Title

Vacuolar H+-translocating pyrophosphatase AVP1 is required for organ development in Arabidopsis

Date of Completion

January 2004

Keywords

Biology, Molecular|Biology, Botany|Biology, Cell|Biology, Plant Physiology

Degree

Ph.D.

Abstract

Vacuolar pyrophosphatase (V-PPase), as well as vacuolar ATPase, generates electro-chemical gradients across the tonoplast to facilitate the transport of ions, sugars, amino acids and other metabolites across the vacuolar membrane. Here we present data gathered via the genetic manipulation of the V-PPase that reveals an unexpected role for this H+-pump in organ development. Overexpression of the Arabidopsis vacuolar pyrophosphatase gene ( AVP1) resulted in larger Arabidopsis plants. The enhanced rosette leaf size and number, petal size and root biomass displayed by AVP1 overexpression plants (AVP1OX) are due to increased cell number. Relative quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of gene expression levels of cyclin D3 and AINTEGUMENT revealed enhanced cell division and meristem competence in AVP1OX plants. Visual analysis of plants transformed with a CycB1::CDBGUS construct confirmed that the cells from the leaves of AVP1OX plants have an enhanced cell division capacity. Another phenotype displayed by the AVP1OX plants became evident when explants were incubated in a shoot induction medium. Explants (i.e., cotyledons, hypocotyls, roots, rosette leaves and inflorescence stems) from AVP1OX lines showed a striking enhancement in their de novo shoot organogenesis capacity. Molecular analysis of the expression patterns of genes relevant to cell division, shoot apical meristem formation, organ differentiation as well as cytokinin signal transduction revealed that AVP1 overexpression is sufficient to induce de novo formation of functional shoot apical meristems. Analysis of loss-of-function mutants in the AVP1 locus further implicated the participation of this vacuolar H+-pump in organ development. Western blot shows that AVP1 translation was abolished in a T-DNA tagged Arabidopsis line (avp1-1). The avp1-1 line showed the opposite phenotypes of AVP1OX lines. The mature rosette of avp1-1 line is severely reduced in size. The root growth is retarded, no lateral roots developed, and these plants cannot survive in soil condition. Shoot organogenesis is completely abolished in root explants of the avp1-1 line. Expression of AVP1 complemented the avp1-1 phenotypes. We conclude that AVP1 plays a pivotal role in plant organ development. Possible mechanisms are discussed. ^