Robert Blanvillain

Less is better: new approaches for seedless fruit production

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).

Paternally inherited transgenes are down-regulated but retain low activity during early embryogenesis in Arabidopsis

We investigated the timing of transgene activation after fertilisation in Arabidopsis following crosses and using two transgenic promoters (from the AtCYCB1 and AtLTP1 genes). Using both a transactivation system and direct transcriptional fusion to drive β‐glucuronidase reporter expression, reciprocal crosses showed a lack of expression of the paternal components. This is consistent with a lack of paternal genome activity previously reported during early seed development in Arabidopsis [Viella‐Calzada et al. (2000) Nature 404, 91–94]. However, transactivation experiments of the BARNASE gene gave evidence that at least some paternal loci retain transcriptional activity, though at a low level, during early embryogenesis.

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.

Stress tolerance to stress escape in plants: role of the OXS2 zinc‐finger transcription factor family

During dire conditions, the channelling of resources into reproduction ensures species preservation. This strategy of survival through the next generation is particularly important for plants that are unable to escape their environment but can produce hardy seeds. Here, we describe the multiple roles of OXIDATIVE STRESS 2 (OXS2) in maintaining vegetative growth, activating stress tolerance, or entering into stress‐induced reproduction. In the absence of stress, OXS2 is cytoplasmic and is needed for vegetative growth; in its absence, the plant flowers earlier. Upon stress, OXS2 is nuclear and is needed for stress tolerance; in its absence, the plant is stress sensitive. OXS2 can activate its own gene and those of floral integrator genes, with direct binding to the floral integrator promoter SOC1. Stress‐induced SOC1 expression and stress‐induced flowering are impaired in mutants with defects in OXS2 and three of the four OXS2‐like paralogues. The autoactivation of OXS2 may be a commensurate response to the stress intensity, stepping up from a strategy based on tolerating the effects of stress to one of escaping the stress via reproduction.

OXIDATIVE STRESS 3 is a chromatin-associated factor involved in tolerance to heavy metals and oxidative stress.

A cDNA expression library from Brassica juncea was introduced into the fission yeast Schizosaccharomyces pombe to select for transformants tolerant to cadmium. Transformants expressing OXIDATIVE STRESS 3 (OXS3) or OXS3-Like cDNA exhibited enhanced tolerance to a range of metals and oxidizing chemicals. OXS3 belongs to a family of proteins that share a highly conserved domain corresponding to a putative N-acetyltransferase or thioltransferase catalytic site. Mutations within this conserved domain abolished the ability of Arabidopsis thaliana OXS3 to enhance stress tolerance in S. pombe, indicating a role in stress tolerance for the presumptive catalytic domain. A stress-sensitive mutant of AtOXS3 and enhanced tolerance of overexpression lines support the role of OXS3 in stress tolerance. The expression of tagged B. juncea and A. thaliana OXS3 proteins in plant cells revealed a subnuclear speckling pattern related to the nucleosome in discrete parts of the chromatin. It is possible that OXS3 might act as a chromatin remodeling factor for the stress response.

EXPORTIN1 Genes Are Essential for Development and Function of the Gametophytes in Arabidopsis thaliana

Gametes are produced in plants through mitotic divisions in the haploid gametophytes. We investigated the role of EXPORTIN1 (XPO1) genes during the development of both female and male gametophytes of Arabidopsis. Exportins exclude target proteins from the nucleus and are also part of a complex recruited at the kinetochores during mitosis. Here we show that double mutants in Arabidopsis XPO1A and XPO1B are gametophytic defective. In homozygous–heterozygous plants, 50% of the ovules were arrested at different stages according to the parental genotype. Double-mutant female gametophytes of xpo1a-3/+; xpo1b-1/xpo1b-1 plants failed to undergo all the mitotic divisions or failed to complete embryo sac maturation. Double-mutant female gametophytes of xpo1a-3/xpo1a-3; xpo1b-1/+ plants had normal mitotic divisions and fertilization occurred; in most of these embryo sacs the endosperm started to divide but an embryo failed to develop. Distortions in male transmission correlated with the occurrence of smaller pollen grains, poor pollen germination, and shorter pollen tubes. Our results show that mitotic divisions are possible without XPO1 during the haploid phase, but that XPO1 is crucial for the maternal-to-embryonic transition.

Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle

Chloroplasts are the sunlight-collecting organelles of photosynthetic eukaryotes that energetically drive the biosphere of our planet. They are the base for all major food webs by providing essential photosynthates to all heterotrophic organisms including humans. Recent research has focused largely on an understanding of the function of these organelles, but knowledge about the biogenesis of chloroplasts is rather limited. It is known that chloroplasts develop from undifferentiated precursor plastids, the proplastids, in meristematic cells. This review focuses on the activation and action of plastid RNA polymerases, which play a key role in the development of new chloroplasts from proplastids. Evolutionarily, plastids emerged from the endosymbiosis of a cyanobacterium-like ancestor into a heterotrophic eukaryote. As an evolutionary remnant of this process, they possess their own genome, which is expressed by two types of plastid RNA polymerase, phage-type and prokaryotic-type RNA polymerase. The protein subunits of these polymerases are encoded in both the nuclear and plastid genomes. Their activation and action therefore require a highly sophisticated regulation that controls and coordinates the expression of the components encoded in the plastid and nucleus. Stoichiometric expression and correct assembly of RNA polymerase complexes is achieved by a combination of developmental and environmentally induced programmes. This review highlights the current knowledge about the functional coordination between the different types of plastid RNA polymerases and provides working models of their sequential expression and function for future investigations.

pH-sensitivity of YFP provides an intracellular indicator of programmed cell death.

BackgroundProgrammed cell death (PCD) is an essential process for the life cycle of all multicellular organisms. In higher plants however, relatively little is known about the cascade of genes and signalling molecules responsible for the initiation and execution of PCD. To aid with the discovery and analysis of plant PCD regulators, we have designed a novel cell death assay based on low cytosolic pH as a marker of PCD.ResultsThe acidification that occurs in the cytosol during plant PCD was monitored by way of the extinction of YFP fluorescence at low pH. This fluorescence was recovered experimentally when bringing the intracellular pH back to 7, demonstrating that there was no protein degradation of YFP. Because it uses YFP, the assay is none-destructive, does not interfere with the PCD process and allows time-lapse studies to be carried out. In addition, changes of sub-cellular localisation can be visualised during PCD using the protein of interest fused to RFP. Coupled to a transient expression system, this pH-based assay can be used to functionally analyse genes involved in PCD, using point mutations or co-expressing PCD regulators. Transfecting mBAX and AtBI-1 in onion epidermal cells showed that the pH shift is downstream of PCD suppression by AtBI-1. In addition, this method can be used to score PCD in tissues of stably transformed transgenic lines. As proof of principle, we show the example of YFP extinction during xylogenesis in Arabidopsis. This demonstrates that the assay is applicable to PCD studies in a variety of tissues.ConclusionsThe observation that YFP fluorescence is lost during the plant PCD process provides a new tool to study the genetic regulation and cell biology of the process. In addition, plant cell biologists should make a note of this effect of PCD on YFP fluorescence to avoid misinterpretation of their data and to select a pH insensitive reporter if appropriate. This method represents an efficient and streamlined tool expected to bring insights on the process leading to the pH shift occurring during PCD.

Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development

Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type.

Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1

The albino pap7-1 mutant of Arabidopsis reveals the relative impact of light and plastid developmental stage on the expression of nuclear genes involved in metabolism and photosynthesis. Plants possessing dysfunctional plastids due to defects in pigment biosynthesis or translation are known to repress photosynthesis-associated nuclear genes via retrograde signals from the disturbed organelles toward the nucleus. These signals are thought to be essential for proper biogenesis and function of the plastid. Mutants lacking plastid-encoded RNA polymerase-associated proteins (PAPs) display a genetic arrest in eoplast-chloroplast transition leading to an albino phenotype in the light. Retrograde signaling in these mutants, therefore, could be expected to be similar as under conditions inducing plastid dysfunction. To answer this question, we performed plastome- and genomewide array analyses in the pap7-1 mutant of Arabidopsis (Arabidopsis thaliana). In parallel, we determined the potential overlap with light-regulated expression networks. To this end, we performed a comparative expression profiling approach using light- and dark-grown wild-type plants as relative control for the expression profiles obtained from light-grown pap7-1 mutants. Our data indicate a specific impact of retrograde signals on metabolism-related genes in pap7-1 mutants reflecting the starvation situation of the albino seedlings. In contrast, light regulation of PhANGs and other nuclear gene groups appears to be fully functional in this mutant, indicating that a block in chloroplast biogenesis per se does not repress expression of them as suggested by earlier studies. Only genes for light harvesting complex proteins displayed a significant repression indicating an exclusive retrograde impact on this gene family. Our results indicate that chloroplasts and arrested plastids each emit specific signals that control different target gene modules both in positive and negative manner.