UDP-Glucose Pyrophosphorylase Is a Novel Plant Cell Death Regulator

Programmed cell death (PCD) is an essential process that functions in plant organ sculpture, tissue differentiation, nutrient recycling, and defense against pathogen attack. A full understanding of the mechanism of PCD in plants is hindered by the limited identification of protein components of the complex signaling circuitry that underpins this important physiological process. Here we have used <i>Arabidopsis thaliana</i> and fumonisin B1 (FB1) to identify proteins that constitute part of the PCD signaling network. We made an inadvertent, but important observation that exogenous sucrose modulates FB1-induced cell death and identified sucrose-induced genes from publicly available transcriptomic data sets for reverse genetic analyses. Using transfer-DNA gene knockout plants, UDP-glucose pyrophosphorylase 1 (<i>UGP1</i>), a sucrose-induced gene, was demonstrated to be a critical factor that regulates FB1-induced PCD. We employed 2D-DiGE to identify proteomic changes preceding PCD after exposure of <i>Arabidopsis</i> to FB1 and used <i>UGP1</i> knockout plants to refine the analysis and isolate downstream candidate proteins with a putative PCD regulatory function. Our results reveal chloroplasts as the predominantly essential organelles in FB1-induced PCD. Overall, this study reveals a novel function of UGP1 as a cell death regulator and provides candidate proteins likely recruited downstream in the activation of plant PCD

Fatty acid analysis of castor tissues and purified lipid X.

<p>Average peak-area percentages of fatty acid methyl ester derivatives are listed. The number of separate sample extractions and analyses are listed in brackets, except for lipid X, where the result from two GC injections of the purified fraction is shown.</p>a<p>Italicised entries are probable fatty acid designations, although the species did not exactly co-chromatograph with standards.</p>b<p>Indicates the identity of the molecular species is not known but likely wax or hydroxylated fatty acid derivatives from pollen protective layers.</p

MS analysis of purified neutral lipids from pollen.

<p>Purified neutral lipids from pollen were analysed by TLC and used for electrospray MS analysis in the presence of lithium (panel A). Ions between 830 and 900 amu in the lipid X fraction (highlighted in red and Panel B) were selected for fragmentation and the diagnostic fragments produced, such as those in Panel C from the 858 mass ion, allowed determination of fatty acid composition of proposed TAG species (Panel D).</p

Pathways of triacylglycerol biosynthesis.

<p>Reactions in the formation of hydroxylated TAGs via the acyl-CoA dependent (solid arrows) and independent (dashed arrows) pathways are shown. Lipid substrates are abbreviated: 18:1, oleic acid; 18:1-OH, ricinoleic acid; LPC, lysophosphatidylcholine; DAG, diacylglycerol. Enzyme abbreviations are: Δ12-OHase, oleate-12-hydroxylase; LPCAT, 1-acylglycerol-3-phosphocholine acyltransferase; PL-A₂, phospholipase A₂; LACS, long chain acyl-CoA synthetase; GPAT, glycerol-3-phosphate acyltransferase; LPAT, lysophosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase; CPT, CDP-choline:diacylglycerol cholinephosphotransferase; PL-C, phospholipase C; PDAT, phosphatidylcholine diacylglycerol acyltransferase.</p

TLC analysis of neutral lipids from castor developing male flowers and pollen.

<p>Lipid extracts were applied to a silica TLC plate which was developed with hexane/diethyl ether/acetic acid (70∶30∶1) before iodine-staining. The position of lipid standard components (lane N) and proposed nature of resolved sample lipids are shown. Lipid X and proposed sterol esters were purified for further analysis.</p

Acyl-CoA levels in developing castor endosperm.

<p>The percentage of the total acyl-CoA peak area represented by specified acyl-CoAs are shown for castor endosperm stages during seed development. Analysis of fluorescent acyl-CoA derivatives was as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030100#pone.0030100-Larson1" target="_blank">[24]</a> and average values from three extractions and analyses for each stage are shown, together with the standard error in brackets (<i>n</i> = 3).</p>a<p>Endosperm samples were staged according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030100#pone.0030100-Greenwood1" target="_blank">[25]</a>.</p

Acyl-CoA analysis of developing castor endosperm.

<p>A chromatogram of fluorescent acyl-CoA derivatives from developing castor endosperm stage III is shown - dark trace. Results from analysis of synthesised 18:1-OH-CoA (green line) and other acyl-CoA standards (red trace) are superimposed to confirm the indicated peak identities in the endosperm sample.</p

Candidate genes that may be important in tri-ricinolein synthesis based on RNA-Seq data.

<p>Candidate genes that may be important in tri-ricinolein synthesis based on RNA-Seq data.</p