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ga20ox1 ga20ox2 ga20ox3-1 Background

To test for significant contributions of AtGA20ox4 and -5 towards promoting growth and development in planta, the two quadruple mutant combinations ga20ox1 ga20ox2 ga20ox3-1

ga20ox4-2 and ga20ox1 ga20ox2 ga20ox3-1 ga20ox5-2 were created and characterised. Both

the ga20ox4-2 and ga20ox5-2 alleles (coming from the Bur-0 and Ler ecotypes, respectively) were introgressed into the Col-0 ecotype by six sequential back-crosses prior to establishing these combinatorial mutant lines. The ga20ox1 ga20ox2 ga20ox3-1 ga20ox4-2 and ga20ox1

ga20ox2 ga20ox3-1 ga20ox5-2 quadruple mutants both display severely-dwarfed phenotypes

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Figure 4.3: Mature rosette phenotypes of ga20ox combinatorial mutants (55 day old plants).

of these new mutant combinations was targeted to characters where differences between

ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 had been previously identified (7-day root length, n =

192; mature rosette diameter and flowering time, n = 48, see section 3.2.2), which might be caused by the continuing presence of AtGA20ox4 or -5. The ga20ox1 ga20ox2 ga20ox3-3 triple mutant was included in these experiments to verify the phenotypic results obtained previously from ga20ox1 ga20ox2 ga20ox3-1.

As previously found (see section 3.2.3), the difference in flowering times between ga20ox1

ga20ox2 ga20ox3-1 and ga1-3 was significant (p < 0.01), as measured both chronologically

(days from sowing, Figure 4.4a) and developmentally (total leaves present at flowering, Figure 4.4b). Flowering time of ga20ox1 ga20ox2 ga20ox3-1 ga20ox4-2 was significantly different from ga20ox1 ga20ox2 ga20ox3-1 (p < 0.01) but not significantly different from

ga1-3 when measured by days, and was significantly different from both of these genotypes (p

< 0.01) when measured by leaf number. In contrast, flowering time of ga20ox1 ga20ox2

ga20ox3-1 ga20ox5-2 was not significantly different from ga20ox1 ga20ox2 ga20ox3-1 on

either scale of measurement, and was significantly different from ga1-3 (p < 0.01) both chronologically and developmentally. This specific result suggests that AtGA20ox4 has some function in promoting the transition to flowering in the absence of AtGA20ox1, -2 and -3. However, the ga20ox1 ga20ox2 ga20ox3-3 phenotype contradicts this conclusion, its chronological flowering time not differing significantly from ga1-3 (p > 0.01). However, when measured on the developmental scale, ga20ox1 ga20ox2 ga20ox3-3 is not significantly different from either ga20ox1 ga20ox2 ga20ox3-1 or ga20ox1 ga20ox2 ga20ox3-1 ga20ox4-2, and like them is significantly different from ga1-3 (p < 0.01). This apparent contradiction

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Figure 4.4: The effect of loss of AtGA20ox4 or -5 on Arabidopsis flowering.

Comparison of flowering time between genotypes, grown under control growth conditions or under GA treatment, as measured by the number of days from sowing (a) and total number of leaves (b) at the first appearance of flower buds.

Bars represent the mean of 8 independent measurements, error bars represent one S.E. Pairwise comparisons were made using a 1% LSD with a significance threshold of 1% between genotypes within the same GA treatment (Days = 1.793; Leaves = 2.610) and between GA treatments within the same genotype (Days = 1.937; Leaves = 2.527). 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 under different GA treatments.

between the phenotypes of the two ga20ox 1 ga20ox2 ga20ox3 triple mutants might be explained by ecotypic differences, the ga20ox3-3 allele having been derived from the Ler background (see section 3.2.1). The growth phenotypes of Col-0 and Ler are quite distinct (see chapter 6), and as such the difference in flowering time between the two triple mutant genotypes may represent pleiotropic effects due to the incorporation of other, Ler ecotype- specific alleles in addition to ga20ox3-3. Both the ga20ox4-2 and ga20ox5-2 alleles were also introduced from other ecotypes (as was the ga1-3 allele), and as such the validity of the results obtained from direct phenotypic comparisons between these ÔhybridÕ mutant genotypes can be questioned. GA treatment caused a significant acceleration in flowering time under both

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scales of measurement for all mutants, although some remain significantly different from GA- treated wild type (Figure 4.4a and b). Ecotypic differences may also explain why flowering of

ga1-3 remains significantly different from both ga20ox1 ga20ox2 ga20ox3-3 and ga20ox1 ga20ox2 ga20ox3-1 ga20ox4-2 (p < 0.01) when measured developmentally (Figure 4.4b).

Alternatively, these two genotypes might retain residual GA20ox activity, either from a known paralogue or potentially an unrelated gene. Despite these reservations, loss of

AtGA20ox4 activity from the ga20ox1 ga20ox2 ga30ox3-1 background does apparently cause

a significant further delay on flowering time, although a definite role for AtGA20ox4 in this process has not been demonstrated conclusively.

Whilst the experimental results regarding flowering time of ga20ox1 ga20ox2 ga20ox3-1 agree with those of earlier characterisation experiments, the phenotypic results obtained for both rosette size and root growth differ from previous findings, in that there were no significant differences observed between ga20ox1 ga20ox2 ga20ox3-1 (or ga20ox1 ga20ox2

ga20ox3-3) and ga1-3 (Table 4.1), indicating that loss of AtGA20ox1, -2 and -3 is sufficient to

replicate the GA-deficient phenotype of ga1-3 and thus excluding further functions for either

AtGA20ox4 or -5. Under control growth conditions all mutant genotypes were significantly

different from wild type for both characters (p < 0.01), but not significantly different from one another. The effect of GA treatment on rosette growth of these mutants corresponds with previous experiments, but the effect of GA treatment on root growth does not (see section 3.2.2), instead causing a reduction in wild-type root growth on this occasion. The different results between these two experiments are therefore likely to be due to technical reasons, potentially through differences in growth conditions. Similar reasons could apply to differences in rosette diameter, or might be due to alterations in the experimental population affecting the statistical analysis.

It can be seen that an additional qualitative rosette phenotype is associated with these ga20ox mutants, in that their rosette leaves routinely exhibit strong curling, a phenotype far less evident in ga1-3 plants (Figure 4.3). Both ga20ox1 ga20ox2 ga20ox3-1 ga20ox4-2 and

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-GA +GA -GA +GA

Wild type (Col-0) 23.83 19.98* 113.80 [10.6630] 134.40* [11.5890] ga20ox1 ga20ox2 ga20ox3-1 14.50 a _17.47 75.38a [8.6690] 135.90* [11.6500] ga20ox1 ga20ox2 ga20ox3-3 16.84 a _20.78* 66.00a [8.1280] 169.40b* [12.9920] ga20ox1 ga20ox2 ga20ox3-1 ga20ox4-2 14.22 a _16.83 66.25a [8.4560] 163.90b,c* [12.8170] ga20ox2 ga20ox2 ga20ox3-1 ga20ox5-1 17.06 a 18.79 71.62 a [8.4560] 145.50* [12.0530] ga1-3 (Col-0) 13.98 a _16.97 74.88a [8.6320] 151.60c* [12.3060] 1% LSD 3.599 (3.673) [0.6197] ([0.6967])

Table 4.1: Phenotypic analysis of ga20ox quadruple mutant vegetative characters.

Values given are means of 16 (roots) and 8 (rosettes) measurements, respectively

Rosette diameter data were analysed on a transformed scale (square root, denoted by square brackets) to meet the assumptions of the statistical model.

ascribed to the presence or absence of a particular paralogue. The phenotypic differences between these and ga1-3 could be due either to residual activity of the single remaining

AtGA20ox paralogue in each case, or potentially to hybrid ecotypic phenotypes in ga1-3.

The floral phenotypes of these mutants were all very similar (Figure 4.5), but also dependent on the inflorescence position examined, as was found previously for ga20ox mutants and ga1-

3 (see sections 3.2.5 and 3.2.7). Comparing floral phenotypes at approximately the 10th flower position (Figure 4.5a) suggests that differences in floral organ size exist between

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Figure 4.5: The effect of loss of AtGA20ox4 or -5 function on floral development.

Comparison between floral phenotypes of ga20ox combinatorial mutants (as shown) and ga1-

3, with mature flower buds taken from approximately the 10th (a) and 15th (b) primary

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(d) Phenotypic rescue of late ga20ox combinatorial mutant and ga1-3 flowers, taken from between inflorescence positions 20-25. Scale bar = 1mm.

reduce floral organ growth, whilst loss of AtGA20ox5 had no apparent effect at this stage. However, the closed nature of these GA-deficient flowers makes it difficult to synchronise development, and the possibility that the ga20ox1 ga20ox2 ga20ox3-1 ga20ox4-2 buds observed may not have been fully developed cannot be completely discounted. Floral buds examined at approximately inflorescence position 15 (Figure 4.5b) did not show these differences, with all ga20ox combinatorial mutant flowers appearing very similar whilst ga1-3 flowers were still apparently smaller. However, in all genotypes studied floral organ growth had increased between these two inflorescence positions, most notably the pistil and petals. Furthermore, pollen from ga1-3 anthers taken from flowers at inflorescence 15 stained as viable (Figure 4.5c), in contrast to similar experiments comparing flowers from earlier inflorescence positions (see section 3.2.4). These results suggest that floral development progresses further in later flowers of ga1-3, consistent with the floral phenotypes presented in section 3.2.7. A more detailed analysis of pollen development in these flowers (Figure 4.6) found tricellular pollen present in all ga20ox combinatorial mutants examined and also in ga1-

3. It should be noted that in the case of ga20ox1 ga20ox2 ga20ox3-1 ga20ox4-2, tricellular

pollen was far rarer than in other genotypes. These results apparently contradict previously published results in which ga1-3 pollen development arrested at the unicellular stage, prior to pollen mitosis I (Cheng et al., 2004). This might be accounted for by ecotypic differences, as the previous study was conducted in the Ler background, or potentially technical differences between experiments such as growth conditions. However, even then, the block on pollen development previously recorded was not absolute, with approximately 6% of microspores containing two or more nuclei, and the stage of flowering from which these results were obtained is not specified. These observations are therefore not incompatible with previously published findings, and suggest that the block on pollen development is relaxed in later

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Figure 4.6: Pollen development in GA-deficient flowers.

Pollen nuclei imaged by DAPI fluorescent stain (Ruzin, 1999). Pollen shown is tricellular in each case, with the nuclei of the vegetative cell (V) and the two sperm cells (S) visible.

flowers even in the absence of bioactive GA. They also provide further evidence for a gradual rescue of floral organ growth independent of GA biosynthesis. Flowers taken from these mutants even later in flowering (inflorescence positions 20-25) were indistinguishable from one another, including those of ga1-3. Flowers by this point developed sufficiently to open, and demonstrated a significant improvement in stamen growth (Figure 4.5d). Fertility of these flowers was not tested experimentally.

The inheritance of the ga20ox4-2 allele between generations was analysed to test whether loss of AtGA20ox4 has any impact on post-anthesis pollen development and fitness. Previous analysis testing the inheritance of AtGA20ox1, -2 and -3 raised the possibility that development of GA-deficient pollen is rescued by GA synthesised by maternal tissues carrying functional copies of all three of these paralogues (see section 3.2.3). In an attempt to unmask any mutant phenotypes in post-anthesis pollen development, this experiment was performed in the ga20ox1 ga20ox2 background, in which GA biosynthesis is reduced whilst fertility remains relatively unaffected (Rieu et al., 2008). The inheritance of both ga20ox3-1 and ga20ox4-2 was scored in the F2 progeny of a self-pollinating ga20ox1 ga20ox2

GA20ox3/ga20ox3-1 GA20ox4/ga20ox4-2 plant (n = 91) grown without chemical GA

treatment. No significant distortion from Mendelian segregation ratios was observed (p = 0.1837, Table 4.2), including the frequency of severely GA-deficient genotypes

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Genotype Expected Freq. (E)

Observed

Freq. (O) O-E (O-E)

2 (O-E)2/E GA20ox3; GA20ox4 5.6875 4 -1.6875 2.848 0.50069 GA20ox3/ga20ox3-1; GA20ox4 11.375 4 -7.3750 54.390 4.78159 ga20ox3-1; GA20ox4 5.6875 5 -0.6875 0.473 0.08310 GA20ox3; GA20ox4/ga20ox4-2 11.375 16 4.6250 21.390 1.88049 GA20ox3; ga20ox4-2 5.6875 6 0.3125 0.098 0.01717 GA20ox3/ga20ox3-1; GA20ox4/ga20ox4-2 22.75 24 1.2500 1.563 0.06868 ga20ox3-1; GA20ox4/ga20ox4-2 11.375 9 -2.375 5.641 0.49588 GA20ox3/ga20ox3-1; ga20ox4-2 11.375 13 1.6250 2.641 0.23214 ga20ox3-1; ga20ox4-2 5.6875 10 4.3125 18.600 3.26992 X2 = 11.3297 (D.f. = 8) p = 0.1837

Table 4.2: Segregation distortion analysis of the ga20ox3-1 and ga20ox4-2 loss-of-function

alleles.

Chi-squared statistical analysis of the segregation of ga20ox3-1 and ga20ox4-2 alleles in the

ga20ox1 ga20ox2 background against the null hypothesis of no fitness penalty for either

allele. Experimental population size = 91.

(ga20ox1 ga20ox2 ga20ox3-1 and ga20ox1 ga20ox2 ga20ox3-1 ga20ox4-2). This result could be taken to indicate that neither AtGA20ox3 nor AtGA20ox4 has a function in post- anthesis pollen development. However, pollen in this experiment was developing on and through maternal tissues carrying functional copies of both of these genes, and as such mutant pollen phenotypes could still be being masked by GA biosynthesis in maternal tissues. The female infertility demonstrated by ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 (see section 3.2.3) prevents this hypothesis from being tested in planta through this methodology.

Despite some evidence of AtGA20ox4 promoting flowering in ga20ox1 ga20ox2 ga20ox3-1, no definite role for either AtGA20ox4 or -5 in promoting plant growth has been conclusively demonstrated in the developmental contexts included in these analyses, characterisation experiments having failed to validate phenotypic differences between ga20ox1 ga20ox2

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development cannot be conclusively ruled out. In particular, broad-scale expression mapping identified the highest expression of AtGA20ox5 in developing siliques, a stage of development not covered in this project. Also, plants for all characterisation experiments in this project were grown under controlled, optimal environmental conditions. In future, AtGA20ox4 or -5 might be found to make significant contributions to growth under sub-optimal conditions.

Together with the observations of section 3.2.7, these results also suggest that stamen developmental arrest seen in severely GA-deficient mutants can be overridden by other factors. The mechanism underlying this is unknown, but seems to be a gradual process that acts across flowering, causing quantitative phenotypic recovery with increasing inflorescence position. The fact that the same phenotypic changes are observed in all ga20ox combinatorial genotypes and ga1-3 argues that this process is independent or downstream of GA

biosynthesis. Expression of AtGID1 has been shown to be feedback-regulated (Griffiths et al., 2006), and AtGID1B expression is significantly up-regulated in ga20ox1 ga20ox2 plant tissues (Rieu et al., 2008). Whether such changes in expression occur in inflorescence tissues during flowering has not been previously addressed.