Production of PR-protein 1 in Mangroves Leaves | Experiment

Production of PR-protein 1 in Mangroves Leaves | Experiment 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 condi tions, 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 c haracteristics 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's 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. Factors in Business Cash Budgeting: With Examples Factors in Business Cash Budgeting: With Examples Contents (Jump to) Task One (p3d) XYZ Traders Cash Budget Task Two (p3abc) Alpha Manufacturing Budgets Task Three (p4abc) Beales Manufacturing – Variances Task Four (p2ab) Beales Manufacturing – Standard Cost Bibliography XYZ Traders Cash Budget Introduction In this report Part A presents a cash budget for XYZ Traders for the six month period from December 2008 to May 2009. Part B comments on the cash flow forecast and outlines the benefits of improved cash flow for the Company and recommends management consider a cash flow improvement program. Part A – Cash Flow Budget December 2008 – May 2009 Table 1 Table 1 shows the cash flow budget based on the following criteria: Cash balance as at 1 December 2008 totals  £2,600 Furniture units sold at  £100 each Furniture units purchased for  £60 each Customer collections on the basis of 50% in month of sale and the balance the following month Purchases paid for in the month following delivery Fixed expenses are  £4,000 per month Loan repayment of  £10,000 due in April 2009 Stock units carried over from October into November 2008 unknown Part B – Budget Cash Flow Commentary The cash flow budget/forecast for the next six months: is cash positive for each of the six months under review will generate sufficient cash for the  £10,000 loan repayment on schedule in April 2009 will allow supplier payment on time one month after receipt of goods has a lowest cash balance of  £1,200 at the end of April following the loan repayment in the same month Recommendation – Cash Flow Improvement Program The cash flow budget indicates that XYZ can continue trading for the next six months without recourse to additional funding. However improving cash-flow can have a number of benefits including: reducing the equity required to finance the business reducing loans and interest payments required for working capital releasing funds for expansion and/or new business activities providing funds for profit distribution to the owners/shareholders We recommend management plan and implement a coordinated program to improve cash-flow. Specific areas for consideration are: minimise inventory by introducing a â€Å"just-in-time† (JIT) arrangement where suppliers orders are placed at the time of customer purchase for receipt a day or two before delivery to the customer eliminate inventory (except for show-room stock) by arranging for suppliers to deliver direct to customers under XYZ Traders instructions and identity negotiate extended payment terms with the suppliers, say, three months credit encourage customers to pay the full purchase price on order placement offer sales on credit with a reputable finance company increase sales volumes with well designed and executed advertising and customer incentive programs reduce overheads and expenses with a cost-reduction program Alfa Manufacturing – Budget Report Introduction This report recommends Alfa Manufacturing introduce a budgetary control system. Section A outlines the purpose and benefits of budgets and Section B describes two possible budget formats, incremental and zero based budgeting and examines the advantages and disadvantages of each method. Section A – Budgets, Purposes and Benefits The primary purpose for introducing a budgeting system is to provide the Company with a powerful tool for planning and monitoring business performance. It improves productive effectiveness and enhances coordination between the various arms of management to achieve the overall Company aims. Budgets represent the primary means of communicating agreed-upon objectives throughout the organization. A budget is a formal written statement of the Companies plans for a specified time period. The principle element of a budget is to plan and predict future income and expenditure against a time-scale, usually on a monthly basis covering a calendar or accounting year. Actual income and expenditure is recorded as it occurs and monitored on a regular basis against the plan or budget. The differences between planned and actual results are monitored, reported and the variances analysed and explained. In summary, the benefits of a budget are that: Management must develop a comprehensive plan for the future. Key objectives are agreed for monitoring and performance evaluation. Potential problems are identified well in advance. Coordination of activities within the business is facilitated. Management is more aware of the Companies overall operations. Each level of management participates in the planning, preparation and monitoring of financial activity. The budget must have the complete support of top management and is an important tool for measuring and evaluating managerial performance. Contemporary budgeting has been defined as a system wherein managers are provided with the flexibility to utilize resources as required, in return for their commitment to achieve certain performance results (Deloitte Touche Tohmatsu, 2008). Section B – Budgeting Systems This section contrasts and compares two basic budgeting methodologies currently in use in industry today, Incremental Budgeting and Zero Based Budgeting. Incremental Budgeting (IB) is the traditional approach to budgeting which relies on historical information and the previous years budget as a basis for the preparing the input and data for the following year’s budget. For example, let’s say last year’s sales budget was for 1000 units at  £500, giving sales revenue of  £500,000. For next year’s budget the market for the product is anticipated to improve by 10% giving unit sales at 1100 thus giving budget sales revenue of  £550,000. Similarly costs would be based on last years budget, modified by projected inflationary factors. Anticipated raw material price increases and labour rates are used in the cost of production and increases in say, rent and utilities would reflect in overheads. Managers will prepare their individual budgets based on a series of pre-determined criteria and assumptions which are normally provided by top management, finance and accounts. The advantages of IB are that it is: relatively easy to implement easy to understand and appreciate less time-consuming to prepare than ZBB a â€Å"top down† approach with the same basic assumptions for all The disadvantages of IB are that it: assumes that the budget methodology and cost structure is correct encourages expectations of inflationary increases predicts sales will reflect the market without competitive analysis encourages departments to spend all of their allocated budget Zero Based Budgeting (ZBB) is an approach to budgeting that starts from the premise that no costs or activities should be factored into the plans for the coming budget period, just because they figured in the costs or activities for the current or previous periods. Rather, everything that is to be included in the budget must be considered and justified. (Chartered Institute for Public Finance and Accounting, 2006). Another definition is the use of budgets which start from a present base of zero and regard all future expenditure as being on new items rather than a continuation of existing ones. In practice this means that a budget has to be justified in full for each year of operation (Steven A. Finkler, 2003). In implementing this process each manager must critically examine his own activities and operations and build his budget from scratch. The advantages of ZBB are that it: questions accepted beliefs focuses on value for money links budgets and objectives involves managers leading to better communication and consensus can lead to better resource allocation is an adaptive approach in changing circumstances The disadvantages of ZBB are that it: is time-consuming and adds to the effort involved in budgeting can be difficult to identify suitable performance measures can be seen as threatening–careful people management is required is about costs and resources of options ignoring current practice can be difficult to comprehend and execute by managers with little financial knowledge and skills Recommendation Since Alpha Manufacturing has no previous experience of budgetary control it is recommended that an Incremental Budgeting program is introduced initially. The budget can be prepared using historical data with guidelines and assumptions provided to each manager by the Finance Department. Beales Manufacturing plc:  Flexible Budgeting Introduction This report examines the budget and actual results for October. It flexes the budget to actual output, provides a variance analysis and identifies possible causes for each negative variance. Managerial accountability for each variance is suggested and possible remedial actions for the unfavourable variances identified. The benefits of using flexible budgets are explained and it is recommended that this technique be introduced as a feature of Beales’ regular budget reviews. Variance Analysis and Explanations Table1 shows the results of the budget and actual output for October, flexed to actual output with each variance examined for possible causes, accountabilities and suggested remedial actions. Benefits of Flexible Budgeting Static budgets have the disadvantage of providing a single specific predicted volume of output. In reality, it very unlikely that the actual output exactly matches the budget. Thus any comparison of actual output to budget suffers from the problem that some of the variances, particularly for variable costs such as labour and materials, will be as a direct result of the differences in the volume of output. Flexible budgets provide an after the facts device to tell what it should have cost for the volume level actually attained (Steven A. Finkler 2003) They are a useful tool for analysing the effects of variations in volume of output against the original budget. Dennis Caplan (2006) suggests that â€Å"the motivation for the flexible budget is to compare apples to apples. If the factory actually produced 10,000 units, then management should compare actual factory costs for 10,000 units to what the factory should have spent to make 10,000 units, not to what the factory should have spent to make 9,000 units or 11,000 units or any other production level.† Recommendation For Beales Manufacturing to make the best use of the budgeting process it is recommended that flexible budgets are prepared each month. Variance analysis as demonstrated above will assist management to implement contingency plans to correct any unfavorable trends and enhance profitability. Beales Manufacturing plc:  Standard Costing Introduction This section defines and describes the principles of Standard Costing. It is an accounting technique which provides a powerful tool for management to analyze business performance and plan improvements. An example of a standard cost is derived from the October budget and the use of variance analysis to identify problem areas and possible remedial actions. Definition Standard costing involves the development of a product or service cost using estimates of both the resources consumed and the prices of those resources. The standard cost may then be increased by an estimated profit margin to produce a standard selling price. These estimates of cost and revenue then provide a foundation for further planning and control (Barrie Mitchinson 2000) Illustration The best way to illustrate the benefits of standard costing is to use the October budget data to arrive at an example of a Standard Cost. This cost can then be compared with the actual unit cost for October and the variances analysed as shown in Table 3 below. Standard Unit Cost vs. Actual (October) From this analysis specific product cost information can be derived. For example, although raw material costs per meter were below standard cost, more material than standard was required to complete the production schedule. Why was that? The manager responsible for production will be able to use the information to investigate the unfavourable variance. Possibly scrap rates were excessive so improved quality control could help reduce or eliminate the problem. Raw material costs were also above standard which will alert the purchasing manager to an overrun of purchasing costs which may require action. Recommendation We recommend that Beales consider the introduction of Standard Costing to provide management with a powerful tool to improve efficiency, productivity and product profitability. Bibliography Deloitte Touche Tohmatsu, 2008, Budgeting Budget Controls  http://www.deloitte.com Chartered Institute for Public Finance and Accounting, 2006, Zero Based Budgeting Briefing Paper, http://www.cipfa.org.uk Steven A. Finkler, 2003, p158, Finance Accounting for Nonfinancial Managers, CCH Tax and Accounting Steven A. Finkler, 2003, p162, Finance Accounting for Nonfinancial Managers, CCH Tax and Accounting Dennis Caplan, 2006, Management Accounting: Concepts and Techniques, OSU College of Business, http://classes.bus.oregonstate.edu Barrie Mitchinson, 2000, Standard Costing and Fixed and Flexed Budgets, Association of Chartered Certified Accountants, http://www.accaglobal.com