Cambial Region Research Articles

Proteolytic activity in the stem cambial region of Pinus sylvestris L. - A contribution to the specific differentiation of secondary xylem and phloem

Proteolytic activity was studied in the differentiating xylem and phloem of Scots pine (<i>Pinus sylvestris</i> L.) to determine the specificity of xylem and phloem differentiation. The activity of autolytic proteases was demonstrated in the differentiating xylem during spring, summer and autumn and it was not detectable during winter. It was initiated with the onset of cambial activity in spring and unchanged during subsequent stages of xylem differentiation. The same proteolytic activity was not detectable in the extract of fresh phloem tissue. It could be detected in phloem after removal of the inhibitor found in the extract. The same pH optimum was determined for proteases extracted from xylem and phloem. However, their identity remains uncertain because of different electrophoretic mobility. On the other hand the presence of protease inhibitor in phloem tissue can be an important factor im determining the specificity of xylem an phloem differentiation.

Hormonal Responses to Water Deficit in Cambial Tissues of Populus alba L.

Changes in the concentrations of bioactive gibberellins and abscisic acid in the cambial region of white poplar (Populus alba L.) were investigated in 1-year-old plants, to highlight how these phytohormone signals are modulated in response to water deficit. Plants were cultivated in pots outdoor and, at the time of maximum cambial growth (T 0), irrigation was withdrawn for 8 days, inducing a mild water deficit, thus mimicking a condition that is recurrent in Mediterranean climates when white poplar attains its maximum growth rate. The water deficit was suspended by resuming irrigation (T max) throughout a recovery period of 2 weeks (T rec). Cambial tissues were sampled at T 0, T max, and T rec. Significant changes of leaf and stem relative water content, leaf water potential, stomatal conductance, transpiration, carbon assimilation, stem shrinkage, and leaf number were induced by soil water shortage, which also negatively affected cambium development. Nevertheless, these responses were almost fully reversed following the resumption of irrigation. Water deficit induced the accumulation of large amounts of abscisic acid in cambial tissues, but the hormone was brought back to pre-stress levels after the recovery period. With regard to bioactive gibberellins, GA1 was several folds more abundant than GA4 and reached the greatest level in the plants recovering from the water status imbalance. The possible functions of gibberellins and abscisic acid in the response of cambial tissues to water deficit are discussed in view of the known physiological roles and molecular mechanisms of action of these hormonal signals.

OnPLS integration of transcriptomic, proteomic and metabolomic data shows multi-level oxidative stress responses in the cambium of transgenic hipI- superoxide dismutase Populus plants

BackgroundReactive oxygen species (ROS) are involved in the regulation of diverse physiological processes in plants, including various biotic and abiotic stress responses. Thus, oxidative stress tolerance mechanisms in plants are complex, and diverse responses at multiple levels need to be characterized in order to understand them. Here we present system responses to oxidative stress in Populus by integrating data from analyses of the cambial region of wild-type controls and plants expressing high-isoelectric-point superoxide dismutase (hipI-SOD) transcripts in antisense orientation showing a higher production of superoxide. The cambium, a thin cell layer, generates cells that differentiate to form either phloem or xylem and is hypothesized to be a major reason for phenotypic perturbations in the transgenic plants. Data from multiple platforms including transcriptomics (microarray analysis), proteomics (UPLC/QTOF-MS), and metabolomics (GC-TOF/MS, UPLC/MS, and UHPLC-LTQ/MS) were integrated using the most recent development of orthogonal projections to latent structures called OnPLS. OnPLS is a symmetrical multi-block method that does not depend on the order of analysis when more than two blocks are analysed. Significantly affected genes, proteins and metabolites were then visualized in painted pathway diagrams.ResultsThe main categories that appear to be significantly influenced in the transgenic plants were pathways related to redox regulation, carbon metabolism and protein degradation, e.g. the glycolysis and pentose phosphate pathways (PPP). The results provide system-level information on ROS metabolism and responses to oxidative stress, and indicate that some initial responses to oxidative stress may share common pathways.ConclusionThe proposed data evaluation strategy shows an efficient way of compiling complex, multi-platform datasets to obtain significant biological information.

Expression of a conifer COBRA-like gene ClCOBL1 from Chinese fir (Cunninghamia lanceolata) alters the leaf architecture in tobacco

The cell wall plays crucial roles in establishing the morphology of the plant cell, defence response to biotic and abiotic stresses, and mechanical properties of organs. The COBRA gene encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein that possesses the ability to modulate cellulose deposition and orient cell expansion in plant cell. We reported here the functional characterization of ClCOBL1, a conifer COBRA-like gene from the differentiating xylem of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook). ClCOBL1 belonged to a woody plant-specific clade of the COBRA protein family with several conserved motifs. Expression pattern demonstrated that ClCOBL1 was constitutively expressed but with high level in cambium region. ClCOBL1 protein was mainly located in the cell wall and plasma membrane. Overexpression of ClCOBL1 in tobacco plants yielded altered leaf adaxial-abaxial patterning and short, swollen corolla tubes. The changed leaf architecture in the ClCOBL1 overexpressors was associated with the differential expression of leaf adaxial-abaxial identity genes. Our results indicated that ClCOBL1 was involved in the determination of leaf dorsoventrality and anisotropic expansion possibly by affecting the expression of adaxial and abaxial identity genes.

Concomitant analysis of cambial abscisic acid and cambial growth activity in poplar

Endogenous levels of cambial region abscisic acid (ABA) were quantified by immunoassay and assessed together with cambial growth activity in poplar (Populus nigra L. × P. maximowiczii Henry, clone Kamabuchi) over the course of a growing season. The level of cambial region ABA increased from spring to late-summer but decreased sharply in autumn. Cambial growth activity, measured as the radial number of undifferentiated cambial cells and enlarging xylem cells, also increased from spring to summer and decreased sharply in autumn, indicating the onset of cambial dormancy. Exogenous ABA, applied laterally to poplar stems at two times within the growing season, enhanced cambial growth activity, as the radial number of undifferentiated cambial cells increased in ABA-treated trees subsequent to the two application times. Xylem cell development was also affected by exogenous ABA as fibre length increased significantly in ABA-treated trees at both application times. The positive correlation of cambial region ABA and cambial growth activity as well as the positive effects of exogenous ABA application thereon sheds new light on the role of this hormonal growth regulator.

The use of cultivars of Raphanus sativus for cytokinin bioassay

Six cultivars of radish (<i>Raphanus sativus</i>) were tested for their usefulness in radish cytokinin bioassay by the method of <i>Letham</i> (1971). The best cultivar was found to be 'Sopel Lodu' which responds well to both zeatin and 2iP over a wide range of concentrations. The fresh weight of cotyledons increased at most by 71.5% (if treated with zeatin) or 101.0% (if treated with 2iP) compared to untreated cotyledons. This cultivar is also sensitive to the partially purified cytokinin-like fraction isolated from the pine (<i>Pinus silvestris</i>) cambial region. The cultivar 'Sopel Lodu' is therefore proposed to be a suitable plant for cytokinin bioassays.

Symplasmic, long‐distance transport in xylem and cambial regions in branches of Acer pseudoplatanus (Aceraceae) and Populus tremula × P. tremuloides (Salicaceae)

The picture of how long-distance transport proceeds in trees is still far from being complete. Beside the apoplasmic pathway, transport undoubtedly also takes place within the system of living cells in the secondary xylem and cambial region. Because detailed, thorough studies of the symplasmic routes in woody branches, using direct localization with fluorescent tracers, had not been done, here we focused on the main routes of long-distance symplasmic transport in xylem and cambial tissues and analyzed in detail tracer distribution in the rays on the extended cambial surface in branches of Acer pseudoplatanus and Populus tremula ×P. tremuloides. Fluorescent tracers were loaded into branches through the vascular system, then their distribution in xylem and cambial regions was analyzed. Tracer signal was present in the symplast of axial and radial xylem parenchyma cells and in both types of cambial cells. The living cells of xylem parenchyma and of the cambium were symplasmically interconnected via xylem rays. Tracer distribution in rays was uneven on the extended cambial surface; cambial regions with intensively or sparsely dyed rays alternated along the vertical axis of analyzed branches. Symplasmic, long-distance transport is present between the living cells of xylem and the cambial region in woody branches. The uneven distribution of fluorescent tracers in cambial rays along the stems is surprising and suggests the presence of an intrinsic pattern caused by an unknown mechanism.

Cellular localization of aquaporin mRNA in hybrid poplar stems

Aquaporins (AQPs) are channel proteins, and their function is mostly associated with transmembrane water transport. While aquaporin genes are known to be expressed in woody poplar stems, little is known about AQP expression at the cellular level. Localization of AQP expression to particular cell and tissue types is a necessary prerequisite in understanding the biological role of these genes. Subsets of plants were subjected to 6 wk of high nitrogen fertilization (high N plants) or to a controlled drought. Experimental treatments affected cambial activity and wood anatomy. RNA in situ hybridization was used to characterize spatial expression of three AQP genes in stem cross sections. The strongest labeling consistently occurred in the cambial region and in adjacent xylem and phloem cells. Expression was also detected in rays. Contact cells exhibited high expression, while expression in other ray cells was more variable. High N plants exhibited a broader band of expression in the cambial region than plants receiving only adequate N fertilization (control plants) and plants subjected to drought. Water channels in stems were expressed in a manner that allows hydraulic coupling between xylem and other tissues that may serve as water reservoirs, including phloem and pith parenchyma. Expression of AQPs in rays may increase radial flow of water from xylem and phloem to the cambial region where AQPs may help sustain rapid cell division and expansion of developing vessel elements.

Oxidative stress proteins as an indicator of a low quality of eucalyptus clones for the pulp and paper industry

Eucalyptus is a genus widely cultivated in many tropical and subtropical regions of the world as one of the main sources of raw materials for the pulp and paper industry. Identification of clones and selection of genotypes with desirable agronomic characteristics would be useful. We assessed eucalyptus full-sibs that varied in wood quality, using a combination of two-dimensional gel electrophoresis and mass spectrometry to identify differentially expressed proteins as candidates for quality markers. Thirty-one differently expressed proteins were identified, including three proteins of clone X1, four of clone X2, and 12 each of clones X3 and X4. These proteins are involved in various biological processes, including polyphosphate biosynthesis, catalytic activity, nucleotide excision repair, cellular metabolic processes, cell redox homeostasis, response to salt stress, response to temperature, oxidation and reduction processes, cellular water homeostasis, and protein phosphorylation. In the cambial region of each clone, the proteins ketol-acid reductoisomerase, uncharacterized protein MG428, receptor-like serine/threonine-protein kinase and a heat shock protein were found in larger quantities in clone X4 than in clone X1. These proteins are known to be related to protection against oxidative stress and biosynthesis of lignin. A high buildup of proteins involved in response to stress in the cambial region of eucalyptus would indicate clones with undesirable characteristics for use in the pulp and paper industry.

Transcript Accumulation Dynamics of Phenylpropanoid Pathway Genes in the Maturing Xylem and Phloem of Picea abies during Latewood Formation

In temperate regions, latewood is produced when cambial activity declines with the approach of autumnal dormancy. The understanding of the temporal (cambium activity vs dormancy) and spatial (phloem, cambial region, maturing xylem) regulation of key genes involved in the phenylpropanoid pathway during latewood formation represents a crucial step towards providing new insights into the molecular basis of xylogenesis. In this study, the temporal pattern of transcript accumulation of 12 phenylpropanoid genes (PAL1, C4H3/5, C4H4, 4CL3, 4CL4, HCT1, C3H3, CCoAOMT1, COMT2, COMT5, CCR2) was analyzed in maturing xylem and phloem of Picea abies during latewood formation. Quantitative reverse transcription-polymerase chain reaction analyses revealed a well-defined RNA accumulation pattern of genes involved in the phenylpropanoid pathway during latewood formation. Differences in the RNA accumulation patterns were detected between the different tissue types analyzed. The results obtained here demonstrated that the molecular processes involved in monolignol biosynthesis are not restricted to the cambial activity timeframe but continued after the end of cambium cell proliferation. Furthermore, since it has been shown that lignification of maturing xylem takes place in late autumn, we argue on the basis of our data that phloem could play a key role in the monolignol biosynthesis process.

Expression pattern of the GRAS gene family during somatic embryogenesis in pine

The GRAS protein family of putative transcription factors, which includes SHORT-ROOT (SHR), SCARECROW (SCR) and SCARECROW-LIKE (SCL) proteins, is involved in root development in Arabidopsisthaliana and other plant species [1]. In forest species, genes with homology to the A. thalianaSCR gene have been involved in the induction of somatic embryogenesis in Picea glauca (Moench) Voss [2] and Pinus taeda L. [3] as well as in the development of radial patterning of roots in Pinus sylvestris L. [4]. Schrader et al[5] also reported the expression of genes with homology to the A. thalianaSHR gene in cambial region of Populus tremula x tremuloides. Increased levels of mRNA of Pinus radiata SHR (PrSHR), Pinus radiata SCARECROW-LIKE1 (PrSCL1) and Castanea sativaSCARECROW-LIKE1 (CsSCL1) have been associated with the early stages of adventitious root induction in Pinus radiata D. Don and Castanea sativa Mill., respectively [6-9]. In addition to PrSHR and PrSCL1, we have identified 13 new GRAS genes belonging to the different GRAS clades in the pine genome. The objective of this work is the analysis of the spatiotemporal expression patterns of the pine GRAS gene family during somatic embryogenesis in Pinus radiata D. Don. Somatic embryogenesis has become the first biotechnology showing great potential for mass propagation of conifers for application in forestry, allowing the implementation of multivarietal forestry (MVF) [10,11]. Despite major advances in clonal regeneration by somatic embryogenesis or organogenesis, many forestry species are recalcitrant [12]. More knowledge of the regeneration process regulation is necessary to improve the capacity of vegetative regeneration. The expression pattern of the genes was analyzed by qRT-PCR following the methodology described by Sanchez et al[6] and Sole et al[7]. For expression analysis, total RNA was extracted from four stages of the somatic embryogenic process: proliferative tissue after 7 and 14 days from the last transference to proliferation medium, somatic embryos at the beginning of differentiation and cotyledonary somatic embryos. In general, the transcripts of the pine GRAS genes accumulated at the highest levels in cotyledonary somatic embryos. In addition, the transcript levels of PrSCR, PrSHR, PrSCL1, PrSCL6, PrSCL8, PrSCL11 andPrSCL12 showed an increase in somatic embryos at the beginning of differentiation. No differences in PrSCL10 transcript levels were found between the four stages analyzed. Transcript levels of PrSCL16 were undetectable at all stages. In situ hybridization for spatial expression analysis will confirm differential expression domains. This work has been funded by the Spanish Ministry of Science and Innovation (AGL-2008-05105-C02-01/FOR). Embryogenic lines were provided by C. Walter (Scion).

Feeding by emerald ash borer larvae induces systemic changes in black ash foliar chemistry

The exotic wood-boring pest, emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), has been threatening North American ash ( Fraxinus spp.) resources, this being recognized since its first detection in Michigan, USA and Ontario, Canada in 2002. Ash trees are killed by larval feeding in the cambial region, which results in disruption of photosynthate and nutrient translocation. In this study, changes in volatile and non-volatile foliar phytochemicals of potted 2-yr-old black ash, Fraxinus nigra Marshall, seedlings were observed in response to EAB larval feeding in the main stem. EAB larval feeding affected levels of six compounds [hexanal, ( E)-2-hexenal, ( Z)-3-hexenyl acetate, ( E)- β-ocimene, methyl salicylate, and ( Z,E)- α-farnesene] with patterns of interaction depending upon compounds of interest and time of observation. Increased methyl salicylate emission suggests similarity in responses induced by EAB larval feeding and other phloem-feeding herbivores. Overall, EAB larval feeding suppressed ( Z)-3-hexenyl acetate emission, elevated ( E)- β-ocimene emission in the first 30 days, but emissions leveled off thereafter, and generally increased the emission of ( Z,E)- α-farnesene. Levels of carbohydrates and phenolics increased overall, while levels of proteins and most amino acids decreased in response to larval feeding. Twenty-three amino acids were consistently detected in the foliage of black ash. The three most abundant amino acids were aspartic acid, glutamic acid, glutamine, while the four least abundant were α-aminobutyric acid, β-aminoisobutyric acid, methionine, and sarcosine. Most (16) foliar free amino acids and 6 of the 9 detected essential amino acids decreased with EAB larval feeding. The ecological consequences of these dynamic phytochemical changes on herbivores harbored by ash trees and potential natural enemies of these herbivores are discussed.

Effect of Water Deficit on Expression of Stress-related Genes in the Cambial Region of Two Contrasting Poplar Clones

In this study we investigated the effect of prolonged water shortage in the cambial region of two poplar clones, Dvina (Populus deltoides) and I-214 (Populus x canadensis) that differ in their response to water deficit. For this purpose we monitored growth parameters in Dvina and I-214 plants under well-watered and water-stress conditions and after rewatering and analyzed the content of malondialdehyde (MDA) and proline and the expression level of genes coding for antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; glutathione reductase, GR) and metallothioneins (MT) by quantitative real-time RT-PCR (RT-qPCR). Water deficit resulted in a significant increase of osmotically active solutes in both clones. No significant increase of MDA level was observed in Dvina, whereas a significant enhancement of lipid peroxidation was detected in I-214, in which also a strong fivefold increase of proline was detected. Transcript analysis of stress-related genes indicated a different ability of the two clones to modulate antioxidant genes under stress. In particular, MT3b was strongly upregulated by water deficit in Dvina thus suggesting an important role for this gene in the maintenance of cellular redox homeostasis in the clone. On the whole, the two clones exhibited changes at the transcription and physiological levels in the cambial region that suggest the occurrence of different strategies of plant protection from prolonged water deficit.

Moisture content and nutrition as selection forces for emerald ash borer larval feeding behaviour

The exotic phloem-feeding emerald ash borer (EAB), Agrilus planipennis, has killed tens of millions of North American ash trees (Fraxinus) since its first detection in the U.S.A. in 2002. Ash trees are killed by larval feeding in the cambial region, which disrupts translocation of photosynthates and nutrients. We observed that EAB larvae feed predominantly downwards in naturally grown green ash trees, a behaviour confirmed in greenhouse-grown black ash seedlings. Furthermore, biomass of larvae feeding downwards was greater than that for larvae feeding upwards. We sought to determine the relative importance of four selection forces (i.e. gravity, moisture content, plant defence, and nutrition) in driving this downward feeding behaviour in this study. The gravity and plant defence (i.e. polyphenols) hypotheses were ruled out because even when seedlings were grown upside down, more EAB larvae moved upwards (towards the root area), and phloem tissue below the feeding site contained higher concentrations of defensive compounds than that above the feeding site.

Sampling cambial region and mature xylem for non structural carbohydrates and starch analyses

This paper describes a procedure to analyze non-structural carbohydrates (NSC) and starch in xylem and cambial region extracted by the scraping method. An application on woody logs of 2-year-old Populus × canadensis Mönch ‘I-214’ during tree-ring formation is reported. Samples are freeze-dried in liquid nitrogen and, successively, the bark is removed. After drying, the breaks between bark and xylem take place in correspondence of differentiating xylem cells. The cambial region with the differentiating phloem and xylem is gently scraped with a razor blade from the inner side of the bark and the outermost side of the stem, respectively. Additionally the mature xylem is milled until obtaining a powder. The amount of the cambial region collected by scraping changes with respect to the width of the differentiating zone (differentiating phloem and xylem), with higher amount of dry matter obtained during the maximum rate of xylem formation and high stem water content. After extraction of the powder in an ethanol solution and centrifugation, the supernatant and the resulting pellet are used for assessing the contents of non structural carbohydrates (NSC) and starch, respectively. NSC are determined using the High-Performance Liquid Chromatography (HPLC) analysis and starch by colorimetric method. In both cambial region and xylem, the major NSC are glucose and fructose which represented together more than 80% of the total NSC during wood formation. However, the total NSC are higher in cambial region compared to xylem, especially when the xylem and phloem are differentiating. The scraping technique provides the possibility to sample cambial region during the year, allowing the investigation of metabolic changes during tree-ring formation.

Seasonal changes in the degree of symplasmic continuity between the cells of cambial region of Acer pseudoplatanus and Ulmus minor

The presence of symplasmic isolation and symplasmic continuity which are functional aspects of cell-to-cell communication, had been studied in cambium of <em>Acer pseudoplatanus</em> and <em>Ulmus minor</em>, with hope that uniqueness of this meristem, exemplified by its morphology and seasonal variations in its activity is also manifested in differences in the efficiency of communication between cambial cells during the year. The degree of symplasmic continuity was estimated by loading the fluorescent symplasmic tracer to the stem and following its distribution in a population of cambial cells observed on tangential, transverse and radial sections. In active cambium the tracer did not enter the rays. This suggested that the ray and fusiform cells, growing and dividing intensively at different rates were specifically isolated from each other. In the state of dormancy the tracer was present also in the rays implying continuity between the two types of cambial cells. Temporal restriction in tracer spreading from secondary xylem to cambial region was observed on transverse sections in both physiological states of the meristem. Higher degree of symplasmic isolation in active cambium is, most probably, associated with functional distinctiveness of ray and fusiform cells. We hypothesize further that the symplasmic continuity in dormant cambium results from the open conformation states of plasmodesmata, because the energy costs of these states are low. It is reasonable strategy when cambial cells do not divide and maintenance of their functional individuality is not necessary.

Overexpression of two cambium‐abundant Chinese fir (Cunninghamia lanceolata) α‐expansin genes ClEXPA1 and ClEXPA2 affect growth and development in transgenic tobacco and increase the amount of cellulose in stem cell walls

Expansins are unique plant cell wall proteins that possess the ability to induce immediately cell wall extension in vitro and cell expansion in vivo. To investigate the biological functions of expansins that are abundant in wood-forming tissues, we cloned two expansin genes from the differentiating xylem of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook). Phylogenetic reconstruction indicated that they belong to α-expansin (EXPA), named ClEXPA1 and ClEXPA2. Expression pattern analysis demonstrated that they are preferentially expressed in the cambium region. Overexpression of ClEXPA1 and ClEXPA2 in tobacco plants yielded pleiotropic phenotypes of plant height, stem diameter, leaf number and seed pod. The height and diameter growth of the 35S(pro) :ClEXPA1 and 35S(pro) :ClEXPA2 transgenic plants were increased drastically, exhibiting an enlargement of pith parenchyma cell size. Isolated cell walls of ClEXPA1 and ClEXPA2 overexpressors contained 30%-50% higher cellulose contents than the wild type, accompanied by a thickening of the cell walls in the xylem region. Both ClEXPA1 and ClEXPA2 are involved in plant growth and development, with a partially functional overlap. Expansins are not only able to induce cell expansion in different tissues/organs in vivo, but they also can act as a potential activator during secondary wall formation by directly or indirectly affecting cellulose metabolism, probably in a cell type-dependent manner.

Immunolocalization indicates plasmodesmal trafficking of storage proteins during cambial reactivation in Populus nigra

Cambium reactivation after dormancy and budbreak in deciduous trees requires a supply of mobilized reserve materials. The pathway and mode of transfer of these materials are poorly understood. Transport of reserve materials during cambium reactivation in Populus nigra was investigated by conventional and immunocytochemical TEM analyses, SDS-PAGE, western blotting and intracellular microinjection of fluorescent dyes. Proteinaceous compounds stored in vacuoles and protein bodies of vascular cells and ray cells disappeared within 3 weeks after cambial reactivation and budbreak. Some of these proteins (32 kDa, 30 kDa and 15 kDa) were labelled by lectin antibodies in SDS-PAGE. The same antibodies were localized to plasmodesmata (PDs) between phloem parenchyma, ray cells and fusiform cambial cells. In addition, proteinaceous particles were localized inside the cytoplasmic sleeves of these PDs during budbreak. During this period, the functional diameter of PDs was about 2.2 nm which corresponds approximately to the Stokes' radius of the detected 15-kDa protein. Lectin-like reserve proteins or their degradation products seem to be transferred through PDs of phloem parenchyma and rays during cambial reactivation and budbreak. PD transfer of storage proteins is a novelty which supports the concept of symplasmic nutrient supply to the cambial region.

Transcriptome changes in the cambial region of poplar (Populus alba L.) in response to water deficit

A transcriptome analysis of the Populus alba cambial region was performed with the aim of elucidating the gene network underlying the response to water deficit within the cambium and differentiating derivative cambial cells. Water stress was induced in 1-year-old P. alba plants by withholding water for 9 days. At that time, leaf predawn water potential fell to -0.8 MPa, resulting in a significant reduction in stomatal conductance, CO(2) assimilation and a consistent increment of stem shrinkage. These effects were almost fully reversed by re-hydration. The water deficit resulted in changes in gene expression that affected several functional categories, such as protein metabolism, cell wall metabolism, stress response, transporters and transcriptional regulation. The function of up- and down-regulated genes is discussed considering the physiological response of the plants to water deficit.

Cloning and endogenous expression of a Eucalyptus grandis UDP-glucose dehydrogenase cDNA

UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis.