Studies on litter characterization using 13C NMR and assessment of microbial activity in natural forest and plantation crops’ (teak and rubber) soil ecosystems of Kerala, India

The leaf litter is the major source of soil organic matter in natural and many plantation crop ecosystems. Quantity and quality of organic matter in a soil ecosystem is of utmost importance in regulating the soil health. Hence assessment of quality of organic matter input, viz., litter is important and is attempted in this study. The leaf litter of rubber (Hevea brasiliensis), pueraria (Pueraria phaseoloides), mucuna (Mucuna bracteata), teak (Tectona grandis) and forest (mixed species) were analyzed using solid state ¹³C nuclear magnetic resonance (NMR) to study the relative abundance of different carbon compounds present. The spectra revealed that litter of all species studied contain relatively larger amounts of polysaccharides compared to other C containing compounds. Also it could be observed that the alkyl-C to O-alkyl-C ratio of rubber litter was much higher compared to that of others. Aromatics and carbonyl compounds were also present in all litter species. The resource quality based on alkyl-C to O-alkyl-C ratio of the litter samples studied can be arranged in the order pueraria > teak > mucuna > forest > rubber. The respiration rate, substrate induced respiration rate and biomass-C (Cmic) of the litter samples were estimated. It could be observed that litter associated microbial activity decreased as alkyl-C to O-alkyl-C ratio increased. Resource quality derived from the NMR spectra and the litter biological properties were complementary. Soil samples (0–15 cm) from the five soil ecosystems (rubber, pueraria, mucuna, teak and forest) were analyzed for respiration rate, substrate induced respiration rate, Cmic, total-C and total-N. The forest soil had higher respiration rate, total-C and total-N compared to cultivated soil systems. Pueraria, mucuna and teak soils were comparable for their biological properties while rubber soil recorded comparatively lower microbial activity.     

Hevea latex lectin binding protein in C-serum as an anti-latex coagulating factor and its role in a proposed new model for latex coagulation

A distinct protein specifically recognized by its strong interaction with Hevea latex lectin (HLL) was detected in the aqueous C-serum fraction of centrifuged fresh latex. This C-serum lectin binding protein (CS-HLLBP) exhibited strong inhibition of HLL-induced hemagglutination. The CS-HLLBP was purified to homogeneity by a protocol that included ammonium sulfate fractionation, size exclusion and ion exchange chromatography. The purified CS-HLLBP had a specific HI titer of 0.23microg ml(-1). Its M(r)s analyzed by SDS-PAGE was ca. 40kDa and that by gel filtration was ca. 204kDa. It has a pI value of 4.7, an optimum activity between pH 6 and10 and was heat stable up to 50 degrees C. The HI activity of CS-HLLBP was abolished upon treatment with chitinase. The CS-HLLBP inhibited HLL-induced rubber particle aggregation in a dose dependent manner. A highly positive correlation between CS-HLLBP activity and rubber yield per tapping was found. The correlations for fresh latex (r=0.98, P<0.01) and dry rubber (r=0.95, P<0.01) were both highly significant. This indicated that the CS-HLLBP might be used as a reliable marker for the mass screening of young seedlings to identify and select clones with potential to be superior producers of rubber. A latex anti-coagulating role of the CS-HLLBP is proposed. The findings described in this 3 paper series have been used to propose a new model of rubber latex coagulation that logically describes roles for the newly characterized latex lectin and the two lectin binding proteins.     

Molecular cloning and characterization of two cDNAs encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Hevea brasiliensis

1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, EC: 1.1.1.267) is the second enzyme in the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway, one of the two pathways in plants that can produce isoprenoids. The MEP pathway is the source of isoprene emitted from leaves, but rubber production is believed to result primarily from the mevalonic acid (MVA) pathway. Two cDNAs for DXR designated HbDXR1 and HbDXR2 were isolated from leaves and latex of rubber tree using RT-PCR based methods. Both cDNAs contain an open reading frame (ORF) of 1416bp encoding 471 amino acids with a molecular mass of about 51kDa. The deduced HbDXRs show extensive sequence similarities to that of other plant DXRs (73-87% identity). Molecular modeling revealed that the two HbDXRs contain all typical characteristics of DXR and share spatial structures, which are very similar to that of Escherichia coli DXR. Phylogenetic and DNA gel blot analyses suggested that a duplication of the DXR gene has occurred in the rubber tree. Semi-quantitative RT-PCR analysis showed that the HbDXR genes are differentially regulated in various tissues of the rubber tree. The HbDXR2 was more highly expressed in clone RRIM 600 than in the wild type, and this is consistent with higher rubber content of this clone. While 2-chloroethane phosphonic acid (ethephon) significantly increased latex yield, it only transiently induced the HbDXR2 gene. The expression of HbDXR2 in the latex suggests its important role in isoprenoid biosynthesis by substrate molecules, indicating that the MEP pathway may have some indirect roles in the biosynthesis of rubber.     

橡胶树胶乳高表达热激蛋白HbHSP90.4基因抗逆功能分析

为了分析热激蛋白90（HSP90）基因在橡胶树（Hevea brasiliensis）逆境胁迫和激素转导中的作用,利用PCR技术从橡胶树品种热研73397胶乳中克隆得到HbHSP90.4基因全长cDNA序列,该基因含有1个2 451 bp开放阅读框（ORF）,编码816个氨基酸。生物信息学分析结果表明,HbHSP90.4含有HSP90 superfamily和HATPase superfamily结构域,属于HSP90家族成员。系统进化分析发现该蛋白与木薯MeHSP90具有较近的亲缘关系。亚细胞定位预测显示HbHSP90.4基因定位在内质网。qRT-PCR结果表明HbHSP90.4基因主要在橡胶树胶乳中表达。干旱、冷胁迫、橡胶树白粉菌侵染、H₂O₂和MeJA处理均可促进胶乳HbHSP90.4基因上调表达,而其在ETH、SA和ABA处理中均呈现显著下调表达。构建植物表达载体HbHSP90.4-mScarlet,为进一步的转基因植物的做成准备了材料。本研究为阐明胶乳HbHSP90.4基因响应橡胶树逆境胁迫过程和植物激素信号传导途径分子调控机制奠定坚实基础。     

杜仲橡胶合成相关酶基因HMGR的克隆与表达分析

为解析HMGR基因在杜仲橡胶生物合成中的作用,本研究以杜仲良种华仲6号叶片和果皮为材料,参考杜仲基因组和转录组数据,克隆了杜仲HMGR基因,命名为Eu HMGR,全长1770 bp,编码590个氨基酸。实时荧光定量PCR测定结果显示,EuHMGR基因在果皮中的表达量于5月中旬达到最大值,而在叶片中的最大值出现在7月中旬。通过索氏提取测定相应时期果皮和叶片的含胶量表明,果皮中的含胶量从4月中旬到5月下旬迅速增加,而叶片中的含胶量一直平稳增长。分析不同发育时期果皮和叶片Eu HMGR基因表达量和含胶增长速率相关性发现,果皮Eu HMGR基因在不同发育时期的相对表达量和含胶增长速率呈显著正相关,而二者在叶片中无明显相关性。因此,推测Eu HMGR基因的表达与杜仲果皮中橡胶生物合成相关。     

Effect of exogenous calcium on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis (rubber tree) friable calli

The influence of CaCl₂ was investigated on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis friable calli which are usually proliferated on maintenance medium (MM) containing 9 mM CaCl₂.Five A. tumefaciens strains (C58pMP90, C58pGV2260, AGL1, LBA4404 and EHA 105) and two binary vectors (pGIN and pCAMBIA2301) were tested and the strain EHA105pC2301 was selected to conduct further experiments. The calli were precultured on MM containing a range of CaCl₂ concentrations, then inoculated with Agrobacterium suspension. Transfer of friable calli from MM containing 9 mM CaCl₂ to calcium-free medium significantly enhanced the transient β-glucuronidase activity. Interestingly, the use of calcium-free Agrobacterium resuspension medium to inoculate friable calli again dramatically increased the transformation efficiency. Induction of Agrobacterium's virulence with acetosyringone remained an important factor to stimulate transformation. Received: 14 October 1999 / Revision received: 3 January 2000 / Accepted: 4 January 2000     

西双版纳热带季节雨林与橡胶林林冠的持水能力

基于2003～2004年的实验室和野外实测数据,采用尺度上推法对西双版纳地区热带季节雨林和橡胶林林冠持水能力进行了研究.结果表明,热带季节雨林林冠持水 能力为0.45～0.79 mm、橡胶林为0.48～0.71 mm,其中林冠木质部分（枝和树皮）的持水能力占较大优势;雾凉季（11月至翌年2月）和干热季（3～4月）的 林冠持水能力高于雨季（5～10月）;林冠各部分达到饱和所需的时间为叶（5 min）＜树皮（2～3 h）＜枝（2.5～4 h）.对两种林分林冠各部分在浸水/风干过程 的分析结果表明,叶易吸水和风干,在历时较短的降雨事件中是林冠截留的主体,在林地中,面积指数较大的枝和树皮吸水和风干较难,在强度大、历时长的降雨事件中可 以较好地发挥截留作用.与橡胶林相比,热带季节雨林林冠持水能力虽较小,但是其多层林冠结构林冠持水能力较强.     

Increased lymphocyte DNA strand breaks in rubber workers

Objective: To study the effect of occupational exposure to rubber processing, smoking, and alcohol drinking on lymphocyte DNA damage. Subjects and Methods: Of 371 employees (197 men and 174 women) from a rubber factory in Guangzhou, 281 were rubber processing workers from five production sections and 90 were managerial workers. Information on occupational exposure, smoking, and drinking was collected by interviews. Blood samples were taken in the morning by venipuncture. DNA damages were measured by the Comet assay. Possible DNA-protein crosslinks were broken down by proteinase K. Tail moment, measured by Komet 4.0 image analysis software, was the measure of DNA damage. Results: The rubber processing workers had larger tail moment than the managerial workers (Geometric mean, 95%CI) [1.77 μm (1.64–1.90) versus 1.52 μm (1.36–1.71), P=0.04]. Both smoking [1.93 μm (1.74–2.13) versus 1.59 μm (1.47–1.71), P=0.003] and alcohol drinking [2.21 μm (1.87–2.62) versus 1.63 μm (1.53–1.74), P<0.001] increased tail moment. Tail moment differed significantly among job categories (F=3.21, P=0.008), the largest was observed in mixers. In the non-smoking and non-drinking workers, rubber processing workers had larger tail moment than managerial workers after adjusting for age (P=0.033). General linear model analysis showed that after adjusting for each other, occupational exposure (P=0.027), smoking (P=0.012), and alcohol drinking (P=0.013) was associated with larger tail moment, whereas age and gender had no effect. Conclusions: Occupational exposure to rubber processing, smoking, and alcohol drinking can cause DNA damage.     

A Study on Manganese Content in Soils and Leaves of Rubber Trees in Rubber Plantation Areas of China

The present paper deals with the status of manganese (Mn) in rubber trees on 110 rubber plantation at 46 counties in the rubber tree growing regions of China. The content of Mn in rubber tree leaves ranges from 31 to 2167 ppm, averaging 390 ppm which is higher than that in the leaves of normal rubber trees (150–300 ppm). Thus it can be concluded that the present status of Mn in China's rubber trees is higher. Nevertheless, the content of Mn of soil of rubber plantations in China varies significantly according to different soil types, fertilization and management. The difference between the highest and the lowest contents of total Mn could be as high as 140 times and an even greater difference of 200–1240-fold between the highest and the lowest contents of replaceable and reducible Mn has been observed. The storage of reducible Mn is generally insufficient in the soil. Therefore, the symptom of leaf yellowing due to deficiency of Mn has developed in rubber trees in certain parts of rubber tree growing regions in China. The essential oure for leaf yellowing of rubber trees is to raise Mn content in the tree by proper application of fertilizer. The procedures, dosage and timing of the fertilizer for leaf yellowing treatments are suggested.