UDP-Glucose Pyrophosphorylase Is a Novel Plant Cell Death Regulator

Article pubs.acs.org/jpr

UDP-Glucose Pyrophosphorylase Is a Novel Plant Cell Death Regulator Stephen Chivasa,† Daniel F. A. Tomé,†,‡ and Antoni R. Slabas*,† †

School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, United Kingdom School of Life Sciences, University of Warwick, Warwick, CV4 7AL, United Kingdom

‡

S Supporting Information *

ABSTRACT: 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 Arabidopsis thaliana 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 (UGP1), 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 Arabidopsis to FB1 and used UGP1 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. KEYWORDS: fumonisin, sucrose, UDP-glucose pyrophosphorylase, proteomics, cell death

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INTRODUCTION Programmed cell death (PCD) in plants is a widespread phenomenon triggered by different biotic and abiotic stimuli. Unlike necrosis, PCD is an active form of cell suicide that is tightly regulated at the genetic level, only being activated when required.1 During growth and development, cell death occurs in many processes such as vascular tissue differentiation,2 senescence,3 and as a result of pathogen attack, when it is known as hypersensitive response (HR) cell death.4 Some abiotic stresses that are capable of initiating PCD are heat stress,5 cold stress,6 ultraviolet radiation,7 and ozone treatment.8 However, the mechanisms by which plants execute PCD are not fully understood. The study of plant PCD has been hampered by the absence of clearly defined core cell death regulators such as BCL-2, APAF1 and caspase families present in animals.9 However, some features of plant PCD share mechanistic similarities with animal apoptosis, suggesting that these cell death effectors might have been conserved between animals and plants throughout evolution.10 Caspases play a critical role in animal apoptosis by specifically cleaving target substrates and orchestrating the proteolytic degradation of the proteome. Treatment of plant cells with caspase inhibitors blocks PCD, suggesting the existence of caspase functional homologues in plants.11 Although plant orthologues of animal caspases have © XXXX American Chemical Society

not been identified after sequencing of the Arabidopsis genome, a closely related group called metacaspases has been suggested as possible regulators of PCD,12 since these metacaspases are specifically required for successful progression of PCD.13 A more recent report identified a metacaspase that regulates developmental PCD by selective cleavage of a target substrate, suggesting metacaspases could indeed function via a similar mechanism to animal caspases.14 In animals, several core cell death factors such as cytochrome c,15 endonuclease G,16 and apoptosis-inducing factor,17 are located inside the mitochondria and only participate in apoptosis when translocated to the cytosol. Although cytochrome c leakage into the cytosol has been associated with some forms of plant PCD, it does not appear to directly activate PCD per se.18 However, the breakdown of plant mitochondrial membrane integrity correlates with cell death induction,19,20 probably due to the release of unknown plant-specific cell death factors. Mutational and reverse genetic analyses have been invaluable in revealing important genetic loci with essential functions in plant cell death. For example, the lsd1 mutant of Arabidopsis displays runaway cell death when challenged with HR-inducing bacteria or directly with superoxide treatment, implicating this Received: November 20, 2012

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dx.doi.org/10.1021/pr3010887 | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

identifying additional genes that regulate PCD in Arabidopsis. We now report that UDP-glucose pyrophosphorylase 1 (UGP1), a sucrose regulated protein, is required for FB1induced cell death. We identify proteins that could play an essential role in regulating cell death downstream of UGP1.

gene as a key suppressor of cell death progression initiated by reactive oxygen species.21 The dnd1 and dnd2 mutants have been identified as specific cyclic nucleotide-gated ion channels that are required to transduce calcium signaling during HR cell death.22,23 Loss-of-function mutants in MLO genes resulted in a spontaneous cell death phenotype in barley24 and members of this gene family are modulated by calcium concentrations via their direct interaction with calmodulin.25 In addition, other lesion mimic mutants have shown a possible involvement of the chloroplast26,27 and sphingolipid signaling28,29 in plant PCD regulation. HR cell death is a form of defensive response that contributes to the prevention of pathogen spread into uninfected plant tissue. It is triggered by pathogen-associated molecular patterns (PAMP’s) such as flagellin30 and bacterial elongation factor Tu31 or by gene-for-gene interactions between plant resistance (R) genes and pathogen avirulence genes.4,32 Host HR cell death can also be caused by pathogen-derived phytotoxic molecules that function as key virulence determinants. Fumonisin B1 (FB1), one such mycotoxin secreted by Fusarium monilliforme, is able to initiate PCD in both plants and animals.33,34 Not only does it activate PCD, but in Arabidopsis FB1 also elicits classical defense responses like production of reactive oxygen species and expression of pathogenesis-related (PR) genes.35 The FB1-induced responses require signaling mediated by reactive oxygen species36 and the phytohormones salicylic acid, jasmonic acid and ethylene.37 To date only few genes specifically involved in FB1-induced PCD have been identified in plants. A forward genetic screen identified FB1 resistant mutants.37 One of these mutants, fbr6, was later characterized and found to have a mutation in the gene AtSPL14, a plant-specific SBP-domain putative transcription factor.38 f br6 mutants displayed altered plant architecture and were not resistant to FB1 in the adult stage, suggesting the resistant phenotype observed is due to tissue specific-expression of AtSPL14 in germinating seedlings.38 This mutant showed no difference to wild type in the susceptibility to avirulent Pseudomonas. syringae pv maculicola, but other mutants have shown a resistant phenotype to virulent P. syringae,35 suggesting FB1-mediated signaling eventually diverges into separate defense and cell death branches. The Arabidopsis vacuolar processing enzyme (vpe)-null mutant, lacking all Arabidopsis VPE genes, is resistant to FB1.39 This resistant phenotype is related to VPE activity being a requirement for the collapse of the tonoplast during FB1 treatment. VPE exhibits caspase-1 activity and treatments with VPE-specific or caspase-1 inhibitors prevented FB1-induced lesions, indicating that caspase activity is required during FB1induced cell death.39 Another study identified the long-chain base 1 subunit of a serine palmitoyltransferase to be required for PCD initiated by FB1.36 This gene is required for ROS production mediated by the free sphingoid bases, demonstrating that sphingolipid-mediated signaling precedes ROS production and is a critical component in FB1 cell death. The use of FB1 with Arabidopsis plants and cell suspension cultures is a very attractive system to study plant cell death. Our group has previously used this system and reported the depletion of extracellular ATP as an important cue for initiating Arabidopsis cell death after exposure to FB1.40 We also found that Arabidopsis ATP synthase β-subunit (At5g08690) is an important factor in cell death regulation as knockout mutants lacking the gene encoding for this protein become insensitive to FB1-induced death.41 The current study was aimed at

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EXPERIMENTAL SECTION

Plant Material and Growth Conditions

Cell suspension cultures of Arabidopsis thaliana var. Landsberg erecta42 were grown at 22 °C under a 16 h photoperiod (100 μmol m−2 s−1) and subcultured into fresh medium (1:10 dilution) every 7 days. Cell cultures were used for experiments in midexponential growth phase (3−4 days postsubculturing). Soil grown-plants were incubated in a growth chamber with a 16 h photoperiod (100−120 μmol m−2 s−1) maintained at 22 °C during the light phase and 18 °C during the dark phase. Plants were used for experiments 4−5 weeks after sowing. Cell Culture Treatment

Stock solutions of FB1 (Sigma, Haverhill, UK), prepared in 70% (v/v) methanol, and 10% (w/v) sucrose stock solutions were filter-sterilized before using to treat cell cultures. All treatments were carried out using cell cultures adjusted to a cell density of 5% (w/v) in a 5 mL culture volume. For viability tests, one set of cell cultures was treated with 4 μM FB1 (FB1) while the second set was treated concurrently with both 4 μM FB1 and 2% (w/v) sucrose (FB1+Suc). Mock treatments were performed with an equivalent dilution of 70% methanol. Cell viability was measured over time using the MTT assay as described previously.43 Comparison of Col-0 and UGP1 Knockout Plant Response to FB1

Discs of 8 mm diameter cored from leaves of Col-0 and UGP1 gene knockout plants (SALK_100183) were floated on 5 μM FB1 or a control solution containing an equivalent volume of methanol in the dark for up to 48 h. Four-replicate samples of Col-0 discs were harvested at 48 h and used for RNA isolation and for protein extraction. Samples of UGP1 knockout (KO) leaf discs harvested at 48 h were used for protein extraction. Each replicate consisted of 10 leaf discs obtained from 10 independent plants which were floated on the treatment solution in a Petri-dish. Two dimensional difference gel electrophoresis (2D-DiGE) was used for proteomic analyses while the RNA samples were used to monitor the transcripts of genes we identified in our unpublished DNA array experiments to respond to FB1 in wildtype Arabidopsis. The marker genes analyzed encode a zinc-finger protein (At1g67030), a pectin esterase (At2g47550), and a glycosyl hydrolase protein (At1g61820). Protein Analyses and Identification

Protein samples from leaf discs were extracted in 10% (v/v) tricholoroacetic acid as described previously44 and labeled for 2D-DiGE using a 2 dye system (Cy3 and Cy5) as described before.45 Labeled protein samples were separated in pH4−7 gradient in 24 cm long first dimension gels and 12% SDSPAGE second dimension gels following a previously published protocol.46 Gel images were acquired using a Typhoon 9400 scanner (GE Healthcare, Buckinghamshire, UK) and image analysis was performed using Progenesis Samespots (NonLinear Dynamics, Newcastle, UK). There were 4 treatment groups as follows: Col-0 control, Col-0 FB1-treated, UGP1 KO control, and UGP1 KO FB1-treated. An analysis of variance B



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statistical test was performed using the normalized spot volumes and ratios for each protein spot generated for comparing relative abundance between untreated Col-0 and UGP1 KO samples and between control and FB1-treated samples within each genotype. Only spots with a statistically significant (p ≤ 0.05) difference in abundance in any one of the 3 comparisons were selected for identification by mass spectrometry. Differentially expressed protein spots were excised from Sypro Ruby-stained (Genomic Solutions, Huntington, UK) preparative gels loaded with 200 μg protein and identified by tandem-MS using the 4800 Proteomic Analyzer (Applied Biosystems, Foster City, USA). The protein spot plugs of 2.0 mm diameter were digested with modified trypsin (Promega, Madison, WI) in a ProGest workstation (Genomic Solutions, Huntington, UK) using the standard overnight digestion protocol supplied by the instrument manufacturers. TOF-MS analysis was performed in the automated data acquisition mode, with processing being controlled by the Applied Biosystems 4000 series Explorer software (version 3.5) using the settings; reflector mode, mass range of 700−4000 m/z, 1000 total laser shots per spectrum, and a laser intensity of 3300 V. The acquired TOF-MS spectra were subjected to noise-correction and peak deisotoping, and then internally calibrated using trypsin autolysis peaks 842.500 and 2211.100 m/z. From each spectrum, up to ten of the most abundant precursor ions were selected by the software for fragmentation and MS-MS analyses using a 1 kV CID fragmentation method collecting 4000 laser shots per spectrum with a laser intensity of 3800 over the mass range. The GPS Explorer software − version 3.6 (Applied Biosystems) generated peak lists of ion masses from the calibrated and deisotoped MS and MS-MS spectra for each sample. Lists of both MS and MS-MS data were used for database searches with MASCOT − version 2.2 (Matrix Science, Boston, MA), against NCBInr 9655479 sequences (released first September 2009). Database search parameters were: digestion enzyme trypsin, single missed cleavage, variable modifications of carboxymethyl cysteine and oxidized methionine, precursor mass tolerance of 50 ppm, and fragment ion tolerance of 0.2 Da. Protein identification was considered positive when the combined protein score, incorporating the MS/MS-derived individual ion scores and the PMF-associated score, was more than 95% (p ≤ 0.05). The cutoff threshold for this score in MASCOT was 82. Where more than one database entry was obtained from a single spot, the spots were excluded because it was impossible to know which of the proteins in the mixture were differentially regulated.

(At1g61820) 5′-TGACAAGACTGCCCCAAAATCGC-3′ and 5′-CCGTATGGCATGGAGCACCGC-3′. Cell Death Assays

Solutions of 5 μM FB1 or 5 μM FB1 + 150 mM sucrose were infiltrated into the apoplast of leaves from the abaxial surface using a needleless syringe. Symptom development was monitored over time and photographs taken every day for 4 days after infiltration. Mock-treated leaves were infiltrated with an equivalent dilution of 70% methanol or 150 mM sucrose. For conductivity assays, discs of 8 mm diameter were cored from leaves of 4-week-old plants and floated on 5 μM FB1 solutions in triplicate Petri-dishes. Each replicate consisted of 10 leaf discs, each originating from one of 10-replicate plants. The discs were incubated in the dark for 48 h to allow uptake of FB1 prior to the onset of cell death. After the dark incubation, the discs were placed under a 16 h photoperiod regime (150 μmol.m−2.s−1) and the conductivity of the underlying solution measured at 24 h intervals using a Jenway conductivity meter (Jenway Ltd., Felsted, UK). The accelerated cell death 2 (acd2) mutant and salicylate hydroxylase gene (nahG)-expressing plants were used as positive and negative controls, respectively, for calibration of the assay. The accelerated cell death 2 (acd2) mutant47 is hypersensitive to FB1 and so dies faster that wildtype plants, while the salicylate hydroxylase gene (nahG)expressing plants48 are resistant to FB1.37

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RESULTS

Sucrose Promotes FB1-induced Cell Death

In the course of studying FB1-induced PCD in Arabidopsis cell suspension cultures, we noticed that their response to FB1 was dependent on the amount of sucrose in the growth medium. Cell cultures with higher levels of sucrose were more sensitive to FB1 than those with reduced sucrose (Figure 1). We designed experiments to test if this observation was an artifact of the cell culture system or a true biological response. Leaves of 4-week old Arabidopsis plants were infiltrated with solutions of FB1 and sucrose and symptom development monitored over

RNA Analysis and PCR Reactions

Total RNA was extracted using RNeasy Plant kit (Qiagen, Crawley, UK), with on-column DNase treatment, according to the manufacturer’s instructions. First-strand cDNA synthesis was performed as previously described46 using oligo-(dT)15 (Progema, Southampton, UK), 3 μg of total RNA and SuperScript III reverse transcriptase (Invitrogen, Paisley, UK). For PCR reactions, the following primer pairs were used: actin 2 (At3g18780) 5′-GGATCGGTGGTTCCATTCTTG-3′ and 5′-AGAGTTGTCACACACAAGTG-3′; zinc-finger protein (At1g67030) 5′-CCGCTTCTCTCCGCCTTCGC-3′ and 5′AGGCTTAAATGGAGGTCCAGCCC-3′; pectin esterase (At2g47550) 5′-ATCCCTCTCACGAGCCCGCC-3′ and 5′TGGCCTTTGGTTGGTCCTGCC-3′; glycosyl hydrolase

Figure 1. Effects of sucrose on the viability of Arabidopsis cell cultures. Viability of cell cultures treated with 4 μM FB1 only or 4 μM FB1 + 2% (w/v) sucrose was monitored over 72 h. Viability values are treatment means as a percentage of the control treatments. An asterisk denotes a significant (p < 0.01) difference in viability of FB1- and FB1+ sucrose treated cells. Values and error bars represent means ± SE (n = 3). C



Article

Effects of UGP1 on FB1-induced Cell Death

time. On exposure to FB1, Arabidopsis leaves develop symptoms that start off as chlorosis, followed by severe water loss leading to shrivelling and death of the entire leaf. Figure 2

We sought to investigate if UGP1 has a role in FB1-induced cell death by using the KO mutants. UGP1 KO mutants used in this study have T-DNA inserted into an intron or exon as schematically depicted in Figure 3a. Inhibition of UGP1 expression in the KO lines was confirmed by RT-PCR using primers designed to flank both insertion sites and amplify nearly the entirety of the UGP1 mRNA. A significant reduction in the level of PCR product in SALK_020808 indicated it to be a partial KO while the complete absence of the expected product in SALK_100183 confirmed it to be a complete KO (Figure 3b). The ability to splice out T-DNA sequences inserted into introns can lead to varying levels of reduced expression in the targeted gene.54 The low level of UGP1 expression of SALK_020808 could be related to inefficient splicing of the intron containing the T-DNA insert. We next established the cell death kinetics of leaf tissue of the UGP1 KO lines against wildtype plants. We used a cell death assay based on measuring the conductivity of a solution containing FB1 on which the leaf tissue being assayed was floated. An increase in the conductivity of the solution is caused by leakage of ions from dying cells, making it a direct measurement of the extent of cell death in the leaf tissue. Tissue from acd2 mutants and nahG-transgenic plants, which are hypersensitive and resistant to FB1, respectively,37 had cell death profiles significantly higher or lower than the wildtype, respectively (Figure 4). This demonstrated the ability of this assay to detect plants dying significantly faster or slower than wildtype. Using this assay, we observed that the extent of cell death was significantly diminished in the UGP1 KO lines (Figure 3c), showing that they were resistant to FB1 treatment. These results show that UDP-glucose pyrophosphorylase is an essential factor that enables FB1 to trigger cell death. Since sucrose increases FB1induced cell death and up-regulates UGP1 expression, we predicted that UGP1 mediates the effects of sucrose on the response of Arabidopsis to FB1. To evaluate the possible role for UGP1 in the sucrose effect, and for subsequent experiments, we used wildtype plants and the complete gene KO plants of the SALK_100183 line. We infiltrated wildtype and SALK_100183 leaves with FB1 only and FB1+sucrose and monitored the progression of cell death (Figure 3d). As predicted, sucrose failed to accelerate cell death in the SALK_100183 line showing that the up-regulation of UGP1 mediates the sucrose effects on FB1-induced cell death. The lack of a functional copy of this gene in the UGP1-null line prevents its sucrose-dependent up-regulation with the result that the symptoms of FB1 and the FB1+sucrose infiltrated leaves treatments become indistinguishable.

Figure 2. Effects of sucrose on FB1-induced cell death. Four-week old Arabidopsis leaves were infiltrated with 150 mM sucrose, 5 μM FB1 or both and photographed at 48, 72, and 96 h after infiltration. Scale bar indicates 1 cm. Experiments were repeated at least twice with similar results.

shows the gradual development of FB1-induced symptoms in an Arabidopsis leaf. However, leaves treated with a combination of FB1 and sucrose rapidly developed severe symptoms and were completely dead within 4 days of treatment (Figure 2). These results suggest that either sucrose promotes FB1-induced cell death signaling or suppresses negative regulators of PCD. In silico Search for Genes Mediating the Sucrose Response

We hypothesized that the effects of sucrose on FB1-induced death were mediated by sucrose-induced changes in gene expression. Indeed, exogenous sucrose has been shown to alter gene expression in plants.49 Therefore, to establish the molecular basis for the sucrose effects, we searched publicly available transcriptomic data sets for Arabidopsis sucroseresponsive genes. We selected 100 genes with the highest magnitude of response to sucrose from transcriptomic data sets generated using Arabidopsis plants50 and Arabidopsis cell cultures.51 The Arabidopsis Information Resource (TAIR) was then searched to determine the size of the gene families to which the 100 genes belonged. The 100 genes were then ordered in a hierarchy, with genes from small gene families placed at the top and those from large gene families at the bottom. Starting from the top of the list, genes with at least two transfer-DNA (T-DNA) KO mutants available in the SALK collection52 were selected for further analysis. One of the genes selected using this criterion was the UDP-glucose pyrophosphorylase 1 (UGP1) gene (At3g03250). This gene belongs to a two member gene family and has two reported T-DNA KO lines, SALK_020808 and SALK_100183. UGP1 expression is up-regulated with sucrose in Arabidopsis leaves.53

Proteomic Analysis of FB1-treated Col-0 and UGP1 KO Plants

In order to refine our understanding of the molecular events that underpin FB1-induced cell death, we used proteomic analyses of wildtype and UGP1 KO mutants. Treatment of Arabidopsis tissues with FB1 does not affect cell viability as long as the tissues are in total darkness, but exposure to light triggers death.35 We decided to compare the total soluble proteomes of wildtype and mutant plants treated with FB1 in the dark so as to identify important molecular responses that precede the onset of cell death. Leaf discs from Col-0 and SALK_100183 KO plants were floated on FB1 in the dark for 48 h. Analysis of selected FB1-responsive marker genes, we identified in unpublished DNA chip experiments, in wildtype plants D



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Figure 4. Cell death profiles of the positive control acd2 and negative control nahG plants. Leaf discs were floated on 5 μM FB1 and the conductivity of the solution was measured every 24 h after a 48 h dark incubation. Values and error bars represent means ± SE (n = 3). An asterisk denotes data points that show a significantly lower means than Col-0 (p < 0.01).

indicated that the absence of light did not hinder at least some known FB1-induced responses. Thus, the suppression of a zincfinger protein gene and the induction of a pectin esterase and a glycosyl hydrolase genes triggered by FB1 treatment were not impeded by the absence of light (Figure 5). Therefore, this experimental system provides direct comparison between FB1resistant KO plants and the wildtype in the absence of secondary effects caused by cell death.

Figure 5. Effects of FB1 on gene expression in the dark. Leaf discs of Col-0 plants were floated on FB1 or a control solution for 48 h in the dark. RNA was extracted from triplicate samples of control and FB1treated tissues and the expression of genes for glycosyl hydrolase, pectin esterase, and a zinc-finger protein analyzed by semiquantitative PCR. Actin-2 was used as a constitutive reference control.

Figure 3. UGP1 is required for FB1-induced cell death. (A) Schematic diagram showing T-DNA insertion sites in two independent KO mutants (SALK_020808 and SALK_100183). The insertion sites are indicated by triangles and the gray and white boxes denote exons and untranslated regions, respectively. The kilobase (Kb) scale shows the relative proportions of the different elements of the gene. (B) RT-PCR amplification of UGP1 in RNA samples derived from Col-0 and the two T-DNA lines. SALK_100183 is shown to be a complete KO while SALK_020808 is a partial knockout. Actin-2 (ACT2) was used as a constitutive reference control. (C) Cell death time-course of leaf discs floated on 5 μM FB1 solutions. The conductivity of the solution was measured every 24 h. Values and error bars represent means ± SE (n = 3). An asterisk denotes data points at which mutants are significantly (p < 0.01) lower than Col-0. (D) Sucrose fails to enhance FB1induced cell death in the UGP1 KO mutant. 4-day old Col-0 and SALK_100183 leaves were infiltrated with either 5 μM FB1 or 5 μM FB1 + 150 mM sucrose and photographed at 48, 72, and 96 h after infiltration. Scale bar indicates 1 cm. Experiments were repeated at least twice with similar results.

The experimental design consisted of 4 treatment groups as follows; mock-treated Col-0 leaf discs, FB1-treated Col-0 discs, mock-treated UGP1 KO discs, and FB1-treated UGP1 KO discs. Four biological replicates of each treatment were generated to mitigate the effects of biological variation, with each biological replicate consisting of 10 leaf discs coming from 10 distinct replicate plants. Use of 2D-DiGE, which utilizes a single pooled sample as an internal standard in each gel obviated the need for technical replicates since any gel-specific technical variation can be identified by cross-gel comparison of the internal standard. The normalized protein spot volumes across the replicates were subjected to analysis of variance and Student’s t test to determine the effect of UGP1 KO on protein expression and comparison of FB1 effects on the KO plants and wildtype plants. E


AtCg00490 At3g54050 At3g12780 AtCg00490 At3g12780 At4g02520 AtCg00490 At2g30860 At5g14740 At3g54050 At1g02920 At1g06680 AtCg00490 AtCg00490 AtCg00120 At3g55800 AtCg00490 AtCg00490 At3g12780 At5g38420 AtCg00490 AtCg00490 At5g38420 At1g32060 At1g23310 At5g66570 At5g49910 At3g01500

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

F

RUBISCO large subunit Chloroplastic fructose-1,6-bisphosphate phosphatase Phosphoglycerate kinase RUBISCO large subunit Phosphoglycerate kinase 1 Glutathione S-transferase phi2 RUBISCO large subunit Glutathione S-transferase phi9 Beta carbonic anhydrase 2 Chloroplastic fructose-1,6-bisphosphate phosphatase Glutathione S-transferase 11 Photosystem II subunit P-1 RUBISCO large subunit RUBISCO large subunit ATP synthase CF1 alpha subunit Sedoheptulose-bisphosphatase RUBISCO large subunit RUBISCO large subunit Phosphoglycerate kinase 1 RUBISCO small subunit 2B RUBISCO large subunit RUBISCO large subunit RUBISCO small subunit 2B Phosphoribulokinase Alanine-2-oxoglutarate aminotransferase 1 33 kDa Oxygen evolving polypeptide 1 Chloroplast heat shock protein 70−2 Beta carbonic anhydrase 1

protein name 443 179 98 481 314 285 355 102 101 87 123 113 497 360 159 120 319 440 231 292 169 426 190 143 88 129 98 147

protein scorec 28% 24% 22% 32% 41% 50% 26% 36% 35% 11% 23% 32% 37% 31% 29% 21% 31% 35% 26% 34% 27% 30% 19% 25% 18% 31% 16% 28%

sequence coverage 13 8 7 15 13 11 11 5 8 5 8 6 22 16 15 10 16 19 10 8 11 15 4 11 10 12 11 10

unique peptidesd 2.20 1.00 1.00 1.30 1.00 1.00 −1.20 −1.30 1.00 1.00 1.20 1.00 −1.20 −1.20 1.00 1.00 −1.20 −1.20 1.00 1.00 −1.20 −1.20 1.00 1.00 1.00 1.00 1.00 1.00

ratio

ratio −1.30 −1.50 −1.60 −1.30 −1.50 1.40 −1.30 1.20 1.50 −1.40 1.30 −1.50 −1.20 −1.20 1.30 −1.40 −1.10 −1.20 −1.30 −1.30 −1.20 −1.20 −1.30 1.40 1.30 −1.30 1.30 1.30

p-value 3.11 × 10−04 0.101 0.859 1.85 × 10−04 0.416 0.081 0.008 0.004 0.216 0.108 0.022 0.782 0.014 0.005 0.519 0.682 0.047 0.013 0.356 0.516 1.19 × 10−04 0.032 0.607 0.555 0.088 0.851 0.928 0.164 1.40 × 0.001 1.75 × 1.58 × 3.44 × 6.00 × 5.95 × 2.10 × 4.59 × 5.25 × 1.00 × 3.00 × 3.11 × 2.83 × 3.00 × 2.36 × 3.30 × 4.58 × 3.14 × 2.00 × 1.00 × 2.00 × 1.40 × 5.16 × 4.89 × 7.00 × 2.00 × 4.00 × 10−04 10−04 10−05 10−03 10−05 10−02 10−05 10−04 10−03 10−03 10−04 10−04 10−03 10−05 10−02 10−04 10−05 10−03 10−03 10−03 10−02 10−04 10−04 10−03 10−03 10−03

10−02

p-value

Col-0f + FB1 1.00 −1.80 1.00 −1.20 −1.30 1.50 −1.40 1.00 1.00 −1.30 1.40 1.00 −1.40 −1.30 1.00 −1.40 −1.30 −1.30 −1.20 1.00 −1.30 −1.40 1.00 1.00 1.00 1.00 1.20 1.20

ratio

0.558 0.002 0.084 0.013 0.043 3.96 × 0.013 0.076 0.699 1.52 × 3.90 × 0.107 0.011 6.09 × 0.076 6.23 × 0.04 0.013 0.015 0.335 3.55 × 0.009 0.949 0.235 0.857 0.223 0.008 0.014

10−05

10−04

10−04

10−04 10−04

10−04

p-value

UGP1 KO+FB1g

Note that some proteins existed as several spots on the 2-D gels. bArabidopsis Genome Initiative locus identifier. cProbability-based score incorporating molecular weight search (MOWSE) score and tandem-MS-derived ion scores. A value above 82 is significant (p < 0.05). dNumber of unique peptides matched to the identified protein. eRatios represent fold-change in mutant relative to Col-0 wildtype without any treatment. Statistically insignificant (p > 0.05) ratios (1.00) are shown in italic bold. fRatios of FB1-treated versus control Col-0 tissues, with statistically insignificant (p > 0.05) changes are shown in italic bold. gRatios of FB1-treated versus control UGP1 KO plant tissues, with statistically insignificant (p > 0.05) changes are shown in italic bold.

a

AGI codeb

spota

mutant/Col-0e

Table 1. Proteins Differentially Expressed in Wildtype and UGP1 KO Mutant Plants Before and After Exposure to FB1

Journal of Proteome Research Article



Article

tation or UDP-glucose feeding, indicating that reduced levels of UDP-glucose are responsible for the observed phenotype.59 Our results now reveal that, in addition to its primary metabolic function, UGP1 has a cell death regulatory role in Arabidopsis.

A total of 28 protein spots positively identified showed a significant (p ≤ 0.05) differential expression either between mock-treated Col-0 and UGP1 KO plants, or between controls and FB1-treated plants in either background (Table 1). Additional data relating to the protein identification are presented as Supporting Information (Table S1). The 28 spots represented 16 unique proteins, with a number of the proteins being found in more than 1 locations, indicating the existence of several post-translational modifications. All the 28 protein spots were differentially expressed in Col-0 plants in response to FB1, while only 17 protein spots (8 unique proteins) were similarly responsive in the UGP1 KO. Thus, 11 protein spots (10 unique proteins) responsive to FB1 in Col-0 did not respond in the KO plants, indicating a difference in molecular response between these plants even before the onset of cell death. Due to the presence of multiple spots of the same protein, one protein (rubisco large subunit) was present in both data sets (Table 1). The fact that there were less protein responses in the KO plants than in Col-0 suggests that the resistance to FB1 seen in the KO plants is likely a result of overall suppression of specific molecular events. Moreover, 11 protein spots (3 unique proteins) showed significant differences in abundance between Col-0 and KO plants even without FB1 treatment. Together, the proteins with altered steady-state levels before and after FB1 treatment define a group of proteins with a potential role in mediating FB1-induced cell death downstream of UGP1. The majority of proteins that responded to FB1 treatments in this study are photosynthetic proteins located in the chloroplast (Table 1). Also represented were cellular detoxification enzymes, energy production proteins, glycolysis proteins, and amino acid metabolism. The basal abundance of the large subunit of rubisco, glutathione S-transferases phi9 and glutathione S-transferase 11 were much higher in control KO plants than in corresponding Col-0 plants (Table 1). The proteins whose response to FB1 was blocked in the KO plants include β-carbonic anhydrase 2, ATP synthase CF1 α-subunit, several Calvin cycle enzymes, and photosystem complex proteins.

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Sucrose Regulates FB1-induced Cell Death

We found sucrose levels to be a critical determinant of the outcome of the response of Arabidopsis to FB1 treatment higher levels of sucrose promoted cell death (Figure 1a). Although soluble sugars, such as sucrose, provide cellular energy and the building blocks for cell growth, they also serve a signaling role in controlling growth and development in plants.49 In Arabidopsis, a signaling role for sugars in the regulation of cell death has been proposed based on genetic studies showing a link between altered sugar signaling/ perception and PCD. Hexokinase 1 (HXK1) is a well characterized sugar sensor that, besides its metabolic function in sugar metabolism, mediates sugar signaling during senescence independently of its primary metabolic function.60 Transgenic tomato plants overexpressing the Arabidopsis HXK1gene displayed an early senescence phenotype61 whereas Arabidopsis HXK1 loss-of-function mutants display a delayed senescence phenotype.60 These studies demonstrate that sugars are important signaling molecules and clearly show that sugar signaling positively regulates PCD via HXK1. In another study, the Arabidopsis hypersenescing mutant hys1, is hyperresponsive to exogenously applied sugars and displays an early senescence phenotype as well as up-regulation of senescence-associated genes.62 The early senescence phenotype in hys1 is independent of HXK1-mediated signaling 63 suggesting multiple pathways mediate sugar sensing and PCD. Our results implicate sugar signaling as an important component of FB1-induced cell death. However, it is not known if FB1 specifically requires HXK1- dependent or -independent signaling since there is no evidence that genes involved in FB1 cell death36,38,39 also regulate sugar signaling. Our observation of the effects of sucrose on FB1-induced cell death provides a new explanation for the effects of light on plant PCD reported in the literature. The requirement for light in PCD has been previously connected to the light-dependent accumulation of toxic chlorophyll products in the chloroplast, such as pheophorbide, red chlorophyll catabolite and uroporphyrinogen III.26,27,64 Some PCD elicitors induced a light-dependent production of excess excitation energy in the chloroplast that could play a role in generation of ROS and activation of PCD in plants.65,66 Our observations suggest the light requirement could also be governed by sucrose concentrations present in the tissue during elicitation. In the light, photosynthetic tissues accumulate higher amounts of sucrose than nonphotosynthetic ones and this correlates with the fact that light treated leaves are more susceptible to FB1 than the same leaves placed in the dark.35 The caveat of reduced levels of sucrose in dark treated tissue is that sucrose signaling is reduced and that could play a significant part in dark-induced resistance to FB1 and other forms of PCD. However this possibility requires further investigation.

DISCUSSION

The UDP-Glucose Pyrophosphorylase is a Novel Cell Death Regulator

In this study, we set out to identify plant cell death regulators using Arabidopsis and the pathogen-derived toxin FB1. A combination of reverse genetics and FB1 treatments established UDP-glucose pyrophosphorylase 1 (UGP1) as a previously unknown regulator of FB1-induced cell death. UGP1 is a key cytosolic enzyme in carbohydrate metabolism that plays an important role in the synthesis of UDP-glucose, a precursor of cellulose, callose and sucrose biosynthesis.55,56 In mature leaves, it is primarily involved in generating sucrose for export whereas in nonphotosynthetic tissues it metabolizes UDP-glucose which is generated from sucrose by sucrose synthase.57 The production of UDP-glucose by UGP1 and its highly homologous family member, UGP2 (At4G17310), is essential for normal growth and development of Arabidopsis. Although single KO mutants for either UGP1 or UGP2 have normal development,58,59 a double KO mutant for both genes had a reduction of 52% in UDP-glucose content and displays a severe reduction in growth, delayed flowering and male sterility.59 The double KO phenotype could be rescued by gene complemen-

Possible Mechanism of UGP1 Regulation of FB1-induced Cell Death

We are currently investigating the mechanism by which UGP1 promotes cell death in Arabidopsis, but a possibility is that UGP1 could have an unknown biochemical activity or signaling properties conferring its secondary function in cell death control. Other proteins have previously been demonstrated to G



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have a secondary function in PCD. For example, cytochrome c is an essential component of the mitochondrial electron transport chain transferring electrons between complex III and IV. However, in animal cells cytochrome c is essential in PCD functionit translocates from mitochondria to the cytosol in order to participate in the assembly of the apoptosome with Apaf-1 and caspase-9 which initiates apoptosis.67 Again, hexokinase has a primary metabolic function in phosphorylation of glucose to glucose-6-phosphate in the commitment step of glycolysis. However, the hexokinase protein has a secondary function of PCD inhibition via binding to the plant mitochondrial membranes.68 In order to figure out the basis for the cell death-regulatory function of UGP1, a close comparison with the other family member UGP2 has to be made. Data presented in this study suggests a lack of gene redundancy between UGP1 and UGP2 in the response to FB1. UGP2 is unable to replace UGP1 in the UGP1 KO lines (Figure 3c), even though it bears similar metabolic functions to UGP1. Both genes code for proteins with conserved amino acid residues essential for their catalytic activity69 and have specific UDP-glucose pyrophosphorylase activity with slight differences in substrate affinity and reaction kinetics.70 Both UGP1 and UGP2 are simultaneously expressed in all plant tissues and genetic studies on ugp1 and ugp2 loss-of function mutants found no differences in the levels of soluble sugars.58 Additionally, no differential morphological phenotype relative to wildtype plants was observed at any stage of development,59 demonstrating metabolic redundancy between the two genes. There are some differences between these genes at the protein level that could be the basis for the observed difference in the effect of UGP1 and UGP2 on cell death. Although UGP1 and UGP2 have a 92% identity at the amino acid level, the slight protein sequence difference results in only 5 out of 8 predicted phosphorylation motifs being shared between the two proteins70 and could account for the apparent differential effects on PCD. Nevertheless, we add a cautionary note that the apparent lack of functional redundancy between UGP1 and UGP2 in PCD regulation may arise from stoichiometry rather than molecular structural differences. If the amount of UGP2 protein is a tiny fraction that of UGP1, then this level of UGP2 predictably fails to compensate for UGP1 loss in UGP1 KO plants. Future experiments will resolve this by testing the ability of UGP2 to restore the wildtype phenotype to UGP1 KO mutants transformed with a vector harboring a UGP2 transgene.

previous study from our group in which we used Arabidopsis cell suspension cultures treated with FB1 in the presence of light.41 However, in the present study, we identified far fewer proteins that responded to FB1. This could be because of either the dominance of rubisco proteins in tissues from whole plants that could potentially mask lower abundance proteins on 2D gels, or due to the absence of light during the treatments. As cell suspension cultures are fed with sucrose, they do not accumulate rubisco to the same levels as soil-grown plants. Thus, the current results isolate the light-independent changes and the use of UGP1 KO plants enabled us to identify potentially important changes downstream of this protein in FB1-mediated PCD signaling. Three observations from the proteomic data provide remarkable insight into plant PCD. First, of the 16 unique proteins that were responsive to FB1 in Col-0 plants, 10 were blocked in UGP1 KO mutants (Table 1). Seven of these are located in the chloroplast and are Calvin cycle enzymes or closely associated with the electron transport chain; rubisco small subunit, phosphoglycerate kinase, phosphoribulokinase, 33 kDa oxygen-evolving protein, ATP synthase CF1 α-subunit, photosystem II subunit P1, and at least one protein spot of rubisco large subunit. Overall, these results suggest that the chloroplast is a major target for FB1. However, the lack of response of most of these photosynthetic proteins in the KO mutants indicates that the photosynthetic machinery was protected from the deleterious effects of FB1 treatment. Disruption of the photosynthesis machinery is bound to impact the cells in at least 2 ways: first energy generation is disrupted leading to growth inhibition and, second the disruption of the chloroplast protein complexes could result in the generation of toxic reactive oxygen radicals and this could explain why the toxic effects of FB1 manifest in the presence of light when electron generation peaks. The second important observation is that in control plants, there was a higher steady-state level of certain cellular detoxification enzymes in the UGP1 KO plants than Col-0 plants, and the subsequent increase in the abundance of these enzymes in response to FB1 was overall higher in the KO plants than in wildtype plants (Table 1). This strongly suggests that the resistance to FB1 seen in these plants could partly relate to their increased capacity to conjugate and detoxify FB1 through the action of glutathione transferases, though this requires experimental proof. Activation of glutathione transferase genes appears to be an essential plant adaptation to survive xenobiotic assault.71 Expression of members of the Arabidopsis family of glutathione S-transferases in a mutant yeast strain with a silenced endogenous glutathione transferase activity is sufficient to restore the yeast’s ability to conjugate and detoxify the fungicide anilazine.72 The third observation is the differential response of βcarbonic anhydrase 1 and β-carbonic anhydrase 2 (Table 1). Carbonic anhydrase catalyzes the hydration of CO2 to bicarbonate and Arabidopsis β-carbonic anhydrase 1 has a chloroplastic transit peptide and is located in the chloroplasts while β-carbonic anhydrase 2 is cytosolic.73 However, tobacco carbonic anhydrase directly binds salicylic acid,74 a plant defense hormone that regulates pathogen-induced HR-cell death, and plants in which carbonic anhydrase gene expression is silenced fail to mount PCD when challenged with bacteria that induce HR-cell death.74 Although similar data do not exist for the Arabidopsis β-carbonic anhydrase 2 protein, Arabidopsis β-carbonic anhydrase 1 binds salicylic acid and T-DNA KO

Proteomic Changes Down-Stream of UGP1

For proteomic studies, we used a strategy that takes advantage of the dependency on light of FB1-induced cell death35 by exposing plant tissues to FB1 in darkness to enable mycotoxin uptake and activation of the synthesis and/or assembly of the cell’s PCD machinery without onset of death. This enabled comparison of Col-0 and UGP1 KO plant proteome responses to FB1, at the same time avoiding masking the important PCD signaling events with secondary effects caused by cell death that would ensue in Col-0 plants. Use of FB1-induced marker genes (Figure 5) demonstrated that FB1 is capable of activating changes in gene expression even in the dark. Using this system, we were able to identify proteome responses to FB1 that are downstream of the position where UGP1 functions. The majority of proteins that were responsive to FB1 treatment are located in the chloroplast and have functions associated with photosynthesis. This is in agreement with a H



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plants of this gene have an altered response to pathogens.75 Collectively, the binding of salicylic acid to carbonic anhydrase proteins and the link with HR-cell death in tobacco and Arabidopsis may serve to rationalize our results. Thus, the complete and partial suppression of β-carbonic anhydrase 2 and β-carbonic anhydrase 1, respectively, in FB1-treated UGP1 KO plants relative to Col-0 plants (Table 1) are consistent with a role of carbonic anhydrase proteins in PCD downstream of UGP1. Reverse genetic analyses will be required to prove if this is so. Finally, among the proteins responsive to FB1 were classical metabolic enzymes, such as photosynthetic enzymes, whose primary functions are not expected to be linked to cell death control. However, there is a growing body of evidence demonstrating secondary functions of enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cell death regulation via a very complex signaling mechanism. Although GAPDH is a classical glycolytic enzyme, it is now known to initiate a cell death cascade when cells receive a cell death stimulus. Diverse apoptotic stimuli activate a nitric oxide synthase-dependent accumulation of NO, which inhibits GAPDH activity via S-nitrosylation but enables it to bind to Siah 1, an E3-ubiquitin ligase.76 Because Siah 1 contains a nucleus localization signal, the GAPDH-Siah 1 complex translocates to the nucleus76,77 where it activates cell death via 2 mechanisms; (i) ubiquitination and degradation of nuclear proteins via its stabilization of Siah 176 and (ii) activation of PCD gene expression. In the latter, the GAPDH-Saih 1 complex forms a super complex with the acetyltransferase p300/CREB binding protein complex, which acetylates GAPDH at Lys-160.78 GAPDH acetylation stimulates p300 autoacetylation (Sen et al 2008) and the GAPDH-p300 complex is thought to directly bind gene promoters and influence expression of apoptosis proteins such as p53, Bax, PUMA and p21.78 In light of this evidence in animal cells, an investigation of a possible role in PCD of primary metabolic enzymes, such as rubisco and the other proteins identified in this study, is warranted.

Author Contributions

S.C. and D.F.A.T. contributed equally to this research. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Joanne Robson for help with mass spectrometry and Colleen Turnbull for maintenance of Arabidopsis cell cultures. This work was supported by BBSRC grant BBH0002831 and a Portuguese government FCT scholarship (SFRH/BD/28814/ 2006) awarded to D.F.A.T.

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CONCLUSIONS This work revealed that FB1-induced PCD in Arabidopsis is regulated by UGP1. This protein integrates primary metabolism and PCD, and sucrose modulates the cell death response via influencing UGP1 gene expression. The resistance to FB1 exhibited by UGP1 gene KO plants is likely to be a combination of glutathione transferase-dependent detoxification, protection of chloroplast metabolism, and inhibition of the assembly or synthesis of the cell death machinery through abolition of an up-surge in the abundance of putative pro-cell death proteins such as GAPDH and β-carbonic anhydrase.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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UDP-Glucose Pyrophosphorylase Is a Novel Plant Cell Death Regulator

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