Hormonal modulation of photomorphogenesis-controlled anthocyanin accumulation in tomato (Solanum lycopersicum L. cv Micro-Tom) hypocotyls: Physiological and genetic studies

Hormones are likely to be important factors modulating the light-dependent anthocyanin accumulation. Here we analyzed anthocyanin contents in hypocotyls of near isogenic Micro-Tom (MT) tomato lines carrying hormone and phytochrome mutations, as single and double-mutant combinations. In order to recapitulate mutant phenotype, exogenous hormone applications were also performed Anthocyanin accumulation was promoted by exogenous abscisic acid (ABA) and inhibited by gibberellin (GA), in accordance to the reduced anthocyanin contents measured in ABA-deficient (notabills) and GA-constitutive response (procera) mutants. Exogenous cytokinin also enhanced anthocyanin levels in MT hypocotyls. Although auxin-insensitive chageotropica mutant exhibited higher anthocyanin contents, pharmacological approaches employing exogenous auxin and a transport inhibitor did not support a direct role of the hormone in anthocyanin accumulation Analysis of mutants exhibiting increased ethylene production (epwastic) or reduced sensitivity (Never ripe), together with pharmacological data obtained from plants treated with the hormone, indicated a limited role for ethylene in anthocyanin contents. Phytochrome-deficiency (aurea) and hormone double-mutant combinations exhibited phenotypes suggesting additive or synergistic interactions, but not fully espistatic ones, in the control of anthocyanin levels in tomato hypocotyls. Our results indicate that phytochrome-mediated anthocyanin accumulation in tomato hypocotyls is modulated by distinct hormone classes via both shared and independent pathways. (C) 2010 Elsevier Ireland Ltd. All rights reserved

Photomorphogenesis and Photoreceptors

Plants use light as their main source of energy and to gather information about their surroundings. The light environment is monitored through an extensive set of photoreceptors and largely dictates plant development through induction of processes such as germination and flowering, entrainment of the circadian clock and photomorphogenic responses. Plants display remarkable phenotypic plasticity upon perception of changes in the light, ranging from seedling de-etiolation to shade avoidance and phototropic responses in competition for light. Here, we describe photomorphogenic responses and their underlying mechanisms such as they occur in a leaf canopy. This shade avoidance review will largely focus on the model plant species Arabidopsis thaliana as the underlying mechanisms controlling shade avoidance are particularly well elucidated in this species.

Convergence of developmental mutants into a single tomato model system: 'Micro-Tom' as an effective toolkit for plant development research

Background: The tomato (Solanum lycopersicum L.) plant is both an economically important food crop and an ideal dicot model to investigate various physiological phenomena not possible in Arabidopsis thaliana. Due to the great diversity of tomato cultivars used by the research community, it is often difficult to reliably compare phenotypes. The lack of tomato developmental mutants in a single genetic background prevents the stacking of mutations to facilitate analysis of double and multiple mutants, often required for elucidating developmental pathways. Results: We took advantage of the small size and rapid life cycle of the tomato cultivar Micro-Tom (MT) to create near-isogenic lines (NILs) by introgressing a suite of hormonal and photomorphogenetic mutations (altered sensitivity or endogenous levels of auxin, ethylene, abscisic acid, gibberellin, brassinosteroid, and light response) into this genetic background. To demonstrate the usefulness of this collection, we compared developmental traits between the produced NILs. All expected mutant phenotypes were expressed in the NILs. We also created NILs harboring the wild type alleles for dwarf, self-pruning and uniform fruit, which are mutations characteristic of MT. This amplified both the applications of the mutant collection presented here and of MT as a genetic model system. Conclusions: The community resource presented here is a useful toolkit for plant research, particularly for future studies in plant development, which will require the simultaneous observation of the effect of various hormones, signaling pathways and crosstalk.

A molecular framework for light and gibberellin control of cell elongation.

Cell elongation during seedling development is antagonistically regulated by light and gibberellins (GAs). Light induces photomorphogenesis, leading to inhibition of hypocotyl growth, whereas GAs promote etiolated growth, characterized by increased hypocotyl elongation. The mechanism underlying this antagonistic interaction remains unclear. Here we report on the central role of the Arabidopsis thaliana nuclear transcription factor PIF4 (encoded by PHYTOCHROME INTERACTING FACTOR 4) in the positive control of genes mediating cell elongation and show that this factor is negatively regulated by the light photoreceptor phyB (ref. 4) and by DELLA proteins that have a key repressor function in GA signalling. Our results demonstrate that PIF4 is destabilized by phyB in the light and that DELLAs block PIF4 transcriptional activity by binding the DNA-recognition domain of this factor. We show that GAs abrogate such repression by promoting DELLA destabilization, and therefore cause a concomitant accumulation of free PIF4 in the nucleus. Consistent with this model, intermediate hypocotyl lengths were observed in transgenic plants over-accumulating both DELLAs and PIF4. Destabilization of this factor by phyB, together with its inactivation by DELLAs, constitutes a protein interaction framework that explains how plants integrate both light and GA signals to optimize growth and development in response to changing environments.

The degradation of HFR1, a putative bHLH class transcription factor involved in light signaling, is regulated by phosphorylation and requires COP1.

All developmental transitions throughout the life cycle of a plant are influenced by light. In Arabidopsis, multiple photoreceptors including the UV-A/blue-sensing cryptochromes (cry1-2) and the red/far-red responsive phytochromes (phyA-E) monitor the ambient light conditions. Light-regulated protein stability is a major control point of photomorphogenesis. The ubiquitin E3 ligase COP1 (constitutively photomorphogenic 1) regulates the stability of several light-signaling components. HFR1 (long hypocotyl in far-red light) is a putative transcription factor with a bHLH domain acting downstream of both phyA and the cryptochromes. HFR1 is closely related to PIF1, PIF3, and PIF4 (phytochrome interacting factor 1, 3 and 4), but in contrast to the latter three, there is no evidence for a direct interaction between HFR1 and the phytochromes. Here, we show that the protein abundance of HFR1 is tightly controlled by light. HFR1 is an unstable phosphoprotein, particularly in the dark. The proteasome and COP1 are required in vivo to degrade phosphorylated HFR1. In addition, HFR1 can interact with COP1, consistent with the idea of COP1 directly mediating HFR1 degradation. We identify a domain, conserved among several bHLH class proteins involved in light signaling , as a determinant of HFR1 stability. Our physiological experiments indicate that the control of HFR1 protein abundance is important for a normal de-etiolation response.

Light-regulated plant growth and development.

Plants are sessile and photo-autotrophic; their entire life cycle is thus strongly influenced by the ever-changing light environment. In order to sense and respond to those fluctuating conditions higher plants possess several families of photoreceptors that can monitor light from UV-B to the near infrared (far-red). The molecular nature of UV-B sensors remains unknown, red (R) and far-red (FR) light is sensed by the phytochromes (phyA-phyE in Arabidopsis) while three classes of UV-A/blue photoreceptors have been identified: cryptochromes, phototropins, and members of the Zeitlupe family (cry1, cry2, phot1, phot2, ZTL, FKF1, and LKP2 in Arabidopsis). Functional specialization within photoreceptor families gave rise to members optimized for a wide range of light intensities. Genetic and photobiological studies performed in Arabidopsis have shown that these light sensors mediate numerous adaptive responses (e.g., phototropism and shade avoidance) and developmental transitions (e.g., germination and flowering). Some physiological responses are specifically triggered by a single photoreceptor but in many cases multiple light sensors ensure a coordinated response. Recent studies also provide examples of crosstalk between the responses of Arabidopsis to different external factors, in particular among light, temperature, and pathogens. Although the different photoreceptors are unrelated in structure, in many cases they trigger similar signaling mechanisms including light-regulated protein-protein interactions or light-regulated stability of several transcription factors. The breath and complexity of this topic forced us to concentrate on specific aspects of photomorphogenesis and we point the readers to recent reviews for some aspects of light-mediated signaling (e.g., transition to flowering).

Phytochrome interacting factors 4 and 5 redundantly limit seedling de-etiolation in continuous far-red light.

Phytochromes are red/far-red photosensors that regulate numerous developmental programs in plants. Among them, phytochrome A (phyA) is essential to enable seedling de-etiolation under continuous far-red (FR) light, a condition that mimics the environment under a dense canopy. The ecological relevance of this response is demonstrated by the high mortality rate of phyA mutant plants that germinate in deep vegetational shade. phyA signaling involves direct interaction of the photoreceptor with phytochrome-interacting factors PIF1 and PIF3, members of the bHLH transcription factor family. Here we investigated the involvement of PIF4 and PIF5 in phyA signaling, and found that they redundantly control de-etiolation in FR light. The pif4 pif5 double mutant is hypersensitive to low fluence rates of FR light. This phenotype is dependent on FR light perception by phyA, but does not rely on alterations in the phyA level. Our microarray analysis shows that PIF4 and PIF5 are part of an inhibitory mechanism that represses the expression of some light-responsive genes in the dark, and that they are also needed for full expression of several growth-related genes in the light. Unlike PIF1 and PIF3, PIF4 and PIF5 are not degraded in response to FR light, indicating that they are light-regulated by a different mechanism. Our genetic analysis suggests that this is achieved through sequestration of these PIFs by the closely related bHLH transcription factor HFR1 (long hypocotyl in FR light).

Full establishment of arbuscular mycorrhizal symbiosis in rice occurs independently of enzymatic jasmonate biosynthesis.

Development of the mutualistic arbuscular mycorrhiza (AM) symbiosis between most land plants and fungi of the Glomeromycota is regulated by phytohormones. The role of jasmonate (JA) in AM colonization has been investigated in the dicotyledons Medicago truncatula, tomato and Nicotiana attenuata and contradicting results have been obtained with respect to a neutral, promotive or inhibitory effect of JA on AM colonization. Furthermore, it is currently unknown whether JA plays a role in AM colonization of monocotyledonous roots. Therefore we examined whether JA biosynthesis is required for AM colonization of the monocot rice. To this end we employed the rice mutant constitutive photomorphogenesis 2 (cpm2), which is deficient in JA biosynthesis. Through a time course experiment the amount and morphology of fungal colonization did not differ between wild-type and cpm2 roots. Furthermore, no significant difference in the expression of AM marker genes was detected between wild type and cpm2. However, treatment of wild-type roots with 50 μM JA lead to a decrease of AM colonization and this was correlated with induction of the defense gene PR4. These results indicate that JA is not required for AM colonization of rice but high levels of JA in the roots suppress AM development likely through the induction of defense.

Design and study of self-assembled functional organic and hybrid systems for biological applications

The focus of self-assembly as a strategy for the synthesis has been confined largely to molecules, because of the importance of manipulating the structure of matter at the molecular scale. We have investigated the influence of temperature and pH, in addition to the concentration of the capping agent used for the formation of the nano-bio conjugates. For example, the formation of the narrower size distribution of the nanoparticles was observed with the increase in the concentration of the protein, which supports the fact that γ-globulin acts both as a controller of nucleation as well as stabiliser. As analyzed through various photophysical, biophysical and microscopic techniques such as TEM, AFM, C-AFM, SEM, DLS, OPM, CD and FTIR, we observed that the initial photoactivation of γ-globulin at pH 12 for 3 h resulted in small protein fibres of ca. Further irradiation for 24 h, led to the formation of selfassembled long fibres of the protein of ca. 5-6 nm and observation of surface plasmon resonance band at around 520 nm with the concomitant quenching of luminescence intensity at 680 nm. The observation of light triggered self-assembly of the protein and its effect on controlling the fate of the anchored nanoparticles can be compared with the naturally occurring process such as photomorphogenesis.Furthermore,our approach offers a way to understand the role played by the self-assembly of the protein in ordering and knock out of the metal nanoparticles and also in the design of nano-biohybrid materials for medicinal and optoelectronic applications. Investigation of the potential applications of NIR absorbing and water soluble squaraine dyes 1-3 for protein labeling and anti-amyloid agents forms the subject matter of the third chapter of the thesis. The study of their interactions with various proteins revealed that 1-3 showed unique interactions towards serum albumins as well as lysozyme. 69%, 71% and 49% in the absorption spectra as well as significant quenching in the fluorescence intensity of the dyes 1-3, respectively. Half-reciprocal analysis of the absorption data and isothermal titration calorimetric (ITC) analysis of the titration experiments gave a 1:1 stoichiometry for the complexes formed between the lysozyme and squaraine dyes with association constants (Kass) in the range 104-105 M-1. We have determined the changes in the free energy (ΔG) for the complex formation and the values are found to be -30.78, -32.31 and -28.58 kJmol-1, respectively for the dyes 1, 2 and 3. Furthermore, we have observed a strong induced CD (ICD) signal corresponding to the squaraine chromophore in the case of the halogenated squaraine dyes 2 and 3 at 636 and 637 nm confirming the complex formation in these cases. To understand the nature of interaction of the squaraine dyes 1-3 with lysozyme, we have investigated the interaction of dyes 1-3 with different amino acids. These results indicated that the dyes 1-3 showed significant interactions with cysteine and glutamic acid which are present in the side chains of lysozyme. In addition the temperature dependent studies have revealed that the interaction of the dye and the lysozyme are irreversible. Furthermore, we have investigated the interactions of these NIR dyes 1-3 with β- amyloid fibres derived from lysozyme to evaluate their potential as inhibitors of this biologically important protein aggregation. These β-amyloid fibrils were insoluble protein aggregates that have been associated with a range of neurodegenerative diseases, including Huntington, Alzheimer’s, Parkinson’s, and Creutzfeldt-Jakob diseases. We have synthesized amyloid fibres from lysozyme through its incubation in acidic solution below pH 4 and by allowing to form amyloid fibres at elevated temperature. To quantify the binding affinities of the squaraine dyes 1-3 with β-amyloids, we have carried out the isothermal titration calorimetric (ITC) measurements. The association constants were determined and are found to be 1.2 × 105, 3.6× 105 and 3.2 × 105 M-1 for the dyes, 1-3, respectively. To gain more insights into the amyloid inhibiting nature of the squaraine dyes under investigations, we have carried out thioflavin assay, CD, isothermal titration calorimetry and microscopic analysis. The addition of the dyes 1-3 (5μM) led to the complete quenching in the apparent thioflavin fluorescence, thereby indicating the destabilization of β-amyloid fibres in the presence of the squaraine dyes. Further, the inhibition of the amyloid fibres by the squaraine dyes 1-3, has been evidenced though the DLS, TEM AFM and SAED, wherein we observed the complete destabilization of the amyloid fibre and transformation of the fibre into spherical particles of ca. These results demonstrate the fact that the squaraine dyes 1-3 can act as protein labeling agents as well as the inhibitors of the protein amyloidogenesis. The last chapter of the thesis describes the synthesis and investigation of selfassembly as well as bio-imaging aspects of a few novel tetraphenylethene conjugates 4-6.Expectedly, these conjugates showed significant solvatochromism and exhibited a hypsochromic shift (negative solvatochromism) as the solvent polarity increased, and these observations were justified though theoretical studies employing the B3LYP/6-31g method. We have investigated the self-assembly properties of these D-A conjugates though variation in the percentage of water in acetonitrile solution due to the formation of nanoaggregates. Further the contour map of the observed fluorescence intensity as a function of the fluorescence excitation and emission wavelength confirmed the formation of J-type aggregates in these cases. To have a better understanding of the type of self-assemblies formed from the TPE conjugates 4-6, we have carried out the morphological analysis through various microscopic techniques such as DLS, SEM and TEM. 70%, we observed rod shape architectures having ~ 780 nm in diameter and ~ 12 μM in length as evidenced through TEM and SEM analysis. We have made similar observations with the dodecyl conjugate 5 at ca. 70% and 50% water/acetonitrile mixtures, the aggregates formed from 4 and 5 were found to be highly crystalline and such structures were transformed to amorphous nature as the water fraction was increased to 99%. To evaluate the potential of the conjugate as bio-imaging agents, we have carried out their in vitro cytotoxicity and cellular uptake studies though MTT assay, flow cytometric and confocal laser scanning microscopic techniques. Thus nanoparticle of these conjugates which exhibited efficient emission, large stoke shift, good stability, biocompatibility and excellent cellular imaging properties can have potential applications for tracking cells as well as in cell-based therapies. In summary we have synthesized novel functional organic chromophores and have studied systematic investigation of self-assembly of these synthetic and biological building blocks under a variety of conditions. The investigation of interaction of water soluble NIR squaraine dyes with lysozyme indicates that these dyes can act as the protein labeling agents and the efficiency of inhibition of β-amyloid indicate, thereby their potential as anti-amyloid agents.

Plant pigments: the many faces of light perception

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Leaf senescence in tomato mutants as affected by irradiance and phytohormones

We explored the interaction between radiation of different wavelength and jasmonic acid (JA) or brassinosteroids (BR) on leaf senescence-induced oxidative stress. Three approaches were used: 1) jasmonic acid insensitive1-1 (jai1-1) and brassinosteroid-deficient [dumpy (dpy)] mutants were treated with red (R) or far-red (FR) radiation; 2) phytochromedeficient aurea (au) and high pigment-1 (hp-1) (radiation exaggerated response) mutants were treated with methyl jasmonate (MeJA) or epibrassinolide (epiBL); and 3) double mutants au jai1-1 and au dpy were produced. Leaf chlorophyll content, lipid peroxidation, and antioxidant enzyme activities were determined. After senescence induction in detached leaves, we verified that the patterns of chlorophyll degradation of hormonal and photomorphogenic mutants were not significantly different in comparison with original cv. Micro-Tom (MT). Moreover, there was no significant change in lipid peroxidation measured as malondialdehyde (MDA) production, as well as catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) activities in the hormonal mutants. Exogenous BR increased CAT and APX activities in MT, au, and hp-1. As concerns the double mutants, severe reduction in H2O2 production which was not accompanied by changes in MDA content, and CAT and APX activities was observed during senescence in au dpy. The results suggest that JA and BR do not participate in light signaling pathway during leaf senescence-induced oxidative stress. © 2013 Springer Science+Business Media Dordrecht.

Photosynthate partitioning and morphoanatomical aspects of photomorphogenic mutants of tomato

The aim of this study was to analyze photosynthate partitioning in tomato photomorphogenic mutants at the ends of the vegetative (40 days after emergence [DAE]) and reproductive (69 DAE) stages and to determine its interaction with morphoanatomical aspects. The mutants aurea (au), phytochrome-deficient, high pigment-1 (hp1), light-exaggerated response, were studied along with the non-mutant Micro-Tom (MT) cultivar. The plants were analyzed at 40 and 68 DAE to identify photosynthate source organs and tissues as well as the target organs of remobilized photosynthate during the reproductive stage. The plants were evaluated for their internal and external morphology as well as the percentage of dry mass of their organs. Photosynthate allocation in the hp1 mutant occurred primarily in the roots and leaves, and allocation in the au mutant occurred primarily in fruits. The au mutant showed a high capacity for photosynthate remobilization to fruit during the reproductive stage, and the predominant sources of these remobilized photosynthates were the leaf spongy parenchyma, the root vascular cylinder and the marrow stem.

Development and Light Response of Leaves of Metasequoia and Close Relatives

Metasequoia glyptostroboides is a useful nearest living relative (NLR) of the Eocene fossil Metasequoia. Research on modern Metasequoia might give us some clues about its fossil counterpart. During this study the leaf anatomy of Metasequoia, Glyptostrobus, Sequoia and Taxodium was investigated with light microscopy and transmission electron microscopy. Metasequoia exhibits several characteristics of typical sciaphilic plants, such as slightly arched outer cell walls in the adaxial epidermal cells, strongly arched outer cell walls in the abaxial epidermal cells, mesophyll composed of spongy cells, chloroplasts with well-developed grana not only in mesophyll cells but in both the adaxial and abaxial epidermis. Based on comparison of leaf morphology and anatomy, we conclude that Metasequoia is best adapted to low light intensities, Sequoia and Taxodium are intermediate, and Glyptostrobus is adapted to higher light intensities. The effects of light intensity on mesophyll plastids of Metasequoia leaves were studied with trees grown under different light intensities. Metasequoia had the ability to synthesize chlorophyll under complete darkness and was stressed under high light. These characteristics would provide adaptive advantages for Metasequoia to adapt to low intensity, low angle, polar light at their Eocene high latitude paleoenvironments, particularly during the polar spring when light levels are exceedingly low. It provides evidence to explain why Metasequoia was the dominant tree species in Eocene high latitudes. The thesis is written as an article to be submitted to the American Journal of Botany.

A phytochrome from the fern Adiantum with features of the putative photoreceptor NPH1

In plant photomorphogenesis, it is well accepted that the perception of red/far-red and blue light is mediated by distinct photoreceptor families, i.e., the phytochromes and blue-light photoreceptors, respectively. Here we describe the discovery of a photoreceptor gene from the fern Adiantum that encodes a protein with features of both phytochrome and NPH1, the putative blue-light receptor for second-positive phototropism in seed plants. The fusion of a functional photosensory domain of phytochrome with a nearly full-length NPH1 homolog suggests that this polypeptide could mediate both red/far-red and blue-light responses in Adiantum normally ascribed to distinct photoreceptors.