Post-occupancy evaluating of low income housing in the Kingdom of Dissertation

Post-occupancy evaluating of low income housing in the Kingdom of Saudi Arabia - Dissertation Example 13 Pictures(3) Showsthe region of Ha’il, with the pleased of the project. 13 Pictures(4) Site plan of the Al-Ghazalah project 13 Pictures(5) Site plan of the Al-Ghazalah project 15 Pictures(6) Ground floor plan 15 Post-Occupancy Evaluation of Low Income Housing in the Kingdom of Saudi Arabia Chapter 1 Introduction Housing demand is one of the most important issues and challenges that face developing and developed countries (Al-klyfh, 2009). These countries have tried to solve their housing demands with different policies. Some policies and proposals have met the needs of different levels of society. One of these economic classes is called the low income group that encompass some Saudi Arabian people with low annual income. To address the issue on housing income, the government tried to establish residential complexes, an introductory concept and still under trial. This paper will focus on low income housing post occupancy evaluation as the main issue. The housing policy will b e assessed using the post-occupancy evaluation method (POE). However, before investigating the Saudi Arabian policy, it is important to investigate the different international policies for low income housing. 1.1. International policies for low income housing Many institutions, international agencies and local communities around the world have for many years now addressed poverty, increase standards of living,as well as provide adequate shelter for the poor (United Nations Centre for Human Settlements, 1987).These UN programs were allocated in 155 countries and have established national centres that work directly with various local government agencies (Tiesdell, 2004). Important processes that have been followed are: a. Secure possession of the land. b. Provide public facilities and services. c. Finance low-cost housing d. Provision of construction materials. e. Community participation. f. Cooperation with the private sector and non-governmental organizations (NGOs) (Tiesdell, 2004) . A review of socio-economic policies indicates that there is a growing interest in the issue of housing for the poor by civil society organizations and governments of many countries. A limited review on Saudi Arabia and the United Kingdom low cost housing will be included due to time constraints. 1.1.1. Affordable Housing in the UK Housing policies in Britain has local planning authorities mandate developers of housing projects to provide a certain percentage of affordable housing for low income groups (Tiesdell, 2004). The two aims are as follows: a. Social: sustain a mixed community in terms of income and social level and reduce illegal settlements that lack basic services b. Economic: shoulder part of the burden of financing in order to allow beneficiaries provided with lower prices than the market price or rent housing (social security) at less than the prevailing costs. UK housing, however, did not meet stated objectives, such as acceptance from high-income and middle classes due to preference

Portfolio Management, Research Paper Example | Topics and Well Written Essays - 1000 words

Portfolio Management, - Research Paper Example The limitations of the study are listed. Economic liberalization and globalization have brought about a new and competitive environment for the common and small investors who are willing to participate in the equity of the corporate sector in our country. Understanding the firm's investment decisions under imperfect market conditions is one of the central issues of the financial economics. Studying firm's investment in such environment can provide insight into the dynamics of its growth as a function of internal and external financial sources. Fazzari et al. (1988) argue that in the presence of financing constraints the firm's investment vary not only with the availability of the profitable investment projects, but also with the internal funds. Consequently, the severity of the financing constraints is proposed to be measured by the magnitude of the cash flow sensitivity of investment. The memo was found at moneycontrol.com. The memo talks about the investment option of a venture capitalist. It provides details of investments in various countries for a period of seven years and advises the manager in taking up an investment decision. The colours in the chart are used appropriately and the chart is easy to read. In the chart, real numbers have been used and in the Y axis instead of real numbers some legend might have been given to represent millions. The lines are used very effectively and the he line in the chart indicates the support level of the investments that the company can afford. I liked the chart and it provides a lot of information and is very effective. 2. Graphic Memo Introduction This memo describes how I spend an average day. First, I list all activities and schedule them. The entire activity is an application of planning. Then after arriving at the activities, a table is prepared showing the minutes of each activity. Finally a chart is drawn illustrating the percentage of time spent. Body The various activities that I perform in an average day includes Travel/Driving, Grooming, Studying, Class Attendance, Leisure Time, Work, Meals, Sleep, Etc. More detailed list is shown in the table. Then time is spent in each activity is calculated after organizing and scheduling the activities in a sequence. Then these activities are put in a table. Finally a pie chart is drawn to display hours. Table 1. List of Activities Activity Start Time End Time Minutes Grooming 7.00 A.M 8.00 A.M 60 Breakfast 8.00 A.M 8.15 A.M 15 Travel/Driving 8.15 A.M 9.00 A.M 45 Studying 9.00 A.M 10.00 A.M 60 Class Attendance 10.00 A.M 1.00 P.M 180 Meals 1.00 P.M 1.30 P.M 30 Leisure Time 1.30 P.M 2.00 P.M 30 Travel/Driving 2.00 P.M 3.00 P.M 60 Work 3.00 P.M 7.00 P.M 240 Travel/Driving 7.00 P.M 8.00 P.M 60 Dinner 8.00 P.M 8.30

Bussiness interrogation Essay Example | Topics and Well Written Essays - 2000 words

Bussiness interrogation - Essay Example An analysis of the foundation of motivation has been regarded as the first step towards the creation of successful organizations. Conversely, Allen and Gilmore (2006, pp.35), indicate that, Herzberg theory indicates that, Herzberg motivation theories of motivation are obliging to a proper understanding of the organization’s functions. In his work, Miner (2007, pp.84) argues that, the attributes that contribute to motivation and demotivation cannot be alienated from an understanding of the work place motivation. Motivation, which emanates from motivators, can be described as an intrinsic process that comes from within the individual. On the other hand, demotivators are extrinsic; that surround the individuals in the work place. Among the demotivators stated included, pay that was regarded as a short term motivator. After a while, employees were seen to be as demotivated as they were at the initiation of the time at the work place. This essay shall focus on the Coca Cola Company and the implication of Herzberg’s theory of motivation. Since the commencement of the company, its success story has always had its background on the motivation of employees in the organization. Through motivating employees, the company has managed to enjoy constant and consistent teamwork form its workers. In return, the employees have never compromised on the quality of products produced by the company. Herzberg’s theory, according to Sapru (2000, pp.76), revolves around two major factors that are inclusive of hygiene factors and the motivators. Hygiene factors are likely to cause dissatisfaction in the workplace if not put under scrutiny. Among the hygiene factors include, aspects like job security, safe working conditions, proper organizational policies as well as, quality of supervision along other factors. Sapru (2000, pp.76) argues that hygiene factors do not necessarily cause demotivation in the workplace, but if they are not incorporated in the work

Final Term Paper Example | Topics and Well Written Essays - 1500 words

Final - Term Paper Example international market regions of Europe, it had to bear varied types of challenges and issues such as barriers in international trade, mitigation as well as investment (Beall, 2010). But due to the support of municipal government, all such international barriers reduced significantly that amplified its position and brand image in the entire globe among other rival players such as General Motors, Toyota etc. As a result of which the total sale and profitability of the organization enhanced that proved effective for the government of the Europe among other neighboring nations. The economic condition of the country enhanced as well as the rate o unemployment and poverty reduced to a significant extent (Ford.com, 2014). Other than this, at the time of expansion in the new market region of Europe, the organization of Ford Motors had to analyze the taste and preferences of the customers so as to improve its prosperity and portfolio in the market (Bradley, 2012). Moreover, due to the active participation of municipal government, the policy related issues such as tariff barriers, subsides to local firms etc reduced significantly that amplified its reputation and market share in the markets of Europe among others (Ford, 2014). The organization of Ford Motors had to bear varied types of political challenges such as tariff rates, trade restrictions due to political instability in the nation of Europe. As a result of which, the brand image and competitiveness of the organization of Ford Motors attained a serious set-back (Kazmi, 2010). Economic factors: inflation is one of the important causes that hindered the total sale and prosperity of the organization of Ford Motors within the region of Europe as compared to many others. As a result, the organization had failed to enhance its profit margin and demand of the products that hindered its portfolio and prosperity in the market among others. Social factors: as the preferences of the customers are changing at a rapid pace so

Prison law and penology Essay Example | Topics and Well Written Essays - 1000 words

Prison law and penology - Essay Example This may lead them to focus more on controlling the inmate population rather than working on their rehabilitation. As previously stated, inmates tend to gather and form groups according to their race (black, Hispanic, Caucasian, etc.) or a race-related reasons (groups such as the neo-Nazis, K.K.K, etc.) and as a result, riots often break due to racial grounds. The real motives behind gangs formation and activity are actually power and wealth, things which they can achieve in various ways, often related to violence and disorder (Summers, 2006). The gangs are becoming increasingly stronger and more dominant ("Violent Gangs", n.d) and are also wise to draft to their ranks some prison guards and others that assist them in exchange for money, favours and more. 1 Therefore, prison authorities and representatives must always be attentive and prepared to thwart gangs attempts to gain more power and hurt others along the way. This undoubtedly shifts the focus of prison authorities from the desire to rehabilitate prisoners to the need to control and monitor them so that they dont cause more trouble and eruption of violence and havoc. Also, there are inmates which obviously overwhelming and something which they cannot escape or resist. These people have no place, power or control in the outside world and are therefore making extreme efforts to make their permanent home, the prison, their own kingdom and domain. This leads them to try and take control over it by forceful means and using violence and terrorizing others. They even develop ways to carry out their deeds using technological innovations and creative ways. For instance, it is claimed that a group of prisoners used a 400-year-old binary code system in order to smuggle notes, using guards as deliverers and smugglers of them (Summers, 2006). This shows the innovation and adaptability of gangs. They are no longer plain and simple, nor do they work in primitive ways. Now, when a

Method of Data Analysis Essay Example for Free

Method of Data Analysis Essay The researcher made use of SPSS version 15. 0 to be able to compute for the statistics required. For the degree of importance and actual performance of Body Shop, the descriptive statistics of means, standard deviations, and frequency distributions were generated. The Pearson product moment correlation coeffiecients were likewise computed; the bigger is the magnitude of the r-value, the stronger is the relationship between two variables. The sign denotes the direction of the relationship – that is, whether they are directly or inversely proportional (Salkind 2000). 3. 1 Conclusion. The present study purposively selected 150 respondents for its telephone interviews. Secondary data were the basis for the literature review and the design of the questionnaire. The latter was both face and content validated. 5 respondents were used to pilot test the instrument to ensure clarity of questions. SPSS 15. 0 was used for the statistical computations. ? Chapter 4 Results and Discussion 4. 1 Introduction This chapter presents the statistical results gathered from the survey questionnaires, and a substantive discussion of these results, culling from the literature in Chapter 2. 4. 2 Results Table 2. Gender: Frequency and Percentage Breakdown. The table shows that all of the respondents were female, accounting for 100% of the sample. Table 3. Age: Frequency and Percentage Breakdown. When asked about their age, majority of the respondets (42. 7%) answered that they were between 21-25 years old. This is followed by the 26-30 years old category (36. 0%); 31-35 years old at 10. 7%; then by 20 years old and 35-40 years old both at 5. 3% Table 4. Have You Purchased a Body Shop Product in the Past 12 Months? All of the respondents likewise expressed that they have purchased at least one Body Shop product over the past 12 months. Table 5. Means and Standard Deviations: Importance of Factors which Affect Cosmetic Product Choice. When asked to rate the importance of the factors that affect their choice of cosmetic products, the items were rated as follows, in descending order: the degree to which the product is environment-friendly (X= 4. 52, sd=. 59); the customer service of the store personnel (X=4. 32, sd=. 65); the sales promotions used for the product (X=4. 31, sd=.65); the values espoused by the company who sells the product (X=4. 16, sd=. 67); Having a globally renowned brand name (X= 4. 16, sd=. 59); trendiness or fashion sense represented by the product (X= 4. 11, sd=. 64); recommendations or positive feedback I garner from my friends, family and acquaintances (X=4. 05, sd=. 68); competitive price of the product (X= 4. 05, sd=. 61); the degree to which the product espouses â€Å"natural† rather than artificial (X=4. 00, sd=. 66); quality of the products (X=3. 88, sd=. 71); the convenience of going to the store location (X=3. 84, sd=.59); the variety of product offerings in the store (X= 3. 59, sd=. 59); Attractiveness of the packaging (X=3. 21, sd=. 52); information on the product’s label (X=3. 17, sd=. 80); and media advertisements of the product (X=2. 73, sd=. 86). Table 6. Means and Standard Deviations: Actual Performance of Factors which Affect Cosmetic Product Choice. On the other hand, when the respondents were asked to rate the actual performance of Body Shop through the same factors, the following are the results in descending order: the degree to which the product is environment-friendly (X=4.57, sd=. 49); The customer service of the store personnel (X=4. 48, sd=. 60); the degree to which the product espouses â€Å"natural† rather than artificial (X=4. 37, sd=. 58); the values espoused by the company who sells the product (X=4. 37, sd=. 67); having a globally renowned brand name (X=4. 20, sd=. 69); quality of the products (X=4. 14, sd=. 74); recommendations or positive feedback I garner from my friends, family and acquaintances (X=4. 11, sd=. 78); the sales promotions used for the product (X=4. 05, sd=. 75); the convenience of going to the store location (X=3. 94, sd=.60); Trendiness or fashion sense represented by the product (X=3. 90, sd=. 72); the variety of product offerings in the store (X=3. 79, sd=. 52); competitive price of the product (X=3. 63, sd=. 70); attractiveness of the packaging (X=3. 58, sd=. 49); information on the product’s label (X=3. 36, sd=. 66); and media advertisements of the product (X=2. 73, sd=. 85). Table 7. 1 Paired Means and Standard Deviations of Factors. Table 7. 2. T-test Results of Pairwise Comparisons of Importance and Actual Performance. The following pairwise comparisons are significant at the . 01 level: Quality of the products (t=-5. 92, p=. 00), the customer service of the store personnel (t=-3. 85, p=. 00); the variety of product offerings in the store (t=-6. 23, p=. 00); the convenience of going to the store location (t=-4. 07, p=. 00); attractiveness of the packaging (t=-7. 69, p=. 00); the degree to which the product espouses â€Å"natural† rather than artificial (t=-6. 12, p=. 00); the values espoused by the company who sells the product (X=4. 37, sd=. 00). In all these items, Body Shop has exceeded the degree of importance attached by the respondent on the factor. In contrast, the following factors are those in which Body Shop did not quite meet client expectations based on the degree of importance they have assigned to each: competitive price of the product (t=8. 66, p=. 00); trendiness or fashion sense represented by the product (t=3. 73, p=. 00); and the sales promotions used for the product (t=3. 98, p=. 00). There are no significant differences on the following items: brand (t=-. 816, p=. 416); the degree to which the product is environment friendly (1. 465, p=. 145); and positive feedback garnered from significant others (t=-1.070, p=. 287). These suggest that Body Shop has met expectations of the respondents based on the degree of importance they have attached to each factor. Table 8. Correlations among Patronage of Environmental Friendly Products, Social Responsibility of a Company, Patronage of Products and Companies that Care for the Environment, and Overall Satisfaction on Body Shop. The correlation results show that patronage of Body Shop products because of their being environment friendly is significantly correlated with their being a socially responsible company (r=. 397, p=.00). This means that patronage of Body Shop products increases as well with positive perceptions of their company as being socially responsible. Moreover, such patronage is also positively and significantly correlated with their expressed support for companies that espouse care for the environment (r=. 533, p=. 00). None of the statements were significantly related to overall satisfaction of Body Shop products, which suggests that there are other determinants of satisfaction apart from espousing environment friendliness and corporate social responsibility.

L. Chinense Growth Conditions and Artificial Pollination

L. Chinense Growth Conditions and Artificial Pollination INTRODUCTION The genus Liriodendron is a distinctive and valuable hardwood that has great ecological and economic values. It grows fast and the wood is light and soft, so it is cultivated in many temperate mountains of the world for wood production [1-4] and it’s recommended for waste landfill remediation [5]. Its a flowering plant with beautiful leaves and hence used for urban landscaping as it provides shading as well. Besides, the tree genus Liriodendron is valued as materials source for honey production, chemical extracts [6-8], biomass and biofuels [9, 10]. The genus Liriodendron survived from the last Ice Age and was distributed in large geographical ranges of North American and East Asian respectively. Currently it comprises only two morphologically similar species, Liriodendron tulipifera L. and Liriodendron chinense (Hemsl.) Sarg. [11]. However L. chinense (Liriodendron chinense (Hemsl.) Sarg.) has been regarded as a rare and endangered plant because it occurs in small, isolated and thinly scattered populations [12]. L. chinense was listed in the IUCN Red List of Endangered Plants in China [13], and has currently been classified as a lower risk or near-threatened species (http://www.iucnredlist.org/). In the process of L. chinense sexual reproduction, the low seed setting percentage is a marked trait. After years of statistics, the setting percentage of L. chinense is not more than 10% in natural state, and it is hard to find the seedling in natural environment [14]. In the last two decades, many researchers have conducted studies, such as examining the relative contribution of the pollen fertility and transfer, availability of resources, flower or seed predation and genetics, to find out why L. chinense only produce few seeds [15-18]. Unfortunately, there has been no consistent conclusion. Pollination, as a key event in reproductive process of plants, especially in rare or endangered plant species like L. chinense that have low seed production, is probably one of the weak links in the reproductive cycle. Any barrier occurring between pollen and stigma interaction will lead to low seed production, however, few studies have focused on the pollination in L. chinense. Zhou and Fan ex amined the pollen quality, pollen germination and growth on stigma using fluorochroma method. The results indicated that in vivo the pollen grains can load on about 64% pistils of the gynoecium, but the rate of pollen tube passing the style is low, only 24% [19]. In addition to few pollen tubes passing the style, the pollen tubes may grow twined or in no direction, suggesting that only a smaller percent of the pollen tubes penetrates the micropyle and enter into ovule [20, 21]. These results show that the interaction between pollen and stigma occurs in different phases after pollen grains loading on stigma, and there are different barriers distributed in stigma surface, style and ovule during pollen tube growth. In self-compatible plants, the pollen-stigma interaction comprises six stages between pollen and pistil: pollen capture and adhesion, pollen hydration, pollen germination, penetration, growth of pollen tube through the stigma and style, pollen tube enter into the ovule and discharge the sperm cells [22]. After the pollen-stigma interaction, the nuclei of two gametes fuse to form the zygote. However, in self-incompatible plants, no matter the barriers occurs in which stage of interaction, there is no formation of a viable zygote. Previous studies in L. chinense showed that many pollen grains germinated on pistils of the gynoecium but few pollen tubes could penetrate the pistil style, and most of the pollen tube couldn’t pass through micropyle and enter into ovule. This phenomenon suggests that there might be other factors affecting pollen-stigma interaction in L. chinense. To verify this hypothesis, we conducted a systematic morphological and proteomic analysis on the pistil of L. chinense during pollination. The result provides new insights in the mechanism underlying sexual reproduction in L. chinense. MATERIALS AND METHODS L. chinense growth conditions and artificial pollination The L. chinense plants was grown in Wuhan Botanical Garden, Chinese Academy of Sciences. During the flowering season, which extends from late April to May, the branches with flower buds which were about to open were cut from the tree and cultivated with half-strength Hoagland’s nutrient solution in greenhouse under 14 h light (400-800ÃŽ ¼molm-2s-1) at 26 ±2oC and 10 h darkness at 20 ±2 oC [23]. The relative humidity was maintained at 60-70% [19]. The flower buds with an opening on top and a probability of opening the following day were chosen and the androecium was emasculated at night before pollination. Artificial pollination was done the next afternoon as follow: Mature pollen grains were harvested from open flowers and then were smeared on the pistils without androecium using a soft brush. This artificially pollinated pistil was cut from the flower 30 minutes after pollination and stored in liquid nitrogen. Similarly, the pistil after 1 h pollination was harvested, sto red in liquid nitrogen. The harvested un-pollinated pistil was stored in liquid nitrogen. All three of these samples were named as S2, S3, and S1 respectively and stored in -80 oC freezer. All three treatments (S1, S2, and S3) were repeated five times respectively. Paraffin section Anthers and pistils were fixed in FAA solution containing 5% glacial acetic acid, 5% formaldehyde, 70% ethanol at room temperature for 24 h. After dehydration and infiltration, the samples were embedded in paraffin and cut into 10- µm-thick sections by Rotary Microtome Leica RM2265 (Germany). Then the sections were sealed by neutral balsam and photographed by Olympus-BX51 (Japan). Gel-based proteomics in L. chinense Protein extraction and 2-DE Proteins of pistils were extracted as previously described [24]. Briefly, 0.25-0.3 g of pistils were ground in 2 ml pre-cooled homogenization buffer which contains 20 mM Tris-HCl (pH7.5), 250 mM sucrose, 10mM EGTA, 1% Triton X-100, 1 mM PMSF, and 1 mM DTT. The homogenate was shifted into a centrifugal tube and centrifuged at 12000Ãâ€"g for 30 min at 4 oC. The supernatant was collected in new centrifugal tube and mixed with 3 volumes cold acetone. The tube was kept at -20 oC at least 2 h, and then centrifuged at 12000Ãâ€"g for 30 min at 4 oC, and the precipitate was collected and washed with cold acetone three times. After centrifugation, the pellet was vacuum-dried. The immobilized pH gradient strips (17 cm, pH 4-7 linear, Bio-Rad, USA) were loaded with 350 ÃŽ ¼l sample buffer containing 800 ÃŽ ¼g sample proteins at room temperature in tray for 16 h. Isoelectric focusing was performed with the PROTEAN IEF system (Bio-Rad, USA) for a total 80000 V-hr. Then the strips were equilibrat ed in equilibration buffer I (6 M urea, 2% SDS, 0.375 M Tris-HCl pH 8.8, 20% glycerol, and 130 mM dithiothreitol) for 15 min and equilibration buffer II (6 M urea, 2% SDS, 0.375 M Tris-HCl pH 8.8, 20% glycerol, and 135 mM iodoacetamide) for 15 min sequentially. After equilibration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with 12% acrylamide gels. The 2-DE gels were stained with Coomassie Brilliant Blue (CBB) R-250. Image analysis of 2-DE gels The 2-DE gels were scanned at 600 DPI resolutions with an EPSON PERFECTIONTM V700 PHOTO scanner (Epson (china) Co., Ltd.). The images were analyzed with PDQuestTM 2-DE Analysis Software (Version 8.0, Bio-Rad, USA). Spot volumes were normalized by total spot volumes per gel to avoid experimental variations among 2-DE gels. Comparisons and statistical analysis were performed using the calculated average values of each biological replicate among the three different treatments. The protein spots with more than a two fold change among treatments and that passed a Student’s t-test (P Protein identification by MALDI-TOF/TOF-MS The significant differentially expressed spots were excised from the gel manually, and washed with double distilled water twice for 20 min, then distained with 100 ÃŽ ¼l of 50 mM NH4HCO3 in 50% v/v acetonitrile (ACN) for 1 h until the gel is mostly colorless at room temperature. The liquid was removed and 50 ÃŽ ¼l ACN was added to dehydrate the gel. After drying the gel, 25 mM NH4HCO3 containing 10 pmol trypsin (Promega, Madison, WI, USA) was added to the tube and kept at 4 oC for 1 h, and then it was kept at 37 oC overnight. The proteins were then digested according to the method described before [25]. The peptides were extracted and collected using three kinds of solution (0.1% TFA/99.9% acetonitrile, 0.1% TFA/99.9% H2O, 0.1% TFA/50% acetonitrile/49.9% H2O) from gel spot. The peptide solution was concentrated to 10 ÃŽ ¼l, and then desalted by ZipTip C18â„ ¢ pipette tips (Millipore, Bedford, MA, USA). After trypsin digestion, the protein peptides were dried by SpeedVac. Then pept ides were dissolved in 0.1% trifluoroacetic acid, and then 1 ÃŽ ¼l of the sample solution was loaded on Anchor Chip Standard (Bruker Daltonics Inc, Germany). After the Anchor Chip drying, the matrix solution (20 g/L HCCA, TA 95%) was loaded on point corresponding to the location of the sample to a target spot. Through ultrafleXtreme (Bruker Daltonics Inc, Germany) Operation, the PMF data was obtained. The instrument parameters for MS acquisition were list as follows: laser intensity was 20%-26%, reflector detector voltage was 2438 V. Protein identification using MS/MS raw data was performed with flexAnalysis software (Bruker Daltonics Inc, Germany) coupled with Mascot Server software (version 2.4.01) based on the NCBI protein database and SwissPort database of green plants. The searching parameters were set as follows: peptide masses were assumed to be monoisotopic, 100 ppm was used as mass accuracy, a maximum of one missing cleavage site, and modifications which included Carbamidom ethy and Oxidation were considered. (The timestamp of NCBI protein database is 2011/11/09, there were 949,856 sequences of Green Plants and 5,512,397,590 redundant total sequences in NCBI database; the timestamp of SwissPort 57.15, there were 28,783 sequences of Green Plants and 515,203 sequences non-redundant total sequences in SwissPort). The proteins which scores greater than 42 (NCBI) or 26 (SwissPort) (P Gel-free proteomics in L. chinense Protein extraction The protein samples for iTRAQ were recovered in lysis buffer (30 mM Tris-HCl, pH 8.5, 7 M urea, 2 M thiourea, and 4% [w/v] CHAPS) by phenol extraction and methanol/ammonium acetate precipitation as described previously [26]. The protein pellets were resuspended in buffer (7 M urea, 2 M thiourea, 4 % CHAPS and 10 mM DTT) in a minimal volume and protein was quantified using BCA protein assay kit (Pierce, USA). Digestion and iTRAQ labeling About 100 ÃŽ ¼g proteins of each sample per tube were prepared. Then it was reduced by adding DTT to a final concentration of 12 mM and incubated for 1 h at 37 oC. Subsequently, iodoacetamide was added to a final concentration of 50 mM, and the mixture was incubated for 1 h at room temperature in the dark. Then the mixture was transferred to centrifugal units (VN01H02, Sartorius, Germany) and centrifuged at 12,000Ãâ€"g for 20 min, and then the filtrate was discarded. Subsequently, 8 mM urea solution was added into the centrifugal units and centrifuged, repeated this step twice. After that, 100 ÃŽ ¼l dilute buffer (50 mM triethylammonium bicarbonate) was added into the centrifugal units and centrifuged. Then 50 ÃŽ ¼l dilute buffer containing 2 ÃŽ ¼g modified trypsin (Promega) was added into the centrifugal units at 37 oC overnight. The resulting peptides were then labeled with iTRAQ reagents (AB Sciex, USA) according to the manufacturer’s instructions. For each time point (i.e ., S1, S2, and S3), each sample was iTRAQ labeled 3 times except S3. (i.e., 113-, 116-, 119-iTRAQ tags for S1 3 replicates. 114-, 117-, 121-iTRAQ tags for S2 3 replicates. 115-, 118- iTRAQ tags for S3 2 replicates.) MS/MS Analysis Then the mixture of labeled peptides was concentrated and acidified to a total volume of 2 mL. Labeled peptides were desalted with C18-solid phase extraction and dissolved in strong cation exchange (SCX) solvent A (25% (v/v) acetonitrile, 10 mM ammonium formate, and 0.1% (v/v) formic acid (pH 2.8). The peptides were fractionated using an Agilent HPLC system 1260 with a polysulfoethylA column (2.1 Ãâ€" 100 mm, 5  µm, 300 Ã…; PolyLC, Columbia, MD, USA). Peptides were eluted with a linear gradient of 0–20% solvent B (25% (v/v) acetonitrile and 500 mM ammonium formate (pH 6.8) over 50 min followed by ramping up to 100% solvent B in 5 min. The absorbance at 280 nm was monitored, and a total of 37 fractions were collected. The fractions were combined into 12 final fractions and lyophilized. A quadrupole time-of-flight (LTQ Orbitrap XL) MS system (Thermo Fisher Scientific, Bremen, Germany) was applied as described previously [27]. It interfaced with an Eksigentnano-LC AS2 syste m (Eksigent Technologies, LLC, Dublin, CA) using high energy collision dissociation (HCD). Each fraction was loaded onto an Agilent Zorbax 300SB-C18 trap column (0.3 mm id Ãâ€" 5 mm length, 5  µm particle size) with a flow rate of 5  µl/min for 10 min. Reversed-phase C18chromatographic separation of peptides was carried out on a pre-packed BetaBasic C18PicoFrit column (75  µm id Ãâ€" 10 cm length, New Objective, Woburn, MA) at 300 nl/min using the following gradient: 5% B for 1 min as an equilibration status; 60% B for 99 min as a gradient; 90% B for 5 min as a washing status; 5% B for 10 min as an equilibration status (solvent A: 0.1% formic acid in 97% water, 3% ACN; solvent B: 0.1% formic acid in 97% ACN, 3% water). Database Search and Quantification The MS/MS data were processed by a thorough search considering biological modification and amino acid substitution against non-redundant NCBI green plants 20131014.fasta (1,544,439 contigs) under the Sequest ®algorithm of Proteome Discoverer.1.4 software (Thermo Fisher Scientific Inc.). Protein function analysis by blast2go software (http://www.blast2go.com/b2ghome) was conducted according to the early literature [28-31]. The search results were passed through additional filters before exporting the data. For protein identification, the filters were set as follows: significance threshold Phttp://mascot-pc/mascot/help/quant_config_help.html); S2/S1 or S3/S1 ratios >2 and