Heterologous expression, and biochemical and cellular characterization of CaPLA1 encoding a hot pepper phospholipase A1 homolog

Abstract

SummaryPhospholipid signaling has been recently implicated in diverse cellular processes in higher plants. We identified a cDNA encoding the phospholipase A1 homolog (CaPLA1) from 5-day-old early roots of hot pepper. The deduced amino acid sequence showed that the lipase-specific catalytic triad is well conserved in CaPLA1. In vitro lipase assays and site-directed mutagenesis revealed that CaPLA1 possesses PLA1 activity, which catalyzes the hydrolysis of phospholipids at the sn-1 position. CaPLA1 was selectively expressed in young roots, at days 4-5 after germination, and rapidly declined thereafter, suggesting that the expression of CaPLA1 is subject to control by a development-specific mechanism in roots. Because transgenic work was extremely difficult in hot peppers, in this study we overexpressed CaPLA1 in Arabidopsis so as to provide cellular information on the function of this gene. CaPLA1 overexpressors had significantly longer roots, leaves and petioles, and grew more rapidly than the wild-type plants, leading to an early bolting phenotype with prolonged inflorescence. Microscopic analysis showed that the vegetative tissues of 35S:CaPLA1 plants contained an increased number of small-sized cells, which resulted in highly populated cell layers. In addition, mRNAs for cell cycle-controlled proteins and fatty acid catabolizing enzymes were coordinately upregulated in CaPLA1-overexpressing plants. These results suggest that CaPLA1 is functionally relevant in heterologous Arabidopsis cells, and hence might participate in a subset of positive control mechanisms of cell and tissue growth in transgenic lines. We discuss possible biochemical and cellular functions of CaPLA1 in relation to the phospholipid signaling pathway in hot pepper and transgenic Arabidopsis plants.

Drought Stress-Induced Rma1H1, a RING Membrane-Anchor E3 Ubiquitin Ligase Homolog, Regulates Aquaporin Levels via Ubiquitination in TransgenicArabidopsisPlants

“…Previous reports indicate that hot pepper genes are fully functional in heterologous Arabidopsis cells (Cho et al, 2006a(Cho et al, , 2006bSeo et al, 2008). Numerous independent T4 transgenic lines that exhibited markedly enhanced levels of the Rma1H1 transcript under normal growth conditions were chosen for further analysis ( Figure 4A).…”

Arabidopsis Small Rubber Particle Protein Homolog SRPs Play Dual Roles as Positive Factors for Tissue Growth and Development and in Drought Stress Responses

“…The percentages of radicle emergence were calculated 1 to 5 d after sowing. Growth of root and leaf tissues was monitored with the image-analyzing program SCIONIMAGE (Scion Corp., Frederic, MD) as described by Seo et al (2008). For the microscopic analysis, 3-d-old roots were stained with propidium iodide and visualized using confocal microscopy (LSM510 META; Carl Zeiss, Oberkochen, Germany) as described by Seo et al (2008).…”

“…U11034), SRP2 (U24879), and SRP3 (U137621) cDNAs were cloned into the binary vector pBI121. SRP-over-expressing transgenic lines were generated as described previously (Seo et al, 2008). The RNAi-mediated knock-down construct of SRP2 (35S:SRP2-RNAi) was introduced into the wild-type and srp1srp3 double mutant plants.…”

“…Growth of root and leaf tissues was monitored with the image-analyzing program SCIONIMAGE (Scion Corp., Frederic, MD) as described by Seo et al (2008). For the microscopic analysis, 3-d-old roots were stained with propidium iodide and visualized using confocal microscopy (LSM510 META; Carl Zeiss, Oberkochen, Germany) as described by Seo et al (2008). For leaf cell layer observation, 5-d-old leaves were cleared with chloral-hydrate solution, and the images were analyzed using light-field microscopy (BX51 fluorescence microscope, Olympus).…”

Purification, Characterization and Application of a Cold- Adapted Phospholipase A1 fromBacillus Cereus Sp.AF-1

“…The ability to work efficiently at low temperatures is environmentally advantageous and energy saving (7). Although several phospholipases isolated from Bacillus cereus which perform efficiently at lower temperature (below 40 °C) have been described (9,15,18,21), the reports do not discuss the properties of these enzymes, other than the low temperature of action and the processes used for effective purification.…”