The Arabidopsis TONNEAU2 gene encodes a putative novel protein phosphatase 2A regulatory subunit essential for the control of the cortical cytoskeleton

In Arabidopsis ton2 mutants, abnormalities of the cortical microtubular cytoskeleton, such as disorganization of the interphase microtubule array and lack of the preprophase band before mitosis, markedly affect cell shape and arrangement as well as overall plant morphology. We present the molecular isolation of the TON2 gene, which is highly conserved in higher plants and has a vertebrate homolog of unknown function. It encodes a protein similar in its C-terminal part to B" regulatory subunits of type 2A protein phosphatases (PP2As). We show that the TON2 protein interacts with an Arabidopsis type A subunit of PP2A in the yeast two-hybrid system and thus likely defines a novel subclass of PP2A subunits that are possibly involved in the control of cytoskeletal structures in plants.     

Expression of antisense chalcone synthase RNA in transgenic hybrid walnut microcuttings. Effect on flavonoid content and rooting ability

Walnut somatic embryos (Juglans nigra × Juglans regia) were transformed with a vector containing a neomycin phosphotransferase II, a -glucuronidase and an antisense chalcone synthase (chs) gene. This antisense construct included a 400 bp cDNA fragment of a walnut chs gene under the control of the duplicated CaMV-35S promoter. Molecular, biochemical and biological characterizations were performed both on transformed embryos propagated by secondary somatic embryogenesis and on microshoots developed by in vitro culture of embryonic epicotyls from somatic embryos. Thirteen transformed lines with the vector containing the antisense chs gene, one line with only the gus and nptII genes and one untransformed line were maintained in tissue culture. Six of the antisense lines were shown to be flavonoid-deficient. They exhibited a strongly reduced expression of chs genes, very low chalcone synthase activity and no detectable amounts of quercitrin, myricitrin, flavane-3-ols and proanthocyanidins in stems. Rooting tests showed that decreased flavonoid content in stems of antisense chs transformed lines was associated with enhanced adventitious root formation. Free auxin and conjugated auxin contents were determined during the latter phase of the micropropagation, and no variations were detected between control and antisense chs transformed lines. The in vitro plants developed a large basal callus and apical necrosis upon auxinic induction and the transformed lines highly deficient in flavonoids were more sensitive to exogenous application of indolebutyric acid (IBA).     

Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin

Plant cells have specific microtubule structures involved in cell division and elongation. The tonneau1 (ton1) mutant of Arabidopsis thaliana displays drastic defects in morphogenesis, positioning of division planes, and cellular organization. These are primarily caused by dysfunction of the cortical cytoskeleton and absence of the preprophase band of microtubules. Characterization of the ton1 insertional mutant reveals complex chromosomal rearrangements leading to simultaneous disruption of two highly similar genes in tandem, TON1a and TON1b. TON1 proteins are conserved in land plants and share sequence motifs with human centrosomal proteins. The TON1 protein associates with soluble and microsomal fractions of Arabidopsis cells, and a green fluorescent protein–TON1 fusion labels cortical cytoskeletal structures, including the preprophase band and the interphase cortical array. A yeast two-hybrid screen identified Arabidopsis centrin as a potential TON1 partner. This interaction was confirmed both in vitro and in plant cells. The similarity of TON1 with centrosomal proteins and its interaction with centrin, another key component of microtubule organizing centers, suggests that functions involved in the organization of microtubule arrays by the centrosome were conserved across the evolutionary divergence between plants and animals.     

Gamma-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. gamma-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, gamma-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 gamma-tubulin genes in plant development and in the formation of microtubule arrays. Isolation of mutants in each gene and analysis of two combinations of gamma-tubulin double mutants showed that the two genes have redundant functions. The first combination is lethal at the gametophytic stage. Disruption of both gamma-tubulin genes causes aberrant spindle and phragmoplast structures and alters nuclear division in gametophytes. The second combination of gamma-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 gamma-tubulin depletion on microtubule arrays in dividing cells using a green fluorescent protein marker. These results establish the central role of gamma-tubulin in the formation and organization of microtubule arrays in Arabidopsis.     

B1‐type cyclins control microtubule organization during cell division in Arabidopsis

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development‐specific requirements of B1‐type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1‐type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1‐type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA‐TUBULIN COMPLEX PROTEIN 3‐INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1‐type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.     

Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase gene in Brassica oleracea.

A receptor-like kinase, SRK, has been implicated in the autoincompatible response that leads to the rejection of self-pollen in Brassica plants. SRK is encoded by one member of a multigene family, which includes several receptor-like kinase genes with patterns of expression very different from that of SRK but of unknown function. Here, we report the characterization of a novel member of the Brassica S gene family, SFR2. RNA gel blot analysis demonstrated that SFR2 mRNA accumulated rapidly in response both to wounding and to infiltration with either of two bacteria: Xanthomonas campestris, a pathogen, and Escherichia coli, a saprophyte. SFR2 mRNA also accumulated rapidly after treatment with salicylic acid, a molecule that has been implicated in plant defense response signaling pathways. A SFR2 promoter and reporter gene fusion was introduced into tobacco and was shown to be induced by bacteria of another genus, Ralstonia (Pseudomonas) solanacearum. The accumulation of SFR2 mRNA in response to wounding and pathogen invasion is typical of a gene involved in the defense responses of the plant. The rapidity of SFR2 mRNA accumulation is consistent with SFR2 playing a role in the signal transduction pathway that leads to induction of plant defense proteins, such as pathogenesis-related proteins or enzymes of phenylpropanoid metabolism.     

The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes

Land plant cells assemble microtubule arrays without a conspicuous microtubule organizing center like a centrosome. In Arabidopsis thaliana, the TONNEAU1 (TON1) proteins, which share similarity with FOP, a human centrosomal protein, are essential for microtubule organization at the cortex. We have identified a novel superfamily of 34 proteins conserved in land plants, the TON1 Recruiting Motif (TRM) proteins, which share six short conserved motifs, including a TON1-interacting motif present in all TRMs. An archetypal member of this family, TRM1, is a microtubule-associated protein that localizes to cortical microtubules and binds microtubules in vitro. Not all TRM proteins can bind microtubules, suggesting a diversity of functions for this family. In addition, we show that TRM1 interacts in vivo with TON1 and is able to target TON1 to cortical microtubules via its C-terminal TON1 interaction motif. Interestingly, three motifs of TRMs are found in CAP350, a human centrosomal protein interacting with FOP, and the C-terminal M2 motif of CAP350 is responsible for FOP recruitment at the centrosome. Moreover, we found that TON1 can interact with the human CAP350 M2 motif in yeast. Taken together, our results suggest conservation of eukaryotic centrosomal components in plant cells.