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Characterization of Differentially Expressed Genes under Salt Stress in Olive.

ABSTRACT: Climate change, currently taking place worldwide and also in the Mediterranean area, is leading to a reduction in water availability and to groundwater salinization. Olive represents one of the most efficient tree crops to face these scenarios, thanks to its natural ability to tolerate moderate salinity and drought. In the present work, four olive cultivars (Koroneiki, Picual, Royal de Cazorla and Fadak86) were exposed to high salt stress conditions (200 mM of NaCl) in greenhouse, in order to evaluate their tolerance level and to identify key genes involved in salt stress response. Molecular and physiological parameters, as well as plant growth and leaves' ions Na⁺ and K⁺ content were measured. Results of the physiological measurements showed Royal de Cazorla as the most tolerant cultivar, and Fadak86 and Picual as the most susceptible ones. Ten candidate genes were analyzed and their complete genomic, CDS and protein sequences were identified. The expression analysis of their transcripts through reverse transcriptase quantitative PCR (RT-qPCR) demonstrated that only OeNHX7, OeP5CS, OeRD19A and OePetD were upregulated in tolerant cultivars, thus suggesting their key role in the activation of a salt tolerance mechanism.

SUBMITTER: Mousavi S

PROVIDER: S-EPMC8745295 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOlea europaea L. is a traditional tree crop of the Mediterranean basin with a worldwide economical high impact. Differently from other fruit tree species, little is known about the physiological and molecular basis of the olive fruit development and a few sequences of genes and gene products are available for olive in public databases. This study deals with the identification of large sets of differentially expressed genes in developing olive fruits and the subsequent computational annotation by means of different software.ResultsmRNA from fruits of the cv. Leccino sampled at three different stages [i.e., initial fruit set (stage 1), completed pit hardening (stage 2) and veraison (stage 3)] was used for the identification of differentially expressed genes putatively involved in main processes along fruit development. Four subtractive hybridization libraries were constructed: forward and reverse between stage 1 and 2 (libraries A and B), and 2 and 3 (libraries C and D). All sequenced clones (1,132 in total) were analyzed through BlastX against non-redundant NCBI databases and about 60% of them showed similarity to known proteins. A total of 89 out of 642 differentially expressed unique sequences was further investigated by Real-Time PCR, showing a validation of the SSH results as high as 69%. Library-specific cDNA repertories were annotated according to the three main vocabularies of the gene ontology (GO): cellular component, biological process and molecular function. BlastX analysis, GO terms mapping and annotation analysis were performed using the Blast2GO software, a research tool designed with the main purpose of enabling GO based data mining on sequence sets for which no GO annotation is yet available. Bioinformatic analysis pointed out a significantly different distribution of the annotated sequences for each GO category, when comparing the three fruit developmental stages. The olive fruit-specific transcriptome dataset was used to query all known KEGG (Kyoto Encyclopaedia of Genes and Genomes) metabolic pathways for characterizing and positioning retrieved EST records. The integration of the olive sequence datasets within the MapMan platform for microarray analysis allowed the identification of specific biosynthetic pathways useful for the definition of key functional categories in time course analyses for gene groups.ConclusionThe bioinformatic annotation of all gene sequences was useful to shed light on metabolic pathways and transcriptional aspects related to carbohydrates, fatty acids, secondary metabolites, transcription factors and hormones as well as response to biotic and abiotic stresses throughout olive drupe development. These results represent a first step toward both functional genomics and systems biology research for understanding the gene functions and regulatory networks in olive fruit growth and ripening.

Project description:Prunus triloba Lindl. is a naturally salt-alkaline-tolerant plant with several unique characteristics, and it can be used as the rootstock of Chinese plum (Prunus salicina Lindl.) in saline-alkaline soils. To comprehensively investigate the alkaline acclimation mechanisms in P. triloba, a series of analyses were conducted under alkaline stress, including analyses of the kinetics of molecular and physiological changes, and leaf microstructure.To understand the kinetics of molecular changes under short-term alkaline stress, we used Illumina HiSeq 2500 platform to identify alkaline stress-related differentially expressed genes (DEGs) in P. triloba. Approximately 53.0 million high-quality clean reads were generated from 59.6 million raw reads, and a total of 124,786 unigenes were obtained after de novo assembly of P. triloba transcriptome data. After alkaline stress treatment, a total of 8948 unigenes were identified as DEGs. Based on these DEGs, a Gene Ontology (GO) enrichment analysis was conducted, suggesting that 28 genes may play an important role in the early alkaline stress response. In addition, analysis of DEGs with the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that pathways were significant at different treatment time points. A significant positive correlation was found between the quantitative real-time PCR (qRT-PCR) results and the RNA-Seq data for seven alkaline-related genes, confirming the reliability of the RNA-Seq results. Based on physiological analysis of P. triloba in response to long-term alkaline stress, we found that the internal microstructures of the leaves of P. triloba changed to adapt to long-term alkaline stress. Various physiological indexes indicated that the degree of membrane injury increased with increasing duration of alkaline stress, affecting photosynthesis in P. triloba seedlings.This represents the first investigation into the physiology and transcriptome of P. triloba in response to alkaline stress. The results of this study can enrich the genomic resources available for P. triloba, as well as deepening our understanding of molecular and physiological alkaline tolerance mechanisms in P. triloba. This will also provide new insights into our understanding of alkaline acclimation mechanisms in Chinese plum (Prunus salicina) trees.

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Characterization of Differentially Expressed Genes under Salt Stress in Olive.

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