Progress 09/01/01 to 08/31/03
Outputs
Integrating proton pumps with growth and adaptation 9/01-8/04130k total. USDA-NRI 2001-35304-10934 PI Heven Sze Proton pumps energize the uptake and release of ions and metabolites, yet we know little about how expression and activity of a multi-subunit vacuolar H+-ATPase are regulated and integrated with growth, signaling and adaptation.The aim was to begin defining in vivo roles of V-ATPase using Arabidopsis. Identifying vacuolar H+-ATPase VHA genes in Arabidopsis and in rice Because of its structural complexity plus the confusing nomenclature, vac-ATPase genes were not easy to identify in the databases in 2000.I initiated a collaborative effort to identify and name all V-ATPase genes (Sze et al.,2002).The entire complement of VHA genes in a higher plant and in a crop,rice,demonstrates for the first time what VHA genes are required to complete a plant life cycle, but even more importantly, it raises specific questions,leads to testable working ideas,and provides
tools to test ideas. Functional Studies Spatial and temporal expression of AtVHA-c in plant to determine the role of multiple genes, we demonstrated that multiple c subunits were differentially expressed in tissues and in a developmental manner. AtVHA-c1 was expressed in all organs whereas AtVHA-c3 was preferentially expressed in a few tissues, such as root tip and pollen. two subunit c genes are differentially influenced by light. Etiolated seedlings demonstrated strong AtVHA-c1:Gus activity in the hypocotyls but not the cotyledons. However AtVHA-c1 was highly expressed in expanded cotyledons, and not in short hypocotyls of light-grown seedlings. Thus AtVHA-c1 promoter may be a downstream target of light-regulated plant development. Function after dsRNA-mediated interference No T-DNA insertional mutants of VHA-c genes are available, so we used double-stranded RNA-mediated interference to silence expression of Vha genes. Our results strongly point to a major role of c1 in cell
expansion: VHA-c1 promoter:GUS activity is high in expanding root, hypocotyl and cotyledon tissues; and reduction of c1 message by dsRNA-mediated silencing reduced root and hypocotyls length relative to wild type plants. Interestingly, vha-c3 mutants were also reduced in root growth. As VHA-c3 expression is high in root cap cells, these results suggest that the activities of cap cells somehow influence root growth. We found that root growth of vha-c1 mutants were more sensitive than WT seedlings to high salt or to 10-40 M AlCl3 unpublished. These experiments would suggest that the V-ATPase is important for conferring tolerance. Significance.The progress resulting from this grant is significant in several ways: The first gene-specific dsRNAi mutants open the door to explore the role of VHA-c, and Vo in plant biology; ii the spatial and temporal expression of VHA-c gives valuable clues to analyze roles in signaling. Expression of VHA-c in pollen, vascular strands, elongating
cells, and floral style suggest that V-ATPase may be important in cells with active vesicle trafficking and in reproduction. Mutant studies support the idea that V-ATPase has roles in conferring tolerance to salt and toxic metals.
Impacts
The progress resulting from this grant is significant in several ways. 1. Studies of a central proton pump using a model plant have been instrumental in identifying related genes in the monocot crop, rice. 2. Functional studies using dsRNA-mediated interference demonstrate roles of the V-ATPase proton pump in growth, signal transduction and in conferring tolerance to stress and toxic metals. 3. Expression of VHA-c in pollen, vascular strands, elongating cells, and floral style suggest that V-ATPase may be important in cells with active vesicle trafficking and in reproduction. Because plant vacuoles serve as a large storage reservoir for nutrients, secondary products and toxic ions, these findings have direct impact on strategies to clean up the environment, and to enhance nutritional quality and stress tolerance of crop plants.
Publications
- Publications resulting from USDA support: Sze H, Schumacher K, Muller ML, Padmanaban S,Taiz L 2002 A simple nomenclature for a complex proton pump: VHA genes encode the vacuolar H+-ATPase. Trends Plant Sci 7(4): 157-161. Padmanaban S, Lin X, Perera I, Kawamura Y, Sze H . 2004 Differential Expression of Vacuolar H+-ATPase Subunit c Genes in Tissues Active n Membrane Trafficking and Their Roles in Plant Growth as Revealed by RNAi. Plant Physiol 134:1514-26.
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Progress 01/01/02 to 12/31/02
Outputs
The vacuolar H+-ATPase (VHA) acidifies intracellular compartments and generates a proton electrochemical gradient to transport ion and metabolite across vacuolar and other membranes. However the physiological functions of this complex pump in plant growth, development and adaptation are not understood. The multiple subunits that form a peripheral V1 complex and an integral Vo complex are encoded by a single gene or by multiple genes in Arabidopsis. Of these, the 16 kDa subunit c of the Vo sector is encoded by the largest gene family with 5 members, Vha-c1 to Vha-c5. We have begun to determine (i) whether the genes are developmentally regulated or expressed in a tissue specific manner using promoter-reporter gene analysis, and (ii) the physiological functions using mutants with reduced V-ATPase. Vha-c1 is highly expressed in most tissues of the root and leaf, whereas Vha-c3 is expressed only in the root tip. In the flower, Vha-c1 is expressed in all flower parts
including sepal, stamen, anther and stigma but not in the petal. In contrast, Vha-c3 is expressed only in pollen. The expression pattern of Vha-c1 is sensitive to light. Vha-c1 is highly expressed in hypocotyls, but not in the cotyledons, of etiolated seedlings. However, in light (blue or far red), Vha-c1 is high in cotyledons but not in hypocotyl. Thus Vha-c1 expression accompanies cell expansion, and its promoter activity is tissue-specific and developmentally regulated. To test in vivo function of each subunit c, homozygous plants containing double-stranded RNA constructs of Vha-c1 or Vha-c3 were analyzed. RT-PCR confirmed that 6 out of 10 transformants showed reduced native RNA level. Relative to wild type, roots of 7 d etiolated seedlings with dsRNA-c1 were shorter by 40%; surprisingly, the hypocotyls were only 10% shorter. Roots of plants with ds-RNA c3 were also inhibited though by only 25%. These results demonstrate that Vha-c1 and Vha-c3 subunits are differentially expressed,
and point to an important role of the V1Vo-ATPase in root growth. Tests are underway to test the roles of the pump in tolerating environmental stresses.
Impacts
(N/A)
Publications
- Maser P, Thomine S, Schroeder JI, Ward JM, Hirschi K, Sze H, Talke IN, Antmann A, Maathius, Sanders D, Harper JF, Tchieu J, Gribskov M, Persans, Salt DE, Kim SA, Guerinot ML (2001) Phylogenetic relationships within cation-transporter families of Arabidopsis thaliana. Plant Physiol 126 (4):1646-1667
- Sze H, Schumacher K, Muller ML, Padmanaban S, Taiz L. (2002) A simple nomenclature for a complex proton pump: VHA genes encode the vacuolar H(+)-ATPase. Trends Plant Sci. 7(4):157-61.
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