Promote Flowering and Both Male and Female Fertility
As discussed in section 1.5.1, GA promotes the transition of Arabidopsis from vegetative growth to the reproductive phase of development under both long day (LD) and short day (SD) conditions. Loss of AtGA20ox1 and -2 significantly delays the transition to flowering under permissive long day conditions (Rieu et al., 2008), but as with other aspects of development the delay is not as severe as that exhibited by ga1-3. In order to establish whether AtGA20ox3 activity is sufficient to explain the difference between these two phenotypes, the point of floral transition (as marked by the appearance of visible flower buds) under LD was scored for each combination of mutants carrying ga20ox1, ga20ox2 and
ga20ox3-1 (n = 216) using both a chronological measure (days from sowing) and a
developmental measure (total number of leaves at the time of flowering).
The Col-0 wild type displayed a mean flowering time of 17.83 days and 13.42 leaves, neither of which was affected by GA treatment (Figure 3.4). As observed by Rieu et al. (2008), flowering of ga20ox1 ga20ox2 is delayed (p < 0.01, 22.50 days and 18.83 leaves), whilst flowering of ga20ox1 ga20ox2 ga20ox3-1 differs significantly from ga20ox1 ga20ox2 on both measures (p < 0.01), displaying a further delay in flowering (31.16 days and 21.21 leaves).
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Figure 3.4: Effect of the loss of AtGA20ox3 on floral transition under long days (LD). Graphs show mean time to flowering as measured by chronological time from sowing (a) and total number of leaves at flowering (b). Values are means of 12 independent measurements. Error bars represent one S.E. Pairwise comparisons were made using 1% LSDs between genotypes within the same GA treatment (Days = 1.818; Leaves = 1.987) and between GA treatments within the same genotype (Days = 1.860; Leaves = 2.003). Letters denote a significant difference from -GA wild type (black) or +GA wild type (grey), respectively. Genotypes marked with different letters are significantly different from one another. Asterisks denote a significant difference between GA treatments within the same genotype.
Comparisons were not made between genotypes in different GA treatments.
However, flowering time of ga20ox1 ga20ox2 ga20ox3-1 is also significantly different from
ga1-3 on both measures (p < 0.01; 33.87 days and 27.93 leaves for ga1-3). These results
imply that, whilst AtGA20ox3 contributes significantly to the promotion of flowering time under long days, the loss of these three paralogues is not sufficient to explain the delay observed in the GA-deficient control. The flowering times of all single mutants and ga20ox1
ga20ox3-1 are not significantly different from wild type on either scale, but ga20ox2 ga20ox3-1 is (p < 0.01). This indicates that, whilst AtGA20ox3 once again acts redundantly
with AtGA20ox1 and -2, AtGA20ox1 alone cannot compensate for the absence of AtGA20ox2 and -3, whilst AtGA20ox2 alone compensate for the loss of AtGA20ox1 and -3. This might be due to homeostatic up-regulation (as with stem elongation, see section 3.1), or could reflect
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differential tissue expression patterns of the three paralogues, as the GA signal associated with floral transition is perceived at the SAM (see section 1.5.1).
Flowering of ga20ox2 ga20ox3-1 is not significantly different from ga20ox1 ga20ox2 when measured chronologically (p > 0.01) but, interestingly, they are significantly different when measured by leaf number (p < 0.01). Similarly, the gap between ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 is far greater in developmental time than chronological time. One possible
explanation is that the rate of organogenesis is altered by GA-deficiency in some mutants, compatible with the role of GA at the SAM (see section 1.5.1). Chemical GA treatment rescued flowering time in all mutants to that of wild type, with the exception of ga1-3 (measured chronologically) and ga20ox1 ga20ox2 (measured developmentally), flowering of which are both accelerated under GA treatment but remain significantly different from the GA-treated wild type.
The concentration of bioactive GA4 across whole plants was quantified in these genotypes (data courtesy of Peter Hedden, Terezie Linhartova and Yuji Kamiya), using the floral transition as a marker to synchronise plant development. All mutant genotypes contained significantly different concentrations of GA4 from wild type at flowering (p < 0.05, Table 3.2). All mutants showed reduced GA4 concentrations, suggesting that loss of any of these genes is sufficient to impair GA biosynthesis. The concentration of GA4 in each ga20ox single mutant is significantly different from the other two (p < 0.05), with ga20ox1 showing the greatest reduction in comparison to wild type, then ga20ox2 and least of all ga20ox3-1, consistent with their respective phenotypes. The concentration of GA4 was not significantly different between ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 (p > 0.05), supporting the hypothesis that AtGA20ox1, -2 and -3 perform the vast majority of GA biosynthesis during vegetative growth. However, this contrasts with the results of the analysis of flowering time (see above), where ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 are significantly different. The GA measurements presented in this study are taken from whole plants, so tissue-specific variations cannot be accounted for, and it may well be that localised GA biosynthesis
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Genotype GA4 Concentration (ng/g dry weight) Mean Rep. 1 Rep. 2 Rep. 3 Rep. 4 Wild type (Col-0) 2.210 2.346 2.222 2.063 -
ga20ox1 1.080a 1.320 1.014 0.991 0.993 ga20ox2 1.360b 1.375 1.408 1.290 1.369 ga20ox3-1 1.969c 2.036 1.988 2.151 1.702 ga20ox1 ga20ox2 0.628d 0.647 0.767 0.613 0.484 ga20ox1 ga20ox3-1 0.553d 0.483 0.622 - - ga20ox2 ga20ox3-1 1.378b 1.337 1.492 1.297 1.385
ga20ox1 ga20ox2 ga20ox3-1 0.154e 0.188 0.133 0.133 0.161
ga1-3 (Col-0) 0.244e 0.202 0.306 0.225 -
5% LSD 0.0171 - - - -
Table 3.2: Concentrations of bioactive GA4 in ga20ox mutants at flowering.
GA4 concentrations were obtained from two to four replicates per genotype (as shown), each
replicate pooling tissue from approximately 24 individual plants. Pairwise comparisons were made between genotypes using 5% LSD, as shown. Superscript letters denote values
significantly different from wild type (p < 0.05). Different letters denote values that are significantly different from one another.
a very low level of GA biosynthesis at the SAM might be sufficient to accelerate flowering in
ga20ox1 ga20ox2 ga20ox3-1.
All double mutants are significantly different from ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 (p < 0.05), the two most GA-deficient genotypes of those examined, supporting the hypothesis that each of these three AtGA20ox paralogues contributes to GA biosynthesis during
vegetative development. GA4 concentrations in ga20ox2 and ga20ox2 ga20ox3-1 tissues are not significantly different, suggesting that AtGA20ox3 function is redundant with AtGA20ox1. Loss of AtGA20ox3 on top of AtGA20ox1 apparently causes a significant difference in GA4 concentration (p < 0.05), reducing the concentration of GA4 to that similar to ga20ox1
ga20ox2. These results supports the hypothesis that AtGA20ox1 and -3 have redundant
functions in vegetative tissues, and highlights the surprisingly mild vegetative phenotype observed in ga20ox1 ga20ox3-1 (see section 3.2.2), which again may emphasise the
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importance of localised GA20ox activity in plant tissues. However, it must be borne in mind that this result is a mean of only two replicates, and so should be treated with some caution.
Beyond the floral transition, flowers develop and open sequentially on the primary inflorescence in place of leaves, resulting in multiple siliques being set by the end of flowering. During the characterisation experiment described above (49 days in length),
ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 did not set seed, although a small number of infertile
flowers were produced by each genotype (Figure 3.5a). The number of siliques produced by single and double mutant combinations carrying ga20ox1 was significantly different from wild type in all cases (p < 0.01, setting fewer siliques before flowering terminated) but not significantly different from one another. Loss of AtGA20ox2 or -3 individually did not affect the number of siliques produced, but loss of both paralogues produced a phenotype similar to the absence of AtGA20ox1. These results indicate that, whilst AtGA20ox1 is more important to promoting flower number, AtGA20ox2 and -3 also function in this process.
As observed by Rieu et al., 2008, early flowers of ga20ox1 ga20ox2 did not set seed (see further discussion in section 3.2.5), but after approximately the 10th inflorescence position all genotypes except ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 reliably set siliques. The silique phenotypes of the different ga20ox mutants were analysed as a measure of fertility, taking siliques beyond the early phase to separate out the two effects. Three mature siliques were examined from each plant (n = 216), taken from between inflorescence positions 20-30, approximately the mid-point of flowering, scoring infertile ga20ox1 ga20ox2 ga20ox3-1 and
ga1-3 flowers as zero values. The mean lengths of mature siliques from all single and double
mutant combinations carrying ga20ox2 were significantly different from wild type (p < 0.01, Figure 3.5b), displaying reduced growth. Loss of both AtGA20ox1 and -2 exacerbated this effect compared to loss of AtGA20ox2 alone, but loss of both AtGA20ox2 and -3 did not have additional effects on silique length. These results concur with the findings of Rieu et al. (2008) that AtGA20ox2 promotes silique outgrowth, with AtGA20ox1 being partially redundant with AtGA20ox2. Loss of AtGA20ox3 has no effect on silique outgrowth in the presence of either AtGA20ox1 or -2. In contrast to these results, loss of individual AtGA20ox
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(a) Mean number of siliques set on the primary inflorescence during flowering. ga20ox1ga20ox2 ga20ox3-1 and ga1-3 only produced infertile flowers which did not set seed
(highlighted in red).
(b) Mean silique length at maturity. Siliques taken from the mid-point of flowering. Pairwise comparisons calculated on a transformed scale. Transformed mean values marked on the appropriate bar.
(c) Mean number of seeds per silique, measured from the siliques in (b).
(d) Photographs of developing seeds within siliques, demonstrating altered seed packing in
ga20ox2, ga20ox1 ga20ox2 and ga20ox2 ga20ox3-1 mutants in the absence of chemical GA
treatment.
Each bar represents the mean of 12 (silique number) and 36 measurements (silique length and seed number) respectively: three siliques per plant were measured. Error bars represent one S.E. Pairwise comparisons were made using a 1% LSD between genotypes within the same GA treatment ((a) 9.832; (b) 0.16952; (c) 9.138) and between GA treatments within the same genotype ((a) 9.877; (b) 0.17387; (c) 9.117). Letters denote a significant difference from -GA wild type (black) or +GA wild type (grey), respectively. Genotypes marked with different letters are significantly different from one another. Asterisks denote a significant difference between GA treatments within the same genotype. Comparisons were not made between genotypes in different GA treatments.
paralogues had far less impact on the number of seeds within these siliques, with only
ga20ox1 ga20ox2 being significantly different from wild type (p < 0.01, Figure 3.5c). This
differential result between silique length and seed number manifests as an altered seed packing phenotype (Figure 3.5d), in which siliques from genotypes lacking AtGA20ox2 present a double row of seeds in each valve rather than the single row observed in wild type. This can be explained through reduced elongation of silique tissues during silique
development. The high expression of AtGA20ox3 found by Rieu et al. (2008) in developing wild-type siliques (see Figure 3.1c) strongly suggests that this paralogue contributes to silique growth and development, but the infertility of ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 precludes further interpretation of AtGA20ox3 function from this dataset.
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Chemical GA treatment restores seed set in both ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 (Figure 3.5c). Interestingly, whilst GA treatment increases silique growth in more severely GA-deficient mutants (Figure 3.5b), it also significantly affects the number of seeds per silique in a number of otherwise fully-fertile genotypes (p < 0.01, Figure 3.5c), reducing seed numbers to values similar to that of untreated ga20ox1. As such, chemical GA treatment has a significant negative impact on seed set, a conclusion also reached by previous studies
(Jacobsen & Olszewski, 1993; Rieu et al., 2008). It has been shown that in vitro pollen tube growth is reduced in the presence of high concentrations of GA (Singh et al., 2002), which could plausibly explain this phenomenon. Another possibility is that chemical GA treatment alters the relative growth of male and female reproductive organs, resulting in a mechanical barrier that reduces pollen transfer to the stigma.
The relatively minor effect of loss of AtGA20ox1 and -2 on seed number (a conclusion supported by Rieu et al., 2008) suggests that AtGA20ox1, -2 and -3 could all act redundantly in promoting fertility, with no one paralogue demonstrating dominance over the others. Seed number is determined by two factors (discounting seed abortion, which was not observed during seed counting): the number of ovules developing within the carpel and the success of pollen in fertilising ovules during the time that they are receptive, a factor governed by pollen germination and pollen tube growth. To directly test these two effects in planta, reciprocal crosses were made between wild-type and ga20ox mutant flowers, subsequently counting the number of seeds set (Figure 3.6). Wild-type pistils manually pollinated with pollen from single or double ga20ox mutants all produced similar numbers of seeds, suggesting that loss of one or two AtGA20ox paralogues out of the three does not impair post-anthesis pollen
development in vivo. Crosses using ga20ox1 ga20ox2 ga20ox3-1 or ga1-3 pollen set practically no seed. However, whilst is was possible to visually confirm transfer of large quantities of pollen in most of the crosses performed for this experiment, pollen proved very difficult to release from anthers of the latter two genotypes. The few seeds that were set may have been the result of contamination of the emasculated pistils by airborne pollen. ga1-3 is published as a male-sterile mutant with a block in pollen development (Koornneef & Van der Veen, 1980; Cheng et al., 2004). Whilst this result cannot be used to directly infer the
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Figure 3.6: Effect of the loss of AtGA20ox3 on male and female fertility.
Graphs show mean number of seeds developed in wild type pistils when pollinated with ga20ox mutant pollen (a) and in ga20ox mutant pistils when pollinated with wild-type pollen (b). Error bars represent one S.E. Pairwise comparisons were made between genotypes using a 5% LSD ((a) 6.286; (b) 6.448). Letters denote means that are significantly different from wild type, with different letters indicating means that are significantly different from each other.
performance of ga20ox1 ga20ox2 ga20ox3-1 pollen, this genotype also appears to be functionally male-sterile.
In the reciprocal experiment, wild-type pollen was crossed to ga20ox mutant pistils (Figure 3.6b). ga20ox1 and ga20ox1 ga20ox3-1 mutant siliques contained a significantly different number of seed from wild type (p < 0.05), though only slightly fewer (means of 55.26 and 48.87, respectively, against 63.50 in wild type), suggesting that loss of AtGA20ox1 may marginally reduce the number of ovules developing in the silique. The ga20ox1 and ga20ox1
ga20ox3-1 mutants do not differ significantly from each other. However, this result contrasts
with that seen in natural self-pollination (Figure 3.5c), in which ga20ox1 and ga20ox1
ga20ox3-1 are not significantly different from wild type. Any effect on ovule number is
therefore likely to be very slight. Also, the number of seeds produced when a ga20ox1
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type control, contrasting both with the results of ga20ox1 and ga20ox1 ga20ox3-1 in this experiment and the situation seen in self-pollination of ga20ox1 ga20ox2 (Figure 3.5c). This result suggests that ga20ox1 ga20ox2 pistils carry a similar number of ovules as wild type. The reduction in seed-set during ga20ox1 ga20ox2 self-pollination may be due either to impaired ga20ox1 ga20ox2 pollen development per se, or to other factors such as floral organ growth restricting access of pollen to the stigma (see section 3.2.5). ga20ox1 ga20ox2
ga20ox3-1 and ga1-3 pistils pollinated with wild-type pollen did not set seed, indicating that
loss of AtGA20ox1, -2 and -3 causes female sterility.
The effects of AtGA20ox1, -2 and -3 on pollen performance post-anthesis were further tested via the segregation of the three mutant alleles in the progeny of a self-pollinating
GA20ox1/ga20ox1 GA20ox2/ga20ox2 GA20ox3/ga20ox3-1 plant (n = 368). This
experimental approach has the additional advantage that the performance of ga20ox1 ga20ox2
ga20ox3-1 pollen can also be assessed. Previous expression studies in rice indicate that GA
biosynthesis in developing pollen grains begins after the separation of haploid microspores (Chhun et al., 2007), and as such pollen phenotypes should reflect the haploid genotype. A statistically significant deviation from the expected frequencies of offspring genotypes was observed (p = 0.008, Table 3.3a), indicating that loss of AtGA20ox paralogues has an impact on gametophyte fitness. Surprisingly, when the results are categorised by phenotype
(grouping the population by homozygous mutant loci), the mutant genotype demonstrating the greatest fitness penalty is incurred by ga20ox3-1 (67.63% of the expected frequency, Table 3.3b), whilst the ga20ox1 ga20ox3-1 and ga20ox2 ga20ox3-1 double mutants are less affected (98.55% and 86.96%, respectively). The mutant genotype demonstrating the next greatest fitness penalty is ga20ox1 ga20ox2 (75.36% of the expected frequency), suggesting that loss of these two paralogues may reduce pollen growth post-anthesis and thus explain the reduced numbers of seed seen in ga20ox1 ga20ox2 self-pollination. However, unexpectedly, the fitness penalty incurred by ga20ox1 ga20ox2 ga20ox3-1 pollen was found to be less than that incurred by ga20ox1 ga20ox2 (86.96% of the expected frequency), suggesting that the effect of the loss of AtGA20ox1, -2 and -3 on pollen performance is negligible.
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(a)
Genotype Expected Freq. (E)
Observed
Freq. (O) O-E (O-E)
2 (O-E)2/E
GA20ox1; GA20ox2; GA20ox3 5.75 12 6.25 39.06 6.793
GA20ox1/ga20ox1; GA20ox2;
GA20ox3 11.5 16 4.50 20.25 1.761
ga20ox1; GA20ox2 ;GA20ox3 5.75 5 -0.75 0.56 0.098
GA20ox1; GA20ox2/ga20ox2;
GA20ox3 11.5 13 1.50 2.25 0.196
GA20ox1; ga20ox2; GA20ox3 5.75 1 -4.75 22.56 3.924
GA20ox1; GA20ox2;
GA20ox3/ga20ox3-1 11.5 17 5.50 30.25 2.630 GA20ox1; GA20ox2; ga20ox3-1 5.75 1 -4.75 22.56 3.924
GA20ox1/ga20ox1; GA20ox2/ga20ox2; GA20ox3 23 21 -2.00 4.00 0.174 GA20ox1/ga20ox1; ga20ox2; GA20ox3 11.5 14 2.50 6.25 0.543 GA20ox1/ga20ox1; GA20ox2; GA20ox3/ga20ox3-1 23 31 8.00 64.00 2.783 GA20ox1/ga20ox1; GA20ox2; ga20ox3-1 11.5 6 -5.50 30.25 2.630 ga20ox1; GA20ox2/ga20ox2; GA20ox3 11.5 17 5.50 30.25 2.630
ga20ox1; ga20ox2; GA20ox3 5.75 4 -1.75 3.06 0.533
ga20ox1; GA20ox2;
GA20ox3/ga20ox3-1 11.5 8 -3.50 12.25 1.065 ga20ox1; GA20ox2; ga20ox3-1 5.75 7 1.25 1.56 0.272
GA20ox1; GA20ox2/ga20ox2; GA20ox3/ga20ox3-1 23 36 13.00 169.00 7.348 GA20ox1; GA20ox2/ga20ox2; ga20ox3-1 11.5 9 -2.50 6.25 0.543 GA20ox1; ga20ox2: GA20ox3/ga20ox3-1 11.5 16 4.50 20.25 1.761 GA20ox1; ga20ox2; ga20ox3-1 5.75 3 -2.75 7.56 1.315
GA20ox1/ga20ox1; GA20ox2/ga20ox2; GA20ox3/ga20ox3-1 46 43 -3.00 9.00 0.196 GA20ox1/ga20ox1; GA20ox2/ga20ox2; ga20ox3-1 23 19 -4.00 16.00 0.696 GA20ox1/ga20ox1; ga20ox2; GA20ox3/ga20ox3-1 23 16 -7.00 49.00 2.130 GA20ox1/ga20ox1; ga20ox2; ga20ox3-1 11.5 12 0.50 0.25 0.022 ga20ox1; GA20ox2/ga20ox2; GA20ox3/ga20ox3-1 23 17 -6.00 36.00 1.565 ga20ox1; GA20ox2/ga20ox2; ga20ox3-1 11.5 10 -1.50 2.25 0.196 ga20ox1; ga20ox2; GA20ox3/ga20ox3-1 11.5 9 -2.50 6.25 0.543 ga20ox1; ga20ox2; ga20ox3-1 5.75 5 -0.75 0.56 0.098
X2 = 46.370
(D.f. = 26)
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(b) Phenotype Expected Freq. Observed Freq. Proportion of Expected (%) Wild type (Col-0) 155.25 189.00 121.74ga20ox1 51.75 47.00 90.82 ga20ox2 51.75 47.00 90.82 ga20ox3-1 51.75 35.00 67.63 ga20ox1 ga20ox2 17.25 13.00 75.36 ga20ox1 ga20ox3-1 17.25 17.00 98.55 ga20ox2 ga20ox3-1 17.25 15.00 86.96
ga20ox1 ga20ox2 ga20ox3-1 5.75 5.00 86.96
Table 3.3: Segregation distortion analysis of ga20ox loss-of-function alleles. (a) Chi-squared statistical analysis of the segregation of progeny of a single
GA20ox1/ga20ox1 GA20ox2/ga20ox2 GA20ox3/ga20ox3-1 parent, against the null
hypothesis of no fitness penalty (1:2:1 segregation) for each allele. Experimental population size = 368.
(b) Summary of population structure, according to the presence of homozygous mutant loci. Genotypes that are wild type or heterozygous at the remaining loci are included in each