Molecular mechanisms of Evening Complex activity in Arabidopsis.

The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX-ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity.

Variations in Circadian Clock Organization & Function: A Journey from Ancient to Recent.

MAIN CONCLUSION: Circadian clock components exhibit structural variations in different plant systems, and functional variations during various abiotic stresses. These variations bear relevance for plant fitness and could be important evolutionarily. All organisms on earth have the innate ability to measure time as diurnal rhythms that occur due to the earth's rotations in a 24-h cycle. Circadian oscillations arising from the circadian clock abide by its fundamental properties of periodicity, entrainment, temperature compensation, and oscillator mechanism, which is central to its function. Despite the fact that a myriad of research in Arabidopsis thaliana illuminated many detailed aspects of the circadian clock, many more variations in clock components' organizations and functions remain to get deciphered. These variations are crucial for sustainability and adaptation in different plant systems in the varied environmental conditions in which they grow. Together with these variations, circadian clock functions differ drastically even during various abiotic and biotic stress conditions. The present review discusses variations in the organization of clock components and their role in different plant systems and abiotic stresses. We briefly introduce the clock components, entrainment, and rhythmicity, followed by the variants of the circadian clock in different plant types, starting from lower non-flowering plants, marine plants, dicots to the monocot crop plants. Furthermore, we discuss the interaction of the circadian clock with components of various abiotic stress pathways, such as temperature, light, water stress, salinity, and nutrient deficiency with implications for the reprogramming during these stresses. We also update on recent advances in clock regulations due to post-transcriptional, post-translation, non-coding, and micro-RNAs. Finally, we end this review by summarizing the points of applicability, a remark on the future perspectives, and the experiments that could clear major enigmas in this area of research.

Uncovering the molecular signature underlying the light intensity-dependent root development in Arabidopsis thaliana.

BACKGROUND: Root morphology is known to be affected by light quality, quantity and direction. Light signal is perceived at the shoot, translocated to roots through vasculature and further modulates the root development. Photoreceptors are differentially expressed in both shoot and root cells. The light irradiation to the root affects shoot morphology as well as whole plant development. The current work aims to understand the white light intensity dependent changes in root patterning and correlate that with the global gene expression profile. RESULTS: Different fluence of white light (WL) regulate overall root development via modulating the expression of a specific set of genes. Phytochrome A deficient Arabidopsis thaliana (phyA-211) showed shorter primary root compared to phytochrome B deficient (phyB-9) and wild type (WT) seedlings at a lower light intensity. However, at higher intensity, both mutants showed shorter primary root in comparison to WT. The lateral root number was observed to be lowest in phyA-211 at intensities of 38 and 75 µmol m - 2 s - 1. The number of adventitious roots was significantly lower in phyA-211 as compared to WT and phyB-9 under all light intensities tested. With the root phenotypic data, microarray was performed for four different intensities of WL light in WT. Here, we identified ~ 5243 differentially expressed genes (DEGs) under all light intensities. Gene ontology-based analysis indicated that different intensities of WL predominantly affect a subset of genes having catalytic activity and localized to the cytoplasm and membrane. Furthermore, when root is irradiated with different intensities of WL, several key genes involved in hormone, light signaling and clock-regulated pathways are differentially expressed. CONCLUSION: Using genome wide microarray-based approach, we have identified candidate genes in Arabidopsis root that responded to the changes in light intensities. Alteration in expression of genes such as PIF4, COL9, EPR1, CIP1, ARF18, ARR6, SAUR9, TOC1 etc. which are involved in light, hormone and clock pathway was validated by qRT-PCR. This indicates their potential role in light intensity mediated root development.

Carbon nanoparticles influence photomorphogenesis and flowering time in Arabidopsis thaliana.

KEY MESSAGE: Inclusion of carbon nanoparticles in growth medium accelerates timing to flower by down-regulating phytochrome B in a CONSTANS-independent but photoperiod-dependent manner in Arabidopsis thaliana. Despite the recognized importance of nanoparticles in plant development over the last decade, the effect of carbon nanoparticles (CNPs) on plant processes such as photomorphogenesis and flowering time control is poorly understood. We explored the uptake, accumulation and effect of CNPs on seedling development and flowering time control in Arabidopsis thaliana (At). CNPs uptake was demonstrated using Raman spectroscopy and light microscopy that affected At seedling growth and flowering time in a dose-dependent manner. The highest accumulation of CNPs was observed in leaves followed by stem and root tissues. CNPs treatment enhanced seed germination, showed elongated hypocotyl, larger cotyledon area and increased chlorophyll content in At seedlings. CNPs treatment induced early flowering in both long-day and short-day growth conditions indicating a photoperiod-dependent effect. CNPs-treated seedlings showed a drastic reduction in the relative abundance of phytochrome B (PHYB) transcript. Further, we analyzed the transcript abundance of at least one major component involved in various pathways that regulate flowering such as (1) photoperiod, (2) gibberellic acid (GA), (3) vernalization and (4) autonomous. An up-regulation of transcript levels of PHYTOCHROME INTERACTING FACTOR 4 (PIF4), GIGANTEA (GI), REPRESSOR OF GIBBERELLIC ACID 1 (RGA1) and LEAFY (LFY) were observed, however, there were no changes in the transcript levels of CONSTANS (CO), VERNALIZATION 1 (VRN1) and FLOWERING CONTROL LOCUS A (FCA). Despite the up-regulation of RGA1, we conclude that the earlier flowering is most likely GA-independent. Here, we demonstrated that the early flowering in CNPs-treated seedlings was PHYB and photoperiod-dependent.

Phosphorylation of CONSTANS and its COP1-dependent degradation during photoperiodic flowering of Arabidopsis.

Seasonal flowering involves responses to changes in day length. In Arabidopsis thaliana, the CONSTANS (CO) transcription factor promotes flowering in the long days of spring and summer. Late flowering in short days is due to instability of CO, which is efficiently ubiquitinated in the dark by the CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase complex. Here we show that CO is also phosphorylated. Phosphorylated and unphosphorylated forms are detected throughout the diurnal cycle but their ratio varies, with the relative abundance of the phosphorylated form being higher in the light and lower in the dark. These changes in relative abundance require COP1, because in the cop1 mutant the phosphorylated form is always more abundant. Inactivation of the PHYTOCHROME A (PHYA), CRYPTOCHROME 1 (CRY1) and CRYPTOCHROME 2 (CRY2) photoreceptors in the phyA cry1 cry2 triple mutant most strongly reduces the amount of the phosphorylated form so that unphosphorylated CO is more abundant. This effect is caused by increased COP1 activity, as it is overcome by introduction of the cop1 mutation in the cop1 phyA cry1 cry2 quadruple mutant. Degradation of CO is also triggered in red light, and as in darkness this increases the relative abundance of unphosphorylated CO. Finally, a fusion protein containing truncated CO protein including only the carboxy-terminal region was phosphorylated in transgenic plants, locating at least one site of phosphorylation in this region. We propose that CO phosphorylation contributes to the photoperiodic flowering response by enhancing the rate of CO turnover via activity of the COP1 ubiquitin ligase.

Balanced activity of microRNA166/165 and its target transcripts from the class III homeodomain-leucine zipper family regulates root growth in Arabidopsis thaliana.

Overexpression of miR166/165 down-regulates target HD - ZIP IIIs and promotes root growth by enhancing cell division and meristematic activity, whereas overexpression of HD - ZIP IIIs inhibits root growth in Arabidopsis thaliana. Post-embryonic growth of higher plants is maintained by active meristems harbouring undifferentiated cells. Shoot and root apical meristems (SAM and RAM) utilize both similar and distinct signalling mechanisms for their maintenance in Arabidopsis thaliana. An important regulatory role in this context has the interaction of microRNAs with their target mRNAs, mostly encoding transcription factors. One class of microRNA166/165 (miR166/165) has been implicated in the maintenance of SAM and vascular patterning. Here, we show that miR166/165 plays an important role in root growth also by negatively regulating its target transcripts, HD-ZIP IIIs, in the RAM. While overexpression of miR166 promotes RAM activity, overexpression of its targets reduces RAM activity. These results reveal a conserved role of miR166/165 in the maintenance of SAM and RAM activity in A. thaliana.

GIGANTEA supresses wilt disease resistance by down-regulating the jasmonate signaling in Arabidopsis thaliana.

GIGANTEA (GI) is a plant-specific nuclear protein that plays a pleiotropic role in the growth and development of plants. GI's involvement in circadian clock function, flowering time regulation, and various types of abiotic stress tolerance has been well documented in recent years. Here, the role of GI in response to Fusarium oxysporum (F. oxysporum) infection is investigated at the molecular level comparing Col-0 WT with the gi-100 mutant in Arabidopsis thaliana. Disease progression, photosynthetic parameters, and comparative anatomy confirmed that the spread and damage caused by pathogen infection were less severe in gi-100 than in Col-0 WT plants. F. oxysporum infection induces a remarkable accumulation of GI protein. Our report showed that it is not involved in flowering time regulation during F. oxysporum infection. Estimation of defense hormone after infection showed that jasmonic acid (JA) level is higher and salicylic acid (SA) level is lower in gi-100 compared to Col-0 WT. Here, we show that the relative transcript expression of CORONATINE INSENSITIVE1 (COI1) and PLANT DEFENSIN1.2 (PDF1.2) as a marker of the JA pathway is significantly higher while ISOCHORISMATE SYNTHASE1 (ICS1) and NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), the markers of the SA pathway, are downregulated in the gi-100 mutants compared to Col-0 plants. The present study convincingly suggests that the GI module promotes susceptibility to F. oxysporum infection by inducing the SA pathway and inhibiting JA signaling in A. thaliana.

Arabidopsis COP1 shapes the temporal pattern of CO accumulation conferring a photoperiodic flowering response.

The transcriptional regulator CONSTANS (CO) promotes flowering of Arabidopsis under long summer days (LDs) but not under short winter days (SDs). Post-translational regulation of CO is crucial for this response by stabilizing the protein at the end of a LD, whereas promoting its degradation throughout the night under LD and SD. We show that mutations in CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a component of a ubiquitin ligase, cause extreme early flowering under SDs, and that this is largely dependent on CO activity. Furthermore, transcription of the CO target gene FT is increased in cop1 mutants and decreased in plants overexpressing COP1 in phloem companion cells. COP1 and CO interact in vivo and in vitro through the C-terminal region of CO. COP1 promotes CO degradation mainly in the dark, so that in cop1 mutants CO protein but not CO mRNA abundance is dramatically increased during the night. However, in the morning CO degradation occurs independently of COP1 by a phytochrome B-dependent mechanism. Thus, COP1 contributes to day length perception by reducing the abundance of CO during the night and thereby delaying flowering under SDs.

Auxin-binding proteins without KDEL sequence in the moss Funaria hygrometrica.

Whereas the important plant growth regulator auxin has multiple effects in flowering plants, it induces a specific cell differentiation step in the filamentous moss protonema. Here, we analyse the presence of classical auxin-binding protein (ABP1) homologues in the moss Funaria hygrometrica. Microsomal membranes isolated from protonemata of F. hygrometrica have specific indole acetic acid-binding sites, estimated to be about 3-5 pmol/mg protein with an apparent dissociation constant (K (d)) between 3 and 5 microM. Western analyses with anti-ABP1 antiserum detected the canonical endoplasmic reticulum (ER)-localised 22-24 kDa ABP1 in Zea mays, but not in F. hygrometrica. Instead, polypeptides of 31-33 and 46 kDa were labelled in the moss as well as in maize. In F. hygrometrica these proteins were found exclusively in microsomal membrane fractions and were confirmed as ABPs by photo-affinity labelling with 5-azido-[7-(3)H]-indole-3-acetic acid. Unlike the classical corn ABP1, these moss ABPs did not contain the KDEL ER retention sequence. Consistently, the fully sequenced genome of the moss Physcomitrella patens, a close relative of F. hygrometrica, encodes an ABP1-homologue without KDEL sequence. Our study suggests the presence of putative ABPs in F. hygrometrica that share immunological epitopes with ABP1 and bind auxin but are different from the classical corn ABP1.

Light and auxin signaling cross-talk programme root development in plants.

Root development in plants is affected by light and phytohormones. Different ranges of light wavelength influence root patterning in a particular manner. Red and white light promote overall root development, whereas blue light has both positive as well as negative role in these processes. Light-mediated root development primarily occurs through modulation of synthesis, signaling and transport of the phytohormone auxin. Auxin has been shown to play a critical role in root development. It is being well-understood that components of light and auxin signaling cross-talk with each other. However, the signaling network that can modulate the root development is an intense area of research. Currently, limited information is available about the interaction of these two signaling pathways. This review not only summarizes the current findings on how different quality and quantity of light affect various aspects of root development but also present the role of auxin in these developmental aspects starting from lower to higher plants.

Shade tolerance in Swarnaprabha rice is associated with higher rate of panicle emergence and positively regulated by genes of ethylene and cytokinin pathway.

This study identifies characteristics of seedling, mature plant phenotypes, changes at genetic and genomic level associated with Swarnaprabha (SP) rice grown under prolonged shade and compared with Nagina 22 (N22). Coleoptile length under low red/far-red was intermediate between that in dark and red light in a 7-days growth frame. Whereas, highest rootlet number was discriminating in seedlings grown for 28 days in hydroponics. In shade, SP and N22 both showed several tolerant mature plant phenotypes, except the panicle length, yield per plant and % grain filling, which were higher in SP. Percentage decrease in yield / plant in shade showed significant positive correlation with increase in NDVI, decrease in panicle length and % grain filling (p ≤ 0.01). Rate of panicle emergence in shade was higher in SP than N22. Expression patterns of PHYTOCHROME INTERACTING FACTOR LIKE-13 and PHYTOCHROME B were contrasting in SP and N22 seedlings under continuous red or red/far-red. Microarray analysis revealed the up-regulation of most of the ethylene and cytokinin pathway genes in shade grown panicles of SP. Significant up-regulation of ETHYLENE RESPONSE ELEMENT BINDING PROTEIN-2, MOTHER OF FLOWERING TIME 1, and SHORT PANICLE1 genes in shade grown panicles of SP could explain its sustainable higher yield in shade.

Aquasorbent guargum grafted hyperbranched poly (acrylic acid): A potential culture medium for microbes and plant tissues.

This study reports the synthesis of an unprecedented bio-based aquasorbent guargum-g-hyperbranched poly (acrylic acid); bGG-g-HBPAA by employing graft-copolymerization and "Strathclyde methodology" simultaneously in emulsion and its possible use as a sustainable nutrient bed for the effective growth of Anabaena cylindrica and Vigna radiata seedlings. The formation of bGG-g-HBPAA and the presence of hyperbranched architectures was confirmed from XRD, FTIR, 13C NMR, solubility, intrinsic viscosity, BET surface area/ pore size, SEM and rheology analyses. The synthesized grade with a branching percent of 65.4% and a swelling percentage of 13,300% facilitated maximum growth of the cultured species as compared to guargum and its linear graft. Semi synthetic bGG-g-HBPAA culture medium was optically transparent, dried at a controlled rate, held a huge amount of water for growth, provided sufficient space for unhindered growth and featured dimensional stability.

Carbohydrates and polyphenolics of extracts from genetically altered plant acts as catalysts for in vitro synthesis of silver nanoparticle.

Eco-friendly biosynthetic approach for silver nanoparticles production using plant extracts is an exciting advancement in bio-nanotechnology and has been successfully attempted in nearly 41 plant species. However, an established model plant system for systematically unraveling the biochemical components required for silver nanoparticles production is lacking. Here we used Arabidopsis thaliana as the model plant for silver nanoparticles biosynthesis in vitro. Employing biochemical, spectroscopic methods, selected mutants and over-expressor plants of Arabidopsis involved in pleotropic functions and sugar homeostasis, we show that carbohydrates, polyphenolics and glyco-proteins are essential components which stimulated silver nanoparticles synthesis. Using molecular genetics as a tool, our data enforces the requirement of sugar conjugated proteins as essentials for AgNPs synthesis over protein alone. Additionally, a comparative analysis of AgNPs synthesis using the aqueous extracts of some of the plant species found in a brackish water ecosystem (Gracilaria, Potamogeton, Enteromorpha and Scendesmus) were explored. Plant extract of Potamogeton showed the highest potential of nanoparticles production comparable to that of Arabidopsis among the species tested. Silver nanoparticles production in the model plant Arabidopsis not only opens up a possibility of using molecular genetics tool to understand the biochemical pathways and components in detail for its synthesis.

Environmental stress influencing plant development and flowering.

Plants are sessile organisms and unlike animals, cannot run away from adverse environmental conditions. Therefore, they have evolved sophisticated signaling and protective systems to overcome sub-lethal stress situations. Although, effect of stress on physiology and morphology were studied earlier, the research on molecular mechanisms of stress response is albeit new. Studies at the molecular level on stress physiology reveal that, many stress-induced pathways converge downstream or interact significantly. Abiotic stress factors regulate the extent and pattern of developmental programme. The timing of transition from vegetative to flowering phase, which is vital for survival and reproductive success, is often altered under various stresses. Unraveling the mechanisms by which different environmental stresses induce their effects and how tolerance to stress is achieved is an active area of research. Enhancing stress tolerance, especially in crop plants is an area of prime importance. In this review, we focus on stress responses induced by temperatures, high and low light intensities, UV radiation, drought and salinity stress and summarize the recent advancements by highlighting the underlying molecular pathways and processes.

Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are essential for a photoperiodic flowering response.

Flowering of Arabidopsis is induced by long summer days (LDs). The transcriptional regulator CONSTANS (CO) promotes flowering, and its transcription is increased under LDs. We systematically misexpressed transcription factors in companion cells and identified several DOF proteins that delay flowering by repressing CO transcription. Combining mutations in four of these, including CYCLING DOF FACTOR 2 (CDF2), caused photoperiod-insensitive early flowering by increasing CO mRNA levels. CO transcription is promoted to differing extents by GIGANTEA (GI) and the F-box protein FKF1. We show that GI stabilizes FKF1, thereby reducing CDF2 abundance and allowing transcription of CO. Despite the crucial function of GI in wild-type plants, introducing mutations in the four DOF-encoding genes into gi mutants restored the diurnal rhythm and light inducibility of CO. Thus, antagonism between GI and DOF transcription factors contributes to photoperiodic flowering by modulating an underlying diurnal rhythm in CO transcript levels.

Constitutive photomorphogenesis 1 and multiple photoreceptors control degradation of phytochrome interacting factor 3, a transcription factor required for light signaling in Arabidopsis.

Light, in a quality- and quantity-dependent fashion, induces nuclear import of the plant photoreceptors phytochrome, promotes interaction of phytochrome A (phyA) and phyB with transcription factors including phytochrome interacting factor 3 (PIF3), and is thought to trigger a transcriptional cascade to regulate the expression of approximately 2500 genes in Arabidopsis thaliana. Here, we show that controlled degradation of the transcription factor PIF3 is a major regulatory step in light signaling. We demonstrate that accumulation of PIF3 in the nucleus in dark requires constitutive photomorphogenesis 1 (COP1), a negative regulator of photomorphogenesis, and show that red (R) and far-red light (FR) induce rapid degradation of the PIF3 protein. This process is controlled by the concerted action of the R/FR absorbing phyA, phyB, and phyD photoreceptors, and it is not affected by COP1. Rapid light-induced degradation of PIF3 indicates that interaction of PIF3 with these phytochrome species is transient. In addition, we provide evidence that the poc1 mutant, a postulated PIF3 overexpressor that displays hypersensitivity to R but not to FR, lacks detectable amounts of the PIF3 protein. Thus, we propose that PIF3 acts transiently, and its major function is to mediate phytochrome-induced signaling during the developmental switch from skotomorphogenesis to photomorphogenesis and/or dark to light transitions.