Prolonged exposure to a synthetic stable auxin increases total protein levels in maize stem tissue.

<p>Average (<i>n</i> = 4, ±SEM) auxin levels of <b>A</b>, indole-3-acetic acid (IAA) and <b>B</b>, 2,4-dichlorophenoxyacetic acid (2,4-D) following a 50 µg application to wounded (W) stems and subsequent harvests at 3, 12, 24, and 48 h after treatment. Average (<i>n</i> = 6, +SEM) total soluble protein extracted at 48 h from maize stem tissue treated with 50 µg <b>C</b>, IAA or <b>D</b>, 2,4-D. Significant differences are indicated by asterisk (Student’s <i>t</i>-test, <i>P</i> < 0.05; n.s.d = no statistical difference).</p

European Corn Borer (<i>Ostrinia nubilalis</i>) Induced Responses Enhance Susceptibility in Maize

<div><p>Herbivore-induced plant responses have been widely described following attack on leaves; however, less attention has been paid to analogous local processes that occur in stems. Early studies of maize (<i>Zea mays</i>) responses to stem boring by European corn borer (ECB, <i>Ostrinia</i><i>nubilalis</i>) larvae revealed the presence of inducible acidic diterpenoid phytoalexins, termed kauralexins, and increases in the benzoxazinoid 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucose (HDMBOA-Glc) after 24 h of herbivory. Despite these rapidly activated defenses, larval growth was not altered in short-term feeding assays. Unexpectedly, ECB growth significantly improved in assays using stem tissue preconditioned by 48 h of larval tunneling. Correspondingly, measures of total soluble protein increased over 2.6-fold in these challenged tissues and were accompanied by elevated levels of sucrose and free linoleic acid. While microarray analyses revealed up-regulation of over 1100 transcripts, fewer individual protein increases were demonstrable. Consistent with induced endoreduplication, both wounding and ECB stem attack resulted in similar significant expansion of the nucleus, nucleolus and levels of extractable DNA from challenged tissues. While many of these responses are triggered by wounding alone, biochemical changes further enhanced in response to ECB may be due to larval secreted effectors. Unlike other Lepidoptera examined, ECB excrete exceedingly high levels of the auxin indole-3-acetic acid (IAA) in their frass which is likely to contact and contaminate the surrounding feeding tunnel. Stem exposure to a metabolically stable auxin, such as 2,4-dichlorophenoxyacetic acid (2,4-D), promoted significant protein accumulation above wounding alone. As a future testable hypothesis, we propose that ECB-associated IAA may function as a candidate herbivore effector promoting the increased nutritional content of maize stems.</p> </div

Consistent with maize endoreduplication, both wounding and ECB stem herbivory treatments promote significant increases both nuclear and nucleolar size as well as total DNA levels.

<p>Light microscopy pictures of nuclei and nucleoli from representative <b>A</b>, control (C), <b>B</b>, wounded (W) and <b>C</b>, larval-conditioned tissues (LCT). Scale bar = 20 µm. Average areas (<i>n</i> = 40, ±SEM) of <b>D</b>, nuclei and <b>E</b>, nucleoli and <b>F</b>, concentration of total DNA (<i>n</i> = 3, +SEM). Different letters (a–b) represent significant differences (all ANOVAs <i>P</i> < 0.01; Tukey test corrections for multiple comparisons, <i>P</i> < 0.05).</p

Validation of ECB-induced microarray probe sets with qRT-PCR.

<p><b>A</b>, Average (<i>n</i> = 3, +SEM) relative gene transcript expression levels for <i>Mpi</i>, <i>Abp20</i>, and <i>GH3</i> which encode the Maize protease inhibitor, Auxin binding protein-20, and a predicted indole-3-acetic acid-amido synthetase. Undamaged control stem tissues (C, white bars) and 48 h larval-conditioned tissue (LCT, black bars). <b>B</b>, Similarly, average (<i>n</i> = 3, +SEM) relative gene transcript expression levels for <i>Pr10</i>, <i>Sh-1</i>, <i>Lox1</i>, and <i>Lox2</i> which encode the proteins Pathogenesis-related 10, Sucrose synthase-1, Lipoxygenase 1 and Lipoxygenase 2. Significant differences indicated by asterisk (Student’s <i>t</i>-test, <i>P</i> < 0.05). <b>C</b>, Average (<i>n</i> = 3, +SEM) relative gene transcript expression levels for <i>Enod93-1</i>, <i>Enod93-2</i>, and <i>Gpc3/4</i> encoding two early nodulin 93 proteins and cytosolic glyceraldehyde-3-phosphate dehydrogenase 3/4, respectively for control (white bars), wound (grey bars), and larval-conditioned tissue (LCT, black bars). Different letters (a–b) represent significant differences (all ANOVAs <i>P</i> < 0.01; Tukey test corrections for multiple comparisons, <i>P</i> < 0.05).</p

ECB oral secretions and frass contain unusually high levels of IAA.

<p><b>A</b>, Average (<i>n</i> = 3, +SEM) concentration of IAA in maize stem tissue collected from control (C), wounded (W), and ECB larvae-conditioned tissues (LCT) after 48 h. <b>B</b>, Average (<i>n</i> = 4, +SEM) IAA levels in ECB OS or recently collected (1 h) frass following larval ingestion of maize leaf or stem tissue for 24 h. <b>C</b>, Average (<i>n</i> = 3, ±SEM) IAA in frass of numerous Lepidopteran pest species (<i>Helicoverpa</i><i>zeae</i>, <i>Heliothis</i><i>virescens</i>, <i>Anticarsia</i><i>gemmatalis</i>, <i>Agrotis</i><i>ipsilon</i>, <i>Pseudoplusia</i><i>includens</i>, <i>Spodoptera</i><i>frugiperda</i>, <i>Trichoplusia ni</i>, <i>Diatraea</i><i>grandiosella</i> and <i>Ostrinia</i><i>nubilalis</i>: ECB) reared on artificial diet. Different letters (a–d) represent significant differences (all ANOVAs <i>P</i> < 0.01; Tukey test corrections for multiple comparisons, <i>P</i> < 0.05).</p

Drought treatment is representative of conditions in which maize plants benefit from elevated [CO₂] induced water conservation.

<p>Drought treatment was imposed on one month old maize by withholding water for five consecutive days. Gas exchange measurements were then taken from the fifth leaf of 1x[CO₂] (400 μmol CO₂ mol^-1 air) or 2x[CO₂] (800 μmol CO₂ mol^-1 air) grown maize. The average (a) stomatal conductance (g_s), (b) percent soil water content and (c) photosynthetic rate (Pn) was estimated for each set of plants with irrigation (+H₂O) or drought (-H₂O) treatment. The statistically significant main effects of differences determined by a 2x2 ([CO₂]xH₂O) analysis of variance (ANOVA) are indicated in the top right hand corner of each graph. Since the interaction between the contributing factors was significant, a Tukey- Kramer honestly significant difference (HSD) test was performed to determine differences between means. Letters above standard error of mean (SEM) bars indicate significant differences (n = 4, <i>P</i><0.01).</p

Influence of 2x[CO₂] and drought (–H₂O) on maize primary metabolite concentrations following <i>F</i>. <i>verticillioides (Fv)</i> inoculation.

<p>The average ± SEM (a) water content, (b) total soluble carbohydrates, (c) starch and (d) total protein in maize stems two days post mock-inoculation (-<i>Fv</i>) or <i>F</i>. <i>verticillioides</i> inoculation (+<i>Fv</i>) under different [CO₂] and drought (–H₂O) was evaluated and compared. Significant contributing factors to differences are indicated in the top right hand corner of each graph (2x2 ([CO₂]x<i>Fv</i>), ANOVA, n = 4, <i>P</i><0.05).</p

Effects of 2x[CO₂] and drought (–H₂O) on maize benzoxazinoid defense metabolites.

<p>The mean ± SEM concentration of (a) DIMBOA-Glc, (a) HDMBOA-Glc and the downstream degradation product of their aglycones, (c) MBOA was determined for both mock (-<i>Fv</i>) or <i>F</i>. <i>verticillioides</i> (+<i>Fv</i>) inoculated stem tissues. Individual and interacting factors significantly contributing to differences are indicated at the top right corner of each graph (2x2x2 ([CO₂]xH₂Ox<i>Fv</i>) ANOVA, n = 4, <i>P</i><0.05).</p

Drought negates the compromising effects of elevated [CO₂] on the accumulation of root terpenoid phytoalexins to <i>Diabrotica balteata</i> feeding damage.

<p>The average concentration ± SEM of (a) jasmonic acid (b) zealexins and (c) kauralexins in roots of maize grown at 1x[CO₂] or 2x[CO₂], with irrigation (+H₂O) or drought (-H₂O) treatment, 2 d post control (-<i>Db</i>) or <i>Diabrotica balteata</i> larvae infestation (+<i>Db</i>) was determined. Statistically significant factors and interactions are included at the top right corner of each graph 2x2x2 ([CO₂]xH₂Ox<i>Fv</i>) ANOVA. If the interaction between the contributing factors was significant, a Tukey- Kramer HSD test was performed to determine differences between means (n = 4, <i>P</i><0.05). If there was no interaction, a <i>t</i>-test was performed on the means of the main effect (n = 16, <i>P</i><0.01). Letters above bars indicate significant differences.</p

2x[CO₂] and drought (-H₂O) increased maize susceptibility to <i>F</i>. <i>verticillioides</i> (<i>Fv</i>) proliferation and fumonisin contamination.

<p>(a) The average <i>Fv</i> biomass in maize stalks seven days post-inoculation grown at 1x[CO₂] or 2x[CO₂] with drought (-H₂O) was estimated as the amount of pg fungal DNA relative to ng maize DNA via qRT-PCR. (b) The mean concentration of fumonisin contaminating stalks grown under different abiotic stress conditions was determined, and (c) the relative amount of fumonisin per pg of <i>Fv</i> DNA was estimated. Values represent averages ± SEM. Letters above bars indicate significant differences (<i>t</i>-test, n = 4, <i>P</i><0.05).</p