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CROP SCIENCE, VOL. 47, SEPTEMBER–OCTOBER 2007 2067

RESEARCH

Assessment of heat tolerance is of primary importance in breeding programs designed to improve heat tolerance in

crop plants. Several methods and approaches are available with the most common being electroconductivity, chlorophyll a fl uor es cence, and triphenyl tetrazolium chloride (TTC) stain-ing. Electroconductivity measures electrolyte leakage from tis-sues subjected to elevated temperatures, thus, it estimates the heat stability of the plasma membrane (Sullivan, 1972; Blum and Ebercon, 1981; Ibrahim and Quick, 2001). Chlorophyll a fl uo-rescence assesses damage to photosystem II (PS II) and thylakoid membranes caused by heat (Krause and Weis, 1984; Ristic and Cass, 1993; Maxwell and Johnson, 2000; Sayed, 2003), and TTC evaluates the mitochondrial electron transport chain (Chen et al., 1982; Krishnan et al., 1989; Fokar et al., 1998).

Despite their reliability and common use, electroconductiv-ity, TTC staining, and chlorophyll a fl uorescence have some limi-tations. Electroconductivity and TTC have limited applications because of the amount of labor involved in variable fi eld conditions. Similarly, measurements of chlorophyll a fl uorescence require use of expensive instrumentation and in some cases necessitates dark adaptation of the leaf tissue, which limits the number of plants that

Correlation between Heat Stability of Thylakoid Membranes and Loss of Chlorophyll

in Winter Wheat under Heat Stress

Zoran Ristic,* Urska Bukovnik, and P.V. Vara Prasad

ABSTRACT

Determining mechanisms associated with heat

tolerance and identifying screening methods

are vital for improvement of heat tolerance

in plants. The objectives of this study were to

investigate the relationship between the heat

stability of thylakoids and loss of chlorophyll in

winter wheat (Triticum aestivum L.) under heat

stress, and to examine whether chlorophyll

loss can be used as an indicator of heat toler-

ance in wheat. We assessed heat tolerance and

measured chlorophyll content in 12 cultivars of

winter wheat at fl owering stage during expo-

sure to 16-d-long heat stress. Heat tolerance

was assessed using fl uorescence to determine

the heat stability of thylakoids, and chlorophyll

content was measured with a chlorophyll meter.

Experiments were conducted under controlled

conditions. Heat stress caused damage to thyla-

koids in all cultivars as indicated by the increase

in the ratio of constant fl uorescence (O) and the

peak of variable fl uorescence (P). Heat stress

also caused a decline in chlorophyll content

in most cultivars. A strong negative correlation

between heat-induced increases in O/P and

chlorophyll content was seen. The results sug-

gest that heat-induced damage to thylakoids

and chlorophyll loss are closely associated in

winter wheat. Measurements of chlorophyll

content with a chlorophyll meter could be useful

for high throughput screening for heat tolerance

in wheat.

Z. Ristic, USDA-ARS, Plant Science and Entomology Research Unit,

4008 Throckmorton Hall, Manhattan, KS 66506; U. Bukovnik and

P.V.V. Prasad, Dep. of Agronomy, Kansas State Univ., Manhattan, KS

66506. Received 20 Oct. 2006. *Corresponding author (zoran.ristic@

gmprc.ksu.edu).

Abbreviations: O/P, ratio of constant fl uorescence and peak of vari-

able fl uorescence; PS II, photosystem II; TTC, triphenyl tetrazolium

chloride.

Published in Crop Sci. 47:2067–2073 (2007).doi: 10.2135/cropsci2006.10.0674© Crop Science Society of America677 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.

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2068 WWW.CROPS.ORG CROP SCIENCE, VOL. 47, SEPTEMBER–OCTOBER 2007

can be screened in a given day. Therefore, there is a need to develop more effi cient, less expensive alternatives for high throughput screening for heat tolerance.

Thylakoid membranes and PS II are considered the most heat-labile cell structures (Santarius, 1974; Schreiber and Berry, 1977). In wheat (Triticum aestivum L.) and related species, for example, thylakoids are more aff ected than the chloroplast envelope, stromal enzymes, or the integrity of cell compartments (Thebud and Santarius, 1982; Monson et al., 1982; Kobza and Edwards, 1987; Sayed et al., 1989; Al-Khatib and Paulsen, 1989). Thylakoids harbor chloro-phyll, a portion of which is associated with the proteins of PS II (Schreiber and Berry, 1977; Vacha et al., 2007). Damage to thylakoids caused by heat could be, therefore, expected to lead to chlorophyll loss. Indeed, heat-induced damage to thylakoid membranes and chlorophyll loss have been observed in many crop plants including wheat (Reynolds et al., 1994; Fokar et al., 1998; Al-Khatib and Paulsen, 1984). However, to our knowledge, the relation-ship between chlorophyll loss and damage to thylakoids has not been clearly established.

The objectives of this study were to (i) investigate the relationship between the heat stability of thylakoid membranes and the loss of chlorophyll in winter wheat under heat stress conditions, and (ii) to test the possibility of using chlorophyll loss, as determined by a chlorophyll meter (Fanizza et al., 1991; Reynolds et al., 1998), as an indicator of heat tolerance in wheat. We assessed thyla-koid stability using fl uorescence and measured chlorophyll content in 12 cultivars of winter wheat under heat stress conditions.

MATERIALS AND METHODS

Plant Material and Experimental ConditionsSeeds of 12 cultivars of winter wheat were obtained from the

Institute of Field and Vegetable Crops, Novi Sad, Serbia (for

cultivar names see Fig. 1). Two independent experiments were

conducted under controlled environment conditions in the

spring of 2006. For each experiment, seeds of each cultivar

were germinated in 4-cm-deep trays containing commercial

Metro Mix 200 potting soil (Hummert Int., Topeka, KS) in a

greenhouse. Ten-day-old seedlings were vernalized at 4°C for

8 wk. Following vernalization, seedlings of each cultivar were

transplanted into 10 pots (three seedlings per pot; pot diameter

at the top and the bottom was 21 and 16 cm, respectively; pot

depth 20 cm) containing Metro Mix 200 potting soil. Plants

were grown in a greenhouse and watered daily. Miracle Gro

fertilizer (24–8–16; Stern’s Miracle-Gro Products, Inc., Port

Washington, NY) was added (according to manufacturer

instructions) once every 7 d during the entire duration of the

experiment. At the beginning of fl owering stage (50% of the

plants at growth stage Feekes 10.5.1 [Large, 1954]), plants of

each cultivar were divided into control (fi ve pots) and high-tem-

perature treatment (fi ve pots) groups. In each group, fi ve plants

were randomly selected (one plant per pot) and one fl ag leaf per

selected plant was randomly chosen and tagged (total of fi ve

fl ag leaves per group were tagged). The tagged fl ag leaves were

later used for assessment of damage to thylakoid membranes

and measurement of chlorophyll content. The control group

was maintained under growth conditions in a greenhouse, and

the treatment group was exposed to heat stress for 16 d (day/

night temperature, 36/30°C; relative humidity, 90–100%; and

photoperiod, 16/8 h; photosynthetic photon fl ux, 280 μmol

m−2 s−1 [Sylvania cool white fl uorescent lamps, Radiant Lamp

Co., Jacksonville, FL]) in a growth chamber (Conviron, Model

CMP4030, Winnipeg, MB). For each cultivar, heat treatment

started when 50% of the plants reached growth stage Feekes

10.5.1 (Large, 1954). Air temperatures, relative humidity, and

light levels were continuously monitored at 10-min intervals

during the entire period of experimentation in the growth

chamber. In the greenhouse data on air temperatures were mea-

sured at hourly intervals (the average daily temperature in the

greenhouse was 22.7 ± 2.8°C). To minimize or avoid possible

dehydration of the leaf tissue during heat treatment, all the pots

including controls were kept in trays containing approximately

1-cm-deep water and irrigation was provided every day as nec-

essary. Plants were assessed for damage to thylakoid membranes

and PS II and chlorophyll loss after 0, 8, 10, 12, 14, and 16 d of

heat stress treatment.

Assessment of Damage to Thylakoid Membranes and Measurement of Chlorophyll ContentDamage to thylakoid membranes was assessed by measuring

chlorophyll a fl uorescence on intact fl ag leaves after 1 h of dark

adaptation (Ristic and Cass, 1993). Fluorescence was measured

halfway between the base and the tip of the blade of the fl ag

leaf. The ratio of constant fl uorescence to the peak of vari-

able fl uorescence, O/P, was used to assess damage to thylakoid

membranes (Krause and Weis, 1984; Ristic and Cass, 1993).

Fluorescence measurements were conducted at room tempera-

ture (25°C) using a pulse modular fl uorometer (Model OS5-

FL, Opti-Sciences, Hudson, NH). Data were analyzed using

two approaches: (i) data from fi ve replicate plants were averaged

and used for statistical analysis, and (ii) individual data were

used for statistical evaluation.

Chlorophyll content was measured in the same fl ag leaves,

in the same blade area that was used for fl uorescence measure-

ments using a self-calibrating SPAD chlorophyll meter (Model

502, Spectrum Technologies, Plainfi eld, IL). Five fl ag leaves per

treatment (control and heat stress) were used for measurements

of each cultivar. Data from fi ve replicates were used for statisti-

cal analysis using the approach described for fl uorescence data.

Statistical AnalysisCorrelation analysis was used to test the relationship between

heat-induced damage to thylakoid membranes and loss of chlo-

rophyll. Data from two independent experiments were ana-

lyzed in two diff erent ways: (i) data from each experiment were

analyzed separately, and (ii) data from two experiments were

averaged and used for correlation analysis. PROC CORR

procedures in Statistical Analysis System (SAS Institute, 2003)

were used to quantify the relationship between the variables.

Similarly, the eff ects of heat stress and cultivars on chlorophyll

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CROP SCIENCE, VOL. 47, SEPTEMBER–OCTOBER 2007 WWW.CROPS.ORG 2069

ences in heat tolerance of cultivars may be partly due to pos-sible diff erential expression of a highly conserved (Bhadula et al., 2001) chloroplast protein, elongation factor EF-Tu. This protein has been shown to play a role in heat tolerance by acting as a molecular chaperone (Rao et al., 2004), and our recent study showed that wheat cultivars that display greater tolerance to heat stress express higher levels of EF-Tu under heat stress conditions (Ristic et al., 2006).

Heat stress also aff ected chlorophyll content in these wheat cultivars. Under control conditions no signifi cant changes in chlorophyll content were observed (data not shown). However, under heat stress conditions all culti-vars, except Ljiljana, showed progressive loss of chlorophyll over time (Fig. 1B). Cultivars that showed chlorophyll loss diff ered in their ability to retain chlorophyll under heat

a fl uorescence and chlorophyll content were analyzed using

PROC ANOVA in SAS with fi ve replications.

RESULTS AND DISCUSSIONWe assessed heat tolerance in 12 cultivars of winter wheat by estimating damage to thylakoid membranes using chlo-rophyll a fl uorescence. Heat stress caused damage to thyla-koids in all wheat cultivars as indicated by increases in O/P (Fig. 1A). However, cultivars diff ered in the extent of the damage. The greatest damage, indicating lowest tolerance to heat stress, was seen in cultivars Zlatka, Stepa, and Rana Niska (O/P > 520% after 16 d of heat stress). Relatively little increase in O/P (O/P < 175% after 16 d of heat stress) was seen in cultivars Proteinka, Ljiljana, Partizanka, Stamena, and Jefi mija (Fig. 1A). We speculate that the observed diff er-

Figure 1. (A) The ratio of constant fl uorescence and the peak of variable fl uorescence (O/P) and (B) chlorophyll content in fl ag leaves from

12 cultivars of winter wheat under heat stress conditions. Chlorophyll a fl uorescence and chlorophyll contents were measured on the

same fl ag leaves after 0, 8, 10, 12, 14, and 16 d of exposure to heat stress. Increases in O/P indicate damage to thylakoid membranes,

the greater the damage the lower the tolerance to heat stress (Ristic and Cass, 1993). Plotted data are from Experiment 1. Bars indicate

±standard errors; n = 5. Similar results were observed in a duplicate experiment.

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2070 WWW.CROPS.ORG CROP SCIENCE, VOL. 47, SEPTEMBER–OCTOBER 2007

stress (Fig. 1B). As indicated by chlorophyll content after 16 d of heat stress, the greatest loss of chlorophyll (>75%) was observed in cultivars Zlatka, Stepa, and Rana Niska, and the least amount of loss (<20%) in cultivars Proteinka, Partizanka, Stamena, and Jefi mija. The cultivar diff er-ences in chlorophyll loss seen in our study are consistent with Wardlaw et al. (1980) and Blum (1986) who demon-strated the presence of genetic variability in chlorophyll content in wheat cultivars when exposed to heat stress.

Chlorophyll loss naturally occurs in plants undergo-ing senescence (Thimann, 1987), and it can also prema-

turely occur in plants experiencing heat stress (Reynolds et al., 1994; Fokar et al., 1998; Al-Khatib and Paulsen, 1984). In our experiments, control plants of all wheat cultivars did not show any signifi cant changes in chloro-phyll content or senescence. Therefore, it is likely that the chlorophyll loss in our heat-stressed plants was primarily due to the eff ects of high temperature rather than natural senescence. The mechanism by which high temperature may have caused chlorophyll loss is, however, unclear. Al-Khatib and Paulsen (1984) and Harding et al. (1990) have suggested that a major eff ect of high temperature

Figure 2. Correlation between the ratio of constant fl uorescence and the peak of variable fl uorescence (O/P) and chlorophyll content

expressed as percentage of control (plants not exposed to heat) in the fl ag leaf from 12 cultivars of winter wheat. Data from fi ve replicate

plants of each cultivar were averaged and used for correlation analysis. Plotted data are from Experiment 1; n = 12. Similar results were

obtained in a duplicate experiment. HS, heat stress.

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CROP SCIENCE, VOL. 47, SEPTEMBER–OCTOBER 2007 WWW.CROPS.ORG 2071

on wheat is acceleration of senescence, which is manifested by an increase in the activity of pro-teolytic enzymes leading to protein degradation and chlorophyll loss. We speculate that this may be the case in our study. Alternatively, chloro-phyll loss in these wheat cultivars may be a con-sequence of heat-induced damage to thylakoid membranes and PS II. Further studies are needed to elucidate the mechanism(s) of chlorophyll loss in wheat under heat stress conditions.

We analyzed the relationship between chlo-rophyll content and damage to thylakoid mem-branes. This analysis was done by expressing chlorophyll content in heat-stressed plants in two diff erent ways and plotting it against O/P. First, we expressed chlorophyll content in heat-stressed plants as a percentage of that in control plants (no heat stress). Chlorophyll content in heat-stressed plants was also expressed as a percentage of the chlorophyll content in the same plants before the beginning of heat stress treatment (Day 0 of heat stress). The chlorophyll content in heat-stressed plants was expressed in two diff erent ways to test the possibility of using chlorophyll content at the beginning of heat stress treatment as a control. This would be useful for measurements of chlo-rophyll content under fi eld conditions where environmental factors including temperature are highly variable, making it diffi cult to have con-trol plants that do not experience heat stress. In both cases, a highly signifi cant negative linear correlation (P < 0.0001) between chlorophyll content and O/P was observed (Fig. 2 and 3, Tables 1 and 2). This correlation was evident in two independent experiments when data were plotted and analyzed for each individual day of heat stress (Fig. 2 and Table 1) as well as when data for all days of stress treatment (Days 8–16) were plotted collectively (Fig. 3 and Table 2). In addition, this correlation was also evident when both averages from fi ve replicate plants (Fig. 2 and 3, and Table 1) and individual data (Table 2) were used for cor-relation analysis.

The observed correlation between chlorophyll content and O/P suggests that under heat stress conditions loss of chlorophyll and damage to thylakoid membranes are closely associated. Moreover, this correlation also suggests that chlo-rophyll loss under heat stress can be used to indicate heat tolerance and that measurements of chlorophyll content using a chlorophyll meter will be useful as a method for high throughput screening for heat tolerance in wheat. This is par-ticularly useful due to the relatively low cost of the chloro-phyll meter compared with fl uorometers. In addition, there is no need for dark adaptation of plants before measurement.

Figure 3. Correlation between the ratio of constant fl uorescence and the peak

of variable fl uorescence (O/P) and chlorophyll content in the fl ag leaves of 12

cultivars of winter wheat. Data represent an average of two experiments in

which in each experiment data from fi ve replicate plants of each cultivar were

averaged. (A) The chlorophyll content in heat-stressed plants, measured on

Days 8, 10, 12, 14, and 16 of heat treatment, was expressed as percentage of

that in control (plants not exposed to heat stress). (B) The chlorophyll content

in heat-stressed plants, measured on Days 8, 10, 12, 14, and 16 of heat

treatment, was expressed as percentage of chlorophyll content measured in

the same plants at the beginning of heat stress (Day 0 of heat stress). Data for

all days of heat stress treatment when fl uorescence and chlorophyll content

were measured are plotted on the same graph (n = 60).

Table 1. Correlation coeffi cients of the relationship between

the ratio of constant fl uorescence and the peak of variable

fl uorescence (O/P) and chlorophyll content in wheat under

heat stress in two separate experiments when chlorophyll

content is expressed as percentage of chlorophyll content

measured in the same plants at the beginning of heat stress

(Day 0 of heat stress). Data from fi ve replicate plants of each

cultivar were averaged and used for correlation analysis.

Days of heat stress

df Experiment 1 Experiment 2

R value P value R value P value

Day 8 10 0.92 0.0001 0.79 0.0002

Day 10 10 0.76 0.004 0.62 0.034

Day 12 10 0.96 0.0001 0.80 0.002

Day 14 10 0.96 0.0001 0.79 0.002

Day 16 10 0.94 0.0001 0.57 0.95

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2072 WWW.CROPS.ORG CROP SCIENCE, VOL. 47, SEPTEMBER–OCTOBER 2007

This study revealed a quantitative relationship between the unitless SPAD chlorophyll meter readings and the physiological state of thylakoid membranes, as determined by chlorophyll a fl uorescence. Such a result is critical for using SPAD meter readings to indicate thermotolerance. Wheat cultivars that lose less chlorophyll under heat stress, as determined by SPAD, can thus be expected to be more heat tolerant than cultivars that lose more chlorophyll. The ability of a plant to retain chlorophyll under stress is generally known as the “stay-green trait” (Reynolds et al., 1997; Thomas and Howarth, 2000), and the identifi cation of plants displaying this trait could aid in producing new wheat cultivars with improved tolerance to heat stress.

Our study also revealed that chlorophyll content at the beginning of heat stress could be used as a “control” for determination of chlorophyll loss and assessment of heat tol-erance. As stated earlier, this may be of particular importance under fi eld conditions where it is diffi cult to have plants that do not experience heat stress. The exact timing (beginning and duration) of chlorophyll content measurements is diffi -cult to predict; however, it is reasonable to suggest that initial measurements should be taken when wheat begins to experi-ence temperatures that are generally considered as heat stress temperatures for wheat (≥28–32°C) (Mullarkey and Jones, 2000). Wheat may experience heat stress and suff er injury during vegetative or reproductive phases depending on the location and season (Kolderup, 1979), but most commonly it encounters stress in the later part of the growing season (Wardlaw et al., 1989), during fl owering. Hence, in most cases measurements of chlorophyll content under heat stress conditions could begin at the beginning of fl owering and continue thereafter for 7 to 21 d. Heat-induced chlorophyll loss will probably depend on environmental conditions or fi eld location and wheat heat tolerance. If fi eld air tempera-ture is not suffi ciently high or the high temperatures do not last for a prolonged period of time, loss of chlorophyll may not be observed. Therefore, it is important that measure-ments of chlorophyll content and assessment of heat tolerance are conducted in hot environments.

In summary, our study revealed a highly signifi cant negative linear correlation between chlorophyll content

and damage to thylakoid membranes and PS II in winter wheat under heat stress. Cultivars of wheat that suff ered more damage to thylakoid membranes (displayed lower tolerance to heat stress) under heat stress lost more chlo-rophyll than cultivars that suff ered less damage (displayed higher tolerance to heat stress). The results suggest that loss of chlorophyll under heat stress, as determined with a chlorophyll meter, could be used as a reliable and high throughput approach or method for screening for heat tol-erance in wheat.

AcknowledgmentsThe authors are grateful to Dr. Novica Mladenov and Dr.

Radivoje Jevtic, Institute of Field and Vegetable Crops, Novi Sad,

Serbia, for generously providing seeds of cultivars of winter wheat.

The authors are also grateful to Dr. Thomas Elthon, University of

Nebraska, Lincoln, NE; Dr. David Horvath, USDA-ARS, Fargo,

ND; Dr. Jeff rey Pedersen, USDA-ARS, Lincoln, NE; and Dr.

Kassim Al-Khatib, Kansas State University, Manhattan, KS, for

suggestions on the manuscript. This publication is approved as

Kansas Agriculture Experiment Station No. 07-90-J. Mention

of a trademark or proprietary product does not constitute a

guarantee or warranty of the product by the U.S. Department of

Agriculture, and does not imply its approval to the exclusion of

other products that may also be suitable.

ReferencesAl-Khatib, K., and G.M. Paulsen. 1984. Mode of high tempera-

ture injury to wheat during grain development. Physiol. Plant.

61:363–368.

Al-Khatib, K., and G.M. Paulsen. 1989. Enhancement of thermal

injury to photosynthesis in wheat plants and thylakoids by

high light intensity. Plant Physiol. 90:1041–1048.

Bhadula, S.K., T.E. Elthon, J.E. Habben, T.G. Helentjaris, S. Jiao,

and Z. Ristic. 2001. Heat-stress induced synthesis of chloro-

plast protein synthesis elongation factor (EF-Tu) in a heat-tol-

erant maize line. Planta 212:359–366.

Blum, A. 1986. The eff ect of heat stresses on wheat leaf and ear

photosynthesis. J. Exp. Bot. 37:111–118.

Blum, A., and A. Ebercon. 1981. Cell membrane stability as a measure

of drought and heat tolerance in wheat. Crop Sci. 21:43–47.

Chen, H.H., Z.Y. Shen, and P.H. Li. 1982. Adaptability of crop

plants to high temperature stress. Crop Sci. 22:719–724.

Fanizza, G., L. Ricciardi, and C. Bagnulo. 1991. Leaf greenness

measurements to evaluate water stressed genotypes in Vitis

vinifera. Euphytica 55:27–31.

Fokar, M., H.T. Nguyen, and A. Blum. 1998. Heat tolerance in

spring wheat: I. Estimating cellular thermotolerance and its

heritability. Euphytica 104:1–8.

Harding, S.A., J.A. Guikema, and G.M. Paulsen. 1990. Photosyn-

thetic decline from high temperature stress during matura-

tion of wheat: I. Interaction with senescence processes. Plant

Physiol. 92:648–653.

Ibrahim, A.M.H., and J.S. Quick. 2001. Genetic control of high

temperature tolerance in wheat as measured by membrane

thermal stability. Crop Sci. 41:1405–1407.

Kobza, J., and G.E. Edwards. 1987. Infl uences of leaf tempera-

ture on photosynthetic carbon metabolism in wheat. Plant

Physiol. 83:69–74.

Table 2. Correlation coeffi cients of the relationship between

the ratio of constant fl uorescence and the peak of variable

fl uorescence (O/P) and chlorophyll content in 12 cultivars of

winter wheat under heat stress. O/P is expressed as percent-

age of that in control (plants not exposed to heat stress).†

Days of heat stress

Experiment 1 Experiment 2

df R value P value df R value P value

(A) Days 8–16 296 0.58 0.0001 292 0.37 0.0001

(B) Days 8–16 296 0.59 0.0001 292 0.36 0.0001

†(A) Chlorophyll content in heat-stressed plants expressed as percentage of that in

control (plants not exposed to heat stress). (B) Chlorophyll content in heat-stressed

plants expressed as percentage of chlorophyll content measured in the same

plants at the beginning of heat stress (Day 0 of heat stress). Correlation coeffi cients

were calculated using individual data.

Repro

duced

from

Cro

pS

cie

nce.

Publis

hed

by

Cro

pS

cie

nce

Socie

tyof

Am

erica.

All

copyrights

reserv

ed.

CROP SCIENCE, VOL. 47, SEPTEMBER–OCTOBER 2007 WWW.CROPS.ORG 2073

Kolderup, F. 1979. Application of diff erent temperatures in three

growth phases of wheat. Acta Agric. Scand. 29:6–10.

Krause, G.H., and E. Weis. 1984. Chlorophyll fl uorescence as a

tool in plant physiology: II. Interpretation of fl uorescence sig-

nals. Photosynth. Res. 5:139–157.

Krishnan, M., H.T. Nguyen, and J.J. Burke. 1989. Heat shock

protein synthesis and thermal tolerance in wheat. Plant

Physiol. 90:140–145.

Large, E.C. 1954. Growth stages in cereals. Plant Pathol. 3:128–129.

Maxwell, K., and G.N. Johnson. 2000. Chlorophyll fl uorescence:

A practical guide. J. Exp. Bot. 51:659–668.

Monson, R.K., M.A. Stidham, G.J. Williams, and G.E. Edwards.

1982. Temperature dependence of photosynthesis in Agropyron

smithii Rydb.: I. Factors aff ecting net CO2 uptake in intact leaves

and contribution from ribulose-1,5-biphosphate carboxylase

measured in vivo and in vitro. Plant Physiol. 69:921–928.

Mullarkey, M., and P. Jones. 2000. Isolation and analysis of ther-

motolerant mutants of wheat. J. Exp. Bot. 51:139–146.

Rao, D., I. Momcilovic, S. Kobayashi, E. Callegari, and Z. Ristic.

2004. Chaperone activity of recombinant maize chloroplast

protein synthesis elongation factor, EF-Tu. Eur. J. Biochem.

271:3684–3692.

Reynolds, M.P., M. Balota, M.I.B. Delgado, I. Amani, and R.A.

Fisher. 1994. Physiological and morphological traits associated

with spring wheat yield under hot, irrigated conditions. Aust.

J. Plant Physiol. 21:717–730.

Reynolds, M.P., S. Nagarayan, M.A. Razzaue, and O.A.A. Ageeb.

1997. Using canopy temperature depression to select for yield

potential of wheat in heat-stressed environments. Wheat Spe-

cial Rep. No. 42. CIMMYT, Mexico.

Reynolds, M.P., R.P. Singh, A. Ibrahim, O.A.A. Ageeb, A.

Larque-Saavedra, and J.S. Quick. 1998. Evaluating physi-

ological traits to complement empirical selection for wheat in

warm environments. Euphytica 100:84–95.

Ristic, Z., and D.D. Cass. 1993. Dehydration avoidance and dam-

age to the plasma and thylakoid membranes in lines of maize

diff ering in endogenous levels of abscisic acid. J. Plant Physiol.

142:759–764.

Ristic, Z., I. Momcilovic, J. Fu, U. Bukovnik, A. Fritz, M.A.

Babar, and P.V.V. Prasad. 2006. Heat tolerance and relative

levels of chloroplast protein synthesis elongation factor, EF-

Tu, in wheat under heat stress conditions. P09036. In Plant

Biology 2006 Final Program, Boston. 5–9 Aug. 2006. Am.

Soc. of Plant Biologists, Rockville, MD.

Santarius, K.A. 1974. Seasonal changes in plant membrane stability

as evidenced by the heat sensitivity of chloroplast membrane

reactions. Z. Pfl anzenphysiol. 73:448–451.

SAS Institute. 2003. User manual for SAS for windows version 8.

SAS Inst., Cary, NC.

Sayed, O.H. 2003. Chlorophyll fl uorescence as a tool in cereal

crop research. Photosynthetica 41:321–330.

Sayed, O.H., M.J. Earnshaw, and M.J. Emes. 1989. Photosynthetic

responses of diff erent varieties of wheat to high temperature:

II. Eff ect of heat stress on photosynthetic electron transport. J.

Exp. Bot. 40:633–638.

Schreiber, U., and J.A. Berry. 1977. Heat-induced changes of chlo-

rophyll fl uorescence in intact leaves correlated with damage of

the photosynthetic apparatus. Planta 136:233–238.

Sullivan, C.Y. 1972. Mechanisms of heat and drought resistance in

grain sorghum and methods of measurement. In N.G.P. Rao

and L.R. House (ed.) Sorghum in the seventies. Oxford and

IPH Publishing Co., New Delhi, India.

Thebud, R., and K.A. Santarius. 1982. Eff ects of high-tempera-

ture stress on various biomembranes of leaf cells in situ and in

vitro. Plant Physiol. 70:200–205.

Thimann, K.V. 1987. Plant senescence: A proposed interaction of

the constituent. p. 1–19. In W.W. Thomson, E.A. Nothnagel,

and R.C. Huff aker (ed.) Plant senescence: Its biochemistry

and physiology. Am. Soc. of Plant Physiologists, New York.

Thomas, H., and C.J. Howarth. 2000. Five ways to stay green. J.

Exp. Bot. 51:329–337.

Vacha, F., F. Adamec, J. Valenta, and M. Vacha. 2007. Spatial loca-

tion of photosystem pigment–protein complexes in thylakoid

membranes of chloroplasts of Pisum sativum studied by chloro-

phyll fl uorescence. J. Luminesc. 122–123:301–303.

Wardlaw, I.F., I.A. Dawson, P. Munchi, and R. Fewster. 1989.

The tolerance of wheat to high temperatures during repro-

ductive growth: I. Survey procedures and general response

patterns. Aust. J. Agric. Res. 40:1–13.

Wardlaw, I.F., I. Sofi eld, and P.M. Cartwright. 1980. Factors limiting

the rate of dry matter accumulation in the grain of wheat grown

at high temperature. Aust. J. Plant Physiol. 7:387–400.