CONSTANS, a HUB for all seasons: How photoperiod pervades plant physiology regulatory circuits.

How does a plant detect the changing seasons and make important developmental decisions accordingly? How do they incorporate daylength information into their routine physiological processes? Photoperiodism, or the capacity to measure the daylength, is a crucial aspect of plant development that helps plants determine the best time of the year to make vital decisions, such as flowering. The protein CONSTANS (CO) constitutes the central regulator of this sensing mechanism, not only activating florigen production in the leaves but also participating in many physiological aspects in which seasonality is important. Recent discoveries place CO in the center of a gene network that can determine the length of the day and confer seasonal input to aspects of plant development and physiology as important as senescence, seed size, or circadian rhythms. In this review, we discuss the importance of CO protein structure, function, and evolutionary mechanisms that embryophytes have developed to incorporate annual information into their physiology.

Peptidomics Methods Applied to the Study of Flower Development.

Understanding the global and dynamic nature of plant developmental processes requires not only the study of the transcriptome, but also of the proteome, including its largely uncharacterized peptidome fraction. Recent advances in proteomics and high-throughput analyses of translating RNAs (ribosome profiling) have begun to address this issue, evidencing the existence of novel, uncharacterized, and possibly functional peptides. To validate the accumulation in tissues of sORF-encoded polypeptides (SEPs), the basic setup of proteomic analyses (i.e., LC-MS/MS) can be followed. However, the detection of peptides that are small (up to ~100 aa, 6-7 kDa) and novel (i.e., not annotated in reference databases) presents specific challenges that need to be addressed both experimentally and with computational biology resources. Several methods have been developed in recent years to isolate and identify peptides from plant tissues. In this chapter, we outline two different peptide extraction protocols and the subsequent peptide identification by mass spectrometry using the database search or the de novo identification methods.

S-acylated and nucleus-localized SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 stabilizes GIGANTEA to regulate Arabidopsis flowering time under salt stress.

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering.

Regulation of floral senescence in Arabidopsis by coordinated action of CONSTANS and jasmonate signaling.

In Arabidopsis, photoperiodic flowering is controlled by the regulatory hub gene CONSTANS (CO), whereas floral organ senescence is regulated by the jasmonates (JAs). Because these processes are chronologically ordered, it remains unknown whether there are common regulators of both processes. In this study, we discovered that CO protein accumulates in Arabidopsis flowers after floral induction, and it displays a diurnal pattern in floral organs different from that in the leaves. We observed that altered CO expression could affect flower senescence and abscission by interfering with JA response, as shown by petal-specific transcriptomic analysis as well as CO overexpression in JA synthesis and signaling mutants. We found that CO has a ZIM (ZINC-FINGER INFLORESCENCE MERISTEM) like domain that mediates its interaction with the JA response repressor JAZ3 (jasmonate ZIM-domain 3). Their interaction inhibits the repressor activity of JAZ3, resulting in activation of downstream transcription factors involved in promoting flower senescence. Furthermore, we showed that CO, JAZ3, and the E3 ubiquitin ligase COI1 (Coronatine Insensitive 1) could form a protein complex in planta, which promotes the degradation of both CO and JAZ3 in the presence of JAs. Taken together, our results indicate that CO, a key regulator of photoperiodic flowering, is also involved in promoting flower senescence and abscission by augmenting JA signaling and response. We propose that coordinated recruitment of photoperiodic and JA signaling pathways could be an efficient way for plants to chronologically order floral processes and ensure the success of offspring production.

Photoperiodic Signaling and Senescence, an Ancient Solution to a Modern Problem?

The length of the day (photoperiod) is a robust seasonal signal originated by earth orbital and translational movements, a resilient external cue to the global climate change, and a predictable hint to initiate or complete different developmental programs. In eukaryotic algae, the gene expression network that controls the cellular response to photoperiod also regulates other basic physiological functions such as starch synthesis or redox homeostasis. Land plants, evolving in a novel and demanding environment, imbued these external signals within the regulatory networks controlling organogenesis and developmental programs. Unlike algae that largely have to deal with cellular physical cues, within the course of evolution land plants had to transfer this external information from the receiving organs to the target tissues, and mobile signals such as hormones were recruited and incorporated in the regulomes. Control of senescence by photoperiod, as suggested in this perspective, would be an accurate way to feed seasonal information into a newly developed function (senescence) using an ancient route (photoperiodic signaling). This way, the plant would assure that two coordinated aspects of development such as flowering and organ senescence were sequentially controlled. As in the case of senescence, there is growing evidence to support the idea that harnessing the reliability of photoperiod regulation over other, more labile signaling pathways could be used as a robust breeding tool to enhance plants against the harmful effects of climate change.

CONSTANS-FKBP12 interaction contributes to modulation of photoperiodic flowering in Arabidopsis.

Flowering time is a key process in plant development. Photoperiodic signals play a crucial role in the floral transition in Arabidopsis thaliana, and the protein CONSTANS (CO) has a central regulatory function that is tightly regulated at the transcriptional and post-translational levels. The stability of CO protein depends on a light-driven proteasome process that optimizes its accumulation in the evening to promote the production of the florigen FLOWERING LOCUS T (FT) and induce seasonal flowering. To further investigate the post-translational regulation of CO protein we have dissected its interactome network employing in vivo and in vitro assays and molecular genetics approaches. The immunophilin FKBP12 has been identified in Arabidopsis as a CO interactor that regulates its accumulation and activity. FKBP12 and CO interact through the CCT domain, affecting the stability and function of CO. fkbp12 insertion mutants show a delay in flowering time, while FKBP12 overexpression accelerates flowering, and these phenotypes can be directly related to a change in accumulation of FT protein. The interaction is conserved between the Chlamydomonas algal orthologs CrCO-CrFKBP12, revealing an ancient regulatory step in photoperiod regulation of plant development.

Ubiquitin carboxyl-terminal hydrolases are required for period maintenance of the circadian clock at high temperature in Arabidopsis.

Protein ubiquitylation participates in a number of essential cellular processes including signal transduction and transcription, often by initiating the degradation of specific substrates through the 26S proteasome. Within the ubiquitin-proteasome system, deubiquitylating enzymes (DUBs) not only help generate and maintain the supply of free ubiquitin monomers, they also directly control functions and activities of specific target proteins by modulating the pool of ubiquitylated species. Ubiquitin carboxyl-terminal hydrolases (UCHs) belong to an enzymatic subclass of DUBs, and are represented by three members in Arabidopsis, UCH1, UCH2 and UCH3. UCH1 and UCH2 influence auxin-dependent developmental pathways in Arabidopsis through their deubiquitylation activities, whereas biological and enzymatic functions of UCH3 remain unclear. Here, we demonstrate that Arabidopsis UCH3 acts to maintain the period of the circadian clock at high temperatures redundantly with UCH1 and UCH2. Whereas single uch1, uch2 and uch3 mutants have weak circadian phenotypes, the triple uch mutant displays a drastic lengthening of period at high temperatures that is more extreme than the uch1 uch2 double mutant. UCH3 also possesses a broad deubiquitylation activity against a range of substrates that link ubiquitin via peptide and isopeptide linkages. While the protein target(s) of UCH1-3 are not yet known, we propose that these DUBs act on one or more factors that control period length of the circadian clock through removal of their bound ubiquitin moieties, thus ensuring that the clock oscillates with a proper period even at elevated temperatures.

Evolution of Daily Gene Co-expression Patterns from Algae to Plants.

Daily rhythms play a key role in transcriptome regulation in plants and microalgae orchestrating responses that, among other processes, anticipate light transitions that are essential for their metabolism and development. The recent accumulation of genome-wide transcriptomic data generated under alternating light:dark periods from plants and microalgae has made possible integrative and comparative analysis that could contribute to shed light on the evolution of daily rhythms in the green lineage. In this work, RNA-seq and microarray data generated over 24 h periods in different light regimes from the eudicot Arabidopsis thaliana and the microalgae Chlamydomonas reinhardtii and Ostreococcus tauri have been integrated and analyzed using gene co-expression networks. This analysis revealed a reduction in the size of the daily rhythmic transcriptome from around 90% in Ostreococcus, being heavily influenced by light transitions, to around 40% in Arabidopsis, where a certain independence from light transitions can be observed. A novel Multiple Bidirectional Best Hit (MBBH) algorithm was applied to associate single genes with a family of potential orthologues from evolutionary distant species. Gene duplication, amplification and divergence of rhythmic expression profiles seems to have played a central role in the evolution of gene families in the green lineage such as Pseudo Response Regulators (PRRs), CONSTANS-Likes (COLs), and DNA-binding with One Finger (DOFs). Gene clustering and functional enrichment have been used to identify groups of genes with similar rhythmic gene expression patterns. The comparison of gene clusters between species based on potential orthologous relationships has unveiled a low to moderate level of conservation of daily rhythmic expression patterns. However, a strikingly high conservation was found for the gene clusters exhibiting their highest and/or lowest expression value during the light transitions.

Evolutionary Analysis of DELLA-Associated Transcriptional Networks.

DELLA proteins are transcriptional regulators present in all land plants which have been shown to modulate the activity of over 100 transcription factors in Arabidopsis, involved in multiple physiological and developmental processes. It has been proposed that DELLAs transduce environmental information to pre-wired transcriptional circuits because their stability is regulated by gibberellins (GAs), whose homeostasis largely depends on environmental signals. The ability of GAs to promote DELLA degradation coincides with the origin of vascular plants, but the presence of DELLAs in other land plants poses at least two questions: what regulatory properties have DELLAs provided to the behavior of transcriptional networks in land plants, and how has the recruitment of DELLAs by GA signaling affected this regulation. To address these issues, we have constructed gene co-expression networks of four different organisms within the green lineage with different properties regarding DELLAs: Arabidopsis thaliana and Solanum lycopersicum (both with GA-regulated DELLA proteins), Physcomitrella patens (with GA-independent DELLA proteins) and Chlamydomonas reinhardtii (a green alga without DELLA), and we have examined the relative evolution of the subnetworks containing the potential DELLA-dependent transcriptomes. Network analysis indicates a relative increase in parameters associated with the degree of interconnectivity in the DELLA-associated subnetworks of land plants, with a stronger effect in species with GA-regulated DELLA proteins. These results suggest that DELLAs may have played a role in the coordination of multiple transcriptional programs along evolution, and the function of DELLAs as regulatory 'hubs' became further consolidated after their recruitment by GA signaling in higher plants.

Evolution of photoperiod sensing in plants and algae.

Measuring day length confers a strong fitness improvement to photosynthetic organisms as it allows them to anticipate light phases and take the best decisions preceding diurnal transitions. In close association with signals from the circadian clock and the photoreceptors, photoperiodic sensing constitutes also a precise way to determine the passing of the seasons and to take annual decisions such as the best time to flower or the beginning of dormancy. Photoperiodic sensing in photosynthetic organisms is ancient and two major stages in its evolution could be identified, the cyanobacterial time sensing and the evolutionary tool kit that arose in green algae and developed into the photoperiodic system of modern plants. The most recent discoveries about the evolution of the perception of light, measurement of day length and relationship with the circadian clock along the evolution of the eukaryotic green lineage will be discussed in this review.

Characterization of the Sucrose Phosphate Phosphatase (SPP) Isoforms from Arabidopsis thaliana and Role of the S6PPc Domain in Dimerization.

Sucrose-phosphate phosphatase (SPP) catalyses the final step in the sucrose biosynthesis pathway. Arabidopsis thaliana genome codifies four SPP isoforms. In this study, the four Arabidopsis thaliana genes coding for SPP isoforms have been cloned, expressed in Escherichia coli and the kinetic and regulatory properties of the purified enzymes analysed. SPP2 is the isoform showing the highest activity, with SPP3b and SPP3a showing lower activity levels. No activity was detected for SPP1. We propose that this lack of activity is probably due to the absence of an essential amino acid participating in catalysis and/or in the binding of the substrate, sucrose-6-phosphate (Suc6P). The expression patterns of Arabidopsis SPP genes indicate that SPP2 and SPP3b are the main isoforms expressed in different tissues and organs, although the non-catalytic SPP1 is the main isoform expressed in roots. Thus, SPP1 could have acquired new unknown functions. We also show that the three catalytically active SPPs from Arabidopsis are dimers. By generating a chimeric SPP composed of the monomeric cyanobacterial SPP fused to the higher plant non-catalytic S6PPc domain (from SPP2), we show that the S6PPc domain is responsible for SPP dimerization. This is the first experimental study on the functionality and gene expression pattern of all the SPPs from a single plant species.

New challenges in microalgae biotechnology.

Photosynthetic protists, also called microalgae, have been systematically studied for more than a century. However, only recently broad biotechnological applications have fostered a novel wave of research on their potentialities as sustainable resources of renewable energy as well as valuable industrial and agro-food products. At the recent VII European Congress of Protistology held in Seville, three outstanding examples of different research strategies on microalgae with biotechnological implications were presented, which suggested that integrative approaches will produce very significant advances in this field in the next future. In any case, intense research and the application of systems biology and genetic engineering techniques are absolutely essential to reach the full potential of microalgae as cell-factories of bio-based products and, therefore, could contribute significantly to solve the problems of biosustainability and energy shortage.

ChlamyNET: a Chlamydomonas gene co-expression network reveals global properties of the transcriptome and the early setup of key co-expression patterns in the green lineage.

BACKGROUND: Chlamydomonas reinhardtii is the model organism that serves as a reference for studies in algal genomics and physiology. It is of special interest in the study of the evolution of regulatory pathways from algae to higher plants. Additionally, it has recently gained attention as a potential source for bio-fuel and bio-hydrogen production. The genome of Chlamydomonas is available, facilitating the analysis of its transcriptome by RNA-seq data. This has produced a massive amount of data that remains fragmented making necessary the application of integrative approaches based on molecular systems biology. RESULTS: We constructed a gene co-expression network based on RNA-seq data and developed a web-based tool, ChlamyNET, for the exploration of the Chlamydomonas transcriptome. ChlamyNET exhibits a scale-free and small world topology. Applying clustering techniques, we identified nine gene clusters that capture the structure of the transcriptome under the analyzed conditions. One of the most central clusters was shown to be involved in carbon/nitrogen metabolism and signalling, whereas one of the most peripheral clusters was involved in DNA replication and cell cycle regulation. The transcription factors and regulators in the Chlamydomonas genome have been identified in ChlamyNET. The biological processes potentially regulated by them as well as their putative transcription factor binding sites were determined. The putative light regulated transcription factors and regulators in the Chlamydomonas genome were analyzed in order to provide a case study on the use of ChlamyNET. Finally, we used an independent data set to cross-validate the predictive power of ChlamyNET. CONCLUSIONS: The topological properties of ChlamyNET suggest that the Chlamydomonas transcriptome posseses important characteristics related to error tolerance, vulnerability and information propagation. The central part of ChlamyNET constitutes the core of the transcriptome where most authoritative hub genes are located interconnecting key biological processes such as light response with carbon and nitrogen metabolism. Our study reveals that key elements in the regulation of carbon and nitrogen metabolism, light response and cell cycle identified in higher plants were already established in Chlamydomonas. These conserved elements are not only limited to transcription factors, regulators and their targets, but also include the cis-regulatory elements recognized by them.

Vacuolar H(+)-Pyrophosphatase AVP1 is Involved in Amine Fungicide Tolerance in Arabidopsis thaliana and Provides Tridemorph Resistance in Yeast.

Amine fungicides are widely used as crop protectants. Their success is believed to be related to their ability to inhibit postlanosterol sterol biosynthesis in fungi, in particular sterol-Δ(8),Δ(7)-isomerases and sterol-Δ(14)-reductases, with a concomitant accumulation of toxic abnormal sterols. However, their actual cellular effects and mechanisms of death induction are still poorly understood. Paradoxically, plants exhibit a natural resistance to amine fungicides although they have similar enzymes in postcicloartenol sterol biosynthesis that are also susceptible to fungicide inhibition. A major difference in vacuolar ion homeostasis between plants and fungi is the presence of a dual set of primary proton pumps in the former (V-ATPase and H(+)-pyrophosphatase), but only the V-ATPase in the latter. Abnormal sterols affect the proton-pumping capacity of V-ATPases in fungi and this has been proposed as a major determinant in fungicide action. Using Saccharomyces cerevisiae as a model fungus, we provide evidence that amine fungicide treatment induced cell death by apoptosis. Cell death was concomitant with impaired H(+)-pumping capacity in vacuole vesicles and dependent on vacuolar proteases. Also, the heterologous expression of the Arabidopsis thaliana main H(+)-pyrophosphatase (AVP1) at the fungal vacuolar membrane reduced apoptosis levels in yeast and increased resistance to amine fungicides. Consistently, A. thaliana avp1 mutant seedlings showed increased susceptibility to this amine fungicide, particularly at the level of root development. This is in agreement with AVP1 being nearly the sole H(+)-pyrophosphatase gene expressed at the root elongation zones. All in all, the present data suggest that H(+)-pyrophosphatases are major determinants of plant tolerance to amine fungicides.

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.

Photoperiodic control of sugar release during the floral transition: What is the role of sugars in the florigenic signal?

Florigen is a mobile signal released by the leaves that reaching the shoot apical meristem (SAM), changes its developmental program from vegetative to reproductive. The protein FLOWERING LOCUS T (FT) constitutes an important element of the florigen, but other components such as sugars, have been also proposed to be part of this signal. (1-5) We have studied the accumulation and composition of starch during the floral transition in Arabidopsis thaliana in order to understand the role of carbon mobilization in this process. In A. thaliana and Antirrhinum majus the gene coding for the Granule-Bound Starch Synthase (GBSS) is regulated by the circadian clock (6,7) while in the green alga Chlamydomonas reinhardtii the homolog gene CrGBSS is controlled by photoperiod and circadian signals. (8,9) In a recent paper(10) we described the role of the central photoperiodic factor CONSTANS (CO) in the regulation of GBSS expression in Arabidopsis. This regulation is in the basis of the change in the balance between starch and free sugars observed during the floral transition. We propose that this regulation may contribute to the florigenic signal and to the increase in sugar transport required during the flowering process.

An Evolutionarily Conserved DOF-CONSTANS Module Controls Plant Photoperiodic Signaling.

The response to daylength is a crucial process that evolved very early in plant evolution, entitling the early green eukaryote to predict seasonal variability and attune its physiological responses to the environment. The photoperiod responses evolved into the complex signaling pathways that govern the angiosperm floral transition today. The Chlamydomonas reinhardtii DNA-Binding with One Finger (CrDOF) gene controls transcription in a photoperiod-dependent manner, and its misexpression influences algal growth and viability. In short days, CrDOF enhances CrCO expression, a homolog of plant CONSTANS (CO), by direct binding to its promoter, while it reduces the expression of cell division genes in long days independently of CrCO. In Arabidopsis (Arabidopsis thaliana), transgenic plants overexpressing CrDOF show floral delay and reduced expression of the photoperiodic genes CO and FLOWERING LOCUS T. The conservation of the DOF-CO module during plant evolution could be an important clue to understanding diversification by the inheritance of conserved gene toolkits in key developmental programs.