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this member of the gene family could therefore be one mediatorof this PCD process. Using overexpressing plants and KOplants, we showed that this metacaspase is required for deathinduced by UV or H2O2. Concerning plant proteases withcaspase-like activity, few have been identified thus far. Inparticular, a caspase-3 activity is required for most examples ofplant PCD, but its identity remain so far unknown. We haveshown that UV radiation can induce PCD and that caspase-1inhibitor, caspase-3 inhibitor and the pancaspase inhibitor p35were able to suppress DNA fragmentation and cell death. Inaddition, a caspase-3 activity cleaving the caspase substrateAsp-Glu-Val-Asp (DEVDase activity) is induced in the earlystages of cell death. We have purified this DEVDase activityand identified one protease present in the active fraction usingtandem-MS. Progress is being made toward the characterisationof this novel protease.

doi:10.1016/j.cbpa.2007.01.446

C2.11Characterisation of a caspase-3-like/DEVDase activityinduced in Papaver pollen by self-incompatibility

M. Bosch, N. Franklin-Tong, (University of Birmingham)

Self-incompatibility (SI) is an important mechanism to preventinbreeding in higher plants. In Papaver rhoeas, the rejection of“self” pollen involves a Ca2+-dependent signalling network thattriggers PCD, providing a neat way to get rid of unwantedincompatible pollen. We previously identified a caspase-3-like/DEVDase activity that is activated by SI specifically inincompatible pollen and demonstrated that it is involved inmediating PCD. The nature of caspase-like proteases in plants isof considerable interest, as no caspase homologues have beenfound in plant genomes. The presence of a caspase-3-like/DEVDase activity is of particular importance as this activity iscritical for the execution phase of apoptosis in mammaliansystems. To study the temporal and spatial aspects of thecaspase-3-like activity in vivo, we utilized specific cell-permeable substrates that only fluoresce after cleavage. TheSI-activated DEVDase activity is cytosolic, but is also localisedin the nuclei. Although peak activity is detected at 5 h after SIinduction, the activity is already significantly increased as earlyas 3 h after induction. Further analysis of the caspase activity incellular lysates provides evidence that SI also activates aVEIDase activity. Both caspase-like activities show a pro-nounced increase of proteolytic cleavage at acidic pH.Importantly, there is no evidence for a metacaspase activityinvolved in SI-induced PCD. This study provides the firstdetailed characterisation of a caspase-3-like/DEVDase activityin higher plant cells using a combination of live-cell imagingand enzyme activity assays.

doi:10.1016/j.cbpa.2007.01.447

C2.12Light-dependent programmed cell death in Arabidopsis

thaliana

G. Drury, P. Gallois, (University of Manchester)

Several instances of light-dependent programmed cell death havebeen reported in plants. Our work aims to contribute to theunderstanding of a light-dependent cascade leading to PCDinduction, particularly in relation to evidence for differentialtranslation requirement. The mechanism of light-dependent PCDis likely to be linked to the chloroplast. UVC radiation causesPCD in wild-type Arabidopsis seedlings when kept in the lightafter irradiation. We show that UVC damage caused increasedfluorescence emission by photosystem II and is accompanied byprogressive chlorophyll degradation. In addition, protoplast cellsshowed an increased production of H2O2 after UVC treatmentsuggesting that chloroplast-produced reactive oxygen species(ROS) are important during PCD induction. To establish whetherchloroplast H2O2 is a death signal, we analysed the radical celldeath (rcd1-1) mutant, which is hypertolerant to superoxide, andH2O2 produced by photosystem I. rcd1-1 showed hypertoleranceto UVC, implicating that chloroplast produced H2O2 duringUVC-induced PCD. rcd1-1 not only shows an interesting ROS-sensitivity phenotype but it is also impaired in ethylene signalling.We are using rcd1-1 along with other mutants in ethylene, methyljasmonate and the transgenic NahG to further understand thepathway to death. We are using ROS scavengers and fluorescentROS labelling compounds to further investigate ROS induction inresponse to UVC, as well as other PCD eliciting treatments.Surprisingly, we have evidence of differential translation require-ments depending of the PCD inducer used.

doi:10.1016/j.cbpa.2007.01.448

C2.13Can chloroplasts regulate plant programmed cell death?

S. Doyle, P. McCabe, (University College Dublin)

Programmed cell death (PCD) is a gene-controlled, tightly regu-lated and vital process in normal plant development and function.The induction of PCDactivates cascades of cellular signals such asreactive oxygen species that result in organized cell death.We use Arabidopsis thaliana cell suspension cultures as modelsystems in which cell death may easily be induced by heat treat-ment. Our research group has previously shown that mitochondriahave a role in the regulation of plant PCD. We intended to deter-mine whether chloroplasts are also involved in PCD regulation.Our results have shown:

– Heat treatment induces significantly more PCD in dark-grown than in light-grown cultures, suggesting that chlor-oplasts may be involved in PCD regulation;

– Culture growth in light suppresses PCD induction in a lightintensity-dependent manner;

S203Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S199–S204

– Suppression of chloroplast biosynthesis in light-growncultures raises PCD induction levels to those found in dark-grown cultures;

– Antioxidant treatment of light-grown cultures also raisesPCD induction levels to those found in dark-grown cultures,suggesting the involvement of chloroplast-produced reactiveoxygen species in PCD regulation;

– Protein synthesis inhibition can suppress PCD, however, thiseffect is eliminated by culture growth in darkness and byantioxidant treatment, suggesting that chloroplast activityand nuclear protein synthesis may interact during PCDregulation.

Our results indicate that chloroplasts may be involved in theregulation of plant PCD. We are currently investigatingwhether this regulation may be mediated by chloroplast-produced reactive oxygen species.

doi:10.1016/j.cbpa.2007.01.449

C2.14The U-box protein AtPUB17 is a functional ortholog of

NtACRE276 and its E3 ubiquitin ligase activity is requiredfor plant cell death and defence

A. Sadanandom, (University of Glasgow)

Previous analysis of transcriptional changes after elicitation of Cf-9-transgenic tobacco by Avr9 peptide revealed a rapidly upregu-lated gene NtACRE276. Here we show that NtACRE276 istransiently induced in leaves within 15 min by wounding, butupon Avr9 elicitor treatment this upregulation is further enhancedand maintained until the onset of cell death in Cf-9 tobacco.Silencing of NtACRE276 using RNAi in tobacco results in the lossof the hypersensitive response (HR) specified by Cf resistancegenes. NtACRE276 RNAi plants are also compromised for HRmediated by the TMV defence elicitor, p50. Furthermore, silencingLeACRE276 in tomato leads to the breakdown of Cf-9-specifiedresistance against leaf mould caused by Cladosporium fulvum. Weconfirmed that NtACRE276 is an E3 ubiquitin ligase and that anintact U-box domain is required for this function. Bioinformaticanalyses revealed that Arabidopsis PUB17 (AtPUB17) and Bras-sica napus ARC1 are the closest homologs of NtACRE276.Transiently expressing AtPUB17 in Cf-9 tobacco plants silencedfor NtACRE276 leads to restoration of the HR whilst a mutantAtPUB17 lacking E3 ligase activity fails to do, demonstrating thatthe E3 ligase activity of AtPUB17 is crucial for defence signalling.AtPUB17 knockout plants are compromised in their RPM1- andRPS4-mediated resistance against Pseudomonas syringae pv. to-mato containing avirulence genes AvrB and AvrRPS4, respectively.Our data identify the presence of a conserved class of U-box ARMrepeat E3 ligases across the Solanaceous and Brassica genera thatact as positive regulators of cell death and defence.

doi:10.1016/j.cbpa.2007.01.450

C2.15Arabidopsis male sterility1 regulates programmed cell

death in the anther tapetum and pollen wall development

Z. Wilson, G. Vizcay Barrena, C. Yang, (University ofNottingham)

Programmed cell death (PCD) processes are crucial to sexualplant reproduction. During anther and pollen development aseries of perfectly timed programmed cell death events takeplace in the tapetum, stomium and other anther tissues, so theanther can dehisce and the mature pollen can be released. Theimportance of this process in the tapetum is evident from thelarge number of reports of male sterility associated withdisturbances in tapetal development and degeneration. TheArabidopsis male sterility1 mutant presents mature anthers thatare devoid of viable pollen. Although the early stages ofdevelopment progress as in the wild-type, soon after microsporerelease, the microspore cytoplasm and tapetum becomeabnormally granular and vacuolated, and degeneration occurs.Ultrastructural studies show severe abnormalities within themutant pollen wall that affect exine and intine formation. Theyalso show abnormal vacuolation in the tapetum. TUNELstaining and ultrastructural observations indicate that PCDoccurs in the wild-type tapetum after microspore mitosis I.However, in the ms1 mutant, no TUNEL signal is detected andthe large autophagic vacuoles and mitochondrial swelling isobserved. This suggest that necrotic-based breakdown of thetapetum is occurring in the ms1 mutant rather than the normal,regulated PCD process. TUNEL staining is, however, detectedin the mutant microspores, indicating that they may go througha PCD-based breakdown as a secondary consequence of theobserved tapetal aberrations. This suggests that MS1 may act asa trigger for the start of PCD in the anther tapetum.

doi:10.1016/j.cbpa.2007.01.451

C2.16A novel peptide that regulates cell death in the Arabidopsissuspensor

B. Young, P. Gallois, (Manchester University); V. Delorme,(UMR-CNRS-IRD-Universite); R. Blanvillain1, (University ofCalifornia Berkeley)

A novel peptide encoding gene has been detected in Arabi-dopsis thaliana using a promoter trap line that displayedspecific reporter expression in the suspensor during earlyembryogenesis. Expression analysis revealed a transcript thatencodes a predicted peptide of 25 amino acid residues.Homozygous KO plants for the gene showed a delay in theinduction of cell death in suspensor cells when compared towild-type plants. The gene was aptly named KISS OF DEATH(KOD) and appropriately lived up to its name by inducing celldeath when overexpressed in plants. Analysis of several