To evaluate the combined effects of 2x[CO2] and -H2O on the maize defense response, we analyzed the concentration of major phytohormones in stem tissues of -Fv and +Fv plants. Consis tent with +H2O maize, the concentration of JA was induced 2 d post +Fv in maize at 1x [CO2]-H2O but not at 2x[CO2]-H2O . However, there was no significant difference between the JA concentration of 1x[CO2]-H2O+Fv and 2x[CO2]-H2O+Fv plants. SA concentration displayed no significant difference among treatments with–H2O , but in comparison to +H2O plants, -H2O plants had significantly lower levels of both JA and SA 2 d post inoculation. Furthermore, SA levels were not reduced with +Fv under conditions of–H2O as observed with +H2O. In contrast to the other phytohormones, ABA levels increased with drought and was highest in plants at 1x[CO2]-H2O . Both [CO2] and [CO2]xH2O were significant factors contributing to differences in ABA concentrations presumably due to the variable level of drought stress in plants at 1x[CO2] and 2x [CO2], which is consistent with the amelioration of drought stress at 2x[CO2]. Additionally, Fv inoculation stimulated the production of ABA at both [CO2] suggesting that 2x[CO2] does not dampen the induction of ABA. In order to further assess the combined effects of elevated [CO2] and drought on the initial burst of JA and SA, phytohormone concentrations were analyzed at several time points over a 1 h time course immediately following +Fv. In combination with -H2O, 2x[CO2] still dampened the early induction of JA . The greatest difference of JA between 1x[CO2]-H2O+Fv and 2x[CO2]-H2O+Fv was observed at the 15 min time point when the concentration of JA in 1x [CO2]-H2O+Fv was double that of plants at 2x[CO2]-H2O+Fv .

The induction of JA was more rapid and JA concentrations reached higher levels in -H2O as compared to +H2O plants.The concentration of SA in plants at 2x[CO2]-H2O was significantly less than 1x[CO2]- H2O at both the 30 min and 60 min time point . At the time points evaluated,plastic planters wholesale -H2O did not appear to significantly influence SA levels when compared to +H2O at the same [CO2] .The even greater susceptibility of maize to Fv proliferation at 2x[CO2]-H2O is consistent with a weaker response of important phytochemicals involved in resistance. Therefore, to evaluate the downstream impact of abiotic stress induced changes in phytohormone signaling, the concentrations of major maize defense metabolites were analyzed and compared.Although the biologically inactive 2– beta-D-glucopyranose and 2–beta-D-glucopyranose are synthesized during development and are primarily thought of as phytoanticipins, it has been suggested that JA is involved in the signal transduction leading to the conversion of DIMBOA-Glc into HDMBOA-Glc in response to pathogen attack. Therefore, compromised JA signaling could also influence this con version. The interaction between [CO2] and H2O did not influence DIMBOA-Glc concentra tions. However, independent of other treatments, [CO2], H2O and Fv did significantly reduce DIMBOA-Glc concentrations . According to the ANOVA model, the concentration of HDMBOA-Glc was significantly influenced by the interaction between [CO2]xH2O . But the slight reduction of HDMBOA-Glc in stems at 2x[CO2]+H2O was only marginally sig nificant in comparison to concentrations 1x[CO2]+H2O using the Tukey-Kramer test. No significant difference was detected in HDMBOA-Glc at 1x[CO2]-H2O and 2x[CO2]- H2O. The unstable biologically active aglycones were not directly quantified, but the concentra tion of their degradation product MBOA was determined . With the exception of plants at 1x[CO2]-H2O, the concentration of MBOA was reduced with +Fv. The concentration of MBOA appeared to follow the inverse pattern of Fv biomass. The highest concentration of MBOA coincided with the least amount of pathogen in 1x[CO2]-H2O plants and the lower concentrations of MBOA coincided with the higher amounts of pathogen in plants at 2x[CO2]. However, 2x[CO2]-H2O+Fv plants which had the greatest amount of Fv biomass contained more MBOA then 2x[CO2]+H2O+Fv. Although the accumulation of terpenoid phytoalexins was significantly influenced by the interaction between [CO2] and Fv at–H2O, the concentration of zealexins and kauralexins was greater in 2x[CO2]-H2O+Fv plants than in 1x[CO2]-H2O+Fv which is opposite of what was observed in corresponding +H2O plants. Treatment with +Fv strongly induced the production of both zealexin and kauralexin families; however, the accumulation was signifi cantly less in comparison to 1x[CO2]+H2O+Fv. Terpenoid phytoalexin accumulation was weakest in stems at 1x[CO2]-H2O where the concentration of zealexins and kauralexins was approximately 34% and 22% the concentration in 1x[CO2]+H2O+Fv stems, respectively. Additionally, the induction of terpenoid phytoalexins was dampened in plants at 2x[CO2] regardless of H2O treatment. The concentration of total zealexins at 2x[CO2] was not further reduced by -H2O, but the amount of total kauralexins in +Fv stem tissues at 2x[CO2]-H2O was 25% less than the concentration in 2x[CO2]+H2O+Fv stems .Since the accumulation of terpenoid phytoalexins in drought stressed maize roots could poten tially influence the induction potential in above ground organs, the concentration of root terpenoid phytoalexins in plants grown at 1x[CO2] and 2x[CO2] was evaluated over a time course of withholding water. Even though the soil volumetric water content of plants at 2x[CO2] did not decline as quickly as plants at 1x[CO2], the induction of terpenoid phyto alexins was not significantly different . The spike of ABA was also not significantly different between the two [CO2] treatments . However, the gradual increase in JA was dampened at 2x[CO2] . At day 6 of withholding water, plant roots at 1x[CO2] contained approximately two times the amount of JA compared on to roots at 2x[CO2] .To distinguish between the potential compromising effects 2x[CO2] on JA signaling in maize roots and the consequence of enhanced water-use efficiency at 2x[CO2], the concentration of the JA and terpenoid phytoalexins in Diabrotica balteata larvae infested and con trol roots grown under the different abiotic stress treatments was measured. The interaction between [CO2] and Db infestation was only marginally significant . At 2 d post infestation, JA levels increased with larval root feeding ; however, the concentration of JA in root tissues at 2x[CO2] was still significantly less than roots at 1x[CO2] . While Db infestation was the only significant factor contributing to differences in root zealexin concentrations , kauralexin concentrations were influenced by all three interacting factors [CO2]xH2OxDb . Kauralexin concentrations increased with Db feeding damage or -H2O stress, and simultaneous root feeding damage and drought made the accumulation of kauralexins even stronger. Irrigated maize exposed to simultaneous 2x[CO2]+Db did not display an increase in kauralexins. However, since the kaur alexin concentration increased in maize roots with -H2O regardless of the compromising effects of 2x[CO2],hydroponic bucket there was no significant difference in root kauralexin levels between plants exposed to simultaneous 1x[CO2]-H2O+Db and 2x[CO2]-H2O+Db.Our research has demonstrated that while the physiological effects of elevated [CO2] provide a photosynthetic advantage to maize under conditions of drought , the plants are more susceptible to Fusarium verticillioides proliferation and prone to higher levels of fumonisin contamination . Recent reviews have summarized the potential effects of future climate scenarios on the development of plant diseases and disease epidemics; however, there is a general consensus that relatively few studies have evaluated the interactive effects of multi ple climate change factors on host-pathogen interactions, and further research is still required particularly with respect to mycotoxigenic pathogens. This report represents an anal ysis of the interactive influence of elevated [CO2] and drought on maize phytochemical defense responses to an economically important mycotoxigenic pathogen. Our findings provide insight into climate change induced host metabolic alterations that lead to variation in crop susceptibility and mycotoxin contamination. The majority of studies investigating the effects of climate change on plant defense are on C3 plants and plant-herbivore interactions. Emerging results suggest that abiotic stress can modulate phytohormone signals leading to differential responses to biotic stress, variation in downstream defense chemistry, and changes in susceptibility. This is consistent with our findings in C4 maize both the defense response to Fv stem inoculation and Db root feeding was altered by the abiotic stress treatments . However, the dampened response of JA regulated defenses and increase in SA regulated defenses in C3 plants under elevated [CO2] are negated in combination with drought. In maize, the accumulation of both JA and SA is compromised under elevated [CO2] and these effects persist even in combination with drought . Therefore, above ground the effects of elevated [CO2] supersede any potential stimulation of JA defenses with -H2O . The initial induction of JA following Fv inoculation was stronger in -H2O stressed plants in comparison to +H2O plants; however, this stimulation did not last . Other literature reports also suggest that, on its own, drought does not adequately stimulate JA defenses in above ground maize tissues. Therefore, the mechanism of phytohormone modulation by which elevated [CO2] and drought alters defenses in C4 maize is different from those previously described for C3 plants and require additional research so that strategies can be developed to ensure the quality and safety of the future maize crop under climate change scenarios. The increase in maize susceptibility to Fv proliferation suggests a compromised defense response. Consistent with this notion, the initial induction of JA and SA phytohormones was dampened at 2x[CO2]-H2O in comparison to 1x[CO2]-H2O . In comparison to 2x[CO2]+H2O plants though, JA levels of 2x[CO2]-H2O plants were only slightly reduced . An even weaker JA signal should have resulted in reduced accumulation of zealexins and kauralexins. Although terpenoid phytoalexin concentrations were greater in infected plants at 2x[CO2]-H2O in comparison to 1x[CO2]-H2O , it is important to consider that the accumulation of these phytoalexins has been shown to be dependent on the amount of pathogen inoculum. Therefore, while the reduction in pathogen biomass could account for the corresponding reduction in terpenoid phytoalexins at 1x[CO2]-H2O in comparison to 1x[CO2]+H2O, the concentration of both zealexin and kauralexin metabolites could be further induced in 2x[CO2]-H2O plants, which had significantly more pathogen biomass. Neverthe less, despite the potential augmented induction of plants at 2x[CO2]-H2O+Fv in comparison to 2x[CO2]+H2O+Fv, only the concentration of kauralexins was significantly less while the concentration of zealexins remained unchanged. However since the pathogen biomass was greater, the accumulation of zealexins was likely also compromised at 2x[CO2]-H2O in com parison to 2x[CO2]+H2O. The redirection of resources to maize roots may have contributed to the weaker response of terpenoid phytoalexins leading to intensified above ground susceptibility to Fv proliferation between +H2O and -H2O plants at 2x[CO2] . The 20 carbon diterpenoid kauralexins require more resources and accumulate to higher concentrations than the 15 carbon ses quiterpenoid zealexins in -H2O stressed maize roots . This could potentially explain the greater effect on kauralexin levels in maize stems in comparison to zealexin levels. Although JA was also reduced in maize roots and the accumulation of terpenoid phytoalexins in response to Diabrotica balteata larval feeding was impaired under conditions of 2x[CO2] , ABA signaling was unaffected and the accumulation of drought induced terpenoid phytoalexins was not inhibited . This is consistent with previous results demonstrating that application of ABA was sufficient to induce the accumulation of maize root zealexins and kauralexins. Conse quently, in contrast to above ground tissues, -H2O induced ABA in maize roots can counterbal ance the compromising effects of 2x[CO2] on the accumulation of zealexins and kauralexins. Therefore, at least with respect to root terpenoid phytoalexins, drought does negate the effects of elevated 2x[CO2]. Glycosylated benzoxazinoid concentrations were also influenced by the abiotic stress treat ments; however, these changes do not appear to be correlated with increased susceptibility to Fv under conditions of 2x[CO2]+H2O or 2x[CO2]-H2O. The decrease in DIMBOA-Glc and corresponding increase in HDMBOA-Glc following Fv inoculation, which has previously been reported with F. graminearum maize stem infection, was not inhibited by 2x[CO2] . The DIMBOA-Glc 4-O-methyltransferase which converts DIMBOA-Glc to HDMBOA-Glc is thought to be regulated by JA, but HDMBOA-Glc can also accumulate de novo in response to pathogen attack. Moreover, even the compromised JA signal was adequate to stimulate HDMBOA-Glc accumulation. In contrast, drought considerably reduced the concentration of HDMBOA-Glc. Benzoxazinoids are suspected to be involved in drought stress tolerance because drought or below ground application of ABA can induce the concentration of DIMBOA in maize leaves. Although the highly reactive aglycones were not quantified in this study, DIMBOA concentrations likely increased in maize stalks with drought and may have partly contributed to the reduction in pathogen biomass at 1x[CO2]-H2O.