Deficient Backgrounds, Even in the Absence of Chemical
GA Treatment
The phenotypic analyses of ga20ox mutant described so far in this chapter were all performed on plants up to 50 days old, by which time all genotypes except ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 had finished development and were entering senescence. However, these latter two mutants demonstrated greater longevity than the other, less GA-deficient genotypes,
potentially due to a far slower rate of growth, reduced fertility and/or arguably increased stress tolerance conferred by their severely-dwarfed vegetative phenotypes. By 50 days these two genotypes had developed far fewer flowers than even ga20ox1 ga20ox2 (Figure 3.5a), so in order to observe later phases of reproductive development these two genotypes were subsequently grown until senescence (90-100 days). Very surprisingly, it was found that substantial rescue of floral organ growth reliably occurred in later flowers on the primary inflorescence (from approximately floral position 20-25) in both genotypes (Figure 3.17), though all floral organs remained far smaller than those exhibited by wild type flowers. This phenotype was observed on several different occasions, including when special precautions were taken to isolate the population to prevent contamination with bioactive GA.
Restoration of stamen growth relative to the pistil was observed in these later flowers, developmental recovery in both ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 proceeding to the point of successful anther dehiscence on some occasions. Some seeds were recovered from
ga20ox1 ga20ox2 ga20ox3-1 plants (Figure 3.17b), and genotyping of the subsequent progeny
confirmed them as homozygous ga20ox1 ga20ox2 ga20ox3-1 mutants (data not shown), indicating that successful self-fertilisation occurred. Despite several attempts, it was not possible to recover seed from ga1-3 plants under circumstances in which there was no possibility of contamination with bioactive GA. On the basis of this evidence, self-
fertilisation of ga1-3 cannot be confirmed or refuted. These results are unexpected because published evidence on the floral development of ga1-3 demonstrates developmental arrest at
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Figure 3.17: Phenotypic rescue of flowers in GA-deficient mutants.
(a) Comparison of floral phenotypes between early and late ga20ox1 ga20ox2 ga20ox3-1 flowers. Late ga20ox1 ga20ox2 ga20ox3-1 flowers demonstrate anther dehiscence, with released pollen grains visible.
(b) Self-fertilised ga20ox1 ga20ox2 ga20ox3-1 silique, containing mature seed from a 95 day- old plant.
(c) Comparison of floral phenotypes between early and late ga1-3 flowers.
floral stage 10 (Koornneef & Van der Veen, 1980; Cheng et al., 2004), and in none of the published work using this genotype as a GA-deficient control are the phenotypes described above reported. Furthermore, under our growth conditions individual ga1-3 plants often displayed bolting, particularly of secondary inflorescences (data not shown), whilst the published phenotype of ga1-3 is that no internode elongation occurs (Koornneef & Van der
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Veen, 1980). These differences can potentially be reconciled either by the age of the plants characterised in this experiment (not specified in previously published phenotypic
characterisations), or through the fact that the ga1-3 (Col-0) line used in these experiments differs phenotypically from the original ga1-3 line in the Ler ecotype. Further investigation is required to establish the precise cause for the phenotypes observed here.
The above observations raise interesting implications for the role of GA in floral development. Firstly, given that a trend of increasing fertility and relative stamen growth was identified in early flowers irrespective of GA treatment, it could be hypothesised that the late recovery seen in ga20ox1 ga20ox2 ga20ox3-1 and ga1-3 could be caused by that same underlying trend. If this is the case, then the results presented here suggest the existence of a pathway independent of GA biosynthesis that can partially restore GA-deficient floral phenotypes. Whilst attempts were made during this experiment to prevent contamination by bioactive GA, the possibility still exists that the phenotype of ga1-3 was influenced by contamination by the GA
intermediate ent-kaurene, which lies downstream of CPS in the GA biosynthesis pathway (Figure 1.3) and which has been shown to transmit between plants as an airborne volatile (Otsuka et al., 2004). Previous GA analyses have identified very small quantities of bioactive GA in ga1-3 tissues (King et al., 2001; Silverstone et al., 2001), though whether due to contamination or residual endogenous CPS function remains undetermined. ga20ox1 ga20ox2
ga20ox3-1, however, is blocked in GA biosynthesis downstream of ent-kaurene, and so this
precursor cannot be responsible for alterations in the phenotype of this mutant. Grafting experiments performed in pea between GA-deficient mutants demonstrated that at least one GA precursor beyond GA12 is transmissible between plant tissues, further radiolabelling experiments suggesting that the mobile precursor is the product of GA20ox activity (in the case of pea, GA20; Proebsting et al., 1992). However, there is no published evidence of C19- GAs transmitting between individual plants.
The recovery of self-fertilised seed from ga20ox1 ga20ox2 ga20ox3-1 plants is very
interesting, because it indicates that both male and female fertility have been restored in later flowers. This is reminiscent of the effect seen in the stamens of early flowers of ga20ox1
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ga20ox2 but at a much later stage of flowering, the simplest hypothesis being that the samemechanism underpins both. The evidence from the ga20ox1 ga20ox2 phenotype suggests that the rescue of stamen development in this genotype is due to AtGA20ox3 activity. One possible explanation is that AtGA20ox4 or -5 accumulates in the ga20ox1 ga20ox2 ga20ox3-1 floral tissues, eventually producing sufficient bioactive GA to restore pollen development. This hypothesis is partially supported by the lack of observed fertility in equivalent ga1-3 plants, but cannot explain the recovery of floral development observed in this mutant. In the case of stamen development, this includes overcoming a developmental block imposed by the absence of GA, presumably by DELLA repression, during pollen development and also potentially during stamen maturation (see section 3.2.5).
As discussed in sections 1.3.2 and 1.5.4, DELLA proteins are a point of integration between GA and other hormone signalling pathways, including auxin (Fu & Harberd, 2003), ABA (Achard et al., 2006), ethylene (Achard et al., 2003; Achard et al., 2007) and jasmonate (JA; Hou et al.. 2010). Of these, both auxin and JA act to promote GA downstream responses. JA signalling in particular is associated with floral development, and has recently been shown to transmit part of the GA signal that triggers GA-dependent growth responses during stamen maturation (Cheng et al., 2009). However, stamen development in JA biosynthetic and signalling mutants proceeds further than those of GA-deficient or insensitive mutants (Stintzi & Browse, 2001, Feys et al., 1994), and chemical JA treatment cannot rescue GA-deficient stamen development (Cheng et al., 2009), suggesting that JA signalling alone is not sufficient to rescue stamen development to the extent observed in ga20ox1 ga20ox2 ga20ox3-1 and ga1-
3. However, it might comprise a significant component, in conjunction with other
mechanisms such as auxin signalling. The role of auxin in stamen development is not well understood, with evidence for both early and late functions in promoting stamen outgrowth and development (Cheng et al., 2006; Cecchetti et al., 2008), and potential interactions with the JA signalling pathway via AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 (Nagpal et al., 2005) in a relationship similar to that between GA and JA. However, the hierarchy between these three signalling pathways during stamen development has not yet been clearly
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defined, and the question of whether combined auxin and JA signalling can overcome a block imposed by the absence of GA has not been addressed.