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.     

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.     

Overexpression of the PtSOS2 gene improves tolerance to salt stress in transgenic poplar plants

In higher plants, the salt overly sensitive (SOS) signalling pathway plays a crucial role in maintaining ion homoeostasis and conferring salt tolerance under salinity condition. Previously, we functionally characterized the conserved SOS pathway in the woody plant Populus trichocarpa. In this study, we demonstrate that overexpression of the constitutively active form of PtSOS2 (PtSOS2TD), one of the key components of this pathway, significantly increased salt tolerance in aspen hybrid clone Shanxin Yang (Populus davidiana × Populus bolleana). Compared to the wild‐type control, transgenic plants constitutively expressing PtSOS2TD exhibited more vigorous growth and produced greater biomass in the presence of high concentrations of NaCl. The improved salt tolerance was associated with a decreased Na⁺ accumulation in the leaves of transgenic plants. Further analyses revealed that plasma membrane Na⁺/H⁺ exchange activity and Na⁺ efflux in transgenic plants were significantly higher than those in the wild‐type plants. Moreover, transgenic plants showed improved capacity in scavenging reactive oxygen species (ROS) generated by salt stress. Taken together, our results suggest that PtSOS2 could serve as an ideal target gene to genetically engineer salt‐tolerant trees.     

Short-term effects of nitrogen availability on wood formation and fibre properties in hybrid poplar

The application of nitrogen-containing fertilisers is one approach used to increase growth rates and productivity of forest tree plantations. However, the effects of nitrogen fertilisation on wood properties have not been systematically assessed. The aim of this work was to document the impacts of nitrogen fertilisation on wood formation and secondary xylem fibre properties. We used three fertilisation treatments in which the level of ammonium nitrate was adjusted to 0, 1 and 10 mM in a complete nutrient solution applied daily over a period of 28 days in standardised greenhouse experiments with clonal material of Populus trichocarpa (Torr and Gray) × deltoides (Bartr. ex Marsh). We showed that there was a short-term and repeatable response in which xylem fibre morphology and secondary cell wall structure adapt to a shift in N availability. Under high-nitrogen exposure, xylem fibres were 17% wider and 18% shorter compared to the adequate nitrogen treatment. A very significant thickening of the fibre cell walls was also observed throughout the stem of trees receiving the high-N treatment. It appeared that cell wall structure was greatly affected by the high-N treatment as fibres developed a modified inner cell wall layer. Histological observations indicated that the internal cell wall layer was enriched in cellulose and chemical determinations showed that wood contained more holocellulose. Together, these results indicate that the response of poplar to nitrogen availability may involve marked effects on secondary xylem formation.     

Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants

Over‐expression of glutamine synthetase (GS, EC 6.3.1.2), a key enzyme in nitrogen assimilation, may be a reasonable approach to enhance plant nitrogen use efficiency. In this work phenotypic and biochemical characterizations of young transgenic poplars showing ectopic expression of a pine cytosolic GS transgene in photosynthetic tissue (Gallardo et al., Planta 210, 19–26, 1999) are presented. Analysis of 22 independent transgenic lines in a 6 month greenhouse study indicated that expression of the pine GS transgene affects early vegetative growth and leaf morphology. In comparison with non‐transgenic controls, transgenic trees exhibited significantly greater numbers of nodes and leaves (12%), and higher average leaf length and width resulting in an increase in leaf area (25%). Leaf shape was not altered. Transgenic poplars also exhibited increased GS activity (66%), chlorophyll content (33%) and protein content (21%). Plant height was correlated with GS content in young leaves, suggesting that GS can be considered a marker for vegetative growth. Molecular and kinetic characterization of GS isoforms in leaves indicated that poplar GS isoforms are similar to their counterparts in herbaceous plants. A new GS isoenzyme that displayed molecular and kinetic characteristics corresponding to the octomeric pine cytosolic GS1 was identified in the photosynthetic tissues of transgenic poplar leaves. These results indicate that enhanced growth and alterations in biochemistry during early growth are the consequence of transgene expression and assembly of pine GS1 subunits into a new functional holoenzyme in the cytosol of photosynthetic cells.     

Rhamnogalacturonan‐I is a determinant of cell–cell adhesion in poplar wood

The molecular basis of cell–cell adhesion in woody tissues is not known. Xylem cells in wood particles of hybrid poplar (Populus tremula × P. alba cv. INRA 717‐1B4) were separated by oxidation of lignin with acidic sodium chlorite when combined with extraction of xylan and rhamnogalacturonan‐I (RG‐I) using either dilute alkali or a combination of xylanase and RG‐lyase. Acidic chlorite followed by dilute alkali treatment enables cell–cell separation by removing material from the compound middle lamellae between the primary walls. Although lignin is known to contribute to adhesion between wood cells, we found that removing lignin is a necessary but not sufficient condition to effect complete cell–cell separation in poplar lines with various ratios of syringyl:guaiacyl lignin. Transgenic poplar lines expressing an Arabidopsis thaliana gene encoding an RG‐lyase (AtRGIL6) showed enhanced cell–cell separation, increased accessibility of cellulose and xylan to hydrolytic enzyme activities, and increased fragmentation of intact wood particles into small cell clusters and single cells under mechanical stress. Our results indicate a novel function for RG‐I, and also for xylan, as determinants of cell–cell adhesion in poplar wood cell walls. Genetic control of RG‐I content provides a new strategy to increase catalyst accessibility and saccharification yields from woody biomass for biofuels and industrial chemicals.     

Water stress-induced xylem hydraulic failure is a causal factor of tree mortality in beech and poplar

Background and Aims

Extreme water stress episodes induce tree mortality, but the physiological mechanisms causing tree death are still poorly understood. This study tests the hypothesis that a potted tree''s ability to survive extreme monotonic water stress is determined by the cavitation resistance of its xylem tissue.

Methods

Two species were selected with contrasting cavitation resistance (beech and poplar), and potted juvenile trees were exposed to a range of water stresses, causing up to 100 % plant death.

Key Results

The lethal dose of water stress, defined as the xylem pressure inducing 50 % mortality, differed sharply across species (1·75 and 4·5 MPa in poplar and beech, respectively). However, the relationships between tree mortality and the degree of cavitation in the stems were similar, with mortality occurring suddenly when >90 % cavitation had occurred.

Conclusions

Overall, the results suggest that cavitation resistance is a causal factor of tree mortality under extreme drought conditions.     

永定河沿河沙地杨树人工林蒸腾耗水特征及其环境响应

杨树是我国北方最常见的人工造林树种之一。一直以来在干旱、半干旱地区,速生杨树用材林和生态防护林的耗水问题备受关注。研究不同生长发育阶段杨树人工林蒸腾耗水及其对各环境因子的响应对于实现杨树人工林可持续经营具有重要价值。采用树干液流法结合微气象观测系统和土壤水分观测,在2010—2011年对位于北京南郊大兴林场、林龄为13a的杨树人工林林分蒸腾耗水和环境因子进行了同步观测,以期能够探究该林分的蒸腾耗水及其对环境因子的响应。结果表明,树干液流密度(Js)日变化呈明显的单峰曲线,单株样木耗水量随着胸径的增加而增大。在半小时尺度上,单株树木Js与浄辐射(Rn)、饱和水气压差(VPD)存在时滞,这种时滞现象随土壤水分条件不同而变化。林分蒸腾耗水总量在2010和2011年生长季内分别为113.7 mm和174.8 mm,占同期降雨的30.2%和36.9%,与该杨树人工林前期研究相比,随着林龄的增长2010—2011年的蒸腾量呈减小趋势。日尺度上,该人工林蒸腾耗水与净辐射(Rn)、饱和水汽压差(VPD)和土壤体积含水率(SWC)显著相关,在不同土壤水分条件下Rn与林分蒸腾的相关关系发生变化,而VPD过高会对林分蒸腾产生抑制。林分月蒸腾和年总蒸腾主要取决于同期降雨量,因此,降雨年际差异较大时,蒸腾的年际变化也相应较大。     

Apparent responses of stomata to transpiration and humidity in a hybrid poplar canopy

It has been proposed that the stomatal response to humidity relies on sensing of the transpiration rate itself rather than relative humidity or the saturation deficit per se. We used independent measurements of stomatal conductance (g_s), transpiration (E), and leaf-to-air vapour pressure difference (V) in a hybrid poplar canopy to evaluate relationships between g_s and E and between g_s and V. Relationships between E, V and total vapour phase conductance or crown conductance (g_c) were also assessed. Conductance measurements were made on exposed and partially shaded branches over a wide range of incident solar radiation. In exposed branches, g_s appeared to decline linearly with increasing E and increasing V at both high and low irradiance. However, in a partially shaded branch, a bimodal relationship between g_s and E was observed in which g_s continued to decrease after E had reached a maximum value and begun to decrease. The relationship between g_s and V for this branch was linear. Plots of g_c against E always yielded bimodal or somewhat variable relationships, whereas plots of g_c against V were invariably linear. It was not possible to derive a unique relationship between conductance and E or V because prevailing radiation partially determined the operating range for conductance. Normalization of data by radiation served to linearize responses observed within the same day or type of day, but even after normalization, data collected on partly cloudy days could not be used to predict stomatal behaviour on clear days and vice versa. An additional unidentified factor was thus also involved in determining operating ranges of conductance on days with different overall radiation regimes. We suggest that the simplest mechanism to account for the observed humidity responses is stomatal sensing of the epidermal or cuticular transpiration rate rather than the bulk leaf or stomatal transpiration rate.     

Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica)

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic proteins found primarily in plants. The barley hva1 gene encodes a group 3 LEA protein and is induced by ABA and water deficit conditions. We report here the over expression of hva1 in mulberry under a constitutive promoter via Agrobacterium-mediated transformation. Molecular analysis of the transgenic plants revealed the stable integration and expression of the transgene in the transformants. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of hva1 in conferring tolerance. The transgenic plants showed better cellular membrane stability (CMS), photosynthetic yield, less photo-oxidative damage and better water use efficiency as compared to the non-transgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants show many fold increase in proline concentration than the non-transgenic plants and under water deficit conditions proline is accumulated only in the non-transgenic plants. Results also indicate that the production of HVA1 proteins helps in better performance of transgenic mulberry by protecting membrane stability of plasma membrane as well as chloroplastic membranes from injury under abiotic stress. Interestingly, it was observed that hva1 conferred different degrees of tolerance to the transgenic plants towards various stress conditions. Amongst the lines analysed for stress tolerance transgenic line ST8 was relatively more salt tolerant, ST30, ST31 more drought tolerant, and lines ST11 and ST6 responded well under both salinity and drought stress conditions as compared to the non-transgenic plants. Thus hva1 appears to confer a broad spectrum of tolerance under abiotic stress in mulberry.     

Construction of a cDNA library of Aphis gossypii Glover for use in RNAi

Aphis gossypii Glover is an important insect pest that functions as a viral vector and mediates approximately 45 different viral diseases. As part of a strategy for control of A. gossypii, we investigated the functions of genes using RNAi. To this end, a cDNA library was constructed for various genes and for selecting appropriate targets for RNAi mediated silencing. The cDNA library was constructed using the Gateway cloning system with site‐specific recombination of bacteriophage λ. It was used to carry out single step cloning of A. gossypii cDNAs. As a result, a cDNA library with a titer of 8.4 × 10⁶ was constructed. Since the sequences in this library carry att sites, they can be cloned into various binary vectors. This library will be of value for various studies. For later screening of selected genes, it is planned to clone the library into virus‐induced gene silencing (VIGS) vectors, which makes it possible to analyze gene function and allow subsequent transfection of plants. Such transfection experiments will allow testing of RNAi‐induced insecticidal activity or repellent activity to A. gossypii, and result in the identification of target genes. It is also expected that the constructed cDNA library will be useful for analysis of gene functions in A. gossypii.     

Overexpression of a NTR1 in transgenic soybean confers tolerance to water stress

Methyl jasmonate (MeJA) is an important plant regulator that involves in plant development and regulates the expression of plant defense genes in response to various stresses such as wounding, drought, and pathogens. In order to determine the physiological role of endogenous MeJA in plants, a NTR1 from Brassica campestris encoding a jasmonic acid carboxyl methyltransferase that produces methyl jasmonate was constructed under the control of CaMV 35S promoter and transformed into soybean [Glycine max (L) Merrill]. The transgenic soybean plants constitutively expressed the NTR1 and accumulated more MeJA levels than wild type plants. Overexpression of the gene in transgenic soybean conferred tolerance to dehydration during seed germination and seedling growth as reflected by the percentage of the fresh weight of seedlings. In addition, the transgenic soybean plants also conferred better capacity to retain water than wild type plants when drought tolerance was tested using detached leaves.     

Genetic analysis of a mutation on appressorium formation in Magnaporthe grisea

The rice blast fungus, Magnaporthe grisea, forms a dome-shaped and darkly pigmented infection structure, an appressorium, to penetrate its host. Differentiation and maturation of appressoria are critical steps for successful infection. A spontaneous developmental mutant (MG01) defective in appressorium formation was found in this fungus. The mutant did not form appressoria either on inductive hydrophobic surfaces or on rice leaves. The addition of cyclic AMP or 1,16-hexadecanediol was not effective in inducing appressorium formation in this mutant. This mutant did not cause lesions on rice when inoculated with conidial suspension by spraying or injecting into the leaf sheath. Genetic analysis of the mutant indicated that the phenotype is under single gene control, designated APP5. Crosses with previously described appressorium defective mutants (app1⁻ and app3⁻) of Magnaporthe grisea suggested that the mutations are at different loci. Bulked segregant analysis was employed to obtain DNA markers linked to the APP5 locus.     

Nutrient resorption and stoichiometric responses of poplar (Populus deltoids) plantations to N addition in a coastal region of eastern China

中国东部沿海杨树人工林养分重吸收和化学计量对氮添加的响应 叶片养分重吸收对土壤养分的变化很敏感。然而,我们尚不清楚氮沉降如何影响植物大量元素重吸收率。杨树(Populus deltoids)是世界上栽培最广泛的阔叶树种之一。本文研究了在氮添加条件下,杨树重吸收率及其化学计量比的规律和主要驱动因素。通过一个4年的氮添加实验,我们探究中国东部沿海两个林分(8和12年)杨树人工林重吸收率及其化学计量比对氮添加的响应。我们测定了在一系列氮添加浓度水平(0、50、100、150、300 kg N ha⁻¹ yr⁻¹)下,土壤和叶片(包括绿叶和落叶)中氮、磷、钾、钙、镁的浓度。研究结果表明,除钙元素重吸收率和钙、镁元素重吸收率化学计量比外,氮添加对两个林分大量元素重吸收率及其化学计量比都没有显著影响。氮、磷重吸收率尺度斜率在不同氮添加水平下均小于1,表明氮添加条件下,氮、磷元素重吸收率解耦。养分重吸收率与绿叶中养分含量显著正相关,表明重吸收主要受到绿叶养分调控。我们的研究结果为中国东部沿海地区12年生杨树人工林的生长受氮限制提供了直接证据。     

Postnatal expression of myostatin propeptide cDNA maintained high muscle growth and normal adipose tissue mass in transgenic mice fed a high-fat diet

Myostatin plays a robust, negative role in controlling muscle mass. A disruption of myostatin function by transgenic expression of its propeptide (the 5'region, 866 nucleotides) results in significant muscle growth (Yang et al., 2001. Mol Rep Dev 60:351-361). Studies from myostatin and the propeptide transgene mRNA indicated that myostatin mRNA was detected at day 10.5 postcoitum in fetal mice. Its level remained low, but increased by 180% during the postnatal fast-growth period (day 0-10). An early, high-level postnatal expression of the transgene was identified as being responsible for a highly muscled phenotype. High-fat diet induces adiposity in rodents. To study the effects of dietary fat on muscle growth and adipose tissue fat deposition in the transgenic mice, we challenged the mice with a high-fat diet (45% kcal fat) for 21 weeks. Transgenic mice showed 24%-50% further enhancement of growth on the high-fat diet compared to the normal-fat diet (P = 0.004) from 17 to 25 weeks of age. The total mass of the main muscles of transgenic mice showed a 27% increase on the high-fat diet compared to the normal-fat diet (P = 0.004), while the white adipose tissue mass of the transgenic mice was not significantly different from that of wild-type mice fed a normal-fat diet (P = 0.434). The high-fat diet induced wild-type mice developed 190% greater mass of white adipose tissues compared to the normal-fat diet (P = 0.008), which primarily resulted from enlarged adipocytes. These results demonstrate that disruption of myostatin function by its propeptide shifted dietary fat utilization toward muscle tissues with minimal effects on adiposity. These results suggest that enhancing muscle growth by myostatin propeptide or other means during the early developmental stage may serve as an effective means for obesity prevention.