An aquaporin gene from halophyte <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis>, <Emphasis Type="Italic">SpAQP1</Emphasis>, increases salt tolerance in transgenic tobacco

Key message

SpAQP1 was strongly induced by salt in an ABA-independent way, promoted seed germination and root growth in transgenic tobaccos and increased salt tolerance by increasing the activities of antioxidative enzymes.

Abstract

Aquaporin (AQP) plays crucial roles in the responses of plant to abiotic stresses such as drought, salt and cold. Compared to glycophytes, halophytes often have excellent salt and drought tolerances. To uncover the molecular mechanism of halophyte Sesuvium portulacastrum tolerance to salt, in this study, an AQP gene, SpAQP1, from S. portulacastrum was isolated and characterized. The amino acid sequence of SpAQP1 shared high homology with that of plant plasma membrane intrinsic proteins (PIPs) and contained the distinct molecular features of PIPs. In the phylogenic tree, SpAQP1 was evidently classified as the PIP2 subfamily. SpAQP1 is expressed in roots, stems and leaves, and was significantly induced by NaCl treatment and inhibited by abscisic acid (ABA) treatment. When heterologously expressed in yeast and tobacco, SpAQP1 enhanced the salt tolerance of yeast strains and tobacco plants and promoted seed germination and root growth under salt stress in transgenic plants. The activity of antioxidative enzymes including superoxide dismutase, peroxidase and catalase was increased in transgenic plants overexpressing SpAQP1. Taken together, our studies suggested that SpAQP1 functioned in the responses of S. portulacastrum to salt stress and could increase salt tolerance by enhancing the antioxidative activity of plants.

     

Drought-induced expression of aquaporin genes in leaves of two common bean cultivars differing in tolerance to drought stress

Aquaporin proteins are part of the complex response of common bean (Phaseolus vulgaris L.) to drought which affects the quality and quantity of yield of this important crop. To better understand the role of aquaporins in common bean, drought-induced gene expression of several aquaporins was determined in two cultivars, the more drought tolerant Tiber and the less tolerant Starozagorski ?ern. The two bean cultivars were selected among 16 European genotypes based on the tolerance to drought determined by time needed for plants to wilt after withholding irrigation and yield at harvest. The expression patterns of two plasma membrane intrinsic proteins, PvPIP1;2 and PvPIP2;7, and two tonoplast intrinsic proteins, PvTIP1;1 and PvTIP4;1 in leaves of 21 day old plants were determined by RT-qPCR in both cultivars under three degrees of drought stress, and under rehydration and control conditions. Gene expression of all four examined aquaporins was down-regulated in drought stressed plants. After rehydration it returned to the level of control plants or was even higher. The responses of PvPIP2;7 and PvTIP1;1 during drought and rehydration were particularly pronounced. The gene expression of PvPIP2;7 and PvTIP4;1 during drought was cultivar specific, with greater down-regulation of these two aquaporins in drought tolerant Tiber. Under drought stress the relative water content and water potential of leaves were higher in Tiber than in Starozagorski plants. The differences in these physiological parameters indicate greater prevention of water loss in Tiber during drought, which may be associated with rapid and adequate down-regulation of aquaporins. These results suggest that the ability of plants to conserve water during drought stress involves timely and sufficient down-regulation of gene expression of specific aquaporins.     

Water use efficiency in the drought-stressed sorghum and maize in relation to expression of aquaporin genes

Zea mays L. is less tolerant to drought than Sorghum bicolor L. In the present study, we investigated the response of both plants to drought stress applied under field conditions by withholding water for 10 d. The plant growth in terms of shoot fresh and dry masses was more severely reduced in maize than in sorghum, consistently with reduction of leaf relative water content. Gas exchange was also more inhibited by drought in maize than in sorghum. The water use efficiency (WUE) of maize fluctuated during the day and in response to the drought stress. In contrast, sorghum was able to maintain a largely constant WUE during the day in the well-watered plants as well as in the stressed ones. Studying the expression of four aquaporin genes (PIP1;5, PIP1;6, PIP2;3, and TIP1;2) revealed that PIP1;5 in leaves and PIP2;3 in roots were highly responsive to drought in sorghum but not in maize, where they might have supported a greater water transport. The expression pattern of PIP1;6 suggests its possible role in CO₂ transport in control but not droughty leaves of both the plants. TIP1;2 seemed to contribute to water transport in leaves of the control but not droughty plants. We conclude that PIP1;5 and PIP2;3 may have a prominent role in drought tolerance and maintenance of WUE in sorghum plants.     

Drastic anthocyanin increase in response to <Emphasis Type="Italic">PAP1</Emphasis> overexpression in <Emphasis Type="Italic">fls1</Emphasis> knockout mutant confers enhanced osmotic stress tolerance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

pap1 - D/fls1ko double mutant plants that produce substantial amounts of anthocyanin show tolerance to abiotic stress.

Abstract

Anthocyanins are flavonoids that are abundant in various plants and have beneficial effects on both plants and humans. Many genes in flavonoid biosynthetic pathways have been identified, including those in the MYB-bHLH-WD40 (MBW) complex. The MYB gene Production of Anthocyanin Pigment 1 (PAP1) plays a particularly important role in anthocyanin accumulation. PAP1 expression in many plant systems strongly increases anthocyanin levels, resulting in a dark purple color in many plant organs. In this study, we generated double mutant plants that harbor fls1ko in the pap1-D background (i.e., pap1-D/fls1ko plants), to examine whether anthocyanins can be further enhanced by blocking flavonol biosynthesis under PAP1 overexpression. We also wanted to examine whether the increased anthocyanin levels contribute to defense against osmotic stresses. The pap1-D/fls1ko mutants accumulated higher anthocyanin levels than pap1-D plants in both control and sucrose-treated conditions. However, flavonoid biosynthesis genes were slightly down-regulated in the pap1-D/fls1ko seedlings as compared to their expression in pap1-D seedlings. We also report the performance of pap1-D/fls1ko seedlings in response to plant osmotic stresses.

     

Over-expression of PIP2;5 aquaporin alleviates gas exchange and growth inhibition in poplars exposed to mild osmotic stress with polyethylene glycol

The effects of mild osmotic stress conditions on aquaporin-mediated water transport are not well understood. In the present study, mild osmotic stress treatments with 20 and 50 g L^?1 polyethylene glycol 6000 (PEG) in Hoagland’s mineral solution were applied for 3 weeks under controlled environmental conditions to transgenic Populus tremula × Populus alba plants constitutively over-expressing a Populus PIP2;5 aquaporin and compared with the wild-type plants. The PEG treatments resulted in growth reductions and triggered changes in net photosynthesis, transpiration, stomatal conductance and root hydraulic conductivity in the wild-type plants. However, height growth, leaf area, gas exchange, and root hydraulic conductivity were less affected by the PEG treatments in PIP2;5-over-expressing poplar lines. These results suggest that water transport across the PIP2;5 aquaporin is an important process contributing to tolerance of mild osmotic stress in poplar. Greater membrane abundance of PIP2;5 was most likely the factor that was responsible for higher root hydraulic conductivity leading to improved plant water flux and, consequently, greater gas exchange and growth rates under mild osmotic stress conditions. The results also provide evidence for the functional significance of PIP2;5 aquaporin in water transport and its strong link to growth processes in poplar.     

Fatty acid composition and <Emphasis Type="Italic">PlFADs</Emphasis> expression related to α-linolenic acid biosynthesis in herbaceous peony (<Emphasis Type="Italic">Paeonia lactiflora</Emphasis> Pall.)

As a new α-linolenic acid (ALA) resource, there has been little known about the relationship between expression levels of fatty acid desaturase (FAD) genes during seed development and fatty acid (FA) composition in herbaceous peony (Paeonia lactiflora Pall.). In this study, oil content and FA composition of nine cultivars were measured at four different stages during seed development. Moreover, five genes including PlFAD2-1, PlFAD2-2, PlFAD3-2, PlFAD6 and PlFAD7 related to the ALA biosynthesis were isolated. Furthermore, the relative expression levels of these genes in seeds were investigated in two cultivars, that is, ‘PL1’ and ‘PL2’ with higher and lower ALA content, respectively. The results showed that oil content was from 17.03 ± 0.15 to 24.51 ± 0.15%; 15 kinds of FA were detected. However, the relative content of ALA was decreased during seed development. Although, the genes PlFAD2-1, PlFAD2-2, PlFAD3-2, PlFAD6 and PlFAD7 were all expressed during the seed development, the expression levels varied significantly. Most of the genes were expressed strongly in the early stage but weakly in the late stage except PlFAD6. Moreover, expression level of gene PlFAD2-2 was the highest while that of PlFAD7 was lowest at S1. In addition, the relative expression level of genes in ‘PL1’ (high ALA content) was higher than those in ‘PL2’ (low ALA content) during the early stage of the seed development apart from gene PlFAD6. This study provides a reliable theoretical basis for improving the contents of ALA by means of genetic engineering in the herbaceous peony seed oil.     

Phosphorus deprivation effects on water relations of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> plant via reducing plasma membrane permeability

Plants grown in phosphorus-deprived solutions often exhibit disruption of water transport due to reduction in root hydraulic conductivity (Lp_r). To uncover the relationship between root Lp_r and water permeability coefficient (P_f) of plasma membrane and the role of aquaporins, we evaluated P_f of plasma membrane and also PIP-type aquaporin gene expression in tobacco (Nicotiana tabacum L.) plant roots after seven days P-deprivation. The results showed significant reduction in sap flow rate (J_v) and osmotic root hydraulic conductivity (L_pr-o) in P-deprived roots. These effects were reversed 24 h after P-resupplying. Interestingly, the P_f of root protoplasts was 57% lower in P-deprived plants compared with P-sufficient ones. The expression of NtPIP1;1 and NtPIP2;1 aquaporins did not change significantly in P-deprived plants compared with P-sufficient ones, but the copy number of NtAQP1 increased significantly in P-deprived plants. P-deprivation did not change Lpr-o significantly in antisense NtAQP1 plants. Taken together, these findings suggest that P-deprivation may play an important role in modulation of root hydraulic conductivity by affecting P_f in transcellular pathway of water flow across roots and aquaporins. Finally, we concluded that dominant water transport pathway under P-deprivation was transcellular one.     

Growth,ionic response,and gene expression of shoots and roots of perennial ryegrass under salinity stress

Growth, ionic responses, and expression of candidate genes to salinity stress were examined in two perennial ryegrass accessions differing in salinity tolerance. The salinity tolerant (PI265349) and sensitive accessions (PI231595) were subjected to 75-mM NaCl for 14 days in a growth chamber. Across two accessions, salinity stress increased shoot dry weight and concentrations of malondialdehyde (MDA) and Na⁺ in the shoots and roots, but decreased shoot Ca²⁺ and root K⁺ concentrations. Salinity stress also increased root expressions of SOS1, PIP1, and TIP1. Plant height and chlorophyll content were unaffected by salinity stress in the tolerant accession but significantly decreased in the sensitive accession. Shoot MDA content did not change in the tolerant accession but increased in the sensitive accession. A more dramatic increase in Na⁺ was found in the roots of the sensitive accession. Relative to the control, salinity stress reduced expression of SOS1, NHX1, PIP1, and TIP1 in the shoots but increased expression of these genes in the roots of the tolerant accession. Expression levels of SOS1 increased in the roots and expression of NHX1 increased in the shoots but decreased in the roots of the sensitive accession under salinity stress. A decline in PIP1 expression in the shoots and dramatic increases in TIP expression in both shoots and roots were found in the sensitive accession under salinity stress. The results suggested maintenance of plant growth and leaf chlorophyll content, lesser Na⁺ accumulation in the roots, and lower lipid peroxidation in the shoots which could be associated with salinity tolerance. The decreased expressions of SOS1, NHX1, and TIP1 in the shoots, and increased expressions of NHX1 and PIP1 in the roots might also be related to salinity tolerance in perennial ryegrass.     

Peanut RNA Helicase <Emphasis Type="Italic">AhRH47</Emphasis> Sustains Protein Synthesis Under Stress and Improves Stress Adaptation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Stress responsive RNA helicases are involved in translation initiation sustain protein synthesis. In this study, a stress responsive DEAD box RNA helicase, AhRH47 from peanut cDNA library was identified and characterised during stress. In silico analysis of AhRH47 showed the nine conserved motifs characteristic of an RNA helicase. The phylogenetic and amino acid sequence alignment analyses revealed that AhRH47 is highly homologous to an important DEAD box RNA helicase (eIF4A), which is involved in translation initiation. AhRH47 is stress responsive, being highly expressed under salinity and moisture stress, which is induced to a lesser extent under PEG and ABA treatments. Constitutive overexpression of AhRH47 in Arabidopsis conferred enhanced tolerance to salinity and mannitol-induced stresses. In addition, the transgenic plants showed improved tolerance under moisture stress and exhibited improved recovery growth on stress alleviation. Overexpressing plants showed increased ¹⁴C-labelled amino acids incorporation in to protein especially under stress condition. The results suggest AhRH47 transgenic lines maintained higher protein synthesis under stress and thus improved adaptation to osmotic and desiccation stresses.     

Identification of a drought responsive gene encoding a nuclear protein involved in drought and freezing stress tolerance in Arabidopsis

Plants have developed adaptive strategies to survive under different abiotic stressors. To identify new components involved in abiotic stress tolerance, we screened unannotated expressed sequence tags (ESTs) and evaluated their cold or drought response in Arabidopsis. We identified a drought response gene (DRG) encoding a 39.5-kDa polypeptide. This protein was expressed specifically in siliques and was induced by drought stress in most tissues. When a DRG-GFP construct was introduced into Arabidopsis protoplasts, GFP signals were detected only in the nucleus. The drg mutant plant was more sensitive to mannitol-induced osmotic stress in agar plates and to drought or freezing stress in soil than the wild-type. Activating the DRG restored the normal sensitivity of drg mutants to abiotic stressors. No differences in drought or freezing tolerance were observed between the wild-type and transgenic plants overexpressing the DRG. When DRG was expressed in a cold-sensitive Escherichia coli strain BX04, the transformed bacteria grew faster than the untransformed BXO4 cells under cold stress. These results demonstrate that DRG is a nuclear protein induced by abiotic stresses and it is required for drought and freezing tolerance in Arabidopsis.