Physiological aspects of raffinose family oligosaccharides in plants: protection against abiotic stress.

Abiotic stresses resulting from water deficit, high salinity or periods of drought adversely affect plant growth and development and represent major selective forces during plant evolution. The raffinose family oligosaccharides (RFOs) are synthesised from sucrose by the subsequent addition of activated galactinol moieties donated by galactinol. RFOs are characterised as compatible solutes involved in stress tolerance defence mechanisms, although evidence also suggests that they act as antioxidants, are part of carbon partitioning strategies and may serve as signals in response to stress. The key enzyme and regulatory point in RFO biosynthesis is galactinol synthase (GolS), and an increase of GolS in expression and activity is often associated with abiotic stress. It has also been shown that different GolS isoforms are expressed in response to different types of abiotic stress, suggesting that the timing and accumulation of RFOs are controlled for each abiotic stress. However, the accumulation of RFOs in response to stress is not universal and other functional roles have been suggested for RFOs, such as being part of a carbon storage mechanism. Transgenic Arabidopsis plants with increased galactinol and raffinose concentrations had better ROS scavenging capacity, while many sugars have been shown in vitro to have antioxidant activity, suggesting that RFOs may also act as antioxidants. The RFO pathway also interacts with other carbohydrate pathways, such as that of O-methyl inositol (OMI), which shows that the functional relevance of RFOs must not be seen in isolation to overall carbon re-allocation during stress responses.

The SUI-homologous translation initiation factor eIF-1 is involved in regulation of ion homeostasis in rice.

Halophytes survive high salinity by using complex adaptive mechanisms. In a search for novel molecular mechanisms involved in salt acclimation, transcript analyses revealed increased expression of a SUI-homologous translation initiation factor eIF-1 in the salt-tolerant grass species Festuca rubra ssp. littoralis but not in rice. Upon analysis of the cell specificity of eIF-1 transcription by in situ polymerase chain reaction (PCR), predominant signals were detected in rice leaf mesophyll. To further examine the role of eIF-1 in salt tolerance, transgenic rice plants were generated that over-express this factor under the control of the CaMV-35S promoter. The eIF-1 over-expressing lines showed improved growth under salt stress that was correlated with maintenance of photosynthetic activity and reduced Na(+) and Cl(-) accumulation in leaves. The transgenic rice lines also activated expression of the vacuolar H(+)-ATPase. In addition, an oxidoreductase that belongs to the aldo/keto reductase family was identified as a gene with modified expression in the eIF-1 over-expressing lines, compared with wild-type rice. Our data suggest that eIF-1 has a central function in salt-stress adaptation in rice by regulating ion accumulation and the intracellular redox status.

Reversible redox control of plant vacuolar H+-ATPase activity is related to disulfide bridge formation in subunit E as well as subunit A.

The plant vacuolar proton pump can be subjected to reversible redox regulation in vitro. The redox-dependent activity change involves disulfide bridge formation not only in Vatp A, as reported for bovine V-ATPase, but also in the stalk subunit Vatp E. Microsomal membranes isolated from barley leaves were analysed for their activity of bafilomycin-sensitive ATP hydrolysis and proton pumping using quinacrine fluorescence quenching in vesicle preparations. ATP hydrolysis and proton pumping activity were inhibited by H2O2. H2O2-deactivated ATPase was reactivated by cysteine and glutathione. The glutathione concentration needed for half maximal reactivation was 1 mmol l-1. The activity loss was accompanied by shifts in electrophoretic mobility of Vatp A and E which were reversed upon reductive reactivation. The redox-dependent shift was also seen with recombinant Vatp E, and was absent following site-directed mutagenesis of either of the two cys residues conserved throughout all plant Vatp E sequences. V-ATPase was also inhibited by oxidized thioredoxin. These results support the hypothesis that tuning of vacuolar ATPase activity can be mediated by redox control depending on the metabolic requirements.

Significance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level.

Two electrogenic H(+)-pumps, the vacuolar type H(+)-ATPase (V-ATPase) and the vacuolar pyrophosphatase, coexist at membranes of the secretory pathway of plants. The V-ATPase is the dominant H(+)-pump at endomembranes of most plant cells, both in terms of protein amount and, frequently, also in activity. The V-ATPase is indispensable for plant growth under normal conditions due to its role in energizing secondary transport, maintenance of solute homeostasis and, possibly, in facilitating vesicle fusion. Under stress conditions such as salinity, drought, cold, acid stress, anoxia, and excess heavy metals in the soil, survival of the cells depends strongly on maintaining or adjusting the activity of the V-ATPase. Regulation of gene expression and activity are involved in adapting the V-ATPase on long- and short-term bases. The mechanisms known to regulate the V-ATPase are summarized in this paper with an emphasis on their implications for growth and development under stress.

Salt-induced expression of the vacuolar H+-ATPase in the common ice plant is developmentally controlled and tissue specific.

For salinity stress tolerance in plants, the vacuolar type H+-ATPase (V-ATPase) is of prime importance in energizing sodium sequestration into the central vacuole and it is known to respond to salt stress with increased expression and enzyme activity. In this work we provide information that the expressional response to salinity of the V-ATPase is regulated tissue and cell specifically under developmental control in the facultative halophyte common ice plant (Mesembryanthemum crystallinum). By transcript analysis of subunit E of the V-ATPase, amounts did not change in response to salinity stress in juvenile plants that are not salt-tolerant. In a converse manner, in halotolerant mature plants the transcript levels increased in leaves, but not in roots when salt stressed for 72 h. By in situ hybridizations and immunocytological protein analysis, subunit E was shown to be synthesized in all cell types. During salt stress, signal intensity declined in root cortex cells and in the cells of the root vascular cylinder. In salt-stressed leaves of mature plants, the strongest signals were localized surrounding the vasculature. Within control cells and with highest abundance in mesophyll cells of salt-treated leaves, accumulation of subunit E protein was observed in the cytoplasm, indicating its presence not only in the tonoplast, but also in other endoplasmic compartments.

Expression and stress-dependent induction of potassium channel transcripts in the common ice plant.

We have characterized transcripts for three potassium channel homologs in the AKT/KAT subfamily (Shaker type) from the common ice plant (Mesembryanthemum crystallinum), with a focus on their expression during salt stress (up to 500 mM NaCl). Mkt1 and 2, Arabidopsis AKT homologs, and Kmt1, a KAT homolog, are members of small gene families with two to three isoforms each. Mkt1 is root specific; Mkt2 is found in leaves, flowers, and seed capsules; and Kmt1 is expressed in leaves and seed capsules. Mkt1 is present in all cells of the root, and in leaves a highly conserved isoform is detected present in all cells with highest abundance in the vasculature. MKT1 for which antibodies were made is localized to the plasma membrane. Following salt stress, MKT1 (transcripts and protein) is drastically down-regulated, Mkt2 transcripts do not change significantly, and Kmt1 is strongly and transiently (maximum at 6 h) up-regulated in leaves and stems. The detection and stress-dependent behavior of abundant transcripts representing subfamilies of potassium channels provides information about tissue specificity and the complex regulation of genes encoding potassium uptake systems in a halophytic plant.

Expression of water channel proteins in Mesembryanthemum crystallinum.

We have characterized transcripts for nine major intrinsic proteins (MIPs), some of which function as water channels (aquaporins), from the ice plant Mesembryanthemum crystallinum. To determine the cellular distribution and expression of these MIPs, oligopeptide-based antibodies were generated against MIP-A, MIP-B, MIP-C, or MIP-F, which, according to sequence and functional characteristics, are located in the plasma membrane (PM) and tonoplast, respectively. MIPs were most abundant in cells involved in bulk water flow and solute flux. The tonoplast MIP-F was found in all cells, while signature cell types identified different PM-MIPs: MIP-A predominantly in phloem-associated cells, MIP-B in xylem parenchyma, and MIP-C in the epidermis and endodermis of immature roots. Membrane protein analysis confirmed MIP-F as tonoplast located. MIP-A and MIP-B were found in tonoplast fractions and also in fractions distinct from either the tonoplast or PM. MIP-C was most abundant but not exclusive to PM fractions, where it is expected based on its sequence signature. We suggest that within the cell, MIPs are mobile, which is similar to aquaporins cycling through animal endosomes. MIP cycling and the differential regulation of these proteins observed under conditions of salt stress may be fundamental for the control of tissue water flux.

Subunit C of the vacuolar H+-ATPase of Hordeum vulgare.

The molecular cloning of the first subunit C of the plant vacuolar H+-ATPase is reported. Tonoplast vesicles were purified from barley leaves by sucrose gradient centrifugation, and the tonoplast polypeptides were separated by two-dimensional (2-D) gel electrophoresis. Using an anti-ATPase holoenzyme antibody, a polypeptide was recognized in the molecular mass range of 40 kDa with an isoelectric point of about 6.0, and tentatively identified as subunit C. The polypeptide spot was excised from about 50 2-D gels and subjected to endo Lys C proteolysis. Two proteolytic peptides were sequenced and the amino acid sequences were used to design degenerated oligonucleotides, followed by PCR amplification with cDNA template and screening of a cDNA library synthesized from Hordeum vulgare poly A mRNA of epidermis strips. The full length clone of 1.5 kbp contains an open reading frame of 1062 bp encoding a polypeptide of 354 amino acids with a molecular mass of 39,982 Da and an isoelectric point of 6.04. Amino acid identity with sequences of SUC from animals and fungi is in the range of 36.7 to 38.5%. Expression of the cloned gene was demonstrated by Northern blotting and RT-PCR.

Subunit D of the vacuolar H+-ATPase of Arabidopsis thaliana.

A 1034 bp cDNA encoding the full length sequence of subunit D of the vacuolar H+-ATPase was cloned from Arabidopsis thaliana. The open reading frame of the cDNA clone vatpD contains 780 bp and codes for a protein of 29.1 kDa with a pI of 9.52. Structural predictions show similarities to subunit gamma of the F-ATP synthases. Identity between subunit D of the vacuolar H+-ATPase of A. thaliana and subunits D from other eukaryotic organisms is in the range of 57% (Bos taurus) to 48% (Candida albicans). Hybridization of genomic DNA with vatpD indicates the existence of one gene copy of subunit D in A. thaliana. Northern blot hybridization and in situ hybridization showed expression of vatpD in all cell types. The expression of subunit D was not modified by salt stress or abscisic acid treatment in A. thaliana.