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In the halotolerant sugar beet co-expression of V-ATPase and a vacuolar Na+/H(+)-antiporter provides a mechanism for vacuolar salt sequestration. To analyze salt-induced changes in the expression of the vacuolar H(+)-ATPase (V-ATPase) a partial cDNA of the proton-channel forming subunit c was cloned by RT-PCR. Southern blot analysis indicated a small gene family. In control plants transcript levels were high in roots and young growing leaves but low in fully expanded leaves. In mature leaves salt exposure (400 mM, 48 h) induced a strong increase in subunit c-mRNA. Transcripts for the catalytic subunit A followed a similar developmental and stress-modulated pattern, indicating a coordinate regulation of transcripts for both V-ATPase subunits. Concomittant with the mRNA increases the amount of V-ATPase protein increased as well.

Cell extension growth in the mesocotyl tip of darkgrown Zea mays L. seedlings is dependent on vacuole enlargement and massive flux of ER and Golgi vesicles. Water flow into the expanding vacuole is driven by ion accumulation, which in turn is energized by the vacuolar H+-ATPase (V-ATPase). The V-ATPase energizes the secondary ion transport into the expanding vacuole. As light exposure leads to a strong inhibition of extension growth, the effect of light on transcript levels for subunits A and c of the V-ATPase was analyzed. Partial homologous cDNAs for subnnit A and two isoforms of snbunit c were cloned by RT-PCR. In dark-grown seedlings transcript levels for both subnnits were much higher in the growing mesocotyl tip than in the fully differentiated mesocotyl tissue. Only in the tip region did light exposure lead to a strong and coordinate down-regulation of both mRNAs whereas in the differentiated mesocotyl only a slight decrease was observed. The results indicate that expression of the 'housekeeping' V-type H +-ATPase is strongly regulated in response to growth rate. maize seedlings was chosen as a well defined experimental system. Here, elongation is the result of directional cell extension and is inhibited by light exposure [10][11][12]. In the etiolated seedling, cell division and elongation in the mesocotyl tip (5 mm) are dependent on intense traffic of ER and Golgi vesicles and vacuole expansion [13]. These processes are in turn dependent on luminal acidification by V-ATPase and, possibly, H---PPiase, which resides on the same membrane [14]. We have studied the expression of V-ATPase genes coding for the catalytic subunit A (part of the peripheral Vl-complex) and the proton channel forming subunit c (part of the membrane-integral Vo-complex) [1]. Transcript levels of both subunits were analyzed in the growing mesocotyl tip (0-5 mm) and the mesocotyl base (5-70 mm) of 6-day-old etiolated Zea mays seedlings before and after light exposure. The results indicate that expression of V-ATPase genes is strongly correlated with extension growth.

The plant V-type H+-ATPase (V-ATPase) does not only serve basic housekeeping functions but is also involved in stress-induced NaCl sequestration during salinity stress. To address the question whether the same isoforms conferring housekeeping functions are equally involved in the response to high salinity, we have isolated cDNA clones for subunits A and c, as representing the peripheral V1 complex and the membrane-integral V0 complex, respectively, from the halotolerant sugar beet (Beta vulgaris L., diploid variety). RNA blot analysis with gene-specific probes revealed a coordinate expression of the cloned subunit A and c isoforms during plant development and in response to high salinity. Also, in rapidly dividing suspension-cultured cells with 10-fold increased transcript amounts as compared to young leaf tissue, the ratio of transcripts for both genes was similar to the ratio found for transcripts in leaves of different age. We have then isolated partial genomic clones (BVA/70 for Beta V-ATPase 70 kDa subunit; BVA/16-1 for Beta V-ATPase 16 kDa subunit), including the promoter regions. Transcription start mapping revealed long 5'-UTR leader sequences (230 and 172 bases, respectively) for both genes. Both promoters contain putative G-box motifs in similar distance to the TATA boxes. For a quantitative comparison of relative promoter strength, the BVA/70 and BVA/16-1 promoters linked to the luciferase reporter gene (LUC) were delivered to sugar beet suspension-cultured cells by particle bombardment. The BVA/16-1 promoter showed a 1.7-fold higher activity as compared with the BVA/70 promoter. Salt treatment induced an increase of BVA/70 (+70%) and BVA/16-1 (+57%) promoter activities, concomitant with increased transcript amounts. The following sequences have been deposited at the EMBL database X98767: Beta vulgaris V-ATPase subunit A, cDNA clone; X98851, B. vulgaris V-ATPase subunit c isoform 1, cDNA clone; Y11038, B. vulgaris V-ATPase subunit A, partial genomic clone; Y11037, B. vulgaris V-ATPase subunit c isoform 1, partial genomic clone.

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