Arabidopsis pumila , an annual ephemeral plant, plays important roles in preventing wind and sand erosion, water and soil conservation, and microhabitat improvement in the North of Xinjiang, China. Studies of adaptive mechanisms in harsh desert environments at the genetic and genomic levels can be used to more effectively develop and protect this species. The quantitative real-time polymerase chain reaction (qRT-PCR) method is one of the essential means to achieve these goals, and the selection of an appropriate reference gene is the prerequisite for qRT-PCR. In this study, 10 candidate reference genes were identified from the full-length transcriptome data of A. pumila , and their expression stabilities under four abiotic stresses (drought, heat, cold and salt) and in seven different tissues (roots, hypocotyl, cotyledon, leaves, stems, flowers and siliques) were evaluated with four programmes geNorm, NormFinder, Bestkeeper and RefFinder. Although the most stable reference genes were variable under different treatments using different software, comprehensive ranking revealed that UEP and HAF1 under drought stress, UBQ9 and GAPDH under heat stress, UBC35 and GAPDH under cold stress, GAPDH and ACT1 under salt stress, and ACT1 and GAPDH in different tissues were the most stable reference genes. Moreover, GAPDH and UBQ9 were the most suitable reference gene combinations for all samples. The expression pattern of the K + uptake permease gene KUP9 further validated that the selected reference genes were suitable for normalization of gene expression. The identification of reliable reference genes guarantees more accurate qRT-PCR quantification for A. pumila and facilitates functional genomics studies of ephemeral plants.
BackgroundArabidopsis pumila is native to the desert region of northwest China and it is extraordinarily well adapted to the local semi-desert saline soil, thus providing a candidate plant system for environmental adaptation and salt-tolerance gene mining. However, understanding of the salt-adaptation mechanism of this species is limited because of genomic sequences scarcity. In the present study, the transcriptome profiles of A. pumila leaf tissues treated with 250 mM NaCl for 0, 0.5, 3, 6, 12, 24 and 48 h were analyzed using a combination of second-generation sequencing (SGS) and third-generation single-molecule real-time (SMRT) sequencing.ResultsCorrection of SMRT long reads by SGS short reads resulted in 59,328 transcripts. We found 8075 differentially expressed genes (DEGs) between salt-stressed tissues and controls, of which 483 were transcription factors and 1157 were transport proteins. Most DEGs were activated within 6 h of salt stress and their expression stabilized after 48 h; the number of DEGs was greatest within 12 h of salt stress. Gene annotation and functional analyses revealed that expression of genes associated with the osmotic and ionic phases rapidly and coordinately changed during the continuous salt stress in this species, and salt stress-related categories were highly enriched among these DEGs, including oxidation–reduction, transmembrane transport, transcription factor activity and ion channel activity. Orphan, MYB, HB, bHLH, C3H, PHD, bZIP, ARF and NAC TFs were most enriched in DEGs; ABCB1, CLC-A, CPK30, KEA2, KUP9, NHX1, SOS1, VHA-A and VP1 TPs were extensively up-regulated in salt-stressed samples, suggesting that they play important roles in slat tolerance. Importantly, further experimental studies identified a mitogen-activated protein kinase (MAPK) gene MAPKKK18 as continuously up-regulated throughout salt stress, suggesting its crucial role in salt tolerance. The expression patterns of the salt-responsive 24 genes resulted from quantitative real-time PCR were basically consistent with their transcript abundance changes identified by RNA-Seq.ConclusionThe full-length transcripts generated in this study provide a more accurate depiction of gene transcription of A. pumila. We identified potential genes involved in salt tolerance of A. pumila. These data present a genetic resource and facilitate better understanding of salt-adaptation mechanism for ephemeral plants.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5106-y) contains supplementary material, which is available to authorized users.
Plasma membrane intrinsic proteins (PIPs) are known to be major facilitators of the movement of a number of substrates across cell membranes. From a drought-resistant cultivar of Oryza sativa (rice), we isolated an OsPIP1;3 gene single-nucleotide polymorphism (SNP) that is mostly expressed in rice roots and is strongly responsive to drought stress. Immunocytochemistry showed that OsPIP1;3 majorly accumulated on the proximal end of the endodermis and the cell surface around the xylem. Expression of GFP-OsPIP1;3 alone in Xenopus oocytes or rice protoplasts showed OsPIP1;3 mislocalization in the endoplasmic reticulum (ER)-like neighborhood, whereas co-expression of OsPIP2;2 recruited OsPIP1;3 to the plasma membrane and led to a significant enhancement of water permeability in oocytes. Moreover, reconstitution of 103His-OsPIP1;3 in liposomes demonstrated water channel activity, as revealed by stopped-flow light scattering. Intriguingly, by patch-clamp technique, we detected significant NO 3 À conductance of OsPIP1;3 in mammalian cells. To investigate the physiological functions of OsPIP1;3, we ectopically expressed the OsPIP1;3 gene in Nicotiana benthamiana (tobacco). The transgenic tobacco plants exhibited higher photosynthesis rates, root hydraulic conductivity (Lp r ) and water-use efficiency, resulting in a greater biomass and a higher resistance to water deficit than the wild-type did. Further experiments suggested that heterologous expression of OsPIP1;3 in cyanobacterium altered bacterial growth under different conditions of CO 2 gas supply. Overall, besides shedding light on the multiple functions played by OsPIP1;3, this work provides insights into the translational value of plant AQPs.
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