Stomatal response of hybrid poplar to incident light, sudden darkening and leaf excision

Responses of abaxial and adaxial stomata of Populus trichocarpa Torr. & Gray. × P. deltoides Bartr. (ex Marsh.) cv. Unal to incident light, sudden darkening and leaf excision in the light and in the dark were studied on 5-year-old trees in the field using diffusion porometry. Stomatal closure in the dark was found to be incomplete in most cases studies. Stomata closed after leaf excision in the dark within 90 min. Stomatal closure after darkening of an entire tree or an entire branch (white the rest of the tree was in the light) was slower, and complete stomatal closure was noticed only for adaxial stomata after 3 h. Adaxial stomata were more reactive and sensitive than abaxial stomata to sudden darkening and leaf excision in the light and the dark. In all treatments, stomatal response was more responsive in mature leaves than in young, still expanding leaves.     

Unique occurrence of pectin-like fibrillar cell wall deposits in xylem fibres of poplar

Using light and electron microscopic techniques, we studied the unique occurrence of fibrillar cell wall deposits in mature xylem fibres from poplar. These cell wall deposits lined the lumen-facing side of the wall, mainly in fibres next to vessel elements. Different lines of evidence point to the pectin-like nature of these fibrillar cell wall deposits. First, specific staining by Alcian Blue 8GX, a dye with high affinity for pectic substances. Second, the strongly reduced staining of the cell wall deposits in microscopic sections treated with pectolytic enzyme. Third, concomitant staining of pits, which are known to consist mainly of pectic substances. Given the pectin-like nature of the fibrillar cell wall deposits as well as their preferred occurrence in fibres neighbouring water-conducting vessel elements, a function for the fibrillar cell wall deposits in lateral water diffusion and stem water storage is hypothesised. The hypothesis is supported by the increased abundance of these cell wall deposits in wood tissue of a drought-sensitive poplar species.     

杨树杂种无性系生长与光合生理遗传变异研究

以美洲黑杨与大青杨的27个杂种无性系1年生盆栽扦插苗为材料,以美洲黑杨无性系I-69为对照,对其生长性状、扦插成活率以及主要光合生理指标进行遗传变异分析,并利用分析结果对杂种无性系进行初步选择.结果表明:(1)27个杂种无性系间苗期生长性状及光合生理等12个性状指标差异极显著,无性系重复力均在0.7以上,最高达0.987,均受强遗传控制.(2)结合生长性状分析不同无性系的净光合速率,发现具有较高净光合速率的无性系,同样具有较大生长量和较高成活率.(3)经主成分分析和聚类分析,初选出7个生长快、成活率较高、光合作用能力较强的无性系(编号依次为135、141、153、155、165、177和193),7个无性系苗高超对照10%~33%,地径超对照11%~24%,成活率超对照30%~48%,净光合速率超对照7%~29%,且干形优美,适应性强,可作为进一步选育的材料.     

Cloning of metallothionein genes from <Emphasis Type="Italic">Arachis hypogaea</Emphasis> and characterization of <Emphasis Type="Italic">AhMT2a</Emphasis>

Seven cDNA sequences putatively encoding metallothionein (MT) proteins were isolated from peanut (Arachis hypogaea L.). We analyzed the sequences of the deduced amino acids and characterized one of these genes, AhMT2a. Northern blot analysis indicates that the accumulation of AhMT2a mRNA was found in stems, young roots, and young leaves, and AhMT2a mRNA levels were up-regulated by cadmium, copper, ABA, H₂O₂, and heat shock. The metal-binding properties of the recombinant protein expressed in E. coli was investigated, and the results show that bacteria harboring AhMT2a exhibited increased tolerance to cadmium, copper, and lead, and the metal accumulation was also higher than the controls. Taken together, our results suggest that the AhMT2a protein might function in both detoxification of heavy metals and scavenging of reactive oxygen species in peanut. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 5, pp. 755–762. The text was submitted by the authors in English.     

Effects of low level O3 exposure on leaf diffusive conductance and water-use efficiency in hybrid poplar

Abstract Young, amphistomatous hybrid poplar (Populus deltoides x trichocarpa) plants were exposed daily to either background (0.025 cm³ m^-3) or elevated (0.125 cm³ m^-3) concentrations of O₃. Levels of abaxial and adaxial leaf conductance were affected interactively by pollutant treatment, leaf age, and photon fluence rate. Consequently, conductance in O₃-treated leaves was sometimes higher and sometimes lower than in comparable control leaves, depending on leaf age or level of photon fluence rate. For example, at low photon fluence rate or in the dark, conductance was greater in O₃-treated than in control plants, while at high photon fluence rate that relationship was reversed. Exposure to O₃ also reduced the water-use efficiency and range of leaf conductance of individual leaves, and altered the relationship between the conductances of the two leaf surfaces (the ratio of abaxial to adaxial leaf conductance was increased). Furthermore, O₃ treatment resulted in diminished stomatal control of water loss; excised O₃-treated leaves had higher conductances and wilted sooner than excised control leaves of identical ages. Overall, the data indicate that exposure to O₃ resulted in impaired stomatal function.     

Molecular characterization and expression of metallothionein from freshwater pearl mussel,Hyriopsis schlegelii

Two metallothionein genes (HsMT1 and HsMT2) were first identified and described from Hyriopsis schlegelii. The open reading frame of HsMT1 and HsMT2 were 216 and 222 bp, encoding a protein of 71 and 73 amino acid residues. The deduced amino acid sequences showed they contained parts of typical MT characteristics, apart from HsMT2 lacked Cys–Cys motifs. The phylogenetic tree showed HsMT1 shared a high similarity with that of other molluscs, but HsMT2 was split into a distinct group separated from known molluscan MTs. HsMT1 exhibited constitutive expression in all examined tissues and the highest expression occurred in hepatopancreas, however, nearly all HsMT2 was just detected in gonad. After Cd exposure, their mRNA levels presented similar expression patterns. The transgenic bacteria of HsMT1 showed higher tolerance than HsMT2 in Cd environment. It was implied that HsMT1 and HsMT2 were involved in metal response but HsMT2 might have other physiological functions.     

Cloning and identification of methionine synthase gene from Pichia pastoris

Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5‘ and 3‘ regions were further cloned by 5‘- and 3‘-rapid amplification of cDNA ends (RACE), respectively. The assembled sequence reveals an open reading frame encoding a polypeptide of 768 residues, and the deduced product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two domains common to MetEs. The active site is located in the C-terminal domain, in which the residues involved in the interaction of zinc with substrates are conserved. Homologous expression of PpMetE in P. pastoris was achieved, and the heterologous expression of PpMetE in the S. cerevisiae strain XJB3-1D that is MetE-defective restored the growth of the mutant on methionine-free minimal media. The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No. AY601648.     

Stimulation of hybrid poplar growth in petroleum-contaminated soils through oxygen addition and soil nutrient amendments

Hybrid poplar trees (Populus deltoides x nigra DN34) were grown in a green-house using hydrocarbon-contaminated soil from a phytoremediation demonstration site in Health, Ohio. Two independent experiments investigated the effect of nutrient addition on poplar growth and the importance of oxygen addition to root development and plant growth. Biomass measurements, poplar height, and leaf color were used as indicators of plant health in the selection of a 10/5/5 NPK fertilizer applied at 1121 kg/ha (112 kg-N, 24.4 kg-P, 46.5 kg-K per ha) to enhance hybrid poplar growth at the Health site. Five passive methods of oxygen delivery were examined, including aeration tubes, gravel addition, and an Oxygen Release Compound (ORC). When ORC was placed in coffee filters above hydrocarbon-contaminated soil, a statistically significant increase of 145% was observed in poplar biomass growth, relative to unamended controls. The ORC in filters also stimulated significant increases in root density. A 15.2-cm interval of soil directly below ORC addition exhibited an increase from 2.6 +/- 1.0 mg/cm3 to 4.8 +/- 1.0 mg/cm3, showing stimulation of root growth in hydrocarbon-stained soil. The positive response of hybrid poplars to oxygen amendments suggests that overcoming oxygen limitation to plants should be considered in phytoremediation projects when soil contamination exerts a high biochemical oxygen demand, such as in former refinery sites.     

Efficient tissue culture and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of haploid poplar derived from anthers

Calli were induced from anthers of Populus simonii × P. nigra. Haploid plants were then regenerated from the callus and multiplied efficiently by culturing leaf explants. The presence of both haploid and diploid cells in the same plant revealed spontaneous chromosome doubling in haploid cells. The haploid plants were transformed with the nptII gene by Agrobacterium-mediated method using leaf explants, and five independent kanamycin-resistant lines were obtained, with a transformation frequency more than 6%. Further PCR test indicated that the exogenous betA gene was transferred into these kanamycin-resistant lines, which were still haploid. Thus, the efficient tissue culture system and transformation of haploid poplar plants were achieved. Our study will contribute to forest improvement via the haploid culture and transgenic technology. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 629–633. The text was submitted by the authors in English.     

Comparative expression analysis of genes encoding metallothioneins in response to heavy metals and abiotic stresses in rice (Oryza sativa) and Arabidopsis thaliana

To get insights into the functions of metallothionein (MT) in plant response to multiple stresses, expressions of 10 rice MT genes (OsMTs) and 7 Arabidopsis MT genes (AtMTs) were comprehensively analyzed under combined heavy metal and salt stress. OsMT1a, OsMT1b, OsMT1c, OsMT1g, and OsMT2a were increased by different heavy metals. Notably, ABA remarkably increased OsMT4 up to 80-fold. Combined salt and heavy metals (Cd, Pb, Cu) synergistically increased OsMT1a, OsMT1c, and OsMT1g, whereas combined salt and H₂O₂ or ABA synergistically increased OsMT1a and OsMT4. Heavy metals decreased AtMT1c, AtMT2b, and AtMT3 but cold or ABA increased AtMT1a, AtMT1c, and AtMT2a. AtMT4a was markedly increased by salt stress. Combined salt and other stresses (Pb, Cd, H₂O₂) synergistically increased AtMT4a. Taken together, these findings suggest that MTs in monocot and dicot respond differently to combined stresses, which provides a valuable basis to further determine the roles of MTs in broad stress tolerance.     

Decomposition of roots of black alder and hybrid poplar in short-rotation plantings: Nitrogen and lignin control

The decomposition of the roots (0–2 mm, 2–5 mm and 5–10 mm) of black alder (Alnus glutinosa (L.) Gaertn.) and hybrid poplar (Populus nigra L. X Populus trichocarpa Torr & Gray) was followed over a 462-day period in pure and mixed plantings in southern Quebec. Small roots of alder had the highest initial concentrations of nitrogen and lignin, and lost 9 and 10% less mass than medium and large roots, respectively. Large roots of poplar had the highest lignin-to-nitrogen ratio and showed the smallest loss of mass over the total incubation period. Slow root decomposition of black alder and hybrid poplar was characterized by a greater proportion of initial root nitrogen immobilized per unit of carbon respired. Lignin concentration in roots of alder and poplar increased rapidly at the beginning of the incubation. Our results suggest that high levels of nitrogen in roots of alder could contribute in slowing the rate of decomposition by allowing the formation of nitrogen-lignin derivatives and low levels of nitrogen in roots of poplar may limit the growth of microorganisms and the rate of root decomposition. A multiple regression was developed using initial nitrogen, lignin concentration and the ratio of lignin to nitrogen to produce an index of the rate of root decomposition. The correlation between the index values and the percentage of residual root mass was significant (r=0.98, p<0.01).     

Cloning,expression and function of phosphate transporter encoded gene in Oryza sativa L.

OsPT6:1,a phosphate transporter encoding gene from the leaf samples of Oryza sativa, was identified through PCR with specifically designed primers.The phylogenetic analysis and the conserved amino acid residue site detection suggested OsPT6:1 a possible high-affinity phosphate transporter encoding gene.In situ hybridization and RT-PCR demonstrated the expression of OsPT6:1 in both roots and leaves.The peak expression signal was observed in mesophyll cells under low phosphorus(P)induction.A homologous recombination study indicated that OsPT6:1 can enhance the Pi uptake efficiency of Pichia pastoris.At the meantime,the introduction of OsPT6:1 was able to complement the Pi uptake function of yeast cells with high-affinity phosphate transporters de- ficient.Those results substantiated our contention that OsPT6:1 encoded a high-affinity phosphate transporter of Oryza sativa.     

Growth and root hydraulic conductivity of several citrus rootstocks under salt and polyethylene glycol stresses

The water relations responses to salt of several important citrus rootstocks such as Swingle citrumelo, sour orange, and Milam lemon have not been studied in detail before. Studies were set up to compare growth and root hydraulic properties of these rootstocks to other citrus rootstocks by exposing them to NaCl and polyethylene glycol (PEG) stresses. Seedlings of 7 citrus rootstocks were irrigated for 5 months with nutrient solutions containing NaCl or PEG that had been adjusted to osmotic potentials of -0.10, -0.20 or -0.35 MPa. The 7 rootstocks studied were sour orange (Citrus aurantium), Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (C. paradisi x P. trifoliata), Carrizo citrange (C. sinensis x P. trifoliata), rough lemon (Citrus jambhiri Lush), Milam lemon (C. jambhiri hybrid), and trifoliate orange (Poncirus trifoliata [L.] Raf.). In both shoot and root growth, Cleopatra mandarin and sour orange were the least sensitive to salt, Milam and trifoliate orange were the most sensitive, and rough lemon, Swingle, and Carrizo were intermediate in sensitivity. Even though the roots were exposed to solutions of equal osmotic potentials, plant growth and root conductivity were reduced more by the PEG treatments than the corresponding NaCl treatments. At -0.10 and -0.20 MPa, shoot and root dry weights were reduced 16 to 55% by NaCl and 24 to 68% by PEG. Shoot root ratio was lowered at the higher concentrations, particularly by PEG. There was a major decrease in root conductivity caused by NaCl at -0.10 MPa (19 to 30% in sour orange and Cleopatra mandarin and 78 to 85% in trifoliate orange and Milam). Conductivity decreased more at -0.20 and -0.35 MPa, but not proportionally as much as at -0.10 MPa. Root weight per unit length increased at the higher salt levels, particularly in trifoliate orange. Water flow rate through root systems followed the same trend as root conductivity; salt affected sour orange and Cleopatra mandarin the least and trifoliate orange and Milam the most. However, reductions in fibrous root length by salt treatment differed. Root lengths of Swingle and Carrizo were least affected by salt while sour orange. Milam, and rough lemon were the most affected. Hence, even though sour orange and Cleopatra mandarin were more tolerant than the other rootstocks in terms of water flow rate or root conductivity, these 2 rootstocks showed a proportionally greater decrease in root length than Carrizo, Swingle, or trifoliate orange.     

纳豆激酶基因的克隆及其在毕赤酵母中的表达

纳豆激酶纳是从日本传统食品纳豆中发现的一类具有溶栓效果的蛋白酶,由于其具有安全,高效,作用时间长,易吸收,廉价等优点,现在正成为一个开发治疗血栓类疾病药物的研究热点。从本实验室保存的一株高溶栓的纳豆杆菌N07出发,提取总基因组DNA,利用PCR手段扩增获得了纳豆激酶长为825bp的成熟肽基因片段。构建重组表达质粒pPICZaA-NK,经EcoR I、Xba I双酶切、PCR、测序验证得出重组表达质粒上的外源基因即为825bp的目的片段;将重组质粒pPICZaA-NK用内切酶Sac I线性化后电击导入毕赤酵母X33,通过含Zeocin的YPDS平板筛选获得重组酵母。重组酵母在BMMY培养基中发酵培养,用1%甲醇诱导目的蛋白表达。用纤维蛋白平板法检测发现发酵上清具有纤溶活性,经硫酸铵盐析、透析、Sephadex-G50过柱等步骤分离得到纳豆激酶蛋白,进行SDS-PAGE鉴定表明,表达的纳豆激酶蛋白分子量为27KD。以尿激酶为标准,实验所得纳豆激酶发酵上清液溶栓活性约为195U/mL。成功的将纳豆激酶成熟肽基因在毕赤酵母X33中表达,为纳豆激酶基因工程进一步研究奠定基础。     

伴矿景天植物螯合肽合酶基因的克隆及功能分析

重金属超积累植物由于长期生长在高浓度的重金属环境中,使得经由植物螯合肽（phytochelatins, PCs）解毒途径来应对重金属毒害代价高昂。我们从Zn/Cd超积累植物伴矿景天（Sedum plumbizincicola）中克隆了植物螯合肽合酶（phytochelatin synthase, PCS）基因SepPCS。该基因在裂殖酵母和拟南芥中表达后都具有PCS活性,而且能够互补它们的PCs缺失突变体的Cd敏感表型。SepPCS在伴矿景天中的表达受到高浓度Cd处理的诱导。与其近亲非超积累生态型东南景天（S. alfredii）相比,虽然伴矿景天地上部PCs与Cd的摩尔比远低于东南景天,但是在高浓度Cd处理时PCs含量以及PCs与Cd的摩尔比急剧增加。我们推测在伴矿景天应对Cd毒害的过程中, PCs起到一定的作用,并且在高浓度Cd胁迫时地上部PCs依赖的解毒作用有所加强。