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
Greer S, Wen M, Bird D, Wu X, Samuels L, Kunst L, Jetter R. 2007. The cytochrome P450 enzyme CYP96A15 is the mid-chain alkane hydroxylase responsible for formation of secondary alcohols and ketones in stem cuticular wax of Arabidopsis thaliana. Plant Physiol. 145:653–67
Hooker TS, Millar AA, Kunst L. 2002. Significance of the expression of the CER6 condensing enzyme for cuticular wax production in Arabidopsis. Plant Physiol. 129:1568– 80
Hooker TS, Lam P, Zheng H, Kunst L. 2007. A core subunit of the RNAprocessing/ degrading exosome specifically influences cuticular wax biosynthesis in Arabidopsis. Plant Cell 19:904–13
Jetter R, Kunst L, Samuels L. 2006. Composition of plant cuticular waxes. Annual Plant Reviews, pp. 145–81
Jenks MA, Ashworth EN. 1999. Plant epicuticular waxes: function, production, and genetics. Hortic Rev 23:1–68.
Luo B, Xue XY, Hu WL, Wang LJ, Chen XY. 2007. An ABC transporter gene of Arabidopsis thaliana, AtWBC11, is involved in cuticle development and prevention of organ fusion. Plant Cell Physiol. 48:1790-1802.
Martre P., Morillon R., Barrieu F., North GB., Nobel PS., Chrispeels MJ. 2002. Plasma membrane aquaporins play a significant role during recovery from water deficit. Plant Physiology 130: 2101 – 2110.
Panikashvili D, Savaldi-Goldstein S, Mandel T, Yifhar T, Franke RB, Höfer R, Schreiber L, Chory J, Aharoni A. 2007. The Arabidopsis DESPERADO/AtWBC11 transporter is required for cutin and wax secretion. Plant Physiol. 145:1345-1360.
Reddy A. R., Chaitanya K. V., Vivekanandan M. 2004 Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants Journal of Plant Physiology. 161: 1189–1202.
Rowland O, Zheng H, Hepworth SR, Lam P, Jetter R, Kunst L. 2006. CER4 encodes an alcohol-forming fatty acyl-Coenzyme A reductase involved in cuticular wax production in Arabidopsis. Plant Physiol. 142:866–77
Samuels L Kunst L Jetter R 2008. Sealing Plant Surfaces: CuticularWax Formation by Epidermal Cells Annu. Rev. Plant Biol. 59:683–707.
Zhang J-Y, Broeckling CD, Blancaflor EB, Sledge M, Sumner LW, Wang Z-Y. 2005. Overexpression of WXP1, a putative Medicago truncatula AP2 domain-containing transcription factor gene, increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa (Medicago sativa). Plant Journal. 42:689–707.
Zhang J-Y., Broeckling C D., Summer L W. 2007. Heterologous expression of two Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response in freezing tolerance” Plant Mol Biol 64: 265 – 278.
Zheng H, Rowland O, Kunst L. 2005. Disruptions of the Arabidopsis enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis. Plant Cell 17:1467–81.