Gene expression in Eucalyptus branch wood with marked variation in cellulose microfibril orientation and lacking G-layers

In response to gravitational stresses, angiosperm trees form tension wood in the upper sides of branches and leaning stems in which cellulose content is higher, microfibrils are typically aligned closely with the fibre axis and the fibres often have a thick inner gelatinous cell wall layer (G-layer). Gene expression was studied in Eucalyptus nitens branches oriented at 45 degrees using microarrays containing 4900 xylem cDNAs, and wood fibre characteristics revealed by X-ray diffraction, chemical and histochemical methods. Xylem fibres in tension wood (upper branch) had a low microfibril angle, contained few fibres with G-layers and had higher cellulose and decreased Klason lignin compared with lower branch wood. Expression of two closely related fasciclin-like arabinogalactan proteins and a beta-tubulin was inversely correlated with microfibril angle in upper and lower xylem from branches. Structural and chemical modifications throughout the secondary cell walls of fibres sufficient to resist tension forces in branches can occur in the absence of G-layer enriched fibres and some important genes involved in responses to gravitational stress in eucalypt xylem are identified.     

Cytosolic glutamine synthetase in higher plants. A comparative immunological study

Cytosolic glutamine synthetase (GS1) was purified to homogeneity from etiolated barley leaves by DEAE-Sephacel and hydroxyapatite chromatography, gel filtration and polyacrylamide gel electrophoresis. Specific antibodies against the purified protein were raised by the immunization of rabbits. Immunoprecipitation experiments demonstrated that cytosolic glutamine synthetases isolated from the leaves of different plant species were very similar proteins. Good recognition of other cytosolic glutamine synthetases from roots, root nodular tissue and seeds by barley GS1 antibodies was obtained, suggesting that they too are all quite similar proteins. In contrast, chloroplast glutamine synthetase (GS2) was considered to be a different protein in view of its low level of recognition by barley GS1 antibodies.     

Molecular characterization of PoGT8D and PoGT43B, two secondary wall-associated glycosyltransferases in poplar

Dicot wood is mainly composed of cellulose, lignin and glucuronoxylan (GX). Although the biosynthetic genes for cellulose and lignin have been studied intensively, little is known about the genes involved in the biosynthesis of GX during wood formation. Here, we report the molecular characterization of two genes, PoGT8D and PoGT43B, which encode putative glycosyltransferases, in the hybrid poplar Populus alba x tremula. The predicted amino acid sequences of PoGT8D and PoGT43B exhibit 89 and 75% similarity to the Arabidopsis thaliana IRREGULAR XYLEM8 (IRX8) and IRX9, respectively, both of which have been shown to be required for GX biosynthesis. The PoGT8D and PoGT43B genes were found to be expressed in cells undergoing secondary wall thickening, including the primary xylem, secondary xylem and phloem fibers in stems, and the secondary xylem in roots. Both PoGT8D and PoGT43B are predicted to be type II membrane proteins and shown to be targeted to Golgi. Overexpression of PoGT43B in the irx9 mutant was able to rescue the defects in plant size and secondary wall thickness and partially restore the xylose content. Taken together, our results demonstrate that PoGT8D and PoGT43B are Golgi-localized, secondary wall-associated proteins, and PoGT43B is a functional ortholog of IRX9 involved in GX biosynthesis during wood formation.     

Influence of over-expression of the FLOWERING PROMOTING FACTOR 1 gene (FPF1) from Arabidopsis on wood formation in hybrid poplar (Populus tremula L. × P. tremuloides Michx.)

Constitutive expression of the FPF1 gene in hybrid aspen (Populus tremula L. × P. tremuloides Michx.) showed a strong effect on wood formation but no effect on flowering time. Gene expression studies showed that activity of flowering time genes PtFT1, PtCO2, and PtFUL was not increased in FPF1 transgenic plants. However, the SOC1/TM3 class gene PTM5, which has been related to wood formation and flowering time, showed a strong activity in stems of all transgenic lines studied. Wood density was lower in transgenic plants, despite significantly reduced vessel frequency which was overcompensated by thinner fibre cell walls. Chemical screening of the wood by pyrolysis GC/MS showed that FPF1 transgenics have higher fractions of cellulose and glucomannan products as well as lower lignin content. The latter observation was confirmed by UV microspectrophotometry on a cellular level. Topochemical lignin distribution revealed a slower increase of lignin incorporation in the developing xylem of the transgenics when compared with the wild-type plants. In line with the reduced wood density, micromechanical wood properties such as stiffness and ultimate stress were also significantly reduced in all transgenic lines. Thus, we provide evidence that FPF1 class genes may play a regulatory role in both wood formation and flowering in poplar.     

A comparison of proteins from the developing xylem of compression and non-compression wood of branches of sitka spruce (Picea sitchensis) reveals a differentially expressed laccase

Soluble and cell wall-associated proteins were extracted from the developing xylem of the compression and non-compression sides of branches of Sitka spruce (Picea sitchensis (Bong) Carr.) by an identical procedure. Equal amounts of proteins were separated by SDS-PAGE, and polypeptides were identified that were more abundant in soluble and cell wall-associated extracts from the developing xylem of either compression or non-compression wood. Two polypeptides (at apparent M(r)s of 48 kDa and 120 kDa) that were more abundant in cell wall-associated extracts of the developing xylem of the compression tissues were selected for amino-terminal protein sequencing. The 48 kDa polypeptide yielded an amino-terminal sequence that had no homology with known protein, gene or EST database sequences. The amino-terminal sequence of the 120 kDa polypeptide was homologous to a number of laccase-type polyphenol oxidases (EC 1.10.3.2) thought to be involved in lignin biosynthesis in trees. Using non-denaturing SDS-PAGE, the 120 kDa laccase was confirmed as a major oxidase activity in extracts of lignifying compression xylem but it was barely detectable in the non-compression extracts where an 85 kDa oxidase was the predominant activity. The differential expression of oxidases in compression and non-compression xylem is discussed.     

Enhanced expression of glutamine synthetase (GS1a) confers altered fibre and wood chemistry in field grown hybrid poplar (Populus tremula X alba) (717-1B4)

Hybrid poplar (Populus tremula X P. alba) genetically engineered to express the pine cytosolic glutamine synthetase gene (GS1a) has been previously shown to display desirable field performance characteristics, including enhancements in growth and nitrogen use efficiency. Analysis of wood samples from a 3‐year‐old field trial of three independently transformed GS1a transgenic hybrid poplar lines revealed that, when compared with wild‐type controls, ectopic expression of GS1a resulted in alterations in wood properties and wood chemistry. Included were significant enhancements in wood fibre length, wood density, microfibre angle, per cent syringyl lignin and elevated concentrations of wood sugars, specifically glucose, galactose, mannose and xylose. Total extractive content and acid‐insoluble lignin were significantly reduced in wood of GS1a transgenics when compared with wild‐type trees. Together, these cell wall characteristics resulted in improved wood pulping attributes, including improved lignin solubilization with no concurrent decrease in yield. Trees with increased GS1a expression have improved characteristics for pulp and paper production and hold potential as a feedstock for biofuels production.     

Characterization of a lignin-specific O-Methyltransferase in aspen wood

O-Methyltransferases were extracted from the differentiating xylem of 10-yr-old Populus euramericana. The enzymes were partially purified by ammonium sulfate precipitation, and column chromatography on DEAE-cellulose, Sephadex G200 and hydroxyapatite. The enzymes were resolved into two peaks by DEAE-cellulose chromatography, and the MWs of the respective enzymes were estimated to be 72 000 and 75 000 by gel filtration chromatography. The enzyme corresponding to the latter peak was unstable and thus only the former peak enzyme was characterized completely. Magnesium ions had no effect, EDTA moderately stimulated and heavy metals and SH group inhibitors strongly inhibited enzyme activity. K_mm values for caffeate and 5-hydroxyferulate were estimated to be 3.8 x 10⁻⁴ and 3.1 x 10⁻⁴ M, respectively. The ratio of V_max/K_m for 5-hydroxyferulate was 5.4 times greater than that for caffeate. The enzyme(s) catalysing the formation of ferulate from caffeate and of sinapate from 5-hydroxyferulate were not separated during the purification or by the disc electrophoresis using polyacrylamide gel. Quercetin, cyanin and catechin were not methylated by the enzyme preparation. The O-methyltransferase of aspen wood, where the phenolic metabolism is almost exclusively directed to lignin biosynthesis, catalyses the methylation of both guaiacyl and syringyl lignin precursors, with preferential utilization of the latter substrate. These findings lead to the conclusion that the enzyme is a typical angiosperm-type O-methyltransferase related to guaiacyl and syringyl lignin biosynthesis in aspen wood.     

Electron Transport Controls Glutamine Synthetase Activity in the Facultative Heterotrophic Cyanobacterium Synechocystis sp. PCC 6803

Glutamine synthetase (GS) from Synechocystis sp. PCC 6803 was inactivated in vivo by transferring cells from light to darkness or by incubation with the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea but not with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. Addition of glucose prevented both dark and 3-(3,4-dichlorophenyl)-1,1-dimethylurea GS inactivation. In a Synechocystis psbE-psbF mutant (T1297) lacking photosystem II, glucose was required to maintain active GS, even in the light. However, in nitrogen-starved T1297 cells the removal of glucose did not affect GS activity. The fact that dark-inactivated GS was reactivated in vitro by the same treatments that reactivate the ammonium-inactivated GS points out that both nitrogen metabolism and redox state of the cells lead to the same molecular regulatory mechanism in the control of GS activity. Using GS antibodies we detected that dark-inactivated GS displayed a different electrophoretic migration with respect to the active form in nondenaturing polyacrylamide gel electrophoresis but not in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possible pathway to modulate GS activity by the electron transport flow in Synechocystis cells is discussed.     

家蚕蛾触角蛋白的双向电泳分析

为探讨家蚕Bombyx mori蛾触角发生发育、结构与功能的分子基础和调控机制, 我们采用双向电泳结合基质辅助质量飞行时间质谱(MALDI-TOF/MS)技术初步探讨了家蚕蛾触角蛋白及其雌雄表达差异。采用ImageMast 6.0软件分析电泳图谱, 在家蚕蛾触角中检测到约550个蛋白点, 主要集中在分子量14～70 kD, 等电点4～8之间。从雌雄蛾触角电泳图谱中分别检测到419和489个蛋白点, 其中雌雄匹配蛋白点有326对, 匹配率为71.81%。雌雄间表达差异点有34个, 雌雄分别所特有的特异蛋白点分别为9和20个。对特异和差异点进行MALDI-TOF/MS鉴定获得其中5种蛋白--成虫原基生长因子(imaginal disk growth factor)、表皮蛋白RR-1基序15(cuticular protein RR-1 motif 15)、硫醇过氧化还原酶(thiol peroxiredoxin)、空泡ATP酶B亚基(vacuolar ATPase B subunit)以及gasp前体(gasp precursor), 它们在雄蛾触角中表达量都比雌蛾高。这为家蚕触角蛋白的进一步研究提供了基本信息。     

New applications for an old lignified element staining reagent

The use is reported of Mirande's reagent in epifluorescence microscopy which permits a clear distinction between cellulosic and lignified tissues. Homogeneous Prespermatophytae and gymnosperm xylem appeared entirely green with Mirande's reagent under ultraviolet excitation, whereas heteroxyled angiosperm wood showed a mixed pink and blue–green colour. This coloration was due to the fluorescence of cellulose, since certain elements in dicotyledonous wood (parenchyma, fibres, xylem rays) are not entirely lignified. Monocotyledonous (Poaceae) lignin showed an intense blue fluorescence due to hydroxycinnamic acids bound to the cell wall.The method showed that lignification occurs first in the middle lamella, and later in the secondary wall of xylem cells. In addition, this staining technique proved useful in the study of lignin and suberin deposition in response to various stress factors.     

A technique of low-pH gel electrophoresis of chromosomal proteins which does not require preliminary removal of DNA.

Fractionation of chromosomal proteins, in particular, of histones, by acetic acid-urea polyacrylamide gel electrophoresis usually requires preliminary removal of DNA from deoxyribonucleoprotein samples to obtain good separation of proteins. We have found that this difficulty can be overcome by addition of cetyltrimethylammonium bromide (CTAB) to the gel and electrode buffers. Since CTAB can readily diffuse into polyacrylamide gels two-dimensional fractionation becomes possible; that is, deoxyribonucleoprotein particles are fractionated in the first dimension followed by immersion of a gel in a CTAB solution and then low-pH gel electrophoresis of proteins in the second dimension.     

Cellulose acetate impregnated with polyacrylamide: a novel medium for electrophoresis

Cross-linked polyacrylamide gel containing a low proportion of methylenebis-acrylamide has been incorporated into cellulose acetate membranes. Unlike on cellulose acetate itself, electrophoresis on these modified membranes enables molecular sieving of proteins under a wide range of conditions. By modifying only part of the membrane, samples can be loaded in the normal way and sharpen as they migrate across the cellulose acetate-polyacrylamide boundary. The thin membranes retain their general ease of handling and speed of staining and destaining.