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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.
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