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a Universidade Federal do Rio Grande do Sul, Faculdade de Agronomia, Departamento de Plantas de Lavoura, Av. Bento Gonçalves, 7712, Cx.P. 776, Porto Alegre, RS 90012-970, Brazil
b USDA-ARS Plant Science Research Unit and Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, 411 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108
c Dep. of Agronomy and Plant Genetics and Plant Molecular Genetics Institute, Univ. of Minnesota, St. Paul, MN 55108
* Corresponding author (rines001{at}umn.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: GA, gibberellic acid • RFLP, restriction fragment length polymorphism
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Yield advantages in oat lines with the Dw6 dwarfing gene have been obtained in some Australian environments (Anderson and McLean, 1989). However, the dwarf phenotype generally limits the use of dwarfing genes in oat breeding because of decreases in seed size, quality, and yield (Brown et al., 1980; Marshall and Murphy, 1981; Meyers et al., 1985; Kibite and Clayton, 2000; Milach and Federizzi, 2001).
Comparison of plant height components of the Dw6 semidwarf line OT207 with its nondwarf progenitor line OT184 revealed a significant shortening in the Dw6 mutant in the length of its three uppermost internodes, particularly the peduncle (Brown et al., 1980). This reduction in peduncle elongation has been associated with the failure of the panicle to emerge fully from the leaf sheath, particularly under nonideal growth conditions. Panicle exertion genes introduced or selected for in lines carrying the Dw6 gene have been found to help ameliorate this problem (Farnham et al., 1990). The internode constitution of the dwarf lines with the Dw7 gene (in line NC2469-3) or the Dw8 gene in Kanota backcross derivatives has not been previously reported. This information is important in understanding how these genes affect plant height and in attempting to adjust for any undesirable effects they might have for cultivar development.
The gibberellic acid (GA) biosynthetic pathway can be dissected by means of dwarfing genes (Hedden and Proebsting, 1999). Dwarf mutants that are defective for different steps in the GA pathway and which respond to the exogenous application of GA have been described in maize (Zea mays L.), rice, and pea (Pisum sativum L.) (Phinney, 1984). The early 13-hydroxylation GA biosynthetic pathway leading to active GA1 occurs in maize, rice and pea (Phinney, 1984). Intermediates of this pathway have been identified in oat by gas-chromatographic spectrometry, indicating that the pathway also occurs in oat (Kaufman et al., 1976). However, other dwarf mutants have been identified in wheat, rye (Secale cereale L.), maize, and rice that are insensitive to the application of GA (Gale and Youssefian, 1985; Borner, 1991; Harberd and Freeling, 1989; Mitsunaga et al., 1994). In these mutants dwarfism appears to be due to causes other than blocks in GA biosynthesis.
In wheat, the presence of GA-insensitive dwarfing genes can be identified by a GA response test conducted at the seedling stage (Yamada, 1990). The genetics of the rht1 and rht2 recessive dwarfing genes were not clearly resolved until the GA-insensitive response assay was used in the genetic analysis (Gale and Youssefian, 1985). Oat counterparts of the wheat Norin 10 genes have not yet been identified, probably because in oat fewer studies have been conducted to develop assays to identify and manipulate these type of genes. Searching for such mutants in oat will likely be a challenge, especially if they are recessive with a moderate effect on plant height. The use of GA assays to identify GA-insensitive oat mutants would enable distinguishing among such mutants and plants that have reduced height because of other mutations or environmental effects.
The Dw6 and Dw7 dwarf mutants in oat have been identified as responsive to GA3 at the seedling stage (Federizzi, 1986). Farnham et al. (1990) compared the response of the Dw6 line OT207 and its progenitor line OT184 with applied GA3 at the boot stage and concluded that OT207 is also GA-sensitive at this stage. Comparisons have not been made between Dw6, Dw7, and Dw8 dwarf mutants and their respective nondwarf counterpart lines for their relative responses to applied GA at the seedling stage.
The objectives of this study were to determine the effect of the Dw6, Dw7, and Dw8 dwarfing genes on plant height components and to investigate the response of these three sources of dwarfism in oat to GA applied at the seedling stage.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Plant Height Components of Dwarf and Nondwarf Lines
Two rows for each of the nondwarf lines, OT184 and Kanota, and the dwarf lines, OT207, AV13/6/7, AV16/8/4, AV-21/5, AV14/5/1, AV17/3/10, AV12/9/9, and AV18/2/4, were planted in field nurseries at St. Paul, MN, in 1992 in a split plot design. Genotypes were the main plots with 10 replications per plot, which consisted of two rows 1.5 m long. The tall NC2469 and the dwarf line NC2469-3 were not included in the first year experiment because seed of the NC2469 line was not available in 1992. At maturity, plants of each genotype were pulled intact from the field plots. Plant height, internode lengths, panicle length, and panicle exertion were measured on the main tiller of each individual plant. The distance between the bottom of the panicle to the flag-leaf ligule was measured as panicle exertion (negative numbers indicating the portion of the panicle inside the leaf sheaf). A similar experiment was conducted in the field in 1993 at St. Paul. The main plots in 1993 had five 1.5-m-long rows and consisted of 40 replications. Plants of each of the nondwarf lines OT184, NC2469, and Kanota and the nine dwarf genotypes included in this study were pulled in the field and measured for the same traits. Means and standard deviations were calculated for each trait in both years. Plant height components of the dwarf lines were compared with their respective nondwarf counterpart lines by paired t-test.
Response to Gibberellic Acid
Five experiments were performed in growth chambers to assay the response of nondwarf and dwarf oat genotypes to exogenous GA applied at the seedling stage. The growth chamber conditions were similar for all experiments with the temperature at 20°C, 95% relative humidity. A 12-h photoperiod and a photon flux density of 300 to 400 µmol m-2 s-1 was supplied at canopy height from an adjustable height fixture containing a mixture of incandescent and cool white fluorescent lamps. Seed germination of nondwarf and dwarf genotypes in all experiments was carried out in 10-cm diam petri dishes with wetted Whatman No. 2 filter paper (Fisher Scientific, Pittsburgh, PA) for 5 d at 4°C, followed by 2 d at room temperature, before transfer to pots or trays for GA seedling treatments. Gibberellic acid in the GA3 form (Sigma, St. Louis, MO) was used in the first two experiments and in the GA1 and GA20 forms (cordially provided by Dr. B.O. Phinney, University of California, Los Angeles, CA) in the last three experiments. Regression analysis was performed for each genotype by PROC REG of SAS 6.03 (SAS Inst., 1990) for all experiments.
Experiments I and II
The methodology used in these experiments was based on Gale and Youssefian (1985). Seedlings of two nondwarf (OT184 and Kanota) and eight dwarf genotypes (OT207, AV13/6/7, AV12/9/9, AV16/8/4, AV21/5, AV14/5/1, AV17/3/10, and AV18/2/4) were transplanted to pots containing only vermiculite. A completely randomized design with two replications (pots) was used in both experiments. Five seedlings per pot were planted for each genotype. Treatments were applied seven days after transplanting to vermiculite by watering the pots with the GA solution. Treatments consisted of 50 mL of MS (Murashige and Skoog, 1962) salt solution or water supplemented with GA3 at concentrations of 0 (control), 50, 100 and 150 mg L-1. In Exp. I, MS solution with GA3 was applied for 5 d, followed by 4 d of water with GA3. In Exp. II, MS solution with GA3 was alternated with water with GA3. Nine days after the treatments were first applied, seedlings were measured for height to the second leaf insertion with a 30-cm-length ruler.
Experiments III, IV, and V
The methodology used in these experiments was based on the modified microdrop assay (Nishijima et al., 1990). The nondwarf genotypes OT184 and Kanota and the dwarfs OT207 and AV13/6/7 were tested in Exp. III and IV. The tall NC2469 and the dwarf NC2469-3 were only tested in Exp. III. The genotypes tested in Exp. V included Kanota and the Dw8 backcross derivatives AV12/9/9, AV16/8/4, AV14/5/1, AV17/3/10, and AV18/2/4. Germinated seedlings were transplanted to 20- by 40-cm trays filled with a mixture of 1 vermiculite: 1 soil. Three trays were treated with GA1 and three with GA20. Each tray had 10 rows with 10 seedlings of the same genotype per row. Treatments consisted of 0 (control), 1, 10, 100, and 1000 mg L-1 of GA1 or GA20. Genotypes and treatments were randomized within GA1 or GA20, so that each tray would be treated with only one type of GA. Treatments were first applied 3 d after transplanting. By means of a calibrated micropipet, a 10-mL drop of water mixed with a wetting agent, Tween 80 (Fisher Scientific, Pittsburgh, PA), and supplemented with GA1 or GA20 was applied to the surface of the first leaf sheath. This procedure was repeated for 2 d and the height to the first-leaf insertion was measured 5 d later.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The 46% reduction in height of NC2469-3 compared with its nondwarf counterpart NC2469 resulted from decreases in both internode number, as the dwarf line did not exhibit internode p-5, and internode elongation (Table 2). Marshall and Murphy (1981) similarly found a 42.5% reduction in the height of NC2469-3 compared with NC2469. However, the authors did not investigate plant height components of these lines. All internodes were significantly reduced in the NC2469-3 line, but the two internodes below the peduncle were the least affected (Table 2). Although NC2469-3 had one fewer internodes compared with its nondwarf counterpart, the reductions in internode lengths accounted for most of the decrease in plant height with the length reduction being most marked in the lower internodes. The compact panicle type of the dwarf line was 38% shorter than the normal and also contributed to reduction in plant height. According to Federizzi and Qualset (1989), the compact panicle of NC2469-3 is due to one additive gene (C) linked (0.08 ± 0.01 recombination frequency) to Dw7 and not to a pleiotropic effect of the dwarfing gene. The lack of panicle exertion was not as intense in this dwarf as in the OT207 line. The Dw7 gene acted differently from Dw6 and appeared to exert an effect on internodes elongated both early and later in plant development.
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The regression coefficients (b) obtained in Exp. I were similar to those from Exp. II for most genotypes, except Kanota (Table 4). OT184 had slightly larger regression coefficients than its dwarf derivative OT207 in Exp. I and II. However, the 95% confidence intervals for the OT184 and OT207 coefficients overlap, indicating that these differences were not significant. Kanota had a regression coefficient significantly larger than the Japanese lines in Exp. I. These differences were not detected in any of the subsequent experiments and resulted from the higher response of Kanota in Exp. I. Even though Kanota and the Japanese lines might differ in the magnitude of response to applied GA, such differences are small when detected in only one out of the five experiments.
These results demonstrated that both nondwarf and dwarf genotypes responded significantly to applied GA3 at the seedling stage. This finding led us to further investigate the possible role of the dwarfing genes in the GA biosynthetic pathway. In the early 13-hydroxylation pathway, the 3 beta-hydroxylation of GA20 leads to GA1, which is the active endogenous GA form in maize, rice, and pea (Phinney, 1984). A mutation in the gene that controls the GA20 to GA1 step results in accumulation of GA20 in the dwarf plant. Such mutants are responsive to GA1 but not GA20, and have been described in species including pea (Ingram et al., 1984), rice, and maize (Phinney, 1984).
Dwarf lines for each of the three dwarfing genes were compared with their corresponding nondwarf progenitor lines for response to applied GA1 and GA20. All genotypes responded to both GA1 and GA20 with similar results obtained for each genotype across two experiments (Table 5). When the other Dw8 lines were tested in a similar experiment, each of them also responded to both GA1 and GA20 (Exp. V, data not shown). These results indicate that none of the oat dwarf lines studied in this paper result from a modification in the step of conversion of GA20 to GA1 although regression coefficients (b) for the response to GA20 were smaller than the ones for GA1 for tall and dwarf genotypes. This result was expected since GA20 is less biologically active than GA1 (Phinney, 1984). Higher amounts of GA20 would need to be applied to obtain a similar response as with GA1.
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In the GA experiments, OT207 responded about the same as its tall counterpart OT184 to applied GA3 at the seedling stage (Table 4). The differences in the magnitude of the OT207 response to GA at the seedling and at the boot stage may result from a differential expression of the Dw6 gene in these distinct developmental stages. Indeed, as we described previously, the Dw6 gene appears to have a major effect in the elongation of the three uppermost oat internodes.
Dwarf lines carrying the Dw7 or Dw8 genes also responded much like their nondwarf counterparts to applied GA (Table 4). Because of the sensitivity of these lines to GA, these genes are also likely to be involved in GA metabolism. However, it appears that different mechanisms are involved in the action of the Dw7 and Dw8 genes. The effect of the Dw7 gene on plant height components was different from the effect of Dw8. Even though both dominant mutant genes similarly reduce the elongation of all plant internodes, only Dw7 affected the number of internodes. It may be possible to adjust the effects of these genes by crossing the dwarf with tall lines that have more internodes and/or overall extended internodes.
A common feature of GA-sensitive mutants is that they are usually recessive and deficient for GA because of a block in the biosynthetic pathway (Phinney, 1984). Recessive genes involve the loss of wild-type function (Herskowitz, 1987). Interestingly, all the oat dwarf genotypes we studied were responsive to applied GA but controlled by dominant dwarfing genes. A lack of identified recessive mutants for GA biosynthesis in hexaploid oat is not surprising because any such mutation in a GA biosynthetic pathway would likely be masked by the action of homoeologous genes in the other progenitor genomes comprising hexaploid oat. A recessive dwarfing gene dw5 has been identified in diploid oat derivatives (Nishiyama, 1957). Dominant or semidominant dwarfing mutations have been identified at the Rht-B1 and Rht-D1 loci of wheat and the d8 locus of maize. These genes have recently been shown to be orthologs of the Arabidopsis gibberellic acid insensitive (GAI) gene (Peng et al., 1999). The GAI gene was proposed to be the basis of a signaling pathway that regulates negatively gibberellin responses (Peng et al., 1997). A deletion mutant in the GA-recognition portion of the GAI negative modulator (repressor) then acts in a dominant manner to produce GA-insensitive repression of growth genes, and thus reduced plant height. The three dominant dwarf oat mutants studied here, however, are GA-sensitive. To explain the dominance effect of these GA-sensitive dwarfing mutants we postulate that the genes affected may participate normally either directly or indirectly in negative regulation (repression) of genes for GA metabolism. GA biosynthesis is known to be regulated by numerous endogenous and environmental signals (Yamaguchi and Kamiya, 2000). Furthermore, individual steps in GA metabolism may be catalyzed by products of gene families with different tissue-specific and developmental patterns of expression (Hedden and Proebsting, 1999). Thus, the multiplicity in regulatory signals and in genes involved in GA metabolism provides several points at which dominant mutations could occur producing not only GA-deficiency but also resulting in various dwarfing phenotypes.
One possibility on the nature of the oat dwarfing mutations was suggested on the basis of observations that seedlings of the Dw7 line NC2469-3 grown in growth chamber and greenhouse environments often were dark green and accumulated anthocyanin (Milach and Federizzi, 2001). Similar coloration and dwarfing phenotypic effects have been observed in transgenic tomato and Arabidopsis plants transformed with an oat phytochrome A (phyA) gene (Boylan and Quail, 1989; 1991) and in tobacco (Nicotiana tabacum L.) plants transformed with a rice phyA gene (Nagatani et al., 1991). The dwarf phenotype also occurs in transgenic Arabidopsis plants overexpressing the Arabidopsis or the rice phytochrome B (phyB) genes (Wagner et al., 1991). Thus, overexpression of either phyA or phyB genes can cause dwarfism. The semidominant nature of the Dw7 mutation would be in line with overexpression of a phytochrome gene. An oat phyA gene and a rice phyB gene were mapped on the hexaploid oat restriction fragment length polymorphism (RFLP) map (O'Donoughue et al., 1995) for comparison to the location of the previously mapped Dw7 gene (Milach et al., 1997). The oat phyA gene mapped to linkage group 28 and the rice phyB gene to linkage group 30 of the oat map while Dw7 was located on linkage group 22 (Milach and Federizzi, 2001). Independence of Dw6, Dw7, and Dw8 from the mapped phytochrome loci indicates that they are not alleles of these phytochrome genes. However, because at least three phytochromes occur in oat (Wang et al., 1991), Dw7 could be a mutation in a phytochrome structural gene not mapped or in a gene regulating phytochrome production.
The dominant oat dwarfing genes Dw6, Dw7, and Dw8 appear to be quite distinct from the wheat Rht1 and maize Dw8 dominant dwarfing genes because of their difference in GA-sensitivity; however, these oat genes may be similar to GA-sensitive dominant genes which have been identified in wheat and rye. The Rht12 mutant of wheat and the Ddw1 mutant of rye are strong dominant GA-sensitive mutant genes mapping to homoeologous regions on the long arms of chromosomes 5A and 5R, respectively (Borner et al., 1998). The semidominant GA-sensitive dwarfing gene Rht8 has been identified in many central European wheats and mapped to the short arm of 2DS (Korzun et al., 1998). Several additional semi- or partially dominant GA-sensitive dwarfing genes in wheat have been reported (Konzak, 1987) but have not been characterized or mapped. The segmental homoeology within hexaploid oat and a lack of common markers between wheat and the regions surrounding the mapped oat dwarfing genes precludes predictions at this time of possible orthology between various oat and wheat dwarfing mutants. Such relationships, however, should be discernible in the future, particularly as candidate genes for dwarfing and regulation of GA metabolism are identified in various plant species.
In summary, the three oat dwarfing genes—Dw6, Dw7, and Dw8—reduced plant height in different ways. The Dw6 gene primarily reduced the length of the three uppermost internodes and did not affect internode number. The Dw7 gene shortened all internodes but particularly the first and the last, and reduced internode number in the dwarf line. The Dw8 gene significantly shortened all internodes but did not affect internode number. The three dwarfing loci investigated in this study are responsive to GA and are likely involved in GA metabolism. The oat dwarf mutants studied respond to both GA1 and GA20 and, therefore, do not result from a modification in the conversion of GA20 to GA1. Based on the GA-sensitivity and the dominant or semidominant inheritance pattern of the Dw6, Dw7, and Dw8 mutants, we suggest that they may be variants in some type of negative regulation of GA metabolism in hexaploid oat. The results indicate that for use of the three dwarfing genes in oat cultivar development different strategies may be needed to adjust for their differing effects on various plant height components.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication June 4, 2001.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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