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Figure 5.15 Overview of pathways in control of floral transition in A. thaliana. Four inputs are indicated, with a selection of the key genes involved. CRY2, CRYPTOCHROME 2; CO, CONSTANS; FLC, FLOWERING LOCUS C; FRI, FRIGIDA; FT, FLOWERING LOCUS T; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS I; FPF1, FLORAL PROMOTING FACTOR 1; API, APETALA 1; LFY, LEAFY; GA, gibberellic acid. Arrows indicate promotion; bars indicate repression. After Mouradov et al. (2002). Copyright American Society of Plant Biologists.

Figure 5.15 Overview of pathways in control of floral transition in A. thaliana. Four inputs are indicated, with a selection of the key genes involved. CRY2, CRYPTOCHROME 2; CO, CONSTANS; FLC, FLOWERING LOCUS C; FRI, FRIGIDA; FT, FLOWERING LOCUS T; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS I; FPF1, FLORAL PROMOTING FACTOR 1; API, APETALA 1; LFY, LEAFY; GA, gibberellic acid. Arrows indicate promotion; bars indicate repression. After Mouradov et al. (2002). Copyright American Society of Plant Biologists.

dark, the CO protein is then unstable, FT expression is not upregulated, and flowering is delayed (Yanovsky and Kay 2003; Hayama and Coupland 2004; Putterill et al. 2004; Schepens et al. 2004).

A surprisingly large part of the genome of A. thaliana undergoes day-night oscillations. A genome-wide transcription-profiling study by Harmer et al. (2000) classified 6% of the probes on an oligonucleotide gene chip as cycling with a period of between 20 and 28 h. Four categories were recognized: (i) light-harvesting centres, photosynthesis genes, phytochromes, and cryptochromes,

(ii) genes involved with photoprotective pigment pathways, such as flavonoids and anthocyanins,

(iii) enzymes involved in resistance to chill and drought, for example catalysing lipid modification, and (iv) enzymes of the carbon, nitrogen, and sulphur pathways. The authors were also able to identify a conserved 9 bp motif in the promoters of 35 cycling genes, which suggests that these genes are regulated by the same transcription factor. A similar study was reported by Schaffer et al. (2001), who used a microarray with 11521 Arabidopsis ESTs showing that 11% of the genes were expressed diurnally. Obviously, such widespread rhythmicity in the genome reflects the pervasive influence of light on the metabolism of phototrophic organisms; at the same time, it reminds experimenters how important it is to standardize the time of the day when harvesting RNA in studies looking at gene expression in plants.

The vernalization pathway for the control of flowering time integrates information from the past temperature regime. A crucial gene in this pathway is FRI (FRIGIDA). This gene promotes the transcription of FLC and so represses the floral transition. Mutations in FRI remove this positive regulation and cause loss of vernalization requirement. The FRI locus is particularly relevant in the ecology of Arabidopsis, because it shows natural variation associated with flowering time. When a comparison is made of flowering times of Arabidopsis accessions of different geographic origin, plants from low latitude tend to flower earlier in a common garden than plants from high latitude. However, this latitudinal cline is only found in ecotypes that have a functional FRI gene (Stinchcombe et al. 2004), suggesting that FRI mediates the transduction of latitudinal information into regulation of flowering time.

An important issue in understanding the response to vernalization has been the question of how vernalized plants can remember a cold treatment and flower several weeks later, even when temperatures are higher for some time after the cold signal. It turns out that cold treatment induces an altered state in the shoot apex which can be passed on through mitotic cell divisions, even in the absence of cold. Recent research has shown that gene silencing due to changes in chromatin structure is the basis of this cellular memory (Bastow et al. 2004; Sung and Amasino 2004).

We know from basic biochemistry that in eukaryotic chromosomes the DNA double helix is wound around groups of small globular proteins, histones, forming nucleosomes. These histones have tails protruding outward from a nucleosome, in which the lysine residues are normally acetylated. Because histones are highly positively charged proteins, acetylation of the tails is nessessary to prevent the formation of aggregates of nucleosomes and to maintain a loose chromatin structure in which DNA is accessible to transcription.

Sung and Amasino (2004) identified a regulatory gene in Arabidopsis called VERNALIZATION INSENSITIVE 3 (VIN3), which, in conjunction with two other vernalization genes, VNR1 and VNR2, inactivates FLC by local deacetylation of histones. Histone deacetylation causes condensation of chro-matin and shuts off DNA from transcription. This process is catalysed by histone deacetylase complex (HDAC), a cluster of molecules involving a DNA-binding protein and an acetyltransferase enzyme. The condensed state of chromatin is transferred to the daughter cells when cells divide and so provides a mechanism of inheritance that does not involve alterations in DNA. This type of inheritance is called epigenetic.

VIN3 is the most upstream component of the vernalization pathway identified so far, but it is still not known how the protein senses cold. Sung and Amasino (2004) suggest that VIN3 might be a receptor for phosphoinositides (a group of phospholipids) in the nucleus, and could perceive changes in the composition of these compounds that occur during cold exposure.

The third pathway for regulating flowering time in Arabidopsis, the autonomous pathway, integrates information from the developmental stage of the plant. In the default state it represses FLC like the vernalization pathway (Fig. 5.15). Arabidopsis mutants of the autonomous pathway are early-flowering but retain the photoperiodic response, and so the flowering signal is independent of environmental cues (hence the use of the term autonomous). However, work by Blazquez et al. (2003) has shown that expression of genes in the autonomous pathway depends on temperature; this would represent a system of thermosensory control of flowering time that acts in parallel to vernalization. The autonomous pathway involves a group of six different genes, which act upon FLC in two different ways. One of the mechanisms involves inactivation of FLC by histone deacetyla-tion, like in the vernalization pathway (He et al. 2003). This is mediated by a locus FLD, which encodes a protein that forms part of an HDAC. Another gene product of the autonomous pathway, FVE, is probably part of the same HDAC (Amasino 2004; Ausin et al. 2004; Kim et al. 2004). In a series of elegant experiments He et al. (2003) were able to show that a specific region in the first intron of FLC acted as a binding site for the HDAC (Fig. 5.16). Mutations in FLD, as well as deletion of a 294 bp region from intron 1, prevented binding of HDAC to the FLC gene, causing continued transcription of FLC and late flowering.

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