The Physiology of Flowering Plants - KHAM PHA MOI

PHYTOCHROME SIGNAL TRANSDUCTION 265phytochrome A and B are initially located in the cytoplasm and thenmove into the nucleus upon activation. Studies of this movementare possible because phytochrome is still physiologically active evenif fused to another protein. Genetically modified plants have beenproduced where the coding region of PHYA or PHYB has been fusedto a reporter gene. One such reporter gene encodes green fluorescentprotein (GFP) which fluoresces green when illuminated withblue light, allowing its position to be determined by microscopy.When plants are grown in darkness, phyA::GFP fusions (orphyB::GFP fusions) are found in the cytoplasm. When treated withlight which will trigger a phytochrome response, the fusion proteinsmove into the nucleus. The movement of phyB::GFP is rapid (itoccurs within 15 minutes), red : far-red reversible and requires thepresence of the chromophore. The phyA::GFP fusion protein entersthe nucleus under illumination conditions which would triggerVLFR and HIR. Having entered the nucleus the phytochromes accumulatein small patches or ‘speckles’. In contrast, phyC, D and E areconstitutively localized in the nucleus, although speckle formationresults from illumination with red light and can be reversed by far-redlight.At least two pathways have been identified by which phytochromecan stimulate nuclear gene expression, and others undoubtedly exist.In both cases, phytochrome enters the nucleus and interacts withother proteins, and these then stimulate gene expression by bindingto short stretches of DNA (light-responsive-elements – LREs) found inthe promoters of light-responsive genes. In this way the expression ofmany different genes can be stimulated by the action of a few signaltransduction chains.The first pathway involves a number of proteins whose role wasfirst identified in mutagenic studies (Fig. 10.14). These include COP1,other members of the cop/det/fus family, and HY5. In the dark, a largemultiprotein complex (referred to as a signalosome) is found in thenucleus which includes the COP1 protein. This interacts with HY5and prevents HY5 from binding to the LREs in the promoters of lightregulatedgenes. In the absence of HY5, transcription does not occur.When phytochrome is activated, it enters the nucleus and breaksdown the association between HY5, COP1 and the signalosome. HY5binds to the LREs and the transcription of light-regulated genes isstimulated, triggering de-etiolation. COP1 leaves the nucleus andenters the cytoplasm, although the rest of the signalosome remains.This model elegantly explains the phenotypes of the variousmutants. Plants which lack phytochrome or HY5 will be etiolated inthe light as the transcription of the light-regulated genes is notstimulated. On the other hand, the absence of COP1, or other componentsof the signalosome, causes the plants to be constitutively deetiolatedas HY5 is always able to stimulate transcription.The second pathway involves PIF3. As well as interacting withphytochrome, PIF3 will also bind to another class of LRE. AlthoughPIF3 will bind to the LRE in both the light and the dark, it formsBox 10.3Green fluorescent protein (GFP) isa naturally fluorescent proteinwhich is found in the Pacificnorthwestern jellyfish (Aequoreavictoria). The advantage of such areporter gene is that the protein itencodes can be visualized withoutdestroying the plant material,allowing the same cells to bestudied over time. Otherapproaches have used reportergenes such as -glucuronidase(GUS), which allows cells to bestained blue. However, the stainingprocess is destructive so it is notpossible to follow the movementof the protein directly over time.