Abstract
Tonoplast Intrinsic Proteins (TIP) are plant aquaporins that are primarily localized in the tonoplast and play a role in the bidirectional flux of water and other substrates across a membrane. In barley, eleven members of the HvTIP gene subfamily have been identified. Here, we describe the transcription profile of the HvTIP genes in the leaves of barley seedlings being grown under optimal moisture conditions, drought stress and a re-watering phase. The applied drought stress caused a 55% decrease in the relative water content (RWC) in seedlings, while re-watering increased the RWC to 90% of the control. Our analysis showed that all HvTIP genes, except HvTIP3;2, HvTIP4;3 and HvTIP5.1, were expressed in leaves of ten-day-old barley seedlings under optimal water conditions with the transcripts of HvTIP2;3, HvTIP1;2 and HvTIP1;1 being the most abundant. We showed, for the first time in barley, a significant variation in the transcriptional activity between the analysed genes under drought stress. After drought treatment, five HvTIP genes, which are engaged in water transport, were down-regulated to varying degrees, while two, HvTIP3;1 and HvTIP4;1, were up-regulated. The HvTIP3;1 isoform, which is postulated as transporting hydrogen peroxide, expressed the highest increase of activity (ca. 5000x) under drought stress, thus indicating its importance in the response to this stress. Re-hydration caused the return of the expression of many genes to the level that was observed under optimal moisture conditions or, at least, a change in this direction Additionally, we examined the promotor regions of HvTIP and detected the presence of the cis-regulatory elements that are connected with the hormone and stress responses in all of the genes. Overall, our results suggest that 7 of 11 studied HvTIP (HvTIP1;1, HvTIP1;2, HvTIP2;1, HvTIP2;2, HvTIP2;3, HvTIP3;1, HvTIP4;1) have an important function during the adaptation of barley to drought stress conditions. We discuss the identified drought-responsive HvTIP in terms of their function in the adaptation of barley to this stress.
Highlights
TIP aquaporin in barley control growth; DAS, days after sowing; DRE, dehydration-responsive element; DS, drought stress; gibberellic acid (GA), gibberellins; LTR, low temperatureresponsive elements; MBS, MYB binding site involved in drought-inducibility; MeJA, methyl jasmonate; MIP, membrane intrinsic proteins; NPA, asparagine-proline-alanine; RW, re-watering phase; RWC, relative water content; TIP, tonoplast intrinsic proteins
The EnsemblePlants database was used to identify the promotor sequences of the HvTIP genes and the mRNA sequences that are deposited in the NCBI database were used as the query
The three main clusters are shown: a): HvTIP4;3, HvTIP4;1, HvTIP4;2 and HvTIP5;1, b): HvTIP1;1, HvTIP1;2, HvTIP3;1 and HvTIP3;2 and c): HvTIP2;1, HvTIP2;2 and HvTIP2;3, which are further subdivided into smaller groups in which the isoforms that have the same main number occur (Fig 2)
Summary
Depending on the membrane location and amino acid sequence, the higher plant MIP, including AQP, are typically divided into five subfamilies: the plasma membrane intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), nodulin-26-like proteins (NIP), small, basic intrinsic proteins (SIP) and the uncategorised X intrinsic proteins (XIP) [5]. According to their molecular structure, all MIP consist of: six transmembrane helices, five inter-helical loops, two short helices that contain the highly conserved Asn-Pro-Ala (NPA) motif that forms the pore and the so-called aromatic/arginine (ar/R) selectivity filter, including four amino acids that act as a size-exclusion barrier because they form the narrowest part of a pore [5, 6, 7]. Four AQP monomers assemble to form a tetrameric holoprotein [5]
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