ABSTRACT
We have used reverse transcription-PCR coupled with 5'- and 3'-RACE to isolate a full length INO1 cDNA (1692 bp with an ORF of 1530) from the resurrection plant Xerophyta viscosa Baker. XvINO1 encodes 510 amino acids, with a predicted MW of 56.7kD and contains four sequence motifs that are highly conserved in plant myo-inositol-1-phosphate synthases (MIPS, EC5.5.1.4), the enzyme that catalyses the first step in the formation of myo-inositol (Ino). Northern and western analyses show that the transcript and protein are constitutively present in leaves but their expression increases, temporarily, in response to both accumulative salt stress (~300 mM NaCl) and desiccation (to 5% relative water content). Leaf Ino concentration increases 40-fold during the first 6 h of salt stress, and levels of this and other carbohydrates (galactinol, sucrose, raffinose, stachyose and hexoses) remain elevated relative to control leaves for the duration of salt stress treatment. The timing and pattern of accumulation of these carbohydrates differ under desiccation stress and we propose that they perform different functions in the respective stresses. These are elaborated in discussion of our data.
ABSTRACT
We have used reverse transcription-PCR coupled with 52- and 32-RACE to isolate a full length INO1 cDNA (1692bp with an ORF of 1530) from the resurrection plant Xerophyta viscosa Baker. XvINO1 encodes 510 amino acids, with a predicted MW of 56.7kD and contains four sequence motifs that are highly conserved in plant myo-inositol-1-phosphate synthases (MIPS, EC5.5.1.4), the enzyme that catalyses the first step in the formation of myo-inositol (Ino). Northern and western analyses show that the transcript and protein are constitutively present in leaves but their expression increases, temporarily, in response to both accumulative salt stress (~300mM NaCl) and desiccation (to 5% relative water content). Leaf Ino concentration increases 40-fold during the first 6h of salt stress, and levels of this and other carbohydrates (galactinol, sucrose, raffinose, stachyose and hexoses) remain elevated relative to control leaves for the duration of salt stress treatment. The timing and pattern of accumulation of these carbohydrates differ under desiccation stress and we propose that they perform different functions in the respective stresses. These are elaborated in discussion of our data.
ABSTRACT
The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.
