Chhandak Basu

Efficient Atmospheric Cleansing of Oxidized Organic Trace Gases by Vegetation

Volatiles Versus Vegetation Plants act as both global sources and sinks of highly reactive volatile organic compounds (VOCs). Models typically treat the uptake and degradation of these compounds as if they are mostly unreactive, like other more commonly studied biogenic gases such as ozone. A study by Karl et al. (p. 816, published online 21 October) suggests that VOCs may be more reactive than expected. By monitoring six field sites representing a range of deciduous ecosystems, several oxidized VOCs were found to have high deposition fluxes. Fumigation experiments in the laboratory confirmed that leaves are capable of oxidizing these compounds, and do so through an enzymatic detoxification or stress-response mechanism. Budgets for VOC flux in the atmosphere suggests that, on a global scale, plants may take up significant levels of VOCs in polluted regions, especially in the tropics. Deciduous trees enzymatically remove oxygenated volatile organic compounds from the atmosphere. The biosphere is the major source and sink of nonmethane volatile organic compounds (VOCs) in the atmosphere. Gas-phase chemical reactions initiate the removal of these compounds from the atmosphere, which ultimately proceeds via deposition at the surface or direct oxidation to carbon monoxide or carbon dioxide. We performed ecosystem-scale flux measurements that show that the removal of oxygenated VOC via dry deposition is substantially larger than is currently assumed for deciduous ecosystems. Laboratory experiments indicate efficient enzymatic conversion and potential up-regulation of various stress-related genes, leading to enhanced uptake rates as a response to ozone and methyl vinyl ketone exposure or mechanical wounding. A revised scheme for the uptake of oxygenated VOCs, incorporated into a global chemistry-transport model, predicts appreciable regional changes in annual dry deposition fluxes.

Real‐time PCR (qPCR) primer design using free online software

Real‐time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT‐PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double‐stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green‐based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real‐time PCR, the basic rules and pitfalls of primer design, and provides a step‐by‐step protocol for designing SYBR® Green‐based primers with free, online software. Biochemistry and Molecular Biology Education Vol. 39, No. 2, pp. 145–154, 2011

The production of the sesquiterpene β-caryophyllene in a transgenic strain of the cyanobacterium Synechocystis.

The plant secondary metabolite, β-caryophyllene, is a ubiquitous component of many plant resins that has traditionally been used in the cosmetics industry to provide a woody, spicy aroma to cosmetics and perfumes. Clinical studies have shown it to be potentially effective as an antibiotic, anesthetic, and anti-inflammatory agent. Additionally, there is significant interest in engineering phototrophic microorganisms with sesquiterpene synthase genes for the production of biofuels. Currently, the isolation of β-caryophyllene relies on purification methods from oleoresins extracted from large amounts of plant material. An engineered cyanobacterium platform that produces β-caryophyllene may provide a more sustainable and controllable means of production. To this end, the β-caryophyllene synthase gene (QHS1) from Artemisia annua was stably inserted, via double homologous recombination, into the genome of the cyanobacterium Synechocystis sp. strain PCC6803. Gene insertion into Synechocystis was confirmed through PCR assays and sequencing reactions. Transcription and expression of QHS1 were confirmed using RT-PCR, and synthesis of β-caryophyllene was confirmed in the transgenic strain using GC-FID and GC-MS analysis.

Leaf Proteome Analysis Reveals Prospective Drought and Heat Stress Response Mechanisms in Soybean

Drought and heat are among the major abiotic stresses that affect soybean crops worldwide. During the current investigation, the effect of drought, heat, and drought plus heat stresses was compared in the leaves of two soybean varieties, Surge and Davison, combining 2D-DIGE proteomic data with physiology and biochemical analyses. We demonstrated how 25 differentially expressed photosynthesis-related proteins affect RuBisCO regulation, electron transport, Calvin cycle, and carbon fixation during drought and heat stress. We also observed higher abundance of heat stress-induced EF-Tu protein in Surge. It is possible that EF-Tu might have activated heat tolerance mechanisms in the soybean. Higher level expressions of heat shock-related protein seem to be regulating the heat tolerance mechanisms. This study identifies the differential expression of various abiotic stress-responsive proteins that regulate various molecular processes and signaling cascades. One inevitable outcome from the biochemical and proteomics assays of this study is that increase of ROS levels during drought stress does not show significant changes at the phenotypic level in Davison and this seems to be due to a higher amount of carbonic anhydrase accumulation in the cell which aids the cell to become more resistant to cytotoxic concentrations of H2O2.

Promoter analysis in transient assays using a GUS reporter gene construct in creeping bentgrass (Agrostis palustris)

Transient expression profiles for several chimeric beta-glucuronidase (GUS) gene constructs were determined in tissues (young leaves, mature leaves and roots) of creeping bentgrass (Agrostis palustris, cv. Penn A4) following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by four different promoters (ubiquitin 3-potato, ubiquitin corn, ubiquitin rice and CaMV 35S). The total number of GUS hits (or transient expression units; TEUs) were determined manually under a dissecting scope after histochemical staining for GUS. Results suggest that the ubiquitin rice promoter is most active in cells of turfgrass, regardless of the developmental stage or tissue-type. The ubiquitin corn promoter was the next best. Of the four promoter used, except for ubiquitin 3-potato, reporter gene activity was dramatically higher in mature leaves compared to young leaves. The relative efficiency of each promoter was about the same in roots and leaves. We have also analyzed uidA (GUS) reporter gene activity following microprojectile bombardment in transient expression assays with callus from two cultivars (Providence or Penn A4) of creeping bentgrass. Differences in the frequency of GUS positive hits were observed between cultivars up to 72 hours post-bombardment. However, this difference between cultivars disappeared after 72 hours post-bombardment. This information describing promoter functionality in bentgrass will be important when designing gene constructs for trait modification and when choosing appropriate cultivars for improvement through gene transfer experiments. This is the first in depth report on organ-specific and developmental gene expression profiles for transgenes in a turfgrass species.

Comparative Analyses of Plant Transcription Factor Databases

Transcription factors (TFs) are proteinaceous complex, which bind to the promoter regions in the DNA and affect transcription initiation. Plant TFs control gene expressions and genes control many physiological processes, which in turn trigger cascades of biochemical reactions in plant cells. The databases available for plant TFs are somewhat abundant but all convey different information and in different formats. Some of the publicly available plant TF databases may be narrow, while others are broad in scopes. For example, some of the best TF databases are ones that are very specific with just one plant species, but there are also other databases that contain a total of up to 20 different plant species. In this review plant TF databases ranging from a single species to many will be assessed and described. The comparative analyses of all the databases and their advantages and disadvantages are also discussed.

IN SILICO ANALYSIS OF TERPENE SYNTHASE GENES IN ARABIDOPSIS THALIANA

Terpenes are defense chemicals found in wide groups of plants. Terpenoids play a large role in plant development and stress response. The terpene synthase family comprises a diverse set of genes, all which contribute to production of terpenoids. We have used tools of bioinformatics and performed an in silico analysis of developmental and tissue specific terpene synthase gene expression in Arabidopsis thaliana, as well as those expressed due to biotic and abiotic environmental stimuli. Using software tools from Genevestigator, a powerful microarray analyzer, we used multiple tool sets to better understand terpene synthase expression in Arabidopsis, which will hopefully open the genetic door to further wet laboratory investigations. The data can be used to predict roles of terpene synthase genes in plant cell division and growth. The data presented here can be used to model for terpene synthesis expression in other plant species and can also be used to integrate basic plant physiology, and ‘omics’ disciplines.

Engineering an Isoprenoid Pathway in Escherichia coli for Production of 2-Methyl-3-buten-2-ol: A Potential Biofuel

Abstract2-Methyl-3-buten-2-ol (MBO) is a natural volatile 5-carbon alcohol produced by several pine species that have the potential to be used as biofuel. MBO has a high energy content making it superior to ethanol in terms of energy output, and due to its volatility and lower solubility in water, MBO is easier to recover than ethanol. Pine’s MBO synthase enzyme utilizes the intermediate dimethylallyl pyrophosphate (DMAPP) produced by the methyl-erythritol-4-phosphate isoprenoid pathway for the production of MBO. In this study, we performed metabolic engineering of Escherichia coli to express an alternate mevalonate dependent pathway for production of DMAPP, along with a codon optimized Pinus sabiniana MBO synthase gene. This heterologous expressed pathway carried out the conversion of an acetyl CoA precursor to DMAPP leading to production of MBO.

Biochemical analysis of ‘kerosene tree’ Hymenaea courbaril L. under heat stress

Hymenaea courbaril or jatoba is a tropical tree known for its medically important secondary metabolites production. Considering climate change, the goal of this study was to investigate differential expression of proteins and lipids produced by this tree under heat stress conditions. Total lipid was extracted from heat stressed plant leaves and various sesquiterpenes produced by the tree under heat stress were identified. Gas chromatographic and mass spectrometric analysis were used to study lipid and volatile compounds produced by the plant. Several volatiles, isoprene, 2-methyl butanenitrile, β ocimene and a numbers of sesquiterpenes differentially produced by the plant under heat stress were identified. We propose these compounds were produced by the tree to cope up with heat stress. A protein gel electrophoresis (2-D DIGE) was performed to study differential expression of proteins in heat stressed plants. Several proteins were found to be expressed many folds different in heat stressed plants compared to the control. These proteins included heat shock proteins, histone proteins, oxygen evolving complex, and photosynthetic proteins, which, we believe, played key roles in imparting thermotolerance in Hymenaea tree. To the best of our knowledge, this is the first report of extensive molecular physiological study of Hymenaea trees under heat stress. This work will open avenues of further research on effects of heat stress in Hymenaea and the findings can be applied to understand how global warming can affect physiology of other plants.

Cross-species PCR and field studies on Paulownia elongata: A potential bioenergy crop

Abstract Paulownia elongata is a short-rotation fast growing tree and is known for high biomass accumulation and carbon sequestration potential. Optimization of protocols for nucleic acid extraction, PCR, RT-PCR, and other molecular biology techniques are required for better understanding of cellulose synthesis and to assess the potential of Paulownia as a biofuel tree. The main objective of this work was to study a putative cellulose synthase amplicon expression under various environmental conditions and evaluate the potentials of Paulownia as a biofuel tree. Using cross-species PCR an amplicon representative of a putative cellulose synthase gene from Paulownia was identified. This 177-bp long DNA sequence was 46% similar with cellulose synthase genes from Arabidopsis as expected. Gene specific primers for this particular Paulownia cellulose synthase gene were designed and reverse transcription PCR was performed to confirm its transcription. We report an inexpensive cDNA dot-blot method to study expression of this gene under various environmental conditions. We observed that cold and, to a lesser extent, heat stress downregulated its expression. This information will help to understand cellulose deposition in plant cell wall under stressful conditions. To the best of our knowledge this is the first characterization of a cDNA sequence from Paulownia elongata.