1. Abstract
One important trait of a plant is its ability to form natural barriers that keep harmful microorganisms away. For instance, mangrove leaves may produce a hormone called salicylic acid to inhibit the growth of fungal pathogens. Such a hormone may induce an antimicrobial substance known as Pathogenesis Related (PR) proteins. A lot of Pathogenesis related protein researches have been done with tobacco and tomatoes leaves. In this study, the aim of the experiment is to determine whether mangroves leaves induce the PR-protein 1. The mangrove leaves are treated with salicylic acid and are left for 7 days in order for leaves to induce proteins. The leaves extraction procedure is carried out by the reference procedure Verlag Paul Parey (1980) and Thierry Niderman (1995). The PR-protein 1 in the leaf extraction solution can be identified by using the SDS-PAGE technique, which the SDS detergent is sticking to the protein during denaturing of the protein and electrophoresis gel can separate the different PR protein family based on their molecular size. The molecular weight of protein bands can be categorized by using reference protein and the molecular weight of PR-protein is known as 14-17 kilo daltons.
Acknowledgement
Initially the gratitude I would like to extend to my supervisor and foremost, Mark Duxbury is of highest praise for his accomplishments in helping me to succeed in our field of study. Through-out my research project there has been a lot of work that i have strived to excel into a project worthy of my subject teachings, the questions that I have asked of him, he has never neglected to reply to in good time.
Collectively with all the staff in the AUT laboratory, I would like to thank for providing all the equipment and chemical supplies and gear that have been used through-out the year in my research project.
2. Introduction
There are approximately 70 different types of mangrove species in the world. Some countries have a variety of mangrove species, however New Zealand has only one species which is called Avicennia marina or Mana, it belongs to the Verbenaceae family that is a cosmopolitan species which is widely distributed along tropical and subtropical coastlines. Mangroves have been growing in New Zealand for more than 19 million years. Most mangroves tend to grow in warmer climates, which influences the diversity and size of growth in the tropics. Therefore, mangroves do not tend to survive in colder climates, and this is why all the mangroves in New Zealand are likely to grow in Northland because it is warmer there. They grow larger in warmer temperatures,so the optimum growth of mangroves in New Zealand is experienced within 60 centimetres of the tide line, and flourish mostly around placid waters with shelving along the shoreline, in and around harbours and estuaries. Otherwise alternative conditions, harsh and rough water waves, can dislodge their roots as well as upset the silt that settles, which they require to grow. They can tolerate salt water even if submerged completely. This tolerance is not permanent, they need to be uncovered for half of either tide.
2-1. Importance of mangroves
It is important to know about mangroves in New Zealand, what their role is in erosion control and what they support. Mangroves have wide a diversity in their roles of food webs and the support they have for animals and plants in their environment. There are four different reasons why the mangroves are important in New Zealand.
Productivity of mangroves
The productivity of Mangroves is measurable with close analysis of their reproductive parts, twigs and leaves, this measurement determines the organic matter of the plant, its energy and its ability to contribute environment.
Food source
The distribution of fresh organic matter is localised, the most important attribute of mangroves in New Zealand, is the dependence the environment has on them. They supports the greater habitat around the nearby harbours and estuaries. Reference to this process is called the food web, when the distribution feeds a range of animals like crabs that digest nutrients from mangrove and return these nutrients into the food web.
Support for other plants
Different varieties grow in different environments, in tropical environments, this supports low density growth on sandy shores and seagrass beds in between trees, and can form new beds with the upper edges of these mangroves from the edge of the forests. It is clear that these enviroments do not support the same growth as in New Zealand.
Support for animals
Mangrove beds are located in harsh conditions so the range of dependency on this plant life is limited, reasons being; decomposition of sediment, oxygen starvation, muddy flats and tidal exposure. The variety of animals that feed off mangroves are somewhat small but they include worms, mud crabs, shellfish, mud snails and whelks – although terrestrial animals (spiders and insects) are also at home around mangroves. The range of inhabitants also varies with the location and nature of the sediment.
2-2 Defence mechanisms of plant
Explanation of the first and second defence mechanisms of plant will follow, in the first defence mechanism the invasion of pathogens is protected by a physical barrier, in the plants developed cell wall, and is composed of cellulose, pectin and lignin etc. The second defence mechanism is different by the use of the plants chemical defence, by alkaloids, saponins and phenols. This process is a constitutive expression of the plants secondary metabolite. The reaction of the defence mechanism involves turning a cascade of genes to produce a plant-pathogen interaction. Production of reactive oxygen species, through cell wall defence and through other group proteins such as PR (pathogenesis-related).
3. Literature review
Many of the PR proteins are diverse in their families, so that they are found in a lot of types of plants such as, 33 in Tobacco, 20 in sugar beets also in spinach. Classification of PR proteins that induce pathological and related states (Van Loon 1985), are a classification of family, through relationships of cellular form, amino acid sequences and biological activity, further families are identified as tobacco and tomatoes with classifying PR proteins. Sequence Homology is the term for the families of PR proteins, but are not defined only by this but also by migration, the specific reaction with antisera, biological activity also determines PR and resistance to interactions. Characters of cDNAs that reveal PR genes are seen in the systematic resistance in plants against pathogens, and incompatible hosts house bacteria, fungi and viruses, elicitors of PR genes derive molecules from pathogens. These proteins are found in different plant types, although healthy plants suffocate the characteristics of PR, multi gene families have the most PR proteins and are proven to inhibit the growth of fungi. Interestingly the use of large groups of PR genes can be characterised and used to sustain healthier responses to biotic and abiotic stress, to the betterment of transduction mechanisms and the measure of stress signals to return greater defence for enhanced engineering of crop plants. Necrotic lesions in plants, can be a result of the resistance produced by the PR.
3-1. Variation of infectious
PR has been identified in the infected tissue of unhealthy plants, as well as in healthy plants, e.g Tomato plants are known to display seven PR&aposs when they are in good health. Varieties of the plants also support PR proteins, in fact PR proteins are usually only present after stress, unlike flowers, pollens, stigma and seeds. Associated stress induced by pathogens is the dominant group PR-1 and is used as a signal for SAR. The research that has produced our current knowledge of PR-1 was undertaken in 1970, limited antifungal activity suggests it is a defence mechanism – but the action it takes, and the relationship with other proteins is a new subject.
4. Pathogenesis-related (PR) proteins
PR proteins induced in a pathological environment allow the role of the proteins defence in the plant, that is the result of a reaction to infection to produce the PR. Antimicrobial proteins attack molecules in the cell wall that are bacterial or fungal. Host plants see the interaction of pathogens that are labelled as hypersensitive reactions, that are the proteins produced in the event of a pathogen attack – there are 17 types discovered (Van Loon,2001)
4-1. PR-1 protein family.
PR protein typically has 160 amino acids in length, although it is not clearly studied in biochemical properties, the most abundant group in the PR-protein family is PR-1 because of its high sensitivity to infection, it induces a very high level, between 1 and 2 percent of the protein in the leaf. There is a molecular marker of the expression of PR-1 that indicates the plants defence response, this particular protein is most studied in tobacco.
Picture 1. Families of PR proteins
The picture 1 showed different types of the PR-protein family and different PR-families that have different molecular sizes. The first family is the PR protein 1 and it has a molecular size of 15 to 17 kDa. The biochemical properties of the PR-1 family is not clearly established, so those stated are unknown. However, it is known that the PR-1 family is the most abundant group of proteins, which induce very high levels of PR Protein when infected (approximately 1 to 2 % of the total leaf protein). They are typically 160 amino acids in length and exist as an acid or base dependent on their functional grouping (Punja, Z. 2004).
4-2. Acidic Pathogenesis Related -1 Proteins
Tomatoes, barley, maize, parsley, as well as other plants of the Graminae, Solanaceae, Chenopodiaceae and Amaranthiceae families have detected acidic pathogenesis related -1 proteins. The high resistance to proteolysis is adapted to the extracellular environment, and the protein in the plant is soluble in acidic buffers with low molecular weights. The protein P14 isoforms have been found in barley, and tomatoes which have a similar protein to tobacco, that respond to different polyclonal antibodies, unlike acidic PR-1 genes that do not target vacuolar peptide sequences form PR-1 has 138 amino acids that synthesize a higher molecular weight that contains N-terminal amino acids that produce K15da mature protein.
4-3. Basic Pathogenesis Related -1 Proteins
Basic Pathogenesis Related -1 protein contains 30 amino acids in the hydrophobic N-terminal region of 30 amino acids, this is a signal peptide that is the translocation of endoplasmic reticulum – the C-terminal peptide also contains the vacuolar targeting signals, for example in tobacco leaf PR-1 proteins are localised in extracellular space responding to TMV infection. The 17KDA are two basic isoforms, and PR-1 has an isoelectric point of 10.5 and 11.0, the exception between the similarities of PR-1 basic and PR-1 acidic is one amino acid sequence and are identified in maize, celery and other cereals.
5. Salicylic acid
The benzoic acid derivative is Salicylic acid (SA), an important phytohormone is involved in the regulation of the plants defense mechanism. The important role that Salicylic acid fills in plant defence for the protection from pathogen attack, recently was proven to be necessary for SAR. Salicylic acid has a role that is observed in the plants defence mechanism was similar to the medication of aspirin in observation, the acid induced resistance to the mosaic virus in tobacco. The accumulation of PR proteins increased in resistance, and assumed as markers of the defence response. Biosynthesis has elucidated in the pathway of salicylic acid and has synthesized from benzoic acid into cinnamic acid, and this reaction catalyzes a function of cytochrome P450 monoxygenase. Salicylic acid is an essential compound in the signal pathways accompanied by an induced excess of acidic pathogens- related protein genes. The production of pathogenesis-related proteins is the role Salicylic acid holds in the resistance to pathogens.
6. SDS – PAGE(Sodium dodecyl sulfate – Polyacrylamide gel electrophoresis).
SDS is anionic detergent consisting of 12 carbon tail attached to sulphate group, which has a negative charge. It also disrupts the non-covalent bond particularly protein and denatures the molecule. The protein can be denatured at certain temperature and lose their shape. At that stage, SDS can stick to the denatured protein due to similar shape and charge ratio as protein. PAGE separates the macromolecule based on their electrophoresis mobility. Smaller polypeptides travel faster and quickly through pores, while large polypeptides travel slower. The polypeptides have similar charge to mass ratio which is dependent only on molecular weight.