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Univ. of Guelph, Dep. of Plant Agriculture Guelph, Ontario, Canada N1G 2W1
* Corresponding author (lizlee{at}uoguelph.ca)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: CER, carbon exchange rate • 
PSII, quantum efficiency of Photosystem II • DAP, days after planting • PPFD, photosynthetic photon flux density • IRGA, infrared gas analyzer • DW, dry weight • CO2, quantum yield of CO2 assimilation
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Substantial efforts have been dedicated to understanding cold tolerance in maize, primarily concentrating on the germination to 3-leaf stage of seedling development, a period of time during which the plant relies in part on seed reserves for its assimilate supply. Numerous cold tolerance discriminators for maize have been examined, covering four aspects of growth and development: (i) germination: the ability to germinate under suboptimal temperature conditions (McConnell and Gardner, 1979), percent emergence, and emergence rate (Mock and McNeill, 1979); (ii) survival: leaf viability and number of dead plants (Aidun et al., 1991); (iii) rate of development: rate of development and shoot length (Aidun et al., 1991); and (iv) dry matter accumulation: photosynthesis and respiration (Miedema and Sinnaeve, 1980), susceptibility to and recovery from photoinhibition (Hetherington et al., 1989; Aguilera et al., 1999), and seedling dry weight (Mock and McNeill, 1979).
Several basic physiological parameters such as CER and its component processes are known to be affected by cold stress. Maize leaf CER decreases substantially when plants are subjected to cold stress (Tollenaar, 1989b; Long et al., 1983; Janda et al., 1998; Ying et al. 2000). The mechanism responsible for the reduction of leaf CER at low temperatures is in dispute, as stomatal closure (Massacci et al., 1995), reduced chlorophyll content (Leipner et al., 1999), reduced ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity (Janda et al., 1999), and reduced quantum efficiency of photosystem II (PSII) (Hetherington et al., 1983; Fracheboud et al., 1999; Leipner et al., 1999) have all been proposed as possible candidates.
The effect of cold stress on dry matter accumulation varies among maize genotypes (Stamp, 1980). Optimal temperature for total dry matter accumulation is between 15 and 23°C, and dry matter partitioning varies with temperature and phase of development (Tollenaar 1989a). Dry matter in young maize is either partitioned to the roots or the shoots. Generally, the root/shoot dry matter ratio increases with decreasing temperature (Miedema, 1982; Hardacre and Eagles, 1986; Tollenaar, 1989a; Richner et al., 1996). A shift to a higher root/shoot ratio at lower temperatures may result in lower dry matter accumulation since a lower partitioning of dry matter to the leaves can result in lower leaf area and lower interception of incident solar radiation.
Evidence of genetic variation has been observed for many of the physiological discriminators examined (Thiagarajah et al., 1979; Miedema, 1982; Massacci et al., 1995; Greaves, 1996; Pietrini et al., 1999; Kingston-Smith et al., 1999; Aguilera et al., 1999; Haldimann, 1999). In this study, we examined a cold stress scenario of favorable early spring temperatures allowing the plants to develop to a stage where they are autotrophs, followed by a cold stress. Field-grown maize may experience such conditions in Ontario during cool spells in late May or early June. Our objectives were to place genetically diverse autotrophic maize plants under cold stress conditions and (i) examine a set of interrelated physiological parameters and (ii) quantify genetic variability in the response to cold stress. Understanding what physiological parameters are being affected in an autotrophic maize plant under cold stress conditions and identifying a useful set of physiological discriminators should lead to developing more efficient breeding schemes for cold tolerance in maize.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Treatments and Experimental Design
The experimental design was a split-plot randomized complete-block design replicated eight times and consisting of two treatments. Temperature treatments were whole units while the inbred lines within a temperature treatment were the subunits. Subunit size was a pot with one plant. Treatments consisted of two temperature regimes, control (25/15°C; 16-h photoperiod) and cold stress (15/3°C; 16-h photoperiod). Fifty maize inbred lines from diverse genetic sources were used in the experiment (Table 1)
. One replication consisted of two growth cabinets each containing a complete set of inbred lines. At the start of a replication, one cabinet was designated control and the other was designated cold stress. Inbred seed was produced in the breeding nursery during the summer of 1999 at the Cambridge Research Station. Beginning approximately 14 days after planting (DAP), the leaf stage of each plant was recorded twice daily. Initially, all plants were grown under control conditions. The number of days from planting to the 7-leaf stage was recorded for each plant. Once a cold treatment plant reached the 7-leaf stage, it was immediately placed in a cabinet set for the cold stress conditions while control treatment plants remained under the 25/15°C temperature regime.
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Leaf Carbon Exchange and Leaf Conductance
Leaf CER and leaf conductance were measured on a 6-cm2 portion of the leaf with a portable, open-flow gas exchange system (LI-6400, LI-COR Inc.). PPFD at the leaf surface was maintained at 600 µmol m-2 s-1 by means of the 6400-02 LED light source. The CO2 concentration at the reference infrared gas analyzer (IRGA) was maintained at 350 µmol mol-1 by means of a 12-g CO2 cylinder and the 6400-01 CO2 injector, with the airflow rate through the chamber maintained at 500 µmol s-1. Block temperature (at the IRGAs) for the control and cold-stress measurements was maintained at 25 and 15°C, respectively. Ambient humidity was used for all measurements. Carbon exchange rate and leaf conductance were recorded when the LI-COR system was stable with a CV of less than 1%. Leaf CER and leaf conductance were calculated by the LI-6400 operating software, according to the method of von Caemmerer and Farquhar (1981). Sample and reference IRGAs were matched after every six measurements within a treatment, or when switching between control and cold stress measurements.
Light Adapted Chlorophyll Fluorescence
Quantum efficiency of PSII was measured with the Photosynthesis Yield Analyzer Mini-PAM (Heinz Walz GmbH, Effeltrich, Germany), according to Genty et al. (1989). Gain, damping, and measuring intensity settings used were 4, 2, and 12, respectively. The measuring light modulation frequency was set at 20 kHz, and a saturating pulse of approximately 4000 µmol m-2 s-1 PPFD and 0.8 s in duration was used to induce maximum fluorescence. Measurements were made on light adapted leaves in the growth cabinet. The fibre optic probe was attached to the 2030-B Leaf-Clip Holder (Heinz Walz GmbH) and held at a 60° angle relative to the leaf sample plane. To ensure a stable PPFD reading, the leaf was held in place in the leaf clip for about 3 s before a measurement was triggered. The 1.5-mm microquantum sensor, located in the center of the measurement area on the Leaf-Clip Holder, was calibrated weekly by means of the LI-COR Point Quantum Sensor (Model LI-185 B). Three separate measurements were made for each leaf and averaged. Two PSII readings were taken on either side of the midrib about halfway between the point of attachment and the leaf tip, and one was taken near the leaf tip.
Chlorophyll Content
The Minolta SPAD-502 Chlorophyll Meter (Minolta Corporation, Ramsey, NJ) was used to quantify chlorophyll content of the leaf. Five measurements, at random locations along the leaf, were taken for each plant and averaged.
Dry Weight and Root-Shoot Ratio
Intact plants were taken out of the pots and the roots were washed to remove the turface. Roots were separated from shoots (leaves and stem), and both roots and shoots were dried for 2 wk at 80°C. Root and shoot DW were measured, and total DW and root/shoot ratios were calculated.
Data Analysis
Data were analyzed by the general linear models procedure (PROC GLM) of SAS (SAS Institute, 1996, Cary, NC). Variance was partitioned into replication, treatment, replication x treatment, genotype, and treatment x genotype effects. Replication and treatment effects were tested by the replication x treatment mean square error (error A), genotype effects were tested using within treatment replication x genotype mean square error (error B). Inbred line CG95 was removed from the analysis because of poor seedling health, leaving 49 inbred lines in the experiment. Simple phenotypic correlations between physiological parameters were computed by the SAS CORR procedure. To allow relative comparison of all traits among genotypes within a treatment, percent deviation from the entry mean for each parameter was calculated as (genotype value - mean of 49 genotypes) x 100/(mean of 49 genotypes).
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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PSII, and leaf conductance values were higher and the ranges were narrower for control plants than for cold stressed plants (Table 3)
. Rate of leaf appearance from the 7- to 8-leaf stage for the control plants was similar to rate of leaf appearance from planting to the 7-leaf stage (0.38 leaves d-1) and rate of leaf appearance in the cold-stress treatment was 0.13 leaves d-1. Rate of leaf appearance from planting to the 7-leaf stage did not differ between the two treatments, indicating that plants of the two treatments were uniform at the onset of the cold stress. Root/shoot ratios, on average, were not affected by cold stress, though among inbred lines some genotypes responded differently to cold stress. This is in contrast to results from several studies that have shown that root/shoot ratios in maize increase with decreasing temperatures (Miedema, 1982; Hardacre and Eagles, 1986; Tollenaar, 1989a). The period of stress in this study may not have been long enough to significantly affect the partitioning pattern of the genotypes.
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Interrelationship between Physiological Traits—Control Plants
Dry Matter Accumulation
Dry weight at a given stage of development is the result of leaf area for photon capture, leaf photosynthesis, measured as leaf CER, and the duration from planting to that stage. Leaf area of inbred lines was not measured. However, the relative value for leaf area among inbred lines may be inferred from the root/shoot ratio, as higher dry matter partitioning to the shoot will, in general, result in a greater leaf area. Dry weight was positively correlated (P < 0.01) with leaf CER (r = 0.63) and days from planting to 7-leaf stage (r = 0.53), and negatively correlated with root/shoot ratio (r = -0.33). The five inbred lines with the highest DW also had higher than average CER, lower than average root/shoot ratio, and higher than average days from planting to 7-leaf stage (Fig. 1)
. Again, this fits the pattern where inbred lines with higher CER, more leaf area, and more time during which to fix CO2 are able to accumulate more dry matter. The same associations, though in opposite directions, were present for the five worst ranking inbred lines with respect to DW. All five inbred lines had lower than average CER and higher than average root/shoot, and four of the five inbred line had lower then average days from planting to the 7-leaf stage.
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PSII, and leaf conductance were measured in this study. There were significant (P < 0.01) positive correlations between CER and SPAD value (r = 0.58), CER and PSII (r = 0.47), and CER and leaf conductance (r = 0.83) for the control treatment. The association between CER and its component processes was apparent for the extremes in CER among the inbred lines (Fig. 2) . The top five inbred lines, ranked for deviation from mean CER, all have higher than average leaf conductance. The trend was less obvious for SPAD and PSII, for which only two of the top five inbred lines had appreciably higher than average values. The five worst inbred lines ranked for percent deviation from mean CER, had consistently lower than average leaf conductance, meaning less CO2 was available to the leaf for carbon fixation. Furthermore, three of the worst five ranking inbred lines had lower than average chlorophyll content, as measured by SPAD value, and all five inbred lines had lower than average PSII. However, the SPAD and PSII percent deviations from the mean for four of these five inbred lines were 10% or less. This may not be surprising, as the ranges of SPAD values and PSII were not very large for plants grown under control treatment conditions (Table 3). It appears that the three components of photosynthesis can independently influence CER in that average values for some factors can be compensated for by higher than average values of others. In other words, despite the overall positive correlation between CER and its components, it is apparent that there are deviations from the rule due to the genetic variability of the 49 inbred lines.
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PSII (r = 0.73), and leaf conductance (r = 0.64) were positively correlated (P < 0.01) in the cold treatment. The correlations between CER and SPAD were higher for the cold than for the control treatment. At low levels of SPAD the amount of chlorophyll may become limiting to CER due to low photon capture. Under optimal conditions, when photon capture by the leaf is close to maximum, a difference in chlorophyll content would have little impact on CER. Because enzyme kinetics are temperature dependent, cold stress can also impact photosynthesis through decreased enzyme activities (Pietrini et al., 1999; Kingston-Smith et al., 1997). The association between CER and its component processes was also apparent for the extremes in CER among the inbred lines under the cold treatment (Fig. 5) . In all cases for the five best inbred lines ranked for CER, the three components of photosynthesis were at least 10% above average. Similarly, the three components of photosynthesis were all below average for the five lowest ranking inbred lines, though for some they were less than 10% below average. W23, CO328, and CG58 were in the top five ranking inbred lines for both control and cold treatments, whereas CO330 and CG74 were in the bottom five ranking inbred lines for both treatments. It seems that, in general, genotypes with low leaf CER under control treatment conditions also have low leaf CER under cold treatment conditions, and conversely, ones with high leaf CER under control treatment conditions usually also have high leaf CER under cold treatment conditions.
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On the basis of percent reduction in leaf CER, only one genotype stood apart from the others. Superficially, CG24 looks to be a cold-tolerant genotype with only a 16.2% reduction in leaf CER. All other genotypes had percent reductions in CER between 44.1 and 74.6%. Under cold conditions, CG24's leaf CER was one of the highest (9.3 µmol m-2 s-1), which is very desirable. However, the scenario under which we are screening for cold tolerance assumes that the suboptimal temperatures will not persist for the entire life cycle of the plant. Therefore, CG24's leaf CER under control conditions should be taken into account. Herein lies the problem. Under control conditions, CG24 had the third lowest ranking CER (11.8 µmol m-2 s-1), and it is that low control CER value that has created the appearance of cold tolerance for the leaf CER variable. Apparent cold tolerance of leaf CER at the expense of performance under optimal conditions is not a desirable avenue for achieving cold tolerance.
Ratio of Quantum Efficiency of Photosystem II (PSII) and Quantum Yield of CO2 Assimilation (CO2)
The ratio of quantum yields of Photosystem II and of CO2 assimilation (PSII/CO2) has been shown to be quite stable at about 12 over a range of physiological conditions in fully expanded maize leaves (Edwards and Baker, 1993; Earl and Tollenaar, 1998), indicating that CO2 assimilation in maize could be predicted from fluorescence analysis. The quantum yield of CO2 assimilation (CO2) can be estimated from CER, respiration in the light (RL), which is assumed here to be equal to the dark respiration of mature maize leaves (RD = 1.98 µmol CO2 m-2 s-1) reported by Earl and Tollenaar (1998), incident PPFD (600 µmol m-2 s-1), and leaf absorptance (ABSL), which is estimated according to the method of Earl and Tollenaar (1997) from SPAD values: CO2 = (CER + RL)/(PPFD x ABSL). Estimated values of CO2 and measured values of PSII were used to compute the PSII/CO2 ratio (data not shown). The mean PSII/CO2 ratio for the control treatment (13.4) was not significantly different from that for the cold treatment (14.1), but the range of the PSII/CO2 ratio for the inbred lines was large for both the control treatment (10.9–17.2) and the cold treatment (10.4–19.8). Some of the variation in the ratio in this study may be associated with possible differences in leaf temperature when measuring leaf CER (when temperature was controlled) and when measuring chlorophyll fluorescence (when temperature was not controlled). It was assumed in the estimation of the PSII/CO2 ratio that respiration of the inbred lines did not differ and the large variation in the ratio among the inbred lines in this study is most likely related to respiration. Lack of significance between the two treatments and large ranges across inbred lines, indicate that PSII per se may not be a good measure to differentiate maize inbred lines for CO2 assimilation.
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Leaf CER response to cold-stress relative to control conditions was useful in discriminating cold tolerance among inbreds, as there were significant treatment x genotype interactions for inbreds. Despite positive correlations between leaf CER and its component processes, any of these processes, taken alone, could not be used to predict accurately a genotype's CER relative to other genotypes. Though enzyme activity was not accounted for in this study, the other three component processes of leaf CER when taken together could often be used to explain the relative CER ranking of genotypes.
Rate of development from 7- to 8-leaf stage under cold stress was the other promising physiological discriminator identified in this study. Rate of development identified a different set of genotypes, than were identified by CER. Inbred lines among the extremes for CER response to cold stress were not necessarily among the extremes for the response to cold stress of duration from 7- to 8-leaf stage. Whether maintenance of CER or rate of development during cold stress is the more desirable trait for cold tolerance during early phases of development may depend on the duration of the cold stress and the recovery time required for these traits upon resumption of optimal conditions.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication October 1, 2001.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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