Generation, Annotation, and Analysis of a Large-Scale Expressed Sequence Tag Library from Arabidopsis pumila to Explore Salt-Responsive Genes

Abstract

Arabidopsis pumila is an ephemeral plant, and a close relative of the model plant Arabidopsis thaliana, but it possesses higher photosynthetic efficiency, higher propagation rate, and higher salinity tolerance compared to those A. thaliana, thus providing a candidate plant system for gene mining for environmental adaption and salt tolerance. However, A. pumila is an under-explored resource for understanding the genetic mechanisms underlying abiotic stress adaptation. To improve our understanding of the molecular and genetic mechanisms of salt stress adaptation, more than 19,900 clones randomly selected from a cDNA library constructed previously from leaf tissue exposed to high-salinity shock were sequenced. A total of 16,014 high-quality expressed sequence tags (ESTs) were generated, which have been deposited in the dbEST GenBank under accession numbers JZ932319 to JZ948332. Clustering and assembly of these ESTs resulted in the identification of 8,835 unique sequences, consisting of 2,469 contigs and 6,366 singletons. The blastx results revealed 8,011 unigenes with significant similarity to known genes, while only 425 unigenes remained uncharacterized. Functional classification demonstrated an abundance of unigenes involved in binding, catalytic, structural or transporter activities, and in pathways of energy, carbohydrate, amino acid, or lipid metabolism. At least seven main classes of genes were related to salt-tolerance among the 8,835 unigenes. Many previously reported salt tolerance genes were also manifested in this library, for example VP1, H+-ATPase, NHX1, SOS2, SOS3, NAC, MYB, ERF, LEA, P5CS1. In addition, 251 transcription factors were identified from the library, classified into 42 families. Lastly, changes in expression of the 12 most abundant unigenes, 12 transcription factor genes, and 19 stress-related genes in the first 24 h of exposure to high-salinity stress conditions were monitored by qRT-PCR. The large-scale EST library obtained in this study provides first-hand information on gene sequences expressed in young leaves of A. pumila exposed to salt shock. The rapid discovery of known or unknown genes related to salinity stress response in A. pumila will facilitate the understanding of complex adaptive mechanisms for ephemerals.

Full-length transcriptome sequences of ephemeral plant Arabidopsis pumila provides insight into gene expression dynamics during continuous salt stress

“…Proline plays a crucial role in oxidative and osmotic responses in higher plants, and proline accumulation is a well-known measure adopted for alleviation of salt stress. We found that genes involved in arginine and proline metabolism were induced under stress, such as P5CS1 (Additional file 11 : Figure S6), which was consistent with our previous report [ 14 ]. Arginine and proline metabolism is one of the central pathways for the biosynthesis of the amino acids arginine and proline.…”

“…In plants, many transcription factors have been identified to confer salt tolerance using transcriptomic approaches, including AP2-EREBP, bHLH, bZIP, C2H2, NAC and WRKY [ 14 , 56 – 58 ]. In this study, 480 TFs were differentially regulated under salt stress, suggesting that TFs play important roles in modulating the acclimation response of A. pumila to salt stress.…”

“…Collection of plant specimens every year is permitted by the Xinjiang Uygur Autonomous Region government, and sampling collection at these locations did not involve protected or endangered species. Seeds of A. pumila were surface sterilized and planted as described by Huang et al (2017) [ 14 ]. After 7 days, the seedlings were transplanted into pots containing peat soil and vermiculite (1:1) and kept in a plant growth chamber with long day conditions (16-h-light/8-h-dark photoperiod, light intensity of 200 μmol photons m − 2 s − 1 , ambient temperature of 22 °C).…”

“…Primers used in this study are listed in Additional file 15 : Table S8. The expression of actin2 gene was used as the internal control [ 14 ]. Real-time PCR was carried out with the SYBR Green PCR Master Mix system (Takara) on an Applied Biosystems 7500/7500 Fast Real-time PCR System (ABI, Foster City, CA, USA).…”

“…An ephemeral plant, Arabidopsis pumila has considerable adaptability to local semi-arid and semi-salinized habitats [ 3 , 12 , 13 ]. We previously constructed a normalized cDNA library and analyzed the potential roles of genes represented by stress-responsive expressed sequence tags [ 3 , 14 ]. To date no genome size information has been reported in A. pumila , RNA-Seq is an ideal method to study gene expression profiles.…”