Study of cucumber mosaic virus gene expression in Capsicum annuum

Cucumber mosaic virus (CMV) is a plant pathogenic virus in the genus Cucumovirus, family Bromoviridae. It has the potential and reputation of having the widest host range of any known plant virus including monocotyledons and dicotyledons, herbaceous plants, shrubs and trees. CMV is one of the major diseases in Capsicum annum (chilli). Chilli plant samples exhibiting virus-like disease symptoms were collected from Taman Pertanian Indera Mahkota (location 1) and Greenhouse 12 of Horticulture Research Centre, Serdang (location 2). Viral disease was detected based on symptoms like mosaic-mottling, yellow ringspots and cholorotic that appeared on the leaves. The isolation of total RNA was done by using Vivantis GF-1 total RNA extraction kit. RT-PCR technique was used to detect the presence of virus disease symptoms gene in chillies. Identification of causal agents was based on cDNA amplified product size, using virus-specific oligonucleotides. Actin was used as the internal PCR control. The product size of the DNA fragment was 315 bp. From RT-PCR, the expression of CMV can be detected in chilli plants that exhibited the virus-like disease symptoms. This research revealed that some of the chilli plants at the Taman Pertanian Indera Mahkota and Greenhouse 12 of Horticulture Research Centre have been affected by this viral disease

Response of Arabidopsis thaliana seedlings to lead exposure: effect of pre-treatment with sodium nitroprusside

Plants are regularly exposed to unfavorable conditions that impose stress. Lead (Pb), is one of the major pollutants in the environment that causes serious public health and environmental concerns. Increasing levels of lead could also have severe consequences for plants. Plants exposed to lead stress initiate signaling pathways, and make specific changes in their cell physiology and metabolism to avoid or tolerate the stress. However, mechanisms to reduce the effects of lead may vary from plant to plant, developmental stage, and culture medium. Nitric oxide is an important signaling molecule in plant development and defense responses. It has been shown to play a major role in plant responses to several abiotic stresses, such as heat, chilling, drought, salt, UV irradiation and ozone exposure. The objective of this study is to examine the physiological responses to Pb exposure of Arabidopsis thaliana seeds pre-treated with sodium nitroprusside (SNP), a nitric oxide donor. All experiments were carried out using 7-day-old seedlings in a laboratory environment. Pb contents were determined using a graphite furnace spectrometer. Antioxidant assays and reactive oxygen species were carried out using a microplate reader. A major finding is that Pb treatment resulted in increased oxidative stress, which was counteracted by SNP pre-treatment. This and other results obtained are discussed in relation to a strategy to tolerate Pb accumulation in plant cells

Gene expression of Anx3 in Pb-treated Nicotiana tabacum using a real-time RT-PCR

Lead (Pb) is one of the highly persistent, toxic, and widely distributed heavy metal pollutants in the environment. This heavy metal has a tendency to enter human food chain, thus affecting public health. One effective way to remove heavy metals pollutant is by using plants, a technology known as phytoremediation. One such plant that is routinely employed as an experimental model for such studies is Nicotiana tabacum. As tobacco plants are not generally consumed by herbivores, it minimizes the possibility of Pb from entering food chain. A number of studies suggest that annexins, a calcium-binding protein, does play a role in plant stress response. The expression of annexin gene in plants appeared to be regulated by tissue-specific developmental and environmental signal. A vacuole-associated annexin from N. tabacum, Anx3, was investigated, to observe the involvement of this gene in Pb-induced stress. Reverse transcription following quantitative real-time 0olymerase chain reaction (qRT-PCR) is a useful analysis to study gene expression. In this analysis, a reference gene that acts as internal control (housekeeping gene) is routinely employed for normalization of qRT-PCR against the target gene (Anx3). The candidate reference genes, L25, EF-1α, and Ntubc2, were evaluated using suitable primer pairs in order to select the most stable reference gene for normalization of qRT-PCR in this study. Using geNorm, NormFinder, and BestKeeper programs, the most suitable reference gene identified in this study was L25. The relative quantification of Anx3 gene expression normalizing against L25 was accomplished by REST software. The expression level of Anx3 in Pb-treated N. tabacum was upregulated by 2.2-fold (p < 0.05). The experimental methods used and the participation of Anx3 in defense against Pb stress will be discussed

Participation of annexin 1 in the response of Arabidopsis thaliana to lead exposure: potential for phytoremediation

Heavy metal pollution has become a serious public health and environmental concern. Lead (Pb) is one of the heavy metals known to bioaccumulate in plants. Phytoremediation is an emerging technology based on the ability of green plants to remove Pb from the environment in a cost-efficient and ecologically sound manner. Currently, an important research focus is to seek a better understanding of the mechanisms of Pb tolerance by plant cells, with the aim of genetically engineering plants with improved tolerance to Pb, and hence better phytoremediation capabilities in the near future. Annexin, a calcium-dependent membrane-binding protein is believed to play a role in many essential cellular processes. It has been shown that expression of annexin genes from Arabidopsis thaliana are differentially regulated in response to a variety of abiotic stresses. Thus annexins are likely be involved in the response of plants to heavy metal stress. This study aimed to obtain new insights into whether annexin 1 (AnnAt1), is involved in Pb tolerance in plant cells. Message levels of AnnAt1 were assessed in response to Pb treatments using quantitative real-time PCR. Expression results were analysed using REST 2008 and normalized against the mitosis protein YLS8. We found that Pb effect on AnnAt1 expression in plants exposed to lower Pb concentrations (25 µM, 50 µM, and 75 µM) was not significantly different from the controls. However, AnnAt1 message levels doubled (2.12-fold, S.E. range is 1.77 - 2.61, p < 0.001) in seedlings treated with 100 µM Pb, in comparison to the control plants. The relative contribution of AnnAt1 in defence against Pb stress will be discussed

Response of Arabidopsis thaliana seedlings to lead exposure

Lead (Pb) is one of the most commonly occurring, highly persistent and widely distributed heavy metal contaminants in the environment. It has a tendency to bioaccumulate in animals and plants, and potentially, it is able to enter the human food chain where it poses a hazard to public health. Generally, conventional remediation technologies applied to decontaminate heavy metals from groundwater and soils are very costly. Hence, phytoremediation has emerged as an ecologically friendly and economically attractive technology that uses green plants to clean up heavy metal contaminated sites. However, a lack of knowledge of the biological processes associated with plant responses to Pb (e.g. Pb uptake, accumulation, translocation, and tolerance) has been a bottleneck for the application of Pb phytoremediation in the field. A model genetic system of higher plants, Arabidopsis thaliana, was selected to further examine the physiological, biochemical and molecular events occuring in plants under Pb stress. The overall aim of this project was to obtain a better understanding of plant responses to Pb contaminants in the early developmental stages of A. thaliana seedlings. This research encompassed the physiological responses of A. thaliana seedlings to Pb exposure, monitoring their antioxidative defence systems, and investigating the participation of annexin 1 in the response to Pb-mediated oxidative stress. This research also assessed the protective effect of nitric oxide on Pb-induced toxicity of A. thaliana seedlings and it isolated a putative Pb tolerant mutant from an EMS-mutagenized M2 population. A multiexperimental approach was adopted to achieve these objectives. A. thaliana seedlings were grown on modified Huang & Cunningham (1996) nutrient solution containing 0.8% (w/v) agar, with and without Pb(NO3)2, under controlled conditions. A. thaliana seedlings were insensitive to Pb during seed germination. In treatments with up to 200 μM Pb(NO₃)₂, morphological changes and inhibition of root growth were observed in the 7-d-old seedlings. A tolerance index revealed that Pb(NO₃)₂ concentration of 75 μM and higher brought about more than 50% root growth inhibition. Pb was predominantly retained in the roots. Analysis using a graphite furnace atomic absorption spectroscopy indicated that the level of Pb accumulation in A. thaliana roots was greatly dependent on the Pb(NO₃)₂ concentrations, but only a small fraction of the accumulated Pb was translocated to the shoots (18 - 43%). Transmission electron microscopy analysis showed that Pb was mainly immobilized in the cell walls and intercellular spaces. This was interpreted as a mechanism that minimizes the entry of Pb into cells and interference with cellular functions. Pb that gained entry into the cytoplasm was sequestered into the vacuoles. The toxicity of Pb in the cytosol of A. thaliana seedlings was studied by measuring the H₂O₂ and lipid hydroperoxide levels using a microplate reader. When the Pb(NO₃)₂ concentration in the growth medium was 100 μM, the 7-d-old seedlings contained 2.2-fold higher H₂O₂ and 9.6-fold higher lipid hydroperoxide than the control without Pb(NO₃)₂. This was followed by an up-regulation of the activity of antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPX), and general peroxidase (POD) by 2.1-, 3.2-, 2.3-, 1.8- and 4.6-fold, respectively, compared with the control. Pb toxicity is known to trigger oxidative stress, but A. thaliana seedlings appeared to be capable of activating cell rescue, defending themselves against harmful oxidative stress and also acclimating to Pb. Data from physiological and biochemical analysis indicate that a combination of avoidance and tolerance mechanisms exists in Pb-treated A. thaliana seedlings to maintain the essential cellular metabolism for survival. Real-time reverse-transcription polymerase chain reaction was used to show the involvement of AnnAt1 in the response of 7-d-old A. thaliana seedlings to a high threshold concentration of Pb. When the seedlings were treated with 100 μM Pb(NO₃)₂, AnnAt1 message levels were up-regulated by 2.12-fold. Pb-mediated oxidative stress may be a component of AnnAt1 gene expression. AnnAt1 potentially could be invoked to reduce the toxic effects of Pb stress by acting as ROS and/or Ca²⁺ signals, as a membrane protector, in detoxification of excessive ROS, or in sequestration of Pb. Pb stress symptoms were less evident in seedlings pre-treated with 1 mM sodium nitroprusside (SNP), a nitric oxide (NO) donor. The present study found that exogenous NO did not alter Pb transport into the plants or efflux pumping of Pb at the plasma membrane. However, NO conferred protection to 7-d-old A. thaliana seedlings primarily by acting as an antioxidant or a signal for actions to scavenge excessive ROS level. The application of exogenous NO before subjecting to 100 μM Pb(NO₃)₂ decreased H₂O₂ back to its original level, and reduced 50% lipid hydroperoxide in the Pb-treated seedlings. As a result, the antioxidative enzyme activities in Pb-exposed seedlings pre-treated with SNP were 23 - 45% lower than those without SNP pre-treatment. Less antioxidative enzyme activities were probably needed to counteract the reduced amount of Pb-induced ROS in A. thaliana seedlings. A post-germination procedure involving prolonged exposure to 150 μM Pb(NO₃)₂ was developed to screen an EMS-mutagenized M2 population of A. thaliana. Potential Pb tolerant mutants were selected based on the ability to grow with their roots penetrating into the medium and maintain purple-green leaves without wilting. A minority of the survivors appeared to go into a resting stage and they seemed to have altered transporters that prevented Pb from entering the cells. Only one putative Pb mutant (M3-1) was recovered from the rescue and set seeds. The M₄ generation of this putative Pb mutant was re-screened for phenotypic confirmation and to determine the regulation of AnnAt1. The 7-d-old putative Pb mutant seemed to display enhanced root and shoot growth in the presence of 150 μM Pb(NO₃)₂ compared to the wild-type seedlings. The transcript level of AnnAt1 in this putative Pb tolerant mutant increased by 2.19-fold when exposed to 150 μM Pb(NO₃)₂

Investigation into participation of annexins in the response of Arabidopsis thaliana roots to lead (Pb) exposure: Preliminary study

As shown in previous studies, annexins are involved in many essential cellular processes and defence against various types of biotic and abiotic stresses. The purpose of this study is primarily to assess the relative contribution of annexins in the antioxidative defence against lead (Pb) stress. It is hypothesized that Pb exposure could result in oxidative burst in Arabidopsis thaliana, particularly the roots, which are in direct contact with the heavy metal. This new insight of how annexins might be involved in Pb tolerance of plant cells will pave way to the development of phytoremediation technology based on this green plants’ ability to remove Pb from the environment in a cost-efficient and ecologically sound manner. The initial correlative studies included studies on the effect of Pb on plant growth, determination of Pb content in plant material, ultrastructural localization of Pb within plant cells, and antioxidative enzyme assays. The severity of these responses increased with increasing concentrations of Pb. On the other hand, the initial manipulative studies showed that the interaction of NO with reactive oxygen species acts as a protective mechanism in Pb stress. All these preliminary experiments will further support the role which annexins could play in the response of plants to heavy metal stress in order to protect the cells from the stress. Primers for annexin 1 (AnnAt1) have been obtained and testing for RT-PCR is now underway