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Jules Beekwilder, Wessel van Leeuwen,
Nicole M van Dam,
Monica Bertossi,
Valentina Grandi,
Luca Mizzi,
Mikhail Soloviev,
Laszlo Szabados,
Jos W Molthoff,
Bert Schipper,
Hans Verbocht,
Ric C H de Vos,
Piero Morandini,
Mark G M Aarts,
Arnaud Bovy
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ABSTRACT: Aliphatic glucosinolates are compounds which occur in high concentrations in Arabidopsis thaliana and other Brassicaceae species. They are important for the resistance of the plant to pest insects. Previously, the biosynthesis of these compounds was shown to be regulated by transcription factors MYB28 and MYB29. We now show that MYB28 and MYB29 are partially redundant, but in the absence of both, the synthesis of all aliphatic glucosinolates is blocked. Untargeted and targeted biochemical analyses of leaf metabolites showed that differences between single and double knock-out mutants and wild type plants were restricted to glucosinolates. Biosynthesis of long-chain aliphatic glucosinolates was blocked by the myb28 mutation, while short-chain aliphatic glucosinolates were reduced by about 50% in both the myb28 and the myb29 single mutants. Most remarkably, all aliphatic glucosinolates were completely absent in the double mutant. Expression of glucosinolate biosynthetic genes was slightly but significantly reduced by the single myb mutations, while the double mutation resulted in a drastic decrease in expression of these genes. Since the myb28myb29 double mutant is the first Arabidopsis genotype without any aliphatic glucosinolates, we used it to establish the relevance of aliphatic glucosinolate biosynthesis to herbivory by larvae of the lepidopteran insect Mamestra brassicae. Plant damage correlated inversely to the levels of aliphatic glucosinolates observed in those plants: Larval weight gain was 2.6 fold higher on the double myb28myb29 mutant completely lacking aliphatic glucosinolates and 1.8 higher on the single mutants with intermediate levels of aliphatic glucosinolates compared to wild type plants.
PLoS ONE 02/2008; 3(4):e2068. · 4.09 Impact Factor
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ABSTRACT: Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is an important signalling lipid in mammalian cells, where it functions as a second-messenger precursor in response to agonist-dependent activation of phospholipase C (PLC) but also operates as a signalling molecule on its own. Much of the recent knowledge about it has come from a new technique to visualize PtdIns(4,5)P(2)in vivo, by expressing a green or yellow fluorescent protein (GFP or YFP) fused to the pleckstrin homology (PH) domain of human PLCdelta1 that specifically binds PtdIns(4,5)P(2). In this way, YFP-PH(PLCdelta1) has been shown to predominantly label the plasma membrane and to transiently translocate into the cytoplasm upon PLC activation in a variety of mammalian cell systems. In plants, biochemical studies have shown that PtdIns(4,5)P(2) is present in very small quantities, but knowledge of its localization and function is still very limited. In this study, we have used YFP-PH(PLCdelta1) to try monitoring PtdIns(4,5)P(2)/PLC signalling in stably-transformed tobacco Bright Yellow-2 (BY-2) cells and Arabidopsis seedlings. In both plant systems, no detrimental effects were observed, indicating that overexpression of the biosensor did not interfere with the function of PtdIns(4,5)P(2). Confocal imaging revealed that most of the YFP-PH(PLCdelta1) fluorescence was present in the cytoplasm, and not in the plasma membrane as in mammalian cells. Nonetheless, four conditions were found in which YFP-PH(PLCdelta1) was concentrated at the plasma membrane: (i) upon treatment with the PLC inhibitor U73122; (ii) in response to salt stress; (iii) as a gradient at the tip of growing root hairs; (iv) during the final stage of a BY-2 cell division. We conclude that PtdIns(4,5)P(2), as in animals, is present in the plasma membrane of plants, but that its concentration in most cells is too low to be detected by YFP-PH(PLCdelta1). Hence, the reporter remains unbound in the cytosol, making it unsuitable to monitor PLC signalling. Nonetheless, YFP-PH(PLCdelta1) is a valuable plant PtdIns(4,5)P(2) reporter, for it highlights specific cells and conditions where this lipid becomes abnormally concentrated in membranes, raising the question of what it is doing there. New roles for PtdIns(4,5)P(2) in plant cell signalling are discussed.
The Plant Journal 01/2008; 52(6):1014-26. · 6.16 Impact Factor
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ABSTRACT: To investigate PtdIns3P localization and function in plants, a fluorescent PtdIns3P-specific biosensor (YFP-2xFYVE) was created. On lipid dot blots it bound specifically and with high affinity to PtdIns3P. Transient expression in cowpea protoplasts labelled vacuolar membranes and highly motile structures undergoing fusion and fission. Stable expression in tobacco BY-2 cells labelled similar motile structures, but labelled vacuolar membranes hardly at all. YFP-2xFYVE fluorescence strongly co-localized with the pre-vacuolar marker AtRABF2b, partially co-localized with the endosomal tracer FM4-64, but showed no overlap with the Golgi marker STtmd-CFP. Treatment of cells with wortmannin, a PI3 kinase inhibitor, caused the YFP-2xFYVE fluorescence to redistribute into the cytosol and nucleus within 15 min. BY-2 cells expressing YFP-2xFYVE contained twice as much PtdIns3P as YFP-transformed cells, but this had no effect on cell-growth or stress-induced phospholipid signalling responses. Upon treatment with wortmannin, PtdIns3P levels were reduced by approximately 40% within 15 min in both cell lines. Stable expression of YFP-2xFYVE in Arabidopsis plants labelled different subcellular structures in root compared with shoot tissues. In addition labelling the motile structures common to all cells, YFP-2xFYVE strongly labelled the vacuolar membrane in leaf epidermal and guard cells, suggesting that cell differentiation alters the distribution of PtdIns3P. In dividing BY-2 cells, YFP-2xFYVE-labelled vesicles surrounded the newly formed cell plate, suggesting a role for PtdIns3P in cytokinesis. Together, these data show that YFP-2xFYVE may be used as a biosensor to specifically visualize PtdIns3P in living plant cells.
The Plant Journal 10/2006; 47(5):687-700. · 6.16 Impact Factor
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ABSTRACT: Phospholipase D (PLD) has been implicated in various cellular processes including membrane degradation, vesicular trafficking and signal transduction. Previously, we described a PLD gene family in tomato (Lycopersicon esculentum) and showed that expression of one of these genes, LePLDbeta1, was induced by treatment with the fungal elicitor xylanase. To further investigate the function of this PLD, a gene-specific RNAi construct was used to knock down levels of LePLDbeta1 transcript in suspension-cultured tomato cells. Silenced cells exhibited a strong decrease in xylanase-induced PLD activity and responded to xylanase treatment with a disproportionate oxidative burst. Furthermore, LePLDbeta1-silenced cell-suspension cultures were found to have increased polyphenol oxidase activity, to secrete less of the beta-d-xylosidase LeXYL2 and to secrete and express more of the xyloglucan-specific endoglucanase inhibitor protein XEGIP. Using an LePLDbeta1-green fluorescent protein (GFP) fusion protein for confocal laser scanning microscopy-mediated localization studies, untreated cells displayed a cytosolic localization, whereas treatment with xylanase induced relocalization to punctuate structures within the cytosol. Possible functions for PLDbeta in plant-pathogen interactions are discussed.
The Plant Journal 03/2006; 45(3):358-68. · 6.16 Impact Factor
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ABSTRACT: Phospholipase D (PLD) has been implicated in various cellular processes including membrane degradation, vesicular trafficking and signal transduction. Previously, we described a PLD gene family in tomato (Lycopersicon esculentum) and showed that expression of one of these genes, LePLDβ1, was induced by treatment with the fungal elicitor xylanase. To further investigate the function of this PLD, a gene-specific RNAi construct was used to knock down levels of LePLDβ1 transcript in suspension-cultured tomato cells. Silenced cells exhibited a strong decrease in xylanase-induced PLD activity and responded to xylanase treatment with a disproportionate oxidative burst. Furthermore, LePLDβ1-silenced cell-suspension cultures were found to have increased polyphenol oxidase activity, to secrete less of the β-d-xylosidase LeXYL2 and to secrete and express more of the xyloglucan-specific endoglucanase inhibitor protein XEGIP. Using an LePLDβ1–green fluorescent protein (GFP) fusion protein for confocal laser scanning microscopy-mediated localization studies, untreated cells displayed a cytosolic localization, whereas treatment with xylanase induced relocalization to punctuate structures within the cytosol. Possible functions for PLDβ in plant–pathogen interactions are discussed.
The Plant Journal 01/2006; 45(3):358 - 368. · 6.16 Impact Factor
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ABSTRACT: Plant cells respond to different biotic and abiotic stresses by producing various uncommon phospholipids that are believed to play key roles in cell signalling. We can predict how they work because animal and yeast proteins have been shown to have specific lipid-binding domains, which act as docking sites. When such proteins are recruited to the membrane locations where these phospholipids are synthesized, the phospholipids activate them directly, by inducing a conformational change, or indirectly, by juxtaposing them with an activator protein. The same lipid-binding domains are present in Arabidopsis proteins. We believe that they represent an untapped well of information about plant lipid signalling.
Trends in Plant Science 09/2004; 9(8):378-84. · 11.05 Impact Factor
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ABSTRACT: The Ac/Ds transposon system from maize was used for insertional mutagenesis in tomato. Marker genes were employed for the selection of plants carrying a total of 471 unique Ds elements. Three mutants were obtained with Ds insertions closely linked to recessive mutations: feebly (fb), yellow jim (yj) and dopey (dp). The fb seedlings produced high anthocyanin levels, developed into small fragile plants, and were insensitive to the herbicide phosphinothricin. The yj plants had yellow leaves as a result of reduced levels of chlorophyll. The dp mutants completely or partially lacked inflorescences. The fb and yj loci were genetically linked to the Ds donor site on chromosome 3. Reactivation of the Ds element in the fb mutants by crosses with an Ac-containing line resulted in restoration of the wild-type phenotypes. Plant DNA fragments flanking both sides of the Ds element in the fb mutant were isolated by the inverse polymerase chain reaction. Molecular analysis showed that phenotypic reversions of fb were correlated with excisions of Ds. DNA sequence analysis of Fb reversion alleles showed the characteristic Ds footprints. Northern and cDNA sequence analysis indicated that transcription of the FEEBLY (FB) gene was impeded by the insertion of Ds in an intron. Comparison of the predicted amino acid sequence of the FB protein with other database sequences indicated that FB is a novel gene.
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ABSTRACT: Quantitative differences in transgene expression between independent transformants are generally ascribed to different integration sites of the transgene (position effect). The contribution of spatial and temporal changes in transgene promoter activity to these position‐induced differences in transgene expression in planta are characterized, using the firefly luciferase ( luc ) reporter system. The activity of three different promoters (Cauliflower Mosaic Virus (CaMV) 35S, modified CaMV 35S and the promoter of an Arabidopsis thaliana Lipid Transfer Protein gene) was shown to vary not only among independent transformants, but also between leaves on the same plant and within a leaf. The differences in local LUC activity between leaves and within a leaf correlated with differences in local luc mRNA steady‐state levels. Imaging of LUC activity in the same leaves over a 50 d period, shows that individual transformants can show different types of temporal regulation. Both the spatial and the temporal type of luc transgene expression pattern are inherited by the next generation. It is concluded that previously reported position‐induced quantitative differences in transgene expression are probably an accumulated effect of differences in spatial and temporal regulation of transgene promoter activity.
