Glyoxalase I expression pattern in Hevea brasiliensis seedlings under varied stress conditions

Drought is one of the most important stress factors which adversely affect plants’ growth and productivity. Global climate change may make this situation more serious in the years ahead. Considering the long time span required for the generation of drought resistant genotypes in Rubber (Hevea brasiliensis) through conventional breeding, molecular interventions to engineer plants to have either drought responsive genes or genes expected to alter osmotic regulation would be very attractive. The glyoxalase pathway involving glyoxalase I and glyoxalase II enzymes is required for glutathionebased detoxification of methylglyoxal. In this study the effects of various abiotic stresses on the upregulation of methylglyoxal levels and glyoxalase I activities in Hevea brasiliensis seedlings were investigated. Most of the stresses caused significant increase in methylglyoxal level and glyoxalase I activity, among which drought caused the highest induction of glx I followed by salinity, 2, 4-D, ABA, methylglyoxal, white light and CdCl2. The stress-induced increases in methylglyoxal and glyoxalase I activity found in the present study suggest an important role of glyoxalase I in conferring drought tolerance. The up-regulation of glyoxalase I under drought stress indicates its future utility in developing tolerance to drought stress in Hevea brasiliensis. In the present study, a partial cDNA sequence coding for glyoxalase I was amplified by PCR using specific primers. The 440 bp cDNA amplicon obtained was sequenced and subjected to online BLAST analysis. The sequence of Hevea brasiliensis glyoxalase I (GenBank Acc. No: GU598520) had six open reading frames. The ORF finder revealed the longest ORF of 336 bp. Glyoxalase I from Ricinus communis had the highest nucleotide sequence homology (90%) compared to the amplified gene. BLASTP analysis also showed high homology between the deduced protein sequence of the amplified gene and glyoxalases from other species. Our results suggest that the multi-stress inducibility of glyoxalase I in the present study may be due to the fact that it might protect the plants against MG that is formed under various stresses including drought and confers tolerance by increasing the GSH-based detoxification system and decreasing lipid peroxidationKeywords: Glyoxalase I, Hevea brasiliensis, Methylglyoxal, drought tolerance, abiotic stres

Effect of Exogenously Applied 24-Epibrassinolide and Brassinazole on Xylogenesis and Microdistribution of Cell Wall Polymers in Leucaena leucocephala (Lam) De Wit

Plant growth regulators play a key role in cell wall structure and chemistry of woody plants. Understanding of these regulatory signals is important in advanced research on wood quality improvement in trees. The present study is aimed to investigate the influence of exogenous application of 24-epibrassinolide (EBR) and brassinosteroid inhibitor, brassinazole (BRZ) on wood formation and spatial distribution of cell wall polymers in the xylem tissue of Leucaena leucocephala using light and immuno electron microscopy methods. Brassinazole caused a decrease in cambial activity, xylem differentiation, length and width of fibres, vessel element width and radial extent of xylem suggesting brassinosteroid inhibition has a concomitant impact on cell elongation, expansion and secondary wall deposition. Histochemical studies of 24-epibrassinolide treated plants showed an increase in syringyl lignin content in the xylem cell walls. Fluorescence microscopy and transmission electron microscopy studies revealed the inhomogenous pattern of lignin distribution in the cell corners and middle lamellae region of BRZ treated plants. Immunolocalization studies using LM10 and LM 11 antibodies have shown a drastic change in the micro-distribution pattern of less substituted and highly substituted xylans in the xylem fibres of plants treated with EBR and BRZ. In conclusion, present study demonstrates an important role of brassinosteroid in plant development through regulating xylogenesis and cell wall chemistry in higher plants

High frequency Agrobacterium mediated genetic transformation in rubber tree via. vacuum infiltration

In a tree species like Hevea brasiliensis, genetic transformation offers a viable approach for crop improvement within a short period. Hevea being highly recalcitrant to in vitro culture, an efficient transformation protocol is necessary for generating large number of transgenic plants with stable foreign gene expression. After several modifications in the initial transformation protocol, the transformation frequency was increased to 14 per cent using proliferated anther callus. Therefore, vacuum infiltration was attempted for enhancing the transformation frequency. In the present work the conditions for vacuum infiltration viz. vacuum pressure and period of infiltration were standardized. Vacuum infiltration at 30 psi pressure for 10 minutes was found to be ideal for getting high frequency transformation in H. brasiliensis. Irrespective of the gene constructs experimented, transformation frequency was significantly improved by adopting vacuum infiltration. Employing this technique, the transformation efficiency of MnSOD gene construct with FMV34S promoter could be enhanced from 14 to 50 percent

Drought tolerance in MnSOD transgenic Hevea brasiliensis in a dry sub-humid environment

One year old bud-grafted plants of MnSOD transgenic Hevea lines (L1 and L2) and an untransformed line of clone RRII 105 were used in the present study to evaluate their physiological performance in a dry sub-humid environment by withholding irrigation and to assess the recovery by re-watering. The dry matter partitioning was more towards the root in transgenic lines (55% and 60% in Ll and L2, respectively) but, was less in the untransformed RRII 105 (43%). After six days of moisture stress in polybags, pre-dawn leaf water potential and relative water content declined in all the lines, however, transgenic line L1 showed higher tissue water content throughout the drought as well as recovery period. Chlorophyll content did not show any significant reduction. Net photosynthetic rate (PN) declined rapidly and it reached near zero on the third day of drought imposition except for line L1, which showed lesser decline in PN. The decline in stomatal conductance (gs) was more rapid than PN in all the lines. On re-watering, recovery of PN and gs was better in the transgenic lines than untransformed RRII 105, which did not recover fully from the drought impact. Antioxidant enzymes, superoxide dismutase and peroxidase did not show a definite trend in their activities in these lines. However, it was found that the transgenic line L1 had better drought tolerant capacity in terms of lesser inhibition of photosynthetic rate under drought and faster recovery on re-watering

Induction of callus and plant regeneration in Vicoa indica

Callus cultures were initiated from the stem and leaf explants of aseptically grown Vicoa indica. A simple method is described for plant regeneration from callus and the rapid multiplication of the plants thus obtained. Callus initiation was optimum in Gamborg B5 (B5) basal medium containing either $2.0 mg\hspace{2mm} l^{-1}$ naphthaleneacetic acid (NAA) with $0.2 mg\hspace{2mm} l^{-1}$ kinetin (Kn) or $2.0 mg\hspace{2mm} l^{-1}$ 6-benzylaminopurine (BAP) with $0.2 mg\hspace{2mm} l^{-1}$ NAA. The calli initiated on B5 medium were able to proliferate on both Murashige and Skoog (MS) and B5 basal medium. Shoot primordia were obtained from greenish callus on passage to B5 basal medium containing $3.0 mg\hspace{2mm} l^{-1}$ BAP and $1.0 mg\hspace{2mm} l^{-1}$ Kn. On further subculture onto B5 medium containing $0.2 mg\hspace{2mm} l^{-1}$ Kn the shoot primordia developed into plantlets

Optimization of RQRT-PCR protocols to measure <img src='/image/spc_char/beta.gif'>-1,3-glucanase mRNA levels in infected tissues of rubber tree (<i style="">Hevea brasiliensis</i>)

492-498RQRT-PCR technique was evaluated for its validity as an alternative to Northern blotting for quantification of plant gene expression in diseased tissues of Hevea. Reliable RT-PCR results could be obtained by co-amplification of housekeeping actin gene as the internal control along with the gene of interest. The product of interest was quantified relative to that of the internal control by measuring net intensity of bands. Expression levels of defense-related -1,3-glucanase gene was studied in the pathogen infected tissues of rubber. The -1,3-glucanase gene was found to be induced in infected leaf tissues and reached a peak at 48 h after inoculation. The -1,3-glucanase gene expression during pathogen infection was determined through Northern blot hybridization also, using 18S RNA as the internal control. RQRT-PCR and Northern hybridization showed almost similar results, thereby validating the use of this technique to study the gene expression in rubber

Hypothermia due to limbic system involvement and longitudinal myelitis in a case of Japanese encephalitis: a case report from India

Santhosh Narayanan,1 NK Thulaseedharan,1 Gomathy Subramaniam,2 Geetha Panarkandy,1 VK Shameer,1 Arathi Narayanan1 1Department of General Medicine, 2Department of Radiodiagnosis, Government Medical College, Kozhikode, Kerala, India Abstract: Japanese encephalitis (JE) is an infectious encephalitis prevalent in Asia. It usually presents with fever, headache, convulsions and extrapyramidal symptoms. Limbic system involvement and hypothermia though common in autoimmune encephalitis have never been reported in JE. We report a case of an 18-year-old girl with no previous comorbidities who presented to us with a history of fever and headache for 1&nbsp;week duration. She developed bilateral lateral rectus palsy and asymmetric flaccid weakness of all four limbs, after 2&nbsp;days of admission, which was followed by altered sensorium and intermittent hypothermia. Neuroimaging revealed longitudinal myelitis extending from pons till the L1 level along with bilateral thalamic hemorrhage in brain. Cerebrospinal fluid (CSF) was positive for IgM antibody to JE virus. She was treated with supportive measures, but she developed intractable hypothermia and seizures and succumbed to illness after 2&nbsp;weeks of admission. Keywords: Japanese encephalitis, hypothermia, limbic syste