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1.
Figure 11.

Figure 11. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

Altered Anatomy of Interfascicular Fibers in Stalks of Overexpression Mutants of pPLAIIIβ Compared with Those of the Wild Type.
(A) to (D) Transverse sections of the stalks of 8-week-old wild-type (A) and OE1 plants (B) stained with toluidine blue showing differences in interfascicular fibers (if).
(C) and (D) Enlargement of the boxed regions indicated in (A) and (B) showing that the fiber cell wall (arrows) in the wild type (C) is thicker than that in OE1 (D).
(E) and (F) Longitudinal sections of the stalk of wild-type (E) and OE1 plants (F). Fiber cells in OE1 are shorter and wider than those in the wild type. Arrows mark the ends of a fiber cell. c, cortex; e, epidermis; if, interfascicular fiber; pi, pith; x, xylem.
[See online article for color version of this figure.]

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
2.
Figure 5.

Figure 5. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

Effect of pPLAIIIβ Alterations on Phospholipid and Galactolipid Content in Arabidopsis.
Lipids from rosettes of 2-week-old soil-grown plants were quantified by ESI-MS/MS. Values are means ± sd (n = 5); each replicate contained at least three plant rosettes. Asterisk indicates significant difference at P < 0.05 compared with the wild type based on Student’s t test.
(A) Phospholipid and galactolipid content of wild-type, KO, OE, and COM mutant rosettes. Phospholipids include PC, PE, PI, PS, PA, and PG; galactolipids include MGDG and DGDG.
(B) Total lipids in wild-type, KO, OE, and COM mutant lines. Total lipids refer to the total amount of PC, PE, PI, PS, PA, PG, MGDG, DGDG, LPC, LPE, and LPG.
(C) Molecular species of phospholipids and galactolipids in wild-type, KO, OE, and COM mutants. Phospholipids include PC, PE, PI, PS, PA, and PG; galactolipids include MGDG and DGDG.

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
3.
Figure 13.

Figure 13. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

A Working Model of the Effects of pPLAIIIβ on Lipid Metabolism, Cellulose Content, Cell Elongation, Cell Morphology, and Mechanical Strength.
pPLAIIIβ hydrolyzes acyl-CoA and phospholipids such as PC and generates FFA and lysophospholipids, such as LPC. The released FFA and lysophospholipids can be reacylated. The hydrolysis catalyzed by pPLAIIIβ may facilitate the lipid metabolic flux and results in the increased synthesis of other phospholipids, such as PC, PE, PG, and PA, and galactolipids, such as MGDG and DGDG. As a result, less carbon from photosynthesis may be partitioned into the synthesis of cellulose for cell wall synthesis, which provides the plant with mechanical strength. DAG, diacylglycerol; FRA2, fragile fiber 2; G-3-P, glycerol-3-phosphate; LPA, lysophosphatidic acid; lysoPL, lysophospholipids; TAG, triacylglycerol.
[See online article for color version of this figure.]

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
4.
Figure 7.

Figure 7. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

Altered Plant Size of Knockout and Overexpression Mutants of pPLAIIIβ.
(A) Morphology of 2-week-old plants. pPLAIIIβ knockout leads to slightly enlarged Arabidopsis plants, and overexpression of pPLAIIIβ leads to significantly smaller plants. The rosette leaves of OE plants are more compact than those of wild-type (WT) plants. KO, T-DNA insertion line; COM1, complementation line 1; COM2, complementation line 2; OE1 to 4, independent overexpression lines 1 to 4.
(B) Individual leaves of 4-week-old plants. The lengths of both blades and petioles in OE1 plants are reduced compared with those of the wild-type plants. From left to right, the leaves are arranged from cotyledons to the youngest leaves.
(C) Morphology of main inflorescence stalks of 8-week-old plants. The stalk of the OE plants is much thicker than that of a wild-type plant.
(D) Siliques of 8-week-old plants. The length of siliques and pedicels in OE plants are shorter than those of wild-type plants. In (C) and (D), (a), wild type; (b), KO; (c), OE1; (d), OE2; (e), COM1; (f), COM2.
(E) Morphology of flowers. The length of flower buds in OE plants is shorter than in wild-type plants.
[See online article for color version of this figure.]

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
5.
Figure 10.

Figure 10. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

Altered Mechanical Strength and Cellulose Content in Knockout and Overexpression Mutants of pPLAIIIβ.
(A) Physical properties of main inflorescence stalks of wild-type (WT) and OE1 plants showing an easily broken stalk, as indicated by the arrow.
(B) Physical properties of branching stalks of wild-type and OE1 plants showing an easily broken branching stalk, as indicated by the arrow.
(C) Measurement of the force required to break the main inflorescence stalks in the bottom segment, showing that the mechanical strength was slightly increased in KO and dramatically reduced in OE stalks. Breaking force is defined as the gram of weight needed to break the stem. Data are the mean values ± se for the bottom stalk segment of 20 plants. Asterisk indicates significant difference at P < 0.05 compared with the wild type based on Student’s t test.
(D) Measurement of the cellulose content of wild-type, KO, OE, and COM plant stalks showing slightly increased but dramatically reduced cellulose content in KO and OE stalks, respectively, when compared with the wild type. Data are means ± se of 10 samples. Asterisk indicates significant difference at P < 0.05 compared with the wild type based on Student’s t test.
[See online article for color version of this figure.]

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
6.
Figure 4.

Figure 4. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

The Effect of pPLAIIIβ Alterations on Lipid Content of FFA and Lysophospholipids in Arabidopsis.
Lipids were extracted from 2-week-old soil-grown plant rosettes and analyzed by ESI-MS/MS as described by Xiao et al. (2010). Asterisk indicates a significant difference between mutant plants and wild-type plants with P < 0.05 in the Student’s t test.
(A) T-DNA insertion site in the pPLAIIIβ gene and the complementation construct introduced into the T-DNA insertion mutant. Boxes denote exons and lines introns.
(B) Determination of pPLAIIIβ transcript in the wild type, knockout line (pPLAIIIβ-KO), overexpression lines (pPLAIIIβ-OE), and complementation line (pPLAIIIβ-COM). The expression levels were normalized in comparison to UBQ10. Values are means ± sd (n = 3 technical replicates).
(C) Total FFAs in wild-type, KO, OE, and COM lines, showing the reduced FFA content of KO and the increased FFA content of OE plants compared with the wild type. Values are means ± sd (n = 5 separate samples).
(D) FFA molecular species in wild-type, KO, OE, and COM mutants. Values are means ± sd (n = 5 separate samples).
(E) Total lysophospholipid content in wild-type, KO, OE, and COM lines. Lysophospholipids include LPC, LPE, and LPG. Values are means ± sd (n = 5 separate samples).
(F) Lysophospholipid molecular species in wild-type, KO, OE, and COM mutants. Lysophospholipids include LPC, LPE, and LPG. Values are means ± sd (n = 5 separate samples).

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
7.
Figure 9.

Figure 9. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

Altered Cell Length of Leaves, Trichomes, and Hypocotyls of Knockout and Overexpression Mutants of pPLAIIIβ Examined by Scanning Electron Microscopy.
Data are means ± se of 20 samples and asterisk indicates significant difference at P < 0.05 compared with the wild type (WT), based on Student’s t test ([B], [C], [E], [F], and [H]).
(A) Leaf epidermal cells of wild-type, KO, OE, and COM plants showing reduced cell length and reduced convolution of epidermal cells in OE plants compared with wild type. Bars = 100 μm.
(B) Leaf epidermal cell width showing little change in KO and OE plants compared with that of the wild type.
(C) Leaf epidermal cell length showing increased and reduced length in KO and OE plants, respectively, compared with the wild type. Seedlings were grown on half-strength Murashige and Skoog vertical plates for 3 d.
(D) Hypocotyl epidermal cells of wild-type, KO, OE, and COM plants showing reduced cell length in the OE plants. Bars = 100 μm.
(E) Hypocotyl cell length showing increased and reduced length in KO and OE plants, respectively, compared with the wild type. Seedlings were grown on half-strength Murashige and Skoog vertical plates for 3 d.
(F) Hypocotyl cell width showing no change in KO but an increase in OE plants compared with wild-type hypocotyl cell widths.
(G) Morphology of trichomes of wild-type, KO, OE, and COM plants showing slightly longer trichome branches in KO and shorter trichome branches in OE plants compared with those of the wild type. Bar = 100 μm.
(H) Length of trichome branches showing a slight increase in KO but a reduction in OE compared with the wild type.

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
8.
Figure 2.

Figure 2. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

Acyl Hydrolyzing Activity of Bacterially Expressed pPLAIIIβ.
(A) Coomassie blue staining of an 8% SDS-PAGE gel loaded with affinity-purified His-tagged pPLAIIIβ (arrow) from E. coli.
(B) FFA released by pPLAIIIβ when 16:0-18:2 PC [1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine] vesicles were used as a substrate. Inset: structure of 16:0-18:2 PC. Values are means ± sd (n = 3 separate samples).
(C) LPC released by pPLAIIIβ when 16:0-18:2 PC vesicles were used as a substrate. Inset: structures of 16:0-LPC and 18:2-LPC. Values are means ± sd (n = 3 separate samples).
(D) Acyl hydrolysis activity of pPLAIIIβ toward various classes of phospholipids and galactolipids. Vesicles made from individual lipid species were incubated with pPLAIIIβ at 30°C for 30 min. After the reaction, lipids were extracted and quantified by ESI-MS/MS (Welti et al., 2002). Values are means ± sd (n = 3 separate samples).
(E) Acyl-CoA substrate decreased after incubation with pPLAIIIβ. 18:3-CoA was hosted in 16:0-18:2 PC vesicles and incubated with pPLAIIIβ for the indicated amount of time. After the reaction, acyl-CoA was quantified by ESI-MS/MS in neutral loss mode as described in Methods. Values are means ± sd (n = 3 separate samples).
(F) FFA released by pPLAIIIβ when 18:3-CoA (6Z,9Z,12Z-octadecatrienoyl CoA), hosted in 16:0-18:2 PC vesicles, was used as a substrate. Vector refers to a control in which proteins from E. coli transformed with an empty vector were isolated using the same immunoaffinity procedure used to isolate pPLAIIIβ-His. Values are means ± sd (n = 3 separate samples).

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
9.
Figure 1.

Figure 1. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

Alignment of 10 pPLAs and Gene Expression of pPLAIIIs.
(A) Alignment of deduced amino acid sequences of plant patatin-like acyl-hydrolase families (pPLAs) in Arabidopsis. Ten Arabidopsis genes encode pPLAs, and these are classified into three groups (Scherer et al., 2010). Group 1 has one gene, pPLAI; group 2 has five genes, pPLAIIα, β, γ, δ, and ε; and group 3 has four genes, pPLAIIIα, β, γ, and δ. The catalytic center is marked; this includes the esterase box GxSxG (in pPLAIIIα, β, γ, and δ, it is GxGxG), the phosphate or anion binding element DGGGxxG, and the catalytic dyad-containing motif DGG or DGA (in pPLAIIIβ and γ, it is GGG). The protein sequences were aligned using the website http://www.ebi.ac.uk/Tools/clustalw2/index.html, and the conserved domains were identified using the website http://www.ncbi.nlm.nih.gov/Structure/cdd.shtml. The gray highlighting indicates the region of conserved motif. The bold highlighting indicates the different amino acids in the conserved motif.
(B) Expression of group 3 pPLAs, pPLAIIIα, pPLAIIIβ, pPLAIIIγ, and pPLAIIIδ, in seven types of Arabidopsis tissues, as quantified by real-time PCR normalized to ubiquitin10 (UBQ10). Values are means ± sd (n = 3 technical replicates).
(C) Expression of group 3 pPLAs, pPLAIIIα, pPLAIIIβ, pPLAIIIγ, and pPLAIIIδ, in seven types of Arabidopsis tissues, as quantified by real-time PCR normalized to β-tubulin. Values are means ± sd (n = 3 technical replicates).
(D) Expression of group 3 pPLAs, pPLAIIIα, pPLAIIIβ, pPLAIIIγ, and pPLAIIIδ, in seven types of Arabidopsis tissues based on data from Genevestigator (http://www.genevestigator.com). Values are means ± sd (n = 5 independent experiments).

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
10.
Figure 6.

Figure 6. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

Subcellular Localization of pPLAIIIβ.
(A) Confocal micrographs of epidermal cells of wild-type leaf ([a] to [d]) and pPLAIIIβ-OE:GFP leaf ([e] to [h]) and chlorophyll fluorescence ([a] and [e]; red) versus GFP ([b] and [f]; green). The green fluorescent signal of GFP-tagged pPLAIIIβ is shown in (f). Transmitted light ([c] and [g]) overlays ([d] and [h]) clarify cell outlines. Bar = 50 μm.
(B) Plasmolysis of root epidermal cells of the pPLAIIIβ-OE:GFP mutant. (a) Before plasmolysis: signal was at the cell surface; (b) 5 min after plasmolysis: signal was plasmolyzed. Bar = 50 μm.
(C) Subcellular fractionation of pPLAIIIβ. Twenty micrograms of soluble protein was loaded per lane, and 5 μg of protein was loaded per lane for membrane fractions. Cytosol, soluble fraction; MM, microsomal membrane fraction; PM, plasma membrane; IM, intracellular membrane.
(D) PC-hydrolyzing activity in pPLAIIIβ-OE membrane fractions from leaves. The same amounts of protein from the membrane fractions from wild-type leaves and pPLAIIIβ-OE leaves were used for the PLA assay. The pPLA activity due to overexpression of pPLAIIIβ is represented as the activity in the pPLAIIIβ-OE fraction minus the activity in the corresponding wild-type fraction (n = 3). Asterisk indicates significant difference at P < 0.05 compared with the cytosol, based on Student’s t test.

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
11.
Figure 8.

Figure 8. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

Altered Organ Length of Leaves, Stalks, Hypocotyls, Primary Roots, and Root Hairs of Knockout and Overexpression Mutants of pPLAIIIβ.
(A) Diagram showing the growth parameters measured, including leaf length, leaf width, petiole length, plant height, and length of 1st, 2nd, and 3rd internodes.
(B) The length and width of leaves. The fifth to eighth leaves of 5-week-old plants were measured. Lengths of leaf blades and petioles are slightly longer in KO and dramatically shorter in OE plants compared with wild-type leaves. Data are means ± se of 20 leaves. Asterisk indicates significant difference at P < 0.05 compared with the wild type, based on Student’s t test.
(C) Measurement of the length and width of main inflorescence stalks. The internodes of 8-week-old plants were measured. The main inflorescence of OE plants is shorter than that of wild-type plants and so is the length of the 1st, 2nd, and 3rd internodes. Data are means ± se of 20 samples. Asterisk indicates significant difference at P < 0.05 compared with the wild type, based on Student’s t test.
(D) Three-day-old seedlings of wild-type, KO, OE, and COM plants showing longer primary roots in KO plants and shorter ones in OE plants compared with the wild type and COM. Seedlings were grown in half-strength Murashige and Skoog agar medium plates in a horizontal orientation for 3 d, and growth parameters were measured.
(E) Root hairs of wild-type, KO, OE, and COM plants, showing increased and reduced length of root hairs in KO and OE plants, respectively, compared with the wild type.
(F) Primary root length in wild-type, KO, OE, and COM plants showing that KO has longer and OE has shorter primary roots in 3-d-old seedlings compared with wild-type and COM plants. Data are means ± se of 20 samples. Asterisk indicates significant difference at P < 0.05 compared with the wild type based on Student’s t test.
(G) Hypocotyl length in wild-type, KO, OE, and COM plants showing that KO has longer and OE has shorter hypocotyls in 3-d-old seedlings compared with the wild type and COM. Data are means ± se of 20 samples. Asterisk indicates significant difference at P < 0.05 compared with the wild type based on Student’s t test.
(H) Root hair length in the primary roots of wild-type, KO, OE, and COM plants showing that KO has longer and OE has shorter root hairs in 3-d-old seedlings compared with the wild type. Data are means ± se of 50 samples. Asterisk indicates significant difference at P < 0.05 compared with the wild type based on Student’s t test.
[See online article for color version of this figure.]

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
12.
Figure 12.

Figure 12. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

Altered Sensitivity of Plant Root Growth of KO and OE1 in Response to Treatment by FFA but Not Lysophospholipids.
(A) Root growth inhibition by FFA (18:3-FFA) showing that primary root growth of KO is less sensitive and OE1 is more sensitive to 50 μM 18:3 (FFA) treatment compared with the wild type (WT). Three-day-old seedlings grown on half-strength Murashige and Skoog plates were transferred to new plates containing the indicated concentrations of FFA for 2 d. The arrows mark the newly emerged primary root after 2 d.
(B) Response of primary root growth to various concentrations of 18:3, showing that the OE1 plant has more and the KO plant has less sensitivity to the treatment compared with the wild type. The difference is particularly significant with the 50 μM 18:3 treatment. Values are means ± se (n = 18). Asterisk indicates significant difference at P < 0.05 compared with the wild type based on Student’s t test.
(C) Response of primary root growth to various types of FFA in wild-type, KO, OE1, and COM1 plants. Three-day-old seedlings were transferred to new plates containing the indicated amount of FFA. After 2 d, the newly elongated primary root length was measured. The root growth in the control plate was set as 100%. Five types of FFAs were applied: 16:0, 18:0, 18:1, 18:2, and 18:3. Data are means ± se of 18 samples. Asterisk indicates significant difference at P < 0.05 compared with the wild type based on Student’s t test.
(D) Response of primary root growth to various concentrations of LPC, showing little difference in wild-type, KO, OE1, and COM1 plant response. Data are means ± se of 18 samples.
(E) Response of primary root growth to various concentrations of LPE showing little difference in wild-type, KO, OE1, and COM1 plant response. Data are means ± se of 18 samples.
(F) Oxylipin levels in wild-type, KO, OE1, and COM1 plant seedlings showing a significant difference between KO and OE1 seedlings. Five-day-old seedlings grown on half-strength Murashige and Skoog plates were harvested for oxylipin measurements. Values are means ± se (n = 5). Inset: P value based on Student’s t test. FW, fresh weight.

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.
13.
Figure 3.

Figure 3. From: Patatin-Related Phospholipase pPLAIII?-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W].

pPLAIIIβ Expression in Arabidopsis.
(A) Production of His-tagged pPLAIIIβ in Arabidopsis. The pPLAIIIβ genomic DNA sequence was cloned into a vector with the constitutive expression promoter 35S and in frame with the GFP-His tag in the C terminus. Leaf proteins extracted from pPLAIIIβ-GFP-His transgenic plants were separated by 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Lanes 1 through 5 represent different transgenic lines carrying the pPLAIIIβ-GFP-His overexpression construct. The pPLAIIIβ-GFP-His–tagged protein was purified from leaves using NTA agarose beads (Qiagen) and then visualized with alkaline phosphatase conjugated to a secondary anti-mouse antibody after blotting with GFP antibody.
(B) Acyl hydrolyzing activity of pPLAIIIβ. pPLAIIIβ-GFP-His, purified from plant leaves using NTA agarose beads, was incubated with vesicles of 16:0-18:2 PC for 30 min at 30°C with gentle shaking. After the reaction, the lipids were extracted, and FFAs were quantified by ESI-MS scans. Values are means ± sd (n = 3) of three independent experiments. Asterisk indicates significant difference at P < 0.05 compared between 0 and 30 min of pPLAIIIβ incubation based on Student’s t test.
(C) Fatty acyl-CoA thioesterase activity of pPLAIIIβ. Purified pPLAIIIβ-GFP-His protein from plant leaves was incubated with equal amounts of linoleoyl CoA and 6Z,9Z,12Z-octadecatrienoyl CoA (18:2-CoA and 18:3-CoA) hosted in 16:0-18:1 PC vesicles for 30 min at 30°C with gentle shaking. Released FFAs were extracted and quantified by ESI-MS scans. Each data point represents the average ± sd from three separate determinations.

Maoyin Li, et al. Plant Cell. 2011 March;23(3):1107-1123.

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