Phosphorylation Dephosphorylation

The central biochemical events that constitute the signal transfer from the photo-activated photoreceptors to their primary signalling partners are subjects of research in many laboratories. Phosphorylation/dephosphorylation of photoreceptors and their signalling intermediates might play an important role in regulating plant growth responses to perceived light. Structural and evolutionary studies indicate that plant phytochromes have evolved from prokaryotic bacteriophytochromes, which resemble two-component His kinases. Whereas crytochromes have no resemblance to kinases, phototropins contain a C-terminal Ser/Thr kinase domain. All phytochromes, cryptochromes and phototropins possess autophophorylation activity.

Because bacteriophytochromes have been demonstrated to posses light-regulated His kinase activity, it was concluded that protein kinase activity is an early and important event in bacteriophytochrome signalling. However, plant phytochromes are most likely not His kinases, since key conserved residues in the HKRD are missing. Oat phyA possesses Ser/Thr protein kinase activity in vitro, with Pfr being more active. The kinase activity of phyA was confirmed after identification of several substrates, such as PSK1 (PHYTOCHROME SUBSTRATE KINASE 1), Aux/IAAs and cryptochromes. Phosphorylation of phyA modulates photoresponses in several ways, by controlling the subcellular localization of phyA, its stability and its affinity towards downstream signal transducers. Several components were identified to regulate phytochrome activity both via phosphorylation (NDPK2, NUCLEOSIDE DIPHOSPHATE KINASE 2) and dephosphorylation (FYPP, FLOWER-SPECIFIC PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE; PAPP5, PHYTO-CHROME-ASSOCIATED PROTEIN PHOSPHATASE 5), which activate and deactivate phyA activity respectively. Little is known about a possible kinase activity of the other plant phytochromes (Bae and Choi 2008).

B light-dependent autophosphorylation of cry1 and cry2 is also important for their function. In the case of cry2, phosphorylation is associated with proteolytic degradation. The C-terminal domain of cry2 fused to GUS results in constitutive signalling activity and constitutive phosphorylation. It has been suggested that light activation of N-terminus of cry1 induces a conformational changes in its C-terminus that allows its autophosphorylation and dimerization, and possible interaction with downstream partner proteins. There are also reports that phyA can phosphorylate cryptochromes in vitro, although the functional relevance of this interaction is not clear (Chen et al. 2004).

Phototropins are perhaps the only photoreceptors with well-established kinase activity. Since recombinant phot1 undergoes light-dependent autophosphorylation in the absence of any other plant proteins, it can be concluded that phot1 is necessary and sufficient for light-regulated protein kinase activity. Recombinant phot2 has similar spectral and protein kinase properties. Structural studies suggest that upon light absorption there are large light-driven structural rearrangements that liberate the protein kinase domain and presumably allow protein kinase activity (Chen et al. 2004).

Light signal transduction is highly regulated by phosphorylation also at the level of photoreceptor direct interaction partner proteins. For instance, rapid degradation of nuclear-localized PIF3 protein requires phyA, phyB and phyD. Although the mechanism of light-induced PIF degradation is not clear, the data suggest that the first step in the light-induced degradation of PIF3 is its phosphorylation after direct physical interaction with phytochromes. Phosphorylated PIF3 may then be degraded by the 26S proteasome (see Sect. 14.5.3). Similar processes were described for additional components of the phytochrome signalling pathway, such as other PIFs/PILs, HFR1 and HY5. In summary, the effect of protein phosphory-lation in light signalling differs from stabilization of the protein (e.g. HY5) to targeting it to proteasome-mediated degradation (e.g. HFR1, PIF3; Jiao et al. 2007).

14.5.3 Ubiquitination/Proteasome-Mediated Proteolysis

Regulation of protein degradation is a fundamental part of light signalling, especially through the 26S proteasome pathway. In order for a protein to be degraded by this system, it is polyubiquitinated through the sequential action of three enzymatic activities, namely an E1 ubiquitin (Ub)-activating enzyme, an E2 Ub-conjugating enzyme and an E3 Ub ligase, which recognizes and recruits both Ub-charged E2 and the target protein to catalyze Ub binding to a specific lysine in the target. From the several cop/det/fus mutants identified, nine have been cloned. Their gene products organize into three complexes in vivo, namely the COP1 complex, COP9 signalosome (CSN) and CDD complex. These three multi-protein complexes are directly involved in the Ub/proteasome-mediated protein degradation pathway. The best characterized component is COP1, a RING finger-type E3 Ub ligase. COP1 is required for the light-dependent degradation of several transcription factors involved in the light-regulated transcriptional network. In the dark, COP1 localizes in the nucleus and is able to bind transcription factors, such as HY5, LAF1, HFR1, and then ubiquinate and target them for degradation by the 26S proteasome. Upon light perception, COP1 is translocated to the cytoplasm, allowing the accumulation of those transcriptional factors necessary for photomorpho-genesis (Fig. 14.3b). COP1 is also required for the accumulation of both PIF3 and FHY1 in the dark, although the mechanism of this regulation remains unclear.

Light-induced change in COP1 nucleocytoplasmic localization is modulated via interactions with CSN. The direct interaction of COP1 with phyA, phyB, cry1 and cry2 indicates an additional light-dependent regulation of COP1 activity, for example, targeting photolabile photoreceptors (phyA and cry2) for light-dependent degradation. Moreover, it was suggested that, upon light exposure, cry1 conformation rapidly changes and antagonizes the COP1 effect on HY5 and other transcription factors necessary for photomorphogenesis (Fig. 14.3b). Integrating different light signals, COP1 provides a point of convergence for photoreceptor-mediated signal transduction pathways, acting as a key regulator of light responsive gene expression (Yi and Deng 2005).

14.5.4 Light-Regulated Transcriptional Networks: Changes in Gene Expression

Transcriptional networks have been implicated in mediating photomorphogenesis. R/FR-sensing phytochromes convert perceived light information into absolute concentrations and/or concentration gradients of the Pfr active form (see Sect. 14.3.1). Based on the hypothesis that Pfr can rapidly modulate gene expression by interacting with different PIFs (see Sect. 14.5.1), it was proposed that upon light perception phytochromes initiate complex transcriptional networks instrumental for the implementation of photomorphogenic responses. In agreement with this hypothesis, global gene expression analyses have shown that phytochrome photoperception is associated with massive alterations in gene expression. Consistently, some studies showed that several PIFs, such as PIF1 and PIF3, have a role in controlling gene expression as transcription factors in a phytochrome-dependent manner. Also, direct target genes of phytochrome action, such as HFR1, PIL1, PAR1 (PHYTOCHROME RAPIDLY REGULATED 1) and ATHB4 (ARABIDOPSIS THALIANA HOMEOBOX 4), are specifically involved in the implementation of SAS responses (Jiao et al. 2007; Roig-Villanova et al. 2007).

Gene expression regulation is also a major signalling mechanism underlying cryptochrome action, as demonstrated by transcriptome analysis. It is not clear as to which of the cryptochrome-regulated genes is directly involved in B light de-etiolation responses and how cryptochromes generally regulate gene expression. The genetic identification of components of cryptochrome signalling suggests that important events might occur not only in the nucleus (e.g. HY5, HYH, HFR1 and PP7) but also in the cytoplasm (e.g. SUB1; Li and Yang 2007).

14.6 Light Interaction with Endogenous Networks

Since many plant hormones regulate the same cell division and expansion growth responses modulated by photomorphogenesis, it is expected that light signals converge at the endogenous mechanisms of growth, providing a means to integrate light environment information with endogenous developmental programs, such as those controlled by phytohormones and the circadian clock.

14.6.1 Hormone Connections

Auxins mediate a diverse range of responses including cell division, expansion and differentiation. In addition, auxins coordinate plant development (affecting organ patterning, tropic responses and the architecture of shoot and root) as signals transmitted from cell to cell or from one organ to another. Asymmetric distribution of specific influx and efflux carriers, encoded by the AUX and PIN genes and located at specific positions in the cell membrane within a cell and organ, provides a simple but effective means to establish polar (directional) transport of auxin. Therefore, light might interact with auxins at different levels, including auxin biosynthesis, transport and responses.

Early work showed a role for phytochromes in regulating auxin levels in corn and oat coleoptiles. Recently, low R:FR perception was shown to increase the concentrations of endogenous auxins, involving the action of TAAl (TRYPTO-PHAN AMINOTRANSFERASE OF ARABIDOPSIS 1) that encodes an auxin bio-synthetic enzyme required for full induction of SAS responses (Tao et al. 2008). Applications of auxin transport inhibitors, combined with genetic analyses, have also involved phytochromes in light control of auxin transport. Phototropins were also suggested to alter the activity or localization of auxin transport carriers. Upon directional light perception, an auxin gradient is rapidly established by the action of auxin efflux carriers such as PIN proteins that transport the hormone out of the cell. It seems highly probable that phototropin signalling leads to an alteration in the activity or localization of auxin transport facilitators, modifying polar movement of the hormone and resulting in phototropic growth in response to light.

At the molecular level, auxin rapidly activates the transcription of three gene families, namely Aux/IAA, SAUR and GH3 genes. Work from several laboratories evidenced that light appears to regulate the expression of members of the Aux/IAA, SAUR and GH3 families, some particularly by phyA and phyB action. Recently, the SAS regulator PARI was described as a direct repressor of SAUR genes, rapidly connecting shade- and auxin-regulated transcriptional networks. Proteolysis may represent another convergence point between auxin and light signalling. Aux/IAAs proteins operate by binding to ARF (AUXIN RESPONSIVE FACTOR) transcription factors to regulate their action negatively, providing a mechanism by which auxin modulates gene expression. Auxin controls Aux/IAA protein levels via its degradation by the 26S proteasome. Genetic analyses showed that mutations that stabilize IAA3, IAA7 and IAA17 result in mild de-etiolated phenotypes in the dark. These observations suggest that normal turnover of Aux/IAAs is important to repress photomorphogenesis in the dark (Halliday and Fankhauser 2003; Roig-Villanova et al. 2007).

Brassinosteroids (BRs) are powerful growth promoters that act synergistically with auxins. The first evidence of possible interaction between light and BR signalling was the identification of the det2 mutant. DET2 encodes an enzyme involved in BR biosynthesis. This and most other BR biosynthesis mutants identified are dwarfed, dark-green and display a de-etiolated seedling phenotype in the dark. Mutants affected in BR biosynthesis show an enhanced phyA-mediated VLFR and reduced HIR and LFR responses, suggesting the involvement of BRs in phytochrome signalling. BRs regulate the expression of some light-signalling genes such as PIF3 and CIP1 (COP1 INTERACTING PROTEIN 1), although the biological relevance of this interaction is unknown. BAS1 (PHYB-4 ACTIVATION-TAGGED SUPPRESSOR 1) gene, which encodes a BR inactivating enzyme, was proposed as a modulator of photomorphogenesis based on the increased hypocotyl response of bas1 mutants to exogenous BRs, specifically in white and FR light. Recent work has proposed a role for the SAS modulator ATHB4 in BR signalling, integrating shade perception and BR-mediated growth (Halliday and Fankhauser 2003; Sorin et al. 2009).

Gibberellins (GAs) control multiple aspects of plant development, including induction of germination, leaf expansion, stem elongation and flowering. Phyto-chromes regulate GA levels and biosynthesis at several stages of plant development. During germination, the expression of genes encoding GA biosynthetic enzymes is regulated by phyB and by the antagonistic action of the bHLH transcription factors SPT (SPATULA) and PIF1/PIL5. During elongation growth, it was demonstrated that end-of-day FR light treatment increases bioactive GA concentrations, specifically in the elongating stem of cowpea, although genetic studies of phyB ga1 double mutants (GA1 encodes an enzyme acting in GA biosynthesis) suggest that phytochrome control also affects GA signalling. Recently, it was shown that some pif mutants have an altered sensitivity to GAs. The molecular basis for this phenotype is dependent on the activity of DELLA proteins, negative regulators of GA signalling. Notably, DELLA proteins physically interact with several members of the PIF family, an interaction that inhibits the ability of PIF proteins to bind to and regulate their target promoters. Therefore, PIF proteins represent another integration point between these two pathways (Alabadi and Blazquez 2009).

Cytokinins (CKs) are hormones involved in cell division that affect a wide range of developmental responses. Photomorphogenic development can be mimicked by exogenous application of high concentrations of CKs to dark-grown seedlings. Consistently, the amp mutant that contains elevated CK concentrations displays some aspects of de-etiolation when grown in the dark. ARR4, an early CK-respon-sive gene that encodes a response regulator, is also induced by R light via phyB perception. ARR4 also interacts with the N-terminus of phyB, stabilizing the active PfrB form, suggesting that CK signalling might enhance phyB signalling by altering the PrB/PfrB ratio. Consistently, plants overexpressing ARR4 show hyper-sentivity to R light. However, this interaction does not explain how CKs promote some aspects of de-etiolation in the dark.

Ethylene is a gaseous hormone that regulates many aspects of plant development, such as the triple response in etiolated seedlings, fruit ripening and senescence. ACC oxidase, a key enzyme in ethylene biosynthesis, is light-regulated in a phytochrome-dependent manner in both Arabidopsis and Sorghum. The maintenance of the apical hook in etiolated seedling requires normal ethylene perception and signalling, while the unhooking response to light requires functional photo-receptors. Experiments in Arabidopsis suggest that perception of ethylene, rather than its production, is light-modulated during de-etiolation (Halliday and Fankhauser 2003). Furthermore, mutation in BIG confers aberrant photomorphogenic pheno-types and altered responses to ethylene, CKs, GAs and auxins. The study of BIG gene suggests a complex, non-linear interaction between photomorphogenesis and multiple hormones (Halliday and Fankhauser 2003).

14.6.2 Light-Clock Signal Integration

In plants, the circadian clock controls daily changes in gene expression, growth, photosynthetic activity and seasonal flowering. This rhythmic mechanism synchronizes internal signalling processes with external light cues, driving a vast array of metabolic and developmental responses. Clock components and photoreceptors have an intimate relationship; light signals transduced by the phytochromes and cryptochromes ensure that the clock is in tune with daily light/dark cycles. This process, known as photoentrainment, is achieved by adjusting the phase and the period of the oscillator relative to the prevailing photoperiod. Analyses of CAB:: LUC (CHLOROPHYLL A/B BINDING PROTEIN::LUCIFERASE) transgenic lines, which display circadian luciferase activity in vivo, in different photoreceptor null mutants indicated a role for phytochromes and cryptochromes in controlling photoentrainment under different light conditions.

Genetic and molecular analyses have identified elf3 (early flowering 3) and tic (time for coffee) mutants, because light induces high levels of CAB::LUC expression during the dark period, a time when this response is suppressed in the wild type. This indicates that ELF3 and TIC participate in the differential regulation of day and night time sensitivity to light, a mechanism known as circadian gating that ensures correct entrainment of the clock to changing dawn and dusk signals. In the case of ELF3, moderation of the phytochrome signal may be direct, since ELF3, which is localized in the nucleus, interacts with phyB in vitro. Photoentrainment is also controlled by ZTL, a putative photoreceptor (see Sect. 14.3.3). ZTL has been shown to control protein levels of TOC1 (TIMING OF CAB 1), a component of the biological clock, via ubiquitination and subsequent proteolysis by the 26S protea-some (Mas 2008).

14.7 Applied Aspects of Photomorphogenic Research

Many traits selected for plant productivity in modern agriculture are greatly influenced by photoreceptors. Because of these observations, photoperception modification has become an appealing target for crop improvement.

14.7.1 What Is Fit Under Natural Conditions Might Be Inadequate for Agriculture

In natural environments, competition for light is central for plant success and survival to reproductive maturity, and activation of SAS developmental programs aims to overgrow or survive putative plant competitors even before the plant is actually shaded. However, activation of SAS responses in most crop species is disadvantageous for plant productivity in modern agriculture because it results in resource allocation to stem growth, at the cost of leaf growth and the development of storage and reproductive structures. As this example illustrates, modern agricultural practices place different constraints on plant growth that have not been necessarily selected for during plant evolution, although the natural environment selects for certain traits. Traditionally, breeding efforts have been focused on optimizing grain yields by modulating those characteristics that affect these traits, such as plant height, branching and time of flowering. With the recent interest in lignocellulosic-based biofuels, however, a new breeding paradigm may emerge to optimize biomass at the expense of grain yield. Therefore, an understanding of light signalling might lead to the judicial manipulation of photoreceptor signalling pathways to improve the characteristics of these new traits.

14.7.2 Classical Breeding for the Development of Agronomical Varieties Has Selected Light-Regulated Traits

14.7.2.1 Plant Height

Dwarfing is a method utilized for increasing yield by reducing the resources allocated to structural growth. Dwarfing also increases yield in cereal crops by increasing the resistance to mechanical flattening ("lodging") by wind, rain or heavy yield production. Indeed, selection of semi-dwarf varieties of wheat and rice has contributed to the so-called green revolution and has become the choice of most growers. Molecular analyses of these cereal dwarf varieties have shown that they carry mutant alleles that affect gibberellin pathways. An alternative way to control this trait in newly developed crops is to reduce stem elongation by specifically suppressing SAS responses, rather than hormone pathways. This approach would suppress stem elongation only under high plant density conditions typical of crop monocultures (Kebrom and Brutnell 2007).

14.7.2.2 Branching

In cereal grasses, plant breeders have selected to attenuate some but not all SAS responses within modern crop varieties. One of the most dramatic effects of shade in the grasses is the production and proliferation of basal auxiliary meristems that develop into tillers (lateral branches). In general, high planting densities (low R/FR) result in increased apical dominance at the expense of tiller development. In species or varieties in which tillering is a component of yield, the tillering component of the SAS has been tempered to permit tiller production in crop monocultures; for example, selection of rice was for high tillering varieties that are more productive than low tillering varieties under long growing seasons. However, tiller proliferation in maize is often a negative component of yield, and genetic variation has been selected to enhance or maintain suppression of tillers (Kebrom and Brutnell 2007).

14.7.2.3 Flowering Time

Many agriculturally important plant species have critical daylength requirements for flowering and fruiting. The optimization of photoperiod has become a standard practice, where crops are sown only in the season with the appropriate daylength, by supplementing with artificial light to extend photoperiod or by covering the plants with black cloths to shorten it. The knowledge from physiological studies of phytochrome action has allowed the substitution of constant light by single pulses in the night period to control flowering in some species. Such practices permit considerable cost savings to growers and have had dramatic impacts on the flower industry. The understanding of the molecular basis of daylength perception and how this is coupled to the corresponding evocation response has allowed the identification of key photoperiod regulators, such as CO. In that context, it has been shown that overexpression of Arabidopsis CO in potato results in delayed tuberization, suggesting a conserved function for CONSTANS activity in unrelated photoperiod-modulated responses (Kobayashi and Weigel 2007). However, there are no reports of biotechnological applications of this information.

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