Phospholipase pPLAIIIα Increases Germination Rate and Resistance to Turnip Crinkle Virus when Overexpressed.

Patatin-related phospholipase As (pPLAs) are major hydrolases acting on acyl-lipids and play important roles in various plant developmental processes. pPLAIII group members, which lack a canonical catalytic Ser motif, have been less studied than other pPLAs. We report here the characterization of pPLAIIIα in Arabidopsis (Arabidopsis thaliana) based on the biochemical and physiological characterization of pPLAIIIα knockouts, complementants, and overexpressors, as well as heterologous expression of the protein. In vitro activity assays on the purified recombinant protein showed that despite lack of canonical phospholipase motifs, pPLAIIIα had a phospholipase A activity on a wide variety of phospholipids. Overexpression of pPLAIIIα in Arabidopsis resulted in a decrease in many lipid molecular species, but the composition in major lipid classes was not affected. Fluorescence tagging indicated that pPLAIIIα localizes to the plasma membrane. Although Arabidopsis pplaIIIα knockout mutants showed some phenotypes comparable to other pPLAIIIs, such as reduced trichome length and increased hypocotyl length, control of seed size and germination were identified as distinctive pPLAIIIα-mediated functions. Expression of some PLD genes was strongly reduced in the pplaIIIα mutants. Overexpression of pPLAIIIα caused increased resistance to turnip crinkle virus, which associated with a 2-fold higher salicylic acid/jasmonic acid ratio and an increased expression of the defense gene pathogenesis-related protein1. These results therefore show that pPLAIIIα has functions that overlap with those of other pPLAIIIs but also distinctive functions, such as the control of seed germination. This study also provides new insights into the pathways downstream of pPLAIIIα.


INTRODUCTION
can be noted that the phosphate or anion binding element DSGGXXG was not 140 completely conserved in the pPLAIIIα protein because the second glycine (G) was 141 replaced with serine (S). 142 Arabidopsis pPLAIIIα thus lacked the canonical phospholipase motif for the catalytic 143 serine found in other characterized pPLAIIIs and one could question whether this 144 protein really possessed phospholipase activity. The recombinant pPLAIIIα protein His-145 tagged at the C-terminal end was therefore expressed in Escherichia coli and purified 146 (Fig. S1). In vitro enzymatic assays showed that the His-tagged pPLAIIIα had an acyl-147 ester hydrolase activity on each of the four major Arabidopsis phospholipid classes, 148 with a slightly higher activity on phosphatidic acid (PA) than on phosphatidylcholine 149 (PC), phosphatidylethanolamine (PE) or phosphatidylglycerol (PG) (Fig. 1C). 150 Taken together, these results show that pPLAIIIα is a noncanonical phospholipase A 151 that hydrolyzes various phospholipids in vitro.   In seedling stages (Fig. 3F), the hypocotyl length of the pplaIIIα mutants was longer 195 than that of controls, but OE lines were shorter than controls (Fig. 3G). The root lengths 196 were only shorter in two of the OE lines and unaltered in the KO mutant lines (Fig. 3H).

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Both OE 6 and 7 could be considered as ectopic lines with similar levels of expression, 198 and OE line 13 was the most highly expressing line. However, OE line 8 was the most 199 moderate line that was perfectly complemented in the KO#1 (Fig. 3G and H).  In the pplaIIIα KO mutant, analysis of the lipid molecular species did not reveal 211 significant differences, including in the phospholipid species potentially localized to the 212 plasma membrane ( Fig. 4C and Fig. S4). In OE lines, total acyl-lipids were reduced 14 ( Fig. 4A). Significant decreases were observed in many lipid classes (Fig. 4B)    showed 19% lower germination rates than the control, and the OE line showed a 10% 277 greater germination rate on average (Fig. 6E). However, all KO seeds germinated by 30 278 h, which indicated that the germination rate was not affected, but that germination was 279 delayed compared to that in WT. Treatment with gibberellic acid (GA) 3 did not alter the 280 germination substantially (Fig. 6F). Since antagonism between abscisic acid (ABA) and

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Numerous data also suggest that ethylene stimulates seed germination by affecting GA     However, a plant immunity study focusing on virus relative to the level of PA has not 331 been previously reported. Considering that plant viruses are pathogens associated with 332 major threats, resistance to turnip crinkle virus (TCV) in Arabidopsis, which is one of 333 the few manipulative plant-virus systems, was tested in mutant and OE lines (Fig. 9).  FFAs. Some of these phenotypes may result from the decrease in one or several of the 397 many molecular lipid species significantly affected in both OE lines ( Fig. 4 and S4).

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The fact that the Arabidopsis protein localizes to the plasma membrane suggests that 26 homeostasis in these organelles.

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In vitro assays on the partially purified pPLAIIIα showed that the protein hydrolyzed 408 various phospholipids, with a slightly higher activity on PA (Fig. 1B). This seems to be 27 ruled out because lipid species from other phospholipid classes are also reduced in OE 431 lines (Fig. 4). 432 Intriguingly, no increases in PA molecular species were detected in pplaIIIa mutants 433 in rice (Liu et al., 2015) or Arabidopsis (Fig. 4). Since PA is mostly generated by the         (Table S1). transformants in staining buffer following a previous report (Kim et al., 2014).

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Seedlings were photographed under a microscope (ZEISS Axio Observer D1, Germany).  (Table S1).   Transcripts synthesized in vitro from a cloned cDNA of the TCV genome using T7 RNA 659 polymerase were used for viral infections as described (Dempsey et al., 1993).

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Resistance and susceptibility were confirmed by RT-qPCR.  Table S1.

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Alignment of pPLAIIIα with other pPLAIIIs and two pPLAIIs from Arabidopsis.

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Amino acid sequences were analyzed using the pairwise sequence alignment program.

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Multiple sequence alignment was performed using the BioEdit program (version 7.1.9).      conditions. n = 120. (F) The germination rate following treatment with 1 μM of GA 3 .