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A secretory phospholipase D hydrolyzes phosphatidylcholine to suppress rice heading time

['Li Qu', 'Shanghai Collaborative Innovation Center Of Agri-Seeds', 'Joint Center For Single Cell Biology', 'School Of Agriculture', 'Biology', 'Shanghai Jiao Tong University', 'Shanghai', 'National Key Laboratory Of Plant Molecular Genetics', 'Cas Center For Excellence In Molecular Plant Sciences', 'Chinese Academy Of Sciences']

Date: 2022-01 

To investigate the role of signal peptide, we observed tobacco leaves transiently expressing spPLD-GFP fusion protein and found that spPLD-GFP is localized between plasma membrane and cell wall (FM4-64 staining was applied to mark the plasma membrane), suggesting the secretion of spPLD ( Fig 1D ). Further detailed observation of various GFP fusion proteins showed the localization between plasma membrane and cell wall of spPLD-GFP and signal peptide-GFP (sp-GFP), while removal of signal peptide (ΔPLD-GFP) resulted in the fluorescence limited to cytoplasm ( S5C Fig ). Additionally, we transiently expressed the fusion proteins in rice protoplast and analyzed the protein accumulation in incubation medium and protoplast homogenate. Results showed that most spPLD-GFP existed in the supernatant, as well as GFP under signal peptide of spPLD, while ΔPLD-GFP was detected in the precipitation only ( Fig 1E ), confirming that spPLD protein is secreted and soluble. All these results indicated the secretory character of spPLD and a crucial role of signal peptide in guiding the secretion of spPLD protein.

A. Sequence alignment was performed with BLAST and the top 30 proteins with protein functional annotation and identity at more than 70% were analyzed. The bootstrap consensus tree inferred from 1000 replicates is generated to represent the evolutionary history of the taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. NCBI accession number, protein ID and species of analyzed protein are shown. B. Protein structural analysis showed the presence of two HKD motifs and one signal peptide (sp) at N-terminus of rice spPLD. C. qPCR analysis revealed the spPLD expression in various tissues including roots, tillers, stems, SAM, leaves, pulvini, flowers and panicles. Expression level was normalized to ACTIN1 transcript and relative expressions were calculated by setting spPLD expression in leaves as 1.0. Experiments were repeated three times and data were shown as mean ± SD (n = 3). D. Secretion of spPLD was confirmed by observing N. benthamiana plants expressing spPLD-GFP fusion protein. FM4-64 was used to highlight the plasma membrane. Plasmolysis was conducted by 1 M mannitol treatment for 10 min. The secreted sections were highlighted by arrows. DIC, bright field. Scale bar = 50 μm. E. Western Blotting analysis confirms the secretion of spPLD. Various fusion proteins were transiently expressed in rice protoplasts. After incubation for 48 h, proteins of supernatant (incubation medium) and precipitation (protoplast homogenate) were extracted and analyzed by Western Blotting using anti-GFP antibody. Arrows highlighted the GFP (sp-GFP) and spPLD-GFP (ΔPLD-GFP) proteins.

Given the importance of secretory PLDs in animals, it is supposed that spPLD may also play an important role in regulating rice growth and development. Phylogenetic analysis of the top 30 proteins with >70% identities by functional annotation showed that spPLD is in an evolutionarily separated branch ( Fig 1A ). Structural analysis revealed the presence of signal peptide (sp) at N-terminus, and two highly conserved HKD motifs [H(X)K(X) 4 D] at middle and C-terminus, of spPLD (Figs 1B and S1 ). Further phylogenetic analysis showed the uniqueness of signal peptide ( S2 Fig ), while HKD motifs are highly conserved in various phospholipases ( S3 and S4 Figs). Quantitative RT-PCR (qPCR) analysis showed that spPLD is transcribed in various tissues, with a relative higher expression in roots and panicles ( Fig 1C ). Further analysis using RiceXPro ( https://ricexpro.dna.affrc.go.jp/ ) showed that spPLD is highly transcribed in leaf during vegetative period especially before 34 days after transplanting (DAT, S5A Fig ), and interestingly, presents diurnal oscillation (lowest in the morning and highest in the evening, S5B Fig ).

To investigate the physiological functions of spPLD, transgenic rice plants overexpressing spPLD (spPLDox) or spPLD removing the signal peptide (ΔPLDox) were generated (confirmed by qPCR, S6 Fig ). Reduced expression of spPLD was achieved by either RNA interference-mediated silencing (spPLD-RNAi, confirmed by qPCR, S6 Fig ) or CRISPR/Cas9 approach (spPLD-Cas9, confirmed by sequencing, Fig 2B ). Phenotypic observation of homozygous lines showed that altered expression of spPLD does not lead to the obvious growth change during vegetative stage, while interestingly, compared with wild-type plants, significantly delayed heading date was observed in spPLDox plants, whereas spPLD-RNAi and spPLD-Cas9 plants presented earlier heading, under long-day (LD) condition in paddy field (Figs 2C , 2D and S7A ). Similar phenotype was also observed under short-day (SD) condition (Figs 2D and S7B ), indicating that spPLD regulates heading time was not dependent on photoperiod. Interestingly, ΔPLDox plants don’t show delayed heading (Figs 2C , 2D and S7 ), which may due to the deficiency of signal peptide and thus the secretory character of spPLD, and suggests the dependence of spPLD function on the secretion. All these results indicate that spPLD is involved in the regulation of floral transition and functions after being secreted into apoplast.

A. Enzymatic assay showed that both spPLD and ΔPLD (spPLD deleting the signal peptide) present PLD activity. Purified spPLD or ΔPLD (1–5 μg) proteins were used for examination and choline was used as substrate. There is no choline in background (as negative control) and positive control is supplied in assay kit. Experiments were repeated three times and data were shown as mean ± SD (n = 3). B. Sequencing confirmed three mutation lines of spPLD (insertion or deletion of bases), by CRISPR/Cas9. The gRNA targeting site and PAM sequence, and the position of gRNA at spPLD gene, are indicated. C. Phenotypic observation of rice plants with altered spPLD expression under natural long-day condition at heading stage. Representative images were shown. Scale bar = 10 cm. D. Rice plants with altered spPLD expression were grown under natural long-day (left) or short-day (right) conditions and heading date were calculated and statistically analyzed using Tukey’s test (**, p < 0.01; ***, p < 0.001). NS, no significance. Days to flowering were scored when first panicle was bolted, and data were shown as mean ± SD (n = 50).

In vitro enzymatic analysis demonstrated that both spPLD and ΔPLD presents hydrolyzing activity on choline ( Fig 2A ), indicating spPLD a functional PLD and whose activity is signal peptide independent. In eukaryotic cells, phospholipids are asymmetrically distributed on the lipid bilayer of plasma membrane and organelle membranes [ 29 ] and coincidentally, phosphatidylcholine (PC) and sphingomyelin (SM) are exposed on the cell surface [ 30 , 31 ], which shed light on the specific location and physiological effects of spPLD by hydrolyzing PC and indicate the significance for the secretory character of spPLD.

spPLD modulates contents of light period predominant PCs in SAM

As an important developmental stage and agricultural trait, heading date is determined by both endogenous signals and environmental cues and recent studies showed that lipid-mediated signaling has emerged as one of the major regulatory pathways [32]. In Arabidopsis, the key factor regulating plant flowering, Flowering Locus T (FT), preferentially interacts with phosphatidylcholine (PC) species containing less unsaturated fatty acids (36:4, 36:3, 36:2, 36:1 and 34:1) that are predominant in light period to promote flowering [25]. Based on the similar phenotype under both SD and LD conditions, we examined the binding of rice Heading date 3a (Hd3a) and Rice Flowering Locus T 1 (RFT1), with phospholipids through Fat-western immunoblot analysis. Results revealed that both Hd3a and RFT1, bind to phospholipids as well (Figs 3A and S8A). Considering the revealed binding sites of FT for PC (R13, D17, R83 and R119) [27] and high homology of FT, Hd3a and RFT1, the Hd3a or RFT1 with mutated PC binding sites (Hd3aM and RFT1M, the R or D of binding site were mutated to A) were generated. Examination of the phospholipids binding showed the significantly decreased PC binding ability of Hd3aM or RFT1M (Figs 3A and S8A).

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TIFF original image Download: Fig 3. Altered PC species under spPLD overexpression or deficiency. A. Fat-western immunoblot analysis revealed the binding of rice Hd3a (left) and Hd3a with mutated PC binding sites (Hd3aM, right) to phospholipids. The phospholipid type of each dot is indicated. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PA, phosphatidic acid; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; S1P, sphingosine 1-phosphate; PI, phosphatidylinositol; PI3P, PI 3-monophosphate; PI4P; PI5P; PI(3,4)P 2 , PI 3,4-bisphosphate; PI(3,5)P 2 ; PI(4,5)P 2 ; PI(3,4,5)P 3 , phosphatidylinositol 3,4,5-trisphosphate. B. Liposome binding assay (left) and quantitative analysis (right) confirmed the preferential binding of Hd3a to PC. His-tag fused Hd3a and Hd3a with mutated PC binding sites (Hd3aM) were purified and incubated with liposomes containing different PC:PA or PE:PA ratios. After collecting the liposomes, the portion of proteins bound to liposomes was detected by western blotting using anti-His antibody. Nonbinding protein was detected in the supernatant (bottom). Band density is measured by Image J and relative density was calculated by setting the intensity under PC:PA ratio 1:1 as 1.0. Data were presented as means ± SD (n = 3, right). C-D. Relative content of predominant PCs with different saturation status, PC (32:1), PC (36:2), PC (36:3) and PC (38:3) (C); PC (36:5), PC (36:6) and PC (38:6) (D), in ZH11 and various lines with altered spPLD expressions. Ten rice shoot apical meristem before bolting were collected and used for lipids extraction. Phospholipids were profiled by a lipidomic approach using mass spectrometry. Experiments were biologically repeated three times and data were shown as mean ± SD (n = 3). Statistical analysis was performed by Tukey’s test (*, p <0.05; **, p < 0.01 ***, p < 0.001, compared to ZH11). https://doi.org/10.1371/journal.pgen.1009905.g003

Further liposome protein association assay showed that along with the increased ratio of PC:PA in liposomes, stronger binding of Hd3a/RFT1 to PC was observed, while the binding of Hd3aM/RFT1M to PC weakened significantly (Figs 3B and S8B), confirming the binding ability of Hd3a/RFT1 to PC and indicating both Hd3a and RFT1 are also PC-binding proteins. Interestingly, in addition to PC, Hd3a and RFT1 bind to phosphatidylethanolamine (PE), however, liposome protein association assay showed their binding to PE was much weaker than PC and the binding with PE was not affected by the mutation of PC binding sites (Figs 3B and S8B), indicating the binding specificity and that Hd3a and RFT1 preferentially bind to PC than PE. In addition, Hd3a binds to various phospholipids including PI5P, PI(4,5)P 2 , PI(3,4,5)P 3 and slightly binds to phosphatidic acid (PA), suggesting a similar and distinct regulatory mechanism of Hd3a/RFT1 by phospholipids.

Considering spPLD functions at apoplast to affect the heading time and PCs are distributed on the outer plasma membrane, whether altered spPLD expression resulted in the changed content of PC was examined by mass spectrometric analysis. Phospholipids profiles of shoot apical stem (SAM, ~ 7 days before bolting) of ZH11 and various transgenic lines under natural SD condition were investigated with a lipidomics approach [33]. Results showed that there was no evident change of content of Hd3a/RFT1-binding phospholipids, PC and PE, in transgenic lines with altered spPLD expressions (S9A and S9C Fig), while the content of PA was reduced in all examined lines (S9B Fig), suggesting a possible feedback regulation to reduce the PA amount since PA is the product of lipid hydrolysis which is a reversible process [1]. Although other phospholipids including PG, PI, PS, LPA and LPC displayed irregular changes (S9 Fig), there is no binding of them with Hd3a/RFT1 and considering the subcellular location of these phospholipids and secretory character of spPLD, it is suggested that these phospholipids might be not directly involved in the heading time regulation through Hd3a/RFT1.

Although there was no significant difference of total PC levels in rice lines with altered expressions of spPLD, considering the roles of distinct PC species in regulating FT activity and flowering, content of different PC species, particularly with different saturation status, were detailed analyzed. Results indeed showed the evident alteration of specific PC species. Contents of less unsaturated PC species (32:1, 36:2, 36:3 and 38:3) that are predominant in light period are decreased in spPLDox, while increased in spPLD-RNAi or spPLD-Cas9 lines (Figs 3C and S10A–S10D); the contents of high unsaturated PC species (36:5, 36:6 and 38:6) that are predominant in night period are not altered in spPLD-RNAi or spPLD-Cas9 lines, whereas significantly increased in spPLDox lines (Figs 3D and S10E–S10G), which may have a close correlation with the diurnal rhythmic expression of spPLD.

Arabidopsis FT preferentially interacts with less unsaturated PC species (particularly 36:2 and 36:3) to promote flowering, while high unsaturated PC species delay flowering [26]. Hd3a and RFT1 bind PCs and further examination of the expressions of Hd3a/RFT1 downstream genes (OsMADS14/15/18/34) by qPCR showed that the transcription of them were significantly decreased in spPLDox lines while increased in spPLD-RNAi or spPLD-Cas9 lines (Fig 4), indicating that decreased Hd3a/RFT1’ activity in spPLDox lines maybe the direct and main reason for delayed heading. In addition, Hd3a functions as a mobile signal and promotes branching through lateral bud outgrowth [34]. Analysis of the tiller numbers consistently showed the significantly increased tillers of spPLD-RNAi or spPLD-Cas9 rice lines (S11A Fig), which confirmed the enhanced Hd3a activity under deficiency/suppression of spPLD.

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TIFF original image Download: Fig 4. Expression of OsMADS14, 15, 18 and 34 were decreased in spPLDox lines while increased in spPLD-RNAi/Cas9 lines. Total RNAs were extracted from SAM (~ 1 cm in length) of five-week-old rice plants (~ 7 days before bolting) and expressions of OsMADSs genes were examined by qPCR analysis. Rice plants were grown under LD condition (14-h light / 10-h dark cycle, 28°C) with 70% humidity. Expression levels of examined genes were normalized to ACTIN1 transcript. Experiments were repeated for three times and data were shown as mean ± SD (n = 3). Statistical analysis was performed by Tukey’s test (*, p <0.05; **, p < 0.01; ***, p < 0.001, compared to ZH11). https://doi.org/10.1371/journal.pgen.1009905.g004

To further explain whether the Hd3a-phospholipid interaction is necessary to promote the heading, transgenic Arabidopsis overexpressing Hd3a (Hd3aox) and Hd3aM (Hd3aMox) were generated (confirmed by qPCR, S12 Fig). Phenotypic analysis showed that compared to that Arabidopsis seedlings overexpressing Hd3a presented early flowering, seedlings overexpressing Hd3aM did not show promoted flowering time as those overexpressing Hd3a under LD condition (Fig 5A), indicating binding with PC is important for Hd3a function in promoting flowering time.

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