Journal of Heredity Advance Access originally published online on August 31, 2005
Journal of Heredity 2005 96(5):614-617; doi:10.1093/jhered/esi102
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Brief Communication |
A Genetic Test of Bioactive Gibberellins as Regulators of Heterosis in Maize
From the Division of Biological Sciences, University of Missouri, 117 Tucker Hall, Columbia, MO 65211. D. L. Auger is currently at the Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007
Address correspondence to James A. Birchler at the address above, or e-mail: birchlerj{at}missouri.edu.
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Abstract |
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This study tested the hypothesis that gibberellin levels were responsible for the superior growth habit of hybrids (i.e., heterosis). If this were true, plants reduced in their capacity to produce gibberellin, such as maize plants homozygous for dwarf1 (d1), should display a lesser heterotic response. The d1 mutation was introgressed into two inbred lines of maize, B73 and Mo17, for seven generations. Plants segregating for the dwarf phenotype were produced both by self-fertilizing the introgressed inbred lines and by making reciprocal crosses between them to produce hybrids. Measurements were made of several physical traits. The results indicated that the hybrid dwarf plants experienced no loss of heterosis relative to their normal siblings. These results exclude the possibility that modulation of bioactive gibberellins is a major underlying basis of the heterotic response.
Heterosis or hybrid vigor refers to the superior performance of hybrid plants relative to the better of the two parents. Traditionally the issue of heterosis has been posed in genetic terms such as dominance and overdominance (Birchler et al. 2003). Ultimately it is necessary to understand the molecular basis of heterosis.
It was proposed that gibberellin levels are responsible for the vigorous plant growth associated with heterosis (Paleg 1965; Rood et al. 1988; Sarkissian et al. 1964). This proposition was supported by the observations that inbred maize plants were more responsive to the application of exogenous gibberellin A3 (GA3), a synthetic analogue of gibberellin A1 (GA1), than are hybrids (Nickerson 1959; Rood et al. 1983, 1990). These results are consistent with the idea that inbreeding depression is due to an insufficiency of GA, while hybrids possess GA at near saturation. It was demonstrated that levels of GA1 in inbreds are much lower than in hybrids (Rood et al. 1988). At the time of these experiments it was believed that GA1 was the only version of GA that was bioactive in the regulation of maize shoot elongation (Phinney 1985). Subsequent evidence indicated that GA3, as well as another gibberellin, GA5, are endogenous to and bioactive in maize (Spray et al. 1996).
Any maize mutant that is deficient for producing or unresponsive to the presence of bioactive GAs is known as a dwarf. The dwarf 1 (d1) gene encodes a factor that is responsible not only for the conversion of GA20 to GA1 (Spray et al. 1984), but also for the conversion of GA20 to GA5 and GA5 to GA3 (Spray et al. 1996). The homozygous recessive d1 mutants are incapable of synthesizing any bioactive version of GA and exhibit a phenotype indicative of a reduced level of bioactive GAs (Spray et al. 1996). The d1 mutants are short, compact plants with shortened internodes, short wide leaves, and short erect tassels that exhibit difficulty in anther extrusion (Neuffer et al. 1997). In this study we tested the hypothesis that genetic modulation of GA levels affects heterosis. We developed near-isogenic lines that segregated for d1 and then produced hybrids that also segregated for d1. If modulation of the GA level is the underlying basis of heterosis, then the d1/d1 hybrids should experience little or no heterotic response relative to d1/d1 inbreds.
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Materials and Methods |
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Inbred lines B73 and Mo17, as well as a stock carrying the recessive d1, were obtained from E. Coe (University of Missouri). The d1 allele was backcrossed into the two inbreds a minimum of seven times. At each generation, plants were self-pollinated and outcrossed to the respective introgression parent. Sand bench screening of the progeny of selfed plants revealed which outcrosses would be segregating for d1 heterozygotes. The outcross progeny from the plants that proved heterozygous for d1 were used in the next generation of introgression. This produced a near-inbred B73 line and a near-inbred Mo17 line that each segregated for d1. Reciprocal crosses were made between the two lines to produce hybrids that segregated for d1/d1 mutants. Hybrid plants where a B73 plant acted as the female were designated B73/Mo17, while hybrids where Mo17 plants were used as the female were designated Mo17/B73. Some of the near-isogenic plants were self-pollinated to produce d1/d1-segregating samples of inbred B73 and inbred Mo17.
Preliminary sand bench screening was used to identify candidate d1 segregating ears. Seed from the four classes—B73 inbred, Mo17 inbred, B73/Mo17 hybrid, and Mo17/B73 hybrid—were planted in 20 kernel families at the University of Missouri–Columbia Genetics Farm on May 22, 2003. Each genotype was represented by six rows (20 kernels/row); the rows were randomized among genotypes. Rows were 914.4 cm (3 ft) apart and plants within the row were spaced 22.9 cm (9 in.) apart. Plants were classified as to whether they were dwarf or normal according to gross phenotype; dwarf plants have a striking phenotype with short wide leaves with essentially no internode elongation. Two families, both B73/Mo17, failed to segregate for dwarf plants and were eliminated from the study.
The dates of anther and silk emergence were noted for each developing plant. The days to first silk or anther emergence were given as the number of days from planting to the emergence event. After plants had completed growth and elongation, measurements were made of plant height, leaf length, and leaf width. Plant height was measured from ground level to the ligule of the uppermost leaf. Leaf length was measured from the ligule to the leaf tip of the leaf that subtended the primary ear. Leaf width was measured across the widest point of that same leaf. The nodes visible above ground were counted, as well as the number of nodes from the ground to the primary ear. Also counted was the number of tassel branches of each plant, including the main spike. After the plants had senesced, ears were harvested and the lengths measured.
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Results |
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Nine different measurements were made of 332 plants that germinated and successfully grew to maturity (Table 1). Attrition was higher among the inbreds. The dwarves segregated at the expected rate of 3:1 and the variation in the segregation rates among the genotypes was not significant per
2 analysis (P > .05). There were a number of instances where some plants could not be evaluated for every measurement because of damage to the plant or a failure of development. The sample sizes are noted in parentheses beside each average measurement in Table 1. The only genotype to have substantial losses was the Mo17 dwarf class.
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Dwarf plants were remarkably reduced in height, leaf length, number of tassel branches, and ear length relative to their normal siblings (Table 1). All of these differences were highly significant (P < .01 per t test). The number of days to silk and anther emergence was higher for dwarves than for their normal siblings (P < .01). In contrast, the average leaf width of dwarves was greater than their normal siblings (P < .05 for Mo17 inbred; all others P < .01). The dwarf phenotype had no significant effect on the total number of nodes or the node location of the primary ear except in the Mo17 inbred, where both traits were significantly less among the dwarves (P < .05).
The effects of heterosis among the normal plants were apparent (Table 1). The only comparisons between an inbred and a hybrid that were not significant (P > .05) were for total nodes and ear node location between the B73 inbred and the B73/Mo17 hybrid and for the number of tassel branches between the B73 inbred and either of the hybrids. The effects of heterosis are also apparent among the dwarves. Like their normal siblings, the B73 dwarves did not show significant differences for the total nodes and ear node location with the B73/Mo17 hybrid. The B73 dwarves also failed to show significant differences for silk emergence with either hybrid. All other differences between the dwarves of either of the inbreds compared to the dwarves of either of the hybrids were significant (P < .05).
The heterotic effects in dwarf genotypes appear to be no less than in the normal plants. Figure 1 shows the average measurements of the hybrids expressed as a ratio relative to the average of the inbreds. The average measurements for the hybrids were obtained by pooling data from all of the hybrids (B73/Mo17 and Mo17/B73) and the averages for the inbreds was obtained by pooling data from all of the inbreds (B73 and Mo17). For each trait, the differences between the means of the pooled hybrids and the inbreds were highly significant (P < .01 per t test). In no case do dwarves appear to suffer a decrease in heterotic effect. If anything, there seems to have been a slight increase in the relative advantage of hybrid dwarves over inbred dwarves. This was not expected since the hybrid dwarves grew in the same rows as their normal siblings that crowded and towered over them more than the normal siblings of the dwarf inbreds.
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Discussion |
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The heterotic response seen among the hybrid dwarves indicates that heterosis occurs without any limitation due to reduced amounts of bioactive GA. This finding is not consistent with the possibility that modulation of GA is a controlling factor in the heterotic response in maize. If modulation of bioactive GAs was the basis of heterosis, then the dwarves would have been severely restricted in the heterotic response.
Another recent study indicated that GAs do not underlie interspecific heterosis in poplars (Pearce et al. 2004). Although Populus trichocarpa with faster elongating shoots has higher GA concentrations than Populus deltoides, the hybrids possessed GA levels similar to the slower growing species, even though they exhibited heterosis for shoot elongation. Interestingly, among the F2 descendents there was a negative correlation between internode length and GA level.
We interpret the previous findings in maize (Paleg 1965; Rood et al. 1988, 1990; Sarkissian et al. 1964) to indicate that GAs may be a target of the heterotic response, if not an underlying mechanism. Considering heterosis at the cellular level, the growth potential, both in terms of cell size and rate of cell division, is controlled by rate-limiting factors. Clearly availability of resources such as energy, water, and essential nutrients are the ultimate limitations, but next in importance is the ability to obtain and metabolize these resources in an efficient manner. Inasmuch as hormones, such as GAs, can stimulate more effective metabolism of these resources, they are potential targets of heterosis. If all of the physiological mechanisms are working near maximum potential, then the application of exogenous GAs should have little stimulatory effect. If the metabolism of GA is reduced to a level where it becomes rate limiting for the processes it regulates, then the application of exogenous GA will have a stimulating effect on those processes. The dramatic effect of the application of exogenous GA to maize inbreds compared to maize hybrids indicates that it was a rate-limiting factor in the inbreds, but not in the hybrids. This interpretation could explain the results of Pearce et al. (2004). The observation that hybrid poplars had increased growth with lower GA concentrations than the faster growing parental genotype indicates that factors other than GA were rate limiting and were acted upon by heterosis. It is possible that, among the hybrid poplars, the levels of GA may have been down-regulated in order to achieve the optimal stimulatory signal. The present experiments indicate that GA modulation is not the major underlying basis of heterosis. Further work is needed to address the basis of hybrid vigor.
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Acknowledgments |
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We thank E. Coe for sharing the starting stocks and C. Ostlie for grammatical review of this manuscript. Research was supported by a grant from the U.S. Department of Energy Biosciences Program.
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Footnotes |
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Corresponding Editor: Susan Gabay-Laughnan
Received March 15, 2005
Accepted July 2, 2005
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References |
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