Drought Stress and Preharvest Aflatoxin Contamination in Agricultural Commodity: Genetics, Genomics and Proteomics

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1 Journal of Integrative Plant Biology 2008, 50 (10): Invited Review Drought Stress and Preharvest Aflatoxin Contamination in Agricultural Commodity: Genetics, Genomics and Proteomics Baozhu Guo 1, Zhi-Yuan Chen 2, R. Dewey Lee 3 and Brian T. Scully 1 ( 1 Crop Protection and Management Research Unit, Agricultural Research Service, US Department of Agriculture, Tifton, Georgia 31793, USA; 2 Department of Plant Pathology and Crop Physiology, Louisiana State University, Baton Rouge, Louisiana 70803, USA; 3 Department of Crop and Soil Sciences, University of Georgia, Tifton, Georgia 31793, USA) Abstract Throughout the world, aflatoxin contamination is considered one of the most serious food safety issues concerning health. Chronic problems with preharvest aflatoxin contamination occur in the southern US, and are particularly troublesome in corn, peanut, cottonseed, and tree nuts. Drought stress is a major factor to contribute to preharvest aflatoxin contamination. Recent studies have demonstrated higher concentration of defense or stress-related proteins in corn kernels of resistant genotypes compared with susceptible genotypes, suggesting that preharvest field condition (drought or not drought) influences gene expression differently in different genotypes resulting in different levels of end products : PR(pathogenesis-related) proteins in the mature kernels. Because of the complexity of Aspergillus-plant interactions, better understanding of the mechanisms of genetic resistance will be needed using genomics and proteomics for crop improvement. Genetic improvement of crop resistance to drought stress is one component and will provide a good perspective on the efficacy of control strategy. Proteomic comparisons of corn kernel proteins between resistant or susceptible genotypes to Aspergillus flavus infection have identified stress-related proteins along with antifungal proteins as associated with kernel resistance. Gene expression studies in developing corn kernels are in agreement with the proteomic studies that defense-related genes could be upregulated or downregulated by abiotic stresses. Key words: abiotic stress; drought stress; host resistance; preharvest aflatoxin contamination. Guo B, Chen ZY, Lee RD, Scully BT (2008). Drought stress and preharvest aflatoxin contamination in agricultural commodity: Genetics, genomics and proteomics. J. Integr. Plant Biol. 50(10), Available online at Throughout the world, aflatoxin contamination is considered one of the most serious safety issues to health. Aflatoxins are well recognized as a cause of liver cancer, and are common contaminants of foods, particularly in the staple diets of many developing countries. This toxin is produced mainly by Aspergillus flavus Link ex. Fries and A. parasiticus Speare during infection of susceptible crops, such as corn, peanuts, cotton seeds and tree Received 14 Mar Accepted 19 Jun Author for correspondence. Tel: ; Fax: ; <Baozhu.Guo@ars.usda.gov>. C 2008 Institute of Botany, the Chinese Academy of Sciences doi: /j x nuts during production, harvest, storage, and food processing, and it is considered by the US Food and Drug Administration (FDA) to be an unavoidable contaminant of foods (Williams et al. 2004). Chronic problems with aflatoxin contamination occur in the southern US. The impact of aflatoxin contamination on the agricultural economy is especially devastating during drought years when aflatoxin affects the more northern areas including Midwestern corn belt regions. The realization of the unique nature of the aflatoxin problem and the need for novel technologies to ameliorate the impact of this problem became a focal point of discussion in 1988 at the first US Aflatoxin Elimination Workshop held in New Orleans, LA, USA. The Annual Aflatoxin Workshops (Table 1) have served as a forum to assemble USDA-ARS (Agricultural Research Service, US Department of Agriculture) scientists, university faculties, representatives of the different commodities and industries, and international participants in a

2 1282 Journal of Integrative Plant Biology Vol. 50 No Table 1. US Department of Agriculture (USDA) Annual Multicrop Aflatoxin Elimination Workshop ( ). Year Location Year Location 1988 New Orleans, LA 1998 St. Louis, MO 1989 Peoria, IL 1999 Atlanta, GA 1990 St. Louis, MO 2000 Yosemite, CA 1991 Atlanta, GA 2001 Phoenix, AZ 1992 Fresno, CA 2002 San Antonio, TX 1993 Little Rock, AR 2003 Savannah, GA 1994 St. Louis, MO 2004 Sacramento, CA 1995 Atlanta, GA 2005 Raleigh, NC 1996 Fresno, CA 2006 Ft. Worth, TX 1997 Memphis, TN 2007 Atlanta, GA unique cooperative effort to develop aflatoxin control strategies through research and development. Drought and hot weather conditions have been associated with increased aflatoxin contamination in the field (Payne 1998). In support of this role for drought and high temperatures, lower soil temperature was found to reduce aflatoxin contamination in peanut (Hill et al. 1983), while increased aflatoxin contamination has been observed in drought-treated peanuts with increased soil temperatures (Cole et al. 1985). Dorner et al. (1989) also concluded that a higher soil temperature favors A. flavus growth and aflatoxin production, and a study on the effect of drought on peanut resistance to A. flavus by Wotton and Strange (1987) found that fungal colonization was inversely related to water supply, as was aflatoxin production. Holbrook et al. (2000) evaluated resistance to preharvest aflatoxin contamination in a set of peanut genotypes that had been documented as having varying levels of drought tolerance, and concluded that tolerant genotypes also had greatly reduced aflatoxin contamination. The effect of drought stress on preharvest aflatoxin contamination in corn was also observed in the field studies (Guo et al. 2005a). Field studies demonstrate that reduction of drought stress by irrigation reduces aflatoxin contamination in general. Drought-tolerant corn varieties were also found to produce significantly less aflatoxins in the field under drought conditions compared with aflatoxin-resistant controls. The approach to enhance host resistance through conventional breeding has gained renewed attention following the discovery of natural resistance to A. flavus infection and aflatoxin production in corn (Widstrom et al. 1987; Campbell and White 1995; Brown et al. 1999; Guo et al. 2005b). Promising sources of resistant peanut germplasm have also been identified from a core germplasm collection, although resistance screening has proven to be a difficult task with this crop (Holbrook et al 1997; Burow et al. 2008). One approach to enhance host resistance is to pyramid insect and fungal resistance genes into commercial germplasm to reduce fungal infection caused by insect damages (Guo et al. 2000). To meet the challenge of preventing preharvest aflatoxin contamination, more detailed understanding of the expression and function of the genetic material in response to biotic/abiotic stresses will be needed. Moreover, the genes that control functions leading to plant reactions to environmental stress and fungal infection must be identified. In this review, we will discuss aflatoxin and health, factors that affect aflatoxin production, host genetic resistance and drought stress, proteomic and genomic tools used to study host resistance and genetic response to drought stress. Proteomic and genomic research will help identify and understand the function and control of genes to improve the desired traits. At the end of this review, we will briefly report a recent study on the effects of preharvest drought stress of developing kernels on gene expression and postharvest fungal infection (Wang et al. 2008), indicating that corn lines that differ in resistance or susceptibility respond differently to preharvest drought stress and result in different preharvest gene expression in developing kernels and postharvest Aspergillus infection in comparison with the same genotype under different preharvest treatments, drought stress or non-stress (irrigation). Aflatoxins, Food Safety, and Health Aflatoxin was first associated with an outbreak of Turkey X disease, which occurred in 1960 near London, England, and killed approximately poults (Blount 1961; Forgacs and Carll 1962). The cause of the disease was later associated with the feeding of peanut (groundnut) meal infested by A. flavus, and thus the toxins were named aflatoxin for the A. flavus toxin. Aflatoxins belong to a family of compounds with difuranocoumarins. Aflatoxins B 1,B 2, G 1, and G 2 (AFB 1, AFB 2,AFG 1, and AFG 2 ) are the four major aflatoxins based on their blue (B) or green (G) fluorescence under ultraviolet light and their relative mobility by thin-layer chromatography on silica gel. Aflatoxin M 1 is a hydroxylated derivative metabolized from aflatoxin B 1 by cows and secreted in milk (Van Egmond 1989). A. parasiticus produces G 1 and G 2 in addition to aflatoxins B 1 and B 2. Preharvest aflatoxin contamination can occur when A. flavus infects peanut pods, corn ears, and cotton bolls with insect or mechanical damage (or if tissues are not damaged). Postharvest aflatoxin contamination can be problematic if grain storage is poorly managed. Outbreaks of acute aflatoxicosis from contaminated food in humans have been documented in Kenya, India, Malaysia, and Thailand as reported by the Council for Agriculture Science and Technology (CAST 2003). For example, an outbreak of severe aflatoxicosis in humans occurred in more than 150 villages in western India in 1974 where 397 persons were affected and 108 persons died (Krishnamachari et al. 1975). The largest and most severe outbreak of acute aflatoxicosis documented worldwide occurred in Kenya during 2004

3 Drought Stress and Preharvest Aflatoxin Contamination 1283 and involved 317 cases and 125 deaths due to consumption of aflatoxin-contaminated corn (CDC 2004; Lewis et al. 2005). Low dose consumption of aflatoxin-contaminated food stuff causes chronic aflatoxicosis resulting in cancer, suppression of immunological responses, and other slow pathological conditions. The liver is the primary target organ by toxic and carcinogenic aflatoxins. Cytochrome P450 enzymes in the liver convert aflatoxins to the reactive 8,9-epoxide form, which is capable of binding to both DNA and proteins (Eaton and Groopman 1994). Aflatoxin B 1 -DNA adducts can result in the GC to TA transversions in the p53, a DNA-repair, tumor-suppressor gene, at codon 249. Inactivation of the p53 tumor suppressor gene leads to the development of primary liver cancer (Bressac et al. 1991; Hsu et al. 1991). Aflatoxin contamination also has a significant economic impact on worldwide agriculture. In the developing countries, food safety is a major problem where detection and decontamination policies are impractical. Due to food shortage in those countries, routine consumption of aflatoxin-contaminated food is widespread. A guideline of 20 ppb (parts per billion) aflatoxin in food or feed substrate is the maximum allowable limit imposed by the US FDA. The European Union has a maximum level of 2 ppb for aflatoxin B 1 and 4 ppb for total aflatoxins. The aflatoxincontaminated commodities are often destroyed if the aflatoxin content is higher than the mandated levels. This results in billions of dollars in yearly losses worldwide. In some parts of the southern US such as in the southeast peanut, southern cotton belt, and mid-south corn farming regions, severe outbreaks of aflatoxin contamination occur frequently and have resulted in enormous economic losses (Robens and Cardwell 2005). The major biochemical pathway steps have been determined and the chemical structures of aflatoxin intermediates have been characterized (Payne and Brown 1998; Yu 2004; Yu et al. 2005). At least 23 enzymatic reactions are involved in aflatoxin formation. No less than 15 structurally-defined aflatoxin intermediates have been identified in the aflatoxin biosynthetic pathway. Aflatoxins are synthesized from malonyl CoA, first with the formation of hexanoyl CoA, followed by formation of a decaketide anthraquinone (Minto and Townsend 1997). There are two fatty acid synthases (FAS) and a polyketide synthase (PKS) involved in the synthesis of the polyketide from acetyl CoA (Watanabe and Townsend 2002). Norsolorinic acid (NOR) is the first stable aflatoxin intermediate identified in the pathway (Bennett 1981). Aflatoxins are formed after a series of oxidation-reduction reactions. The generally accepted aflatoxin biosynthetic pathway scheme is: a hexanoyl CoA precursor norsolorinic acid, NOR averantin, AVN hydroxyaverantin, HAVN Oxoaverantin, OAVN averufin, AVF hydroxyversicolorone, HVN versiconal hemiacetal acetate, VHA versiconal, VAL versicolorin B, VERB versicolorin A, VERA demethyl-sterigmatocystin, DMST sterigmatocystin, ST O-methylsterigmatocystin, OMST aflatoxin B 1 and aflatoxin G 1. After the VHA step, there is a branch point in the pathway that leads to aflatoxins B 2 and G 2 formation. Factors Affecting Aflatoxin Biosynthesis and Preharvest Aflatoxin Contamination Biotic and abiotic factors, either nutritional or environmental, are known to affect aflatoxin production in toxigenic Aspergillus species and host plants, although the molecular mechanisms for these effects are still unclear (Payne and Brown 1998; Guo et al. 2005a,b). Nutritional factors such as carbon, nitrogen, amino acid, lipid, and trace elements affect aflatoxin production (Payne and Brown 1998; Cuero et al. 2003). Simple sugars such as glucose, sucrose, and maltose support aflatoxin formation, but peptone, sorbose, or lactose do not promote the toxin. Nitrogen source affects aflatoxin formation in varying ways and production levels are different when Aspergillus spp. is on a nitrate versus a nitrite medium. Recent studies using A. flavus show that tryptophan inhibits aflatoxin formation, whereas tyrosine spikes aflatoxin production (Wilkinson et al. 2007). Micronutrients (metal ions) were also reported to affect aflatoxin pathway gene expression (Cuero et al. 2003). Lipids have tremendous effects on aflatoxin formation, not only as a nutritive source but also as substrates metabolized for acyl- CoA starter units (Maggio-Hall et al. 2005) and as signaling molecules (Brodhagen and Keller 2006). Temperature, ph, water activity (drought stress) and other stresses are external environmental factors that can affect aflatoxin production (Cotty 1988; Payne and Brown 1998; Guo et al. 2005b; Sobolev et al. 2007). Studies suggest that aflr transcription is responsive to a G-protein signaling cascade that is mediated by protein kinase A (Hicks et al. 1997). Aflatoxin production is closely related to ph changes where biosynthesis in A. flavus occurs in acidic media, but is inhibited in alkaline media (Cotty 1988). The field environment is a logical place to search for clues that will assist in the identification of causes for poor plant health, reduced vigor, and other symptoms of abnormal development expressed by the growing plant. The major environmental influences will be discussed here individually, with the intent of assessing the relative importance of each, fully realizing that all are intricately interrelated in the imposition of stress on the plant, and in the development of preharvest aflatoxin contamination. High temperature and drought, which often occur together during the growing season and likely contribute to poor kernel development, have been reported to increase growth of the fungus and toxin production (Payne 1998). Irrigation not only relieved drought stress, but also reduced soil temperature. Increased aflatoxin contamination was observed in droughttreated peanuts with increased soil temperatures (Cole et al. 1985). McMillian et al. (1985) conducted a 6 year study in

4 1284 Journal of Integrative Plant Biology Vol. 50 No which the 3 years with the highest contamination also had the highest average daily temperatures during the growing season. Similarly, in a 5 year study, a significant positive correlation between aflatoxin contamination and temperatures was obtained only during the 2 years with exceptionally high concentrations of aflatoxin (Widstrom et al. 1990). Studies of aflatoxin and fumonisin contamination of corn grown under high or moderate heat stress (Abbas et al. 2002) demonstrate that heat stress also plays an important role in the susceptibility of corn to both aflatoxin and fumonisin contamination. Host Resistance, Genetics, and Preharvest Aflatoxin Contamination Various approaches have been suggested for genetic control of preharvest aflatoxin contamination including the development and use of crops with resistance to insects, resistance to plant stress (especially for tolerance to drought and high temperatures). Several sources of resistant germplasm were identified and released for crop genetic improvement (McMillian et al. 1993; Williams and Windham 2001; Guo et al. 2007a; Holbrook et al. 2008). Crop resistance to aflatoxin contamination may be achieved by the three strategies: (i) resistance to fungal invasion; (ii) inhibition of aflatoxin formation; and (iii) resistance to abiotic stress such as drought. Using a combination of genetic, genomic and proteomic approaches to elucidate crop defense mechanisms and their genetic regulation will significantly improve the efficiency of genetic breeding for better crop cultivars (Chen et al. 2004; Guo et al. 2005a; Guo et al. 2006). Recently, resistant lines have been developed from the US resistant maize population GT-MAS:gk as a result of repeated self-pollination by Guo et al. (2001, 2002, 2007a). This population was derived from a commercial hybrid ear (a Pioneer hybrid) visibly segregating for fungal infection by A. flavus and selected for resistance to the fungal infection and reduction of aflatoxin contamination (Widstrom et al. 1987). McMillian et al. (1993) released the maize population GT-MAS:gk as a source of resistance to aflatoxin accumulation. To use the resistance traits from GT-MAS:gk, such as physical pericarp wax (Guo et al. 1995, 1996; Russin et al. 1997) and antifungal proteins (Guo et al. 1997, 1998; Chen et al. 1998), efforts of self-pollination and selection have been made since 1996 for reduced aflatoxin contamination. By evaluating S1 families, Guo et al. (2001) demonstrated that considerable variation among the individual plants within the population GT-MAS:gk was detectable using random amplified polymorphic DNA (RAPD) markers and a laboratory aflatoxin bioassay. Guo et al. (2002) also evaluated the S5 generation using 113 restriction fragment length polymorphism (RFLP) probes for genetic variation and conducted 2 year field tests for aflatoxin contamination. The aflatoxin concentrations and maturity data among the S5 selfed lines were significantly different (Guo et al. 2002). Two inbred lines, GT601 (AM-1) (PI ) and GT602 (AM-2) (PI ), selected from GT-MAS:gk population have been released (Guo et al. 2007a). GT601 (AM-1) flowers about 1 week earlier than GT602 (AM-2). GT601 (AM-1) has a colorless pericarp, white cob, and browning silk, P-wwb; and GT602 (AM-2) has a colorless pericarp, red cob, and browning silk, P-wrb. GT601 (AM-1) had also been used in genetic quantitative trait locus (QTL) mapping studies for silk maysin production (Butrón et al. 2001) and A. flavus infection (Widstrom et al. 2003). Near isogenic lines with combined resistance traits from both the US resistant inbred lines and the Africa lines with resistance to ear rot diseases and aflatoxin accumulation have also been developed at the International Institute for Tropical Agriculture (IITA) (Menkir et al. 2006). Five elite tropical inbred lines have been crossed with four US maize lines with proven resistance to aflatoxin accumulation in Ibadan, Nigeria. These five Africa lines were selected for their resistance to ear rot caused by Aspergillus, Botrydiplodia, Diplodia, Fusarium, and Macropomina, and their potential resistance to aflatoxin accumulation (Brown et al. 2001; Menkir et al. 2006). The F1 crosses were backcrossed to their respective US inbred lines and selfpollinated thereafter. Sixty-four of the resulting S4 lines were screened in the laboratory, five pairs of the near isogenic lines were found to be significantly different in aflatoxin resistance (Chen et al. 2005). Proteomics of Corn Kernel Total Proteins and Host Resistance Brown et al. (1993) demonstrated the existence of a subpericarp resistance in maize kernels and that the expression of this resistance requires viable embryo, indicating that biochemical factors may play a major role in resistance. Guo et al. (1996, 1997) found pre-imbibition significantly increased aflatoxin resistance of susceptible maize genotypes. Further investigation revealed that susceptible genotypes were able to induce the same antifungal proteins as resistant lines upon fungal infection, but at a slower pace or at lower levels compared with resistant maize lines (Guo et al. 1997; Chen et al. 2001). This work also suggested that susceptible lines have the ability to induce an active defense mechanism if they were given enough time to imbibe water and induce antifungal proteins. A more rapid and stronger induction of the pathogenesis-related (pr1 and pr5) genes in maize leaves has also been observed in a resistant reaction when compared with a susceptible reaction upon pathogen infection (Morris et al. 1998). In another investigation, a 14kDa trypsin inhibitor (TI) protein was constitutively produced at high levels in resistant lines but at low levels or was missing in susceptible ones (Chen et al. 1998). This protein demonstrated antifungal activity against A. flavus and several other pathogenic fungi (Chen et al. 1999), possibly through inhibition of fungal

5 Drought Stress and Preharvest Aflatoxin Contamination 1285 α-amylase activity and production. This could limit the availability of simple sugars needed for fungal growth and aflatoxin production (Woloshuk et al. 1997). Further investigation found that both constitutive and inducible proteins are required for kernel resistance to A. flavus infection and aflatoxin production (Chen et al. 2001). It also showed that one major difference between resistant and susceptible genotypes is that resistant lines have higher constitutive levels of stress-related proteins, antifungal proteins, and highly-hydrophilic storage proteins compared with susceptible lines. Therefore, constitutive produced proteins have been the focus of a number of important investigations. Through proteome analysis, Chen et al. (2002) identified unique or elevated levels of maize kernel proteins in association with resistance to aflatoxin contamination, recovered these peptide spots from preparative 2-D gels and sequenced them using electronic spray ion/mass spectrometry (ESI-MS)/MS. These proteins can be grouped into three categories based on their peptide sequence homology: (i) storage proteins, such as globulin proteins (GLB1, GLB2), and late embryogenesis abundant proteins (LEA3, LEA14); (ii) stress-responsive proteins, such as aldose reductase (ALD), a glyoxalase I protein (GLX I), and a 16.9 kda heat shock protein; and (iii) antifungal proteins (TI and PR10). A proteomic analysis of domestic aflatoxin-resistant and susceptible corn lines identified several stress-related proteins and antifungal proteins (such as GLX I and PR-10) (Chen et al. 2002). High level expression of some stress-related proteins has been reported to not only confer stress-tolerance, but also enhance disease resistance. For example, the role of glyoxalase in stress-tolerance is highlighted in a study using transgenic tobacco plants overexpressing a Brassica juncea glyoxalase I (Veena et al. 1999). Chen et al. (2004) suggested a direct role for glyoxalase I in host resistance in corn against aflatoxin accumulation through removal of its aflatoxin-inducing substrate, methylglyoxal. Transgenic tobacco plants expression pr-10 showed increased inhibitory effort on A. flavus growth in vitro (Chen et al. 2006). Further studies using RNA interference (RNAi) confirmed the direct involvement of both glx I and pr-10 in host resistance (Chen et al. 2008). Genomics and Gene Expression Profiles Macroarray (nylon-based) and microarray (glass slide-based) screening methods allow the simultaneous determination of expression levels of thousands of genes, making it possible to obtain a global view of the transcriptional state in a cell or tissue, and to associate genes with functions or specific physiological conditions. Recently, microarray technology has been demonstrated in a small scale pilot study of peanut expression in response to drought stress and Aspergillus infection in peanuts (Luo et al. 2005a, 2005c). Expressed sequence tag (EST)-derived cdna microarrays of 400 unigenes were probed under different conditions. Luo et al. (2005c) identified 25 ESTs that were potentially associated with drought stress or that responded to A. parasiticus challenge, and further study is warranted. Likewise, 56 upregulated transcripts were identified and confirmed by real-time polymerase chain reaction (PCR) upon infection with Cercosporidium personatum (Luo et al. 2005b). A long oligonucleotide microarray consisting of more gene-elements is under development for peanuts (Guo et al. 2007b). In corn, the inbred line Tex6 has been used in a gene expression profile study to compare drought stress with irrigation (B Guo et al., unpubl. data, 2008). Gene expression profiles were analyzed across a temporal sampling interval of 25, 30, 35, 40, and 45 d after pollination (DAP) under both suboptimal and optimal irrigation regimens using the 70-mer maize oligonucleotide arrays (version 1.2, 58 k) from the Maize Oligonucleotide Array Project. These results indicate that 35 to 45 DAP was a unique time-point when the developing kernels entered a distinct phase related to disease defense. The most responding genes to drought stress appeared at the 35 DAP and the 40 DAP (Figure 1). Several defense genes have been downregulated, which might play more important roles in Aspergillus resistance in the later stage of kernel development. The difference in aflatoxin contamination in preharvest kernels between the resistant and the susceptible lines not only depends on genetic aspect (constitutive), but also on the response aspect (induced) of stress or defense-related genes to drought stress. Preliminary study of diverse inbreds (Figure 2) suggests that preharvest drought stress could affect postharvest host- A. flavus interaction, and different genetic genotypes respond to drought stress in the field differently and result in different levels of stress-induced anti-fungal or anti-stress proteins, which in turn affect postharvest response to A. flavus infection or Number of selected gene Up-regulated gene Down-regulated gene D after pollination Figure 1. Numbers of up-regulated and down-regulated genes in Tex6 under drought stress using maize oligo microarray. Up-regulated genes: log 2 ratio>1; down-regulated genes: log 2 ratio <1; ratio: normalized spot intensity under drought stress/normalized spot intensity under normal conditions.

6 1286 Journal of Integrative Plant Biology Vol. 50 No Figure 2. Aspergillus flavus sporulation ratings (%) using laboratory kernel screening assay of diverse inbreds grown under drought stress or irrigation in the field and the effects on postharvest host-fungal interaction. Fungal sporulation was rated as follows: 0, mycelium visible on kernel surface but no sporulation; 1, 1% 20%; 2, 21% 40%; 3, 41% 60%; 4, 61% 80%; and 5, 81% 100%; each with six replications. colonization (B Guo et al., unpubl. data, 2008). Research is in progress to focus on oligo-macroarray profiling of developing corn kernel gene expression under drought stress (Wang et al. 2008) using the genes identified in microarray studies (Figure 1). Two sites of genes have been synthesized, including 211 and genes related to drought stress and defense, respectively. These genes have been spotted on membranes as macroarray for corn germplasm assessment and evaluation.

7 Drought Stress and Preharvest Aflatoxin Contamination 1287 Functions of Storage and Stress-related Proteins Storage and stress-related proteins may play important roles in enhancing stress tolerance of host plants. The expression of storage protein GLB1 and LEA3 has been reported to be stressresponsive and abscisic acid (ABA)-dependant (Thomann et al. 1992). Transgenic rice overexpressing a barley LEA3 protein HVA1 showed significantly increased tolerance to water deficit and salinity (Xu et al. 1996). The role of GLX I (glyoxalase I) in stress-tolerance was first highlighted in an earlier study using transgenic tobacco plants overexpressing a Brassica juncea glyoxalase I (Veena et al. 1999). GLX I may play an important role in maize resistance to aflatoxin accumulation by reducing the levels of methylglyoxal, which was shown to induce aflatoxin production in vitro (Chen et al. 2004). PER1, a 1-cys peroxiredoxin antioxidant identified in corn (Chen et al. 2007), was demonstrated to be an abundant peroxidase, and may play a role in the removal of reactive oxygen species (ROS). The PR10 expression during kernel development was induced in the resistant line GT-MAS:gk, but not in susceptible Mo17 in response to fungal inoculation (Chen et al. 2006). Members of the LEA genes family have been associated with plant responses to many different stresses including drought, salt, cold, heat, and wounding (Thomann et al. 1992). Transgenic expression of an LEA protein from barley demonstrated increased tolerance to water and salt stress in rice (Xu et al. 1996). Aside from heat stress, HSP (heat shock protein) are also induced by other stresses such as cold, drought, or salinity (Sabehat et al. 1998). The role of glyoxalase in stress tolerance is also highlighted in a recent study using transgenic tobacco plants overexpressing a Brassica juncea glyoxalase I (Veena et al. 1999). Further investigation suggests a direct role for GLX I in corn resistance against aflatoxin accumulation through the removal of its aflatoxin inducing substrate, methyglyoxal (Chen et al. 2004). An increasing body of evidence suggests that a subset of plant responses to biotic and abiotic stress is shared, such as the generation of ROS, the activation of mitogen-activated protein kinases (MAPKs), and hormone modulations. ROS production is recognized as a common event in plant response to biotic and abiotic stresses (Mithöfer et al. 2004). The mechanism of how ROS leads to downstream responses is still not clear; however, the requirement of specific MAPKs has been implicated (Kovtun et al. 2000), possibly mediated through a serine/threonine kinase (OXI1) in Arabidopsis (Rentel et al. 2004). The activity of this kinase was induced in vivo by H 2 O 2 and its expression was upregulated by a wide range of H 2 O 2 -generating stimuli (Rentel et al. 2004), suggesting that this kinase is an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses. All plant MAPKs have a Thr Glu Tyr activation motif, except members of subfamily V, where Glu is replaced by Asp (Zhang and Klessig 2001). One of the mechanisms by which different stimuli converge onto one MAPK is believed to involve several unrelated kinases that function as MAPKKKs to initiate the MAPK cascade (Widmann et al. 1999). Based on the homology of the kinase domain, several plant kinases have been identified as MAPKKKs, including EDR1 and NPK1/ANPs (Zhang and Klessig 2001). A recent review by Hammond-Kosacky and Parkerz (2003) provides a comprehensive list of MAPKs identified from different plant-pathogen systems. Studies of transcriptional activation of some stress responsive genes have also led to the identification of cis-acting elements ABRE (ABA responsive element) and DRE (dehydration responsive element)/crt (C-repeat) that function in ABA-dependent and ABA-independent gene expression in response to stress, respectively (Seki et al. 2003). Transcription factors belonging to the ethylene-responsive element binding factor family that bind to DRE/CRT were also isolated (Liu et al. 1998). Similar transcription factors (DREB2A and DREB2B) are also induced by dehydration and promote the expression of various genes involved in drought stress tolerance (Liu et al. 1998). The expression of a new DNA-binding protein DBF1 that specifically interacts with the DRE2 cis-element of a corn rab17 gene promoter is induced by ABA, dehydration and high salinity (Kizis and Pages 2002). A variety of plant hormones, including salicylic acid (SA), jasmonate (JA), ethylene (ET), and abscisic acid, have been implicated in mediating responses to a wide range of biotic and abiotic stresses (Audenaert et al. 2002; Diaz et al. 2002). The roles of these hormones are dependent on the particular host pathogen interaction. On the basis of the interactions that have been studied, a general rule for hormonal action has been proposed in which resistant responses to biotrophs require SA, whereas responses to necrotrophs require JA and ET (Feys and Parker 2000). In tomato, ET, JA, and SA all independently contribute to its resistance to Botrytis cinerea (Diaz et al. 2002). Also in tomato, the host plant actively regulates the Xanthomonas campestris pv vesicatoria-induced disease response via the sequential action of at least three hormones (JA, ET, and SA), which promote expansive cell death of its own tissue (O Donnell et al. 2003). Further, the effect of phytohormones is also regulated by other factors. For example, the MAPK kinase kinase, EDR1, negatively regulates SA-inducible defenses (Frye et al. 2001), whereas MAPK 4 appears to differentially regulate SA and JA signals (Petersen et al. 2000). These findings also suggest that MAPK modulates cross-talk between different plant defense pathways (Hammond-Kosacky and Parkerz 2003). Concluding Remarks To reduce and eliminate aflatoxin contamination in preharvest field crops and postharvest grains is a serious challenge facing scientists today (Yu et al. 2008). Research efforts to understand

8 1288 Journal of Integrative Plant Biology Vol. 50 No host resistance mechanisms to A. flavus infection and aflatoxin contamination in the past indicated that maize kernel proteins, especially stress-related proteins and antifungal proteins play a role in host resistance. Enhancing the expression of these proteins can be an effective approach to improve genetic resistance to abiotic stress such as drought and reduce preharvest aflatoxin contamination in susceptible crops. Evidence from field studies, from proteomic comparisons of differences between resistant and susceptible corn genotypes, from gene expression analysis of plants in response to biotic and abiotic stresses, and from examination of signal transduction components involved in biotic and abiotic stress responses indicates the existence of an association between stress tolerance and disease resistance. 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