Descripción del proceso de implementación del proyecto

The transition from vegetative to reproductive development is caused by a change of identity at the apical meristem, from the vegetative Shoot Apical Meristem (SAM) which produces vegetative organs (leaves), to the reproductive Inflorescence Meristem (IM), which instead of leaves produces floral meristems (FM) that go on to produce floral organs and thus constitute a flower. The transition to flowering is subject to regulation by endogenous and

27

& Coupland, 2004). These cues act through a number of different pathways (reviewed in Boss et al., 2004), which are integrated by regulating the expression of a number of key genes that confer IM identity: FLOWERING LOCUS T (FT), SUPPRESSOR OF

OVEREXPRESSION OF CONSTANS 1 (SOC1) and LEAFY (LFY). LFY is also important for

subsequently determining FM identity (Weigel et al., 1992; Weigel & Nilsson, 1995) through antagonising the expression of TERMINAL FLOWER 1 (TFL1) in conjunction with the floral identity gene APETALA 1 (AP1, Weigel et al., 1992; Ratcliffe et al., 1999).

GA signalling promotes flowering, but its importance is dependent on other factors. Flowering is promoted under a long day (LD) photoperiod in Arabidopsis through the LD pathway which acts via light signalling and circadian regulation to promote expression of

CONSTANS (CO), which in turn promotes both SOC1 and FT (Searle & Coupland, 2004).

Under short days (SD), which are not permissive to flowering, the failure of the GA-deficient mutant ga1-3 to flower in contrast to Ler wild type (Wilson et al., 1992, Rieu et al., 2008) demonstrates that under SD conditions GA is essential for the floral transition to occur. Under LD, ga1-3 eventually flowers without GA treatment (though it is still delayed compared to Ler), demonstrating that the reliance of the floral transition on GA is reduced under LD. The failure of ga1-3 to flower under SD is due to an absence of LFY expression in this mutant, constitutive expression of which can restore flowering, whereas under LD LFY expression in

ga1-3 is reduced compared to Ler, but not abolished (Bl‡zquez et al. 1998). Under SD, GA

promotes expression of SOC1 (Moon et al., 2003), constitutive expression of which can also rescue flowering in ga1-3. SOC1 has since been shown to directly activate LFY expression (Lee et al., 2008), and as such GA may act indirectly to regulate LFY through this pathway. Interestingly, although SOC1 integrates signals from the autonomous/vernalisation pathway (Moon et al., 2003), loss of GA biosynthesis in the ga1-3 mutant does not inhibit flowering responses to vernalization in vernalization-sensitive Arabidopsis backgrounds (Michaels & Amasino, 1999).

The delayed flowering of GA-deficient mutants under LD demonstrates that GA still acts to promote flowering under these conditions. As well as the links to LFY (see above) GA

28

treatment has been shown to promote FT expression in ga1-3 under LD (Hisamatsu & King, 2008). FT expression is up-regulated under LD conditions by combined CONSTANS (CO) and light signalling to promote flowering (Boss et al. 2004). The inductive effect on FT by GA treatment was found to be far greater under LD than SD (Hisamatsu & King, 2008), suggesting that FT is repressed by the photoperiod pathway under SD (i.e. non-permissive) conditions, even in the presence of GA. Hisamatsu and King (2008) also found evidence that GA promotes flowering under LD independently of FT regulation: GA treatment accelerated flowering of a ft mutant under LD conditions. However, as mentioned in section 1.3.1, two reports on the Arabidopsis gid1a gid1b gid1c GA-insensitive mutant indicate that this mutant does not flower even under inductive LD conditions (Iuchi et al., 2007; Willige et al., 2007), implying an absolute reliance of Arabidopsis on GA signalling to flower. These contrast with a third report (Griffiths et al., 2006), which describes flowering (albeit delayed) of a gid1a

gid1b gid1c mutant under LD. This discrepancy has yet to be resolved. Two of these

conflicting reports (Griffiths et al., 2006; Iuchi et al., 2007) utilise the same triple mutant, so the described phenotypic differences are unlikely to be due to allelic variation and may instead be due to differing growth conditions between the two studies, given the influence that environment has in the making the decision to flower.

High levels of GA4 have been recorded at the SAM just prior to the floral transition under SD conditions, but the expression of GA biosynthesis genes at the SAM at this time do not correlate with this (Eriksson et al., 2006), suggesting that the SAM is not the source of GA synthesis to induce flowering. Eriksson et al. (2006) demonstrated through radiolabelling studies that GA4 can travel from rosette leaves to the SAM, which means that it is feasible for remote vegetative tissues to promote the floral transition via GA. A precedent for this type of signalling already exists, with the photoperiod inductive signal originating in rosette leaves rather than the SAM (Zeevaart, 1976). Furthermore, AtGA2ox4 has been shown through mutant analysis to delay the transition to flowering under SD conditions (Rieu et al., 2008a). This GA2ox paralogue is expressed in stem tissues directly beneath the SAM (Jasinski et al., 2005, Figure 1.6), and so it may act to delay flowering by inactivating remotely-synthesised GA4, preventing it from reaching the SAM. Similarly, expression of GA-deactivating

29

enzymes (GA2oxÕs and a putative 16,17-epoxidase) beneath the SAM has been observed during vegetative growth of Lolium temulentum (King et al., 2008) and rice (Sakamoto et al., 2001a; Zhu et al. 2006).

GA promotes organogenesis and antagonises meristematic identity (Hay et al., 2002),

necessitating its exclusion from the SAM. The KNOX gene SHOOTMERISTEMLESS (STM), which is necessary for maintenance of the meristematic indeterminate cell fate (Endrizzi et al., 1996), acts to repress AtGA20ox1 expression within the SAM (Hay et el., 2002) and to upregulate AtGA2ox expression at the SAM boundary, this latter function acting through upregulation of cytokinin (CK) biosynthesis and signalling (Jasinski et al., 2005, Figure 1.6). The repression of GA20ox expression by KNOX proteins has been observed in other species (Sakamoto et al., 2001; Chen et al., 2004). Conversely, AtGA20ox1 is expressed in

developing leaf primordia outside the SAM (Hay et al., 2002). Interestingly, at a similar stage of development AtGA3ox1 is reported as being expressed in the SAM (Mitchum et al., 2006), though primarily in the rib meristem beneath the indeterminate meristematic cells. This contradiction has yet to be reconciled, but may hinge on the availability of GA9 for GA3ox to convert into bioactive GA4. A similar antagonistic relationship between KNOX and GA is likely to exist between the IM and developing floral primordia: weak stm alleles demonstrate that STM is required for the maintenance of inflorescence and floral meristems (Endrizzi et al., 1996) and the severity of the floral phenotype is enhanced when CK signalling is also impaired (Jasinski et al., 2005). Bartrina et al. (2011) further demonstrate that inhibiting degradation of CK in the meristematic results in a larger IM, whilst overexpressing CK degrading genes reduces IM size. The expression pattern of AtGA20ox1 in the reproductive context is not reported, but consistent with this model AtGA3ox1 is expressed in early floral primordia and not in the IM (Hu et al., 2008).