Full-length transcript sequencing and comparative transcriptomic analysis to evaluate the contribution of osmotic and ionic stress components towards salinity tolerance in the roots of cultivated alfalfa (Medicago sativa L.)

Abstract

BackgroundAlfalfa is the most extensively cultivated forage legume. Salinity is a major environmental factor that impacts on alfalfa’s productivity. However, little is known about the molecular mechanisms underlying alfalfa responses to salinity, especially the relative contribution of the two important components of osmotic and ionic stress.ResultsIn this study, we constructed the first full-length transcriptome database for alfalfa root tips under continuous NaCl and mannitol treatments for 1, 3, 6, 12, and 24 h (three biological replicates for each time points, including the control group) via PacBio Iso-Seq. This resulted in the identification of 52,787 full-length transcripts, with an average length of 2551 bp. Global transcriptional changes in the same 33 stressed samples were then analyzed via BGISEQ-500 RNA-Seq. Totals of 8861 NaCl-regulated and 8016 mannitol-regulated differentially expressed genes (DEGs) were identified. Metabolic analyses revealed that these DEGs overlapped or diverged in the cascades of molecular networks involved in signal perception, signal transduction, transcriptional regulation, and antioxidative defense. Notably, several well characterized signalling pathways, such as CDPK, MAPK, CIPK, and PYL-PP2C-SnRK2, were shown to be involved in osmotic stress, while the SOS core pathway was activated by ionic stress. Moreover, the physiological shifts of catalase and peroxidase activity, glutathione and proline content were in accordance with dynamic transcript profiles of the relevant genes, indicating that antioxidative defense system plays critical roles in response to salinity stress.ConclusionsOverall, our study provides evidence that the response to salinity stress in alfalfa includes both osmotic and ionic components. The key osmotic and ionic stress-related genes are candidates for future studies as potential targets to improve resistance to salinity stress via genetic engineering.Electronic supplementary materialThe online version of this article (10.1186/s12870-019-1630-4) contains supplementary material, which is available to authorized users.

Morphological, Physiological, and Genetic Responses to Salt Stress in Alfalfa: A Review

“…Several transcriptome-based and proteome studies have been conducted to understand salinity stress in alfalfa [49,54,66,[81][82][83][84][85][86][87]. After a three-day salt treatment, a proteomic study on the roots of two-week-old seedlings found 83 differentially expressed proteins in alfalfa cultivars with contrasting tolerances to salinity (Table 1) [81].…”

“…Transcriptomic approaches have been employed for alfalfa to understand the gene expression associated with salt stress (Table 1) [49,66,[83][84][85]87]. Eighty-two unique transcripts were found from the salt-stressed seedlings of alfalfa by sampling at different time intervals from 10 min to 24 h, including 24% that were proteins related to plant metabolism and 9% that were related to abiotic stress [87].…”

“…The transcriptome responses of salt-tolerant ("211609") and salt-intolerant ("Xinjiang Daye") alfalfa cultivars revealed significantly higher expression levels of NHX1, ZFG, CBF4, and HSP23 genes in "211609" than in "Xinjiang Daye" [66]. In addition, a transcriptomic analysis of alfalfa roots under~25 dS m −1 NaCl identified 8861 NaCl-regulated DEGs in alfalfa [84]. These DEGs were categorized into 13 gene ontology categories: oxidoreductase activity, oxidation-reduction process, structural constituent of cytoskeleton, hydrolase activity, carbohydrate metabolic process, negative regulation of catalytic activity, polysaccharide catabolic process, iron ion binding, transmembrane transporter activity, cytoskeleton, trehalose biosynthetic process, protein polymerization, and ion homeostasis [84].…”

“…In addition, a transcriptomic analysis of alfalfa roots under~25 dS m −1 NaCl identified 8861 NaCl-regulated DEGs in alfalfa [84]. These DEGs were categorized into 13 gene ontology categories: oxidoreductase activity, oxidation-reduction process, structural constituent of cytoskeleton, hydrolase activity, carbohydrate metabolic process, negative regulation of catalytic activity, polysaccharide catabolic process, iron ion binding, transmembrane transporter activity, cytoskeleton, trehalose biosynthetic process, protein polymerization, and ion homeostasis [84]. In previous transcriptomic studies, an individual alfalfa genotype was sampled as a replicate for RNA-Seq analysis.…”

Coordinated mechanisms of leaves and roots in response to drought stress underlying full-length transcriptome profiling in Vicia sativa L

“…After PCR amplification, the BluePippin Size Selection System (Sage Science, Beverly, MA, USA) was used to select product sizes, and then three libraries were produced, corresponding to fragments of 1-2, 2-3 and 3-6 kb for each sample length. The amplified cDNA products were then subjected to the construction of SMRTbell Template libraries according to the Iso-Seq protocol [87,88]. Further, Qubit2.0 and Agilent 2100 were used to confirm accurate quantification and library size, respectively; only when the library size met the expected criteria was sequencing performed.…”