Lipid Profiling Demonstrates That Suppressing Arabidopsis Phospholipase Dδ Retards ABA-Promoted Leaf Senescence by Attenuating Lipid Degradation

Li W (2013) Lipid Profiling Demonstrates That Suppressing Arabidopsis Phospholipase Dd Retards ABA-Promoted Leaf Senescence by Attenuating Lipid Degradation. PLoS ONE 8(6): e65687. doi:10.1371/journal.pone.0065687 Lipid Profiling Demonstrates That Suppressing Arabidopsis Phospholipase Dd Retards ABA-Promoted Leaf Senescence by Attenuating Lipid Degradation Yanxia Jia 0 Faqing Tao 0 Weiqi Li 0 Diane Bassham, Iowa State University, United States of America 0 1 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Science , Kunming , China , 2 The Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences , Kunming , China , 3 University of Chinese Academy of Sciences , Beijing , China Senescence is the last phase of the plant life cycle and has an important role in plant development. Degradation of membrane lipids is an essential process during leaf senescence. Several studies have reported fundamental changes in membrane lipids and phospholipase D (PLD) activity as leaves senesce. Suppression of phospholipase Da1 (PLDa1) retards abscisic acid (ABA)-promoted senescence. However, given the absence of studies that have profiled changes in the compositions of membrane lipid molecules during leaf senescence, there is no direct evidence that PLD affects lipid composition during the process. Here, we show that application of n-butanol, an inhibitor of PLD, and N-Acylethanolamine (NAE) 12:0, a specific inhibitor of PLDa1, retarded ABA-promoted senescence to different extents. Furthermore, phospholipase Dd (PLDd) was induced in leaves treated with ABA, and suppression of PLDd retarded ABA-promoted senescence in Arabidopsis. Lipid profiling revealed that detachment-induced senescence had different effects on plastidic and extraplastidic lipids. The accelerated degradation of plastidic lipids during ABA-induced senescence in wild-type plants was attenuated in PLDd-knockout (PLDd-KO) plants. Dramatic increases in phosphatidic acid (PA) and decreases in phosphatidylcholine (PC) during ABA-induced senescence were also suppressed in PLDd-KO plants. Our results suggest that PLDd-mediated hydrolysis of PC to PA plays a positive role in ABA-promoted senescence. The attenuation of PA formation resulting from suppression of PLDd blocks the degradation of membrane lipids, which retards ABA-promoted senescence. - Funding: The research was supported by grants from the National Natural Science Foundation of China (NSFC 30670474, 30870571, and 31070262), Fund of State Key Laboratory of Phytochemistry and Plant Resources in West China (0807B01211 and 097C1211Z1), Kunming Institute of Botany (KSCX2-EW-J-24), Germplasm Bank of Wild Species, and CAS Innovation Program of Kunming Institute (540806321211), 100-Talents Program of CAS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Leaf senescence, the last stage of leaf development, is an integral and important part of the plant life cycle [1], and greatly affects crop productivity [14]. For example, the delay of senescence by only a few days before harvest can improve the yields of certain forage crops significantly [5]. Leaf senescence can be induced by at least three conditions. One is natural senescence, which occurs as plants or leaves age. Another is stress-induced senescence, which results from stresses, such detachment, nutrient deficiency, darkness, and disease [1,2]. The third is hormone-promoted senescence. Application of either ABA or ethylene to detached leaves can promote their senescence [6]. Leaf senescence is a complex process that manifests at the levels of morphology (e.g., yellowing of leaf edges), physiology (e.g., reduction of photosynthesis), cell composition (e.g., deterioration of chlorophyll and membranes), biochemistry (e.g., changes in levels of hormones), and molecular genetics (e.g., altered expression of more than 800 senescence-associated genes) [2]. Manipulation of certain genes, such as tmr [7], IPT [8], SAG101 [9], and YUCCA6 [10], delays senescence. Leaf senescence is a highly regulated process, in which events occur in an ordered manner. Whereas leaf yellowing and chlorophyll deterioration are early events, the degradation of nucleic acids and proteins occurs midway through the process [11], and destruction of the plasma membrane occurs at the end of the process [12]. The changes in membrane lipids that occur in senescing leaves were reported decades ago [13], and the activity of PLD, phosphatidic acid phosphatase, lipolytic acyl hydrolase, and lipoxygenase in senescence-related lipid metabolism are well documented [13]. Nonetheless, little is known about many important aspects of leaf senescence, such as the roles of lipolytic enzymes in mediating specific changes in membrane lipids and how these changes affect membrane deterioration. Hydrolysis of membrane phospholipids by PLD produces PA and a free head group. The PLD family comprises 12 members, which are classified into six types, PLDa (3), b (2), c (3), d, e, and f (2). The PLDa1 and PLDd isoforms are two of the most abundant PLDs. All six types have been characterized well in Arabidopsis [14]. Most plant PLDs have distinctive molecular and biochemical properties that are associated with diverse cellular and physiological roles [15]. Through genetic approaches, it has been demonstrated that PLDs play important roles in stress responses, such as those to drought, cold, and salinity [16,17]. By exploiting the specific inhibition of n-butanol on the PLD-mediated PA [18], it has been shown that PLDs have roles in microtubule organization [19,20], salicylic acid signaling [21], seedling development [19,22], and pollen tube growth [23]. By comparing the effects of n-butanol and N-acylethanolamines (NAEs), which are specific inhibitors of PLDa1 [24], it was proposed that different isoforms of PLD might have differential regulatory roles in seedling development and growth [22]. The hydrolytic activity of PLD has been analyzed in senescing rose petals and bean cotyledons [25,26]. Microarray analysis showed that during natural senescence in Arabidopsis, levels of expression of PLDa1 and PLDd increased 1.75- and 2.94-fold, respectively (http://www.expressionbrowser.com/). In Arabidopsis, application of ABA or ethylene increases PLDa1 activity, and suppression of PLDa1 retards ABA- and ethylene-promoted leaf senescence [27]. It has been proposed that PLDa1 plays a key role in lipid metabolism in senescing membranes by producing PA, which destabilizes membranes and activates other lipid-degrading enzymes, resulting in the loss of both membrane integrity and functionality of membrane-associated proteins [13,27]. However, there is no direct evidence that PLDa1 produces PA and affects lipid composition during leaf senescence. It re (...truncated)