Valérie Delorme

Plant programmed cell death: A common way to die

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.

The Arabidopsis Root Transcriptome by Serial Analysis of Gene Expression. Gene Identification Using the Genome Sequence

Large-scale identification of genes expressed in roots of the model plant Arabidopsis was performed by serial analysis of gene expression (SAGE), on a total of 144,083 sequenced tags, representing at least 15,964 different mRNAs. For tag to gene assignment, we developed a computational approach based on 26,620 genes annotated from the complete sequence of the genome. The procedure selected warrants the identification of the genes corresponding to the majority of the tags found experimentally, with a high level of reliability, and provides a reference database for SAGE studies in Arabidopsis. This new resource allowed us to characterize the expression of more than 3,000 genes, for which there is no expressed sequence tag (EST) or cDNA in the databases. Moreover, 85% of the tags were specific for one gene. To illustrate this advantage of SAGE for functional genomics, we show that our data allow an unambiguous analysis of most of the individual genes belonging to 12 different ion transporter multigene families. These results indicate that, compared with EST-based tag to gene assignment, the use of the annotated genome sequence greatly improves gene identification in SAGE studies. However, more than 6,000 different tags remained with no gene match, suggesting that a significant proportion of transcripts present in the roots originate from yet unknown or wrongly annotated genes. The root transcriptome characterized in this study markedly differs from those obtained in other organs, and provides a unique resource for investigating the functional specificities of the root system. As an example of the use of SAGE for transcript profiling in Arabidopsis, we report here the identification of 270 genes differentially expressed between roots of plants grown either with NO3- or NH4NO3 as N source.

The Arabidopsis peptide KISS OF DEATH is an inducer of Programmed Cell Death

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.

ATR3 encodes a diflavin reductase essential for Arabidopsis embryo development

*The Arabidopsis genome possesses two confirmed Cytochrome P450 Reductase (CPR) genes, ATR1 and ATR2, together with a third putative homologue, ATR3, which annotation is questionable. *Phylogenetic analysis classified ATR3 as a CPR-like protein sharing homologies with the animal cytosolic dual flavin reductases, NR1 and Fre-1, distinct from the microsomal CPRs, ATR1 and ATR2. Like NR1 and Fre-1, ATR3 lacks the N-terminal endoplasmic reticulum (ER) anchor domain of CPRs and is localized in the cytoplasm. Recombinant ATR3 in plant soluble extracts was able to reduce cytochrome c but failed to reduce the human P450 CYP1A2. *Loss of ATR3 function resulted in early embryo lethality indicating that this reductase activity is essential. A yeast 2-hybrid screen identified a unique interaction of ATR3 with the homologue of the human anti-apoptotic CIAPIN1 and the yeast Dre2 protein. *This interaction suggests two possible roles for ATR3 in the control of cell death and in chromosome segregation at mitosis. Consistent with these results, the promoter of ATR3 is activated during cell cycle progression. Together these results demonstrated that ATR3 belongs to the NR1 subfamily of diflavin reductases whose characterized members are involved in essential cellular functions.

Identification of protein factors and U3 snoRNAs from a Brassica oleracea RNP complex involved in the processing of pre-rRNA.

We report on the structural characterization of a functional U3 snoRNA ribonucleoprotein complex isolated from Brassica oleracea. The BoU3 snoRNP complex (formerly NF D) binds ribosomal DNA (rDNA), specifically cleaves pre-rRNA at the primary cleavage site in vitro and probably links transcription to early pre-rRNA processing in vivo. Using a proteomic approach we have identified 62 proteins in the purified BoU3 snoRNP fraction, including small RNA associated proteins (Fibrillarin, NOP5/Nop58p, Diskerin/Cbf5p, SUS2/PRP8 and CLO/GFA1/sn114p) and 40S ribosomal associated proteins (22 RPS and four ARCA-like proteins). Another major protein group is composed of chaperones/chaperonins (HSP81/TCP-1) and at least one proteasome subunit (RPN1a). Remarkably, RNA-dependent RNA polymerase (RdRP) and Tudor staphylococcal nuclease (TSN) proteins, which have RNA- and/or DNA-associated activities, were also revealed in the complex. Furthermore, three U3 snoRNA variants were identified in the BoU3 snoRNP fraction, notably an evolutionarily conserved and variable stem loop structure located just downstream from the C-box domain of the U3 sequence structures. We conclude that the BoU3 snoRNP complex is mainly required for 40S pre-ribosome synthesis. It is also expected that U3 snoRNA variants and interacting proteins might play a major role in BoU3 snoRNP complex assembly and/or function. This study provides a basis for further investigation of these novel ribonucleoprotein factors and their role in plant ribosome biogenesis.

Redundancy and crosstalk within the thioredoxin and glutathione pathways a new development in plants.

Thioredoxins (Trx) and glutaredoxins (Grx) are major disulfide reduction enzymes occurring in all living organisms that regulate the redox state of thiol groups of proteins. Initially discovered as a reductant of ribonucleotide reductase (RNR), an enzyme necessary for DNA synthesis, it is now established that they are involved in various biological processes. Trx and Grx have their own reduction system: typically, in most organisms and in the cytosol and mitochondria of plants the Trx pathway (NTS) comprises a redox cascade including NADPH, Trx reductase (NTR), and Trx, while the Grx pathway (NGS) is composed of NADPH, glutathione reductase (GR), glutathione (GSH), and Grx. These two systems act in parallel and have several common target proteins as shown by biochemical and genetic studies. Recent genetic studies in Arabidopsis show that the cytosolic Trx and Grx reduction systems are in fact more complex. In the cytosol, in absence of NTR, Trxs are reduced by a GSH-dependent pathway, while in the absence of GR oxidized glutathione (GSSG) is reduced by the NTR Trx pathway. By contrast in the chloroplast, Trxs have evolved a specific function of control of the light dark metabolism.