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Abstract Global losses in agricultural production due to abiotic stresses have been estimated to be $120 billion. In the wake of shrinking arable land and rampant changes in climate, a second green revolution is important to meet the food needs of the rapidly growing population. This warrants radical changes in research strategies. Genomics approaches such as transcriptome profiling have led to the identification of gene networks important for many different stresses. However, under natural conditions plants are challenged by simultaneous occurrence of two or more stresses. Several studies have recently analyzed the transcriptional responses to two stresses simultaneously and are discussed here. For plant biotechnologies to deliver the promise of a second green revolution, a systems biology approach of examining multiple stresses at various developmental stages is necessary.

21.1 Introduction

Food security is a major issue in the global policy agenda. One of the targets of United Nations Millennium Development goal adopted in 2000 is to halve the proportion of people suffering from hunger by the year 2015 (The World Bank Group 2003). Meeting this food security goal is going to be a major challenge (Rosegrant and Cline 2003). In the next 40 years, demand for cereal production is predicted to increase by 60% as the population rises from the current 6.6 billion to 8.7 billion by the year 2050 (Bengtsson et al. 2006). In a world where population growth exceeds food supply (Malthus 1817), a second green revolution is necessary. Opportunities for plant biotechnologies to contribute to the second green revolution have been widely recognized (Fedoroff and Brown 2004).

N. Jambunathan, M. Puckette, and R. Mahalingam () Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, 74078, USAe-mail: ramamurthy.mahalingam@okstate.edu

Chapter 21Transcriptomic Analysis of Multiple Enviornmental Stresses in Plants

Niranjani Jambunathan, Michael Puckette, and Ramamurthy Mahalingam

S.M. Jain and D.S. Brar (eds.), Molecular Techniques in Crop Improvement,DOI 10.1007/978-90-481-2967-6_21, © Springer Science+Business Media B.V. 2010

512 N. Jambunathan et al.

Three major yield-increasing strategies have been suggested – (1) Increasing harvest index [ratio of grain to total crop biomass], (2) increasing plant biomass, and (3) improving stress tolerance (Cassman 1999; Evans 1998). Nearly 82% of the potential yield of major crop losses occur every year due to abiotic stresses (Boyer 1982). Hence we consider improving stress tolerance in plants as the most impor-tant yield-increasing strategy. Further improving tolerance to stresses can also improve the harvest index, as well as biomass. Among the abiotic stresses, water deprivation or drought account for more than 50% of the estimated losses (Araus et al. 2002; Boyer 1982) and according to recent estimates accounts for more than $120 billion annually (Dhalmini et al. 2005). Plant water status is a central compo-nent of several abiotic stresses including drought, freezing and salinity (Verslues et al. 2006). In the wake of a global scarcity of water resources, drought stress is undoubtedly the major abiotic factor detrimental to agriculture.

The moisture status prevailing in soil environment in turn depends on the ambient environmental conditions such as temperature, light, humidity. Recent reports indicate that increase in global surface temperature is a major indicator of global warming (Van Vuuren et al. 2008). This raise in mean global temperature is attributed to increases in the greenhouse gases such as carbon dioxide and air pollutants such as ozone that has been brought about by anthropogenic activities. Thus plants not only have to deal with scanty water resources but may also have to deal with increasing temperatures, carbon dioxide and ozone. Even more worrisome is the fact some of these environmental variables can occur simultaneously, or successively, or in different times within a growing season. This has been elegantly illustrated in the form of a stress matrix depicting the combinations of agriculturally important biotic and abiotic stresses taking into consideration two different stresses at a time (Mittler 2006).

21.2 Physiological Studies of Combined Stresses

Practical agriculturists have long known that in field conditions plants face several distinct abiotic stresses either simultaneously or at different times during the grow-ing season (Tester and Bacic 2005). Indeed breeding for tolerance to a combina-tion of abiotic stresses is a common strategy for developing new crop varieties. Physiological studies of simultaneous exposure to different stresses in different plant species have been reported. Interactive effects of drought and heat shock stress represent the conditions encountered by plants growing in arid regions (Mittler et al. 2001; Moffat 2002). Analysis of these two stresses in Arabidopsis and tobacco plants showed that combination of heat and drought affected plants differently from drought or heat stress applied individually with reference to photosynthesis, respiration, stomatal conductance and leaf temperature (Rizhsky et al. 2002, 2004). In a study on sorghum, a combination of heat and drought stress during late stages of development [booting and flowering] caused nearly 87% yield reduction while the same stress treatment during vegetative stages had no impact

51321 Transcriptomic Analysis of Multiple Enviornmental Stresses in Plants

on yields (Craufurd et al. 1993). In another study, a combination of ozone and drought stress in two Norway spruce clones had varying effects on biomass depending on the vigour of the clone. In the faster growing clone, ozone reduced the biomass but also protected the plants from drought stress (Karlsson et al. 1997). A combina-tion of high light and ozone in Phaseolus vulgaris was reported to exacerbate the detrimental effects of the pollutant on photosynthesis (Guidi et al. 2000). Modelling studies on the interactive effects of drought and ozone using yield forecasting pre-dicted a general drought-induced reduction of crop sensitivity to ozone and it varied between crops, regions and years (King 1988). These physiological stud-ies clearly illustrate that the abiotic stress combinations represent a new stress scenario and is not just a sum of the two independent stresses. Hence researchers working on improving stress tolerance in plants need to expand their areas of study to include stress combinations.

21.3 Molecular Genetics and Genomics

It is well known that adaptive responses to environmental stresses in plants are coordinated by molecular networks (Vinocur and Altman 2005). These networks help in re-establishing homeostasis and repair damaged proteins and membranes (Wang et al. 2003). In contrast to plant resistance to biotic stresses, which is mostly monogenic, abiotic stresses are multigenic traits and hence pose a formidable challenge for engineering resistance. The popular candidate gene-by-gene approach using genes from signalling and regulatory pathways (Seki et al. 2003; Shinozaki et al. 2003), proteins conferring stress tolerance (Wang and Huang 2004), enzymes leading to production of structural and functional metabolites (Park et al. 2004; Rontein et al. 2002) have met with some success in improving stress tolerance (Fig. 21.1). However many of these studies were short-term treatments considering a single stress and may not represent stress tolerance of crops in field conditions (Vinocur and Altman 2005).

Recently, the genomics approaches have radically changed the view about engineering stress tolerance in plants. The ability to investigate changes in thou-sands of genes simultaneously using microarray technology has led to a flurry of papers in both model systems as well as crop plants, in response to several different stresses (Kreps et al. 2002; Mahalingam et al. 2005, 2006; Puckette et al. 2008; Rossel et al. 2002; Seki et al. 2001). This is based on the premise that the set of genes that are expressed in a cell determines the state of the cell, how it is built, what it is capable of doing and perhaps what it is not capable of doing. There is a tight link between the function of a gene product and its expression pattern. Each gene is expressed in specific cells under specific conditions, and this regulation of when, where, and how much is fine-tuned by the process of natural selection. Promoters of genes operate as molecular switches by regulating transcription of specific genes in response to input information from external factors and internal milieu of a cell. These features of regulation of gene expression at the level of

514 N. Jambunathan et al.

transcript abundance have been attributed to the wide popularity of microarray expression profiling (Brown and Botstein 1999).

Multiprotein Bridging Factor 1c (MBF1c) a common transcriptional coactivator recruits transcriptional machinery to transcription factor bound promoter (Naar et al. 2001). Based on the knowledge that transcription factors play a crucial role in regulating stress responses, MBF1c over-expressing plants were shown to give improved tolerance to environmental stresses (Suzuki et al. 2005).

The analysis of transcriptomes at various times following the stress treatment provides useful information about the temporal progression of thousands of genes.

Fig 21.1 A general signal transduction pathway in response to abiotic stress in plants. Perturbations in the external environment leads to rapid changes in redox, osmotic and ionic bal-ance in the plant cells. These changes are sensed by redox and osmosensors in conjunction with second messengers such as Calcium, ROS, RNS and phytohormones. These changes lead to acti-vation of specific TFs which inturn leads to activating response genes that ultimately leads to stress tolerance or resistance. Inadequate responses at one or more steps in the signaling and gene activation process will result in irreversible changes in cellular homeostasis, lead to membrane and protein damage and ultimately cell death (Modified from Vinocur and Altman, 2005)

Peroxiredoxins, Osmosensors (AtHK1), Phospholipid-cleaving enzymes(PLD), Second messengers (Ca2+, PtdOH, ROS),MAP kinases, Ca2+ sensors (SOS3), calcium dependentProtein kinases (CDPKs), Hormones

Transcription factors (WRKY, CBF/DREB, ABFHSF, bZIP, MYC/MYB) MBF1c

Gene activation

Chaperone functions(HSP, SP1, LEA, COR)

Water & ion movement(aquaporin, ion transporter,SLAC1)

Detoxification(GSTs, SODs, PXs

Osmoprotection (proline,GlyBet, Sugar polyols)

Stress tolerance or resistance

Re-establishment of cellular homeostasis, functionaland structural protection of proteins and membranes

Signal sensing, perceptionand transduction

Transcriptionalcontrol

Stress-responsivemechanisms

DroughtColdSalinityHeatChemical pollution

Secondary stress

Osmotic stress

Oxidative stressChanges in redox, osmotic and ionic homeostasisDamage to membrane, functional, structural proteins

Abiotic Stresses

51521 Transcriptomic Analysis of Multiple Enviornmental Stresses in Plants

In fact compendium of microarray data of entire Arabidopsis transcriptome in response to several stresses is easily accessible by programs like Genevestigator (Zimmermann et al. 2004). Using this compendium approach, a set of Multiple Stress Genes (MSTs) was identified in Arabidopsis (Kant et al. 2008). A subset of these genes belonging to the gene ontology category of transcription regulation was described as Multiple Stress Regulatory Genes (MSTR). Mutational analysis of several of the genes identified by this strategy confirmed the ability of these MSTR genes to confer resistance to multiple stresses (Kant et al. 2008). These studies again are examining a single stress at a time and suffer from the short-term nature of the treatments. As pointed out earlier, simultaneous occurrences of stresses is a common phenomenon in field conditions. Below we discuss several studies examining the transcriptomes following combined application of two stresses that are more likely to occur in realistic field conditions.

21.4 Analysis of Combined Stresses

21.4.1 Heat and High Light

Heat or high temperature stress adversely affect several physiological processes including photosystem II activity (Havaux et al. 1991), carbohydrate partitioning (Lafta and Lorenzen 1995), reactive oxygen metabolism (Larkindale et al. 2005), membrane fluidity (Kim and Portis 2005). Heat stress can also hamper growth processes (Beator and Kloppstech 1992; Cheikh and Jones 1994; Pressman et al. 2002; Wallwork et al. 1998). Excess light can be detrimental to photosynthesis, and lead to excess ROS (Niyogi 1999). Under natural conditions, a combination of high temperatures and high light are more likely to occur. Suppression subtraction hybridization technique was used to identify genes altered in response to combined heat and light stress in Arabidopsis (Nishizawa et al. 2006). Of the 76 genes identified in this study, was a heat shock transcription factor HsfA2. Constitutive overexpression of this gene led to enhanced resistance to combined heat, light and methyl viologen stress (Nishizawa et al. 2006).

In sunflower plants transcriptome analysis following high light, high tempera-ture, and a combination stress showed that 105, 55 and 129 cDNAs were showing significant changes in gene expression, respectively (Hewezi et al. 2008). Significant differences were reported in the vegetative and storage tissues in com-bined stress treatment for the genes involved in photochemical reactions, carbon dioxide acquisition, pyruvate accumulation, lipid degradation, defense responses, protein folding and transcriptional gene regulation (Hewezi et al. 2008). This study also showed that the combined stress of heat and light induced the largest number of genes that were not altered when the stresses were applied singly (Fig. 21.2a). This study supports the notion that the combined heat and light stress represents a new type of stress.

516 N. Jambunathan et al.

21.4.2 Drought and Heat

Simultaneous drought and heat stress makes a case for a model combination stress. In a meta-analysis of all major US weather disasters between 1980 and 2004, it was reported that a combination of drought and heat led to loss of more than $120 billions in the United States alone (Mittler 2006). Drought and heat stress combination has been well studied in several crop plants and grasses (Craufurd et al. 1993; Heyne and Brunson 1940; Jagtap et al. 1998; Jiang and Huang 2001; Perdomo et al. 1996; Savage and Jacobson 1935; Savin and Nicolas 1996; Wang and Huang 2004). Physiological studies indicated that starch breakdown coupled with energy production in the mitochondria play a key role in plant metabolism during combined drought and heat stress (Rizhsky et al. 2002, 2004). Transcriptome analysis of drought, heat and combination of drought and heat indicated that the stress combination required more than 770 transcripts for acclimation and these were not altered by drought or heat stress (Rizhsky et al. 2004) (Fig. 21.2b).

65

24

64 1

733

High light

High light + Heat

Heat

23

a

Beetles Ozone + CO2

1126

11

2076

272 1315

Beetles + CO2 + ozone

3062

15

e

460 98

601

1176

900 378

Drought

Drought + Ozone

Ozone

840

c

76 57

213

202

18 394

Virus

Virus + Ozone

Ozone

231

d

765

77

208

729 255

Drought

Drought + Heat

Heat

772

b

Fig. 21.2 Transcriptomic analysis of various combined stresses in plants. Figures shown here is summarized from data in a Hewezi et al., 2008; b Rizhsky et al., 2004; c Jambunathan et al., 2008; d Bilgin et al., 2008; e Casteel et al., 2008. Numbers in bold represent number of transcripts dif-ferentially expressed only in the combined stresses

51721 Transcriptomic Analysis of Multiple Enviornmental Stresses in Plants

21.4.3 Drought and Ozone

Ozone the most abundant air pollutant reduces plant biomass (Heagle 1989) by affecting allocation of assimilates and induces senescence process in plants (Miller et al. 1999; Pell et al. 1997). Highest ozone concentrations usually occur around mid-day and during summer season (Lorenzini et al. 1994). Field experiments on com-bined ozone and drought in Norway spruce revealed that ozone affected growth and biomass very differently in two different clones of this tree (Karlsson et al. 1997).

Since the flux of ozone into the plant is through the stomata, it has been argued that differences in sensitivity of plants to this pollutant are partly due to differences in stomatal conductance (Reich 1987). This dogma was challenged by elegant patch-clamping experi-ments to demonstrate that ozone directly affected guard cells by targeting inward potas-sium channels, which inhibited stomatal opening (Torsethaugen et al. 1999). Based on the ozone uptake models, drought induced stomatal closure will limit the ozone uptake into the leaves and hence protect the plants from ozone stress (Grunehage and Jager 2003; Panek and Goldstein 2001; Panek et al. 2002). However, this simplistic model has been confronted by the observation that ozone can cause stomatal “sluggishness” that lead to incomplete closure of the stomata and hence exacerbate the effects of drought (Grulke et al. 2003, 2005; Karnosky et al. 2005). The presence of nighttime ozone in rural loca-tions (McCurdy 1994) can also greatly reduce the biomass of plants (Winner et al. 1989), especially if ozone can hinder the opening of the guard cells after a dark exposure (Torsethaugen et al. 1999). These studies show that impact of ozone and water deprivation together on plants is complex and merits attention given that concentrations of ozone in the troposphere will continue to increase in the future (Ashmore 2005).

In the model legume Medicago truncatula we have identified an accession JE154, which is resistant to a combination of chronic ozone and drought stress (Puckette et al. 2007). In the same study we also identified that the popular commercial cultivar Jemalong is sensitive to ozone and drought stress singly and in combination. Temporal transcriptome analysis in response to ozone in these two lines showed more than 2,000 genes were differentially expressed (Puckette et al. 2008). A striking finding was the rapid transcriptional reprogramming mounted by the resistant JE154 in stark contrast to the delayed response in sensitive Jemalong (Puckette et al. 2008). Recently we have examined the combination of chronic ozone and water deprivation stress in Jemalong using the Affymetrix gene chip technology. This analysis revealed that a combination of ozone and water stress simultaneously evoked a massive transcriptional reprogramming when compared with ozone or drought stress alone (Jambunathan et al. 2008) (Fig. 21.2c).

21.4.4 Carbon Dioxide and Ozone

Rising atmospheric [CO2] and [O

3] have been recognized as important to crop

production for only the last 30 or 40 years. The [CO2] levels are reported to increase

from the current levels of 380 umol mol−1 to nearly 550 umol. mol−1 by the year

518 N. Jambunathan et al.

2050 (IPCC 2007). Tropospheric O3 levels in industrialized countries are currently

60 nmol. mol-1 and is predicted to increase by another 20% by 2050 (IPCC 2007). The most important concern related to improving plant tolerance to global climate change factors is that these are complex quantitative traits and hence are more difficult to control and engineer. To make the matters worse, ozone concentration is spatially and temporally heterogeneous owing to its short life and the fact its synthesis is dependent on the abundance of its pollutant precursors, sunlight and water vapour. The physiological consequences of elevated CO

2 and O

3 have been

succinctly discussed in a recent review (Ainsworth et al. 2008).Transcriptome analysis in aspen trees following long-term exposure to elevated

CO2 and tropospheric ozone, revealed that 238 genes were consistently differen-

tially expressed in years 2001 and 2002 (Gupta et al. 2005). In the elevated CO2

treatment, small number of genes was altered while a combination of both gases resulted in maximum number of changes in transcript levels. Transcripts related to photosynthesis were up regulated in the CO

2 treatment and was in support of the

physiological studies showing elevated photosynthetic rates in these Aspen clones (Noormets et al. 2001). The elevated expression of xyloglucan endotransglycosylase responsible for cell wall loosening and wall expansion (Fry et al. 2002) may contribute to larger leaf size in response to increased CO

2 (Ferris et al. 2001).

Downregulation of drought-induced aquaporin gene was taken as evidence for better water management in elevated CO

2 conditions. Elevated ozone alone leads to the

down regulation of photosynthesis related genes and has been reported in several plant species (Mahalingam et al. 2005, 2006; Puckette et al. 2008). Under the com-bined treatment, a number of photosynthesis related genes were down regulated indicating that elevated CO

2 was not able to ameliorate the effects of elevated O

3.

Again, the combined treatment of the two global climate change gases led to differential expression of a larger number of transcripts compared with either of them singly (Gupta et al. 2005).

21.4.5 Ozone and Biotic Stress Interactions

Ozone enters the plant through the stomata and are rapidly broken down into vari-ous reactive oxygen species at the apoplast (Kangasjarvi et al. 2005). By mimicking the oxidative burst that is observed in response to avirulent pathogens, plants sensi-tive to ozone develop hypersensitive response like localized lesions (Puckette et al. 2007; Rao and Davis 2001; Wohlgemuth et al. 2002). The ozone-induced lesions are regulated by the phytohormones such as salicylic acid, jasmonic acid, ethylene and abscisic acid (Ahlfors et al. 2004; Kangasjarvi et al. 2005; Overmyer et al. 2003; Rao et al. 2000, 2002). The changes in phytohormone levels in conjunction with the ROS levels leads to massive reprogramming of the transcriptome in response to elevated ozone (Li et al. 2006; Mahalingam et al. 2005, 2006; Puckette et al. 2008; Tosti et al. 2006). Some of the commonly induced transcripts in differ-ent plant species in response to elevated ozone include those belonging to the

51921 Transcriptomic Analysis of Multiple Enviornmental Stresses in Plants

category of stress/defense signaling. This begs the question of what happens to plant pathogen interactions in the wake of elevated ozone? In a study of soybean mosaic virus (SMV) in the presence of elevated levels of ozone it was reported that increased level of this pollutant reduced the systemic spread of the virus and reduced the overall disease development (Bilgin et al. 2008). This was attributed to the non-specific defence response caused by elevated levels of pathogenesis-related genes such PR-1, PR-5, PR-10 and EDS-1 and genes involved in flavonoid biosyn-thesis pathways. Overall the combined ozone and virus stress led to differential expression of larger number of transcripts compared to ozone or virus alone as the stresses progressed for a longer time (Fig. 21.2d).

21.5 Future Directions

From the above studies on examining two stresses jointly it is very clear that the combined stresses represents a new scenario to the plant and is simply not the sum of the two stresses. Secondly, large-scale transcriptomic studies have shown that in every case analysed to date, the combined stress treatments evoked the differential expression of larger number of transcripts compared to stresses applied singly. This again ratifies the observation that plants perceive combined stresses as something different. In the wake of these studies it is clear that genomics based approaches to improving stress tolerance in plants should move towards the next level of complexity. This entails analysis of multiple stresses simultaneously to simulate the conditions that usually operate in the field conditions. In fact, such a study has recently been reported wherein the impact of elevated CO

2 and elevated O

3 together

was analysed for their impact on the resistance of soybean plants to the Japanese beetles (Casteel et al. 2008) (Fig. 21.2e). The combination of increased ozone and CO

2 along with the beetles, altered the transcript levels of more than 3,000 genes,

elevated ozone and CO2 changed the levels of 2,000 genes, while the beetle damage

singly altered 1,100 genes (Casteel et al. 2008). Large scale transcriptomics data certainly provide a snap shot of the changes in the steady-state mRNA levels. Recent studies have indicated that post-transcriptional gene regulation plays a crucial role in plant stress signalling (Liu et al. 2008; Shukla et al. 2008; Sunkar et al. 2007). The dawn of the genomics era has led to emphasis on integrated analy-sis of stress response in whole plants tying together physiological and phenotypic observations with information on gene complement, transcript changes, protein complexes and metabolite levels (Bohnert et al. 2006). Combining transcriptomics of cell-and development stage specific profiling in roots in response to abiotic stress, it was elegantly demonstrated that transcriptional state of a cell is predomi-nantly a reaction to environmental conditions regulated by a core set of genes that determines the cell identity (Dinneny et al. 2008). Thus it is apparent that to engineer tolerance to multiple traits that operate under realistic field conditions, plant molecular biologists and plant biotechnologists have to embrace the systems biology approaches to integrate data from multiple platforms.

520 N. Jambunathan et al.

Acknowledgements The authors thank Dr. Yuhong Tang, Samuel Roberts Noble Foundation for her assistance with the microarray analysis of combined ozone and drought stress in Medicago truncatula. Research in R.M. laboratory was supported by funding from USDA-NRI grant no. 2007-02635-0210696 and Oklahoma Agricultural Experiment Station.

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