Chloroplasts are the particular class of plastids in plant cells where photosynthesis takes place. They are subcellular organelles that perform various specific functions in plant cell and algae.
Many research studies have confirmed the fact that evolution of chloroplasts occurred from cyanobacterium through the process of endosymbiosis (Raven and Allen, 2003). This has been revealed by studying the genetic sequence of cyanobacteria (Raven and Allen, 2003). There are many kinds of plastids in plants but chloroplasts are concerned with photosynthesis. One kind of plastids is etioplasts, a primitive stage of cholorplasts, and found in abundance in the leaves of plants that grow in darkness. However, these are instantaneously converted into chloroplasts when they come in contact with sunlight.
Chloroplasts are present in cytoplasm of a cell bears the green pigment chlorophyll. Chlorophyll absorbs sunlight to provide energy for photosynthesis. These are present in autotrophic plants (Briscoe).
Many theories have been suggested for the evolution of chloroplasts. The chloroplasts are monophyletic. Their genome and gene structure matches the genetic sequence of cyanobacterium (Raven and Allen, 2003).
In chloroplasts evolution the most important theory is endosymbiotic gene transfer theory. Theory defines that throughout the stage of evolution endosymbiont events take place and gene transfers from symbionts to the host. The intergenomic transfers and genetic interactions between the nucleus and organelles are highly regulated (Raven and Allen, 2003).
Plastids are derived from cyanobacteria by process of endosymbiosis was first hypothesized by Mereschkowsky (1905). However, further studies in molecular genetics, biochemical, and microscopic studies have revealed that plastids contain ribosomes, DNA/RNA that responsible for inheritance of certain characteristics (Briscoe). The genome in plastids replicates as normal genome, and carry out all other functions of transcription and translation (Briscoe).
Plastids are evolved from blue-green algae (cyanobacteria). A number of studies have confirmed this fact that cyanobacteria are the closest bacterial homolog of plastids. They have a system of sunlight absorbing, oxygen production and water split same that of cholorplasts (Molnar, 1999).
All proteins that are actively involved in plastids functions are encoded by genes. This is said to be the result of evolution of cyanobacteria and gene transfer (Doolittle et al 2003). During this transfer certain genes are moved while others are retained.
“It also becomes possible to see clearly the algal ancestry of cells that have vestigial and otherwise unrecognizable plastids, and even to discern the unmistakable genomic footprint of plastids long lost from organisms one might never imagine to have descended from plants.” (Zhou et al 2006)
Genomes in plastids encode above 100 of proteins (50-200), however there are inmureaable nuclear encoded products found in cytoplasms. Cyanobacterium gneome codes 1500 proteins.
Many similarities have been found between proteome of cyanobacterium and other organelles of cells. It is said that the genes of cyanobacteria evolutes from endosymbiotic pre-plastids (Briscoe). When genes transfer from one location to another it is most probably that some of it will be lost, some will be transferred and some will be retained.
“The process of transfer of genes to the nucleus would have involved duplication of each plastid gene, and a nuclear copy of the gene becoming able to produce a functional product in the cytosol or, with appropriate targeting sequences, in other compartments.” (Zhou et al 2006)
It has been hypothesized that transfer from cyanobacterial genome into the plant genome took place in horizontal gene transfer. Different metabolic pathways are determined for encoding purpose of chloroplast genome and nuclear genome.
Zhou et al (2006) compared the photosynthetic networks of chloroplasts and cyanobateria. It was postulated that compound reactions in chloroplasts are less than cyanobacteria, the path length of metabolic network was longer, less dense, with localized high density areas in chloroplasts.
Enzyme proteins are encoded by genomes. These enzymes actively participate in different cellular functions. Chloroplast’s modular organization is organized in a much better way than cyanobacteria.
Zhou et al (2006) concluded that as there are metabolic network differences between the two, cholorplasts and cyanobacteria, it shows that modifications may occur during evolutionary period during the process of endosymbiosis. Photosynthetic process of light absorption, efficiency of energy absorption, water split and oxygen release depends on the overall metabolic network.
Clegg et al (1996) studied different patterns of genes that facilitate evolution. During the process of evolution nucleotide replacement.
“The chloroplast genome (cpDNA) of plants has been a focus of research in plant molecular evolution and systematics. Several features of this genome have facilitated molecular evolutionary analyses. First, the genome is small and constitutes an abundant component of cellular DNA. Second, the chloroplast genome has been extensively characterized at the molecular level providing the basic information to support comparative evolutionary research. And third, rates of nucleotide substitution are relatively slow and therefore provide the appropriate window of resolution to study plant phylogeny at deep levels of evolution.” (Clegg et al 1996)
DNA sequencing in chloroplasts is a new technique and depends upon how reliable a method is for DNA sequencing. Researches across the United States have developed very latest and sophisticated techniques for DNA sequencing. However, the process is very delicate, that even very minor error can disregard the whole process. Scientific labs have new latest technology implemented and DNA sequencing is made very comprehensive, easy and done locally as well.
With new changes and advances in genetic engineering and its implication in plant genomics and evolution, it has become mandatory to further research in this field, as this can become strong scientific evidence. DNA sequencing is gaining popularity because of its unique and un-identical pattern, which is unique in different plastids. With DNA sequencing use in the research, its fair and valid use is important.
Other problem in studying evolution and DNA sequencing is a factor of contamination. The DNA samples collected from the chloroplasts can get contaminated. There might be contamination of bacteria, dust or organic material in the samples. DNA can also be degraded if left for long period at the labs. Degraded DNA can give false positive or false negative increasing the probability of error in the results.
The allele frequencies are often used for studying evolution in chloroplasts because chances of random match are very great and not appropriate for small number of plastids and it does not affect probability of being similar or dissimilar. Errors produced in first case does not leave enough samples for further sequencing in many cases which cause much of the frustration to scientists and law professionals
Evolutionary studies show that various benefits for DNA sequencing are that DNA patterns are very unique and complete sequencing will differentiate chloroplasts from several other kinds of plastids. DNA when acted upon with polymerase can be amplified and hence smaller sample sizes are sufficient to use for sequencing. Because on single DNA strand can be split into many pieces several samples can be used for sequencing. Any cyanobacterium cell can be examined for DNA sequencing as every cell is nucleated. This gives preference of DNA sequencing of chloroplasts over other sequencing techniques. DNA sequencing also gives it preference over protein sequencing as it is more resistant to degradation. “Modular structures differ among different organisms. The similarity of overall modular structure among chloroplasts, photosynthetic bacteria, E.coli, Arabidopsis thaliana and Cyanidioschyzon merolae has been calculated and is shown as a dendrogram in Figure 2 (see “Methods” section for detailed description of the similarity measurements of modules). Remarkably, all cyanobacteria exhibit very similar modular organization and are different from chloroplasts. Arabidopsis thaliana and Cyanidioschyzon merolae are clustered together with high similar modular structure. This result is consistent with the topological results (Table 1) that chloroplast metabolic network shows different characteristics.” (Zhou et al 2006)
With new changes and advances in genetic engineering and its implication in plant evolution, it has become mandatory to further investigate and research in this field, as this can become strong scientific evidence. DNA sequencing is gaining popularity because of its unique and un-identical pattern, which is different in every different kinds of plant cells. With DNA sequencing use in the research, its fair and valid use is important. What genetic information is valid as evidence in chloroplasts evolution?
It is of immense importance to understand that the two bands of DNA are a match even if they’re not aligned with each other. However, it is clear to the experts that band shifting requires additional analysis for interpretation of the correct results. There are so many intricacies in DNA profiling that with a slight mistake with a false similarity in different plastids. Possibilities lies in the fact that band shift can shift either more like a match or away from match.
“Only comparative studies of molecular sequences have the resolution to reveal this underlying complexity. A complete description of the complexity of molecular change is essential to a full understanding of the mechanisms of evolutionary change and in the formulation of realistic models of mutational processes.” (Clegg et al 1996)
Band shifting is one of the major problems in evolutionary studies today’s research face while making decisions and this has to be understood and checked for reliability in the chloroplasts evolution. In addition, they are developing special probes. In another study probe was conducted and evidence was provided even when bands were shifted but later the evidence was withdrawn because in a second probe a correction factor was found. In many such cases DNA evidence was not accepted because of the discrepancies in the results.
DNA typing is becoming more popular in the chloroplasts evolution. The Geneticists and law professional will need to work strong relation to implement DNA evidence in conviction cases. Researchers must get trained and educated in DNA patterning and sequencing system to decide for an authentic results. There is need to set standards for the procedures and techniques used. Absence of any universally adopted system may produce discrepancies in the results. Technicians and the professionals hired for this purpose must have skilled and experienced background. In many research cases it was found that the professional involved in Lab for sequencing purpose were not skilled and had forged credentials. It is very important to update current research studies with new technology to get more and more reliable method of scrutinizing results in chloroplast evolutionary studies. However, it must be reliable and useful to investigate previous researches as well.
“The work of Brinkman et al.  re-examines the processes that have led to the high proportion of proteins of a bacterial human pathogen, Chlamydia, that are similar to those of plants. This similarity was formerly attributed to horizontal gene transfer from plants, or plant-like host organisms, to the bacterium. Brinkman et al.  point out that such gene transfer is unlikely since all extant Chlamydiaceae are obligate intracellular parasites of animals. Instead, the analysis by Brinkman et al.  shows that the majority of the plant-like genes in Chlamydia are, in plant cells, targeted to the chloroplast. But the conclusion that this targeting of proteins to chloroplasts is necessarily a function of their origin from a plastid ancestor is not always sound. Furthermore, Martin et al.  did not find much similarity between Chlamydia and Arabidopsis (see Figure 1 in ). Clearly, further investigation is needed.” (Raven and Allen)
One of the many problems cited by professionals in the plant evolution fields are the evidentiary flaws in the studies. There are some technologies available to extract evidence from such as leaves, but they are not routinely in use. Unfortunately, these fluids lack a “definitive set of useful genetic markers” that proves a regular handicap for a conclusive test of DNA that results in usable evidence. In watching the results a viewer can easily assume that this type of evidence is easy to collect and test, and that it would be used in a case.
The chloroplast genome has been a major focus in studying plant evolution and plant genetics (Golenberg et al., 1993; Clegg et al. 1994; Morton, 1995; Clegg et al. 1997; Morton, 1999; Stoebe and Kowllik, 1999). It is now commonly believed that chloroplasts are the consequence of an endosymbiotic event between a eukaryotic host cell and an ancestor of the cyanobacteria (Curtis and Clegg, 1984; Delwiche et al. 1995; Barbrook et al., 1998; Turmel et al. 1999). Plastids developed either from a primary endosymbiotic event or from a secondary event. One of the main points of conjecture is the whether all plastids are monophyletic or polyphyletic.
The evidence appears to overwhelmingly support a monophyletic origin (Delwiche et al., 1995), yet some cases are not so clear cut (Penny and O’Kelly, 1991; Lockhart et al. 1992). Plastids in the red algae appear to be of polyphyletic origin relative to the green plastid lineage. On the other hand the green algae, from which green plants evolved, later acquired their plastids from a different cyanobacterial species, in which case they would be polyphyletic to the rhodophytes. Ultimately, all plastids are monophyletic — assuming there was only one universal ancestor to all life.
Throughout evolution, chloroplasts (and mitochondria) appear to have lost most of their ancestral genes. If chloroplasts are descendents from free living cyanobacteria, then there has been a major reduction in the genome sizes since their endosymbiotic origin.
Some chloroplast genes are thought to be transferred to the nucleus, while some genes are thought to have been transferred to the mitochondria as well (Gray and Joyce, 1989; Menaud et al., 1998). For example, in Arabidopsis thaliana, a gene coding for methionyl-tRNA synthetase in the mitochondrial genome may have originated in the chloroplast (Menaud et al. 1998). Therefore the mitochondrial genome is a mosaic of genes with different origins (Gray and Joyce, 1989) — as is the nuclear genome, and possibly the chloroplast genome.
Creating transgenic crops, determining the gene flow, determining the inheritance patterns, etc will be the most highlighted issues. Another fact may be the events in transgenic crops that a mechanism might be elucidated by catching a transfer event in the act, which could shed some light on the early evolution of endosymbionts with their hosts.
“Despite a conservative rate of evolution and a relatively stable gene content, comparative molecular analyses reveal complex patterns of mutational changes. Non-coding regions of cpDNA diverge through insertion/deletion changes that are sometimes site dependent. Coding genes exhibit different patterns of codon bias that appear to violate the equilibrium assumptions of some evolutionary models. Rates of molecular change often vary among plant families and orders in a manner that violates the assumption of a simple molecular clock. Finally, protein-coding genes exhibit patterns of amino acid change that appear to depend on protein structure, and these patterns may reveal subtle aspects of structure/function relationships.” (Clegg, 1994)
Restriction Fragment Length Polymorphism (RFLP) which is a technique that analyzes the lengths of DNA fragments using an enzyme that cuts a specific sequence that carries a particular recognition site. The presence or absence of the sire identifies the DNA sequence in the sample. RFLP is one of the original applications, though it is in use to a lesser degree as newer techniques prove more efficient in DNA analysis and because it is more easily contaminated than newer techniques.
Polymerase Chain Reaction (PCR) Analysis is capable of amplifying tiny samples of DNA and allows for degraded samples to be analyzed. It is also easily contaminated so great care must be taken in the collection and preservation of the samples.
Short Tandem Repeat (STR) Analysis evaluates specific regions of nuclear DNA because the variability of the regions are used to distinguish DNA profiles from one another. It is with this technology that the FBI uses a standard set of 13 regions to match DNA profiles to individuals in real cases as well as on television shows using the national CODIS database. (HGP, 2004)
Therefore, only highly skilled, educated and trained professionals should be hired for this purpose. Skilled professionals, reliable procedures, well working equipments should be used. Scientists should be trained and educated about the new techniques and methods and their validity so that they may take accurate decisions.
While discussing such an important topic, it’s very crucial to understand the mechanisms of Gene transfer. Here the question arises why would genes be transferred to the nucleus in the first place? What selective advantage could there be etc. One opinion in this regard is that when a gene moves from the chloroplast to the nucleus, there is a change in context from an asexual to a sexual genome. Recombination can then take place to reduce genetic load (Race et al. 1999). The case in plants is, however, different, i.e. much lower.
Concluding the matter, it can be stated that, understanding chloroplast genomes and mechanisms of gene regulation will be of utmost importance in future. Though a lot of work has already been done but there’s still the room for improvement.
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