C07-02-0119.Burow x.indd

An irradiation-induced bloomless mutant of sorghum [Sorghum bicolor (L.) Moench.], KFS2021, which visually exhibits an absence of white fl uffy 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 3to 6-fold higher nighttime transpiration rates relative to F2 bloom individuals based on nighttime conductance. Correlation analysis showed signifi cant 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 effi ciency of sorghum by regulating nighttime water loss. USDA-ARS, Plant Stress & Germplasm Development Unit, Cropping Systems Research Laboratory, 3810 4th St., Lubbock, TX 79415. G.B. Burow and C.D. Franks 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. Received 2 Mar. 2007. *Corresponding author ([email protected]). Abbreviations: DW, dry weight; EW, epicuticular wax; EWL, epicuticular wax load; FW, initial fresh weight; RWC, relative water content; TW, turgid weight. Published in Crop Sci. 48:41–48 (2008). doi: 10.2135/cropsci2007.02.0119 © Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA 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. R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . 42 WWW.CROPS.ORG CROP SCIENCE, VOL. 48, JANUARY–FEBRUARY 2008 genes, with bloomless (bm) and sparse bloom (h) recognized as genetically distinct (Ayyangar and Ponnaiya, 1941). A comparison of the phenotypes and a more detailed crossing study revealed that more genes could be involved depending on the crossing scheme and the mutants used in the study (Peterson et al., 1982). 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 signifi cance 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 fi eld study, genetic variation and diff erences 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 modifi ed cuvette apparatus ( Jordan et al., 1984). They estimated that an EW load of about 0.067 g m–2 could provide an eff ective 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 effi ciency 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 eff ects 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 eff ects. 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 artifi cial methods in inducing stomatal closure were used. The eff ect 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 eff ects of this mutation using a defi ned 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. MATERIALS AND METHODS Plant Materials and Genetic Analysis To study the genetics and physiology of the bloom–bloomless trait in sorghum, an F2 population was developed by crossing (via hand emasculation) the mutant line KFS2021 to BTx623. KFS2021 was developed by gamma irradiation of the cultivar Tx7078 by the late Dr. Keith F. Schertz. Tx7078 is an established restorer line characterized by early maturity, prefl owering drought tolerance, and good combining ability. BTx623 is a widely adapted maintainer line that has been used extensively in sorghum genetics study. F1 seeds were harvested, planted one plant per pot, grown in 8-liter pots fi lled with commercial soil mix (SunGro Professional Mix #1, Bellevue, WA), in the greenhouse in Lubbock, Texas, under a temperature regime of 28°C day and 25°C night, with a relative humidity of 44 to 48%. Plants were maintained under well-watered conditions with automatic drip irrigation and fertilized with Osmocote (Scotts Co., Marysville, OH) slow-release formulation. The F1 plants were found to exhibit the bloom phenotype, indicating that bloom is a dominant phenotypic trait. The F2 seeds from a single confi rmed F1 plant were harvested, planted in 100 pots (with 2 seeds per pot), and grown in the same greenhouse under the conditions described above. Bloom–bloomless phenotypes were scored at 22 d after planting, at which time one random seedling per pot was removed and used for the seedling water loss study. The remaining 100 F2 individuals were grown to maturity and used for detailed characterization of physiological features related to epicuticular wax load and self-pollinated to produce the F2:3 generation. Twenty random seeds from each of the F2:3 families were planted in 2-liter pots in the greenhouse to further examine the bloom–bloomless phenotypic and genotypic scores in the F2 generation. Determination of Seedling Water Loss Seedling water loss rates were determined from the 100 F2 thinned seedlings described above and fi ve 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 fi lter 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 AB104S, 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 R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . CROP SCIENCE, VOL. 48, JANUARY–FEBRUARY 2008 WWW.CROPS.ORG 43 Conductance Measurement Preliminary measurements of nighttime conductance were performed to determine whether variation existed among three diff erent leaf positions (second, third, and fi fth leaf ) in 10 plant samples (representing fi ve each of bloom and bloomless phenotypes) between diff erent portions of the leaves using a portable leaf poromoter (Decagon Inc., Pullman, WA). Results showed that nighttime conductance was not aff ected by leaf position and that measurement in diff erent 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 fl ag leaf between 1100 and 1500 hours on three diff erent 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 Effi ciency Characterization of water use effi ciency was performed using a modifi ed 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 fi lled with a measured amount of commercial soil mix as described in the plant materials section and watered with 0.5X Miracle-Gro (ScottsMiracle 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 fi nal pot weight (including root) was weighed. Water used was calculated by subtracting the fi nal pot weight from the initial weight. Transpiration effi ciency was calculated by dividing the total dry matter with the water used. Statistical Analysis A Chi-square analysis for goodness of fi t of the F2 and F2:3 phenotypic distributions to a 3:1 and 1:2:1 genetic ratio, respectively, 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 fi nal dry weights (DW). Relative water content was calculated as: RWC = [(FW − DW])/[TW − DW)] × 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 fl ag 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). Briefl y, 10 leaf discs from the fourth leaf (minus fl ag 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. R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . 44 WWW.CROPS.ORG CROP SCIENCE, VOL. 48, JANUARY–FEBRUARY 2008 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 signifi cance were calculated according to formula by Cochran and Cox (1957). All statistical analyses were conducted using SYSTAT 7.0 (SPSS, Chicago, IL).