Patatin-related phospholipase A, pPLAIIIα, modulates the longitudinal growth of vegetative tissues and seeds in rice

Abstract

Patatin-related phospholipase A (pPLA) hydrolyses glycerolipids to produce fatty acids and lysoglycerolipids. The Oryza sativa genome has 21 putative pPLAs that are grouped into five subfamilies. Overexpression of OspPLAIIIα resulted in a dwarf phenotype with decreased length of rice stems, roots, leaves, seeds, panicles, and seeds, whereas OspPLAIIIα-knockout plants had longer panicles and seeds. OspPLAIIIα-overexpressing plants were less sensitive than wild-type and knockout plants to gibberellin-promoted seedling elongation. OspPLAIIIα overexpression and knockout had an opposite effect on the expression of the growth repressor SLENDER1 in the gibberellin signalling process. OspPLAIIIα-overexpressing plants had decreased mechanical strength and cellulose content, but exhibited increases in the expression of several cellulose synthase genes. These results indicate that OspPLAIIIα plays a role in rice vegetative and reproductive growth and that the constitutive, high activity of OspPLAIIIα suppresses cell elongation. The decreased gibberellin response in overexpressing plants is probably a result of the decreased ability to make cellulose for anisotropic cell expansion.

Keywords: Cellulose; dwarf; gibberellin; longitudinal growth; phospholipase A; phospholipids; rice seeds..

Fig. 1.

Phylogenic relationship of pPLAs in Oryza sativa and other species, and sequence alignments. (A) The cladogram of putative pPLAs from Oryza sativa and their relationship with pPLAs from other species based on protein sequences was produced with MEGA4. AtPLAI, IIα, IIIα, Arabidopsis patatin-like acyl hydrolase I, IIα, and IIIα (At1g61850, At2g26560, At2g39220, Arabidopsis thaliana); StPatatin, a potato class I patatin precursor (P11768, Solanum tuberosum); Hs-iPLA2, human intracellular membrane-associated calcium-independent phospholipase A2 (EAL24384, Homo sapiens); RpPL-protein, a patatinB1 precursor from the bacterium Rickettsia prowazekii (CAA15046); SpPL-protein, a putative patatin-like protein from fission yeast (CAB16355, Schizosaccharomyces pombe); Nc-iPLA2, an iPLA2-related protein from Neurospora crassa (CAE76294); the nomenclature of OsPLA is shown in Supplementary Table S1 at JXB online. (B) Alignment of amino acid sequences of rice pPLAIII subfamilies with Clustal W2. The conserved motifs are marked with boxes; the phosphate- or anion-binding element is DGGGx(xx)G, the esterase box is GxGxG but not GxSxG, and the catalytic dyad-containing motif is GGG, SGG, TGG, TGG, or DGG.

Fig. 2.

OspPLAIIIα T-DNA insertion mutant and overexpression in rice. (A) Schematic map showing the T-DNA insertion location (PEG_3A-11040.R) in OspPLAIIIα. (B) PCR verification of the OspPLAIIIα transcript level in the mutant. RNA was extracted from the same stage rice leaves from mutant and wild-type seedlings grown in a greenhouse. The RNA level was normalized to that of β-actin. (C) Transcript level of OspPLAIIIα in wild-type and overexpressing leaves at the tillering stage. The transcript level was measured by real-time PCR and normalized to the level of β-actin. Values are means ±SD (n=3). (D) Immunoblotting of pPLAIIIα in overexpressing and wild-type rice leaves. pPLAIIIα was fused to a Flag tag and, after SDS–PAGE separation, protein was immunoblotted with anti-Flag antibodies and visualized by alkaline phosphatase activity. The upper band was unique to OE transgenic plants.

Fig. 3.

Reduced longitudinal growth in OspPLAIIIα-overexpressing plants in rice. (A–D) Morphology of seedlings, panicles, flag leaves, and roots from two independent OspPLAIIIα-overexpression lines, OE-1 and OE-2, knockout (KO), and wild type (WT) plants. Except for panicles that were collected at the maturation stage, all other tissues were sampled at the tillering stage grown in the field. (E) Plant height of wild-type, KO, and OE plants at the maturation stage. Values are means ±SD (n _WT=90, n _KO=90, n _OE-1=4, n _OE-2=4). (F and G) Length and number of first branches of the panicle. Values are means ±SD (n _WT=60, n _HM=60, n _OE-1=28, n _OE-2=28). (H and I) Flag leaf length and width at the maturation stage. Values are means ±SD (n=12) (J) Root length of primary roots of WT, KO, and OE plants. Values are means ±SD (n=12). ** indicates a significant difference compared with the wild type according to Student’s t-test, P<0.01.

Fig. 4.

Effect of OspPLAIIIα alterations on seeds. (A) Seed shape of WT, KO, and OE plants. (B) Seed length and width of WT, KO, and OE plants grown in the field. (C) Quantitation of length and width of 20-grain and 100-grain weight of WT, KO, and OE seeds. Values are means ±SD (n=6). ** indicates a significant difference compared with the wild type according to Student’s t-test, P<0.01.

Fig. 5.

Cell size in roots of OspPLAIIIα-overexpressing lines and the wild type. (A) Leaf epidermis cells of WT, KO, and OE plants at the tillering stage grown in the field. Scale bar=30 μm. (B) Leaf stomata guard cells at the tillering stage. Scale bar=10 μm. (C) Sheath cells at the four-leaf stage. Scale bar=50 μm. (D) Primary root tip cells at the maturation stage. Sale bar=30 μm. (E and F) Quantitation of leaf epidermis cell length and width at the four-leaf stage. Values are means ±SD (n=40). (G and H) Sheath cell length and width. Values are means ±SD (n=50). ** indicates a significant difference compared with the wild type according to Student’s t-test, P<0.01.

Fig. 6.

Decreased mechanical strength and cellulose content in OspPLAIIIα-overexpressing tissues. (A and B) Brittle stems and nodes of OspPLAIIIα-OE plants. (C) Breaking force of WT, KO, and OE stems. Values are means ±SD (n=20). (D) Cellulose content in mature leaves. Values are means ±SD (n=3). ** indicates a significant difference compared with the wild type according to Student’s t-test, P<0.01. (E) Transcript levels of cellulose synthase (CesA) genes of leaves at the tillering stage of plants grown in the field. The level was measured by real-time PCR and normalized to β-actin. Values are means ±SD (n=3). (This figure is available in colour at JXB online.)

Fig. 7.

The involvement of OspPLAIIIα in gibberellin (GA) response. (A) The growth response of WT, KO, and OE seedlings grown in liquid medium without and with 500 μM GA₃ for 14 d. (B) The plant height of OspPLAIIIα-alterated seedlings treated with and without GA₃ for 14 d. Values are means ±SD (n=6). (C) Transcript level of genes involved in GA synthesis, degradation, and response. The level was measured by real-time PCR and normalized to β-actin. a, b, c, and d indicates different expression levels between treated and untreated plants within each genotypes according to ANOVA, P<0.05. Values are means ±SD (n=3). (This figure is available in colour at JXB online.)

Fig. 8.

The effect of OspPLAIIIα on glycerolipid content and composition.. (A) Contents of different lipid classes from fully expanded leaves at the tillering stage of plants grown in the field. Values are means ±SD (n=3). (B) PA molecular species of rice leaves. Values are means ±SD (n=3). (C) PC molecular species of rice leaves. The numbers on the x-axis in (B) and (C) denote total acyl carbons:total number of acyl carbon double bonds of PA species. * and ** indicate a significant difference between OE plants and wild-type plants with P<0.05 and P<0.01 in the Student’s t-test. Values are means ±SD (n=3).