GRAB proteins,novel members of the NAC domain family,isolated by their interaction with a geminivirus protein

Geminiviruses encode a few proteins and depend on cellular factors to complete their replicative cycle. As a way to understand geminivirus-host interactions, we have searched for cellular proteins which interact with viral proteins. By using the yeast two-hybrid technology and the wheat dwarf geminivirus (WDV) RepA protein as a bait, we have isolated a family of proteins which we termed GRAB (for Geminivirus Rep A-binding). We report here the molecular characterization of two members, GRAB1 and GRAB2. We have found that the 37 C-terminal amino acids of RepA are required for interaction with GRAB proteins. This region contains residues conserved in an equivalent region of the RepA proteins encoded by other viruses of the WDV subgroup. The N-terminal domain of GRAB proteins is necessary and sufficient to interact with WDV RepA. GRAB proteins contain an unique acidic C-terminal domain while their N-terminal domain, of ca. 170 amino acids, are highly conserved in all of them. Interestingly, this conserved N-terminal domain of GRAB proteins exhibits a significant amino acid homology to the NAC domain present in proteins involved in plant development and senescence. GRAB1 and GRAB2 mRNAs are present in cultured cells and roots but are barely detectable in leaves. GRAB expression inhibits WDV DNA replication in cultured wheat cells. Our studies highlight the importance that the pathway(s) mediated by GRAB proteins, as well as by other NAC domain-containing proteins, might have on geminivirus DNA replication in connection to plant growth, development and senescence pathways.     

Biochemical characterization of xylan xylosyltransferases involved in wood formation in poplar

The major polysaccharides in dicot wood biomass are cellulose and xylan. Although wood-associated cellulose synthase genes responsible for cellulose biosynthesis have been characterized, wood-associated xylan synthase genes have not been biochemically identified. A recent report by Lee et al. (2012) provides the first biochemical evidence that two functionally non-redundant Arabidopsis GT43 members are xylosyltransferases (XylTs) that function cooperatively in the elongation of the xylan backbone. We further extend this finding in the current report demonstrating that two poplar (Populus trichocarpa) GT43 glycosyltransferases, PtrGT43B and PtrGT43C, are xylan XylTs involved in wood formation. We show that microsomes from transgenic tobacco BY2 cells coexpressing PtrGT43B and PtrGT43C exhibited a high XylT activity capable of generating β-(1,4)-linked xylooligosaccharides, whereas little XylT activity was detected in microsomes with expression of PtrGT43B or PtrGT43C alone. These findings indicate that poplar GT43 members are XylTs that act cooperatively in catalyzing the successive transfer of xylosyl residues during xylan backbone biosynthesis, which provides further support of the hypothesis that the biochemical functions of GT43 members in vascular plants are evolutionarily conserved.     

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.     

Asymmetric expression of a poplar ACC oxidase controls ethylene production during gravitational induction of tension wood

Ethylene is produced in wood-forming tissues, and when applied exogenously, it has been shown to cause profound effects on the pattern and rate of wood development. However, the molecular regulation of ethylene biosynthesis during wood formation is poorly understood. We have characterised an abundant 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene (PttACO1) in the wood-forming tissues of Populus tremula (L.) x P. tremuloides (Michx). PttACO1 is primarily expressed in developing secondary xylem, and is specifically upregulated during secondary wall formation. Nevertheless, according to GC-MS analysis combined with tangential cryosectioning, the distribution of ACC was found to be fairly uniform across the cambial-region tissues. Gravitational stimulation, which causes tension wood to form on the upper side of the stem, resulted in a strong induction of PttACO1 expression and ACC oxidase activity in the tension wood-forming tissues. The ACC levels increased in parallel to the PttACO1 expression. However, the increase on the upper (tension wood) side was only minor, whereas large amounts of both ACC and its hydrolysable conjugates accumulated on the lower (opposite) side of the stem. This suggests that the relatively low level of ACC on the tension wood side is a result of its conversion to ethylene by the highly upregulated PttACO1, and the concurrent accumulation of ACC on the opposite side of the wood is because of the low PttACO1 levels. We conclude that PttACO1 and ACC oxidase activity, but not ACC availability, are important in the control of the asymmetric ethylene production within the poplar stem when tension wood is induced by gravitational stimulation.     

Calcium nutrition has a significant influence on wood formation in poplar

To test the effects of calcium on wood formation, Populus tremula x Populus tremuloides clones were supplied with Hoagland solution modified in its calcium contents. Energy-dispersive X-ray analysis (EDXA) revealed an increase in calcium in the phloem, the cambium and the xylem elongation zone with increasing Ca(2+) supply in the nutrient solution. Using light and electron microscopy, a strong impact was shown on the cambial and the elongation zones under calcium starvation. Using Fourier transform infrared (FTIR) spectroscopy on wood and bark cells formed under calcium starvation, we detected a reduction of some absorptions, such as carbonyl and methoxy groups from S-lignin. Also, a significant reduction in fiber length was detected with decreasing calcium supply in the nutrient solution. High-performance liquid chromatography (HPLC) analysis revealed a large increase in sugar concentrations in the leaves, but reduced concentrations in the bark under Ca(2+) deficiency. In conclusion, our results show a significant influence of calcium on the structure, chemistry and physiology of wood formation. Thus, efficient Ca(2+) supply has to be considered a decisive factor in wood formation.     

Role of membrane potential in protein folding and domain formation during secretion in Escherichia coli

The synthesis and processing of the periplasmic components of the leucine transport system of E coli have been studied to determine the role played by transmembrane potential in protein secretion. Both the leucine-isoleucine-valine binding protein and the leucine-specific binding protein are synthesized as precursors with 23 amino acid N-terminal leader sequences. The processing of these precursors is sensitive to the transmembrane potential. Since the amino acid sequence and the crystal structure have been determined for the leucine-isoleucine-valine binding protein, it and the closely related leucine-specific binding protein represent convenient models in which to examine the mechanism of protein secretion in E coli. A model for secretion has been proposed, suggesting a role for transmembrane potential. In this model, the N-terminal amino acid sequence of the precursor is assumed to form a hairpin of two helices. The membrane potential may orient this structure to make it accessible to processing. In addition, the model suggests that a negatively charged, folded domain of the secretory protein may electrophorese toward the trans-positive side of the membrane, thus providing an additional role for the transmembrane potential.     

Evaluation of lignocellulolytic activities of ten fungal species able to degrade poplar wood

The study of wood decay fungi that naturally biodegrade lignocellulosic polymers has been steadily increasing during the past two decades due to their industrial and innovative applications. In this work, we compare ten species of lignicolous macrofungi which develop fruiting bodies on poplar in relation to their capacity for growing on poplar wood chips and sawdust and of secreting cell wall degrading enzymes. All the fungi studied appeared to be able to grow well in these conditions and to secrete cellulase and hemicellulase, Mn-peroxidase and cellobiose dehydrogenase, while Li-peroxidase and laccase were produced by seven and six out of the ten species, respectively. Variability in the levels of all these enzymatic activities was assessed. Two species, never investigated before, showed the best performances as regards production of cellulolytic and hemicellulolytic activities (Lenzites warnieri) and Mn-peroxidase (Perenniporia meridionalis). The highest laccase level was detected in the well known plant pathogen Fomes fomentarius, and the brown-rot Daedalea quercina proved to be the best producer of lignin peroxidase and cellobiose dehydrogenase.