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a USDA-ARS, Plant Stress & Germplasm Development Unit, Cropping Systems Research Laboratory, 3810 4th St., Lubbock, TX 79415
b contributed equally to this work. Inquiries about seeds and the population used in this study should be addressed to Dr. C.D. Franks. Mention of trade name does not constitute endorsement of the product to the exclusion of similar products by the USDA
* Corresponding author (gburow{at}lbk.ars.usda.gov).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: DW, dry weight • EW, epicuticular wax • EWL, epicuticular wax load • FW, initial fresh weight • RWC, relative water content • TW, turgid weight
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication March 2, 2007.
a USDA-ARS, Plant Stress & Germplasm Development Unit, Cropping Systems Research Laboratory, 3810 4th St., Lubbock, TX 79415
b contributed equally to this work. Inquiries about seeds and the population used in this study should be addressed to Dr. C.D. Franks. Mention of trade name does not constitute endorsement of the product to the exclusion of similar products by the USDA
* Corresponding author (gburow{at}lbk.ars.usda.gov).
An irradiation-induced bloomless mutant of sorghum [Sorghum bicolor (L.) Moench.], KFS2021, which visually exhibits an absence of white fluffy epicuticular wax in leaf and sheath, was characterized using a combination of genetic and physiological approaches. Study of the phenotypic segregation for the bloomless trait in F2 and F2:3 populations from a cross between KFS2021 and BTx623 (a cultivar with bloom showing profuse deposition of white epicuticular wax) suggests that bloomless is controlled by a single nuclear recessive gene. The bloomless parent (KFS2021) and F2 individuals had lower frequency of guttation, leakier epidermal layer (based on percentage of chlorophyll leaching), and higher rate of seedling water loss than the BTx623 and F2 bloom individuals. Bloomless F2 individuals showed 3- to 6-fold higher nighttime transpiration rates relative to F2 bloom individuals based on nighttime conductance. Correlation analysis showed significant negative associations between leaf epicuticular wax load with epidermal permeability and nighttime conductance, which indicate the important role of epicuticular wax in these traits. These results suggest that epicuticular wax may enhance water use efficiency of sorghum by regulating nighttime water loss.
Abbreviations: DW, dry weight • EW, epicuticular wax • EWL, epicuticular wax load • FW, initial fresh weight • RWC, relative water content • TW, turgid weight
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evidence for the role and contribution of EW to tolerance to abiotic stress had been alluded to earlier in several studies that focused on the significance of EW to drought tolerance. Blum (1975) proposed that thicker EW in sorghum could lead to reduced cuticular transpiration and possibly enhance stomatal control of water loss. In a field study, genetic variation and differences in combining abilities for EW production were observed in 14 normal bloom sorghum lines (Jordan et al., 1983). Furthermore, EW load was found to increase with drought stress (Jordan et al., 1983). In a related study, a comparison of 38 near isogenic lines (19 normal bloom, 14 bloomless, and 5 sparse bloom) of sorghum showed that a decrease in EW load from 0.1 to 0.03 g m–2 resulted in an increase in cuticular transpiration using detached leaves and a mass water transpiration method measured with a modified cuvette apparatus (Jordan et al., 1984). They estimated that an EW load of about 0.067 g m–2 could provide an effective barrier to water loss through the cuticle (Jordan et al., 1984). Furthermore, in a comparative study of three bloomless and one sparse bloom mutant lines with their corresponding isogenic siblings under greenhouse conditions, Premachandra et al. (1994) reported that water use efficiency was positively correlated with epicuticular wax load under both irrigated and nonirrigated conditions. Jenks et al. (1994) showed that bloomless mutation increased cuticular transpiration with pleiotropic effects and increased the susceptibility to the fungal pathogen Exserohilum turcicum. Results from these studies suggest that sorghum EW could play an important role in possibly reducing cuticular transpiration and could have other pleiotropic effects. However, in these studies, determination of cuticular transpiration was based on a detached leaf method (except for the study by Jenks et al., 1994), and artificial methods in inducing stomatal closure were used. The effect of a bloomless mutation on nighttime transpiration, when stomata are generally thought to close under normal conditions, has never been examined.
The main objective of this research was to characterize the physiological and genetic features of an irradiated bloomless mutant of sorghum (EW mutant) and analyze the physiological effects of this mutation using a defined F2 population under controlled greenhouse conditions. This study also determined the relationship between cuticular wax and nighttime leaf transpiration of sorghum in a pedigreed population.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Determination of Seedling Water Loss
Seedling water loss rates were determined from the 100 F2 thinned seedlings described above and five plants from each parental line. Before harvest, the plants were scored visually for presence or absence of epicuticular wax. Seedlings were cut at the base of the shoot, wrapped in moist filter paper, placed in labeled plastic bags, and transported to the laboratory. The cut surface was covered with transparent tape to prevent water loss through the base of the stem. At the beginning of the experiment, initial fresh weight (FW-i) of each seedling was determined to within 0.1 mg (Mettler Toledo model AB104-S, Mettler Toledo Inc., Columbus, OH). Seedlings were hung from the attached tape on paper clips in an open area in the laboratory in consecutive harvest order and allowed to transpire freely. Fresh weights were measured at hourly intervals for 8 h. Water loss rate was expressed as the rate of change in fresh weight relative to original FW-i.
Guttation Frequency and Relative Water Content
Guttation was determined from the remaining 100 F2 greenhouse-grown plants at mid-vegetative stage. Guttation was scored as present or absent based on dew formation on the margins of two to three fully expanded leaves. Observations for guttation were made every other day during the morning hours of 0700 to 0900. The frequency of guttation was calculated on the basis of observations over 5 d, and frequency of guttation for the parental lines and the 100 F2 progenies were calculated.
Relative water content (RWC) was measured based on previously described methods (Barrs and Weatherly, 1962). Five leaf discs (0.8 cm in diameter) were obtained from parents and from 24 F2 individuals (12 each of bloom and bloomless F2 plants). Leaf discs were weighed immediately and then immersed in 5 mL of distilled deionized H2O for 8 h at room temperature; subsequently, turgid weight (TW) was determined and leaf discs were dried at 65°C for 24 h to determine final dry weights (DW). Relative water content was calculated as: RWC = [(FW – DW])/[TW – DW)] x 100. Relative water contents were also determined from the parents and the same 24 F2 plants at anthesis before measurement of conductance.
Epicuticular Wax Load Determination
Gravimetric determination of wax load in leaves was conducted using 20 leaf discs (0.8 cm diam.) from each of the 5 parental plants and from the 100 F2 individuals. Leaf discs were collected from the third leaf (minus flag leaf) at anthesis. The samples were transported to the laboratory in closed plastic boxes, and epicuticular wax was extracted from leaves according to previously described methods (Ebercon et al., 1977). Epicuticular wax was extracted from both abaxial and adaxial sides of leaves using 5 mL of gas-chromatography grade chloroform by swirling the discs in the solvent for 30 s. Measurement of gravimetric amount of wax was performed using a balance with sensitivity of 0.1 mg (Mettler Toledo model AB104-S, Mettler Toledo Inc., Columbus, OH). Spectrophotometric or chemical assay of leaf wax was also performed by dissolving wax in 0.5 mL of 0.016M potassium dichromate in 96% sulfuric acid as wax reagent, transferred to 1.5 mL microcentrifuge tubes and heated at 90°C for 30 min in a dry bath under the hood as described by Ebercon et al. (1977). Absorbance was measured at 590 nm using Beckman UV-VIS DU-640 (Beckman Coulter, Fullerton, CA). Calculation of wax load was based on a standard using a standard curve of a known amount of sorghum wax from 0.5 to 12 mg. Wax load from sheaths was determined using the same protocols as described for leaf blades except that only 10 discs were used for sheath assay.
Gravimetric and chemical assay of leaf wax was found to be 95% correlated with each other. Results from the gravimetric assay are presented in this report.
Determination of Epidermal Permeability
Cuticle epidermal permeability based on chlorophyll leaching was analyzed using previously described protocols (Lolle et al., 1997). Briefly, 10 leaf discs from the fourth leaf (minus flag leaf) were collected at 70 d after planting both parents and 100 F2 individuals. Leaf discs were immersed in 10 mL of 80% ethanol, and chlorophyll was allowed to leach into the solvent. The samples were placed in the dark, and absorbance at 645 and 667 nm were measured at hourly intervals for 6 h after collection. Total chlorophyll leached at 24 h after immersion was determined, and leaching was expressed as a percentage of the total chlorophyll at 24 h.
Conductance Measurement
Preliminary measurements of nighttime conductance were performed to determine whether variation existed among three different leaf positions (second, third, and fifth leaf) in 10 plant samples (representing five each of bloom and bloomless phenotypes) between different portions of the leaves using a portable leaf poromoter (Decagon Inc., Pullman, WA). Results showed that nighttime conductance was not affected by leaf position and that measurement in different portions of the leaves showed similar values. Preliminary measurements were also conducted to compare conductance values of the adaxial and abaxial sides of the leaves at night for the parental lines and 10 representative F2 individuals (5 plants each of bloom and bloomless phenotype). Our results showed that conductance values for the adaxial side of the leaves were very low and probably beyond the detection of the instrument used in the study. However, conductance values from the abaxial side of the leaves were high and within the range of values reported for sorghum (Jordan et al., 1983, 1984; Muchow and Sinclair, 1989; Premachandra et al., 1994; Jenks et al., 1994). From these results, we decided to focus on measurement of conductance from the abaxial side of the leaf.
Daytime stomatal conductance was measured on the third leaf below the flag leaf between 1100 and 1500 hours on three different days. Nighttime conductance was measured on the same leaf at night between 2000 and 2400 hours on three different nights in the same week. Leaves were carefully handled so as not to reduce EW in the leaves and sheaths throughout the study. On each set of readings (whether day or night), the two parents and all 100 F2 individuals were measured, and each sampling date was considered as a replication.
Water Use Efficiency
Characterization of water use efficiency was performed using a modified lysimetry method. Seeds from the parents were planted in plastic pots with a 15.2-cm diameter and 17.8-cm depth holding approximately 2 L of potting mix. The pots were filled with a measured amount of commercial soil mix as described in the plant materials section and watered with 0.5X Miracle-Gro (Scotts-Miracle Gro Co., Marysville, OH) until dripping from the bottom. Three seeds were planted per pot. After planting, the pots were covered with a layer of dry potting mix to reduce water loss from the soil surface. One week after emergence, each pot was thinned to one plant, and the pot was covered from both ends with 2 Mil poly bags (S-3478, Uline, Waukegan, IL), which are permeable to air but impermeable to water. A hole was cut in the top of the bag to allow the plant to protrude. The hole was further sealed with strong packing tape and covered with a layer of dry potting mix to prevent water loss through the opening in the top of the bag. Pots with soil and a small seedling were weighed as initial weight. When plants reached permanent wilt, the shoots were harvested and the final pot weight (including root) was weighed. Water used was calculated by subtracting the final pot weight from the initial weight. Transpiration efficiency was calculated by dividing the total dry matter with the water used.
Statistical Analysis
A Chi-square analysis for goodness of fit of the F2 and F2:3 phenotypic distributions to a 3:1 and 1:2:1 genetic ratio, respectively, was conducted. Since this study involved an F2 population with only two phenotypes being compared, unpaired t tests between the two classes for the traits measured were conducted. The t tests were performed with due consideration of unequal variance and computed t values for testing significance were calculated according to formula by Cochran and Cox (1957). All statistical analyses were conducted using SYSTAT 7.0 (SPSS, Chicago, IL).
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RESULTS |
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Genetic Analysis of Mutation
The segregation data for the 100 F2 individuals used in the study is presented in Table 2
. The data fits a 3:1 ratio, indicating that the bloomless mutation in this study is under the control of a single recessive nuclear gene. Further analysis of segregation in the F2:3 families confirmed the segregation pattern found in the F2 generation, which indicated that a single nuclear recessive gene could account for the bloomless mutation in KFS2021 (Table 2).
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, a combination of genetic and physiological approaches were used to analyze how leaf and, to some extent, sheath EW are related to specific physiological traits of sorghum. We used an F2 generation from a cross between an irradiated bloomless mutant KFS2021 and a wild-type cultivated parent line BTx623. KFS2021 exhibited almost no visible wax in its aerial parts, and this study verified that this mutant line had a very small amount of detectable epicuticular wax when subjected to both gravimetric and spectrophotometric analysis compared with BTx623. At present this mutant and its F2:3 progenies are being studied in detail at the biochemical and molecular levels. Genetic analysis using the F2 generation provides information comparable to that found using near-isogenic lines of chemically induced bloomless mutants of sorghum (Jenks et al., 1994). The segregation data from this study show that the bloomless mutation in KFS2021 is controlled by a single nuclear recessive gene. Further genetic studies using genetic mapping and molecular biology technologies will facilitate the identification of the actual gene involved in this mutation. Genetic mapping studies and molecular analyses of the wax pathway using the population described here are underway.
Most of the physiological traits analyzed in this study, including seedling water loss, chlorophyll leaching, guttation, and day and night leaf conductances are generally considered as complex traits and are likely controlled by multiple genes. This hypothesis is supported by the histogram shown for each of the traits. However, an overall trend for modification toward a bimodal distribution in the F2 population, in contrast to a continuous distribution based on phenotypic classes, suggests that EW could have significant effect on each of these complex traits determined. The relationships between EW and the physiological traits are further supported by correlation analysis.
Genetic correlation analyses from the F2 generation for leaf EW and related traits indicated strong associations among traits. A reduction in EW was found to be associated with high seedling water loss, in agreement with previous reports in sorghum (Chatterton et al., 1975; Jenks et al., 1994; Premachandra et al., 1994). More interestingly, EW was found to be positively associated with guttation and negatively correlated with epidermal permeability. Characterization of these two physiological traits has never been reported in sorghum before, and these data provide further support for the involvement of EW in reducing night transpiration of sorghum. Leaf EW of sorghum could serve as a barrier for leaching of molecules out of the leaves, as exemplified by the relatively higher levels of chlorophyll leaching associated with reduction in EW. The observed association between guttation and EW was interesting. It was consistently found that only bloomless F2 individuals showed decreased frequency of guttation, which is related to the role of EW in controlling cuticular transpiration. This decrease in guttation in bloomless plants is proposed to be a result of higher nighttime transpiration, which prevents these plants from building positive hydrostatic or root pressure and thus precluding the occurrence of guttation. Nobel (1991) previously described this relationship between transpiration and guttation. Consequently, as was observed in this study, F2 plants with bloom or high EW had lower nighttime transpiration, had a higher chance of developing positive root pressure in the evening, and thus exhibited increased guttation frequencies.
Nighttime conductance measurements can be thought of as an estimate of cuticular transpiration since the effects of stomatal conductance were presumably negligible. It is generally assumed that at night plants close their stomata and only lose water via the cuticle (Nobel, 1991). Plants in this study were grown in the greenhouse, where humidity, airflow, and temperature were controlled, thus allowing for a good estimate of cuticular transpiration. The analysis of 100 F2 individuals for conductance was greatly facilitated by the use of a handheld porometer. The values for nighttime conductance were highly consistent for each individual progeny and also across the different days of measurement. (In this study, RWC of the leaves was also measured to rule out the effects of possible differences in leaf hydration on conductances.) These data from the F2 population indicate that EW plays an important role in regulating nighttime cuticular transpiration in sorghum. The epicuticular wax load is probably one important factor that could contribute to the greater water efficiency and drought tolerance of sorghum. This study suggests that the high levels of EW in sorghum and its possible role in reducing cuticular transpiration may play a very important role in sorghum's drought avoidance and tolerance characteristics in the greenhouse. Further field study using an advanced generation of the population developed in this study will provide additional evidence to the importance of EW in drought response of sorghum.
The extent to which EW can affect nighttime transpiration has not been explored fully. Information on nighttime transpiration is limited, and study of this physiological activity is generally in its infancy (Snyder et al., 2003; Caird et al., 2007; Howard and Donovan, 2007). Based on the results from this study, significant EW reduction or possible elimination could result in significant increase in nighttime transpiration, which could be equivalent to 30% of daytime water loss. When compounded over the course of the growing season, this represents a significant amount of water loss that has never received ample attention. Given the present condition of dwindling water supplies, increased attention to the levels of EW in a system such as sorghum could provide a good entry point toward manipulation of nighttime water loss in agricultural systems. The implications of these results could also be important for development of crop varieties that can perform well under sustainable agricultural systems.
The authors would like to thank Dr. Robert Klein, SPARC, USDA-ARS, College Station, TX, for generously providing the mutant seeds used in the study. The authors also acknowledge Halee Hughes, Lindsey Fox, Charles Woodfin, Naomi Kaskela, and Lance Layton for excellent technical assistance.
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 March 2, 2007.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  P. J. Peters, M. A. Jenks, P. J. Rich, J. D. Axtell, and G. Ejeta Mutagenesis, Selection, and Allelic Analysis of Epicuticular Wax Mutants in Sorghum Crop Sci., June 26, 2009; 49(4): 1250 - 1258. [Abstract] [Full Text] [PDF]
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