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Endoplasmic reticulum (ER) is an important organelle responsible as protein synthesis regulator in plant. High salinity can also lead to the activation of ER stress, caused by the accumulation of misfolded protein. This could lead to a stress response mechanism, unfolded protein response (UPR). Failure of UPR to reverse the effect of protein misfolding will activate Programmed Cell Death (PCD). Metacaspase genes regulate programmed cell death (PCD) in plants. The present study was focused on comprehensive gene analyses of the expression patterns of type II rice metacaspase (OsMC) genes in response to the endoplasmic reticulum (ER) and salinity stress in rice leaf and OsMC4 in callus. A strong evidence of unfolded protein response (UPR) during tolerance to both ER and salinity stress was found in the present study. Overexpression of OsMC4 in rice callus as a fusion protein with TagRFP and controlled by the CaMV35 promoter caused major changes in the expression of the stress ER-marker genes, protein disulfide isomerase (PDI) and Binding immunoglobulin Protein (BiP), and OsMC4 in overexpressing calli. These expression analyses of the OsMC family provide valuable information for further functional studies on the biological roles of OsMCs in PCD related to ER and salinity stress responses.

Metacaspases and paracaspases are proteases that were first identified as containing a caspase-like structural fold (Uren et al., 2000). Like caspases, metacaspases and paracaspases are multifunctional proteins regulating diverse biological phenomena, such as aging, immunity, proteostasis, and programmed cell death. The broad phylogenetic distribution of metacaspases and paracaspases across all kingdoms of life and large variation of their biochemical and structural features complicate classification and annotation of the rapidly growing number of identified homologs. Establishment of an adequate classification and unified nomenclature of metacaspases and paracaspases is especially important to avoid frequent confusion of these proteases with caspases—a tenacious misnomer that unfortunately does not appear to decline with time. This Letter represents a consensus opinion of researchers studying different aspects of caspases, metacaspases, and paracaspases in various organisms, ranging from microbes to plants and animals., This work was supported by the Knut and Alice Wallenberg Foundation.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.

The sustained increase in atmospheric [CO2] over the past two centuries has brought a series of global challenges in plant-environment interactions. However, genetic mechanisms in botanical adaption and feedback to environmental alteration remain elusive. Here we collected and analysed transcriptome sequencing data from leaf tissues of maize Zea mays conditioned under high [CO2] stress for 0, 7 or 14 d. A total of 1390 genes, either up- or down-regulated, differed significantly in these conditions. Gene Ontology (GO) enrichment terms and KEGG metabolism pathways included protein phosphorylation, protein ubiquitination, oxidation-reduction, plant organ development, cellular response to endogenous response and MAPK signaling. We also identified three densely connected gene clusters: TCP/PYE, WRKYs and MYC/JAZ from significant DEGs. In conclusion, we provided a genetic database for biologists in botany, agronomy and ecology to further investigate the molecular machinery by which C4-plants respond to elevated atmospheric [CO2] and accompanying global climate change.

Programmed cell death (PCD) is used by plants for development and survival to biotic and abiotic stresses. The role of caspases in PCD is well established in animal cells. Over the past 15 years, the importance of caspase-3-like enzymatic activity for plant PCD completion has been widely documented despite the absence of caspase orthologues. In particular, caspase-3 inhibitors blocked nearly all plant PCD tested. Here, we affinity-purified a plant caspase-3-like activity using a biotin-labelled caspase-3 inhibitor and identified Arabidopsis thaliana cathepsin B3 (AtCathB3) by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Consistent with this, recombinant AtCathB3 was found to have caspase-3-like activity and to be inhibited by caspase-3 inhibitors. AtCathepsin B triple-mutant lines showed reduced caspase-3-like enzymatic activity and reduced labelling with activity-based caspase-3 probes. Importantly, AtCathepsin B triple mutants showed a strong reduction in the PCD induced by ultraviolet (UV), oxidative stress (H2O2, methyl viologen) or endoplasmic reticulum stress. Our observations contribute to explain why caspase-3 inhibitors inhibit plant PCD and provide new tools to further plant PCD research. The fact that cathepsin B does regulate PCD in both animal and plant cells suggests that this protease may be part of an ancestral PCD pathway pre-existing the plant/animal divergence that needs further characterisation.

The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

International audience; Autophagy is essential for protein degradation, nutrient recycling, and nitrogen remobilization. Autophagy is induced during leaf ageing and in response to nitrogen starvation, and is known to play a fundamental role in nutrient recycling for remobilization and seed filling. Accordingly, ageing leaves of Arabidopsis autophagy mutants (atg) have been shown to over-accumulate proteins and peptides, possibly because of a reduced protein degradation capacity. Surprisingly, atg leaves also displayed higher protease activities. The work reported here aimed at identifying the nature of the proteases and protease activities that accumulated differentially (higher or lower) in the atg mutants. Protease identification was performed using shotgun LC-MS/MS proteome analyses and activity-based protein profiling (ABPP). The results showed that the chloroplast FTSH (FILAMENTATION TEMPERATURE SENSITIVE H) and DEG (DEGRADATION OF PERIPLASMIC PROTEINS) proteases and several extracellular serine proteases [subtilases (SBTs) and serine carboxypeptidase-like (SCPL) proteases] were less abundant in atg5 mutants. By contrast, proteasome-related proteins and cytosolic or vacuole cysteine proteases were more abundant in atg5 mutants. Rubisco degradation assays and ABPP showed that the activities of proteasome and papain-like cysteine protease were increased in atg5 mutants. Whether these proteases play a back-up role in nutrient recycling and remobilization in atg mutants or act to promote cell death is discussed in relation to their accumulation patterns in the atg5 mutant compared with the salicylic acid-depleted atg5/sid2 double-mutant, and in low nitrate compared with high nitrate conditions. Several of the proteins identified are indeed known as senescence- and stress-related proteases or as spontaneous cell-death triggering factors.

Phytaspases are plant cell death-related proteases of the subtilisin-like protease family that possess an unusual aspartate cleavage specificity. Although phytaspase activity is widespread in plants, phytaspase of Arabidopsis thaliana (L.) Heynh. has escaped detection and identification thus far. Here, we show that a single gene (At4 g10540) out of 56 A. thaliana subtilisin-like protease genes encodes a phytaspase. The recombinant phytaspase was overproduced in Nicotiana benthamiana Domin leaves, isolated, and its substrate specificity and properties were characterised. At pH 5.5, at physiological mildly acidic reaction conditions, the Arabidopsis phytaspase was shown to be strictly Asp-specific. The strongly preferred cleavage motifs of the enzyme out of a panel of synthetic peptide substrates were YVAD and IETD, while the VEID-based substrate preferred by the tobacco and rice phytaspases was almost completely resistant to hydrolysis. At neutral pH, however, the Arabidopsis phytaspase could hydrolyse peptide substrates after two additional amino acid residues, His and Phe, in addition to Asp. This observation may indicate that the repertoire of Arabidopsis phytaspase targets could possibly be regulated by the conditions of the cellular environment. Similar to tobacco and rice phytaspases, the Arabidopsis enzyme was shown to accumulate in the apoplast of epidermal leaf cells. However, in stomatal cells Arabidopsis phytaspase was observed inside the cells, possibly co-localising with vacuole. Our study thus demonstrates that the Arabidopsis phytaspase possesses both important similarities with and distinctions from the already known phytaspases, and is likely to be the most divergent member of the phytaspase family.

Programmed cell death (PCD) is a critical component of plant development, defense against invading pathogens, and response to environmental stresses. In this chapter, we provide detailed technical methods for studying PCD associated with plant development or induced by abiotic stress. A root hair assay or electrolyte leakage assay are excellent techniques for the quantitative determination of PCD and/or cellular injury induced in response to abiotic stress, whereas the lace plant provides a unique model that facilitates the study of genetically regulated PCD during leaf development.

Programmed cell death (PCD) is a critical component of plant development, defense against invading pathogens, and response to environmental stresses. In this chapter, we provide detailed technical methods for studying PCD associated with plant development or induced by abiotic stress. A root hair assay or electrolyte leakage assay are excellent techniques for the quantitative determination of PCD and/or cellular injury induced in response to abiotic stress, whereas the lace plant provides a unique model that facilitates the study of genetically regulated PCD during leaf development.

Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term 'apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during tissue and organ formation and elimination, whereas necrosis is typically found under abiotic stress. Some examples of plant PCD cannot be ascribed to either major class and are therefore classified as separate modalities. These are PCD associated with the hypersensitive response to biotrophic pathogens, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined.

Programmed cell death (PCD) has a key role in defence and development of all multicellular organisms. In plants, there is a large gap in our knowledge of the molecular machinery involved at the various stages of PCD, especially the early steps. Here, we identify kiss of death (KOD) encoding a 25-amino-acid peptide that activates a PCD pathway in Arabidopsis thaliana. Two mutant alleles of KOD exhibited a reduced PCD of the suspensor, a single file of cells that support embryo development, and a reduced PCD of root hairs after a 55°C heat shock. KOD expression was found to be inducible by biotic and abiotic stresses. Furthermore, KOD expression was sufficient to cause death in leaves or seedlings and to activate caspase-like activities. In addition, KOD-induced PCD required light in leaves and was repressed by the PCD-suppressor genes AtBax inhibitor 1 and p35. KOD expression resulted in depolarization of the mitochondrial membrane, placing KOD above mitochondria dysfunction, an early step in plant PCD. A KOD∷GFP fusion, however, localized in the cytosol of cells and not mitochondria.

Community genetics examines how genotypic variation within a species influences the associated ecological community. The inclusion of additional environmental and genotypic factors is a natural extension of the current community genetics framework. However, the extent to which the presence of and genetic variation in associated species influences interspecific interactions (i.e., genotype x genotype x environment [G x G x E] interactions) has been largely ignored. We used a community genetics approach to study the interaction of barley and aphids in the absence and presence of rhizosphere bacteria. We designed a matrix of aphid genotype and barley genotype combinations and found a significant G x G x E interaction, indicating that the barley-aphid interaction is dependent on the genotypes of the interacting species as well as the biotic environment. We discuss the consequences of the strong G x G x E interaction found in our study in relation to its impact on the study of species interactions in a community context.

Tracheary elements (TEs) are cells in the xylem that are highly specialized for transporting water and solutes up the plant. TEs undergo a very well-defined process of differentiation that involves specification, enlargement, patterned cell wall deposition, programmed cell death and cell wall removal. This process is coordinated such that adjacent TEs are joined together to form a continuous network. Expression studies on model systems as diverse as trees and cell cultures have contributed to providing a flood of candidate genes with potential roles in TE differentiation. Analysis of some of these genes has yielded important information on processes such as patterned secondary cell wall deposition. The current challenge is to continue this functional analysis and to use these data and build an integrated model of TE development.

The occurrence of senescence (Evans blue staining) and programmed cell death (Hoechst staining/TUNEL reaction) was studied in the colonial cyanobacteria Anabaena flos-aquae and Microcystis flos-aquae during the late summer bloom in a eutrophic lake. Algae were analysed over a seven-day period (three sampling days) within mixed phytoplankton samples obtained from the upper epilimnion (trawl-net sample) and lower epilimnion (sediment-trap sample). Death of Anabaena in the trawl-net population was attributed to widespread infection by a chytrid fungus, resulting in breakdown and positive Evans blue staining of most filamentous colonies. Associated akinetes were a mixture of senescent cells (30% of total, Evans blue stained) and viable cells (unstained) plus a few dead cells (no contents). Sedimentation resulted in loss of filamentous colonies, leaving groups of akinetes with about 30% nonviable cells (senescent and dead). Over the sampling period, 20–50% of Microcystis colonies in the trawl-net samp...

Caspases are key proteases for the initiation and completion of animal apoptosis, and caspase enzymatic activities can be detected in plant cell extracts. In addition, various caspase inhibitors block plant programmed cell death (PCD), suggesting that the proteases involved are required for completion of the process. Yet plants do not have caspase orthologous genes in their genomes, and the proteases phylogenetically closest to caspases, the metacaspases, do not have caspase activities. A major challenge has therefore been to identify the plant proteases that are responsible for caspase-like activities in order to understand how, or indeed if, they are related to animal caspases. We now know three proteases with caspase-like activity: vacuolar processing enzyme (VPE), a legumain that cleaves caspase-1 synthetic substrates; phytaspase, a subtilisin-like protease that cleaves caspase-6 synthetic substrates; and the proteasome subunit PBA1 that has caspase-3-like activity. Although these proteases participate in plant PCD, they are unrelated to animal caspases and do not directly form a protease cascade. Additional plant proteases with caspase-like enzymatic activity remain to be uncovered.

Several studies have shown that protease inhibitors can suppress programmed cell death in various plant species and plant tissues. This is especially true of caspase inhibitors that can block programmed cell death and its marker DNA laddering. There are up to six different caspase-like activities that can be measured in plant extracts, the most prominent being caspase1-like and caspase3-like. These activities can be located in vacuoles and also in the nucleus or the cytoplasm. This represents a striking apparent similarity with animal programmed cell death. Because there are no caspase orthologue in plant genomes, a major challenge is to identify these proteases. Recently two proteases with caspase-like activities have been recognized as belonging to two different protease families that are not closely related to animal caspases. Various other protease families have been implicated and this suggests that complex protease networks have been recruited for the plant cell demise.

The pOp/LhG4 transcription factor system was used to determine whether the synthetic pOp promoter, integrated at one position in the Arabidopsis genome, could be efficiently and faithfully activated by the heterologous transcription factor, LhG4, expressed in a variety of different patterns. This is a precondition for the development and exploitation of large collections of LhG4 activation lines that direct predictable tissue-specific expression of transgenes. We selected a pOp-GUS reporter insertion that was efficiently activated after crossing to an activator line that expressed the synthetic transcription factor LhG4 from the Cauliflower Mosaic Virus 35S promoter. This reporter line, pOp-GUS(g2), was then combined with activator loci that expressed LhG4 from one of seven different promoters, each with a different tissue specificity. pOp-GUS(g2) was activated faithfully in combination with six of these seven activator constructs, but generated an unexpected expression pattern in combination with the seventh construct, a fusion to a cyclin promoter (CYC-LhG4). The aberrant expression pattern could be attributed to the pOp-GUS(g2) insertion site, as the CYC-LhG4 activator lines directed the expected pattern of expression from a second pOp-GUS insertion. These results show that it is feasible to construct an activator collection in which LhG4 is expressed from diverse promoters or enhancer traps, but that individual pOp reporter loci can vary in their competence to respond to certain activator patterns. We discuss the implications for the design and use of mis-expression technology in Arabidopsis.

Plant cells, like cells from other kingdoms, have the ability to self-destruct in a genetically controlled manner. This process is defined as Programmed Cell Death (PCD). PCD can be triggered by various stimuli in plants including by endoplasmic reticulum (ER) stress. Research in the past two decades discovered that disruption of protein homeostasis in the endoplasmic reticulum (ER) could cause ER stress, which when prolonged/unresolved leads cells into PCD. ER stress-induced PCD is part of several plant processes, for instance, drought and heat stress have been found to elicit ER stress-induced PCD. Despite the importance of ER stress-induced PCD in plants, its regulation remains largely unknown, when compared with its counterpart in animal cells. In mammalian cells, several pro-apoptotic proteases called caspases were found to play a crucial role in ER stress-induced PCD. Over the past decade, several key proteases with caspase-like enzymatic activity have been discovered in plants and implicated in PCD regulation. This review covers what is known about caspase-like enzymatic activities during plant ER stress-induced PCD and discusses possible regulation pathways leading to the activation of relevant proteases in plants.

Genetically controlled expression of a toxin provides a tool to remove a specific structure and consequently study its role during a developmental process. The availability of many tissue-specific promoters is a good argument for the development of such a strategy in plants. We have developed a conditional system for targeted toxin expression and demonstrated its use for generating embryo phenotypes that can bring valuable information about signalling during embryogenesis. The BARNASE gene was expressed in the Arabidopsis embryo under the control of two promoters, one from the cyclin AtCYCB1 gene and one from the AtLTP1 gene (Lipid Transfer Protein 1). One-hundred percent seed abortion was obtained with the cyclin promoter. Surprisingly however, the embryos displayed a range of lethal phenotypes instead of a single arrested stage as expected from this promoter. We also show that BARNASE expression under the control of the AtLTP1 promoter affects the basal pole of the globular embryo. Together with reporter expression studies, this result suggests a role of the epidermis in controlling the development of the lower tier of the embryo. This defect was not embryo-lethal and we show that the seedlings displayed a severe shoot phenotype correlated to epidermal defects. Therefore, the epidermis does not play an active role during organogenesis in seedlings but is important for the postgermination development of a viable plant.

Embryogenesis is a long-standing field of interest for plant scientist as recorded in the ‘notes’ of the French Science Academy. This either with fundamental or applied points of view. Since the beginning of the century techniques and questions have co-evolved, from microscope and fate map to laser ablation and cell–cell signalling. So far in plant embryogenesis, a limited use has been made of the whole range of approaches generally available to study development. This is due to technical limitations when working with plant embryos. Novel mutant screens and techniques are now at hand and are expected to unravel further the nature of cell interactions underlying embryo development. This in turn will modify the focus of our questioning.

In the last few years programmed cell death in plants inspired many studies in development and environmental stresses. Some of these studies showed that hallmarks of animal programmed cell death were found at cellular or molecular level in plant cells in different experimental systems. Additionally the effect of over-expression in plants of animal genes implicated in programmed cell death has been tested, and some plant homologues of these genes have been found. This suggests that, despite some differences, plants and animals could share at least some common components of a core mechanism used to carry out programmed cell death in eukaryotes. In this review, we will concentrate on the last findings that suggest similarity between plant programmed cell death and its better known counterpart in animals.

Almost all the nuclear genes of four Gramineae (maize, wheat, barley, rice) and pea are located in DNA fractions covering only a 1–2% GC range and representing between 10 and 25% of the different genomes. These DNA fractions comprise large gene-rich regions (collectively called the ‘gene space’) separated by vast gene-empty, repeated sequences. In contrast, in Arabidopsis thaliana , genes are distributed in DNA fractions covering an 8% GC range and representing 85% of the genome. Here, we investigated the integration of a transferred DNA (T-DNA) in the genomes of Arabidopsis and rice and found different patterns of integration, which are correlated with the different gene distributions. While T-DNA integrates essentially everywhere in the Arabidopsis genome, integration was detected only in the gene space, namely in the gene-rich, transcriptionally active, regions of the rice genome. The implications of these results for the integration of foreign DNA are discussed.

Introduction Arabidopsis AtEm1 and AtEm6 proteins were overex- In most higher plants the later phase of seed maturationpressed in E. coli and used to raise antibodies which is characterized by a desiccation phase during which awere used to analyse Em expression at the protein number of proteins distinct from the storage proteins arelevel. One of the sera is specific for AtEm1 protein accumulated in embryos. According to their accumulationwhereas the second reacts with both AtEm1 and pattern it has been suggested that these particular pro-AtEm6 proteins. These antibodies were used to ana- teins, called LEA for Late Embryogenesis Abundant,lyse expression of Em genes at the protein level and could be involved in seed desiccation tolerance (Dureto complete previous studies at the mRNA level. During et al., 1989; Hughes and Galau, 1989; Dure 1993; Leprinceseed maturation, AtEm1 protein accumulates earlier et al., 1993; Xu et al., 1996; Kermode, 1997). Over thethan AtEm6, in parallel with the corresponding mRNA last 15 years many studies have revealed the widespreadbut with a 3 d delay. During germination, AtEm1 pro- distribution of LEA among several plant species and attein undergoes two successive cleavages before being least five major groups of these proteins have beendegraded. Both proteins are much more stable than described on the basis of their amino acid sequencethe corresponding mRNA. Soaking of dormant seeds homologies (Dure et al., 1989; Delseny et al., 1993). Inindicates that imbibition is sufficient to induce Em addition to their expression during seed desiccation, manyprotein degradation and that germination per se is not of the genes coding for LEA can be precociously inducedrequired. AtEm1and AtEm6 mRNA canbeprecociously in immature seeds or activated in vegetative tissues follow-induced by ABA in immature siliques, but protein accu- ing ABA application, osmotic or water stresses (Skrivermulation could not be observed. A similar observation and Mundy, 1990; Jakobsen et al., 1994), indicating thatwas made with leaves of transgenic plants ectopically they are regulated essentially at the transcriptional level.expressing ABI3. These results establish clearly that During the last few years this laboratory has focusedEm protein accumulation is also tightly controlled by on the analysis of expression of LEA group 1 genes, thepost-transcriptional mechanisms in addition to tran- so-called Em. The first Em (Early methionine labelled)scriptional ones. protein was observed, as the most strongly labelled poly-peptide in wheat embryos incubated in the presence ofKey words: Late Embryogenesis Abundant protein, post- [35S] Met, by Cuming and Lane (1979). Since this initialtranscriptional regulation. discovery, several Em coding genes have been identifiedand characterized from wheat (Cuming, 1984; Williamsonet al., 1985; Futers et al., 1993) and from many otherplant species such as cotton (Baker et al., 1988; Galau

With a view to studying programmed cell death in plants at the molecular level, we report here for the first time that apoptotic-like changes are induced by UV radiation in plant nuclei. In Arabidopsis thaliana seedlings a UV-C dose of 10–50 kJ/m2 induces an oligonucleosomal DNA fragmentation which is reminiscent of the apoptotic DNA ladder described in animal cells. This DNA fragmentation was also detected in situ in protoplast nuclei as soon as 2 h after UV-C treatment. Moreover, UV-C induced a nuclear morphology characteristic of animal apoptotic nuclei. We propose that UV-C induction constitutes a powerful tool to compare the cellular response to irreversible UV damage in plants to that in animals and to study programmed cell death in A. thaliana.

Programmed cell death or apoptosis is a process in which unwanted cells are eliminated during growth and development. In mammals, several genes have been identified whose products are necessary to prevent entry into the apoptotic process. We have isolated a clone from an Arabidopsis thaliana cDNA library whose predicted translation product shows highly significant similarity to the mammalian defender against apoptotic death 1 (DAD1) protein. Transformation of the mutant hamster tsBN7 cells, which undergo apoptosis at restrictive temperature, demonstrates that the plant protein is as efficient as human DAD1 in rescuing these hamster cells from apoptosis. In contrast to mammals, Southern hybridisation and genomic data indicate that there are probably two genes in Arabidopsis thaliana. Northern blot analysis shows that AtDAD transcripts are present in all tissues examined, although the abundance of the transcripts is reduced in siliques during the maturation and desiccation phase of the seed. This is the first experimental proof that a homologue of an animal gene involved in apoptosis exists in plants and the first demonstration of complementation of a vertebrate mutant by a plant cDNA. Our results suggest that this process of suppression of apoptosis has been conserved in animals and plants.

An Arabidopsis thaliana cDNA encoding a new RNA-binding protein (RBP37) was cloned from a silique cDNA library. The predicted amino acid sequence corresponds to a RBP containing two RNA recognition motifs (RRM) and a basic domain. An affinity for nucleic acids was confirmed in binding assays using in vitro synthesised AtRBP37 protein. In situ hybridisation experiments on sections of flowers and siliques showed expression only in growing organs: gynoecium, petals, filaments and during early-embryogenesis expression is located in the embryo proper and the suspensor up to late heart stage. Expression is not detected in the embryo during maturation. This results suggests an expression pattern correlated with dividing cells.

A protocol is described for simultaneous histochemical detection of LacZ and Gus activity in plant tissues after microprojectile bombardment. The suitability of two different Gus substrates (Salmon-glcA, Magenta-β-d-glcA) is compared. This detection system is used to assay the number of cells expressing either or both reporter gene. This technique is used as a qualitative assay to demonstrate the tissue specificity of a Hrgp promoter in maize embryos, and to measure ABA responsiveness of a Lea promoter in Arabidopsis. The promoter to be studied is linked to the gus reporter gene and the lacZ reporter gene linked to the CaMV 35S promoter is used as a constitutive internal control. The use of an internal control drastically reduces the data variation inherent to microprojectile bombardment.

In Arabidopsis thaliana we demonstrate that dying root hairs provide an easy and rapid in vivo model for the morphological identification of apoptotic-like programmed cell death (AL-PCD) in plants. The model described here is transferable between species, can be used to investigate rates of AL-PCD in response to various treatments and to identify modulation of AL-PCD rates in mutant/transgenic plant lines facilitating rapid screening of mutant populations in order to identify genes involved in AL-PCD regulation.

Programmed cell death (PCD) has a key role in defence and development of all multicellular organisms. In plants, there is a large gap in our knowledge of the molecular machinery involved at the various stages of PCD, especially the early steps. Here, we identify kiss of death (KOD) encoding a 25-amino-acid peptide that activates a PCD pathway in Arabidopsis thaliana. Two mutant alleles of KOD exhibited a reduced PCD of the suspensor, a single file of cells that support embryo development, and a reduced PCD of root hairs after a 55°C heat shock. KOD expression was found to be inducible by biotic and abiotic stresses. Furthermore, KOD expression was sufficient to cause death in leaves or seedlings and to activate caspase-like activities. In addition, KOD-induced PCD required light in leaves and was repressed by the PCD-suppressor genes AtBax inhibitor 1 and p35. KOD expression resulted in depolarization of the mitochondrial membrane, placing KOD above mitochondria dysfunction, an early step in plant PCD. A KOD∷GFP fusion, however, localized in the cytosol of cells and not mitochondria.

We report on the characterization of the interaction between reactive oxygen species signalling and abscisic acid (ABA)-mediated gene network in ozone (O(3)) stress response. To identify the stress-related signalling pathways and possible cross-talk controlled by an ABA-negative regulator, the protein phosphatase 2C abscisic acid insensitive1 (ABI1), we performed a genome-wide transcription profiling of O(3)-treated wild-type and ABI1 knockout (abi1td) plants. In addition, to better understand ABA signalling and the interactions between stress response pathways, we performed a microarray analysis of drought-treated plants. Functional categorization of the identified genes showed that ABI1 is involved in the modulation of several cellular processes including metabolism, transport, development, information pathways and variant splicing. Comparisons with available transcriptome data sets revealed the extent of ABI1 involvement in both ABA-dependent and ABA-independent gene expression. Furthermore, in O(3) stress the ABA hypersensitivity of abi1td resulted in a significant reduction of the ABA level, ethylene (ET) over-production and O(3) tolerance. Moreover, the physical interaction of ABI1 with ACC synthase2 and ACC synthase6 was shown. We provide a model explaining how ABI1 can regulate both ABA and ET biosynthesis. Altogether, our findings indicate that ABI1 plays the role of a general signal transducer linking ABA and ET biosynthesis as well as signalling pathways to O(3) stress tolerance.

Promoter trapping is a particular gene trap strategy that represents a valuable tool for the discovery of specific cell-type markers. The principle is to generate a collection of transgenic lines with random insertions of a promoter-less reporter gene and to screen for specific reporter activity in the domain of interest. The use of beta-glucuronidase (GUS) as a reporter gene provides a simple and sensitive assay that allows identification of very restricted expression patterns and makes the promoter trap appropriate to study embryogenesis. Plant embryogenesis starts at the fertilization of the egg cell encapsulated in the maternal tissue and leads to the establishment of a new organism capable of an autonomous life. Uncovering genes specifically expressed in sub-domain of the embryo during its development represents a major technical challenge due, in part, to size and accessibility limitations. Promoter trapping approaches have been successfully used to overcome these problems. The trapped activity represents thereafter a useful genetic marker of the uncovered cell type, which is expected to reveal the properties of a specific promoter shedding light on a new gene function. In this chapter, protocols for examining and documenting GUS reporter gene activities in the embryo are described. Methods for the amplification of sequences flanking insertions and subsequent molecular and genetic characterization are provided.

International audience; The extent of conservation in the programmed cell death pathways that are activated in species belonging to different kingdoms is not clear. Caspases are key components of animal apoptosis; caspase activities are detected in both animal and plant cells. Yet, while animals have caspase genes, plants do not have orthologous sequences in their genomes. It is 10 years since the first caspase activity was reported in plants, and there are now at least eight caspase activities that have been measured in plant extracts using caspase substrates. Various caspase inhibitors can block many forms of plant programmed cell death, suggesting that caspaselike activities are required for completion of the process. Since plant metacaspases do not have caspase activities, a major challenge is to identify the plant proteases that are responsible for the caspase-like activities and to understand how they relate, if at all, to animal caspases. The protease vacuolar processing enzyme, a legumain, is responsible for the cleavage of caspase-1 synthetic substrate in plant extracts. Saspase, a serine protease, cleaves caspase-8 and some caspase-6 synthetic substrates. Possible scenarios that could explain why plants have caspase activities without caspases are discussed.

Promoter trapping is a particular gene trap strategy that represents a valuable tool for the discovery of specific cell-type markers. The principle is to generate a collection of transgenic lines with random insertions of a promoter-less reporter gene and to screen for specific reporter activity in the domain of interest. The use of beta-glucuronidase (GUS) as a reporter gene provides a simple and sensitive assay that allows identification of very restricted expression patterns and makes the promoter trap appropriate to study embryogenesis. Plant embryogenesis starts at the fertilization of the egg cell encapsulated in the maternal tissue and leads to the establishment of a new organism capable of an autonomous life. Uncovering genes specifically expressed in sub-domain of the embryo during its development represents a major technical challenge due, in part, to size and accessibility limitations. Promoter trapping approaches have been successfully used to overcome these problems. The trapped activity represents thereafter a useful genetic marker of the uncovered cell type, which is expected to reveal the properties of a specific promoter shedding light on a new gene function. In this chapter, protocols for examining and documenting GUS reporter gene activities in the embryo are described. Methods for the amplification of sequences flanking insertions and subsequent molecular and genetic characterization are provided.

Programmed cell death (PCD) is a genetically controlled cell death that is regulated during development and activated in response to environmental stresses or pathogen infection. The degree of conservation of PCD across kingdoms and phylum is not yet clear; however, whereas caspases are proteases that act as key components of animal apoptosis, plants have no orthologous caspase sequences in their genomes. The discovery of plant and fungi metacaspases as proteases most closely related to animal caspases led to the hypothesis that metacaspases are the functional homologues of animal caspases in these organisms. Arabidopsis thaliana has nine metacaspase genes, and so far it is unknown which members of the family if any are involved in the regulation of PCD. We show here that metacaspase-8 (AtMC8) is a member of the gene family strongly up-regulated by oxidative stresses caused by UVC, H(2)O(2), or methyl viologen. This up-regulation was dependent of RCD1, a mediator of the oxidative stress response. Recombinant metacaspase-8 cleaved after arginine, had a pH optimum of 8, and complemented the H(2)O(2) no-death phenotype of a yeast metacaspase knock-out. Overexpressing AtMC8 up-regulated PCD induced by UVC or H(2)O(2), and knocking out AtMC8 reduced cell death triggered by UVC and H(2)O(2) in protoplasts. Knock-out seeds and seedlings had an increased tolerance to the herbicide methyl viologen. We suggest that metacaspase-8 is part of an evolutionary conserved PCD pathway activated by oxidative stress.

A method has been developed for the regeneration of transformed plants of the commercially important crop sugarbeet (Beta vulgaris L.), using Agrobacterium tumefaciens. Binary vectors were used, carrying both screenable and selectable genes. Plant regeneration from shoot-base tissues was found to be relatively rapid and frequent compared with petioles or leaf tissue. Inoculation of cultured shoot-base tissues resulted in the production of transformed plants, as determined by (1) introduced resistance to kanamycin, (2) introduced CAT or GUS activity, and (3) Southern blot analysis to show the integration of foreign DNA. The transformation frequency was found to be dependent upon expiant source, plant genotype and selection conditions used.

When plants experience an imbalance between the absorption of light energy and the use of that energy to drive metabolism, they are liable to suffer from oxidative stress. Such imbalances arise due to environmental conditions (e.g. heat, chilling or drought), and can result in the production of reactive oxygen species (ROS). Here, we present evidence for a novel protective process — feedback redox regulation via the redox poise of the NADP(H) pool. Photosynthetic electron transport was studied in two transgenic tobacco ( Nicotiana tabacum ) lines — one having reduced levels of ferredoxin NADP + -reductase (FNR), the enzyme responsible for reducing NADP + , and the other reduced levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the principal consumer of NADPH. Both had a similar degree of inhibition of carbon fixation and impaired electron transport. However, whilst FNR antisense plants were obviously stressed, with extensive bleaching of leaves, GAPDH antisense plants showed no visible signs of stress, beyond having a slowed growth rate. Examination of electron transport in these plants indicated that this difference is due to feedback regulation occurring in the GAPDH but not the FNR antisense plants. We propose that this reflects the occurrence of a previously undescribed regulatory pathway responding to the redox poise of the NADP(H) pool.

A very important goal is to determine which molecular components may be used in the execution of programmed cell death (PCD) in plants, which have been conserved during evolution and which are plant specific. Using A. thaliana we have shown that UV radiation can induce apoptotic-like changes at the cellular level and that an UV experimental system was relevant to the study of PCD in plants. UV induction of PCD requires light and a protease cleaving the caspase substrate Asp-Glu-Val-Asp (DEVDase activity) is induced within 30 minutes and peaks at one hour. This DEVDase appears related to animal caspases at the biochemical level, being insensitive to broad-range cysteine protease inhibitors. In addition, caspase1, caspase-3 inhibitors and the pancaspase inhibitor p35 were able to suppress DNA fragmentation and cell death. These results suggest that a YVADase (Tyr-Val-Ala-Asp) activity and an inducible DEVDase activity are possibly mediating DNA fragmentation during plant PCD induced by UV overexposure. Progress is being made towards the biochemical characterisation of the proteases involved.

High ozone concentration generates oxidative stress in plants. To investigate the detailed transcriptional regulation of Arabidopsis thaliana genes encoding antioxidant enzymes upon ozone stress, we performed a microarray analysis using Affymetrix GeneChip technology. Our transcription profiling revealed a differential expression equal or greater than 2-fold change for 2385 genes (at confidence 99%) in response to 350 ppb ozone dose after 3 and 6 hours of treatment. Among these, we chose 38 genes to be oxidative stress related in ozone treatment: 29 of them were 2 times up-regulated and 9 were shown to be down-regulated in at least one of the time points. Our study revealed a new transcription pattern for catalase genes and showed the first detailed transcriptional analysis of phenylopropanoid-related genes in ozone stress conditions.

The pOp/LhG4 transcription factor system was used to determine whether the synthetic pOp promoter, integrated at one position in the Arabidopsis genome, could be efficiently and faithfully activated by the heterologous transcription factor, LhG4, expressed in a variety of different patterns. This is a precondition for the development and exploitation of large collections of LhG4 activation lines that direct predictable tissue-specific expression of transgenes. We selected a pOp-GUS reporter insertion that was efficiently activated after crossing to an activator line that expressed the synthetic transcription factor LhG4 from the Cauliflower Mosaic Virus 35S promoter. This reporter line, pOp-GUS(g2), was then combined with activator loci that expressed LhG4 from one of seven different promoters, each with a different tissue specificity. pOp-GUS(g2) was activated faithfully in combination with six of these seven activator constructs, but generated an unexpected expression pattern in combination with the seventh construct, a fusion to a cyclin promoter (CYC-LhG4). The aberrant expression pattern could be attributed to the pOp-GUS(g2) insertion site, as the CYC-LhG4 activator lines directed the expected pattern of expression from a second pOp-GUS insertion. These results show that it is feasible to construct an activator collection in which LhG4 is expressed from diverse promoters or enhancer traps, but that individual pOp reporter loci can vary in their competence to respond to certain activator patterns. We discuss the implications for the design and use of mis-expression technology in Arabidopsis.

International audience; Plants, animals, and several branches of unicellular eukaryotes use programmed cell death (PCD) for defense or developmental mechanisms. This argues for a common ancestral apoptotic system in eukaryotes. However, at the molecular level, very few regulatory proteins or protein domains have been identified as conserved across all eukaryotic PCD forms. A very important goal is to determine which molecular components may be used in the execution of PCD in plants, which have been conserved during evolution, and which are plant-specific. Using Arabidopsis thaliana, we have shown that UV radiation can induce apoptosis-like changes at the cellular level and that a UV experimental system is relevant to the study of PCD in plants. We report here that UV induction of PCD required light and that a protease cleaving the caspase substrate Asp-Glu-Val-Asp (DEVDase activity) was induced within 30 min and peaked at 1 h. This DEVDase appears to be related to animal caspases at the biochemical level, being insensitive to broad-range cysteine protease inhibitors. In addition, caspase-1 and caspase-3 inhibitors and the pan-caspase inhibitor p35 were able to suppress DNA fragmentation and cell death. These results suggest that a YVADase activity and an inducible DEVDase activity possibly mediate DNA fragmentation during plant PCD induced by UV overexposure. We also report that At-DAD1 and At-DAD2, the two A. thaliana homologs of Defender against Apoptotic Death-1, could suppress the onset of DNA fragmentation in A. thaliana, supporting an involvement of the endoplasmic reticulum in this form of the plant PCD pathway.

Previously, we described two cDNA clones corresponding to a new family of cold regulated genes, pBnC24a and pBnC24b homologous to the human BBC1 (Breast Basic Conserved) gene. In order to further analyze the function and regulation of these two genes we have prepared antibodies against a recombinant fusion protein, MBP–BnC24A and used them to study expression at the protein level. In contrast to the increased mRNA accumulation induced by cold, immuno-blot analysis showed that the quantity of the BnC24 protein was not correlated with the accumulation of BnC24 transcripts and is identical in both control and low temperature treated (4°C) plants, suggesting a translational or post-translational regulation. This was confirmed by overexpressing BnC24 in transgenic Arabidopsis thaliana plants. Despite a substantial increase in mRNA, the BnC24 protein level is unchanged. In addition, we demonstrated by subcellular fractionation and immunodetection that a significant fraction of BnC24 protein is located in the nucleus. Using a GUS fusion construction and biolistic experiments it was found that a portion of the amino terminal region is sufficient to target this protein to the nucleus. These results are consistent with the recent finding that BBC1 and its homologues code for the ribosomal large subunit protein L13. In addition they illustrate the difficulty of correlating accumulation of a given mRNA in response to a given stimulus with a biologically significant role in the adaptation to a new environment.

We are very grateful to P. This for helpful discussion on seedless fruits. We thank G. Hull and J. Timmis for critically reading and improving the manuscript before submission. R. Blanvillain is funded by an EC grant (EPEN BIO4-CT96-0689).