Jean-Luc Evrard

γ-Tubulin Is Essential for Microtubule Organization and Development in Arabidopsis

The process of microtubule nucleation in plant cells is still a major question in plant cell biology. γ-Tubulin is known as one of the key molecular players for microtubule nucleation in animal and fungal cells. Here, we provide genetic evidence that in Arabidopsis thaliana, γ-tubulin is required for the formation of spindle, phragmoplast, and cortical microtubule arrays. We used a reverse genetics approach to investigate the role of the two Arabidopsis γ-tubulin genes in plant development and in the formation of microtubule arrays. Isolation of mutants in each gene and analysis of two combinations of γ-tubulin double mutants showed that the two genes have redundant functions. The first combination is lethal at the gametophytic stage. Disruption of both γ-tubulin genes causes aberrant spindle and phragmoplast structures and alters nuclear division in gametophytes. The second combination of γ-tubulin alleles affects late seedling development, ultimately leading to lethality 3 weeks after germination. This partially viable mutant combination enabled us to follow dynamically the effects of γ-tubulin depletion on microtubule arrays in dividing cells using a green fluorescent protein marker. These results establish the central role of γ-tubulin in the formation and organization of microtubule arrays in Arabidopsis.

The Plant TPX2 Protein Regulates Prospindle Assembly before Nuclear Envelope Breakdown

The Targeting Protein for Xklp2 (TPX2) is a central regulator of spindle assembly in vertebrate cells. The absence or excess of TPX2 inhibits spindle formation. We have defined a TPX2 signature motif that is present once in vertebrate sequences but twice in plants. Plant TPX2 is predominantly nuclear during interphase and is actively exported before nuclear envelope breakdown to initiate prospindle assembly. It localizes to the spindle microtubules but not to the interdigitating polar microtubules during anaphase or to the phragmoplast as it is rapidly degraded during telophase. We characterized the Arabidopsis thaliana TPX2-targeting domains and show that the protein is able to rescue microtubule assembly in TPX2-depleted Xenopus laevis egg extracts. Injection of antibodies to TPX2 into living plant cells inhibits the onset of mitosis. These results demonstrate that plant TPX2 already functions before nuclear envelope breakdown. Thus, plants have adapted nuclear–cytoplasmic shuttling of TPX2 to maintain proper spindle assembly without centrosomes.

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.

A LIM motif is present in a pollen-specific protein.

We have recently described a sunflower cDNA sequence coding for a pollenspecific protein (SF3) with putative zinc finger domains (Baltz et al., 1992). In a more recent analysis we have found that these domains correspond to the conserved LIM motif identified so far only in a family of metal binding, cysteine-rich proteins from animais. This motif, n~55 amino acids long, is characterized by a unique organization of cysteine and histidine residues into two adjacent putative zinc fingers. LIM motif-containing proteins include developmental regulators such as the rat insulin gene enhancer binding protein ISL-1 (Karlsson et al., 1990), the Caenorhabdifis elegans proteins LIN-11 (Freyd et al., 1990) and MEC-3 (Way and Chalfie, 1988), the Drosophila APTEROUS protein (Cohen et al., 1992), the XenopusXLIM-1 protein (Tairaet al., 1992), and the mammalian oncoproteins TTG-1 and TTG-2 (also known as RHOMQ) of the rhombotin family (McGuire et ai., 1989; Boehm et al., 1990, 1991; Royer-Pokora et al., 1991). The mammalian cysteine-rich proteins CRlP (Birkenmeyer and Gordon, 1986), hCRP (Liebhaber et al., 1990; Wang et al., 1992), and ESP-l (Nalik et al., 1989), all of which are of yet unknown function, also contain LIM motifs. LIM motifs are found either alone (in CRIP, TTG-1, TTGP, ESP-1, and hCRP) or in association with a homeodomain (in MEC-3, ISL-1, LIM-11, XLIM-1, and APTEROUS). Figure 1 shows an alignment of the LIM motifs of the pollen-specific protein SF3 with those of the animal LIM proteins. ConSeNed residues are shown in bold type. Aclose examination of a number of semiconserved positions (see boxed residues) shows evidence for the existence of two subfamilies of LIM proteins: subfamily A, which includes SF3, hCRP, CRlP and ESP-1, and subfamily B, which comprises the seven other proteins. The most frequently occurring metalchelating residues in the potential zinc fingers are cysteines and histidines. However, in the majority of the LIM proteins, aspartate (D) is the last residue in the second finger (position 57). This is not necessarily surprising because aspartate has been identified as a metal-chelating residue in zinc-containing enzymes (Vallee and Auld, 1990). As potential zinc finger domains, the LIM motifs could be directly involved in DNA binding, although a possible role in protein-protein interactions has been

Molecular and expression analysis of a LIM protein gene family from flowering plants.

Abstract. LIM-domain proteins participate in important cellular processes in eukaryotes, including gene transcription and actin cytoskeleton organization. They are predominantly found in animals, but have also been identified in yeast and plants. Following the characterization of a LIM-domain protein in sunflower pollen, we carried out an extensive search for these proteins in flowering plants. We have isolated and studied cDNAs and/or genomic sequences for two novel LIM-domain proteins from sunflower, three from tobacco, and one from Arabidopsis. The plant proteins are structurally related to the cytoskeleton-associated CRP class of LIM proteins in animals, but show several distinctive features, including a second, atypical, LIM domain. We have performed comparative expression studies of these genes, as well as of one other gene from tobacco and two additional Arabidopsis genes whose sequences are available from databases. These studies, carried out by RT-PCR in the presence of gene-specific primers, showed that, in sunflower and tobacco, pollen grains and sporophytic tissues express different sets of LIM proteins. With the exception of one Arabidopsis gene – which has two introns – all the genes analyzed contain four introns at conserved positions, indicating that the ancestral gene from which the various copies evolved in higher plants already had this split structure.

Expression of plastid and nuclear genes during chromoplast differentiation in bell pepper (Capsicum annuum) and sunflower (Helianthus annuus)

SummaryThe expression of genes involved in plastid differentiation is subject to developmental control. To understand better the molecular basis of this regulation, we have analyzed in parallel the changes in relative levels of four chloroplast and two nuclear transcripts during differentiation of chloroplasts into chromoplasts in ripening bell pepper fruits and in maturing sunflower petals, as well as in the case of chemically-induced chromoplast formation in leaves. Amongst the prominent features, we noted that transcripts of nuclear genes coding for chloroplast proteins, such as the major chlorophyll a/b-binding protein (encoded by cab) and the small subunit of ribuloses-1,5-bisphosphate carboxylase (encoded by rbcS), were detected in chromoplast-containing bell pepper leaves and sunflower petals, whilst they disappeared in bell pepper fruit chromoplasts. Transcripts of the plastid gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase were detected during the whole differentiation process. The steady-state level of the mRNA corresponding to the 32 kDa herbicide-binding protein increased in fruit and petal chromoplasts. Changes in the relative transcriptional activities of plastid genes and in their relative mRNA levels occurred during bell pepper fruit ripening. However, no significant change in the overall transcriptional activity was found when comparing both types of plastids, whereas dramatic changes in translational activity occur, indicating the prevalence of translational control of plastid gene expression in the chromoplast differentiation process.

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.

Anther-specific, developmentally regulated expression of genes encoding a new class of proline-rich proteins in sunflower.

We have used RNA gel blot analysis to demonstrate the anther-specific expression of three genes in sunflower. Expression of these genes was first detected shortly before flower opening, which occurs sequentially on the sunflower inflorescence, and continues during pollination. In contrast, these genes are not expressed (or only weakly expressed) in a male-sterile line in which anther development aborts. In situ hybridization experiments showed that these genes are only expressed in the single cell layer of the sunflower anther epidermis. In the case of one of these genes, which codes for an abundant mRNA, we report the peptide sequences deduced from the sequence of two similar but non identical cDNAs. These proteins contain a potential signal peptide and are characterized by the presence of a proline-rich region which reads KPSTPAPPPPPP(PP)K. Our results also suggest that several proline-rich proteins of unknown functions are specifically synthesized during the maturation of anthers in sunflower.

A class-I intron in a cyanelle tRNA gene from Cyanophora paradoxa: phylogenetic relationship between cyanelles and plant chloroplasts

Cyanelles are photosynthetic organelles which are considered as intermediates between cyanobacteria and chloroplasts, and which have been found in unicellular eukaryotes such as Cyanophora paradoxa. The nucleotide sequence of a 667-bp region of the cyanelle genome from Cyanophora paradoxa containing genes coding for tRNA(UUCGlu) and tRNA(UAALeu) has been determined. The gene coding for tRNA(UAALeu) is split by a 232-bp intron which has a secondary structure typical for class-I structured introns and which is closely related to the intron located in the corresponding gene from liverwort and higher plant chloroplasts. It appears therefore that these tRNA(UAALeu) genes are all derived from one common ancestral gene which already contained a class-I intron.

A transmitting tissue- and pollen-expressed protein from sunflower with sequence similarity to the human RTP protein

Abstract A novel pistil- and pollen-expressed gene ( sf21 ) encoding a 352 amino acid long polypeptide was isolated by differential screening of a floral cDNA library from sunflower ( Helianthus annuus L.) and characterized. The deduced polypeptide is structurally related to the human protein RTP and the mouse protein Ndr1. It also shares significant sequence homology with an unidentified polypeptide from human cerebellum (N-terminus) and with the ligand binding region of the vertebrate inositol 1,4,5 trisphosphate (IP3) receptor (C-terminus). The sf21 gene is expressed in young and mature florets of an open inflorescence but not at the floral bud stage. In the sunflower pistil, expression is restricted to the transmitting tissue and the phloem cells. The same location of expression, with an increasing concentration gradient of SF21 transcripts towards the ovule, is observed during specific stages in tobacco pistils. The transcripts gradually disappear as the pollen tubes grow through the transmitting tissue. These observations suggest that the protein acts in pollen-pistil interactions.