Patterns and Timing in Expression of Early Auxin-Induced Genes Imply Involvement of Phospholipases A (pPLAs) in the Regulation of Auxin Responses

CorinnaLabusch 0 1 MariaShishova 0 1 YunusEffendi 0 1 MaoyinLi 0 1 XueminWang 0 1 Gnther F.E.Scherer 0 1 0 a Leibniz Universitt Hannover, Institut fr Zierpflanzenbau und Gehlzwissenschaften , Abt. Molekulare Ertragsphysiologie, Herrenhuser Str. 2, D-30419 Hannover, Germany b Department of Plant Physiology and Biochemistry, State University of St. Petersburg Universitetskaya em. 7/9 , St. Petersburg, 199034, Russia c Department of Biology, University of Missouri , St Louis, MO 63121 , USA d Donald Danforth Plant Science Center , St Louis, MO 63132, USA 1 Labusch et al. Auxin-Induced Genes in Phospholipase AMutants While it is known that patatin-related phospholipase A (pPLA) activity is rapidly activated within 3 min by auxin, hardly anything is known about how this signal influences downstream responses like transcription of early auxin-induced genes or other physiological responses. We screened mutants with T-DNA insertions in members of the pPLA gene family for molecular and physiological phenotypes related to auxin. Only one in nine Arabidopsis thaliana ppla knockdown mutants displayed an obvious constitutive auxin-related phenotype. Compared to wild-type, ppla-III mutant seedlings had decreased main root lengths and increased lateral root numbers. We tested auxin-induced gene expression as a molecular readout for primary molecular auxin responses in nine ppla mutants and found delayed upregulation of auxin-responsive gene expression in all of them. Thirty minutes after auxin treatment, up-regulation of up to 40% of auxin-induced genes was delayed in mutant seedlings. We observed only a few cases with hypersensitive auxin-induced gene expression in ppla mutants. While, in three ppla mutants, which were investigated in detail, rapid upregulation (as early as 10 min after auxin stimulus) of auxin-regulated genes was impaired, late transcriptional responses were wild-type-like. This regulatory or dynamic phenotype was consistently observed in different ppla mutants with delayed up-regulation that frequently affected the same genes. This defect was not affected by pPLA transcript levels which remained constant. This indicates a posttranslational mechanism as a functional link of pPLAs to auxin signaling. The need for a receptor triggering an auxin response without employing transcription regulation is discussed. - INTRODu CTION The generation of lipid messengers like free fatty acids and lysolipids by phospholipase A2 (PLA2) enzymes is an important step in early plant signal transduction because they regulate distinct proteins or downstream processes. In plants, two main PLA2 gene families function in signal transduction, patatinrelated phospholipase A, and secreted PLA2 ( Scherer et al., 2010). In Arabidopsis thaliana, the pPLA family is represented by 10 genes that can be classified into three sub-groups (IIII) based on sequence comparisons (Holk etal., 2002; Ryu, 2004; Scherer etal., 2010). Group I consists of the single gene pPLA-I that has clear homology to animal Ca2+-independent PLA2 (iPLA) enzymes (Balsinde and Balboa, 2005). The remaining nine group II and III pPLAs have only rudimentary sequence homology to animal sequences, indicating that group I pPLA-I is evolutionarily ancient (Holk et al., 2002). pPLA-I T-DNA insertion mutants exhibit reduced basal levels of jasmonic acid. However, pathogen or wounding-induced jasmonic acid levels are indistinguishable from wild-type (Yang etal., 2007). In addition, we found that auxin-inducible gene expression and shade-induced elongation growth are affected in ppla-I insertion mutants (Effendi etal., unpublished). Group II consists of five genes (pPLA-IIpPLA-II). pPLA-II transcription is up-regulated by abiotic and biotic stresses (Matos etal., 2001; Rietz etal., 2004; La Camera etal., 2005). The phenotypic analysis of ppla-II and ppla-II suggested a function in root architecture regulated by auxin and ABA (Rietz etal., 2010). The members of group III (pPLA-IIIpPLA-III) are plantspecific and differ in their intron/exon structures and catalytic centers from the other subfamilies (Scherer etal., 2010). Interestingly, only one of the insertion mutants (ppla-III) displayed a subtle morphological auxin response phenotype (Li etal., 2011). The above-described phenotypes together with rapid activation of pPLAs 25 min after auxin treatments (Scherer and Andr, 1989; Paul etal., 1998) indicate a possible function of pPLA enzymes in auxin signaling. However, in recent years, additional evidence in support of this line of argumentation has accumulated. Pharmacological approaches, for example, revealed that pPLA inhibitors block auxin-induced elongation growth (Scherer and Arnold, 1997; Holk etal., 2002) and auxin-induced gene expression (Scherer etal., 2007). On the biochemical level, we could recently show that pPLA-II and pPLA are activated by CPK3 (Rietz etal., 2010). To substantiate a possible role of pPLAs in early auxin signaling, we here aimed to systematically analyze the auxin inducibility of classic auxin response genes in pPLA mutant backgrounds. In contrast to classic physiological assays, such a rapid response biotest is able to show the direct effect of pPLAs on the primary molecular auxin response clearer than in developmental tests taking days. The classical example for this principle is the hypocotyl and coleoptile elongation test with etiolated tissues where a response can be detected after about 10 min. We included various auxin response genes in this analysis, some of which are induced within minutes independent of de novo protein synthesis (e.g. the IAA, GH3, and SAUR families) (Hagen and Guilfoyle, 2002; Abel etal., 1994, 1995; Abel and Theologis, 1996; Guilfoyle etal., 1998a, 1998b; Paponov et al., 2008). Thus, these genes are referred to as early or primary auxin response genes. IAA proteins are unstable transcriptional repressors and developmental responses to auxin are sensitive to the levels of these proteins (Dreher etal., 2006; Mockaitis and Estelle, 2008). Mutations of several IAA genes like MASSAGU2 ( MSG2/IAA19) (Tatematsu et al., 2004), SUPPRESSOR OF HY2 ( SHY2/IAA3) (Abel et al., 1995; Kim etal., 1996; Soh etal., 1999; Tian and Reed, 1999; Reed, 2001; Tian etal., 2003) or SOLITARY ROOT (SLR/IAA14) (Abel et al., 1995; Fukaki et al., 2002, 2005; Vanneste et al., 2005) reduce multiple auxin responses. Especially, they have defects in auxin-induced lateral root formation and reduced cell cycle activity (Fukaki etal., 2006; Mockaitis and Estelle, 2008). The GH3 gene family in Arabidopsis consists of three subfamilies and several of the members encode IAAamido synthetases (Hagen and Guilfoyle, 2002; Staswick et al., 2005). A rapid transcriptional activation leads to a higher amount of IAA amido synthetase, which then converts auxin to amino acid conjugates that are either inactive or become degraded. Thus, a function of GH3 is to maintain IAA homeostasis (Staswick etal., 2005). From SAUR, genes (...truncated)