| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a ExSeed Genetics L.L.C., 2901 South Loop Dr. Bldg #3, Suite 3360, ISU Research Park, Ames, IA 50010
b Department of Agronomy and Plant Genetics, 411 Borlaug Hall, 1991 Upper Buford Circle, Univ. of Minnesota, St. Paul, MN 55108
* Corresponding author (jones012{at}tc.umn.edu)
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Abbreviations: DAP, d after pollination • HTT, high temperature treatment • SEM, scanning electron micrograph
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
10% in the U.S. Corn Belt (Thompson, 1966). Whole plant and in vitro kernel culture studies have shown that as little as 4 d of high temperature (35°C) imposed during cell division in the endosperm (i.e., the lag phase) reduced kernel mass by 35 to 40%, and significantly increased kernel abortion (Cheikh and Jones, 1994, 1995). To stabilize yield of this important crop, it is important to understand the mechanisms by which environmental perturbations like high temperature disrupt kernel development. Our previous investigations have shown that elevated temperature (4–6 d at 35°C) caused reductions in kernel mass and increased kernel abortion. Such high temperature-induced disruption of kernel development is due mainly to reduced kernel sink capacity, that is, the intrinsic ability of kernels to attract and store assimilate (Jones et al., 1984, 1985). Kernel sink capacity in maize is principally a function of the number of endosperm cells and starch granules established during the first 10 to 14 DAP (Capitanio et al., 1983; Reddy and Daynard, 1983; Jones et al., 1985, 1996). Genotypic difference in kernel sink capacity has also been shown to be maternally regulated (Jones et al., 1996). High temperature-induced reductions in kernel sink capacity and the subsequent decrease in kernel growth are not due to an inability of kernels to take up sucrose (Cheikh and Jones, 1995). Circumstantial evidence suggests that a high temperature-induced shift in the hormonal balance (cytokinins/abscisic acid), due mainly to a precipitous decline in endogenous cytokinin levels, is the principal mechanism involved (Cheikh and Jones, 1994). Likewise, Mambelli and Setter (1998) have shown that an increasing concentration of ABA in maize endosperm negatively affects cell division. Also, cytokinins have been shown to regulate cell and plastid division in plant tissue (Davies, 1987). Thus, there is compelling evidence to surmise that hormones play an important role in kernel development through regulation of kernel sink capacity.
Recently, Wilhelm et al. (1999) surveyed the response of seven maize inbred lines (representing various heterotic groups) to exposure to high temperatures during the linear-phase of kernel development. They demonstrated that high temperature during this latter stage of grain filling only moderately affected seed storage processes and the activity of key starch metabolism enzymes across all inbred lines studied. However, similar information is lacking on how genotypes vary in their response to high temperature during the early kernel development. The majority of our earlier investigations were conducted using a single maize genotype, A619 x W64A. To the best of our knowledge, no study has focused on determining whether genotypes differ in stability of their kernel sink capacity during and after exposure to high temperature. Jorgensen et al. (1992) compared the inbred lines B73 and Mo17 for thermotolerance during vegetative development and found that Mo17 possessed greater tolerance to high temperature than B73 (based on electrolyte leakage studies). It is not known, however, if the thermotolerance properties of vegetative organs of these inbred lines are retained and expressed in a comparable fashion in reproductive structures like developing kernels. Therefore, the current study was undertaken with the principal objective of better understanding the mechanisms by which high temperature disrupts kernel development in maize by comparing the response of B73 and Mo17, two genotypes differing in their tolerance to high temperatures. Genotypic differences were assessed by investigating the effects of HTTs on changes in kernel morphology, kernel sink capacity, and kernel mass. It is well known that the detrimental effect of high temperature on kernel development in maize and other cereal crops is via a direct effect on the grain itself (Ford et al., 1976; Wardlaw et al., 1980). Therefore, to facilitate our studies of genotypic responses of maize kernels to high temperature, we used an in vitro culture technique (Jones et al., 1981; Gengenbach and Jones, 1993) and a recently-developed ear-heating system to apply HTT directly to kernels on intact ears of field-grown plants. Unfortunately, both of these techniques preclude the ability to study high temperature-induced kernel abortion as it occurs in a field environment, which usually is initiated in the apical portion of the ear. However, both approaches offer the advantage of facilitating the study of the direct effects of high temperature on kernel development without the confounding impact of other environmental factors.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
In Vitro Culture Study
At 3 DAP, uniform ears were removed from field-grown plants, and kernels were removed under sterile conditions and placed into an in vitro culture, described by Gengenbach and Jones (1993). Kernels were allowed to acclimate for 1 d in incubators maintained at 25°C continuously. After which (5 DAP), a portion of the kernels were exposed to HTTs (35°C, day/night) for either 4 or 6 d. At the end of each HTT, kernels were transferred back to 25°C (day/night) and retained there until dry matter accumulation ceased. The remainder of the kernels were used as controls, and thus maintained at 25°C (day/night) throughout the study. Three replicate samples per treatment were taken at periodic intervals for measurement of kernel dry weight and the number of endosperm cells and starch granules.
Field Study
High temperature treatments were imposed on the kernels developing on intact ears of field-grown plants. The HTTs were generated using a 24-V alternating current electrical system ear-heating device consisting of proportional, integral, and derivative type electronic temperature controllers (Model E5CS-X, Omron Corporation, Japan), a bimetal thermocouple temperature sensor (Type K, Omega Corporation, Stamford, CT), and a 12.7 by 15.2-cm silicon rubber insulated thermo-foil heating mat (Minco Products Inc., Minneapolis, MN). At 5 DAP, HTTs were imposed as follows: A thermocouple wire was carefully inserted between the innermost layer of the husk and the kernels, to the midpoint of the each ear, as shown in Fig. 1. The thermocouple served as a thermostat, and thus was used to sense and maintain a constant temperature of 35°C surrounding the kernels. To facilitate uniform heat distribution, the ears were covered with aluminum foil, leaving both ends of the foil open, and the heating mat was wrapped around the ear and secured by rubber bands (Fig. 1). Each ear-heating device was protected from weather damage by covering it with a pollen collection bag. Two temperature control units were constructed by rack mounting 10 of the electronic temperature controllers in each of two metal cabinets, thus providing the potential to heat 20 ears concurrently. The two-temperature control units were placed in a weatherproofed camping trailer that was positioned adjacent to the field plots. For each of the 20 units, all electrical connections situated outside the mobile lab were waterproofed. The 35°C set point was maintained with a precision of ± 0.1°C. The control ears were also fitted with an ear-heating device, but not connected to a temperature controller, and were thus maintained at near ambient temperature. At the end of each HTT, the ear-heating devices were removed and the ears allowed to develop at ambient temperature until kernel dry matter accumulation ceased.
|
Determination of the Number of Endosperm Cells and Starch Granules
Endosperm cell numbers were counted as described by Jones et al. (1985). Three replicate samples of five kernels each were fixed in a solution containing three parts of 95:5 EtOH:H2O and one part propionic acid (3:1, v/v) for 24 h at room temperature, and then stored at -20°C in 70:30 EtOH:H2O. Prior to analysis, kernels were placed in 50:50 EtOH:H2O for 5 min, and then in ddH2O for 5 min. Endosperm cells were isolated and placed in test tubes containing 1 mL of 1 M HCL at 0 to 4°C for 30 min, and then transferred to a 60°C water bath for 16 min. After rinsing endosperm cells with ddH2O, nuclei were stained with 2 mL of Feulgin's reagent for 4 h in the dark. Endosperm cells were rinsed twice in ddH2O and digested by adding 1 mL of 20 mg L-1 cellulysin (cellulase, Cal Biochem-Behring, La Jolla, CA) in 0.1 M NaOAc buffer (pH 4.7) at 37°C overnight. Nuclei were dispersed in the suspension with a Pasteur pipette and counted visually under a reverse phase microscope using a hemocytometer, as described by Jones et al. (1985).
Starch granule number was counted by flow cytometry (Jones et al., 1992). Endosperm cells were digested at 37°C overnight with 1 mL of 20 g L-1 cellulysin in 0.1 M NaOAc buffer (pH 4.7). The suspension was filtered through 150-µm nylon mesh to separate starch granules from cellular debris. Starch granules were suspended in a known volume of 20 g L-1 sodium dodecyl sulfate solution, vortexed, and sonicated for 5 min at room temperature. A flow cytometer (MDADS II, Coulter, Hialeah, FL) equipped with an argon blue laser adjusted to 488 nm at a light-stabilized beam power of 200 mW was used for counting starch granule number. A known number of fluorescent microspheres (Coulter, Hialeah, FL) were added to each sample in a predetermined volume to determine the analysis volume, and thus estimate starch granule number per endosperm. Three replicate samples of each treatment were analyzed, and errors due to any cellular debris were deleted by gating.
In all the studies, the experimental design was a randomized complete block, and statistical analysis was performed by ANOVA.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
40% and 79%, respectively (Fig. 3A). Whereas, in field-grown B73, the 4- and 6-d HTTs reduced kernel dry weights by 65% and 93%, respectively (Fig. 3B).
|
30% of the dry weights of the field-grown controls (Fig. 3D). These data support the previous observation of Gengenbach (1977), that some maize genotypes are less amenable than others to growth under in vitro culture conditions. The basis for this differential response is not known.
Nevertheless, the 4- and 6-d HTTs had similar effects on MO17 when grown in vitro or under field conditions (Fig. 3C, D). Dry weights of in-vitro-grown Mo17 were reduced by 92% with both 4- and 6-d HTTs (Fig. 3C). Similarly, in the field-grown Mo17, the 4- and 6-d HTTs reduced kernel dry weights by 90 to 95%, as compared with the controls (Fig. 3C, D). These data indicate that high temperatures during the endosperm cell division stage were detrimental to both genotypes studied, although the effects were more pronounced in Mo17. For example, the 4-d HTT reduced dry matter accumulation by 40 to 65% in B73, whereas it completely disrupted kernel growth and development in Mo17. Hence, these results clearly demonstrate that B73 is more tolerant than is Mo17 to brief periods of high temperature during the endosperm cell division phase of kernel development. This response is in contrast to that reported for these two genotypes when exposed to high temperature during vegetative development (Jorgensen et al., 1992).
Morphological Changes in the Kernels Exposed to High Temperatures
To investigate the nature of the genotypic differences in kernel responses to HTTs, structural changes were monitored by comparing scanning electron micrographs (SEMs) of longitudinal sections of field-grown kernels sampled at 11 DAP. Micrographs of representative sections from both genotypes exposed to each HTT are shown in Fig. 4. The kernel cavities of both B73 and Mo17 controls (25°C) were occupied with endosperm cells by 11 DAP (Fig. 4A and D). The embryo was prominent and the nucellar tissue was no longer present in control kernels indicating normal progression toward completion of the endosperm cell division stage. The basal and central endosperm cells appeared larger than those located in the peripheral layers. These observations are in agreement with the investigations by Kiesselbach (1949), documenting that in a developing maize kernel, cell division in the basal endosperm occurs for a short time after cellularization (4 DAP). Thereafter, it is limited to the outer zone, but continues to divide for several cell layers deep, filling up the entire kernel cavity (Kiesselbach, 1949). This rapid expansion of endosperm replaces the nucellus and ultimately compresses any nucellar cells to the outer edge of the kernel cavity by 12 DAP, as observed in the control kernels of both B73 (Fig. 4A) and Mo17 (Fig. 4D). Therefore, although the basal endosperm cells are the oldest, cell division in the periphery of the endosperm is essential for filling up the kernel cavity and also for establishing maximum sink capacity. From SEMs of kernels exposed to the 4- and 6-d HTTs, it appears that HTTs affected peripheral endosperm cell divisions in both B73 and Mo17, although the effect was more severe in the latter (Fig. 4). In B73, the 4-d HTT appeared to have resulted in: (i) an elongated but narrower endosperm with relatively fewer cells compared with the controls, (ii) reduced embryo size, and (iii) increased thickness of the pericarp (Fig. 4B). These effects were similar but more pronounced in B73 kernels exposed to HTT for 6 d (Fig. 4C). In these kernels, the size of the endosperm was severely reduced, the embryo was not detectable, and the bulk of the nucellar tissue was still present at 11 DAP. Therefore, in the inbred line B73, increased duration of HTT during endosperm cell division increased the extent of the disruption of kernel ultrastructure. Recently, we reported similar high temperature-induced changes to kernel ultrastructure in the single cross hybrid A619 x W64A (Commuri and Jones, 1996, 1999). In contrast to B73, exposure of Mo17 to a 4- or 6-d HTT had an even more detrimental impact on the kernel ultrastructure (Fig. 4E, F). For both treatments, the size of endosperm was significantly reduced, embryos were malformed, thickness of the pericarp was increased, and the bulk of the nucellar tissue was still present in both the treatments (Fig. 4E, F). Basal endosperm cells were enlarged, but the initiation of peripheral endosperm cell division (Fig. 4E, F) was not detected in SEMs of Mo17 kernels from these treatments.
|
Effect of High Temperature on Kernel Sink Capacity
Endosperm Cell Number
Kernel sink capacity in maize kernels is a function of the number of endosperm cells formed and starch granules initiated (Capitanio et al., 1983; Reddy and Daynard, 1983; Jones et al., 1985, 1996). The SEMs of B73 and Mo17 provided visual evidence that sink capacity of Mo17 is more severely disrupted by exposure to HTT than B73 (Fig. 4). We also wanted to attain a quantitative assessment of this apparent genotype specific, high temperature-induced reduction in kernel sink capacity. Thus, the number of endosperm cells was counted at 12 and 16 DAP, and the number of starch granules was assessed as kernels approached maximum dry matter accumulation (Fig. 5). Field-grown kernels of both genotypes were compared with those grown in vitro. At 12 DAP, the 4-d HTT reduced the number of endosperm cells formed in B73 by only 8% in the in vitro experiment (Fig. 5A), but by 26% in the field study (Fig. 5C). In contrast, at 12 DAP the 6-d HTT reduced the number of endosperm cells formed by 51 and 63% with in vitro and field-grown B73 kernels, respectively (Fig. 5A and C). Relative to 12 DAP, a significant increase in the number of endosperm cells formed was observed at 16 DAP in the field-grown B73 kernels, regardless of HTT (Fig. 5A). For those grown in vitro and exposed to 4 or 6 d of HTT, however, the number of endosperm cells was reduced 13 and 38%, respectively, compared with controls (Fig. 5A). By 16 DAP, in the field-grown kernels exposed to 4- or 6-d HTT (Fig. 5C), endosperm cell number recovered to a level that was not significantly different from the control. Hence, these data suggest that at 12 DAP, under both environments, endosperm cell division in B73 was reduced significantly by HTT. Moreover, subsequent (at 16 DAP) endosperm cell division in both in vitro and field-grown kernels was able to recover completely from the detrimental effects of high temperature, thus resulting in no significant effect on the number of endosperm cells formed. The physiological basis for this response is unclear, but may have been due in part to the fact that B73 appeared to sustain less damage to endosperm cell ultrastructure than Mo17 when exposed to a 4-d HTT. Moreover, the apparent ability of endosperm cell number to recover to control levels in both experiments may suggest that this response is independent of the way kernels were grown and the methods by which HTTs were imposed. This response is in contrast to that previously reported in wheat (Triticum aestivum L.). Studies by Hawker and Jenner (1993) and Jenner (1970) showed that under very similar HTTs (35°C), wheat kernels grown in vitro were more adversely affected than those developed on heads of intact plants. Therefore, in maize, in vitro studies are useful in studying certain aspects of the response of kernel development to supraoptimal temperature.
|
64 and 75% at 12 DAP, and by 47 and 80% at 16 DAP, respectively (Fig. 5B). Similarly, 4 and 6 d of HTT applied to field-grown Mo17 kernels reduced endosperm cell numbers by 84 and 74% at 12 DAP, and by 85 and 79% at 16 DAP, respectively (Fig. 5D). Hence, in contrast to B73, these data suggest that endosperm cell division in Mo17 is generally more sensitive to high temperature, since it was reduced by both the 4- and 6-d HTT. This genotype-specific response may be associated with differences in other physiological processes that are important in kernel development. We have shown that the magnitude of DNA endoreduplication is progressively reduced and peak mitotic index is delayed in the endosperm cells of kernels exposed to 4- and 6-d HTTs (Engelen-Eigles and Jones, 2000). Whether or not there are genotypic differences in DNA endoreduplication that are also associated with a difference in tolerance to high temperature awaits future investigations.
Starch Granule Number
Starch granule number, another important component of kernel sink capacity and final kernel mass, was estimated as kernel dry matter accumulation ceased. In both field and in vitro-cultured kernels, HTTs reduced starch granule number more severely than endosperm cell number in both genotypes studied (Fig. 6). High temperature for 4 and 6 d reduced starch granule numbers in B73 by 22 and 61 in vitro, and 74 and 98% in field-grown kernels, respectively (Fig. 6A and C). On the other hand, high temperatures for 4 and 6 d reduced starch granule numbers in Mo17 by 82 and 98% in vitro, and 83 and 99% in field-grown kernels, respectively (Fig. 6B and D). The degree of reduction in starch granule number varied between the two growth environments, but in both genotypes, the effect was more severe under field conditions (Fig. 6). In agreement with previous investigations (Cheikh and Jones, 1994, 1995; Commuri and Jones, 1996, 1999), it is apparent that both the 4- and 6-d HTTs during early formative stages significantly reduced kernel dry matter accumulation by reducing starch granule number irrespective of the genotype, growing condition, or the methods by which HTTs were imposed. It is important, however, to note again that the effects were more severe in Mo17.
|
Starch granule number is a function of the number of amyloplasts formed. The processes of plastid biogenesis and the conversion of proplastids to amyloplasts are not well understood. Our previous studies, however, suggested that HTT during endosperm cell division can cause significant delays in this important process, resulting in a significant reduction in the number of amyloplasts and subsequently the number of starch granules formed (Commuri and Jones, 1999). These data suggest that processes associated with the initiation of amyloplasts in B73 and the subsequent deposition of starch into these organelles (Fig. 4) are clearly more heat labile than is endosperm cell division (Fig. 3). In Mo17, however, both endosperm cell division and amyloplast initiation appears to be equally heat labile. Hence, thermal stability of kernels exposed to high temperature is dependent upon not only the number of endosperm cells formed, but also on the number of starch granules established. The physiological basis for the greater thermotolerance of B73 in response to HTT during endosperm cell division is unclear, but may be associated with genotypic differences in maintaining hormonal balance, particularly with respect to endogenous cytokinin levels. We have shown that endogenous cytokinin levels decline 70 to 100% in response to a 4- or 8-d HTT imposed during endosperm cell division, resulting in increased kernel abortion (Cheikh and Jones, 1994). When plants exposed to a HTT were infused with the synthetic cytokinin benzyladenine, however, both kernel abortion and reduction in kernel mass were averted. These data provided circumstantial evidence that maintaining kernel endogenous cytokinin levels above some minimum threshold is critical to stabilizing grain yield of maize against environmental perturbations like high temperature. Therefore, it may be reasonable to surmise that the increased heat tolerance observed for B73 may be associated with the maintenance of higher cytokinin levels during and after exposure to high temperature. Although this conjecture was recently supported by results from preliminary investigations (Jones and Setter, 2000), more detailed studies are needed.
|
CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
Received for publication August 6, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. A. Dong, J. L. Bufford, Y. Oono, K. Church, M. Q. Dau, K. Michels, M. Haughton, and G. Tallman Heat Suppresses Activation of an Auxin-Responsive Promoter in Cultured Guard Cell Protoplasts of Tree Tobacco Plant Physiology, October 1, 2007; 145(2): 367 - 377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. DeVries, A. S. Goggi, and K. J. Moore Determining Seed Performance of Frost-Damaged Maize Seed Lots Crop Sci., September 1, 2007; 47(5): 2089 - 2097. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Monjardino, A. G. Smith, and R. J. Jones Heat Stress Effects on Protein Accumulation of Maize Endosperm Crop Sci., May 27, 2005; 45(4): 1203 - 1210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Brugiere, S. Jiao, S. Hantke, C. Zinselmeier, J. A. Roessler, X. Niu, R. J. Jones, and J. E. Habben Cytokinin Oxidase Gene Expression in Maize Is Localized to the Vasculature, and Is Induced by Cytokinins, Abscisic Acid, and Abiotic Stress Plant Physiology, July 1, 2003; 132(3): 1228 - 1240. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| The SCI Journals | Agronomy Journal | Vadose Zone Journal | |||
| Journal of Natural Resources and Life Sciences Education |
Soil Science Society of America Journal | ||||
| Journal of Plant Registrations | Journal of Environmental Quality |
The Plant Genome | |||
0.01 level.
