Phytochrome genes in higher plants: Structure,expression, and evolution

© 2006 Springer. All Rights Reserved. Phytochromes play critical roles in monitoring light quantity, quality, and periodicity in plants and they relay this photosensory information to a large number of signaling pathways that regulate plant growth and development. Given these complex functions, it is not surprising that the phytochrome apoproteins are encoded by small multigene families and that different forms of phytochrome regulate different aspects of photomorphogenesis. Over the course of the last decade, progress has been made in defining the number, molecular properties, and biological activities of the photoreceptors that constitute a plant R/FR sensing system. This chapter summarizes our current understanding of the structure of the genes that encode the phytochrome apoproteins (the PHY genes), the expression patterns of those genes, the nature of the phytochrome apoprotein family, and PHY gene evolution in seed plants. Phytochrome was discovered and its basic photochemical properties were first described through physiological studies of light-sensitive seed germination and photoperiodic effects on flowering (Borthwick, et al., 1948, Borthwick, et al., 1952). The pigment itself was initially isolated from extracts of dark-grown (etiolated) plant tissue in 1959 (Butler, et al., 1959), but it was not until much later that phytochrome was purified to homogeneity in an undegraded form (Vierstra and Quail, 1983). DNA sequences of gene and cDNA clones for oat etiolated-tissue spectroscopically in planta and purified in its native form, this dark-tissue phytochrome (now called phyA) remains the most completely biochemically and spectroscopically characterized form of the receptor. At various times throughout the first 40 years of the study of the abundant etiolated-tissue phytochrome, evidence for the presence and activity of additional forms of phytochrome, often referred to as green-tissue or light-stable phytochromes, was obtained. Initially, in physiological experiments, it was sometimes not possible to correlate specific in vivo phytochrome activities with the phytochrome provided the first complete descriptions of the apoprotein (Hershey et al., 1985). Because it accumulates to levels that permit it to be assayed known spectroscopic properties of the molecule. Later, direct evidence for multiple species of phytochrome in plants and in plant extracts was obtained using both spectroscopic and immunochemical methods (reviewed in Pratt, 1995). The molecular identities of these additional phytochrome forms were ultimately deduced from cDNA clones that were isolated by nucleic acid similarity to etiolated-tissue phytochrome sequences (Sharrock and Quail, 1989). More recently, analysis of a large number of complete and partial PHY gene or cDNA sequences from a broad sampling of plant phylogenetic groups and sequencing of several plant genomes have resulted in a much clearer and more general picture of what constitutes a higher plant R/FR photoreceptor family. It is likely that the major types of long-wavelength photosensing pigments have now been identified and the challenge that lies ahead is to understand how the signalling mechanisms, expression patterns, and interactions of these molecules contribute to plant responses to the R/FR environment. Extending the investigation of phytochrome gene families and their functions to additional angiosperm and gymnosperm genera will be an integral component of this effort and of our ability to utilize this growing understanding of phytochrome function to modify the agricultural properties of plants and to better understand the history of land plants

Alternative Splicing Diversifies the Transcriptome during Early Photomorphogenesis and Responds to Metabolic Signals in Arabidopsis

Light is an important source of both information and energy for plants. Diurnal rhythms and seasonal changes, as well as surrounding competition are detected by the available light quality and quantity. Sensing changing light conditions to adjust metabolism and control development is cruicial for survival. Here, we analyse transcriptome-wide changes in gene expression and alternative splicing (AS) in dark-grown (etiolated) seedlings as they transition to growth in white, blue, or red light, undergoing photomorphogenesis. We find changes in expression levels for about 20 % of all genes and changes in splicing patterns in hundreds of transcripts. In more than 60 % of the light-regulated splicing events involving an assumed non-coding variant, production of a presumably protein-coding variant is increased in light, while levels of the other variant carrying Nonsense-mediated decay (NMD)-triggering features decline. Following this pattern, AS of the red light signalling component and putative splicing regulator REDUCED RED-LIGHT RESPONSES IN CRY1CRY2 BACKGROUND 1 (RRC1) shifts in favour of the functional variant upon light exposure. While AS of splicing regulator Ser/Arg-rich protein (SR) 30 also favours the protein-coding variant in light, the alternative variant is not degraded by NMD, and we explore potential other biological functions of this AS event. Furthermore, aiming to elucidate upstream signalling components, we find light-dependent AS to be unaffected in the photoreceptor mutant phyA phyB exposed to white light, indicating that photoreceptor signalling only plays a minor role upstream of AS in white light. Interestingly, sucrose supply and light alter the AS output similarly, suggesting that the changes in AS correlate with the plant energy status.Pflanzen nutzen Licht als Energiequelle und Informationsträger um den Tag/Nacht Zyklus, die Jahreszeiten sowie konkurrierende Pflanzen in ihrer unmittelbaren Umgebung zu detektieren. Das Erkennen veränderter Lichtbedingungen ist für Pflanzen essentiell um ihren Metabolismus den äußeren Bedingungen anzupassen und ihre Entwicklungsprozesse zu steuern. In dieser Arbeit haben wir transkriptomweite Änderungen der Genexpression und des Alternativen Spleißens (AS) in dunkel gezogenen (etiolierten) Keimlingen untersucht, die sich unter blauem, rotem oder weißem Licht morphologisch den veränderten Lichtbedingungen anpassen, d. h. den Prozess der Photomorphogenese durchlaufen. Unsere Analyse zeigt, dass sich hierbei die Expressionslevel nahezu 20 % aller Gene, sowie die Spleißmuster hunderter Vorläufer-mRNAs (prä-mRNAs) ändern. Licht führt in mehr als 60 % der lichtregulierten Fälle mit AS zu einer vermutlich nicht-proteinbildenden Spleißvariante zur verstärkten Bildung der wahrscheinlich proteinkodierenden Spleißform. Die Varianten, von denen hierbei im Ausgleich weniger gebildet wird, tragen Merkmale, die voraussichtlich zum Abbau dieser mRNAs durch den RNA-Qualitätskontrollmechanismus Nonsense-mediated decay (NMD) führen. Entsprechend zeigen wir für den mutmaßlichen Spleißfaktor REDUCED RED-LIGHT RESPONSES IN CRY1CRY2 BACKGROUND 1 (RRC1), dessen Rolle in der Lichtsignalgebung in einer früheren Arbeit beschrieben wurde, dass in Licht das AS der prä-mRNA zugunsten der funktionellen Variante verschoben wird. Außerdem untersuchen wir die biologische Funktion des lichtabhängigen AS von dem Spleißfaktor Ser/Arg-rich protein (SR) 30, dessen alternative Variante trotz vorhandener NMD Merkmale nicht durch NMD abgebaut zu werden scheint. Um die für lichtabhängiges AS notwendige Signalgebung besser zu verstehen haben wir Bedeutung von Photorezeptoren in diesem Zusammenhang untersucht. Die Analysen zeigen, dass die Reaktion auf Weißlicht in der Rotlichtrezeptormutante phyA phyB unverändert ist. Dies deutet auf eine untergeordnete Rolle von Photorezeptoren für die Regulation von lichtabhängigem AS hin. Zudem führen interessanterweise die Gabe von Saccharose zu sehr ähnlichen Änderungen im AS wie Licht, was auf eine Kopplung des AS mit dem Energiestatus der Pflanze hinweist

Plastid tubules in higher plants: an analysis of form and function

Besides photosynthesis, plastids are responsible for starch storage, fatty acid biosynthesis and nitrate metabolism. Our understanding of plastids can be improved with observation by microscopy, but this has been hampered by the invisibility of many plastid types. By targeting green fluorescent protein (GFP) to the plastid in transgenic plants, the visualisation of plastids has become routinely possible. Using GFP, motile, tubular protrusions can be observed to emanate from the plastid envelope into the surrounding cytoplasm. These structures, called stromules, vary considerably in frequency and length between different plastid types, but their function is poorly understood. During tomato fruit ripening, chloroplasts in the pericarp cells differentiate into chromoplasts. As chlorophyll degrades and carotenoids accumulate, plastid and stromule morphology change dramatically. Stromules become significantly more abundant upon chromoplast differentiation, but only in one cell type where plastids are large and sparsely distributed within the cell. Ectopic chloroplast components inhibit stromule formation, whereas preventing chloroplast development leads to increased numbers of stromules. Together, these findings imply that stromule function is closely related to the differentiation status, and thus role, of the plastid in question. In tobacco seedlings, stromules in hypocotyl epidermal cells become longer as plastids become more widely distributed within the cell, implying a plastid density-dependent regulation of stromules. Co-expression of fluorescent proteins targeted to plastids, mitochondria and peroxisomes revealed a close spatio-temporal relationship between stromules and other organelles. Stromule and plastid fusion could not be induced under conditions which promote substantial fusion of mitochondria. Data are presented suggesting that organelles may be able to pass between cells, and an experiment was designed to test this possibility in the C4 photosynthetic cells of maize. Inhibitor studies have shown that stromule and plastid movement is dependent on the actin cytoskeleton and the ATPase activity of myosin. An Arabidopsis gene, CHUP1, is responsible for chloroplast relocation in response to light intensity and encodes a chloroplast-localised actin-binding protein. To assess whether this protein is involved in stromule movement, CHUP1 was down-regulated with RNAi. Whilst plants with reduced CHUP1 expression exhibited a chup1 mutant phenotype, no significant effect on stromules was discovered. It was thus concluded that chloroplast relocation and stromule formation are two independent processes that employ different actin-dependent mechanisms. It is proposed that stromules act primarily to increase the plastid surface area in response to a number of developmental and environmental factors

Plastid tubules in higher plants: an analysis of form and function

Besides photosynthesis, plastids are responsible for starch storage, fatty acid biosynthesis and nitrate metabolism. Our understanding of plastids can be improved with observation by microscopy, but this has been hampered by the invisibility of many plastid types. By targeting green fluorescent protein (GFP) to the plastid in transgenic plants, the visualisation of plastids has become routinely possible. Using GFP, motile, tubular protrusions can be observed to emanate from the plastid envelope into the surrounding cytoplasm. These structures, called stromules, vary considerably in frequency and length between different plastid types, but their function is poorly understood. During tomato fruit ripening, chloroplasts in the pericarp cells differentiate into chromoplasts. As chlorophyll degrades and carotenoids accumulate, plastid and stromule morphology change dramatically. Stromules become significantly more abundant upon chromoplast differentiation, but only in one cell type where plastids are large and sparsely distributed within the cell. Ectopic chloroplast components inhibit stromule formation, whereas preventing chloroplast development leads to increased numbers of stromules. Together, these findings imply that stromule function is closely related to the differentiation status, and thus role, of the plastid in question. In tobacco seedlings, stromules in hypocotyl epidermal cells become longer as plastids become more widely distributed within the cell, implying a plastid density-dependent regulation of stromules. Co-expression of fluorescent proteins targeted to plastids, mitochondria and peroxisomes revealed a close spatio-temporal relationship between stromules and other organelles. Stromule and plastid fusion could not be induced under conditions which promote substantial fusion of mitochondria. Data are presented suggesting that organelles may be able to pass between cells, and an experiment was designed to test this possibility in the C4 photosynthetic cells of maize. Inhibitor studies have shown that stromule and plastid movement is dependent on the actin cytoskeleton and the ATPase activity of myosin. An Arabidopsis gene, CHUP1, is responsible for chloroplast relocation in response to light intensity and encodes a chloroplast-localised actin-binding protein. To assess whether this protein is involved in stromule movement, CHUP1 was down-regulated with RNAi. Whilst plants with reduced CHUP1 expression exhibited a chup1 mutant phenotype, no significant effect on stromules was discovered. It was thus concluded that chloroplast relocation and stromule formation are two independent processes that employ different actin-dependent mechanisms. It is proposed that stromules act primarily to increase the plastid surface area in response to a number of developmental and environmental factors

Ultraviolet – B -mediated control of PHYTOCHROME INTERACTING FACTOR (PIF) transcription in Arabidopsis thaliana

Plants, as sessile autotrophic organisms, rely on light cues not only as a source of energy, but also to regulate developmental responses to cope with their everchanging environment. Physiological changes triggered by light vary according to the light quality that is perceived by specific specialized photoreceptors, including phytochromes, cryptochromes and UV RESISTANCE LOCUS 8 (UVR8). These photoreceptors transduce the light cues to regulate PHYTOCHROME-INTERACTING FACTORS (PIFs). PIFs are a small subset of transcription factors of the basic helix-loop-helix (bHLH) subfamily, which act as a cellular signalling hub that integrates multiple signals, including light and temperature, to regulate plant morphogenesis. The mechanisms underlying transcriptional regulation of PIFs are poorly understood in comparison to their posttranscriptional regulation. This thesis examines the transcriptional regulation of PIFs in response to low dose ULTRAVIOLET-B (UV-B) light. UV-B is shown to suppress the transcript abundance of PIF3, PIF4 and PIF5 by inhibition of promoter activity, in a UVR8- dependent manner. Evidence supporting a role for COP1 in the suppression of PIF4 and PIF5transcript abundance in UV-B is also presented. Three different mechanisms controlling UV-B - mediated suppression of PIF transcript abundance are investigated. The first involves the plant hormones, brassinosteroids (BR). This thesis shows that BR signals are not involved in the UV-B - mediated suppression of PIF4 transcript at high temperatures, but support a role for BR signalling in the UV-B-mediated suppression of thermomorphogenesis. The second involves a potential autoregulatory loop involving UV-B-mediated degradation of PIF protein. Data suggest that UV-B - mediated PIF4 degradation may occur via an alternative pathway to PIF5. The third investigates the role of MYB30 in regulating PIF transcript abundance. Data show that MYB30 is suppressed by UV-B in a UVR8-dependent manner and promotes PIF7 transcription in white light. In addition, MYB30 regulates shade-avoidances responses to green shade.<br/

UVR8 mediated spatial differences as a prerequisite for UV-B induced inflorescence phototropism

In Arabidopsis hypocotyls, phototropins are the dominant photoreceptors for the positive phototropism response towards unilateral ultraviolet-B (UV-B) radiation. We report a stark contrast of response mechanism with inflorescence stems with a central role for UV RESISTANCE LOCUS 8 (UVR8). The perception of UV-B occurs mainly in the epidermis and cortex with a lesser contribution of the endodermis. Unilateral UV-B exposure does not lead to a spatial difference in UVR8 protein levels but does cause differential UVR8 signal throughout the stem with at the irradiated side 1) increase of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), 2) an associated strong activation of flavonoid biosynthesis genes and flavonoid accumulation, 3) increased GA2oxidase expression, diminished gibberellin1 levels and accumulation of DELLA protein REPRESSOR OF GA1 (RGA) and, 4) increased expression of the auxin transport regulator, PINOID, contributing to local diminished auxin signalling. Our molecular findings are in support of the Blaauw theory (1919), suggesting that differential growth occurs trough unilateral photomorphogenic growth inhibition. Together the data indicate phototropin independent inflorescence phototropism through multiple locally UVR8-regulated hormone pathways

Tree Peony Species Are a Novel Resource for Production of α-Linolenic Acid

Tree peony is known worldwide for its excellent ornamental and medical values, but recent reports that their seeds contain over 40% α-linolenic acid (ALA), an essential fatty acid for humans drew additional interest of biochemists. To understand the key factors that contribute to this rich accumulation of ALA, we carried out a comprehensive study of oil accumulation in developing seeds of nine wild tree peony species. The fatty acid content and composition was highly variable among the nine species; however, we selected a high- (P. rockii) and low-oil (P. lutea) accumulating species for a comparative transcriptome analysis. Similar to other oilseed transcriptomic studies, upregulation of select genes involved in plastidial fatty acid synthesis, and acyl editing, desaturation and triacylglycerol assembly in the endoplasmic reticulum was noted in seeds of P. rockii relative to P. lutea. Also, in association with the ALA content, transcript levels for fatty acid desaturases (SAD, FAD2 and FAD3), which encode for enzymes necessary for polyunsaturated fatty acid synthesis were higher in P. rockii compared to P. lutea. We further showed that the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and α-linolenic acid content, respectively and modulated their final ratio in the seed oil. In conclusion, we identified the key steps that contribute to efficient ALA synthesis and validated the necessary desaturases in P. rockii that are responsible for not only increasing oil content but also modulating 18:2/18:3 ratio in seeds. Together, these results will aid to improve essential fatty acid content in seeds of tree peonies and other crops of agronomic interest