Published online 31 May 2007
Published in Crop Sci 47:1125-1130 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP PHYSIOLOGY & METABOLISM-NOTES
Soluble Peroxidase Activity in Maize Endosperm Associated with Maize Weevil Resistance
Silverio García-Laraa,
John T. Arnasonb,
David Díaz-Pontonesc,
Elvira Gonzalezc and
David J. Bergvinsona,*
a CIMMYT, Apdo Postal 6-641, 06600 Mexico D.F
b Dep. of Biology, Univ. of Ottawa, Ottawa, ON, Canada, K1N 6N5
c Dep. Ciencias de la Salud, Univ. Autonoma Metropolitana, Apdo Postal 55-5350, 09340 Mexico D.F
* Corresponding author (dbergvinson{at}gmail.com).
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ABSTRACT
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Plant peroxidases (PODs) are involved in resistance to pathogens and insects. This study investigated the role of POD in maize (Zea mays L.) resistance to the maize weevil, Sitophilus zeamais (Motsch.). Insect bioassays were performed under controlled conditions to assess maize weevil resistance. Peroxidase activity was measured in the major grain tissues using guaiacol and peroxide. Significant variation (P > 0.001) among genotypes was observed in both the insect bioassay traits and POD activity. Peroxidase was detected in the embryo, endosperm, and pedicel, but it was not detected in the pericarp. Significant correlations were found between endosperm POD activity and maize weevil resistance (r = 0.89, P < 0.001). Histological staining confirmed POD activity in the vascular cylinder of the embryo, while activity in the endosperm was restricted to the aleurone layer. This study shows that POD activity is correlated with maize weevil resistance and may be used as a potential biochemical marker.
Abbreviations: CIMMYT, International Maize and Wheat Improvement Center • MW, maize weevil • POD, peroxidase.
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INTRODUCTION
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PEROXIDASES (PODs) are monomeric hemoproteins with glycosylation, and a subclass of oxidoreductases that catalyze the oxidation of compounds using hydrogen peroxide as an oxygen acceptor (Penel et al., 1992). These enzymes are distributed within three cellular environments: soluble, membrane-bound, and cell wall–bound (Boyes et al., 1997). Plant PODs are widespread in the plant kingdom and include several isozymes whose pattern of expression is dependent on tissue, development stage, and environmental stimuli (Penel et al., 1992; Valério et al., 2004). Plant PODs are involved in numerous physiological functions, including defense against pathogens (Bestwick et al., 1998; Lamb and Dixon, 1997; López-Curto et al., 2006) and insects (Dowd and Lagrimini, 1997).
The role of plant POD in resistance to biotic stresses is well known (Dowd and Lagrimini, 1997). Increases in POD activity during pathogen attack have been associated with an incorporation of phenolic compounds within the cell wall (Lamb and Dixon, 1997). Peroxidases catalyze the cross-linking of cell wall components such as extensins, phenolics, and polysaccharides (Brisson et al., 1994; Fry et al., 2000). Reinforcement of the cell wall through these cross-linkages can act as a mechanical barrier to penetration by pathogens (Brisson et al., 1994). Reinforcement in mature pericarp cell walls of maize (Zea mays L.) also functions in maize weevil (MW) [Sitophilus zeamais (Motsch.)] resistance (García-Lara et al., 2004). In addition, POD can contribute to insect resistance by quinone oxidation in the developing grain pericarp, which can bind to proteins to reduce digestibility in insects (Dowd, 1994; Dowd and Lagrimini, 1997). In mature maize grain, numerous isozymes of peroxidase are present; their genetic loci have been previously documented (Brewbaker et al., 1985). However, there is no direct evidence that PODs are associated with MW resistance in mature maize grain.
Recently, maize germplasm with MW resistance was reported (García-Lara et al., 2004). The resistant varieties had elevated levels of cell wall cross-linking components in the pericarp. The principal cell wall components associated with resistance were simple phenolic acids, diferulates, and extensins (Sen et al., 1994; García-Lara et al., 2004). Using these varieties and the MW resistance data, we extend our previous work to better understand the physiological function of maize grain POD activity with MW resistance. The objectives were (i) to quantitatively measure POD activity in the major grain tissue fraction (embryo, endosperm pericarp, and pedicle) of nine maize genotypes, (ii) to calculate the correlation between POD activities and MW resistance, and (iii) to localize at the histological level the POD activity within maize grain tissues.
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MATERIALS AND METHODS
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Maize Germplasm
Nine maize genotypes available at the International Maize and Wheat Improvement Center (CIMMYT) with a wide range in MW resistance were used in this study (Table 1). Their features were described by García-Lara et al. (2004). The seed was increased during 2001 at CIMMYT's experiment station at Tlaltizapán, Morelos, Mexico (18°41' N lat., 940 meters above sea level). A randomized complete block planting design blocked by variety type (line or full-vigor population or hybrid) was planted in three replicates. Each plot consisted of two 5-m rows with an interrow spacing of 0.75 m and a plant spacing of 0.25 m to provide an overall plant population of 56000 plants ha–1. Populations were increased by bulk pollination of individual plants; inbred lines (Muneng-8128 C0 HC1-18-2-1-1 and CML290) were selfed and shelled in bulk, and F2 seed derived from population crosses and single-cross hybrids were produced by bulk pollination of 20 plants. Ears from hand pollinations were selected to eliminate off-types and those with ear rot, leaving a minimum of 15 ears per replicate. Ears from each replicate were handled separately for use within insect bioassays and biochemical characterization. Ears were sun dried for 2 d in an insecticide-free environment, air dried at 35°C using a forced air dryer for 3 d, shelled, and stored at 13% grain moisture and 15°C for 1 mo before being used in insect bioassays and biochemical assays. Biochemical analysis and insect bioassays were run concurrently.
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Table 1. Maize weevil, Sitophilus zeamais, resistance traits for nine maize (Zea mays L.) genotypes with different levels of weevil resistance.
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Insect Bioassays
For insect bioassays, grain was equilibrated for 30 d at 27 ± 1°C and 70 ± 5% relative humidity. Three replicates containing 30 g of maize grain were then infested with 25 unsexed adult weevils no older than 7 d. Adults were removed after 1 wk. Infested grain was then stored under controlled conditions (27°C, 70% relative humidity) with a photoperiod of 12:12 (dark:light). Bioassays ran for 45 d at which time percentage grain weight loss and damaged kernels showing tunneling or emergence holes, number of adult progeny, and flour production were recorded. Resistance was determined according to the method developed by Dobie (1977).
Enriched Tissue Fractions
For all genotypes, grain tissue samples were prepared from three separate replicates generated from 20 kernel samples for each genotype. Studies were conducted using individual tissues (embryo, endosperm, pericarp, and pedicle) separated by hand dissection. The pedicle was first removed from the intact seed by a transversal cut of the kernel cap area. This structure consisted of the tip cap and a small section of the pericarp, endosperm, and embryo. Each tissue was removed by hand after imbibing seeds in distilled water for 10 min at 4°C. The samples were dried and ground to a fine powder using a cyclone mill fitted with a 1-mm screen.
Peroxidase Extraction and Assay
The peroxidase extraction procedure was modified from Bestwick et al. (1998). Ground samples of grain tissue (0.125 g) were hydrated (1 h, 4°C) with 1.5 mL of extraction buffer containing 100 mM sodium phosphate buffer of pH 6.8. The sample was homogenized for 1 min and then centrifuged at 12000 g for 30 min at 4°C. Supernatants were stored at 4°C before analysis. Protein concentrations were determined by the method of Bradford (1976), and bovine serum albumin (1mg mL–1) was used as a standard of concentration. Peroxidase assays were performed in 1-mL volumes at 25°C in a UV–visible light spectrophotometer (Beckman, model DU-640, Fullerton, CA). Peroxidase activity was measured with guaiacol at 470 nm with a minor modification to the method reported by Bestwick et al. (1998). The assay mixture contained 880 µL of 10 mM guaiacol in 50 mM potassium phosphate buffer, pH 6.58; 110 to 120 µL of extracted supernatant; 80 to 90 µL of sterile water; and 20 µL of 3% H2O2 to initiate the assay. Total activity was expressed in units per gram (U g–1) of dry weight, while specific activity was expressed in units per gram (U g–1) of protein content. One activity unit was considered as the oxidation of 1 mg of guaiacol per minute per mg of protein or mg of dry weight of sample.
Peroxidase Localization
Histological staining to monitor the presence of POD activity was developed using guaiacol (Bestwick et al., 1998). Mature maize kernels of genotype P84c2, a MW resistant line, were imbibed for 12 h in distilled water at 4°C to optimize POD activity. Hand sections of kernel tissues were immediately incubated for 15 min at 25°C in a potassium phosphate buffer (50 mM, pH 6.58) containing 10 mM guaiacol and 3% of H2O2. Sections were then washed with distilled water for 1 min and dried on filter paper. Brown staining appears at the sites where POD activity was present. The control was incubated using the same tissues under the same conditions but without H2O2.
Statistical Analysis
Peroxidase activity and insect bioassay data were subjected to ANOVA using the statistical software Statistix v.8 (Analytical Software, Tallahassee, FL), and differences among means were compared by the LSD test at P = 0.05. A mixed model was used whereby replications were considered random and genotypes were fixed effects. Orthogonal comparison of means was conducted for two contrast groups, resistant vs. susceptible, leaving out the moderately susceptible group (Sokal and Rohlf, 1981).
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RESULTS
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Insect Bioassay Data
Significant variation was observed in insect bioassay traits (P < 0.001) among genotypes evaluated during 2001 (Table 1) and show a considerable difference between resistant and susceptible groups by orthogonal comparison (P < 0.001). The Dobie index, number of weevil progeny, grain weight loss, and number of damaged kernels were highly correlated (r > 0.9, P < 0.001, n = 4). On the basis of previous testing, the Dobie index was used to establish three major groups: (i) resistant, (ii) moderately resistant, and (iii) highly susceptible. Reevaluation of the nine genotypes is reported in Table 1.
Peroxidase Activity
A significant difference in POD activity was observed between maize varieties and between the structures analyzed. Peroxidase activity was identified in the embryo, endosperm, and pedicle (Table 2). The method of extraction used in this study did not permit detection of POD activity in the pericarp.
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Table 2. Mean of total and specific peroxidase activity for the main kernel structures from nine maize (Zea mays L.) genotypes with different level of resistance to the maize weevil, Sitophilus zeamais.
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Embryo POD represented more than 86% of total activity, while activity in the endosperm and pedicle represented 0.5% and 13%, respectively. There was a significant difference between susceptible and resistant groups based on orthogonal comparisons in POD activity within a given tissue (Table 2). However, only in a few cases (total and specific activity in the endosperm and specific activity in the pedicle) did resistant genotypes have a higher POD activity. Maternal effects on POD activity were observed in the crosses between P84 (resistant) and P47 (susceptible). P84 x P47 showed a higher level of POD than its reciprocal cross, but this difference was only significant (P < 0.05) using a t test comparison for specific and total activity in the embryo and pedicle (data not shown).
Relationship between Insect Bioassay and Peroxidase Activity
Correlations among insect bioassay traits with POD activity are shown in Table 3. Each of the five MW resistance parameters were positively correlated (P < 0.05) with total and specific POD activity in the endosperm. Total and specific activities within the embryo and pedicle were positively correlated but not significant.
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Table 3. Pearson correlations between maize weevil, Sitophilus zeamais, resistance traits and peroxidase activity for the main structures from nine maize genotypes.
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Peroxidase Localization
Localization of POD activity was conducted on mature maize grain from P84c2, resistant to the MW (Fig. 1). Histological staining with guaiacol and peroxide demonstrated that POD was present in the embryo and endosperm but not in the pericarp; however, without peroxide, no staining occurred in these tissues. Embryo POD activity was found in the scutellum behind the radicle and was localized in the vascular cylinder. In the endosperm POD activity was restricted to the aleurone layer.
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Figure 1. Hand sections of maize grain stained for peroxidase activity. A and C: longitudinal sections of total grain incubated with guaiacol alone (controls) demonstrating no brown staining in the endosperm (Ed), embryo (Em), or pericarp (Pr). B and D: same sections as in A and C with H2O2 added, demonstrating brown staining in the aleurone layer (Al), embryo root (Er), and scutellum (Sc). E: transverse section of radicle stained with guaiacol and H2O2 demonstrating brown staining in vascular xylem (Xi), phloem (Ph), endodermis (En), and cortex (Cx).
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DISCUSSION
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Peroxidase has been previously detected in maize grain (Brewbaker et al., 1985; Boyes et al., 1997), and certain isozymes are known to be confined to particular tissues within the grain. In this study maize genotypes resistant to the MW showed elevated levels of POD activity, with levels of MW resistance being comparable to an early study (García-Lara et al., 2004). Since the resistant varieties used in this study were developed through intrapopulation improvement and are rather heterogeneous compared with the two susceptible checks, one may consider heterogeneity to be important in MW resistance. However, several heterogeneous CIMMYT tropical maize populations developed using similar breeding methodology showed diverse levels of MW resistance (Arnason et al., 1993), as did homogeneous hybrids evaluated against the same storage pest (Kim and Kossou, 2003). Endosperm POD activity had a highly significant and positive correlation with MW resistance parameters. The current study focused on the soluble POD enzyme fraction that is the most abundant fraction in maize grain (Boyes et al., 1997), while membrane and cell wall–bound PODs were not considered. Peroxidase activity was localized in the vascular cylinder of the embryo axis, scutellum, and aleurone layer of the endosperm, which is in agreement with POD activity previously reported for maize grain (Brewbaker et al., 1985; Kim et al., 1996). However, pericarp POD activity was not detected in mature grain using our extraction method. As 99% of the POD activity was localized within the embryo and pedicle, it is likely that POD activity plays an important role in other functions, such as maintaining seed viability, based on preliminary results (Garcia-Lara, unpublished data, 2007).
Associations between POD activity and their role in plant defense against pathogen attack have been demonstrated in maize plants (Penel et al., 1992; Dowd and Lagrimini, 1997; Caruso et al., 2001). In this study, endosperm-soluble POD was highly correlated with MW resistance parameters, suggesting that POD activity or its catalytic products contribute to MW resistance. It has been established that the aleurone layer in maize is composed of living cells that contain phenolic compounds (Sen et al., 1994). Quinones derived from phenolics can bind to proteins or carbohydrates and have been reported to reduce the digestibility of maize in insects and make protein sources less nutritious (Dowd, 1994; Dowd and Lagrimini, 1997). Additionally, different protein inhibitors have been recognized in maize kernels that reduce insect and disease infection (Figueira et al., 2003). However, the only example of POD in grain for defense was shown by Caruso et al. (2001) in wheat, where they reported that a basic POD from the kernel provides protection against fungal pathogens by inhibiting germ tube elongation. Proteome analysis should be conducted to isolate and characterize maize PODs and define their role in MW resistance.
Using maize populations developed for MW resistance and comparing them with MW susceptible varieties, we showed the correlation of endosperm POD activity with MW resistance. The sources of MW resistance used in this study should be used in future breeding efforts to improve MW resistance, with POD activity serving as a biochemical marker to accelerate the selection process.
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ACKNOWLEDGMENTS
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We gratefully acknowledge the support of the Canadian International Development Agency (CIDA) for the project titled "Reducing Postharvest Losses in Maize" and a scholarship to S. García-Lara by CONACYT, Mexico. Editorial and technical reviews of the manuscript by D. Beck, M. Banziger, A. Krivanek, and M. Listman are gratefully acknowledged.
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NOTES
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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication October 27, 2006.
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ABSTRACT
INTRODUCTION
