New Insights into the Roles of Osmanthus Fragrans Heat-Shock Transcription Factors in Cold and Other Stress Responses

Sweet osmanthus (Osmanthus fragrans) is an evergreen woody plant that emits a floral aroma and is widely used in the landscape and fragrance industries. However, its application and cultivation regions are limited by cold stress. Heat-shock transcription factor (HSF) family members are widely present in plants and participate in, and regulate, the defense processes of plants under various abiotic stress conditions, but now, the role of this family in the responses of O. fragrans to cold stress is still not clear. Here, 46 OfHSF members were identified in the O. fragrans genome and divided into three subfamilies on the basis of a phylogenetic analysis. The promoter regions of most OfHSFs contained many cis-acting elements involved in multiple hormonal and abiotic stresses. RNA-seq data revealed that most of OfHSF genes were differentially expressed in various tissues, and some OfHSF members were induced by cold stress. The qRT-PCR analysis identified four OfHSFs that were induced by both cold and heat stresses, in which OfHSF11 and OfHSF43 had contrary expression trends under cold stress conditions and their expression patterns both showed recovery tendencies after the cold stress. OfHSF11 and OfHSF43 localized to the nuclei and their expression patterns were also induced under multiple abiotic stresses and hormonal treatments, indicating that they play critical roles in responses to multiple stresses. Furthermore, after a cold treatment, transient expression revealed that the malondialdehyde (MDA) content of OfHSF11-transformed tobacco significantly increased, and the expression levels of cold-response regulatory gene NbDREB3, cold response gene NbLEA5 and ROS detoxification gene NbCAT were significantly inhibited, implying that OfHSF11 is a negative regulator of cold responses in O. fragrans. Our study contributes to the further functional characterization of OfHSFs and will be useful in developing improved cold-tolerant cultivars of O. fragrans.

Brassinosteroid Biosynthetic Gene SlCYP90B3 Alleviates Chilling Injury of Tomato (Solanum lycopersicum) Fruits during Cold Storage

Tomato is susceptible to chilling injury during cold storage. In this study, we found that low temperature promoted the expression of brassinosteroid (BR) biosynthetic genes in tomato fruits. The overexpression of SlCYP90B3 (SlCYP90B3-OE), a key BR biosynthetic gene, alleviated the chilling injury with decreased electrical conductivity and malondialdehyde. In SlCYP90B3-OE tomato fruits, the activities of antioxidant enzymes, including ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), were markedly increased, while the activity of membranous lipolytic enzymes, lipoxygenase (LOX), and phospholipase D (PLD), were significantly decreased when compared with the wild-type in response to cold storage. Furthermore, the expression level of the cold-response-system component, SlCBF1, was higher in SlCYP90B3-OE fruits than in the wild-type fruits. These results indicated that SlCYP90B3 might be involved in the chilling tolerance of tomato fruits during cold storage, possibly by regulating the antioxidant enzyme system and SlCBF1 expression.

What can the cold-induced transcriptomes of Arctic Brassicaceae tell us about the evolution of cold tolerance?

By studying the molecular basis of cold response in plants adapted to some of the world’s coldest biomes, we can gain insight into the evolution of cold tolerance - an important factor in determining plant distributions worldwide.Although cold tolerance in temperate plants have been extensively studied, little is known about the evolutionary changes needed to transition from temperate to the more extreme polar zones.Here, we conducted a time series experiment to examine the transcriptional responses of three Arctic Brassicaceae to low temperatures. RNA was sampled before onset of treatment, and after 3h, 6h, and 24h with 2 °C. We identified sets of genes that were differentially expressed in response to cold and compared them between species, as well as to published data from the temperate Arabidopsis thaliana.We found that the cold response is highly species-specific. Among thousands of differentially expressed genes, ∼200 genes were shared among the three Arctic species and A. thaliana, and only ∼100 genes were specific to the three Arctic species alone. This pattern was also reflected in the functional comparison.Our results show that the cold response of Arctic plant species has mainly evolved independently, although it likely builds on a conserved basis found across Brassicaceae. The findings also confirm that highly polygenic traits, such as cold tolerance, may show less repeatable patterns of adaptation than traits involving only a few genes.

Gene Expression Analysis in Cold Stress Conditions Reveals BBX20 and CLO as Potential Biomarkers for Cold Tolerance in Almond

Late spring frosts can become one of the limiting factors for the expansion of cultivation area towards a harsher climate for the almond [Prunus amygdalus Batsch syn P. dulcis (Mill.) D.A. Webb] crop as spring frost can damage up to 90% of the harvest. In order to identify key genes favoring cold tolerance in almonds, branches from three late-blooming genotypes: ‘Guara’, ‘Soleta’ and ‘Belona’ were exposed at −4 °C during 24 h in a constant climate chamber. Phenotype analysis showed that ‘Guara’ and ‘Soleta’ had a greater acclimation capacity to cold than ‘Belona’. The qRT-PCR BioMark System technology was used to monitor the relative expression of 30 candidate genes with a potential relation to cold response, which are either involved in the ICE-CBF-COR pathway or the independent CBF pathway, and also genes not yet characterized or with unknown function in almond genome. Differences in the gene expression profiles were found among the three studied genotypes and the three time-points of cold exposure (0, 2 and 24 h). BBX20 and CLO genes behaved as differentiator genes between tolerant and susceptible genotypes in cold stress response in almond pistils. In addition, the differences of expression among the tolerant genotypes suggested the intervention of different mechanisms responding to cold stress in almonds.

Data-Independent Acquisition-Based Proteome and Phosphoproteome Profiling Reveals Early Protein Phosphorylation and Dephosphorylation Events in Arabidopsis Seedlings upon Cold Exposure

Protein phosphorylation plays an important role in mediating signal transduction in cold response in plants. To better understand how plants sense and respond to the early temperature drop, we performed data-independent acquisition (DIA) method-based mass spectrometry analysis to profile the proteome and phosphoproteome of Arabidopsis seedlings upon cold stress in a time-course manner (10, 30 and 120 min of cold treatments). Our results showed the rapid and extensive changes at the phosphopeptide levels, but not at the protein abundance levels, indicating cold-mediated protein phosphorylation and dephosphorylation events. Alteration of over 1200 proteins at phosphopeptide levels were observed within 2 h of cold treatment, including over 140 kinases, over 40 transcriptional factors and over 40 E3 ligases, revealing the complexity of regulation of cold adaption. We summarized cold responsive phosphoproteins involved in phospholipid signaling, cytoskeleton reorganization, calcium signaling, and MAPK cascades. Cold-altered levels of 73 phosphopeptides (mostly novel cold-responsive) representing 62 proteins were validated by parallel reaction monitoring (PRM). In summary, this study furthers our understanding of the molecular mechanisms of cold adaption in plants and strongly supports that DIA coupled with PRM are valuable tools in uncovering early signaling events in plants.

Genome-Wide Characterization of Glutamine Synthetase Family Genes in Cucurbitaceae and Their Potential Roles in Cold Response and Rootstock-Scion Signaling Communication

Glutamine synthetase (GS; EC 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is the key enzyme responsible for the primary assimilation and reassimilation of nitrogen (N) in higher plants. There are two main isoforms of GS in higher plants, classified as cytosolic GS (GS1) and chloroplastic GS (GS2) by their size and subcellular localization. In order to improve the stress tolerance, quality, and yield of cucurbit crops such as cucumbers (Csa, Cucumis sativus L.), pumpkins (Cmo, Cucurbita moschata var. Rifu) are often used as rootstocks. Here, the GS family of the two species were comprehensively analyzed using bioinformatics in terms of aspects of the phylogenic tree, gene structure, chromosome location, subcellular localization, and evolutionary and expression patterns. Seven and four GS gene family members were screened in pumpkin and cucumber, respectively. GS family genes were divided into three groups (one for GS2 and two for GS1) according to their homology and phylogenetic relationships with other species. The analysis of gene ontology annotation of GS family genes, promoter regulatory elements, and tissue-specific expression patterns indicates the potential different biological roles of GS isoforms in Cucurbitaceae. In particular, we have identified a potentially available gene (GS1: CmoCh08G004920) from pumpkin that is relatively highly expressed and tissue-specifically expressed. RT-PCR analysis showed that most CmoGSs are induced by low temperature, and long-term (day 2 to day 9) cold stress has a more obvious effect on the RNA abundance of CmoGS. Our work presents the structure and expression patterns of all candidate members of the pumpkin and cucumber GS gene family, and to the best of our knowledge, this is the first time such work has been presented. It is worth focusing on the candidate genes with strong capacity for improving pumpkin rootstock breeding in order to increase nitrogen-use efficiency in cold conditions, as well as rootstock-scion communication.

Novel insights into the cold resistance of Hevea brasiliensis through coexpression networks

Hevea brasiliensis, a tropical tree species from the Amazon rainforest, is the main source of natural rubber worldwide. Due to the high pressure of fungal diseases in hot, humid regions, rubber plantations have been moved to escape areas, which are dryer and have lower temperatures during the winter. Here, we combined gene expression data of a primary (GT1) and a secondary (RRIM600) young rubber tree clones, which present different cold tolerance strategies, to analyze rubber tree gene expression regulation during 24 h of cold exposure (10 degrees Celsius). Together with traditional differential expression approaches, a RNA sequencing (RNA-seq) gene coexpression network (GCN) comprising 27,220 genes was established in which the genes were grouped into 832 clusters. In the GCN, most of the rubber tree molecular responses to cold stress were grouped in 26 clusters, which were divided into three GCN modules: a downregulated group comprising 12 clusters and two upregulated groups comprising eleven and three clusters. Considering the three modules identified, the general Hevea response to short-term cold exposure involved downregulation of gibberellin (GA) signaling, complex regulation of jasmonic acid (JA) stress responses and programmed cell death (PCD) and upregulation of ethylene responsive genes. The hub genes of the cold-responsive modules were subsequently identified and analyzed. As a result of the GCN strategy applied in this study, we could not only access individual DEGs related to the Hevea cold response, but also provide insights into a deeper cascade of associated mechanisms involved in the response to cold stress in young rubber trees. Our results may represent the genetic stress responses of the species, developed during its evolution, since the varieties chosen for this work are genotypes that were selected during the early years of rubber tree domestication. The understanding of H. brasiliensis cold response mechanisms can greatly improve the breeding strategies for this crop, which has a narrow genetic base, is impacted by climate change and is the only source for large-scale rubber production.

Gene Expression under Short-Term Low Temperatures: Preliminary Screening Method to Obtain Tolerant Citrus Rootstocks

Climate change and global warming are leading to a change in weather patterns toward hot and cold waves. Citrus fruits are a tropical or subtropical crop whose growth is altered by changes in weather patterns. Thus, in the present work, two experiments are evaluated to obtain a screening method to select citrus rootstocks that help us to select new low-temperature-tolerant plant materials. One cold experiment was carried out with the Poncirus trifoliata and Citrus macrophylla rootstocks at 4 °C for 4, 8, 24 and 56 h. A second experiment was performed at 4 °C for 5 days with subsequent acclimatization lasting 0, 5, 10 and 24 h. The expression of the cold response genes CAMTA1, CAMTA3, CAMTA5, CBF1, ICE1 and COR413 IM1 was quantified. The results showed that the best rootstock selection strategy was the second experiment, as a higher expression of the genes CAMTA3, CAMTA5, CBF1 and COR413 IM1 was seen in the tolerant genotype P. trifoliata. We quantified the gene expression of proline biosynthesis P5CS1, dOAT and the proline transporters PROT1 and PROT2; the concentration of the amino acid proline in leaves was also quantified. These results once again showed that the best experiment to differentiate between tolerant and sensitive rootstocks was the second experiment with acclimation time.

Physiological and transcriptome analysis of Magnolia denudata leaf buds during long-term cold acclimation

Background Magonlia denudata is an important perennial tree species of the Magnoliaceae family, known for its ornamental value, resistance to smoke pollution and wind, role in air purification, and robust cold tolerance. In this study, a high-throughput transcriptome analysis of leaf buds was performed, and gene expression following artificial acclimation 22 °C, 4 °C and 0 °C, was compared by RNA sequencing. Results Over 426 million clean reads were produced from three libraries (22 °C, 4 °C and 0 °C). A total of 74,503 non-redundant unigenes were generated, with an average length of 1173.7 bp (N50 = 1548). Based on transcriptional results, 357 and 235 unigenes were identified as being upregulated and downregulated under cold stress conditions, respectively. Differentially expressed genes were annotated using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses. The transcriptomic analysis focused on carbon metabolism and plant hormone signal transduction associated with cold acclimation. Transcription factors such as those in the basic helix-loop-helix and AP2/ERF families were found to play an important role in M. denudata cold acclimation. Conclusion M. denudata exhibits responses to non-freezing cold temperature (4 °C) to increase its cold tolerance. Cold resistance was further strengthened with cold acclimation under freezing conditions (0 °C). Cold tolerance genes, and cold signaling transcriptional pathways, and potential functional key components for the regulation of the cold response were identified in M. denudata. These results provide a basis for further studies, and the verification of key genes involved in cold acclimation responses in M. denudata lays a foundation for developing breeding programs for Magnoliaceae species.