microRNA Microarrays - Biocompare Buyer's Guide For Life Scientists

microRNA Microarrays - Biocompare Buyer's Guide For Life Scientists

Tears of Humanity over Siirt Incidents

It was not very long time ago; last week, Turkey was shocked by the news targeting our hearts from deep inside. Two sisters, aged 14 and 16, from a poor family were raped by dozens of men for more than two years. Elder sister reported to the police that the rapers aged between 14 and 70, and include classmates, deputy principal at the girls’ school, storeowners, a policeman, a military personnel and a number of people from prestigious families in the province. As the time passed since this incidence was first publically available by a group of brave and honorable media agencies, there have been deep debates of social dissociation, sexual harassment, rape and vendetta. Unfortunately, top government officials recently warned the media over recent coverage of rape incidents in the province of Siirt. By this way, the motto of “big fish eats little fish” was proven one more time, and some secret powers has pushed the button to squelch the silent scream.
While the country has been boiling with the news of this rape and digesting the consequences of forceful media silence, shocking news shook Turkey from east to west two days ago. Turkish dailies reported on Monday that eight minors aged 13 and 14 raped two toddlers aged 2 and 3, killing one of them, last year in Siirt. Prime Minister Recep Tayyip Erdoğan and Education Minister Nimet Çubukçu talked to reporters on Monday about the issue; with both saying the media should be more careful about covering the incidents.

The worst news was not the rape and death of toddlers by other children, but the fact that this incidence had happened one year ago and had been tried to be kept secret by paying a blood money to the families of toddlers. Mayor of Siirt indicated that the inhabitants of the city are all relatives as belonging to a big tribe. Not only the families, but also police, governor, mayor, leaders of political parties and district prosecutor were all aware of the incidence. According to the Mayor, these people kept the incidence as a secret because they did not wanted the name of Siirt be remembered with bad.

PM Erdoğan mentioned that “The issue is brought to the agenda again. This is not what journalism is about. This is not the rationale of the media. This does not go with media ethics. On the contrary, this causes psychological damage to people related to Siirt and the people who are involved. I think we need to be very careful about this.” What a shame on a prime minister, the primary duty of whom should be “the protection of weak”.

These incidents indicate the probable strengths and weaknesses of “tribe” knowledge in Anatolia. It is worth to mention here that whatever the tribe council takes a decision, the members of the tribe should follow it. This time tribe in Siirt decided on paying the blood money to the families of those two innocent toddlers and keeping this secret forever to protect the name of their city being called with rape. However, they forgot that a secret is not a secret any longer if two people know it.

Finally, it is impossible for me to understand those people who show themselves as Muslim to accept a sexual rape as a natural thing and not mentioning it any longer even if rape is totally banned in Quran, and the rapers were cursed. This is like turkeys voting for Christmas.

I hope these Siirt incidences will be announced as the milestones of humanity and help us to develop our country over strong bricks of non-tribe based, secular ideas in which humanity and human rights always come first.

A small final note:

On May 9, Mother’s Day, Siirt inhabitants are planning to protest the media for their brave journalism about the secret of a tribe and questioning of humanity in Turkey.

References:

http://www.todayszaman.com/tz-web/news-208220-100-15-arrested-dozens-more-involved-in-siirt-rape-case.html

http://www.todayszaman.com/tz-web/news-208658-101-government-warns-media-over-coverage-of-siirt-rape-incidents.html

http://www.hurriyetdailynews.com/n.php?n=from-the-bosphorus-straight---what-kind-of-future-for-our-children-2010-04-22

Can Dostum

Amerika maceramin ilk bir bucuk yilinda yaptigim gozlemler sonucunda ogrendigim en buyuk bilgilerden birisidir Turk insaninin samimiyeti. Bunu karsindakinin gozunden, yuzunden, vucut hal ve davranislarindan anlarsin. Hissedersin karsindaki iyi mi, kotu mu. Sana zarari dokunur mu, dokunmaz mi. Hersey apacik ortadadir. Kendini gizlemez, gizleyemez... Seni seviyorsa hissedersin. Daha da yaklasmak istersin. Sana karsi yaptiklarinda, davranislarinda hicbir yapaylik yoktur. Yapmacik degildir.

Amerika'da ise hersey yuzeyseldir. Dostuk, ask, sevgi... Hepsi yuzeyseldir. Cikarlar dogrultusunda yonetilirler. Beyin vardir bu isin icerisinde. Kalbi bir yana birakmistir. Sanki hicbirisinin can kiriklari olmamistir. Kalpleri nasir baglamis, adeta metal bir kabin icerisinde gizlenmistir. O metal kabi delmek neredeyse imkansizdir. Metalin soguklugu yuzlerine, guluslerine ve sozlerine yansir.

Burada tek gercek zorunluluktur. Cikarlarin dogrultusunda kurulmus bir sevgi yumagi ve insan iliskileri agi soz konusudur. Yumagi yonlendiren, iliskileri denetleyen kalp degildir. Cunku o metal kabin icinde hapsolmustur. Beyin kontrolu ele almis, kisisel cikarlar dogrultusunda insanlari bir siralamaya koyar. Birisinden cikari ne kadar yuksekse ona daha fazla yaklasir. Samimi olur (tabi sozde, ozde degil). Gittiginiz her isletmede her calisan size selam verir, nese ile siritip hatrinizi sorar. Ama bu sadece birkac saniyelik bir iletisimdir. Cikarlari soz konusudur. Siz musterisinizdir ve size iyi davranmakla sorumludur o calisan. Hatri sorulmayan bir musteri her zaman potansiyel bir tehdit olabilir. Gidip calisani magaza mudurune iyi gunler dilemegidi icin sikayet edebilir. Iste budur Amerikali'nin soguk gulumseyisi. Cikarlarindan gelir.

Mesela okulda, cikari varsa arkadasin senin yanina gelir. Cikari ugruna seninle konusur, selam verir, belki bir kahve icer. Donem biter, cikar iliskisi kalmayinca arkadasinin beyni seni siler atar. Paylasilanlar unutulur. Bir donem sonra seni asansorde gorunce selam vermez, yuzune bile bakmaz....

Bu cikar iliskileri toplumun tumune yayilmistir. Hatta samimiyetin, gulumsemenin, kalpten gelen bir sirt oksamanin ayip ve apes kactigi bir aile yasamlari vardir. Anne-baba cocuklarin gozunde para makinesi ve karin doyurucudur. Cikarlari budur. Cikarlari dogrultusunda anne-babaya gunaydin denir. Sevgi sozcukleri dile getirilir. Sonra anne-baba bir gun para vermezse, cocuk ondan nefret ettigini dile getirmekten cekinmez. Farkli bir acidan, cocugun acisindan da konu tipki ayni noktaya cikar. Cocuk universite yasina gelince anne-babasina kulfet gelmeye baslar. Cunku artik kendi ayaklari uzerinde durabilir. Evde oldugu her an ailesini somurdugu dusunulur. Bu yuzden de aile tarafindan dislanabilir.

Uzun lafin kisasi, yozlasmis bir insan toplulugudur Amerikali. Aile ve arkadas baglari cikarlar dogrultusunda beyin tarafindan yonlendirilir. Sevgi, ask gibi terimler coktan anlamini yitirmis, hepsi birer birer unutulup gitmistir. Iste bu yuzden, Turk toplum yapisi, aile baglari, arkadas iliskileri her zaman Amerikalilara degisik, yabanci ve bir o kadar da anlamsiz gelmistir. Her konusmamda konu ne zaman buraya gelse, beni anlayamiyorlar. Islerine gelmiyor. Beyinleri hep eksi puan veriyor...

Bitirirken de canim dostuma gelsin Haluk Leven'tin "Can Dostum" adli sarkisi... Cok ozledim seni, can dostum!

"Bir şarkı yazmak istedim, içinde dostluk olsun


Birden sen geldin aklıma, can dostum

Bu şarkıda seni ne çok özlediğimi

Anlatmak istedim sana bir kere olsun

Bir sen kaldın bana, sakın bırakma

Al yollarına hisset yanında

Dostum, dostum, dostum, can dostum



Burda herşey sahte dostum gülümsemeler bile

Burda herşey sahte dostum sevmeler bile



Şimdi yanımda olamasanda

Seni yaşamamı engelleyemez hiç birşey asla

Şu anda çok uzakta olsan da

Sen aslında benimlesin yanı başımda

Sarıl yine bana al yollarına

Hisset yanında agla gözyasımda

Dostum,dostum,dostum,can dostum



Burda herşey sahte dostum gülümsemeler bile

Burda herşey sahte dostum sevmeler bile"

23rd April - International Children's Day in Turkey

This national day (23 April National Sovereignty and Children's Day) in Turkey is a unique event. The founder of the Turkish Republic, Mustafa Kemal Atatürk, dedicated April 23 to the children of the country to emphasize that they are the future of the new nation. It was on April 23, 1920, during the War of Independence, that the Grand National Assembly met in Ankara and laid down the foundations of a new, independent, secular, and modern republic from the ashes of the Ottoman Empire. Following the defeat of the Allied invasion forces on September 9, 1922 and the signing of the Treaty of Lausanne on July 24, 1923, Ataturk started his task of establishing the institutions of the new state. Over the next eight years, Ataturk and his followers adopted sweeping reforms to create a modern Turkey, divorced from her Ottoman past. In unprecedented moves, he dedicated the sovereignty day to the children and entrusted in the hands of the youth the protection of this sovereignty and independence.

Every year, the children in Turkey celebrate this "Sovereignty and Children's Day" as a national holiday. Schools participate in week-long ceremonies marked by performances in all fields in large stadiums watched by the entire nation. Among the activities on this day, the children send their representatives to replace state officials and high ranking bureaucrats in their offices. The President, the Prime Minister, the Cabinet Ministers, provincial governors all turn over their positions to children's representatives. These children, in turn, sign executive orders relating to educational and environmental policies. On this day, the children also replace the parliamentarians in the Grand National Assembly and hold a special session to discuss matters concerning children's issues.

Over the last two decades, the Turkish officials have been working hard to internationalize this important day. Their efforts resulted in large number of world states' sending groups of children to Turkey to participate in the above stated festivities. During their stay in Turkey, the foreign children are housed in Turkish homes and find an important opportunity to interact with the Turkish kids and learn about each other's countries and cultures. The foreign children groups also participate in the special session of the Grand National Assembly. This results in a truly international Assembly where children pledge their commitment to international peace and brotherhood.

A better recreational way of enjoying the life in Aggieland

As being a member of Texas Aggies and been living in College Station for more than 1.5 years, I have already explored the amusement and attractive nature of Brazos Valley. One of my favorite places in Aggieland is The Lake Bryan. Located 5 miles west of Bryan, the lake serves as a coolant reservoid of the power plant. The area is surrounded by hundreds of live oaks. It is one of the resting spots of many migrating birds including green headed duck (Anas platyrhynchos). Moreover, the lake is the home of a number of different fish species such as catfish, crappie and sunfish.

The lake is used for recreational and musical events. There is a restaurant by the lake. Especially I recommend to go there and grab a beer while observing the sunset over the lake and the dalliance of tree shadows on the water.

There is also a human-made beach over the lake. I saw lots of people come to relax, sunbath and swim in the summer, esspecially at the weekends. Many people visit the lake only one day, but there is always a possibility of camping near the lake. Some bring their RVs and enjoy the nature whithout concede of the luxury. If you are one of those camping fans, then you need the following list of equipments:












Links:

http://www.tpwd.state.tx.us/fishboat/fish/recreational/lakes/bryan/

Proposal: An Alternative Way of the Improvement of Drought Tolerant Wheat Cultivars Under High Temperature Environments with Water Limiting Conditions

INTRODUCTION

Wheat is the most important grain crop grown in our country providing greatest part of the daily nutritional requirement. Drought, salinity, freezing, pests, bacterial and fungal diseases are the most important biotic and abiotic factors affecting quality and yield of wheat varieties used for the production of both bread and pasta flour. To supply the growing nutritional needs of rising human populations, similar increase in the rate of production should be achieved. In 21st Century, drought and water depletion stress is expected to be the most important limiting factors due to the global warming and inappropriate land utilization (Reddy et al., 2004). For this reason it is important to improve the cultivars for drought tolerance which can survive in the marginal lands. Plants experience drought stress when the water supply to roots becomes difficult or when the transpiration rate becomes very high. Plants have evolved different mechanisms to adapt short- and long-term drought conditions. One of those mechanisms, thickening of leaf cuticular wax layer on surface of leaves has been studied for a decade. Plant cuticle is a complex hydrophobic matrix coating the epidermis of aerial organs. It serves as a barrier for the flux of gases and water vapor, and also has some protective roles in biotic and abiotic stress tolerance. By this way, cuticular waxes help maintain reduced transpiration rates and loss of water in high temperature environments with water limiting conditions. Previous studies demonstrated the connection between increased wax accumulation on leaves and drought stress in many plants, including common and durum wheat, thyme, cotton, rose, pea, peanut, tree tobacco, oat, soybean and sorghum. Many of those studies focused on physiological and biochemical effects of drought on cuticular wax accumulation. Mutant screening and characterization of genes responsible for wax biosynthesis and secretion were all carried out in model plant Arabidopsis thaliana. These studies lead our understanding of cuticular wax biosynthesis in epithelial cells, secretion from plasma membranes and their regulation. However, there has not been any progress in the development of drought tolerant crop plants by increased cuticular wax accumulation. Adjustment of drought tolerance in wheat cultivars by increased amounts of wax deposition on leaves will be an important step for the challenge of market adapted, industrial welfare of our country.

The long term goal of our research focuses on the development of drought and heat tolerant wheat plants with a specific emphasis on cuticular wax layer improvements. As the first step of our research, the objective of this proposed project is to describe the effects of increased expression of cuticular wax on the improvement of tolerant wheat cultivars under high temperature environments with water limiting conditions. Previously, the decrement of total wax accumulation in different wax-related gene mutants of Arabidopsis was shown (Goodwin et al., 2005). Among those mutant lines, two of them (cer5 and cer6) showed the two highest reductions in total wax accumulation. CER6 is a β- keto acyl- CoA synthase, which works in early steps of cuticular wax biosynthesis, and CER5 is an ABC type transporter, which localizes on the plasma membrane of epidermal cells and secretes cuticular waxes into cuticle. We hypothesize that co-expression of CER5 and CER6 in wheat inflorescence will increase the accumulation of cuticular waxes on leaves under high temperature environments with drought conditions. We further hypothesize that this method will cause morphological, physiological and biochemical changes in overall wheat plants, and will increase the crop yield under drought conditions. It is already known that the co-expression of two consecutive genes of a specific pathway can cause hyper-accumulation of end products. Hence, increased cuticular wax accumulation in transgenic wheat plants that co-express CER5 and CER6 would be a probable consequence of our hypothesis. Moreover, according to a previous study heterologous expression of two Medicago truncatula wax-related transcription factors in Arabidopsis led to increased leaf wax accumulation and improvement of drought tolerance (Zhang et al., 2007). Thus, increased leaf wax accumulation and improvement of drought tolerance will be obtained by our proposed project.

The following objectives have been identified as the driving forces in attaining the long term goals.

1. Develop wheat genotypes over-expressing both CER5 and CER6 genes and characterize their morphological, physiological, biochemical and molecular changes under drought and heat stress conditions.
2. Determine the connection between increased expression of CER5 and CER6 on soil plant atmosphere continuum in wheat and high temperature and drought stresses.
3. Characterize the effects of increased expression of leaf cuticle on the improvement of wheat yield under high temperature environments with water limiting conditions.

Our strategy of co-expression of CER5 and CER6 in wheat cultivars is unique in terms of its probable high efficiency.

BRIEF LITERATURE REVIEW

Introduction

Plant cuticle is a complex hydrophobic matrix coating the epidermis of aerial organs. It serves as a barrier for the flux of gases and water vapor, and also has some protective roles against biotic and abiotic stresses (Jenks and Ashworth, 1999). Plant cuticle is composed of two lipophilic substances, namely cutin and cuticular waxes. Composed of mid-chain C16 and C18 fatty acids, cutin serves as the structural backbone of cuticle and resists tension. On the other hand, cuticular waxes are composed of mixture of C20 to C60 long aliphatic chains and serves as the barrier to non-stomatal excessive water loss. They may also include secondary metabolites such as terpenoids, phenylpropanoids, and flavonoids (Jetter et.al., 2006). Waxes embedded in the cutin are called as intracuticular waxes whereas epicuticular waxes are found on the surface of cutin layer (Samuels et.al., 2008).

Biosynthesis of Wax Polymers

Synthesis of wax elements start in epidermal leucoplasts by connecting acetyl coenzyme A containing C2 fatty acids to produce straight-chain polymers up to 18 carbon atoms. When the size of the polymer chain reaches to 18, fatty acids are transferred into endoplasmic reticulum (ER), where very-long-chain fatty acids with C20-C34 chains are generated. Later, these chains of fatty acid are modified in ER to produce the end products of cuticular waxes such as alcohols, esters, aldehydes and ketones (Samuels et.al., 2008).

Several wax biosynthesis pathway genes have been identified by Arabidopsis eceriferum (cer) and maize glossy (gl) mutant analysis. These genes include, CER6/CUT1 (Millar et al. 1999; Fiebig et al. 2000), CER10 (Zheng et al., 2005), CER4 (Rowland et al., 2006), GL1 (Hansen et al. 1997; Sturaro et al. 2005), GL2 (Tacke et al. 1995), GL8 (Xu et al. 1997), GL15 (Moose and Sisco 1996), KCS1 (Todd et al. 1999), WSD1 (Samuels et.al., 2008) and MAH1 (Greer et al., 2007). Although single mutations in these genes resulted in decreased wax accumulation in Arabidopsis leaves, double cer mutants showed redundant genetic operations in wax biosynthesis (Goodwin et al., 2005).

Secretion of Cuticular Wax

Synthesized in the ER, wax constituents are delivered to the plasma membrane by two hypothetical pathways (Samuels et.al., 2008). According to the first hypothesis, wax constituents are targeted to plasma membrane through golgi mediated secretory vesicles. On the other hand, second hypothesis indicates the non-vesicular movement of wax components from ER to plasma membrane. However, further investigation is required to understand the mechanism of cuticular wax delivery to the plasma membrane.

The wax molecules transported to the plasma membrane are exported from it by help of two transporters, CER5 (Pighin et al. 2004) and WBC11 (Luo et al., 2007). Both proteins belong to the ATP binding cassette (ABC) protein family and they were shown to function in cuticular wax accumulation in Arabidopsis (Panikashvili et al., 2007).

Regulation of Cuticular Wax Biosynthesis

As the amount and structure of cuticular waxes vary dramatically with developmental and environmental factors, their formation is tightly regulated by transcription factors. For example, both light and osmotic stresses induce CER6 transcription and wax accumulation on leaves of Arabidopsis (Hooker et al., 2002).

Over-expression of the only known transcription factor to regulate wax biosynthesis, namely WAX INDUCER (WIN) 1/SHINE 1 (SHN1) in Arabidopsis and its homolog WAX PRODUCTION 1 (WXP1) in Medicago truncatula, induced leaf cuticular wax accumulation and this resulted in increased drought tolerance of transgenic plants (Aharoni et al, 2004; Broun et al., 2004; Zhang et al., 2005). Expression of wax-related genes such as CER1 and CER2 increased in plants over-expressing WIN1/SHN1, which indicates this transcription factor in upstream of those genes and regulates their expression (Broun et al., 2004).

Another control mechanism of cuticular wax biosynthesis is the degradation of a transcription repressor of CER3 by CER7 ribonuclease, the core subunit of the exosome (Hooker et al., 2007). By this way, CER3 expression and wax biosynthesis are induced by a ribonuclease.

Because of the complex nature of fatty acid biosynthesis, there are many different members of CER family proteins which are involved in wax production. Three genes, CER1 (Aarts et al. 1995), CER2 (Negruk et al. 1996; Xia et al. 1996), and CER3 (Hannoufa et al. 1996), have been isolated, but they were not been able to characterized.


SIGNIFICANCE OF THE PROJECT

It is envisaged that global warming, over-irrigation and the other reasons such as wrong land usage would accelerate water limitations and accordingly the desertification. This situation undoubtedly will lead to reduced yield. However the reality of the world population is growing rapidly reveals the need for more nutrients. One of the methods to obtain enough quantities of plant products to feed this population is to foster the effective farming in marginal areas. For this purpose, one of the widely viewed ways is to increase stress resistance of plants by transferring a variety of genes.

In the last decade, both global warming and significant reduction in seasonal rainfall in our country poses the problem of drought. In such a situation, the strategies that will be implemented contain conscious irrigation efforts, as well as the development of wheat varieties that can grow in arid environments should look like. Unless these strategies are implemented, the nutritional needs of the population would be hardly met and the foreign dependence would grow. For these reasons, improving the drought tolerance of wheat varieties used in our country is of great importance in terms of both food requirements as far as security and freedom of our country. This proposed project will provide a significant input into our agriculture and industry.

SPECIFIC OBJECTIVES OF THE PROPOSAL

The following three objectives will be analyzed in this project.

1.Develop wheat genotypes over-expressing both CER5 and CER6 genes and characterize their morphological, physiological, biochemical and molecular changes under drought and heat stress conditions.

By this specific objective, we hypothesize that over-expression of CER5 and CER6 genes in wheat cultivars would change the morphological, physiological, biochemical and molecular nature of wax accumulation in drought and heat stress conditions. To test this specific hypothesis, CER5 and CER6 genes will be co-expressed in wheat cultivars and morphological and physiological effects of over-expression of both genes together in wheat cultivars will be analyzed. Then, specific biochemical and molecular experiments will be conducted to understand the specific downstream effects of co-expression of CER5 and CER6 in wheat cultivars.

2.Determine the connection between increased expression of CER5 and CER6 on soil plant atmosphere continuum in wheat and high temperature and drought stresses.

The hypothesis for this specific objective is over-expression of CER5 and CER6 would increase the cuticular wax accumulation over leaves under drought and heat stresses. This in turn would limit stomatal conductance and leaf transpiration rate by increasing the leaf water potential. To test this hypothesis, leaf water potential, soil water potential, stomatal conductance, leaf transpiration rates of transgenic plants will be analyzed under high temperature plus drought and normal temperature plus well-watered conditions.

3.Characterize the effects of increased expression of leaf cuticle on the improvement of wheat yield under high temperature environments with water limiting conditions.

This specific objective originates from the hypothesis of obtainment of higher wheat yields under high temperatures with water limiting conditions. To test this hypothesis, both the yield of cereal per unit area of land under cultivation, and the seed generation of the plant itself will be measured for CER5 and CER6 over-expressing wheat cultivars in comparison with control plants grown under normal and high temperature plus drought stress conditions. Also, gluten amounts in wheat seeds will be quantified to understand the changes in seed quality in these plants.

EXPERIMENTAL DESIGN OF THE PROJECT

Plant Materials & Growth of Plants

Triticum aestivum cv. Yüreğir-89 and Triticum turgidum spp. durum cv. Kızıltan-91 will be used as wheat varieties for transformation purposes in this study. Arabidopsis thaliana Colombia (Col0) will be used to obtain genes of CER5 (At1g51500) and CER6 (At1g68530). Arabidopsis seeds will be grown on MS medium for 7 days and then total RNA will be extracted by TriZol method. Then, it will be converted to cDNA.

Development of Transformation Vectors, Plant Transformation and Post-Transformation Analysis

For the over-expression of CER5 and CER6, constitutive promoter, ubi1, and inducible promoter, rd29a, and nos terminator will be obtained from specified vectors. Then, they will be cloned into multiple cloning site of pPZP201 plazmid. rd29a promoter containing construct will be prepared as an alternative way to understand the effect of drought induction on gene expression levels of CER5 and CER6 in transgenic wheat cultivars. By using Gateway® cloning system, the following constructs will be transformed into inflorescence of two wheat varieties, which are grown in greenhouse conditions for 10 days. Transformation will be conducted by Gene Gun.

Ubi1 promotor – CER5 CDT – CER6 CDT - NOS 1
Ubi1 promotor – NOS 2
RD29A Promotor – CER5 CDT – CER6 CDT – NOS 3
RD29A Promotor – NOS 4

Constructs with numbers 2 and 4 will be used as negative controls in the following experiments. Transgenic plants will be selected on mannose media and successful transformations will be further analyzed by GFP expression. Integration of whole constructs will be analyzed by PCR amplification.

Drought and High Temperature Stress Applications

Transformed plants will be grown in tissue culture and then healthy plants will be further grown in greenhouse to obtain T1 seeds. T0 plant leaves will be used for Southern and Northern analysis of gene transfer success rates. Successfully transformed T1 seeds will be germinated on petlit and will be watered by ½ Hoagland’s solution for 10 days. At the end of 10th day, plants will be watered by ½ Hoagland’s solution containing 10% polyethylene glycol (PEG 6000) for the induction of drought stress. Both control and stress-treated plants will be grown in three different temperatures (24°C, 32°C and 44°C) for 1, 3, 7 and 14 days. Then, samples will be collected for further analysis. The plants grown under 24°C will be used as controls.

Morphological, Physiological, Biochemical and Molecular Analysis of Transgenic Wheat Plants

Leaf Area, Leaf Fresh, Dry and Wet Weight, Leaf Length and Relative Water Content

Leaf length and fresh weight will be measured from freshly collected samples and those leaves will be kept in distilled water for 3 hours to obtain wet weight. Then, those leaves will be dried in an oven overnight to obtain dry weight. Then, relative water content will be calculated according to its formula and will be given in percentage. For the leaf area measurements, fresh leaves will be photographed by a digital camera and surface areas will be calculated by a specific computer program.

Morphological Changes on Surface and Integral Parts of the Leaves

Surface and the integral parts of the leaves of both control and stress-treated plants will be analyzed by TEM and SEM, respectively. Plant leaves will be detached and then will be fixed and samples for electron microscopy will be prepared according to manufacturer’s protocols. Developmental differences of palisade parenchyma, epidermal differentiation and under-developed trichome formations between CER5-6 transgenic plants and vector controls under stress conditions and control conditions are expected. Also, thickness of cuticular layers over and in between epidermal cells will be analyzed.

Chlorophyll Leaching and Chlorophyll Fluorescence Assays

Epidermis permeability will be measured using chlorophyll leaching assay. Three true leaves will be collected under dim light from 4-week old plants and amount of chlorophyll extracted into 80% ethanol solution will be measure by spectrophotometrically at 664nm as described by Zhang et al. (2007). In dark adapted leaf tissue, “Fv/Fm” test, which indicates the maximum photochemical yield of PSII, will be performed by using OS5-FL Modulated Fluorometer according to the manufacturer’s protocol (Opti-Sciences, 2003).

Wax Identification and Quantification

Leaf waxes will be extracted by chloroform. Cuticular waxes will be analyzed by gas chromatography – mass spectrometry (GC-MS). Mass spectra and the retention time of individual peaks will be used to determine the identity of wax components. Quantification will be done based on peak areas.

Microarray Analysis of Transgenic Wheat Plants

To analyze the effects of CER5 and CER6 co-expression in wheat under drought and heat stresses, total RNA will be isolated from stressed and non-stressed control plants which are grown for 10 days and they will be converted into cRNA and will be hybridized on Wheat GeneChip according to GeneChip Expression Analysis Technical Manual, Affymetrix. These microarray experiments will be repeated three times. Analysis of differentially expressed genes under drought and heat stresses will be made with GeneSpring GX 10.0 (Agilent) by using RMA data normalization. The results will be given as fold induction with respect to control plants.

RT-qPCR analysis of Downstream Genes

Microarray results will be proven by RT-qPCR analysis of the first 50 highly expressed genes. Later, some interesting genes from the microarray results will be selected for their expression analysis in different time points of stress applications.

RT-qPCR analysis of Drought – and Temperature - Related Genes

In addition to interesting genes selected from microarray experiments, a set of drought and heat related genes will be selected and their expression patterns will be analyzed under different treatments of drought and heat stresses.

Analysis of Plant Atmosphere Continuum in Transgenic Wheat Cultivars Under High Temperature and Drought Stresses

Stomatal Conductance and Leaf Transpiration Rate

The stomatal conductance and the leaf transpiration rates will be determined on the youngest fully expanded leaves in a special growth chamber, to which an infrared CO2/H2O analyzed is attached. Required measurements will be monitored according to Martre et al. (2002).

Leaf Water Potential, Soil Water Potential and Osmotic Pressure

Leaf water potential will be determined using a special type of pressure chamber. After the balance pressure is determined, the leaf will be frozen in liquid nitrogen. After thawing, the tissue will be squeezed and the osmolality of the expressed liquid will be measured with a vapor pressure osmometer. Then, this value will be converted to osmotic pressure according to Van’t Hoff relation. Soil water potential will be determined on soil samples collected in the center of the pot using a dew-point hygrometer (Martre et al,. 2002).

Characterization of Wheat Yields under High Temperature Environments with Water Limiting Conditions

T2 plants will be grown in the field and the yield of cereal per unit area of land under cultivation will be measured for stressed and non-stressed control plants Also, gluten amounts in T2 wheat seeds will be quantified using Elisa Gliadin kits to understand the changes in seed quality in these plants.

REFERENCES

Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A. 2004. The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell 16:2463–2480
Broun P, Poindexter P, Osborne E, Jiang C-Z, Riechmann JL. 2004. WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. PNAS 101:4706–4711
Goodwin SM, Rashotte AM, Rahman M, Feldmann KA, Jenks MA. 2005. Wax constituents on the inflorescence stems of double eceriferum mutants in Arabidopsis reveal complex gene interactions. Phytochemistry 66:771–780
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Iron (Fe) Acquisition By Plant Roots and Fe Signaling

Introduction - Iron

Iron (Fe) is required by nearly all organisms for continued existence. Fe is a crucial component of redox systems because of its ability of donating and accepting electrons. In plants, it works as a cofactor in many metalloproteins and is found in active sites of Fe-S clusters, which are essential for electron transport chains in photosynthesis and respiration. Fe is also required for DNA and hormone biosynthesis, nitrogen fixation, sulfate assimilation, and chlorophyll biosynthesis. Limited Fe solubility is one of the major determinants of its phytoavailability to plants. Fe solubility in an oxygenated medium at pH 7 is hundred times less than the essential iron concentration for survival of a plant because of the ability of Fe to produce ferric oxides and ferric hydroxides in aerobic environments (Guerinot, 1994; Lindsay, 1995; Palmer and Guerinot, 2009). Fe deficiency in plants generally occurs on well-oxygenated and alkaline soils such as calcareous soils (Frossard et al., 2000). These soils cover one third of agricultural land in the world (White and Broadley, 2009). Fe-deficiency induces intercostal leaf chlorosis due to the limited chlorophyll biosynthesis. This in turn, causes significant yield losses of crops in the field. Fe deficiency also compromises human health since plants are the most common source of dietary iron. Iron deficiency is the most common cause of anemia worldwide. In the United States, 7% of toddlers ages 1 to 2 years old and 9-16% of menstruating women are iron deficient (Clark, 2008). 30-70% of the people have iron deficiency anemia in the underdeveloped countries in the world (Yip and Ramakrishnan, 2002). Moreover, it is estimated that iron-deficiency anemia affects some two billion people, causing almost one million deaths each year (WHO, 2002). Understanding the molecular mechanisms of Fe uptake and regulation of these processes is important for the correction of nutritional disorders in both plants and humans.

Iron Acquisition by Roots

Plants have developed two distinct mechanisms to mobilize insoluble iron in the rhizosphere and uptake it through plasma membrane (Romheld, 1987). In Strategy II plants, grasses and cereals, phytosiderophores are released into the rhizosphere, where they complex with ferric iron (Fe3+) and then the complex is taken up into root cells via Yellow-stripe1 (YS1) transporters (Curie et al., 2001; Kim and Guerinot, 2007). In Strategy I plants, which includes all dicots and non-graminaceous monocots such as Arabidopsis thaliana, Fe is taken up by a set of three activities on plasma membrane of root epidermal cells. First, rhizosphere is acidified by a plasma membrane (PM) H+-ATPase-mediated proton extrusion mechanism (Schmidt et al., 2003). In Arabidopsis, rhizosphere acidification under Fe-deficiency is mediated by AHA2, a member of PM H+-ATPase family. On the other hand, another member of the same family, AHA7 functions in root hair development (Santi and Schmidt, 2009). Rhizosphere acidification is followed by reduction of insoluble Fe3+ to soluble Fe2+ by ferric reductase oxidase (FRO) that possesses the transmembrane electron transport ability. To date, FRO encoding genes have been identified in different Strategy I species such as pea (Waters et al., 2002), tomato (Li et al., 2004), cucumber (Waters et al., 2007) and Arabidopsis (Robinson et al., 1999). In Arabidopsis, FRO family consists of eight members (Jeong and Connolly, 2009). The first identified member of the family, FRO2 is localized to PM of root epidermal cells. Members of FRO family show various specificities of tissue expression and subcellular localization, and some of them are Fe-regulated (Robinson et al., 1999; Feng et al., 2006; Mukherjee et al., 2006; Jeong et al., 2008). All of this information indicates multiple roles of FROs in metal acquisition, distribution and homeostasis in plants. Reduced iron is taken up by a high-affinity transporter, iron regulated transporter 1 (IRT1), which is highly expressed in epithelial root cells and localized to PM (Eide et al., 1996; Varotto et al., 2002; Vert et al., 2002). IRT1 belongs to ZIP metal transporter family. Both FRO2 and IRT1 are under transcriptional and post-transcriptional regulation in Fe-limited conditions because their mRNA and protein levels increase in response to Fe-deficiency (Connolly et al., 2002; Kerkeb et al., 2008). There are other transporters that function in subcellular mobilization of Fe such as AtNRAMP3/4, which are two transporters localized on tonoplast. Its expression increases under Fe deficiency and it functions in iron remobilization from vacuole. Fe toxicity is as important as Fe deficiency as excessive amounts of iron can cause over-production of reactive oxygen species in Fenton reaction (Moller et al., 2007). Therefore, Fe homeostasis is strictly regulated (Martinoia et al., 2007). Excessive Fe is mobilized into vacuole by AtVIT1, which is Arabidopsis ortholog of yeast transporter CCC1 (Kim et al., 2006). Fe mobilization into chloroplast is achieved by AtPIC1, which is an ortholog of cyanobacterial Fe transporter (Duy et al., 2007), and AtFRO7 (Jeong et al., 2008). Excessive Fe is sequestered by ferritins in plastids. Arabidopsis ferritins are encoded by four genes (Ravet et al., 2009). AtFER1 is the most highly expressed in response to Fe excess while AtFER2 is the only gene expressed in seeds (Petit et al., 2001; Ravet et al., 2009).

Iron Signaling in Plants


According to recent studies of Fe deficiency induced signaling in Arabidopsis, a basic helix-loop-helix (bHLH) transcription factor called Fe-deficiency-induced transcription factor 1 (FIT1 or bHLH29/FRU) was identified as the regulator of 71 Fe-deficiency induced genes including FRO2, IRT2, AHA7 and nicotinamine synthase 1 (NAS1) (Colangelo and Guerinot, 2004). FIT1 is Arabidopsis ortholog of tomato LeFER gene and belongs to bHLH family of transcription factors (Ling et al., 2002; Yuan et al., 2005). The transcript levels of FIT1 and four other bHLH genes (bHLH38, bHLH39, bHLH100 and bHLH101) change under Fe deficiency and FIT1 forms heterodimers with bHLH38 and bHLH39 (Wang et al., 2007; Yuan et al., 2008). This indicates the presence of upstream components in Fe signaling pathway that mediates sensing and signaling of Fe deficiency. Moreover, Arabidopsis FER1 promoter contains a 15-bp cis-acting element called iron-dependent-regulatory-sequence (IDRS), which is involved in the repression of AtFER1 expression under Fe deficiency (Petit et al., 2001; Tarantino et al., 2003). Root growth inhibitory conditions such as cytokinin, ABA, mannitol or salt treatments suppress the iron deficiency response genes regardless of the plant iron status (Seguela et al., 2008).

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