The proton pump interactor (Ppi) gene family of Arabidopsis thaliana: expression pattern of Ppi1 and characterisation of knockout mutants for Ppi1 and 2

Abstract

Plant plasma membrane H+-ATPases (PM H+-ATPase) are essential for establishing a proton electrochemical gradient across the cell plasma membrane. Their regulation is poorly understood, except for the role of 14-3-3 proteins, which relieve autoinhibition from the C-terminal domain. A novel protein interacting with this domain was recently identified in Arabidopsis and named PPI1 (Proton Pump Interactor 1). PPI1 stimulates PM H+-ATPase activity in vitro. Here, we analyse the expression pattern of Ppi1 using beta-glucuronidase as a reporter. Expression is strong in root and shoot vascular systems, particularly in meristematic and sink tissues, as well as in pollen, stigmas and siliques, but not in developing embryos. Removal of the first intron decreased GUS expression 45-fold. We also analysed the transcription of Ppi2, another gene in the family, and demonstrated that Ppi2 is expressed in seedlings, cultured cells and flowers. We reassessed Ppi2 gene structure based on RT-PCR amplifications, cDNA data and similarity to other Ppi genes. Insertional mutants for both Ppi1 and Ppi2 were isolated. Two different mutants of Ppi1 showed aberrant mRNAs and lacked any detectable protein and are therefore true knockouts. Interestingly, one mutation inhibited the splicing of one intron at a considerable distance (>700 bp) from the T-DNA insertion site, but not the splicing of a proximal intron (29 bp) or of any other intron. At the plant level, neither of the single mutants nor the double ppi1ppi2 mutant showed an altered phenotype in standard growth conditions under acid load or salt stress.

AtHsp70‐15‐deficient Arabidopsis plants are characterized by reduced growth, a constitutive cytosolic protein response and enhanced resistance to TuMV

“…Stomatal opening is largely driven by the activity of plasma membrane H + -ATPases , which is highly regulated. One potential positive regulator of plasma membrane H + -ATPases, the proton pump interactor PPI2 (At3g15340) (Morandini et al, 2002;Anzi et al, 2008), is up-regulated in hsp70-15 knockout plants, which might explain activation of H + -ATPase activity. Up-regulation of heat shock response-related genes in Hsp70-15-deficient plants is similar to that found in HsfA2 over-expressing plants (Nishizawa et al, 2006) and wildtype plants under heat stress treatment (Rizhsky et al, 2004;Swindell et al, 2007).…”

Characterization of StPPI1, a proton pump interactor from Solanum tuberosum L. that is up-regulated during tuber development and by abiotic stress

“…C Control, rRNA ribosomal RNA, Chit A chitinase A the time points analyzed. Publicly available microarray data from Arabidopsis (AtgenExpress website jsp.weigelworld.org/expviz/expviz.jsp) revealed that PPI1 expression increases under salt and cold stress, similarly to StPPI1 (Anzi et al 2008), although there are differences in the response time between both species, probably due to differences in the stress conditions applied. As mentioned before, it has been shown that salt and cold stress increase PM H ?…”

“…PPI1 is a member of a gene family apparently consisting of six members in A. thaliana that share 20-50% sequence identity at the protein level and do not resemble any protein of known function (Morandini et al 2002). The expression analysis using reporter gene constructs revealed that PPI1 is strongly expressed in root and shoot vascular systems, particularly in meristematic and sink tissues, as well as in pollen, stigmas and siliques, but not in developing embryos (Anzi et al 2008). The main part of PPI1 is localized at the endoplasmic reticulum, from which it might translocate to the PM for interaction with the H ?…”

Proton pump and plant cell elongation

“…PPI1 is not homologous to any of the proteins with a known function and, potentially, might be a representative of a new family of plant regulatory proteins. At least five Arabidopsis genes and EST sequences (expressed sequence tags) from different plant species encode proteins substantially similar to PPI1 (Morandini et al, 2002;Anzi et al, 2008). Intensive expression of genes encoding the PPI1 protein has been demonstrated in most plant organs -in the vascular tissues of roots and shoots, in meristematic tissues, in pollen, stigma of pestles, and pods (Anzi et al, 2008).…”

“…At least five Arabidopsis genes and EST sequences (expressed sequence tags) from different plant species encode proteins substantially similar to PPI1 (Morandini et al, 2002;Anzi et al, 2008). Intensive expression of genes encoding the PPI1 protein has been demonstrated in most plant organs -in the vascular tissues of roots and shoots, in meristematic tissues, in pollen, stigma of pestles, and pods (Anzi et al, 2008). The PPI1-binding site at the C-terminus of the enzyme molecule has a different localization than the binding site of 14-3-3 proteins.…”