Guy Houlné

A Ubiquitous Plant Housekeeping Gene, PAP, Encodes a Major Protein Component of Bell Pepper Chromoplasts

We have isolated a cDNA (PAP) corresponding to a single nuclear gene that encodes an approximately 30-kD major protein of bell pepper (Capsicum annuum L.) fruit chromoplasts. RNA and protein analyses revealed that, although at a low level, this gene is also expressed in every organ of the plant, the amount of the corresponding transcript and protein dramatically increasing in the latter stages of fruit development. Western-blot and immunocytochemical analyses of purified chloroplasts from leaves and fruits and of chromoplasts from red fruits showed that the encoded protein is the major component of plastoglobules and fibrils and is localized on the outer surface of these lipid structures. Analyses of PAP in plants belonging to different taxa revealed that it is expressed and highly conserved in both monocotyledonous and dicotyledonous plants. The presence of the protein in plastids not differentiating into chromoplasts indicates that PAP is expressed irrespective of the ontogeny of various plastid lines. In light of our results and since the encoded protein, identical to that previously named ChrB or fibrillin, is present in plastoglobules from several species and accumulates in the fibrils of bell pepper chromoplast, we propose to designate it as a plastid-lipid-associated protein.

The GCP3-Interacting Proteins GIP1 and GIP2 Are Required for γ-Tubulin Complex Protein Localization, Spindle Integrity, and Chromosomal Stability

The stabilization of a robust mitotic spindle is required for correct chromosome segregation. GIP proteins interact with microtubule nucleation complexes and localize on mitotic microtubule arrays. The analysis of knockdown mutants suggests that GIP proteins act in both the recruitment of these complexes at nucleation sites and the maintenance of spindle efficiency. Microtubules (MTs) are crucial for both the establishment of cellular polarity and the progression of all mitotic phases leading to karyokinesis and cytokinesis. MT organization and spindle formation rely on the activity of γ-tubulin and associated proteins throughout the cell cycle. To date, the molecular mechanisms modulating γ-tubulin complex location remain largely unknown. In this work, two Arabidopsis thaliana proteins interacting with GAMMA-TUBULIN COMPLEX PROTEIN3 (GCP3), GCP3-INTERACTING PROTEIN1 (GIP1) and GIP2, have been characterized. Both GIP genes are ubiquitously expressed in all tissues analyzed. Immunolocalization studies combined with the expression of GIP–green fluorescent protein fusions have shown that GIPs colocalize with γ-tubulin, GCP3, and/or GCP4 and reorganize from the nucleus to the prospindle and the preprophase band in late G2. After nuclear envelope breakdown, they localize on spindle and phragmoplast MTs and on the reforming nuclear envelope of daughter cells. The gip1 gip2 double mutants exhibit severe growth defects and sterility. At the cellular level, they are characterized by MT misorganization and abnormal spindle polarity, resulting in ploidy defects. Altogether, our data show that during mitosis GIPs play a role in γ-tubulin complex localization, spindle stability and chromosomal segregation.

Fruit-Specific Expression of a Defensin-Type Gene Family in Bell Pepper (Upregulation during Ripening and upon Wounding)

We have isolated a 454-bp cDNA that encodes a novel fruit-specific defensin from bell pepper (Capsicum annuum). The encoded 75-amino-acid polypeptide contains an N-terminal domain characteristic of a signal peptide and a 48-amino-acid mature domain named J1. The mature protein, from which the N-terminal amino acid sequence was determined, contains eight cysteines that form four intramolecular disulfide bridges, suggesting a monomeric form for J1. In healthy fruits J1 is undetectable at the green stage but high levels accumulate during ripening. In wound areas of the green fruit the accumulation of J1 dramatically increased, suggesting a role for J1 in the plants defense response. Moreover, we have demonstrated that J1 possesses an antifungal activity. We have isolated and characterized the corresponding two homologous genes (j1–1 and j1–2) that exist in the bell pepper genome. Both genes are interrupted by the insertion, at the same position, of one intron of 853 bp for j1–1 and 4900 bp for j1–2. Northern blot and reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analyses revealed that j1–1 transcripts are present only in fruits, only in trace amounts in mature green fruits, and that they accumulate to high levels in fully ripe fruits, whereas no j1–2 transcripts were detected in the samples monitored.

Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation

Seedling explants of three tomato (Lycopersicon esculentum) and four bell pepper (Capsicum annuum) cultivars consisting of the radicle, the hypocotyl and one cotyledon were obtained after removing the primary and axillary meristems. After 14 days of incubation on solid Murashige and Skoog (MS) medium without growth regulators, explants of both species regenerated multiple shoots on the cut surface (2.9–5.3 shoots per explant for tomato and 1.2–2.2 for bell pepper cultivars). After excision, the shoots were rooted on solid MS medium and acclimated to the greenhouse. This method was highly efficient in tomato and, particularly, in bell pepper, where plant regeneration is especially difficult. We used these explants to transform tomato with Agrobacterium tumefaciens containing a 35S-GUS-intron binary vector. As shown by GUS expression, 47% of the tomato explants produced transformed meristems, which differentiated into plants that exhibited a low (3%) tetraploidy ratio. Southern blots and analysis of inheritance of the foreign genes indicated that T-DNA was stably integrated into the plant genome. The use of this technique opens new prospects for plant transformation in other dicotyledoneous plants in which genetic engineering has been limited, to date, due to the difficulties in developing an efficient in vitro regeneration system.

Characterization of a family of genes encoding a fruit-specific wound-stimulated protein of bell pepper (Capsicum annuum): identification of a new family of transposable elements

Using a fruit-specific cDNA as a probe we isolated and sequenced the two corresponding homologous genes (Sn-1 and Sn-2) of the bell pepper (Capsicum annuum) genome. Both genes have a single intron and numerous unusual long inverted repeat sequences. The introns share 87% homology and Sn-2 contains one 450 bp additional sequence with structural features of a transposable element, which is highly repetitive in the bell pepper genome. Surprisingly, analysis in data banks showed that genes encoding the potato starch phosphorylase (EC 2.4.1.1) and patatin contain a similar element, named Alien, in their 5′-upstream region. Alien elements are characterized by a conserved 28 bp terminal inverted repeat (TIR), small size, high AT content, potential to form stable DNA secondary structures and they have probably been inserted in TA target sites. Interestingly, the TIR of the Alien elements shares high homology with sequences existing in the TIR of extrachromosomal linear pSKL DNA plasmid of Saccharomyces kluyveri. Northern blot analyses detected Sn-1 transcripts principally in the red fruit whereas no Sn-2 transcripts were detected in neither of the samples monitored. Western blot analyses detected a 16.8 kDa Sn protein principally in the ripe red fruit and wounded areas of green unripe fruit. A comparison of the deduced amino acid sequence of Sn-1 with protein sequences in data banks revealed a significant homology with proteins likely involved in the plants disease resistance response. Analyses at the subcellular level showed that Sn-1 is localized in the membrane of vacuoles.

Characterization and gene expression of an annexin during fruit development in Capsicum annuum

Several lines of evidence indicate that annexins, as calcium‐dependent phospholipid‐binding proteins, are involved in a variety of plant cellular processes. We were interested in determining if annexins are implicated in the highly regulated fruit development of bell pepper. By differential screening of several cDNA libraries, we isolated a full‐length cDNA of 1180 bp encoding an annexin. Northern blot analyses show a differential expression pattern of the transcripts during the early stages of development and during ripening. Immunoblots using antiserum raised against p33/p35 from maize reveal that cross‐reactive polypeptides of about 30 kDa are present at each stage of fruit development in bell pepper. We partially purified the annexins from seedlings and green fruits. At least one annexin of 32 kDa is present in these plant tissues.

Characterization of cDNA sequences for LHCI apoproteins in Euglena gracilis: The mRNA encodes a large precursor containing several consecutive divergent polypeptides

SummaryScreening of a λgt11 cDNA expression library with antibodies directed against LHCII allowed the detection of several clones which differ markedly from the previously described cDNAs encoding LHCII apoproteins. A Northern analysis revealed a transcript size of 4.2 kb, whereas clones encoding LHCII hybridize with mRNAs in the range of 7.5 kb. Nucleotide sequencing of 2 clones showed open reading frames of 530 and 331 codons, respectively. Within these reading frames, 5 analogous motifs can be delimited corresponding to coding regions for around 180 amino acids (molecular weight, 18–19 kDa). The 5 segments share between 50% and 80% homology. Comparison with a tomato LHCI sequence indicates conserved regions at the two ends, and a central part highly divergent and containing a large deletion. By hybrid-selected translation, followed by immunoprecipitation with a monoclonal antibody directed against LHCI apoprotein, a protein of around 100 kDa is obtained. In vivo, the same antibody recognizes peptides of around 20 kDa. These results, coupled with our previous observations concerning LHCII, confirm that in Euglena at least, some chloroplast proteins encoded in the nucleus are synthesized by large mRNAs containing the information for several consecutive divergent peptides. Implications for processing and chloroplast import are discussed.

Regulation of biosynthesis and cellular localization of Sp32 annexins in tobacco BY2 cells.

Annexins interact in a calcium-dependent manner with membrane phospholipids. Although their exact function is not known, annexins have been proposed to be involved in a variety of cellular processes. To determine whether plant annexins are implicated in cell division, we have isolated cDNAs encoding annexin from TBY2 cells. Based on sequence analysis, these cDNAs fall into two families, differing mainly by deletions or insertions in their 5′- and 3′- untranslated regions. The two annexins Ntp32.1 and Ntp32.2 encoded by these cDNAs are homologous to p32 from bell pepper (Cap32.1): we propose that these Solanaceae annexins constitute a distinct type which we call Sp32 annexins. There are two genes (Ntan.1 and Ntan.2) derived from the separate progenitor species of Nicotiana tabacum and analysis of Southern blots is consistent with the presence of these two genes. We show that Sp32 transcript amounts are developmentally modulated in tobacco plants: RNA levels are highest in growing and dividing tissues. Sp32 annexin gene expression is also regulated in TBY2 cultured cells: transcripts and proteins are detected only in exponentially growing cells. In synchronized TBY2 cells, Sp32 annexin transcripts are expressed at the G2/M transition, in the M phase and at the G1/S transition. These results are the first evidence that the expression of plant annexins is modulated during the cell cycle. The Sp32 annexin proteins accumulate during the cell cycle and peak at the end of mitosis. Immunolocalization shows that the majority of Sp32 annexins is present in intercellular junctions, forming a ring structure under the plasma membrane. Since annexins are known to bind secretory vesicles during exocytosis, their localization at cell junctions suggests that annexins could be involved in cell wall maturation.

The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana.

During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs) are nucleated from γ-Tubulin Complexes (γ-TuCs) located at the surface of the nucleus. The molecular mechanisms of γ-TuC association to the nuclear envelope (NE) are currently unknown. The γ-TuC Protein 3 (GCP3)-Interacting Protein 1 (GIP1) is the smallest γ-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active γ-TuCs at interphasic and mitotic MT nucleation sites. Arabidopsis gip1gip2 mutants are impaired in establishing a fully functional mitotic spindle and exhibit severe developmental defects. In this study, gip1gip2 knock down mutants were further characterized at the cellular level. In addition to defects in both the localization of γ-TuC core proteins and MT fiber robustness, gip1gip2 mutants exhibited a severe alteration of the nuclear shape associated with an abnormal distribution of the nuclear pore complexes. Simultaneously, they showed a misorganization of the inner nuclear membrane protein AtSUN1. Furthermore, AtGIP1 was identified as an interacting partner of AtTSA1 which was detected, like the AtGIP proteins, at the NE. These results provide the first evidence for the involvement of a γ-TuC component in both nuclear shaping and NE organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum.

Ribonucleotide Reductase Regulation in Response to Genotoxic Stress in Arabidopsis

Ribonucleotide reductase (RNR) is an essential enzyme that provides dNTPs for DNA replication and repair. Arabidopsis (Arabidopsis thaliana) encodes three AtRNR2-like catalytic subunit genes (AtTSO2, AtRNR2A, and AtRNR2B). However, it is currently unclear what role, if any, each gene contributes to the DNA damage response, and in particular how each gene is transcriptionally regulated in response to replication blocks and DNA damage. To address this, we investigated transcriptional changes of 17-d-old Arabidopsis plants (which are enriched in S-phase cells over younger seedlings) in response to the replication-blocking agent hydroxyurea (HU) and to the DNA double-strand break inducer bleomycin (BLM). Here we show that AtRNR2A and AtRNR2B are specifically induced by HU but not by BLM. Early AtRNR2A induction is decreased in an atr mutant, and this induction is likely required for the replicative stress checkpoint since rnr2a mutants are hypersensitive to HU, whereas AtRNR2B induction is abolished in the rad9-rad17 double mutant. In contrast, AtTSO2 transcription is only activated in response to double-strand breaks (BLM), and this activation is dependent upon AtE2Fa. Both TSO2 and E2Fa are likely required for the DNA damage response since tso2 and e2fa mutants are hypersensitive to BLM. Interestingly, TSO2 gene expression is increased in atr versus wild type, possibly due to higher ATM expression in atr. On the other hand, a transient ATR-dependent H4 up-regulation was observed in wild type in response to HU and BLM, perhaps linked to a transient S-phase arrest. Our results therefore suggest that individual RNR2-like catalytic subunit genes participate in unique aspects of the cellular response to DNA damage in Arabidopsis.