Plant Physiol. (1998) 116: 1003-1011

Photoperiod Control of Gibberellin Levels and Flowering in Sorghum¹

In-Jung Lee², Kenneth R. Foster³, and Page W. Morgan^*

Department of Soil and Crop Sciences, Texas A&M University, College Station, Texas 77843-2474

	ABSTRACT

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Regulation of rhythmic peaks in levels of endogenous gibberellins (GAs) by photoperiod was studied in the short-day monocot sorghum (Sorghum bicolor [L.] Moench). Comparisons were made between three maturity (Ma) genotypes: 58M (Ma₁Ma₁, Ma₂Ma₂, phyB-1phyB-1, and Ma₄Ma₄ [a phytochrome B null mutant]); 90M (Ma₁Ma₁, Ma₂Ma₂, phyB-2phyB-2, and Ma₄Ma₄); and 100M (Ma₁Ma₁, Ma₂Ma₂, PHYBPHYB, and Ma₄Ma₄). Plants were grown for 14 d under 10-, 14-, 16-, 18-, and 20-h photoperiods, and GA levels were assayed by gas chromatography-mass spectrometry every 3 h for 24 h. Under inductive 10-h photoperiods, the peak of GA₂₀ and GA₁ levels in 90M and 100M was shifted from midday, observed earlier with 12-h photoperiods, to an early morning peak, and flowering was hastened. In addition, the early morning peaks in levels of GA₂₀ and GA₁ in 58M under conditions allowing early flowering (10-, 12-, and 14-h photoperiods) were shifted to midday by noninductive (18- and 20-h) photoperiods, and flowering was delayed. These results are consistent with the possibility that the diurnal rhythm of GA levels plays a role in floral initiation and may be one way by which the absence of phytochrome B causes early flowering in 58M under most photoperiods.

	INTRODUCTION

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The maturity gene Ma₃ contributes to the timing of floral initiation in sorghum (Sorghum bicolor [L.] Moench). This gene has three known alleles, Ma₃, ma₃, and ma₃^R (Quinby, 1973). Plants containing ma₃^R differ from those homozygous for the other alleles in that they flower early, are relatively insensitive to a wide range of photoperiodic conditions, and elongate more rapidly and tiller less than non-ma₃^R plants. In contrast, genotypes containing Ma₃ or ma₃ are very sensitive to photoperiod, and flowering is delayed by long (12-h) days and hastened by short (10-h) days (Pao and Morgan, 1986). Other than relatively small differences in flowering dates, Ma₃ and ma₃ are phenotypically indistinguishable.

Recently, Ma₃ was shown to code for phytochrome B and ma₃^R was shown to contain a deletion mutation that results in a transcription stop codon upstream of the proposed second dimerization site (Childs et al., 1997). Therefore, ma₃^R is an apparent null mutant and the three maturity gene alleles were renamed: Ma₃ to PHYB, ma₃^R to phyB-1 (Childs et al., 1997), and ma₃ to phyB-2 (present paper). Abbreviations for phytochrome genes are according to the proposal of Quail et al. (1994).

Increasing the photoperiod increases GA levels in many long-day plants, presumably as the result of daylength perception by phytochrome (Metzger and Zeevaart, 1980a, 1980b; Gianfagna et al., 1983; Davies et al., 1986; Rood et al., 1986). Photoregulation of GA metabolism may be the result of the presence or absence of light, the transition between light and dark, the duration or alteration of photoperiod length, the quality of the light (particularly red and far-red light), and the quantity of light.

Several of the steps in the early C-13-hydroxylation pathway have been reported to be regulated by the light environment, including those for steps upstream of GA₁₂ (Zeevaart et al., 1993), GA₁₂GA₅₃ (Davies et al., 1986), GA₅₃GA₄₄ (Graebe, 1987), GA₁₉GA₂₀ (Metzger and Zeevaart, 1980b; Gianfagna et al., 1983), and GA₂₀GA₁ (Campell and Bonner, 1986; Gilmour et al., 1986). Assays of GA₁ at the end of the 8-h light period and at the end of the 16-h dark period showed that its level is diurnally regulated in long-day spinach (Talon et al., 1991). The same pattern for GA₁ was observed when 8-h high-light days were extended with 16 h of dim far-red light, but GA₂₀, the precursor to GA₁, continued to increase during the extension, and the highest levels occurred at the end of the daily far-red light period.

GA biosynthesis in sorghum is diurnally regulated (Foster and Morgan, 1995). Under 12-h photoperiods, rhythmic patterns of GA₁₂ and GA₅₃ levels in phyB-1 and non-phyB-1 genotypes were similar, peaking at midday and having their minima at night. However, GA₂₀ and GA₁ exhibited a different pattern between phyB-1 and non-phyB-1 genotypes; levels peaked near dawn in phyB-1 and at midday in the non-phyB-1 genotypes (Foster and Morgan, 1995). This reveals that relative GA₁ level differences between phyB-1 and non-phyB-1 genotypes depend on the time of day; the maximum difference occurs around dawn. The differences in GAs between genotypes are not in the absolute level, but in a shift in the timing of rhythmic peaks or pulses of GA₂₀ and GA_1. The timing of the rhythmic pulse of GA₁ is altered or possibly uncoupled in 58M, and this change is correlated with the absence of a functional phytochrome B, altered photoperiodic sensitivity, early flowering, inhibition of tillering, and promotion of shoot growth (Pao and Morgan, 1986; Childs et al., 1991, 1992, 1995, 1997).

Production of a plant hormone in rhythmic diurnal pulses is not a unique discovery. Ethylene has been shown to be produced in rhythmic, often diurnal pulses in a number of studies (Lipe and Morgan, 1973; Rikin et al., 1984; Morgan et al., 1990; Ievinsh and Kreicbergs, 1992; Machácková et al., 1997). However, the observations on GA₂₀ and GA₁ are the first, to our knowledge, in which the rhythmic levels of hormones have been observed in a context that suggests a possible linkage with both a physiological process (promotion of floral initiation) and a regulatory mechanism (phytochrome perception of daylength) (Talon et al., 1991; Foster and Morgan, 1995). The existence of diurnal rhythms of GA levels in sorghum, and the change in the rhythm of the levels of GA₂₀ and GA₁ in plants deficient in phytochrome B, offer opportunities to further our understanding of photoperiodic control of both GA metabolism and flowering.

If the distinctive pattern of GA₂₀ and GA₁ levels seen under 12-h photoperiods is linked to early floral initiation, that pattern should be altered under long-day conditions, which delay floral initiation in the phyB-1 genotype. It should be of interest to determine whether the pattern of GA concentration rhythms associated with floral induction and noninduction are the same for phyB-1 and non-phyB-1 genotypes. We report here the characterization of the photoperiodic regulation of GA content in genotypes containing phyB-1 (58M) and those without phyB-1 (phyB-2 [90M] and PHYB [100M]).

In our studies with sorghum (for review, see Morgan, 1994), photoperiods have been presented as high-intensity light periods of varying length because photoperiodicity of sorghum had already been established when the work began. In addition, brief daylength extensions with far-red light had been shown to hasten flowering (Lane, 1963; Williams and Morgan, 1977), making the use of dim far-red light to extend a basic photoperiod questionable for this species. Sorghum also appears to be strongly thermoperiodic, because synchrony of photoperiods with thermoperiods influences flowering date (Morgan et al., 1987). Thus, to avoid confusion in interpreting data and to provide a strong, unambiguous cue to the plant, the experiments reported here involved variations in the length of the light period, which conformed to the timing of a 30°C thermoperiod. Unless specified otherwise, we use the term photoperiod to refer to treatments of varying duration of a high-intensity light period with a matching high-temperature period (30°C).

	MATERIALS AND METHODS

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Plant Materials and Growth Conditions

2

1

Harvests started at the beginning of the light period 14 d after seeding at 3-h intervals for 24 h. When sampling times decreased at the transition between lights-on and lights-off, samples were taken immediately after lights-on at the beginning of the photoperiod and immediately before lights-off at the end of the photoperiod. For harvests during the dark period, plants were removed from the growth chamber in complete darkness and cut under a dim-green safelight. Plants were cut at the root-shoot junction and at the top of the tallest leaf collar. Samples containing the part of the culm between the root crown and the tallest leaf collar were obtained from each genotype. The basal three leaf blades and sheaths were removed (two at harvest and the third after freeze-drying). Because samples of 58M grown under 18- and 20-h photoperiods were tall and had more expanded leaves, a fourth leaf was removed. Two replicate samples were obtained at each harvest time. Samples were frozen in liquid N₂ immediately after harvesting. After lyophilization, samples were stored at 20°C until extracted for GAs. To minimize the variation due to differential moisture absorption by tissue during storage, every sample was relyophilized just before weighing and extraction.

Gas Analysis

As discussed by Beall et al. (1991) and Foster et al. (1994), sorghum contains the early C-13-hydroxylation pathway GAs demonstrated to occur in maize (Phinney, 1984), which presumably follow the biosynthetic pathway established in maize: precursor  GA₁₂ GA₅₃  GA₄₄  GA₁₉  GA₂₀  GA₁  GA₈. Analysis focused on these compounds, but because the amounts of GA₄₄ detected were very small, these data were not reported.

Experimental Design and Replication

View larger version (30K): [in this window] [in a new window]   Figure 1. Diurnal regulation of GA levels in shoots of 14-d-old seedlings of 58M (), 90M (), and 100M () grown under 10-h photoperiods. The dark period is indicated by the solid bars at the top and bottom of the figure. GA levels were measured by GC-MS selected ion monitoring using deuterated internal standards. Data are the means of two replicate samples. Error bars show sd. DW, Dry weight.

View larger version (31K): [in this window] [in a new window]   Figure 3. Diurnal regulation of GA levels in shoots of 14-d-old seedlings of 58M (), 90M (), and 100M () grown under 16-h photoperiods. The dark period is indicated by the solid bars at the top and bottom of the figure. GA levels were measured by GC-MS selected ion monitoring using deuterated internal standards. Data are the means of two replicate samples. Error bars show sd. DW, Dry weight.

View larger version (26K): [in this window] [in a new window]   Figure 5. Diurnal regulation of GA levels in shoots of 14- () and 19-d-old seedlings () of 58M, a phytochrome B mutant, grown under 20-h photoperiods. The dark period is indicated by the solid bars at the top and bottom of the figure. GA levels were measured by GC-MS selected ion monitoring using deuterated internal standards. Data for 19-d-old seedlings are the means of two replicate samples, and data for 14-d-old seedlings are single samples. Error bars show sd. DW, Dry weight.

The experiment reported in Figure 5 involved only 58M to determine whether the patterns of GA₁ levels exhibited by 58M under 18-h photoperiods persisted with a longer photoperiod (20 h) and with older plants (19 d old). For this 20-h-photoperiod experiment, we grew 58M in two different growth chambers to verify that microenvironmental differences in different growth chambers did not have a major effect on the rhythms of GA levels. Some of the samples from one set of 14-d-old plants (58M, 2:00 am, Fig. 1) were lost after harvest. The data shown in Figure 6 are averages of all values in Figures 1-4, and sd values are plotted (see legend of Fig. 6).

View larger version (26K): [in this window] [in a new window]   Figure 6. Levels of endogenous GAs in shoots of 14-d-old seedlings of 58M (), 90M (), and 100M () under four different photoperiodic conditions. GA levels were measured by GC-MS selected ion monitoring using deuterated internal standards. Data are the average of 18 samples; two samples each were harvested every 3 h for 24 h. Error bars show sd. DW, Dry weight.

	RESULTS AND DISCUSSION

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The results of earlier experiments suggest that GA₁₂ levels are regulated by photoperiod in 58M (phyB-1phyB-1), 90M (phyB-2phyB-2), and 100M (PHYBPHYB) (Foster and Morgan, 1995). In all three genotypes, GA₁₂ levels were high during the light periods and low during the dark periods. The absence of phytochrome B (Ma₃ gene product) in 58M (Childs et al., 1992, 1997) had no effect on the rhythmic fluctuation of GA₁₂ levels, since the timing of the maximum and minimum levels of GA₁₂ were similar between phyB-1 and non-phyB-1 genotypes. In the present study the pattern of pulses of GA₁₂ levels was not changed to a major degree by different photoperiod and thermoperiod durations ranging from 10 to 18 h (Figs. 1-4). The pattern was expressed most strongly in a 10-h photoperiod/thermoperiod (Fig. 1).

View larger version (29K): [in this window] [in a new window]   Figure 2. Diurnal regulation of GA levels in shoots of 14-d-old seedlings of 58M (), 90M (), and 100M () grown under 14-h photoperiods. The dark period is indicated by the solid bars at the top and bottom of the figure. GA levels were measured by GC-MS selected ion monitoring using deuterated internal standards. Data are the means of two replicate samples. Error bars show sd. DW, Dry weight.

View larger version (31K): [in this window] [in a new window]   Figure 4. Diurnal regulation of GA levels in shoots of 14-d-old seedlings of 58M (), 90M (), and 100M () grown under 18-h photoperiods. The dark period is indicated by the solid bars at the top and bottom of the figure. GA levels were measured by GC-MS selected ion monitoring using deuterated internal standards. Data are the means of two replicate samples. Error bars show sd. DW, Dry weight.

With 14- and 16-h photoperiods, GA₁₂ levels tended not to vary much during the day, especially in the non-phyB-1 genotypes under 14 h, and if levels did vary discernibly, peaks occurred later in the photoperiod. The daily peaks in GA₁₂ were a little more strongly expressed in an 18-h photoperiod, although still peaking late in the day. Genotypes 58M, 90M, and 100M all possess a working circadian clock, as demonstrated by rhythmic pulses in chlorophyll a/b binding protein mRNA that continue in constant light (Childs et al., 1995). Zeevaart and Gage (1993) have demonstrated that ent-kaurene synthesis is regulated by photoperiod, and this may be a result of phytochrome action. ent-Kaurene levels were not measured in this study, and the participation of phytochrome in the control of GA-related metabolism upstream of GA₁₂ or ent-kaurene was not evaluated.

However, if a phytochrome is responsible for diurnal regulation of GA₁₂ level or biosynthetic steps upstream of GA₁₂, it is unlikely to be phytochrome B, which is missing in 58M. The reason for this conclusion is that the pattern of GA₁₂ levels is similar in phyB-1 and non-phyB-1 genotypes across photoperiods ranging from 10 to 18 h, except for the differences previously noted at 14 h (Figs. 1-4).

Levels of GA₅₃ also showed a daytime peak in all three genotypes and all photoperiods tested (Figs. 1-4). This pattern is similar to that of GA₁₂ and suggests that GA₅₃ levels are determined by the levels of its precursor. However, the changes in levels of GAs observed in this study were not demonstrated to be caused by synthesis and could also result from breakdown or conjugation reactions. Levels of GA₅₃ increased gradually after lights-on, peaked later in the light period than GA₁₂ levels, and decreased to minimum levels at the end of the dark period. These diurnal patterns were lost as the presumed pathway of metabolism proceeded through GA₄₄ to GA₁₉. Neither genotype exhibited a distinctive pattern of GA₁₉ levels. It should be noted that pool sizes of GA₁₉ are several times higher than those of GA₁₂ and GA₅₃; therefore, daily pulses in the metabolism of GA₅₃ and GA₁₉ might have less impact on the total GA₁₉ pool size. These patterns of GA₁₂ and GA₅₃ and the absence of a daily peak in GA₁₉ levels are consistent with the observed patterns of these GAs under a 12-h photoperiod (Foster and Morgan, 1995).

In plants that are wild type (Quinby, 1973; Pao and Morgan, 1986) for all aspects of phenotype except flower initiation dates in some environments (90M and 100M), GA₂₀ levels followed a discernible diurnal pattern for all photoperiods tested (Figs. 1-4). The peaks in GA₂₀ levels, although small in magnitude, occurred 6 to 9 h after lights-on. Note, however, that a peak in levels of GA₂₀ did not occur in 90M under 10-h photoperiods, making its GA₂₀ pool behave more like 58M at the 2:00 and 5:00 pm harvests. The uniformity of the data for these non-phyB-1 genotypes indicates that the increase in GA₂₀ is probably controlled by photoperiod. Although the rhythmic pattern of GA₂₀ levels was not altered by variation of the photoperiod (10-18 h) in 90M and 100M, it was altered by photoperiod in the phytochrome B null mutant 58M.

In photoperiods of 16 h or less (Figs. 1-3), GA₂₀ levels in 58M decreased gradually during the day, but in photoperiods of 18 h or more (Figs. 4 and 5), GA₂₀ levels increased gradually to a peak and then declined. These results were reinforced by the rhythmic pattern of GA₂₀ levels (increasing during the day) of 19-d-old 58M under a 20-h photoperiod (Fig. 5). Another way of viewing the pattern of GA₂₀ levels is that under photoperiods of 16 h or less, the highest level during the light period occurred at the time of lights-on in 58M and 6 to 9 h later in 90M and 100M (Figs. 1-3).

Under the 18-h photoperiod, the pattern of GA₂₀ levels was similar in all three genotypes (Fig. 4). The phase shift in the regulation of GA₂₀ levels in 58M compared with 90M and 100M strongly suggests that the missing phytochrome B is required for proper wild-type regulation of the metabolism controlling the GA₂₀ pool size (Figs. 1-3). This reaction in the mutant 58M was affected by photoperiod duration; a noninductive, long photoperiod (18 and 20 h) altered this pattern to one similar to that of wild type (100M) (Figs. 4 and 5).

Floral initiation of 58M was delayed by very long days (Childs et al., 1995). Under 18-h photoperiods, 58M initiated a floral meristem at about d 50 (Foster et al., 1997), whereas under 12-h photoperiods, it initiated at d 20 (Pao and Morgan, 1986). In this study under 18-h photoperiods, 58M did not initiate florally until d 60, and four to five internodes elongated before floral initiation. The same response was noted by Childs et al. (1995). It is interesting that in 58M under the 18-h photoperiod (Fig. 4), in which floral initiation was delayed, the pattern of GA₂₀ levels was changed to one similar to that of 90M and 100M (wild type), which normally exhibit delayed floral initiation with a photoperiod only 12 h long. However, the correlation with flowering appears not to be absolute because in some experiments floral initiation is delayed by 16-h photoperiods (Childs et al., 1995), and that condition did not shift the GA₂₀ content pattern here (Fig. 3).

In this study GA₂₀ levels were regulated in a different manner compared with the levels of GA₅₃ and GA₁₉. Assuming that biosynthesis is a major factor in the fluctuation in GA₂₀ levels, the results here may imply that different enzymes catalyzed the steps before and after GA₁₉.

There is considerable evidence indicating that the steps catalyzed by GA 20-oxidase are important regulatory steps in GA biosynthesis. In maize seedlings bioactive GAs were shown to regulate their own biosynthesis through feedback control of GA 20-oxidase activity (Hedden and Croker, 1992). In addition, expression of GA 20-oxidase genes in Arabidopsis thaliana was remarkably reduced after the application of GA₃ (Phillips et al., 1995). Higher levels of accumulation of GA₂₀ precursors (GA₁₉ and GA₅₃) in non-phyB-1 genotypes (Figs. 1-4) may be the result of the low activity of GA 20-oxidase. Furthermore, it may be speculated that the apparent higher enzyme activity of this enzyme in the mutant phyB-1 may partially account for the rapid growth of this genotype. Correlation between an increase in GA₂₀ and a decrease in its precursor, GA_19, was fairly well kept in 58M (Figs. 1-4). However, there was no clear relationship between these two GAs in 90M and 100M.

The level of GA₁ also appears to be controlled by photoperiod. In the non-phyB-1 genotypes (100M and 90M), rhythmic production of GA₁ occurs in plants grown under 10-h photoperiods (Fig. 1), which is different from that of plants grown under longer photoperiods (Figs. 2-4; see Foster and Morgan [1995] for data on 12-h photoperiods). In photoperiods of 12 h or longer, GA₁ levels consistently increased during the early portion of the light period, with the peak concentration occurring 6 h or occasionally 9 h after lights-on. Under a 10-h photoperiod, GA₁ levels were highest at lights-on and decreased gradually during the day. In 90M this downward trend started after 3 h rather than at lights-on (Fig. 1). This decreasing pattern is similar to that observed in 58M under 12-h photoperiods, when it flowers early (Foster and Morgan, 1995).

Under 10-h photoperiods, there are no differences in floral initiation between these three genotypes (Pao and Morgan, 1986). In the present study, 58M, 90M, and 100M initiated floral primordia at d 20, 23, and 22, respectively, under a 10-h photoperiod. In contrast to the behavior of the non-phyB-1 genotypes under 10-h photoperiods, the patterns of GA₁ levels in 58M grown under photoperiods of 18 h or more (Figs. 4 and 5) were different from those of plants grown under photoperiods of 16 h or less (Figs. 1-3; Foster and Morgan, 1995), with GA₁ levels starting low and increasing during the day in the former cases of 18-h or longer photoperiods.

Under photoperiods of 16 h or less, GA₁ levels in 58M decreased during the day. In addition, this decreasing pattern of GA₁ during the first 3 to 6 h of the light period was sharper in the 10-h photoperiod (Fig. 1) than in the other photoperiods (Figs. 2-4). Again, these patterns can also be viewed as a peak level during the light period, at either lights-on or 6 to 9 h later. This decreasing pattern (morning peak) of GA₁ in 58M under a 10-h photoperiod eventually changed to an increasing pattern (midday peak) observed under an 18-h photoperiod. In 58M, GA₁ levels in all photoperiods followed the pattern of GA₂₀ levels. However, GA₁ levels in 100M and 90M grown under a 10-h photoperiod did not follow the GA₂₀ pattern. Absolute levels of GA₁ in 90M and 100M were slightly lower than those observed for GA₂₀. The magnitude of the differences in levels of GA₁ between peaks and valleys is often small, and the pattern is less distinct at mid-range photoperiods (14 and 16 h) than at those that distinguish differences in genotypes (10 and 18 h).

In some cases, the GA levels exhibited in the first sample (8:00 am, 14 d after seeding) were not reproduced in the sample taken 24 h later (8:00 am, 15 d after seeding). This was most often expressed in low levels of GA₂₀ and GA₁ in 58M at the end of the dark period, as shown in Figures 1-5. This was also noted earlier when the harvest period was 36 h, ending at the end of a light period rather than a dark period (i.e. after two daily patterns were exhibited) (Foster and Morgan, 1995). Possible explanations for this inconsistency have been discussed previously (Foster and Morgan, 1995). There are no data to explain this occasional difference; however, the decreased levels may be related to the frequent opening of the growth chamber during sampling, which might briefly change the temperature, or to a change in the light environment as plant density changed because of thinning of the population during successive harvests. In fact, at the first 8:00 am sampling the growth chamber had not been opened for several hours, but at the last sampling it had been opened every 3 h for 24 h.

The patterns of GA levels found in this study, (a) being high at lights-on and declining or low at lights-off, and (b) increasing to a peak during the day, occurred in multiple experiments in harvests at 14 and 19 d in this study (Fig. 5) and at 14 and 25 d in a previous study (Foster and Morgan, 1995), which indicates that the results are not unique to a certain plant age. Thus, the occasional failure of GA levels to track back to a starting level from 24 h previously is concluded to be of less significance than the more dependable occurrence of pulses or peaks in levels of GA₁₂, GA₅₃, GA₂₀, and GA₁, which are altered by phyB-1 and by the duration of photoperiod (Figs. 1-4; Foster and Morgan, 1995).

The phyB-1 mutant 58M was initially classified as a GA overproducer based on the GA content of sorghum samples collected during the first few hours after lights-on (Beall et al., 1991; Childs, 1993; Foster et al., 1994). The discovery of rhythmic peaks in GA concentrations at different times of the day indicated that this overproducer classification was an oversimplification of the effect of the absence of phytochrome B (Foster and Morgan, 1995). This conclusion is strongly supported by the present study when the data from nine sampling points (two replications) for 1 d were averaged for each photoperiod (Fig. 6). Some of the trends do not represent unquestionable differences, as indicated by the overlap of the sd values. However, if the data for GA₁₂ are set aside, the overall pattern is for the non-phyB-1 genotypes to contain higher levels of early-pathway GAs (GA₅₃ and GA₁₉), whereas the average levels of GA₂₀ are clearly higher in the phyB-1 genotype (58M). Differences in levels of GA₁ between 58M and the non-phyB-1 genotypes tend to follow those of GA₂₀, except at 10 h, when average levels in all three genotypes are about the same. These results suggest that the conversion of GA₁₉ to GA₂₀ is a rate-limiting step in sorghum and may be a major control point of phytochrome B.

Both shoot length and dry weight are known to be higher in 58M than in 90M and 100M when grown under 12-h photoperiods (Pao and Morgan, 1986; Beall et al., 1991). However, under long photoperiods (23 or 24 h) shoot elongation is inhibited (compared with 12-h photoperiods) in 90M and 100M but not in 58M (Childs et al., 1995; K.L. Childs and P.W. Morgan, unpublished data). In the present study increasing daylength promoted shoot dry weight until 18 h, when the dry weight of 90M and 100M but not 58M was reduced (Fig. 7).

View larger version (29K): [in this window] [in a new window]   Figure 7. Effect of photoperiod on shoot dry weight (DW) of 14-d-old seedlings of 58M, 90M, and 100M. Dry weight represents the shoot between the root crown and tallest leaf collar without the basal three leaves. Error bars show sd.

These inhibitory effects of very long photoperiods on shoot growth of wild-type plants is not explained by average GA₁ levels, which do not differ at 18 h from those at 14 or 16 h (Fig. 6). Under shade, shoot growth is favored at the expense of root growth (Smith, 1992), but 58M plants (and other phytochrome B mutants; Childs et al., 1997) grow as if they were continuously in shade. The phyB-1-containing 58M exhibits a higher shoot-to-root ratio of dry weight than the non-phyB-1 genotypes (Lee, 1996; Morgan et al., 1996). The data shown in Figure 7 suggest the possibility that extremely long photoperiods may have the opposite effect of shade on partitioning of photosynthate in the wild type.

It has been clearly demonstrated in a number of rosette long-day plants that photoperiodic control of stem elongation is mediated by GAs (Jones and Zeevaart, 1980; Metzger and Zeevaart, 1980b; Gianfagna et al., 1983; Talon et al., 1990, 1991; Zeevaart et al., 1993). The promotive effect of long days on stem growth and floral initiation appears to be the result of enhanced levels of endogenous GAs, especially the GAs of the late steps of the early C-13-hydroxylation pathway (GA₂₀ and GA₁). However, in short-day sorghum the anticipated increase in height with increasing photoperiod length (Childs et al., 1995) did not parallel the average GA₁ level. This suggests that part of the height advantage that 58M has over 90M and 100M is the result of increased sensitivity to GAs (Weller et al., 1994; Reed et al., 1996) or to a non-GA-mediated mechanism.

As in 58M, short days shifted the peak GA₁ concentration in 100M from midday to near dawn (Fig. 1). As in 90M and 100M, long days shifted GA₂₀ and GA₁ peaks in 58M from early morning to midday (Fig. 4). Thus, GA metabolic steps leading to active GAs appear to be controlled differently in the phytochrome B mutant 58M. These results are consistent with the hypothesis that the rhythm of bioactive GA production may play a role in flowering. The pulses of GAs (especially GA₁) may have different effects on floral initiation according to the time of day that they occur. Plant hormones, including GAs, are well known to regulate gene expression (Fox and Jacobs, 1987; Jacobsen and Gubler, 1992). Changing the timing of a rhythmic peak in the level of a hormone has the potential to alter its relationship to transcription or translation regulators rhythmically produced by circadian oscillator genes.

The actual differences in levels of GAs in this study at different times of day and night are small. This suggests that the pulses or peaks are not physiologically important. On the other hand, it is possible that the mass of tissue harvested and extracted masks larger differences in GA levels in the apical meristems and young leaves. If large differences in GA pulse levels and timing do occur, then the relations noted in this paper between photoperiod and behavior of GA levels in the early-flowering 58M and the late-flowering 90M and 100M may be physiologically important.

	FOOTNOTES

   Received June 2, 1997; accepted November 19, 1997.

	ACKNOWLEDGMENTS

We thank Drs. Fred Miller and Bill Rooney for supplying the sorghum seed needed for these experiments.

	LITERATURE  CITED

Top Abstract Introduction Methods Results & Discussion References

Beall FD, Morgan PW, Mander LN, Miller FR, Babb KH (1991) Genetic regulation of development in Sorghum bicolor. V. The ma₃^R allele results in gibberellin enrichment. Plant Physiol 95: 116-125 [Abstract/Free Full Text]

Campell BR, Bonner BA (1986) Evidence for phytochrome regulation of gibberellin A₂₀ 3-hydroxylation in shoots of dwarf (lele) Pisum sativum L. Plant Physiol 82: 909-915 [Abstract/Free Full Text]

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