Volume 8, Issue 6, Pages 911-921 (June 2015) Targeted Lipidomics Studies Reveal that Linolenic Acid Promotes Cotton Fiber Elongation by Activating Phosphatidylinositol and Phosphatidylinositol Monophosphate Biosynthesis  Gao-Jun Liu, Guang-Hui Xiao, Ning-Jing Liu, Dan Liu, Pei-Shuang Chen, Yong-Mei Qin, Yu-Xian Zhu  Molecular Plant  Volume 8, Issue 6, Pages 911-921 (June 2015) DOI: 10.1016/j.molp.2015.02.010 Copyright © 2015 The Author Terms and Conditions

Figure 1 Targeted Lipodomics Analyses of Glycerophospholipids from 10-dpa WT Fibers, WT Ovules, and fl Ovules Using the Reverse-Phase UPLC/ESI-MS/MS Method. (A) Analyses of PI from 10-dpa WT fibers, WT ovules and fl ovules. (B) Analyses of PA from 10-dpa WT fibers, WT ovules and fl ovules. (C) Analyses of PS from 10-dpa WT fibers, WT ovules and fl ovules. (D) Analyses of PC from 10-dpa WT fibers, WT ovules and fl ovules. (E) Analyses of PE from 10-dpa WT fibers, WT ovules and fl ovules. (F) Analyses of PG from 10-dpa WT fibers, WT ovules and fl ovules. PI, phosphatidylinositol; PA, phosphatidic acid; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol. Lipid contents (dw, dry weight) were calculated based on internal standards. The amount of each glycerophospholipid class represents the sum of individual lipid species. The experiments were repeated three times using independent cotton materials. Error bars represent SD, n = 3. Statistical significance was determined using a one-way analysis of variance (ANOVA) combined with the Tukey test. **P < 0.01; ***P < 0.001. WT, wild-type; fl, fuzzless-lintless mutant. Molecular Plant 2015 8, 911-921DOI: (10.1016/j.molp.2015.02.010) Copyright © 2015 The Author Terms and Conditions

Figure 2 The Effectiveness of Different Classes of Glycerophospholipid on Promoting Cotton Fiber Growth in Ovule Culture Experiments. WT ovules were collected at 1 dpa and cultured for 6 d with 2 μM various soybean glycerophospholipids added to the culture media. Fiber lengths were averaged from three independent experiments with at least 20 ovules in each individual culture (Shi et al., 2006; Li et al.,2007; Pang et al.,2010); data are shown as the mean ± SD in mm. CK, medium only; PI, phosphatidylinositol; PA, phosphatidic acid; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol. See Figure 1 legend for details of statistics. Molecular Plant 2015 8, 911-921DOI: (10.1016/j.molp.2015.02.010) Copyright © 2015 The Author Terms and Conditions

Figure 3 The Linolenic Acid Moiety of Phosphatidylinositol Promotes Fiber Elongation. (A) MS/MS spectrum of PI 34:3, indicating that it contains two fatty acyl chains C16:0 and C18:3. CPS, counts per second. (B) Liver PI, which lacks the C18:3 moiety, has no effect on fiber elongation. (C) C18:3, but not C18:2, C16:0, glycerol, or inositol, promoted significant fiber growth. (D) Quantitative analysis of long-chain FAs in 10-dpa WT fibers, WT ovules, and fl ovules by GC/MS. Heptadecanoic acid (C17:0) was used as an internal standard. The experiments were repeated three times using independent cotton materials. PI, phosphatidylinositol. See Figure 1 legend for details of statistics. Molecular Plant 2015 8, 911-921DOI: (10.1016/j.molp.2015.02.010) Copyright © 2015 The Author Terms and Conditions

Figure 4 The Linolenic Acid Moiety of PIP Promotes Fiber Cell Elongation. (A) Quantification of PIPs extracted from 10-dpa WT fibers, WT ovules, and fl ovules. (B) MS/MS spectrum of PIP 34:3 that showed two fatty acyl chains of C16:0 (m/z 255.3) and C18:3 (m/z 277.4). (C) Comparisons of different fatty acid components of PIPs extracted from 10-dpa fibers and from fl ovules. (D) PIPs extracted from 10-dpa fiber cells, 34:3 prepared from PIPs of 10-dpa WT fibers or fl ovules were able to promote significant fiber growth. PIP, phosphatidylinositol monophosphate. See Figure 1 legend for details of statistics. Molecular Plant 2015 8, 911-921DOI: (10.1016/j.molp.2015.02.010) Copyright © 2015 The Author Terms and Conditions

Figure 5 qRT–PCR Analyses of Transcripts Involved in Biosynthesis of C18:3, PI and PIP. (A) Biosynthetic pathway from linoleic acid to phosphatidylinositol monophosphate. (B) qRT–PCR analysis of the expression of genes encoding Δ15FAD during various developmental stages in WT as well as the fl mutant. (C) qRT–PCR analysis of the expression of genes encoding PI synthases during various developmental stages in WT as well as the fl mutant. (D) qRT–PCR analysis of the expression of genes encoding PI kinases during various developmental stages in WT as well as the fl mutant. qRT–PCR analyses were performed in triplicate using independent cotton materials. See Figure 1 legend for details of statistics. Molecular Plant 2015 8, 911-921DOI: (10.1016/j.molp.2015.02.010) Copyright © 2015 The Author Terms and Conditions

Figure 6 Exogenous PIs Reverted the Growth Inhibition of Carbenoxolone and 5-Hydroxytryptamine, but Not that of Wortmannin Whereas PIPs Nullified the Effects of all Three Growth Inhibitors. (A) Carbenoxolone inhibited fiber growth. (B) Quantification of C18:3, PI 34:3, and PIP 34:3 extracted from 0-dpa wild-type ovules cultured in the medium for 6 days in the presence of 0.5 μM carbenoxolone. (C) The growth inhibition of carbenoxolone in ovule culture can be easily overcome by exogenous C18:3, PI, or PIP. (D) 5-Hydroxytryptamine inhibited fiber growth. (E) Quantification of C18:3, PI 34:3, and PIP 34:3 extracted from 0-dpa wild-type ovules cultured in the medium for 6 days in the presence of 0.5 μM 5-hydroxytryptamine. (F) The growth inhibition of 5-hydroxytryptamine can be overcome by exogenous PI or PIP, but not C18:3. (G) Wortmannin inhibited fiber growth. (H) Quantification of C18:3, PI 34:3, and PIP 34:3 extracted from 0-dpa wild-type ovules cultured in the medium for 6 days in the presence of 0.5 μM wortmannin. (I) Only exogenous PIP, but not PI or C18:3, was able to overcome the fiber growth inhibition caused by adding wortmannin. The amounts of C18:3, PI 34:3, and PIP34:3 are presented as a percentage of the non-treated samples. See Figure 1 legend for details of statistics. Molecular Plant 2015 8, 911-921DOI: (10.1016/j.molp.2015.02.010) Copyright © 2015 The Author Terms and Conditions

Figure 7 Silencing of Either Δ15FAD, PIS, or PIK Genes via VIGS Resulted in Significantly Shortened Cotton Fibers. (A) Phenotypes of WT and gene-silenced cotton plants. WT transformed by an empty vector was used as control. Inset shows the leaf and boll positions on various cotton plants as indicated. (B) qRT–PCR analyses of transcript levels of the target genes in 10-dpa fibers of silenced or non-silenced cotton plants. (C) qRT–PCR analyses of transcript levels of the target genes in 10-dpa ovules of silenced or non-silenced cotton plants. (D) qRT–PCR analyses of transcript levels of the target genes in leaves of silenced or non-silenced cotton plants after CLCrV infection at 30 d after infiltration. (E) Analysis of fiber lengths of WT and gene-silenced cotton plants. WT + vector, WT plants infiltrated with Agrobacterium containing an empty plasmid for Cotton Leaf Crumple Virus (pCLCrV). WT + pCLCrV, Δ15FAD; WT + pCLCrV, PIS; and WT + pCLCrV, PIK. WT plants infiltrated with Agrobacterium containing pCLCrV, Δ15FAD; pCLCrV, PIS; and pCLCrV, PIK plasmids. See Figure 1 legend for details of statistics. Molecular Plant 2015 8, 911-921DOI: (10.1016/j.molp.2015.02.010) Copyright © 2015 The Author Terms and Conditions