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Identification of salt-responsive genes from C4 halophyte Suaeda nudiflora through suppression subtractive hybridization and expression analysis under individual and combined treatment of salt and elevated carbon dioxide conditions.

ABSTRACT: Salinization of soil is a prime abiotic stress that limits agriculture productivity worldwide. To Study the mechanisms that halophytes take up to survive under high salt condition is important in engineering salinity stress tolerance in sensitive species. Suaeda nudiflora is a halophyte plant that grows in the saline environment and extreme high tidal belt. The species have high capability to produce high protein biomass in salty soils due to C4 photosynthesis. The physiological and biochemical changes in S. nudiflora under salinity stress were studied by measuring chlorophyll content, electrolytic leakage, level of lipid peroxidation and total soluble sugars. Increased lipid peroxidation and electrolytic leakage was observed in salt stressed S. nudiflora compared to control plants. A suppression subtractive hybridization strategy was employed to identify differentially expressed genes under salt treatment in S. nudiflora. A total of 333 positive clones were identified and screened. Of these, 250 expressed sequence tags were identified. cDNA subtraction library resulted in 33 contigs and 138 singletons. The functional annotation and metabolic pathways identification were performed using the Blast2GO program. In addition, we analyzed the expression patterns of 18 genes associated with salt stress-responsive pathways by semi-quantitative PCR under salt and elevated carbon dioxide (CO₂) conditions. Several of the analyzed genes showed an increase in expression levels under different time points of salt treatment and at different concentrations of salt. When the same genes were studied for its expression under elevated CO₂ concentrations, most of the known salt responsive genes showed higher expression under the combined treatment of elevated CO₂ concentrations (500 ppm) and NaCl treatment (200 mM) compare to ambient condition. This implies that salt responsive genes are enhanced at elevated CO₂ concentrations.

SUBMITTER: Jothiramshekar S

PROVIDER: S-EPMC7036381 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Identification of salt-responsive genes from C4 halophyte <i>Suaeda nudiflora</i> through suppression subtractive hybridization and expression analysis under individual and combined treatment of salt and elevated carbon dioxide conditions.

Jothiramshekar Saranya S Benjamin Jenifer Joseph JJ Krishnasamy Rani R George Suja S Swaminathan Rajalakshmi R Parida Ajay A

Physiology and molecular biology of plants : an international journal of functional plant biology 20191128 1

Salinization of soil is a prime abiotic stress that limits agriculture productivity worldwide. To Study the mechanisms that halophytes take up to survive under high salt condition is important in engineering salinity stress tolerance in sensitive species. <i>Suaeda nudiflora</i> is a halophyte plant that grows in the saline environment and extreme high tidal belt. The species have high capability to produce high protein biomass in salty soils due to C4 photosynthesis. The physiological and bioch ...[more]

PMID: 32158127

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Project description:BackgroundDespite wealth of information generated on salt tolerance mechanism, its basics still remain elusive. Thus, there is a need of continued effort to understand the salt tolerance mechanism using suitable biotechnological techniques and test plants (species) to enable development of salt tolerant cultivars of interest. Therefore, the present study was undertaken to generate information on salt stress responsive genes in a natural halophyte, Suaeda maritima, using PCR-based suppression subtractive hybridization (PCR-SSH) technique.ResultsForward and reverse SSH cDNA libraries were constructed after exposing the young plants to 425 mM NaCl for 24 h. From the forward SSH cDNA library, 429 high quality ESTs were obtained. BLASTX search and TIGR assembler programme revealed overexpression of 167 unigenes comprising 89 singletons and 78 contigs with ESTs redundancy of 81.8%. Among the unigenes, 32.5% were found to be of special interest, indicating novel function of these genes with regard to salt tolerance. Literature search for the known unigenes revealed that only 17 of them were salt-inducible. A comparative analysis of the existing SSH cDNA libraries for NaCl stress in plants showed that only a few overexpressing unigenes were common in them. Moreover, the present study also showed increased expression of phosphoethanolamine N-methyltransferase gene, indicating the possible accumulation of a much studied osmoticum, glycinebetaine, in halophyte under salt stress. Functional categorization of the proteins as per the Munich database in general revealed that salt tolerance could be largely determined by the proteins involved in transcription, signal transduction, protein activity regulation and cell differentiation and organogenesis.ConclusionThe study provided a clear indication of possible vital role of glycinebetaine in the salt tolerance process in S. maritima. However, the salt-induced expression of a large number of genes involved in a wide range of cellular functions was indicative of highly complex nature of the process as such. Most of the salt inducible genes, nonetheless, appeared to be species-specific. In light of the observations made, it is reasonable to emphasize that a comparative analysis of ESTs from SSH cDNA libraries generated systematically for a few halophytes with varying salt exposure time may clearly identify the key salt tolerance determinant genes to a minimum number, highly desirable for any genetic manipulation adventure.

Project description:BackgroundImprovement of crop production is needed to feed the growing world population as the amount and quality of agricultural land decreases and soil salinity increases. This has stimulated research on salt tolerance in plants. Most crops tolerate a limited amount of salt to survive and produce biomass, while halophytes (salt-tolerant plants) have the ability to grow with saline water utilizing specific biochemical mechanisms. However, little is known about the genes involved in salt tolerance. We have characterized the transcriptome of Suaeda fruticosa, a halophyte that has the ability to sequester salts in its leaves. Suaeda fruticosa is an annual shrub in the family Chenopodiaceae found in coastal and inland regions of Pakistan and Mediterranean shores. This plant is an obligate halophyte that grows optimally from 200-400 mM NaCl and can grow at up to 1000 mM NaCl. High throughput sequencing technology was performed to provide understanding of genes involved in the salt tolerance mechanism. De novo assembly of the transcriptome and analysis has allowed identification of differentially expressed and unique genes present in this non-conventional crop.ResultsTwelve sequencing libraries prepared from control (0 mM NaCl treated) and optimum (300 mM NaCl treated) plants were sequenced using Illumina Hiseq 2000 to investigate differential gene expression between shoots and roots of Suaeda fruticosa. The transcriptome was assembled de novo using Velvet and Oases k-45 and clustered using CDHIT-EST. There are 54,526 unigenes; among these 475 genes are downregulated and 44 are upregulated when samples from plants grown under optimal salt are compared with those grown without salt. BLAST analysis identified the differentially expressed genes, which were categorized in gene ontology terms and their pathways.ConclusionsThis work has identified potential genes involved in salt tolerance in Suaeda fruticosa, and has provided an outline of tools to use for de novo transcriptome analysis. The assemblies that were used provide coverage of a considerable proportion of the transcriptome, which allows analysis of differential gene expression and identification of genes that may be involved in salt tolerance. The transcriptome may serve as a reference sequence for study of other succulent halophytes.

Project description:Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful analytical technique for the measurement of gene expression, which depends on the stability of the reference gene used for data normalization. Suaeda aralocaspica, an annual halophyte with heteromorphic seeds and possessing C4 photosynthesis pathway without Kranz anatomy, is an ideal plant species to identify stress tolerance-related genes and compare relative expression at transcriptional level. So far, no molecular information is available for this species. In the present study, six traditionally used reference genes were selected and their expression stability in two types of seeds of S. aralocaspica under different experimental conditions was evaluated. Three analytical programs, geNorm, NormFinder and BestKeeper, were used to assess and rank the stability of reference gene expression. Results revealed that although some reference genes may display different transcriptional profiles between the two types of seeds, β-TUB and GAPDH appeared to be the most suitable references under different developmental stages and tissues. GAPDH was the appropriate reference gene under different germination time points and salt stress conditions, and ACTIN was suitable for various abiotic stress treatments for the two types of seeds. For all the sample pools, β-TUB served as the most stable reference gene, whereas 18S rRNA and 28S rRNA performed poorly and presented as the least stable genes in our study. UBQ seemed to be unsuitable as internal control under different salt treatments. In addition, the expression of a photosynthesis-related gene (PPDK) of C4 pathway and a salt tolerance-related gene (SAT) of S. aralocaspica were used to validate the best performance reference genes. This is the first systematic comparison of reference gene selection for qRT-PCR work in S. aralocaspica and these data will facilitate further studies on gene expression in this species and other euhalophytes.

Project description:Background:Brazil has the largest commercial herd of ruminants with approximately 211 million head, representing 15% of world's beef production, in an area of 170 million hectares of grasslands, mostly cultivated with Brachiaria spp. Although nutrient reduction due to increased atmospheric carbon dioxide (CO2) concentration has already been verified in important crops, studies evaluating its effects on fiber fractions and elemental composition of this grass genus are still scarce. Therefore, a better understanding of the effects of elevated CO2 on forage quality can elucidate the interaction between forage and livestock production and possible adaptations for a climate change scenario. The objective of this study was to evaluate the effects of contrasting atmospheric CO2 concentrations on biomass production, morphological characteristics, fiber fractions, and elemental composition of Brachiaria decumbens (cv. Basilisk). Methods:A total of 12 octagonal rings with 10 m diameter were distributed in a seven-ha coffee plantation and inside each of them, two plots of 0.25 m2 were seeded with B. decumbens (cv. Basilisk) in a free air carbon dioxide enrichment facility. Six rings were kept under natural conditions (?390 ?mol mol-1 CO2; Control) and other six under pure CO2 flux to achieve a higher concentration (?550 ?mol mol-1 CO2; Elevated CO2). After 30 months under contrasting atmospheric CO2 concentration, grass samples were collected, and then splitted into two portions: in the first, whole forage was kept intact and in the second portion, the leaf, true stem, inflorescence and senescence fractions were manually separated to determine their proportions (%). All samples were then analyzed to determine the fiber fractions (NDF, hemicellulose, ADF, cellulose, and Lignin), carbon (C), nitrogen (N), potassium (K), calcium (Ca), sulfur (S), phosphorus (P), iron (Fe), and manganese (Mn) contents and N isotopic composition. Results:Elevated atmospheric CO2 concentration did not influence biomass productivity, average height, leaf, stem, senescence and inflorescence proportions, and fiber fractions (p > 0.05). Calcium content of the leaf and senescence portion of B. decumbens were reduced under elevated atmospheric CO2 (p < 0.05). Despite no effect on total C and N (p > 0.05), lower C:N ratio was observed in the whole forage grown under elevated CO2 (p < 0.05). The isotopic composition was also affected by elevated CO2, with higher values of ?15N in the leaf and stem portions of B. decumbens (p < 0.05). Discussion:Productivity and fiber fractions of B. decumbens were not influenced by CO2 enrichment. However, elevated CO2 resulted in decreased forage Ca content which could affect livestock production under a climate change scenario.

Dataset Information

Identification of salt-responsive genes from C4 halophyte Suaeda nudiflora through suppression subtractive hybridization and expression analysis under individual and combined treatment of salt and elevated carbon dioxide conditions.

Publications

Identification of salt-responsive genes from C4 halophyte <i>Suaeda nudiflora</i> through suppression subtractive hybridization and expression analysis under individual and combined treatment of salt and elevated carbon dioxide conditions.

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