The Patatin-Containing Phospholipase A pPLAIIα Modulates Oxylipin Formation and Water Loss in Arabidopsis thaliana

Wen-Yu Yang 0 1 Yong Zheng 0 1 Sung Chul Bahn 0 1 Xiang-Qing Pan 0 1 Mao-Yin Li 0 1 Hieu Sy Vu 0 1 Mary R. Roth 0 1 Brad Scheu 0 1 Ruth Welti 0 1 Yue-Yun Hong 0 1 Xue-Min Wang swang@danforthcenter 0 1 0 org, fax 314-587-1519. The Author 2012. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE , SIBS, CAS. doi: 10.1093/mp/ssr118, Advance Access publication 18 January 2012 Received 17 October 2011 ; accepted 12 December 2011 1 a Department of Biology, University of Missouri , St Louis, MO 63121 , USA b Donald Danforth Plant Science Center , St Louis, MO 63132 , USA c Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, HuaZhong Normal University , Wuhan 430079, China d Kansas Lipidomics Research Center, Division of Biology, Kansas State University , Manhattan, KS 66506 , USA e National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University , Wuhan 430070, China The patatin-related phospholipase A (pPLA) hydrolyzes membrane glycerolipids to produce monoacyl compounds and free fatty acids. Phospholipids are cleaved by pPLAIIa at the sn-1 and sn-2 positions, and galactolipids, including those containing oxophytodienoic acids, can also serve as substrates. Ablation of pPLAIIa decreased lysophosphatidylcholine and lysophosphatidylethanolamine levels, but increased free linolenic acid. pPLAIIa-deficient plants displayed a higher level of jasmonic acid and methyl jasmonate, as well as the oxylipin-biosynthetic intermediates 13-hydroperoxylinolenic acid and 12-oxophytodienoic acid than wild-type (WT) plants. The expression of genes involved in oxylipin production was also higher in the pPLAIIa-deficient mutant than in WT plants. The mutant plants lost water more quickly than WT plants. The stomata of WT and mutant plants responded similarly to abscisic acid. In response to desiccation, the mutant and WT leaves produced abscisic acid at the same rate, but, after 4 h of desiccation, the jasmonic acid level was much higher in mutant than WT leaves. These results indicate that pPLAIIa negatively regulates oxylipin production and suggest a role in the removal of oxidatively modified fatty acids from membranes. - INTRODUCTION The release of fatty acids from cellular membrane glycerolipids has been implicated in various cellular processes including the production of signaling messengers, membrane remodeling, membrane deterioration during senescence, and stress damage (Wang, 2004; Scherer et al., 2010). The patatin-related phospholipase A (pPLA) is a major family of enzymes that hydrolyze membrane glycerolipids to produce lysolipids and free fatty acids. Patatin refers to a group of storage glycoproteins that were originally discovered in potato tubers (Galliard, 1971); patatin-related proteins were later found in other plant species and tissues, as well as in animal cells (Holk et al., 2002). The wellcharacterized mammalian cytosolic PLA2s contain a patatin domain that serves as catalytic site (Schrag and Cygler, 1997; Hirschberg et al., 2001). The Arabidopsis pPLA family consists of 10 genes that are grouped into three subfamilies, pPLAI, pPLAII (a, b, c, d, e), and pPLAIII (a, b, c, d), based on gene structures and deduced protein sequences (Scherer et al., 2010). pPLAI contains 1257 amino acid residues whereas the size of pPLAIIs and IIIs ranges from 382 to 526 amino acid residues. pPLAs are involved in various processes, including fungal and bacterial pathogen infection, phosphate deprivation, auxin response, cellulose deposition, cell elongation, and grain yield (La Camera et al., 2005; Yang et al., 2007; Rietz et al., 2010; Li et al., 2011). However, the metabolic and cellular mechanism by which pPLAs affect plant growth and stress response remain elusive. One of potential mechanisms of action for pPLAs starts with the hydrolysis of membrane lipids, releasing polyunsaturated fatty acids (PUFAs), such as linolenic acid that serves as a substrate for the synthesis of jasmonic acid (JA) in response to stress. JA and other cyclopentenone derivatives are synthesized via the octadecanoic pathway from linolenic acid in plants. These oxylipins modulate plant response to various stresses, including pathogen infection, insect attack, and drought (Staswick, 1992; Wasternack and Parthier, 1997; Reymond and Farmer, 1998; Farmer, 2001). One PLA1 was identified as being involved in JA production, pollen maturation, and anther dehiscence in Arabidopsis (Ishiguro et al., 2001), but the identity of the enzyme(s) involved in stress-induced production of PUFAs for oxylipin production it is still unclear. pPLAI has been implicated in the production of basal levels of JA, but not pathogen or wounding-induced JA production (Yang et al., 2007). pPLAI plays a positive role in Arabidopsis resistance to Botrytis cinerea (Yang et al., 2007), whereas the suppression of pPLAIIa was reported to render Arabidopsis plants more resistant to fungal and bacterial infection (La Camera et al., 2005). This negative effect of pPLAIIa on plantpathogen interactions appears to argue against a role of pPLAIIa in promoting JA production. The expression of pPLAIIa is induced in response to various biotic and abiotic stresses, including pathogens, low temperature, high salinity, abscisic acid, salicylic acid, methyl jasmonate, ethylene, iron, and phosphate deficiency (Narusaka et al., 2003; Rietz et al., 2004). These observations raise intriguing questions regarding the role of pPLAIIa in JA production and plant stress responses. Recent studies have shown that complex membrane lipids, such as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and phospholipids, are oxidatively modified (Andersson et al., 2006; Buseman et al., 2006). Oxidized (ox-) MGDG and DGDG containing 12-oxophytodienoic acid (OPDA) and dinor-oxophytodienoic acid (dnOPDA), including Arabidopside A (OPDA/dnOPDA MGDG), B (OPDA/ OPDA MGDG), C (OPDA/dnOPDA DGDG), and D (OPDA/OPDA DGDG), are produced in response to wounding, bacterial and fungal infection, dark, aging, and osmotic stress (Andersson et al., 2006; Buseman et al., 2006; Kourtchenko et al., 2007; Glauser et al., 2008; Maeda et al., 2008; Xiao et al., 2010). pPLAI has been shown to use arabidopsides as substrates, and to prefer them as substrates to unoxidized, normal MGDG and DGDG (Yang et al., 2007). In this study, we analyzed pPLAIIas lipid-hydrolyzing activity and effect on JA formation. The results suggest that this enzyme negatively regulates oxylipin production, possibly via participating in membrane repair that includes removal of oxidatively modified lipids. pPLAIIa Hydrolyzes Glycerolipids at the sn-1 and sn-2 Positions The full-length cDNA of pPLAIIa gene encodes a protein of 407 amino acids with a predicted pI of 5.57 and molecular weight of 44 239. pPLAIIa contains the conserved serine hydrolase motif GXSXG at residue (...truncated)