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Heat Stress during Grain Filling in Maize: Effects on Kernel Growth and Metabolism

THE AVERAGE TEMPERATURE the during grain-filling period of maize in the U.S. Corn Belt is above optimum (22.5 [degrees] C) for maximum dry matter accumulation in kernels, resulting in a reduction in grain yield (Thompson, 1986). An estimation based on crop production and meteorological records, indicates that a 6 [degrees] C rise in temperature from 22 to 28 [degrees] C during grain filling results in a yield loss of [approximately equals]10% in the U.S. Corn Belt (Thompson, 1966). In a field study of maize, Muchow (1990) did not see a yield loss associated with high temperature during grain filling. Muchow (1990) found that during five growing seasons in Northern Australia, yield was unaffected by temperatures, which ranged from 25.4 to 31.6 [degrees] C during the period from pollination to 80% maximum grain size. A growth chamber study by Badu-Apraku et al. (1983) shows a more dramatic yield loss associated with high temperatures during the period of grain filling. They observed a 42% loss in grain weight per plant when day/night temperature from 18 d post-silking to maturity was increased from 25/15 to 35/15 [degrees] C, a 6 [degrees] C rise in average daily temperature.

The interaction of heat stress with other environmental factors in the field, such as drought stress, makes it difficult to study the effect of high temperature on maize yield in isolation. Furthermore, it may be difficult to separate the effects of heat stress occurring during grain filling from a previously occurring heat stress. The use of controlled environments makes it possible to study more precisely how high temperature treatment affects maize grain filling. However, controlled-environment studies should strive to mirror conditions in the field as closely as possible. As described below, conditions of root temperature and photyosynthetically active radiation (PAR) intensity can differ between the field and controlled environment and have been shown to be important factors that help determine how plants respond to high temperature.

Roots growing under a canopy of maize plants in the field are well buffered from extremes in air temperature during grain filling (J.K. Radke, 1995, personal communication). The buffering capacity of the soil to extremes in air temperature is well illustrated by Radke's average daily maximum/minimum temperature recordings during the month of August in 1992 and 1994 at the canopy level (29.3/13.9 [degrees] C) and at soil depths of 5 cm (24.7/ 17.8 [degrees] C), 15 cm (21.8/19.3 [degrees] C), and 50 cm (19.6/19.2 [degrees] C). In contrast, the root temperature of plants grown in pots will reflect the ambient temperature unless efforts are made to control soil temperature independently of temperature experienced by aerial portions of the plant. Furthermore, roots have been shown to be highly sensitive to high temperature (e.g., [is greater than] 25 [degrees] C), especially compared with plant shoots. Kuroyanagi and Paulsen (1988) studied the effects of four root/shoot temperatures (25/ 25, 35/25, 25/35, and 35/35 [degrees] C) during grain filling on dry weight accumulation of developing wheat (Triticum aestivum L.) grains. Grain dry weight of plants with roots held at 35 [degrees] C was dramatically reduced when compared with plants with roots at 25 [degrees] C. However, in plants with roots at the lower temperature, a 10 [degrees] C shift in shoot temperature had a smaller effect on grain growth. Roots of tepary bean (Phaseolus acutifolius Gray) and common bean (Phaseolus vulgaris L.) were also found to be more sensitive to high temperature than plant shoots (Udomprasert et al., 1995).

Plants exposed to the low PAR intensities occurring under growth chamber conditions are less tolerant of high temperature stress. Wardlaw (1970) demonstrated that low PAR intensity during grain filling of wheat significantly reduced grain dry weight, with the effect most pronounced at high temperature. Spiertz (1977) exposed wheat plants grown in a growth chamber to 12 combinations of temperature and PAR following anthesis. The study showed that low PAR intensities decreased grain growth by a greater amount under high temperature than under normal temperature. For the aforementioned reasons, root temperatures and PAR intensity comparable to the field should be incorporated into an experimental design to portray realistic effects of high temperature stress on maize plants.

The mechanisms in cereals that are affected by heat stress and limit kernel growth are not well understood. Mature kernel dry weight is determined by the product of the rate and duration of grain growth, both of which are influenced by temperature (Tashiro and Wardlaw, 1989). Generally, in lower temperature ranges ([approximately equals] 10-25 [degrees] C), cereals respond to increasing temperature with an increase in the rate of grain filling per unit of time. At temperatures greater than a critical maximum ([approximately equals]2535 [degrees] C), the gain in the rate of grain filling begins to diminish, and at supra-optimal temperatures ([approximately equals]4045 [degrees] C) the grain-filling rate drops precipitously. Rising temperatures also produce a progressive decline in grain-filling duration. Hence, at high temperatures, yield losses are the result of a loss in both grain-filling rate and duration.

Grain-filling duration may be determined by a number of factors including sucrose availability to the kernel (Afuakwa et al., 1984) and activity levels of enzymes involved in sugar and starch metabolism in the kernel (Singletary et al., 1994). Similarly, the rate of grain filling may be affected by sucrose concentration in the kernel (Jenner, 1970) and activity levels of enzymes in the pathway of starch biosynthesis (Jenner et al., 1993; Keeling et al., 1993, 1994).

The effects of temperature on starch biosynthesis in the kernel have received much attention because starch accounts for most of the dry weight in cereal grains. In wheat grains, losses in starch accumulation caused by heat stress are believed to be linked to a reduction in the activity of soluble starch synthase (SSS) (Hawker and Jenner, 1993; Jenner et al., 1993; Keeling et al., 1993). The mechanisms limiting starch synthesis in chronically heat-stressed maize kernels are not so well understood. Singletary et al. (1994) studied 13 enzymes of sugar and starch metabolism in maize kernels grown in vitro and exposed to a range of chronic heat stresses. The activities of ADPglucose pyrophosphorylase (AG-Pase) and SSS were reduced the most, and their activities were prematurely terminated compared with other enzymes. The authors concluded that reductions in starch synthesis under heat stress are closely tied to the duration of activity for these enzymes. Keeling et al. (1994) assayed 11 enzymes of starch synthesis extracted from kernels exposed to a short-term (3 h) high temperature stress in vitro. The activity of SSS was reduced most by high temperature and reached a maximal rate at 25 [degrees] C. Other enzymes (with the exception of branching enzyme) increased in activity up to a temperature of 45 [degrees] C. Reductions in the rate of SSS were similar to losses in the rate of starch synthesis caused by heat stress.

The objective of this study was to extend our understanding of the effects of chronic high temperature during grain filling on kernel growth, composition, and starch metabolism in maize plants. Unlike previous controlled-environment studies, we independently regulated root zone temperatures and PAR to simulate field conditions. To do this, plants were greenhouse-grown during summer and early fall for a high PAR level, and root zone temperature was maintained at day/night temperatures of 25/20 [degrees] C for both temperature treatments. Seven inbred lines, representing different heterotic groups, were selected for the study.


Plant Materials and Treatment

Plants of the seven maize inbred lines (ICI04, ICI12, ICI63, ICI66, ICI94, ICI95, and ICI98) were grown individually in 10-L pots in greenhouses at the ICI Seeds research site in Slater, IA and were exposed to a 25/20 [degrees] C day/night temperature regime prior to initiation of the temperature treatments. Plants were fertilized at planting and at 30-d intervals thereafter with 15 g of controlled-release 19-6-12 Osmocote (Grace Sierra, Milipitas, CA), 5 g of 0-46-0 (Lange-Stegman, St. Louis, MO). 1.67 g of 0-0-62 (Lange-Stegman, St. Louis, MO), and 1.6 g of Sprint 330 (10% chelated Fe) (Ciba-Geigy, Greensboro, NC) per pot. To prevent the growth of soil pathogens, 0.225 g of Banrot (5-ethoxy-3-trichioromethyl-1, 2, 4-thiadiazole and thiophanate-methyl) suspended in 0.5 L of water was applied to each pot at 3 and 6 wk after planting. Sibling pollinations were performed at 1 and 3 d following the extrusion of silks, and only plants with well-filled ears were used in the temperature treatments. Plants were kept well watered and relative humidity was maintained between 50 and 80% to avoid drought stress. The two temperature treatments began at 15 DAP and lasted until maturity. At 15 DAP, six plants of good health were chosen from each inbred line and divided equally between a high temperature (mean daily temperature of air: 33.5/25 [degrees] C, and root zone: 25/20 [degrees] C) greenhouse and an adjacent control (mean daily temperature of air: 25/20 [degrees] C, and root zone: 25/20 [degrees] C) greenhouse (Fig. 1). Temperature treatments were based on a 16-h photo/thermal period. They are hereafter referred to as 34/25 [degrees] C and 25/20 [degrees] C. The high temperature treatment represented a chronic heat stress of greater duration than normally experienced in the U.S. Corn Belt, but which may occur in maize producing areas in the Southern United States. The high temperature treatment was established to produce a noticeable temperature effect while remaining in a range of agronomic importance. The control treatment was representative of a near-optimal grain-filling temperature (Thompson, 1966). Root zone temperature was established at 25/20 [degrees] C on the basis of estimates of actual root zone temperatures that normally occur under a canopy of maize during grain filling (see above).


Root zone temperature was controlled by growing plants in specially designed "rootboxes" constructed of two layers of 5-cm-thick polystyrene insulation boards fashioned into a 3.5 by 0.6 by 0.6 m rectangular box. The lids of each box had eight 10-cm-diameter openings that allowed the aerial portion of the plant to grow outside of the box. Lids were divided in half for access to the pots. On one end of each rootbox was a 15-cm-diameter opening for circulating air into the box. Air from the control house was delivered to rootboxes in both the control house and the adjacent high temperature house through a manifold located in the control greenhouse. The manifold was a 2 by 0.6 by 0.6 m box, constructed of the same material used for rootboxes, with five blowers mounted on top. The manifold was connected to rootboxes with insulated tubing. In both greenhouses, a sensor to monitor air temperature was placed at ear height and a sensor to monitor soil temperature was buried at the 5-cm depth in one pot. The sensors were connected to a LI-COR (LI-COR, Lincoln, NE) model LI-1000 data logger.

Analyses of variance were conducted for each variable of response in a split-plot experimental design. The main plot was a greenhouse divided by a glass wall into a control temperature room and a high temperate room. Temperature treatments (two) were assigned to the main plots, and inbreds (seven) were assigned to subplots. Subplots consisted of three plants per inbred, and analyses of variance were determined using average values of the three plants. Plants within each temperature treatment were randomly placed and rerandomized weekly. The main plots were replicated across three planting dates in 1994:6 May, 22 June, and 27 July. A total of 123 plants were used in this study.

Kernel Sampling and Analyses

Kernels were collected from maize ears at three dates during grain filling determined by the accumulation of HU following pollination (Table 1). The daily accumulation of HU was calculated with the following formula:

[1] Accumulated HU = (ADT - 10 [degrees] C)D

where ADT is the average daily temperature recorded on an hourly basis (constant during the experiment), 10 [degrees] C is the base temperature for maize growth (Cross and Zuber, 1972), and D is the accumulation of time in days. A fourth and final harvest was collected following black-layer formation.

Table 1. Kernel collection schedule. For both treatments, harvest times were based on the accumulation of heat units (HU) following pollination.
                                    Days after pollination
           Accumulated HU
Harvest   (both treatments)   25/20 [degrees] C   34/25 [degrees] C

1                190                 15                  15
2                250                 20                  18
3                320                 25                  21
4             maturity            maturity            maturity

Kernels were collected according to the procedure of Duncan and Hatfield (1964). For each sampling date, at -8 h into the light cycle, a strip of husk was peeled from the proximal end of the ear to expose two rows of kernels, and 10 to 15 kernels were removed from a single row in the central portion of the ear. Kernels were immediately frozen in liquid N and stored at -80 [degrees] C. Ears were sprayed with 0.440 g [L.sup.-1] Benlate 50W (methyl 1-[(butylamino)carbonyl)]-1H-benzimidazol-2ylcarbamate(CA)) fungicide to prevent fungal growth, and the husk was returned to its original place and secured to the ear with rubber bands.

The rate and duration of grain filling was calculated according to Johnson and Tanner (1972). Rate was calculated on the basis of HU (mg [kernel.sup.-1] [HU.sup.-1]) and on the basis of time (mg [kernel.sup.-1] [d.sup.-1]). Duration was expressed as total HU accumulated and as total days.

Density of mature grains was measured with a multi-pycnometer, model MVP-1 (Quantachrome Corp., Syosset, NY). Protein, oil, and starch concentrations of mature kernels were determined with an Instalab 600 NIR product analyzer, model 15299-1810V5 (Dickey-John Corp., Auburn, IL).

Enzyme Extraction and Assay

All chemicals and enzymes were obtained from Sigma Chemical Co., St. Louis, MO. The radiochemical adenosine diphospho-D-[[U.sup.-14]C]Glc (1.06 x [10.sup.13] Bq [mol.sup.-1]) was obtained from Amersham Corporation, Arlington Heights, IL. All tissue used in enzyme assays was from kernels collected at the second harvest (250 postpollination accumulated HU). After removing the pericarp and embryo from frozen kernels with forceps, the endosperm was lyophilized, finely ground in liquid N with a mortar and pestle, and returned to -80 [degrees] C.

The enzyme extract was prepared by adding 100 mg of endosperm tissue to 2 mL of extraction buffer [50 mM Hepes-NaOH (pH 7.5), 5 mM Mg[Cl.sub.2], 1 mg [mL.sup.-1] bovine serum albumin, 1 mM dithiothreitol], and homogenizing at 25 000 rpm for 20 s (PowerGen 700, Omni International, Warrenton, VA). The homogenate was centrifuged (30 000 x g, 20 min., 4 [degrees] C) and the supernatant saved for assay of soluble enzymes. The activities of ATP-dependent fructokinase, UTP-dependent fructokinase, glucokinase, and sucrose synthase were measured in dialyzed extracts, with dialysis conducted overnight in extraction buffer at 4 [degrees] C.

Assays used to measure ATP-linked fructokinase, UTP-linked fructokinase, glucokinase, sucrose synthase, PPi-linked phosphofructokinase, ATP-linked phosphofructokinase, and AGPase were described by Singletary and Below (1990), with the modification of 40 mM UDP for the sucrose synthase reaction mix. Phosphoglucomutase, phosphoglucoisomerase, and UDPglucose pyrophosphorylase were assayed by the procedures of Doehlert et al. (1988) using 100 mM Hepes (pH 7.7), 1 mM UDP-Glc, 50 [micro]M Glc-l,6-bisphosphate, and 2.5 U [mL.sup.-1] of the coupling enzymes. With the exception of sucrose synthase, these enzyme activities were measured by mixing reactions in a microtiter plate and monitoring the change in absorbance at 340 nm with a BIO-TEK Instruments (Winooski, VT) Model EL 340 automated microplate reader. Substrate-independent background rates were measured and subtracted from the complete assay to obtain the enzyme activity. To minimize variability between triplicate assays run in micro titer wells, Tween-20 (2 x [10.sup.-5] mL [mL.sup.-l] final concentration) was included in reaction mixtures.

The activity of SSS was determined using 50 [micro]L of undiluted extract as part of a 200-[micro]L reaction mix [100 mM Bicine (pH 8.3), 500 mM sodium citrate, 5 mM EDTA (ethylenediaminetetraacetic acid), 10 mM glutathione, 10 mg [mL.sup.-1] rabbit liver glycogen, and 1 mM ADP-Glc ([sup.14]C), 220 disintegrations [min.sup.-1] [nmol.sup.-1]]. Radiolabeled ADP-Glc was added to start the reaction. The reaction was stopped at 10 min by boiling for 2 min. Unreacted ADP-Glc [sup.14]C] was separated from radio-labeled glucan by passing the reaction mix through OH- ion exchange resin columns as described by Jenner et al. (1995). Radioactivity was measured using a Beckman (Fullerton, CA) model LS 5000TD scintillation counter. Background counts were determined by running the assay with boiled extract.

Sucrose synthase and SSS assays were performed in duplicate. Enzyme activities of both temperature treatments were measured at 25 [degrees] C and reported. For the high temperature treatment, values were also reported after adjustment with the temperature coefficient ([Q .sub.10]) of respective enzymes to estimate the in vivo catalytic activity in heat-stressed kernels. The [Q.sub.10] adjustment was made according to the following formula (adapted from Hochachka and Somero, 1973):


where [VE.sub.31] is an estimate of the enzyme activity at 31 [degrees] C (the average daily temperature in the high temperature treatment) and [V.sub.25] is the measured enzyme activity. The individual enzyme [Q.sub.10] values used in this calculation were obtained from Wilhelm (1995) using the average [Q.sub.10] value measured in 5 [degrees] C increments across the range of 20 to 35 [degrees] C (Table 2). Enzyme activity is reported in units with one unit of enzyme activity defined as the formation of 1 [micro] mol of product per minute at 25 [degrees] C.

Table 2. Average temperature coefficients ([Q.sub.10) of maize endosperm enzymes in the range of 20 to 35 [degrees] C (Wilhelm, 1995).
Enzyme                           [Q.sub.10] (20-35 [degrees]
                                           C range)

Sucrose synthase                            1.90
UDP glucose pyrophosphorylase               1.52
Glucokinase                                 1.53
ATP-linked fructokinase                     2.62
UTP-linked fructokinase                     1.41
Phosphoglucomutase                          1.62
Phosphoglucoisomerase                       2.35
ATP-linked phosphofructokinase              1.33
PPi-linked phosphofructokinase              0.97
ADP glucose pyrophosphorylase               1.32
Soluble starch synthase                     1.33


Kernel Growth

Analysis of variance of the split-plot design reveals that there were significant treatment responses to temperature (P [is less than or equal to] 0.09) and inbred (P [is less than or equal to] 0.04) for all grain quality and growth parameters measured (Table 3). This verifies that the contrasting temperatures and germplasm chosen for this study produced a notable impact on maize seed growth and development. In contrast to temperature and inbred effects, tests for significance of the inbred x temperature interaction revealed P values [is greater than]0.10, except for grain-fill rate (Table 3). Hellewell et al. (1996) also noted that kernel growth of oat (Avena sativa L.) cultivars responded the same to contrasting temperature regimes. Nevertheless, the absence of a genotype x temperature interaction in our study and in that by Hellewell et al. (1996) is more the exception than the rule. Cultivars of wheat (Wardlaw et al., 1989a, 1989b; Wardlaw and Moncur, 1995), barley (Hordeum vulgate L.; Savin et al., 1996), and rice (Oryza sativa L.; Yoshida and Hara, 1977) commonly display differences in seed growth under conditions of supra-optimal grain fill temperatures as opposed to normal temperatures. The same has also been reported for maize. Lu et al. (1996) exposed developing maize kernels to high temperature stress independently of the rest of the plant and found a striking difference in growth response of different inbreds. We believe that in making genetic comparisons related to kernel heat-stress tolerance it is important to recognize that temperature stress applied in different fashions can produce disparate degrees of genotype sensitivity. For instance, maize kernels of eight inbreds displayed similar levels of growth susceptibility to high temperature when grown in vitro (Singletary and Banisadr, 1992), but when plants of the same inbreds were raised in growth chambers and heat stressed during grain filling (25 vs. 35 [degrees] C; root temperature not controlled) genotypic differences in kernel heat-stress tolerance were much more pronounced. Hence, in view of mixed results that are reported for genotypic heatstress tolerance, it is important to emphasize that the results of our study do not rule out the possibility that developing kernels of some maize genotypes may tolerate high temperatures better than others. Nevertheless, because the inbreds we chose represented several heterotic groups and maturities, and because of the difference in the way root temperature was controlled in our work vs. other indoor studies with maize (see discussion below), we are led to conclude that the effect of heat stress on developing kernels of maize probably does not differ widely among elite inbreds.

Table 3. Summary of analysis of variance mean squares (MS) and corresponding P values (in parentheses) of nine grain traits for the inbred, temperature, and inbred x temperature (I x T) sources of variation.
                                              Source of variation

Grain trait                                          Inbred

                                                  MS       P

Dry weight, mg [kernel.sup.-1]                  1563     (0.01)
Starch content, mg [kernel.sup.-1]               536     (0.01)
Protein content, mg [kernel.sup.-1]               65     (0.01)
Oil content, mg [kernel.sup.-1]                   10.7   (0.01)
Density (x 10 4)([dagger])                        43     (0.01)
Grain fill rate, mg [kernel.sup.-1]                4.8   (0.01)
Grain fill rate (x [10.sup.3]), mg                17     (0.01)
 [kernel.sup.-1] [HU.sup.-1]([double dagger])
Grain fill duration, d                            23     (0.04)
Grain fill duration, HU                         7336     (0.01)

Grain trait                                       Temperature

                                                  MS        P

Dry weight, mg [kernel.sup.-1]                   1751     (0.06)
Starch content, mg [kernel.sup.-1]                642     (0.07)
Protein content, mg [kernel.sup.-1]                62     (0.06)
Oil content, mg [kernel.sup.-1]                     3.2   (0.06)
Density (x 10 4)([dagger])                         52     (0.09)
Grain fill rate, mg [kernel.sup.-1]                21.6   (0.01)
Grain fill rate (x [10.sup.3]), mg                178     (0.01)
 [kernel.sup.-1] [HU.sup.-1]([double dagger])
Grain fill duration, d                            318     (0.01)
Grain fill duration, HU                         52047     (0.01)

Grain trait                                         I x T

                                                 MS       P

Dry weight, mg [kernel.sup.-1]                   95     (0.91)
Starch content, mg [kernel.sup.-1]               37     (0.94)
Protein content, mg [kernel.sup.-1]               4     (0.64)
Oil content, mg [kernel.sup.-1]                   0.2   (0.91)
Density (x 10 4)([dagger])                        2     (0.57)
Grain fill rate, mg [kernel.sup.-1]               0.6   (0.12)
Grain fill rate (x [10.sup.3]), mg                2     (0.09)
 [kernel.sup.-1] [HU.sup.-1]([double dagger])
Grain fill duration, d                            4     (0.81)
Grain fill duration, HU                         831     (0.87)

([dagger]) Actual values equal reported value multiplied by the number in parentheses.

([double dagger]) HU = heat unit.

Kernel dry weight was reduced an average of 7% under high grain-fill temperature (Table 4). Such losses would be of considerable economic importance to Midwestern U.S. grain producers. This abatement in seed growth occurred when average daily temperature increased by 7.5 [degrees] C (23.5 vs. 31 [degrees] C), and it is indicative of how plants may respond to a similar type and level of stress in the field. In fact, results of our study are comparable with the level of yield loss caused by a 6 [degrees] C increase in grain-fill temperature during August, as estimated by Thompson (1966) using maize production records for the U.S. Corn Belt. We believe this is because we simulated a heat stress as it would naturally occur in the field by applying heat stress to the entire aerial portion of the plant under natural sunlight conditions, while buffering roots from extreme temperature changes. Maize plants can tolerate moderately high ambient temperatures (35 [degrees] C) during grain fill and still produce kernels of normal or near-normal size if the rooting profile is not adversely heat stressed (Table 4; Muchow, 1990). Kuroyanagi and Paulsen (1988) demonstrated the adverse effect of high root temperature, as opposed to shoot temperature, on wheat grain growth several years ago, but their work has received little attention and many researchers continue to ignore the impact of temperature treatments on roots. The low PAR under artificial lighting may also reduce the ability of plants to tolerate higher temperatures (Spiertz, 1977). Thus, the large effect (nearly 50% reduction) of high temperature on maize kernel dry weight as found in previous controlled-environment studies performed under artificial lighting and without root-temperature control (BaduApraku et al., 1983; Singletary and Banisadr, 1992) may be misleading. Based on these studies and our data, we hypothesize that proper control of root temperature and lighting is needed when studying the effects of heat stress on aerial portions of the plant. Likewise, we theorize that heat stress is more harmful to kernels treated in vitro than for those stressed as part of the aerial portion of the plant. Singletary et al. (1994) and Duke and Doehlert (1996) found that the deposition of starch, the primary constituent of the seed, was reduced 20 to 45% for maize kernels grown in vitro at 30 vs. 25 [degrees] C. Our data show that growth of kernels experiencing a daily average temperature of 31 vs. 23.5 [degrees] C was only reduced by 7% (Table 4). For wheat, Hawker and Jenner (1993) found that continuous heating of intact wheat heads at 35 [degrees] C (vs. 19 [degrees] C) for 7 d reduced kernel dry weight 11 to 20%, but the rate of starch synthesis in isolated kernels heat stressed in vitro at 35 vs. 20 [degrees] C was reduced 43% (Jenner et al., 1993). The interaction between plant and seed appears to be important in determining how the seed responds to changes in temperature, and we believe that keeping the seed intact with the whole plant in temperature studies is vital for determining the true effects of temperature stress on the seed.

Table 4. Mature kernel dry weight, starch, protein, and oil content across all genotypes as affected by two temperature regimes during grain filling.
Grain trait       25/20 [degrees] C   34/25 [degrees] C   Difference

                            mg [kernel.sup.-1]            %     P

Dry weight               180                168           -7   0.06
Starch content           120                112           -7   0.07
Protein content           24.4               21.9        -10   0.06
Oil content                7.4                6.9         -7   0.06

In addition to evaluating mature kernel mass, we also examined the effect of high temperature on the dynamics of seed growth. Immature grain in both temperature treatments was collected after the accumulation of a specific number of HU in order to isolate kernels at equivalent stages of physiological development. Kernel growth was also measured as a function of time to compare with previously published work. In the latter regard, the rate of grain filling per day was increased 19 % and the day-based duration of grain filling was reduced 22% at high temperatures (Table 5). This agrees with previously reported work in maize (Tollenaar and Bruulsema, 1988; Muchow 1990), rice, and wheat (Sofield et al., 1977; Tashiro and Wardlaw, 1989). Interpreting the rate and duration data on a day basis suggests that high temperature reduces kernel size through a reduction in the duration of the fill period and the lack of a compensatory increase in grain-filling rate. However, the importance of the effects of high temperature on the efficiency of fill rate may equally explain why high temperature reduces final seed dry weight. This is illustrated when rate and duration of grain filling are calculated on a HU basis. The HU basis was used to put the data on a normalized basis, inasmuch as temperature, not days, is the primary factor that drives the progression of crop and seed growth. Thus, on an accumulated HU basis, high temperature reduced the rate of grain filling 24% and increased the duration of dry matter accumulation 21% (Table 5). On this basis, the larger reduction in rate of grain filling, as compared with duration, was responsible for the heat-related reduction in seed mass. These heat unit-based responses are in contrast to what we have observed with maize kernels grown in vitro. In culture, heat stress has a small effect on the rate of kernel growth per HU and instead largely causes a premature termination in the duration of storage product deposition (Singletary et al., 1994). However, the data from our study and published data (Wardlaw and Moncur, 1995) converted to a HU basis show that when maize and wheat plants experience supra-optimal temperatures during grain fill, the rate of seed growth is decreased and the duration is increased, but the increase in duration is not large enough to compensate for the decrease in rate. Therefore, the data, when interpreted on a HU basis, indicate that anabolic seed metabolism operates for a longer thermal period in the presence of heat stress, but at greatly reduced biochemical efficiency. Overall, a reduced mature seed size is the result of a decrease in duration without a compensatory increase in rate when expressed on the basis of days or is the result of a decrease in rate without a compensatory increase in duration when expressed on a HU basis. Hence, we recognize the importance of mechanisms that control rate as well as those that control duration, either of which may explain the losses in dry matter accumulation in this study.

Table 5. The rate and duration of dry matter accumulation in the kernel across all genotypes as affected by the two temperature regimes during grain filling.
Grain filling
characteristics                                   25/20 [degrees] C

Grain fill rate, mg [kernel.sup.-1] [HU.sup.-1]         0.55
Grain fill duration, HU                               333
Grain fill rate, mg [kernel.sup.-1] [d.sup.-1]          7.4
Grain fill duration, d                                 24.4

Grain filling
characteristics                                   34/25 [degrees] C

Grain fill rate, mg [kernel.sup.-1] [HU.sup.-1]         0.42
Grain fill duration, HU                               403
Grain fill rate, mg [kernel.sup.-1] [d.sup.-1]          8.8
Grain fill duration, d                                 19.4

Grain filling
characteristics                                   Difference


Grain fill rate, mg [kernel.sup.-1] [HU.sup.-1]    -24(**)
Grain fill duration, HU                             21(**)
Grain fill rate, mg [kernel.sup.-1] [d.sup.-1]      19(**)
Grain fill duration, d                             -22(**)

(**) Significant at P [is less than or equal to] 0.01.

Grain Quality

Average kernel density was reduced by heat stress from 1.275 to 1.253 g [cm.sup.-3] (P = 0.09), as similarly reported by Lu et al. (1996), but starch, protein, and oil concentrations in the kernel were not changed. This resulted in a decrease in content of starch, protein, and oil (P = 0.07, 0.06, and 0.06) similar to reductions in kernel dry weight (Table 4). We are not aware of any other studies that have examined cereal seed starch, protein, and oil constituents simultaneously to examine their responses to heat stress applied specifically during grain filling. Protein and oil concentration does not seem to be affected by August temperature in the U.S. Corn Belt (Earle, 1977). Similarly, high temperatures in a greenhouse study did not affect protein concentration in maize (Lu et al., 1996). In rice, protein percentage in grains declined or was not affected by exposure to high temperature beginning 16 d from heading or later (Tashiro and Wardlaw, 1991).

The parallel decreases in starch, protein, and oil contents caused by chronic heat treatments suggest that losses in kernel dry weight may be produced by one or more mechanisms that broadly affect whole kernel growth. Assimilate transport could be one such mechanism (Wardlaw et al., 1995). The photosynthetic system is another mechanism that may be affected by high temperature during grain filling. For example, Harding et al. (1990) demonstrated that high temperature during reproductive development in wheat seems to affect kernel growth rate and duration by initially accelerating thylakoid component breakdown. Mechanisms of transport and photoassimilation must be considered in studies of heat stress, but perhaps equally important is the effect of high temperature on starch biosynthesis. Numerous studies involving maize (Jones et al., 1984; Keeling et al., 1994; Singletary et al., 1994), wheat (Bhullar and Jenner, 1985), and barley (MacLeod and Duffus, 1988) have shown a negative effect of heat stress on starch deposition in the kernel. A similar series of data showing a negative relationship of protein and oil syntheses to high grain-fill temperature does not exist. We speculate that the negative influence of heat stress on kernel growth in cereals relates to perturbation of starch biosynthesis, but heat stress can also affect the syntheses of other storage products through an indirect effect via interconnected metabolism. It has been reported that coordinated transcriptional regulation between starch and protein syntheses occurs in maize seed (Giroux et al., 1994) and potato (Solanum tuberosum L.) tubers (Muller-Rober et al., 1992), which could account for the like response of starch and protein contents to heat stress. Similarly, the ratio of maize endosperm to embryo weight within a specific genotype remains relatively constant regardless of kernel size (Paddick and Sprague, 1939). Because starch is the primary constituent of the endosperm and oil is found mainly in the embryo, it is possible that a drag on starch biosynthesis could concomitantly restrict development of scutellar cells or diminish the deposition of oil in the kernel.

Enzyme Analysis

A survey of enzymes in the pathway of sugar and starch metabolism was performed to determine if the effect of heat stress on kernel growth was associated with altered rates of enzymatic activity. Endosperms were collected (250 postpollination accumulated HU) during the period when kernel growth rate was also being measured and, hence, entailed a stage of temperature-induced growth impairment. Enzyme activity in crude extracts was measured at 25 [degrees] C for kernels of both temperature treatments. On this basis, high temperature had little effect on most of the enzymes studied, but significantly reduced the endosperm activities of AGPase, glucokinase, sucrose synthase, and SSS (Table 6). Although enzyme activities were measured at 25 [degrees] C, enzymes in kernels growing at the elevated temperature presumably would have functioned at increased catalytic rates, based upon the temperature coefficient (i.e., [Q.sub.10]). To obtain a better comparison between enzymatic activities in control and heat-stressed kernels, estimates of in vivo activities in the heat-stressed kernels were calculated using [Q.sub.10] values previously determined for the same maize endosperm enzymes according to the same reaction conditions described herein (Table 2; Wilhelm, 1995). Under this premise, activities in developing heat-stressed kernels were equal to or greater than corresponding activities in control kernels in nearly all cases. The only exception was AGPase, where the [Q.sub.10]adjusted activity was 15% less than control kernels (Table 6).

Table 6. The activity of 11 enzymes of starch and sugar metabolism extracted from heat-treated and control maize endosperm tissue collected at 250 heat units after pollination. Enzyme activity was measured at 25 [degrees] C for both treatments. For the heat-treated tissue, activites are also reported as a percentage of the control without and after adjustment to the mean of the high temperature treatment (31 [degrees] C) using the [Q.sub.10] from Table 2.
Enzyme                           25/20 [degrees] C

                                 units per endosperm

Sucrose synthase                      0.91
UDP glucose pyrophosphorylase        13.3
Glucokinase                           0.018
ATP-linked fructokinase               0.0065
UTP-linked fructokinase               0.019
Phosphoglucoisomerase                 1.06
Phosphoglucomutase                    6.1
ATP-linked phosphofructokinase        0.025
PPi-linked phosphofructokinase        0.26
ADP glucose pyrophosphorylase         0.20
Soluble starch synthase               0.056

Enzyme                           34/25 [degrees] C

                                 units per endosperm

Sucrose synthase                      0.78
UDP glucose pyrophosphorylase        12.7
Glucokinase                           0.013
ATP-linked fructokinase               0.0057
UTP-linked fructokinase               0.019
Phosphoglucoisomerase                 1.05
Phosphoglucomutase                    5.6
ATP-linked phosphofructokinase        0.023
PPi-linked phosphofructokinase        0.28
ADP glucose pyrophosphorylase         0.14
Soluble starch synthase               0.049

                                  Heat stress-induced difference

Enzyme                           Unadjusted   [Q.sub.10] Adjusted

                                        units per endosperm

Sucrose synthase                     86(*)            138
UDP glucose pyrophosphorylase        95               130
Glucokinase                          72(*)            100
ATP-linked fructokinase              88               182
UTP-linked fructokinase             100               132
Phosphoglucoisomerase                99               189
Phosphoglucomutase                   92               133
ATP-linked phosphofructokinase       92               112
PPi-linked phosphofructokinase      108(*)            104
ADP glucose pyrophosphorylase        70(*)             85
Soluble starch synthase              88(*)            107

(*) Significant at P [is less than or equal to] 0.05.

Despite numerous studies in recent years, determining the aspects of sugar and starch metabolism that are presumably sensitive to conditions of high temperature stress and responsible for the restriction of starch accumulation in heat-stressed seed has remained difficult. Still, it is fair to state that SSS and AGPase are currently given the most credit for restricting starch synthesis under supra-optimal temperatures.

Studies linking specific enzymes with the rate of starch deposition in cereals have suggested SSS as a key control point in the pathway. Catalytically, SSS activity measured in vitro becomes less efficient with increasing temperature (Hawker and Jenner, 1993; Jenner et al., 1995), and the enzyme is known to be labile when it, or isolated kernels it is extracted from, are held in vitro at high temperature for short periods (Keeling et al., 1993). Also, the flux control coefficients of other enzymes in the pathway of sugar and starch metabolism in cereal grains, such as branching enzyme, AGPase, and sucrose synthase (Singletary et al., 1997) are much lower than that reported for SSS (Hawker and Jenner, 1993; Jenner et al., 1993; Keeling et al., 1993). Hence, SSS seems to be responsible for the limitation in starch synthesis that occurs under high temperatures. However, in spite of these factors, there are several points that call into question the role of SSS in the temperature sensitivity of starch biosynthesis. Most importantly, simple genetic comparisons of wheat cultivars that show varying degrees of tolerance to heat stress have failed to relate whole-plant differences to catalytic differences in SSS activity measured at high temperatures (Jenner et al., 1993). We have confirmed this lack of correlation using the same cultivars tested by Jenner et al. (1993) and also after comparing SSS and growth data from heat-stressed kernels of several maize inbreds (unpublished results). We also found that starch synthesis in isolated (i.e., in vitro) wheat kernels shows large sensitivities of SSS to temperature (Keeling et al., 1993), but the same was not true for kernels heat treated on the plant (Kuroyanagi and Paulsen, 1988; Hawker and Jenner, 1993). This raises a concern over the agronomic relevance of the in vitro heat treatments, as we have already noted. It is noteworthy that the activity profile of SSS is compressed in time and amount very similarly to AGPase, and the in vitro measured activity of SSS is low relative to most enzymes of sugar and starch biosynthesis (Caley et al., 1990; Singletary et al., 1994). In our study, [Q.sub.10]corrected SSS activity was slightly increased, which may directly contribute to the small increase in grain-fill rate. Similarly, heat induces a gradual decline in SSS activity, which can lead to a premature end to starch biosynthesis (Singletary et al., 1994).

The response of AGPase is, in some ways, the opposite of SSS in response to high temperatures. In vitro, AGPase activity is stable at high temperature (Kennedy and Isherwood, 1975; Keeling et al., 1993) and its activity profile during development of maize under heat stress is more reduced in magnitude and timing than that of SSS (Ou-Lee and Setter, 1985; Singletary et al., 1994). Work by Duke and Doehlert (1996) involving heat-stressed kernels of maize also demonstrates that AGPase activity is constricted more than several other enzymes of sugar metabolism. In addition, only 5 d of growth at 30 [degrees] C, compared with 25 [degrees] C, reduced the expression of AGPase genes Brittle-2 and Shrunken-2 by 50 and 70%, respectively. On the other hand, simple genetic comparisons of wheat cultivars known to vary in heat-stress tolerance failed to relate AGPase activity to whole-plant differences, similar to SSS (Jenner et al., 1993). In our study of 11 enzymes, [Q.sub.10]-adjusted AGPase activity was the most reduced by high temperature. This suggests that high temperature affects AGPase more than the other enzymes, which may contribute to a reduction in the efficiency of starch and dry matter accumulation or cause a premature termination of grainfilling (Singletary et al., 1994). However, previous studies indicate that reductions in starch accumulation generally do not occur without a decrease in AGPase activity of 40% or more (Singletary et al., 1994, 1997). This calls into question the importance of a 15% reduction in AGPase activity in reducing final seed size, as in our study.

The roles of glucokinase and sucrose synthase in determining starch accumulation in heat-treated tissues are less clear. In our study, high temperature had an adverse effect on the activity of both of these enzymes before [Q.sub.10]-adjustment. Previous studies have shown high-temperature sensitivity of glucokinase in maize endosperm tissue (Singletary et al., 1994) and sucrose synthase in both maize (Singletary et al., 1994) and barley (MacLeod and Duffus, 1988), and reductions in activity of these enzymes may be linked to a reduction in starch biosynthesis.

In conclusion, the seed growth response to heat stress was not significantly different across seven maize genotypes. A heat stress of moderate magnitude, but of much greater duration than normally occurs in the U.S. Corn Belt, reduced kernel dry weight by a small but significant amount (7%). Reductions in kernel dry weight were attributable to proportional losses in starch, protein, and oil contents, indicating that a mechanism affecting whole grain metabolism is responsible for the loss. Grain-filling duration in HU increased under heat stress, but could not compensate for the decrease in grainfilling rate, producing kernel dry weight losses. The chronic high temperature significantly reduced the activities of four enzymes of the starch biosynthetic pathway: AGPase, SSS, glucokinase, and sucrose synthase, but only AGPase activity was reduced when activities were [Q.sub.10]-corrected to the growth temperature. These results indicate that these enzymes are closely linked to reductions in starch content and may explain the reductions in mature seed dry weight.


We would like to acknowledge the significant funding provided by ICI Seeds, Slater, IA. We also thank David Entz for his assistance in the project, and Dr. Jerry Radke and Paul Doi for the soil temperature data that they provided.

Abbreviations: AGPase, adenosine diphosphate-glucose pyrophosphorylase; DAP, days after pollination; HU, heat units: PAR, photosynthetically active radiation; SSS, soluble starch synthase.


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E. P. Wilhelm,(*) R. E. Mullen, P. L. Keeling, and O. W. Singletary

E.P. Wilhelm and P.L. Keeling, ExSeed Genetics L.L.C., 1573 Food Sciences Building, Iowa State University, Ames, IA 50011-2062; G.W. Singletary, Pioneer Hi-Bred International, Inc., P.O. Box 1004, Johnston, IA 50131-1004; R.E. Mullen, Dep. of Agronomy, Iowa State Univ., Ames, IA 50011. The research was funded as a joint collaboration between ICI Seeds (presently Garst Seed Co.), 2369 330th St., P.O, Box 500, Slater, IA 50244 and Iowa State University. Journal Paper no. J-18085 of the Iowa Agric. and Home Econ. Exp. Sta., Ames, IA, Project no. 2775, and supported by Hatch Act and State of Iowa. Received 2 Oct. 1998. (*)Corresponding author (edwilhelm@
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Author:Wilhelm, E. P.; Mullen, R. E.; Keeling, P. L.; Singletary, G. W.
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Date:Nov 1, 1999
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