel gene loci and 95,291 new/novel isoforms compared with the reference database. More differentially expressed genes (DEGs) were discovered in cold-sensitive cultivar (ZH12) than cold-tolerant cultivar (SLH), while more alternative splicing events were found in SLH compared to ZH12. Gene Ontology (GO) analyses of the common DEGs showed that the "response to stress", "chloroplast part", and "transcription factor activity" were the most enriched GO terms, indicating that photosynthesis process and transcription factors play crucial roles in cold stress response in peanut. We also detected a total of 708 differential alternative splicing genes (DASGs) under cold stress compared to normal condition. Intron retention (IR) and exon skipping (ES) were the most prevalent alternative splicing (AS) events. In total, 4,993 transcription factors and 292 splicing factors were detected, many of them had differential expression levels and/or underwent AS events in response to cold stress. Overexpression of two candidate genes (encoding trehalose-6-phosphatephosphatases, AhTPPs) in yeast improves cold tolerance. This study not only provides valuable resources for the study of cold resistance in peanut but also lay a foundation for genetic modification of cold regulators to enhance stress tolerance in crops.

Rapeseed (Brassica napus L.) is an important oilseed crop worldwide. However, its productivity is significantly affected by various abiotic stresses, including cold stress. Among various stresses, cold stress is an important abiotic factor affecting plant growth, yield, and quality. The calcium channels are regarded as key pathways affecting cold tolerance in plants. Thus, improvement in cold tolerance is of great significance for crop improvement. The current study was designed to examine the beneficial role of exogenous inositol in improving cold stress tolerance in rapeseed. From the RNA-seq results, we identified 35 differently expressed genes encoding different inositol enzymes. The results show that inositol (a cyclic polyol) positively regulated cold tolerance by increasing calcium ion (Ca2+) influx in rapeseed. Furthermore, we found that the expression of calcineurin B-like (CBL1) gene was inhibited by inositol. On the other hand, overexpressed plant mediated the Ca2+ flux under cold stress suggesting the key role of inositol-Ca2+ pathway in cold tolerance. Moreover, the overexpression of BnCBL1-2 in Arabidopsis represented that transgenic plants mediated the Ca2+ flux highlighting the vital role of the inositol-Ca2+ pathway in conferring cold stress. Our study provides new insights into rapeseed cold tolerance mechanism and introduces a feasible method to improve the cold tolerance of rapeseed quickly.

Japanese apricot (Prunus mume) is remarkably valuable for its high ornamental and economic importance due to its distinctive features. Low temperature is a serious environmental constraint for this species, restricting its cultivation and dispersal in the north of China. To address this issue, breeding requires an understanding of the molecular mechanisms underlying responses to cold stress. We examined the leaf physiological and transcriptome profile by RNA sequencing in ‘Bungo’ scion cultivar grafted onto Prunus mume (cold-sensitive) and Prunus armeniaca (cold-tolerant) rootstocks at 4 °C for 0, 6, and 24 h. Our results revealed that the increased MDA concentration in the leaves of P. mume cultivar (cold-sensitive) suggests that cold stress might cause oxidative damage and increased sensitivity. Moreover, the cold-tolerant cultivar (P. armeniaca) considerably enhances the enzyme activities (i.e., SOD, POD, and CAT), as well as osmo-protectants (soluble sugars and proline) compared with sensitive cultivar, which helps plants to withstand oxidative damage caused by cold stress. Additionally, differentially expressed genes were shown to be enriched in plant hormone signal transduction, ribosome, MAPK signaling, and circadian rhythm pathway. After 24 h of cold stress, genes related to PYL4, histidine kinase 1, SAUR36, bHLH130, bHLH123, TIFY 6B-like, WRKY 40, WRKY 57, and 60S acidic ribosomal protein P1 were differentially expressed, implying that these DEGs involved in multiple pathways are involved in cold tolerance in Japanese apricot. This study improved our current understanding of the mechanism of cold tolerance in Japanese apricot, and the findings could be utilized for other related fruit species.

Cold stress is a significant factor limiting plant growth and development. Pomegranate is particularly susceptible to low temperatures. Calmodulin-binding transcriptional activators (CAMTAs) are key regulators of cold stress tolerance in plants. In this study, we conducted a comprehensive analysis of the CAMTA family proteins across 12 species, including Punica granatum (pomegranate), using bioinformatic methods. Pomegranate CAMTA3 (PgCAMTA3) was isolated and characterized, and it demonstrated enhanced cold tolerance when expressed in Arabidopsis thaliana. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression of PgCAMTA3 was up-regulated under cold and ABA treatments in pomegranates. Two A. thaliana transgenic lines, OE1 and OE2, which overexpress PgCAMTA3, were generated through genetic transformation. The overexpression of PgCAMTA3 enhanced the cold stress tolerance in transgenic A. thaliana. OE1 and OE2 exhibited higher survival rates under cold stress. Furthermore, enzymatic activity assays revealed enhanced peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) in OE lines. These antioxidant enzymatic activities collectively contribute to better cold stress tolerance by providing more effective reactive oxygen species (ROS) scavenging and cellular protection mechanisms, which was confirmed by lower levels of malondialdehyde (MDA) and ROS production. In addition, the overexpression of PgCAMTA3 led to the upregulation of the expression levels of AtCBF2, AtNCED3, and AtWRKY22, which were modulated by CAMTA3. In summary, we report the significant role of PgCAMTA3 in plant cold tolerance. Our findings provide valuable insights into the CAMATA family in plants and offer new perspectives on the molecular mechanisms underlying cold tolerance in pomegranates.

The objectives that were outlined in our original proposal have largely been achieved or will be so by the end of the project in February 1995 with one exception; that of mapping cold tolerance loci based on the segregation of tolerance in the BC1 progeny population. Briefly, our goals were to 1) construct a densely populated linkage map of the citrus genome: 2) map loci important in cold and/or salt stress tolerance; and 3) characterize the expression of genes responsive to cold land salt stress. As can be seen by the preceding listing of accomplishments, our original objectives A and B have been realized, objective C has been partially tested, objective D has been completed, and work on objectives E and F will be completed by the end of 1995. Although we have yet to map any loci that contribute to an ability of citrus to maintain growth when irrigated with saline water, our very encouraging results from the 1993 experiment provides us with considerable hope that 1994's much more comprehensive and better controlled experiment will yield the desired results once the data has been fully analyzed. Part of our optimism derives from the findings that loci for growth are closely linked with loci associated with foliar Cl- and Na+ accumulation patterns under non-salinization conditions. In the 1994 experiment, if ion exclusion or sequestration traits are segregating in the population, the experimental design will permit their resolution. Our fortunes with respect to cold tolerance is another situation. In three attempts to quantitatively characterize cold tolerance as an LT50, the results have been too variable and the incremental differences between sensitive and tolerant too small to use for mapping. To adequately determine the LT50 requires many plants, many more than we have been able to generate in the time and space available by making cuttings from small greenhouse-grown stock plants. As it has turned out, with citrus, to prepare enough plants needed to be successful in this objective would have required extensive facilities for both growing and testing hardiness which simply were not available at University of Florida. The large populations necessary to overcome the variability we encountered was unanticipated and unforeseeable at the project's outset. In spite of the setbacks, this project, when it is finally complete will be exceedingly successful. Listing of Accomplishments During the funded interval we have accomplished the following objectives: Developed a reasonably high density linkage map for citrus - mapped the loci for two cold responsive genes that were cloned from Poncirus - mapped the loci for csa, the salt responsive gene for glutathione peroxidase, and ccr a circadian rhythm gene from citrus - identified loci that confer parental derived specific DNA methylation patterns in the Citrus X Poncirus cross - mapped 5 loci that determine shoot vigor - mapped 2 loci that influence leaf Na+ accumulation patterns under non-saline conditions in the BC1 population - mapped 3 loci that influence leaf Na+ accumulation paterns during salt sress - mapped 2 loci that control leaf Cl- accumulation patterns under non-saline conditions - mapped a locus that controls leaf Cl- accumulation patterns during salt stress Screened the BC1 population for growth reduction during salinization (controls and salinized), and cold tolerance - determined population variation for shoot/root ratio of Na+ and Cl- - determined levels for 12 inorganic nutrient elements in an effort to examine the influence of salinization on ion content with emphasis on foliar responses - collected data on ion distribution to reveal patterns of exclusion/sequestration/ accumulation - analyzed relationships between ion content and growth Characterization of gene expression in response to salt or cold stress - cloned the gene for the salt responsive protein csa, identified it as glutathione peroxidase, determined the potential target substrate from enzymatic studies - cloned two other genes responsive to salt stress, one for the citrus homologue of a Lea5, and the other for an "oleosin" like gene - cold regulated (cor) genes belonging to five hybridization classes were isolated from Poncirus, two belonged to the group 2 Lea superfamily of stress proteins, the others show no significant homology to other known sequences - the expression of csa during cold acclimation was examined, and the expression of some of the cor genes were examined in response to salt stress - the influence of salinization on cold tolerance has been examined with seedling populations - conducted protein blot studies for expression of cold stress proteins during salt stress and vice versa

Leaf transcriptomic response mediated by cold stress in two maize inbred lines with contrasting tolerance levels

Gupta, K. J., Hinch, D. K., Mur, L. A. (2011). NO way to treat a cold. New Phytologist, 189, (2), 360-363. IMPF: 06.64

This study was carried out to better understand the role of salicylic acid (SA) applied before cold stress in the cold tolerance mechanism. Two barley (Hordeum vulgare) cultivars, cold-sensitive (Akhisar) and cold-tolerant (Tokak), were used and 0.1 mM SA was applied to 7-d-old barley seedlings growing under control conditions (20/18 °C). The seedlings were transferred to cold chamber (7/5 °C) at the age 14, 21, and 28 d. After three days, the leaves were harvested to determine the activities of apoplastic antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) and ice nucleation activity and electrophoretic patterns of apoplastic proteins. Cold treatment decreased the activities of all enzymes in cold-sensitive cultivar, however, it increased CAT and POX activities in cold-tolerant cultivar. Exogenous SA increased enzyme activities in both cultivars. Ice nucleation activity increased by cold treatment, especially in 17-d-old seedlings in both cultivars. In addition, SA treatment increased ice nucleation activity in all examined samplings in both cultivars. SA treatment caused accumulation or de novo synthesis of some apoplastic proteins. The results of the present study show that exogenous SA can improve cold tolerance by regulating the activities of apoplastic antioxidative enzymes, ice nucleation activity, and the patterns of apoplastic proteins.

Enhancement of cold tolerance in tea plants (Camellia sinensis) by glycine betaine accumulation through CsBADH overexpression.

The growth and development of taproots are inhibited by cold stress in radish (Raphanus sativus L.). Ethylene-responsive element binding factors (ERF) are key participators in the cold stress response and growth regulation of plants. However, the function of ERF genes in cold tolerance and root development in radish remains elusive. Here, we showed that the secondary growth of radish taproots was inhibited by cold stress. Comparative transcriptome analysis demonstrated that the RsERF40 gene is an important regulator of the cold stress response and root growth regulation. The cold tolerance of transgenic Arabidopsis plants overexpressing the RsERF40 gene was significantly improved. Overexpressing RsERF40 in the cold-sensitive radish genotype and silencing RsERF40 in the cold-tolerant radish genotype indicated that RsERF40 was beneficial for alleviating oxidative damage under cold stress in radish. Transgenic Arabidopsis seedlings showed an increase in the elongation and radial growth of dark-grown roots. RT-qPCR analysis showed that the expression of the cold-related genes (CORs) RsCOR78 and RsCOR413PM1 and the cell wall strengthening-related genes RsCESA6 and RsEXPB3 was upregulated in transgenic Arabidopsis seedlings. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays (DLA) revealed that RsERF40 directly regulates RsCOR78, RsCOR413PM1, RsCESA6 and RsEXPB3 expression, illustrating that RsERF40 enhances cold tolerance and taproot growth by modulating osmotic adjustment and cell wall mechanical strength in radish. In this study, the RsERF40-regulon was firstly found to be a new cold response pathway independent of the CBF-COR pathway conferring cold stress tolerance with increasing radish taproot growth. These results provided novel insight into the molecular mechanism underlying cold stress response and would facilitate the genetic improvement of cold tolerance in radish and other root vegetable crops.

Quantitative genetic analysis of tomato response to salt or cold stress during seed germination and vegetative growth indicated that both salt and cold tolerance were complex traits, controlled by more than one gene and highly influenced by environmental variation. Molecular marker analyses indicated that at each stage of plant development salt tolerance or cold tolerance was generally controlled by the effects of a few major QTLs (quantitative trait loci) which acted in concert with a number of smaller-effect QTLs. At the seed germination stage, two types of QTLs were identified: those which affected germination under both stress and nonstress conditions, and thus were called stress-nonspecific QTLs, and those which contributed to rapid seed germination only under specific stress conditions, and thus were called stress-specific QTLs. Generally, the stress-nonspecific QTLs exhibited larger effects than the stress-specific QTLs. Consistent with this observation, selection for either salt or cold tolerance during germination resulted in progeny with improved germination under salt and cold stress as well as nonstress conditions. Comparison of salt tolerance during germination and vegetative growth indicated that mostly different QTLs contributed to tolerance at these two developmental stages; furthermore, selection for salt tolerance during germination did not affect progeny salt tolerance during vegetative growth. Similar results were obtained when cold tolerance during germination and vegetative growth were compared. The overall results indicate that, in tomato, stress tolerance during germination is independent of stress tolerance during vegetative growth. However, simultaneous improvement of plants for stress tolerance at multiple stages of plant development should be feasible through marker-assisted selection and breeding.

Increasing the vulnerability of plants especially crops to a wide range of cold stress reduces plant growth, development, yield production, and plant distribution. Cold stress induces physiological, morphological, biochemical, phenotypic, and molecular changes in plants. Transcription factor (TF) is one of the most important regulators that mediate gene expression. TF is activated by the signal transduction pathway, together with cis-acting element modulate the transcription of cold-responsive genes which contribute to increasing cold tolerance in plants. Here, AP2/ERF TF family is one of the most important cold stress-related TF families that along with other TF families, such as WRKY, bHLH, bZIP, MYB, NAC, and C2H2 interrelate to enhance cold stress tolerance. Over the past decade, significant progress has been found to solve the role of transcription factors (TFs) in improving cold tolerance in plants, such as omics analysis. Furthermore, numerous studies have identified and characterized the complexity of cold stress mechanisms among TFs or between TFs and other factors (endogenous and exogenous) including phytohormones, eugenol, and light. The role, function, and relationship among these TFs or between TFs and other factors to enhance cold tolerance still need to be clarified. Here, the current study analysed the role of AP2/ERF TF and the linkages among AP2/ERF with MYB, WRKY, bZIP, bHLH, C2H2, or NAC against cold stress tolerance.

CsERF21-l-CsASA2 module positively regulates cold tolerance by promoting tryptophan biosynthesis in tea plants.