A Meta-Analysis of Transcriptomic Footprints - Research Paper Example

Meta Analysis The major findings and conclusions from the meta analysis are presented below. Firstly, reactive oxygen species are constantly produced during most of the metabolic activities of the cell. When photosynthesis takes place in plants, one of the by products is molecular oxygen, and the concomitant companions of aerobic life are reactive oxygen species. The reactive oxygen species are generally partially reduced or activated derivatives of oxygen, such as singlet oxygen, superoxide anion, hydrogen peroxide and hydroxyl radical, which are all reactive and toxic and thereby can lead to the destruction of cells. These species also have a signalling role and are very important in carrying out this signalling function, which is evident during various developmental processes such as allelopathic plant-plant interactions, cell elongation and division, programmed cell death. It may also be noted during environmental processes as well as biotic and abiotic stress responses. Since a major part of metabolic activities are highly oxidizing, or have very intensive rates of electron flow; as a result, they tend to produce high levels of ROS. The signalling role is important because it produces the control and regulation of various biological processes and the ROS appear to have a dual role for these ROS in plant biology; both as the toxic by-products of aerobic metabolism as well as regulators of the various cell based processes associated with growth, development as well as patterns of defense. Hence, at the outset, the significance of this study lies in the fact that the importance of reactive oxygen species has been identified, i.e., as a toxic byproduct of the biological processes which tends to function as a signal to regulate the various cell processes. Moreover, another aspect that could also be deduced in relation to this study and the findings of other researchers was outlined in the study that the ROS activity can lead to the oxidative destruction of cells. This is important because it shows that in so far as the evolution of aerobic organisms is concerned, the development of efficient ROS scavenging mechanisms are likely to be a causal factor in such evolution. The oxidative destruction of cells is caused by the partially reduced or activated derivatives of oxygen such as hydrogen peroxide and hydroxyl radical. The genome wide microarrays provide the means to access the changes in transcripts arising out of an alternation in specific types of ROS. For example, when light stress occurs, there is an expression of heat shock proteins, which can be controlled by cystolic H2O2. Despite the existing knowledge about the signalling properties of the reactive oxygen species however, the analysis of the various studies concludes that very little is known about the mechanisms governing the process, such as the manner in which stimuli are perceived, or the manner in which these stimuli are transferred into specific cellular responses. The reasons for this lack of knowledge lie in the fact that until recently, there were no accurate and quantifiable means available which could be used to apply experimental methods to detect ROS systems. Another conclusion in the study is that the basal levels of the reactive oxygen species are carefully controlled. The control of these basal levels occurs through the operation of a complex gene network that is able to function in all the sub cellular components. If the signalling actions by these species are to function in a manner that is efficient in the various kinds of developmental and environmental processes they function in, it is necessary that they possess a certain extent of specificity in that they must be able to target the necessary areas where the process is to work and selectivity in that they should be able to act according to the needs generated by the process in question. The signalling role of the reactive oxygen species became evident because hydrogen peroxide, when identified early, has been found to orchestrate an early hypersensitive response, and the signalling response is extremely important during the different processes which have been identified earlier. The objective of the study by Gadjev et al (2006) was to gain some insight into the response offered by plants to oxidative stress. In the process, several ROS were determined which had signalling properties, i.e., H2O2, singlet oxygen (1O2), hydroxyl radical (OH_), and superoxide anion radical (O2_). In this study, transgenic Arabidopsis plants were produced which had compromised levels of certain anti-oxidant enzymes. This in turn produced a mutation wherein the conditional and fluorescent mutant functioned as the means and the tools and provided the basis to assess specific effects of the ROS signalling, because they provided quantifiable measures. The fluorescence was akin to a transcriptomatic footprint that could be used to detect the individual ROS scavenging enzymes. These transriptomatic footprints, as they travelled downstream, were evident because the enzymes used to generate them went through specific alterations in their expression by the ROS and highlighted the pathways which serve as the points where ROS mediated plant responses occur. The objective of the experiment was to derive a comparative analysis on various sets of ROS related data and this data was compiled from all the relevant publicly available or the in-house stored microarray experiments that were dependent on ROS data. While the main subject of the experiments was related to Arabidopsis plants, only those genome wide profiles that were derived using Affymetrix ATH1 or Agilent Arabidopsis2 oligonucleotide arrays that had a high technical quality were used. The objective was to generate an oxidative blast that led to the production of lesions that are associated with cell death and the formation of these oxidative blasts were accelerated by the breakdown of ozone in the apoplast. This study is therefore significant in that it undertook an examination of stress dependent expression of transcript in plants, as metabolic reactions were taking place. When the levels of ROS are raised, a transcriptomatic footprint is produced of the specific signalling capacity than an individual reactive oxygen species has. But as the study also points out, identifying the specific components of the ROS which are responsible for the selectivity and specificity in the signalling tendencies still remains a challenge that needs to be overcome through further study. The conclusion that the authors arrived at was that despite the limitations and need for further research identified in this study, the findings nevertheless make an invaluable contribution to potential further research into the impact of specific ROS signals in environmental conditions, as well as in providing further clarifications and insight into the molecular mechanisms which run concurrently with the oxidative stress response in plants. The study also showed that among the nine ROs generating systems which were used in the study, there was a specific ROS that was derived from each of the specific experiments that was used. In the experiment using flu mutant, oxygen was the major ROS derived; for the KD-SOD experiment, O2- , for the O3 fumigation experiment, the ROS which were identified were ozone, oxygen and hydrogen peroxide. Applying the experiment using CAT2HP1 +HL, hydrogen peroxide was the ROS derived, with the AT spray, hydrogen peroxide, similarly with LOH2 and AAL toxin combined as well, hydrogen peroxide was the derived ROS. The treatment/growth conditions were also varied in accordance with the nature of the experiment, as were the time points, although the two microarray platforms which were primarily used were ATH1 and Arab2. The study also found that in those cases where the flu mutant was used, a rapid response was provoked, but in the case of exogenously applied MV, more time was required for the substance to penetrate the leaves and perform the necessary actions associated with the reaction, to generate the ROS. The strongest reactions were also generated in the case of the mutant flu, AAL toxin and the use of ozone experiments. The experiment was a valuable one because it was able to use several different experimental variables in order to derive the ROS. The data was sub divided into three different horizontal clusters – the first cluster with the MV, flu and ozone based experiments, the second cluster with the KD-Sod and the KO-Apx1 transgenics, while the third cluster comprised the AAL toxin, AT and CAT2HP1 experiments. The findings in the study also showed that five transcripts were up-regulated more than five fold times in at least seven of the eight experiments that were conducted. The oxidative stress response generated these transcripts as their hallmark states, which occurred irrespective of what kind of ROs was involved and also what was the sub cellular site of its production. The oxidative cellular damage appeared to be a condition that was produced as signals which generated transcripts. Yet another important discovery in this experiment was that the expression of two of the transcripts were proteins of unknown function. One of these two proteins were At1g19020, while the other was a TIR (Toll Interleukin-1) class disease resistant protein. While the At1g class of protein showed results far below the imposed threshold of five fold difference, the TIR class protein showed a 4.6 fold increase in expression in the KD-SOD plants. The identification of these TIR proteins was significant because they are the key players in recognition of pathogens and their defense response, and both of these processes are closely linked to oxidative stress as well as ROS signalling. The appearance of stress is an indication that a particular cell is experiencing some form of a disease, to which it responds with a defense reaction. 66 transcripts were found to be common between and exclusive for the flu, MV and ozone experiments and a possible explanation for these transcripts arises in the cell death, which also produces secondary effects that affect gene expression. These transcripts were considered to be hallmarks for oxidative stress that was characterized by the chemical identity of the ROS that was produced, as well as the sub cellular site where it was produced. Another important finding of the experiment was the activity at the thylakoid membranes; decreasing the extent of Cu/ZnSOD activity at the site of these membranes leads to an up-regulation of chloroplastic transcripts, thereby suggesting a highly specific oxygen sensor mechanism in the thylakoids. The results from the study also produced an inference that the transcripts involved in the production of anthocyanins were coagulated in response to oxygen, while hydrogen peroxide impinges negatively on the expression of these transcripts; hence the inverse correlation between the effects of oxygen and hydrogen peroxide is a demonstration of the clear signalling capacities of particular ROS. The study also provided an inference to explain the accumulation of ROS; i.e., the ROs accumulation could represent the toxic by products of stress, while at the same time it could also represent important transduction molecules, with said molecules being responsible for the activation of defense mechanisms. Relatively, there was an abundance of oxygen and hydrogen peroxide transcripts in plant tissues which were subjected to different abiotic stressors. The study found that there was a complex pattern of different ROS transcripts that were evident during all stress conditions, which suggested that there are a range of different ROS stressors which arte generated in cells when they are subjected to abiotic stressors. Those transcripts which are responsive to oxygen represent the largest fraction of genes induced that are related to ROS during these stresses. As opposed to this, the hydrogen peroxide stressors were mainly repressed during the abiotic stressors. Based upon these results, the study drew an inference that different abiotic stress conditions tend to stimulate the production of different kinds of ROS, and depending upon the transcriptome profiling analysis, it was also possible to predict the extent of involvement of a a particular kind of ROS during a specific stress condition. On the basis of the above findings, the manor conclusions of the study were that the expression of transcripts is influenced by the elevated ROs and it also impacts upon the kind of transcriptomatic footprint that is produced, which thereby provides an indication about the specific signalling capacities of the ROS signalling. Conclusions: On the basis of the above, it may be concluded that the analysis which was conducted has proved to be a valuable exercise, but only to a limited extent. The analysis has identified two significant components of the role played by reactive oxygen specifies, i.e, firstly they function as toxic products but this makes them important in terms of functioning as signalling devices, which advertise how the cell processes are taking place. The signalling function is also important because it does regulate the various different processes which take place in the cell, including cell division and elongation, environmental processes and abiotic and biotic stress responses. It may be noted that before this study was conducted, there was very little that was known about reactive oxygen species apart from the fact that they existed and the vague suggestion that they also provided signalling activity. This study has also been very useful because it enabled a process which was able to derive a transcriptomatic footprint which provided a distinct and measurable means of identifying the signalling activity which was taking place. It provided quantifiable measures by developing fluorescent “footprints” which could be used as a means to detect the activity of the ROS; as a result it is useful because it was able to show clearly that signalling does take place and to do it in such a manner that it was evident, visible and noticeable and capable of being measured. As a result, it is significant because it enables a definitive conclusion to be drawn, i.e, that these ROS do exist and that they have a signalling function, because this signalling function has been quantified and put forward in a manner that can be measured. The study was not only able to detect signalling activity; it was also able to provide a basic explanation for how such activity occurring in cells could be detected by measuring the activity of these species in the context of biotic and abiotic stress responses through the detection of fluorescent footprints. The study is however, limited because while it was able to detect signalling activity, it was not able to actually determine the actual mechanisms underlying the activities and how they were being carried out. The study remained unable to determine exact components of ROS which were responsible for the signalling activities; therefore the value of their contributions might be limited. Moreover, the experiment s that were performed in this instance using flu mutants or MV were tested through different experiments; as a result there was no homogeneity in the experimental set-ups, which did not provide a good basis for comparison. The method used for the comparison of the data was thus a relative one, which was appropriate in this instance because the objective was to try and identify the different kinds of ROS; however due to the lack of homogeneity, it makes it difficult to compare the results effectively and arrive at definitive conclusions. The use of relative expression data also gave rise to possible false positives or negatives associated with this approach. As a result, the applicability of these results on a widespread basis would be quite difficult. Reference Gadjev I., Vanderauwera S., Gechev T.S., Laloi C., Minkov I.N., Shulaev V., Apel K., Inzé D., Mittler R. & Van Breusegem F. (2006) Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis. Plant Physiology 141, 436-445. Read More