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1.  Nitric oxide and phytohormone interactions: current status and perspectives 
Nitric oxide (NO) is currently considered a ubiquitous signal in plant systems, playing significant roles in a wide range of responses to environmental and endogenous cues. During the signaling events leading to these plant responses, NO frequently interacts with plant hormones and other endogenous molecules, at times originating remarkably complex signaling cascades. Accumulating evidence indicates that virtually all major classes of plant hormones may influence, at least to some degree, the endogenous levels of NO. In addition, studies conducted during the induction of diverse plant responses have demonstrated that NO may also affect biosynthesis, catabolism/conjugation, transport, perception, and/or transduction of different phytohormones, such as auxins, gibberellins, cytokinins, abscisic acid, ethylene, salicylic acid, jasmonates, and brassinosteroids. Although still not completely elucidated, the mechanisms underlying the interaction between NO and plant hormones have recently been investigated in a number of species and plant responses. This review specifically focuses on the current knowledge of the mechanisms implicated in NO–phytohormone interactions during the regulation of developmental and metabolic plant events. The modifications triggered by NO on the transcription of genes encoding biosynthetic/degradative enzymes as well as proteins involved in the transport and signal transduction of distinct plant hormones will be contextualized during the control of developmental, metabolic, and defense responses in plants. Moreover, the direct post-translational modification of phytohormone biosynthetic enzymes and receptors through S-nitrosylation will also be discussed as a key mechanism for regulating plant physiological responses. Finally, some future perspectives toward a more complete understanding of NO–phytohormone interactions will also be presented and discussed.
doi:10.3389/fpls.2013.00398
PMCID: PMC3793198  PMID: 24130567
nitric oxide; plant hormones; auxin; cytokinin; gibberellin; abscisic acid; ethylene; S-nitrosylation
2.  Modelling and experimental analysis of hormonal crosstalk in Arabidopsis 
An important question in plant biology is how genes influence the crosstalk between hormones to regulate growth. We have developed the first hormonal crosstalk network in Arabidopsis by iteratively combining modelling with experimental analysis.We have revealed that the POLARIS gene interacts with the ethylene receptor and regulates both auxin transport and biosynthesis.Our modelling analysis has reproduced all known mutants. With new experimental data, it has provided new insights into how the POLARIS gene regulates auxin concentration for root development in Arabidopsis, by controlling the relative contribution of auxin transport and biosynthesis and by integrating auxin, ethylene and cytokinin signalling.Modelling and experimental analysis have revealed that a bell-shaped dose–response relationship between endogenous auxin and root length is established via POLARIS.
Hormone signalling systems coordinate plant growth and development through a range of complex interactions. The activities of plant hormones, such as auxin, ethylene and cytokinin, depend on cellular context and exhibit interactions that can be either synergistic or antagonistic. An important question regarding the understanding of those interactions is how genes act on the crosstalk between hormones to regulate plant growth.
Previously, we identified the POLARIS (PLS) gene of Arabidopsis, which transcribes a short mRNA encoding a 36-amino acid peptide that is required for correct root growth and vascular development (Casson et al, 2002). Experimental evidence shows that there is a link between PLS, ethylene signalling, auxin homeostasis and microtubule cytoskeleton dynamics (Chilley et al, 2006). Specifically, mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered sensitivity to microtubule inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. The expression of PLS is itself repressed by ethylene and induced by auxin. It was also shown that pls mutant roots are hyper-responsive to exogenous cytokinins and show increased expression of the cytokinin inducible gene ARR5/IBC6 compared with the wild type (Casson et al, 2002). Therefore, PLS may also be required for correct auxin–cytokinin homeostasis to modulate root growth.
In this study, we model PLS gene function and crosstalk between auxin, ethylene and cytokinin in Arabidopsis.
Experimental evidence suggests that PLS acts on or close to the ethylene receptor ETR1, and a mathematical model describing possible PLS–ethylene pathway interactions is developed, and used to make quantitative predictions about PLS–hormone interactions. Modelling correctly predicts experimental results for the effect of the pls gene mutation on endogenous cytokinin concentration. Modelling also reveals a role for PLS in auxin biosynthesis in addition to a role in auxin transport (Figures 1 and 4).
The model reproduces available mutants, and with new experimental data provides new insights into how PLS regulates auxin concentration, by controlling the relative contribution of auxin transport and biosynthesis and by integrating auxin, ethylene and cytokinin signalling. Modelling further reveals that a bell-shaped dose–response relationship between endogenous auxin and root length is established via PLS.
In summary, we developed the first hormonal crosstalk model in Arabidopsis and revealed a hormonal crosstalk circuit through PLS and the downstream of ethylene signalling. Our study provides a platform to further integrate hormonal crosstalk in space and time in Arabidopsis.
An important question in plant biology is how genes influence the crosstalk between hormones to regulate growth. In this study, we model POLARIS (PLS) gene function and crosstalk between auxin, ethylene and cytokinin in Arabidopsis. Experimental evidence suggests that PLS acts on or close to the ethylene receptor ETR1, and a mathematical model describing possible PLS–ethylene pathway interactions is developed, and used to make quantitative predictions about PLS–hormone interactions. Modelling correctly predicts experimental results for the effect of the pls gene mutation on endogenous cytokinin concentration. Modelling also reveals a role for PLS in auxin biosynthesis in addition to a role in auxin transport. The model reproduces available mutants, and with new experimental data provides new insights into how PLS regulates auxin concentration, by controlling the relative contribution of auxin transport and biosynthesis and by integrating auxin, ethylene and cytokinin signalling. Modelling further reveals that a bell-shaped dose–response relationship between endogenous auxin and root length is established via PLS. This combined modelling and experimental analysis provides new insights into the integration of hormonal signals in plants.
doi:10.1038/msb.2010.26
PMCID: PMC2913391  PMID: 20531403
hormonal crosstalk; mathematical model; POLARIS gene; root development
3.  Jasmonates: An Update on Biosynthesis, Signal Transduction and Action in Plant Stress Response, Growth and Development 
Annals of Botany  2007;100(4):681-697.
Background
Jasmonates are ubiquitously occurring lipid-derived compounds with signal functions in plant responses to abiotic and biotic stresses, as well as in plant growth and development. Jasmonic acid and its various metabolites are members of the oxylipin family. Many of them alter gene expression positively or negatively in a regulatory network with synergistic and antagonistic effects in relation to other plant hormones such as salicylate, auxin, ethylene and abscisic acid.
Scope
This review summarizes biosynthesis and signal transduction of jasmonates with emphasis on new findings in relation to enzymes, their crystal structure, new compounds detected in the oxylipin and jasmonate families, and newly found functions.
Conclusions
Crystal structure of enzymes in jasmonate biosynthesis, increasing number of jasmonate metabolites and newly identified components of the jasmonate signal-transduction pathway, including specifically acting transcription factors, have led to new insights into jasmonate action, but its receptor(s) is/are still missing, in contrast to all other plant hormones.
doi:10.1093/aob/mcm079
PMCID: PMC2749622  PMID: 17513307
Oxylipins; jasmonic acid; jasmonate metabolites; enzymes in biosynthesis and metabolism; signal function
4.  Cytokinin cross-talking during biotic and abiotic stress responses 
As sessile organisms, plants have to be able to adapt to a continuously changing environment. Plants that perceive some of these changes as stress signals activate signaling pathways to modulate their development and to enable them to survive. The complex responses to environmental cues are to a large extent mediated by plant hormones that together orchestrate the final plant response. The phytohormone cytokinin is involved in many plant developmental processes. Recently, it has been established that cytokinin plays an important role in stress responses, but does not act alone. Indeed, the hormonal control of plant development and stress adaptation is the outcome of a complex network of multiple synergistic and antagonistic interactions between various hormones. Here, we review the recent findings on the cytokinin function as part of this hormonal network. We focus on the importance of the crosstalk between cytokinin and other hormones, such as abscisic acid, jasmonate, salicylic acid, ethylene, and auxin in the modulation of plant development and stress adaptation. Finally, the impact of the current research in the biotechnological industry will be discussed.
doi:10.3389/fpls.2013.00451
PMCID: PMC3833016  PMID: 24312105
cytokinin; stress; hormonal crosstalk; salicylic acid; abscisic acid
5.  Disease resistance or growth: the role of plant hormones in balancing immune responses and fitness costs 
Plant growth and response to environmental cues are largely governed by phytohormones. The plant hormones ethylene, jasmonic acid, and salicylic acid (SA) play a central role in the regulation of plant immune responses. In addition, other plant hormones, such as auxins, abscisic acid (ABA), cytokinins, gibberellins, and brassinosteroids, that have been thoroughly described to regulate plant development and growth, have recently emerged as key regulators of plant immunity. Plant hormones interact in complex networks to balance the response to developmental and environmental cues and thus limiting defense-associated fitness costs. The molecular mechanisms that govern these hormonal networks are largely unknown. Moreover, hormone signaling pathways are targeted by pathogens to disturb and evade plant defense responses. In this review, we address novel insights on the regulatory roles of the ABA, SA, and auxin in plant resistance to pathogens and we describe the complex interactions among their signal transduction pathways. The strategies developed by pathogens to evade hormone-mediated defensive responses are also described. Based on these data we discuss how hormone signaling could be manipulated to improve the resistance of crops to pathogens.
doi:10.3389/fpls.2013.00155
PMCID: PMC3662895  PMID: 23745126
abscisic acid; auxin; hormone crosstalk; pathogens; salicylic acid; trade-off; virulence factor
6.  Plant Haemoglobins, Nitric Oxide and Hypoxic Stress 
Annals of Botany  2003;91(2):173-178.
It is now known that there are several classes of haemoglobins in plants. A specialized class of haemoglobins, symbiotic haemoglobins, were discovered 62 years ago and are found only in nodules of plants capable of symbiotic nitrogen fixation. Plant haemoglobins, with properties distinct from symbiotic haemoglobins were discovered 18 years ago and are now believed to exist throughout the plant kingdom. They are expressed in different organs and tissues of both dicot and monocot plants. They are induced by hypoxic stress and by oversupply of certain nutrients. Most recently, truncated haemoglobins have been shown to also exist in plants. While hypoxic stress‐induced haemoglobins are widespread in the plant kingdom, their function has not been elucidated. This review discusses the recent findings regarding the function of these haemoglobins in relation to adaptation to hypoxia in plants. We propose that nitric oxide is an important metabolite in hypoxic plant cells and that at least one of the functions of hypoxic stress‐induced haemoglobins is to modulate nitric oxide levels in the cell.
doi:10.1093/aob/mcf115
PMCID: PMC4244985  PMID: 12509338
Review; haemoglobins; nonsymbiotic haemoglobins; flooding tolerance; nitric oxide; hypoxic stress
7.  Haemoglobin modulates NO emission and hyponasty under hypoxia-related stress in Arabidopsis thaliana  
Journal of Experimental Botany  2012;63(15):5581-5591.
Nitric oxide (NO) and ethylene are signalling molecules that are synthesized in response to oxygen depletion. Non-symbiotic plant haemoglobins (Hbs) have been demonstrated to act in roots under oxygen depletion to scavenge NO. Using Arabidopsis thaliana plants, the online emission of NO or ethylene was directly quantified under normoxia, hypoxia (0.1–1.0% O2), or full anoxia. The production of both gases was increased with reduced expression of either of the Hb genes GLB1 or GLB2, whereas NO emission decreased in plants overexpressing these genes. NO emission in plants with reduced Hb gene expression represented a major loss of nitrogen equivalent to 0.2mM nitrate per 24h under hypoxic conditions. Hb gene expression was greatly enhanced in flooded roots, suggesting induction by reduced oxygen diffusion. The function could be to limit loss of nitrogen under NO emission. NO reacts with thiols to form S-nitrosylated compounds, and it is demonstrated that hypoxia substantially increased the content of S-nitrosylated compounds. A parallel up-regulation of Hb gene expression in the normoxic shoots of the flooded plants may reflect signal transmission from root to shoot via ethylene and a role for Hb in the shoots. Hb gene expression was correlated with ethylene-induced upward leaf movement (hyponastic growth) but not with hypocotyl growth, which was Hb independent. Taken together the data suggest that Hb can influence flood-induced hyponasty via ethylene-dependent and, possibly, ethylene-independent pathways.
doi:10.1093/jxb/ers210
PMCID: PMC3444272  PMID: 22915746
Ethylene; flooding; haemoglobin; hyponastic growth; hypoxia; nitric oxide (NO)
8.  Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry 
Plant Methods  2011;7:37.
Background
Plant hormones play a pivotal role in several physiological processes during a plant's life cycle, from germination to senescence, and the determination of endogenous concentrations of hormones is essential to elucidate the role of a particular hormone in any physiological process. Availability of a sensitive and rapid method to quantify multiple classes of hormones simultaneously will greatly facilitate the investigation of signaling networks in controlling specific developmental pathways and physiological responses. Due to the presence of hormones at very low concentrations in plant tissues (10-9 M to 10-6 M) and their different chemistries, the development of a high-throughput and comprehensive method for the determination of hormones is challenging.
Results
The present work reports a rapid, specific and sensitive method using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) to analyze quantitatively the major hormones found in plant tissues within six minutes, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized for the determination of all plant hormones and are summarized in a schematic extraction diagram for the analysis of small amounts of plant material without time-consuming additional steps such as purification, sample drying or re-suspension.
Conclusions
This new method is applicable to the analysis of dynamic changes in endogenous concentrations of hormones to study plant developmental processes or plant responses to biotic and abiotic stresses in complex tissues. An example is shown in which a hormone profiling is obtained from leaves of plants exposed to salt stress in the aromatic plant, Rosmarinus officinalis.
doi:10.1186/1746-4811-7-37
PMCID: PMC3253682  PMID: 22098763
UPLC/ESI-MS/MS; Phytohormones; Auxins; Abscisic acid; Cytokinins; Gibberellins; Salicylic acid; Jasmonic acid; 1-amino-cyclopropane-1-carboxyic acid; Rosmarinus officinalis
9.  Open or Close the Gate – Stomata Action Under the Control of Phytohormones in Drought Stress Conditions 
Two highly specialized cells, the guard cells that surround the stomatal pore, are able to integrate environmental and endogenous signals in order to control the stomatal aperture and thereby the gas exchange. The uptake of CO2 is associated with a loss of water by leaves. Control of the size of the stomatal aperture optimizes the efficiency of water use through dynamic changes in the turgor of the guard cells. The opening and closing of stomata is regulated by the integration of environmental signals and endogenous hormonal stimuli. The various different factors to which the guard cells respond translates into the complexity of the network of signaling pathways that control stomatal movements. The perception of an abiotic stress triggers the activation of signal transduction cascades that interact with or are activated by phytohormones. Among these, abscisic acid (ABA), is the best-known stress hormone that closes the stomata, although other phytohormones, such as jasmonic acid, brassinosteroids, cytokinins, or ethylene are also involved in the stomatal response to stresses. As a part of the drought response, ABA may interact with jasmonic acid and nitric oxide in order to stimulate stomatal closure. In addition, the regulation of gene expression in response to ABA involves genes that are related to ethylene, cytokinins, and auxin signaling. In this paper, recent findings on phytohormone crosstalk, changes in signaling pathways including the expression of specific genes and their impact on modulating stress response through the closing or opening of stomata, together with the highlights of gaps that need to be elucidated in the signaling network of stomatal regulation, are reviewed.
doi:10.3389/fpls.2013.00138
PMCID: PMC3652521  PMID: 23717320
stomata; guard cells; phytohormones; abiotic stress; ABA; jasmonic acid; crosstalk
10.  Hormonal regulation in green plant lineage families 
The patterns of phytohormones distribution, their native function and possible origin of hormonal regulation across the green plant lineages (chlorophytes, charophytes, bryophytes and tracheophytes) are discussed. The five classical phytohormones — auxins, cytokinins, gibberellins (GA), abscisic acid (ABA) and ethylene occur ubiquitously in green plants. They are produced as secondary metabolites by microorganisms. Some of the bacterial species use phytohormones to interact with the plant as a part of their colonization strategy. Phytohormone biosynthetic pathways in plants seem to be of microbial origin and furthermore, the origin of high affinity perception mechanism could have preceded the recruitment of a metabolite as a hormone. The bryophytes represent the earliest land plants which respond to the phytohormones with the exception of gibberellins. The regulation by auxin and ABA may have evolved before the separation of green algal lineage. Auxin enhances rhizoid and caulonemal differentiation while cytokinins enhance shoot bud formation in mosses. Ethylene retards cell division but seems to promote cell elongation. The presence of responses specific to cytokinins and ethylene strongly suggest the origin of their regulation in bryophytes. The hormonal role of GAs could have evolved in some of the ferns where antheridiogens (compounds related to GAs) and GAs themselves regulate the formation of antheridia.
During migration of life forms to land, the tolerance to desiccation may have evolved and is now observed in some of the microorganisms, animals and plants. Besides plants, sequences coding for late embryogenesis abundant-like proteins occur in the genomes of other anhydrobiotic species of microorganisms and nematodes. ABA acts as a stress signal and increases rapidly upon desiccation or in response to some of the abiotic stresses in green plants. As the salt stress also increases ABA release in the culture medium of cyanobacterium Trichormus variabilis, the recruitment of ABA in the regulation of stress responses could have been derived from prokaryotes and present at the level of common ancestor of green plants. The overall hormonal action mechanisms in mosses are remarkably similar to that of the higher plants. As plants are thought to be monophyletic in origin, the existence of remarkably similar hormonal mechanisms in the mosses and higher plants, suggests that some of the basic elements of regulation cascade could have also evolved at the level of common ancestor of plants. The networking of various steps in a cascade or the crosstalk between different cascades is variable and reflects the dynamic interaction between a species and its specific environment.
doi:10.1007/s12298-008-0003-5
PMCID: PMC3550668  PMID: 23572871
Green plant families; Bryophytes; Abscisic acid; Auxin; Cytokinin; Ethylene; Antheridiogens; miRNA; Origin of hormonal regulation
11.  Transcription Reprogramming during Root Nodule Development in Medicago truncatula 
PLoS ONE  2011;6(1):e16463.
Many genes which are associated with root nodule development and activity in the model legume Medicago truncatula have been described. However information on precise stages of activation of these genes and their corresponding transcriptional regulators is often lacking. Whether these regulators are shared with other plant developmental programs also remains an open question. Here detailed microarray analyses have been used to study the transcriptome of root nodules induced by either wild type or mutant strains of Sinorhizobium meliloti. In this way we have defined eight major activation patterns in nodules and identified associated potential regulatory genes. We have shown that transcription reprogramming during consecutive stages of nodule differentiation occurs in four major phases, respectively associated with (i) early signalling events and/or bacterial infection; plant cell differentiation that is either (ii) independent or (iii) dependent on bacteroid differentiation; (iv) nitrogen fixation. Differential expression of several genes involved in cytokinin biosynthesis was observed in early symbiotic nodule zones, suggesting that cytokinin levels are actively controlled in this region. Taking advantage of databases recently developed for M. truncatula, we identified a small subset of gene expression regulators that were exclusively or predominantly expressed in nodules, whereas most other regulators were also activated under other conditions, and notably in response to abiotic or biotic stresses. We found evidence suggesting the activation of the jasmonate pathway in both wild type and mutant nodules, thus raising questions about the role of jasmonate during nodule development. Finally, quantitative RT-PCR was used to analyse the expression of a series of nodule regulator and marker genes at early symbiotic stages in roots and allowed us to distinguish several early stages of gene expression activation or repression.
doi:10.1371/journal.pone.0016463
PMCID: PMC3029352  PMID: 21304580
12.  Nitric oxide in plants: an assessment of the current state of knowledge 
AoB Plants  2013;5:pls052.
Nitric oxide (NO) is a plant signal contributing to plant stress responses and development. We here review some of the key advances in this field but also highlight certain key aspects of plant NO biology that require further attention.
Background and aims
After a series of seminal works during the last decade of the 20th century, nitric oxide (NO) is now firmly placed in the pantheon of plant signals. Nitric oxide acts in plant–microbe interactions, responses to abiotic stress, stomatal regulation and a range of developmental processes. By considering the recent advances in plant NO biology, this review will highlight certain key aspects that require further attention.
Scope and conclusions
The following questions will be considered. While cytosolic nitrate reductase is an important source of NO, the contributions of other mechanisms, including a poorly defined arginine oxidizing activity, need to be characterized at the molecular level. Other oxidative pathways utilizing polyamine and hydroxylamine also need further attention. Nitric oxide action is dependent on its concentration and spatial generation patterns. However, no single technology currently available is able to provide accurate in planta measurements of spatio-temporal patterns of NO production. It is also the case that pharmaceutical NO donors are used in studies, sometimes with little consideration of the kinetics of NO production. We here include in planta assessments of NO production from diethylamine nitric oxide, S-nitrosoglutathione and sodium nitroprusside following infiltration of tobacco leaves, which could aid workers in their experiments. Further, based on current data it is difficult to define a bespoke plant NO signalling pathway, but rather NO appears to act as a modifier of other signalling pathways. Thus, early reports that NO signalling involves cGMP—as in animal systems—require revisiting. Finally, as plants are exposed to NO from a number of external sources, investigations into the control of NO scavenging by such as non-symbiotic haemoglobins and other sinks for NO should feature more highly. By crystallizing these questions the authors encourage their resolution through the concerted efforts of the plant NO community.
doi:10.1093/aobpla/pls052
PMCID: PMC3560241  PMID: 23372921
13.  Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany 
Annals of Botany  2013;111(6):1021-1058.
Background
Jasmonates are important regulators in plant responses to biotic and abiotic stresses as well as in development. Synthesized from lipid-constituents, the initially formed jasmonic acid is converted to different metabolites including the conjugate with isoleucine. Important new components of jasmonate signalling including its receptor were identified, providing deeper insight into the role of jasmonate signalling pathways in stress responses and development.
Scope
The present review is an update of the review on jasmonates published in this journal in 2007. New data of the last five years are described with emphasis on metabolites of jasmonates, on jasmonate perception and signalling, on cross-talk to other plant hormones and on jasmonate signalling in response to herbivores and pathogens, in symbiotic interactions, in flower development, in root growth and in light perception.
Conclusions
The last few years have seen breakthroughs in the identification of JASMONATE ZIM DOMAIN (JAZ) proteins and their interactors such as transcription factors and co-repressors, and the crystallization of the jasmonate receptor as well as of the enzyme conjugating jasmonate to amino acids. Now, the complex nature of networks of jasmonate signalling in stress responses and development including hormone cross-talk can be addressed.
doi:10.1093/aob/mct067
PMCID: PMC3662512  PMID: 23558912
Jasmonic acid; oxylipins; enzymes in biosynthesis and metabolism; perception; JA signalling; JAZ; SCF; COI1; responses to herbivores and pathogens; symbiotic interaction; light regulation; JA in development
14.  Seed-specific elevation of non-symbiotic hemoglobin AtHb1: beneficial effects and underlying molecular networks in Arabidopsis thaliana 
BMC Plant Biology  2011;11:48.
Background
Seed metabolism is dynamically adjusted to oxygen availability. Processes underlying this auto-regulatory mechanism control the metabolic efficiency under changing environmental conditions/stress and thus, are of relevance for biotechnology. Non-symbiotic hemoglobins have been shown to be involved in scavenging of nitric oxide (NO) molecules, which play a key role in oxygen sensing/balancing in plants and animals. Steady state levels of NO are suggested to act as an integrator of energy and carbon metabolism and subsequently, influence energy-demanding growth processes in plants.
Results
We aimed to manipulate oxygen stress perception in Arabidopsis seeds by overexpression of the non-symbiotic hemoglobin AtHb1 under the control of the seed-specific LeB4 promoter. Seeds of transgenic AtHb1 plants did not accumulate NO under transient hypoxic stress treatment, showed higher respiratory activity and energy status compared to the wild type. Global transcript profiling of seeds/siliques from wild type and transgenic plants under transient hypoxic and standard conditions using Affymetrix ATH1 chips revealed a rearrangement of transcriptional networks by AtHb1 overexpression under non-stress conditions, which included the induction of transcripts related to ABA synthesis and signaling, receptor-like kinase- and MAP kinase-mediated signaling pathways, WRKY transcription factors and ROS metabolism. Overexpression of AtHb1 shifted seed metabolism to an energy-saving mode with the most prominent alterations occurring in cell wall metabolism. In combination with metabolite and physiological measurements, these data demonstrate that AtHb1 overexpression improves oxidative stress tolerance compared to the wild type where a strong transcriptional and metabolic reconfiguration was observed in the hypoxic response.
Conclusions
AtHb1 overexpression mediates a pre-adaptation to hypoxic stress. Under transient stress conditions transgenic seeds were able to keep low levels of endogenous NO and to maintain a high energy status, in contrast to wild type. Higher weight of mature transgenic seeds demonstrated the beneficial effects of seed-specific overexpression of AtHb1.
doi:10.1186/1471-2229-11-48
PMCID: PMC3068945  PMID: 21406103
15.  Evolutionary trace analysis of plant haemoglobins: implications for site-directed mutagenesis 
Bioinformation  2007;1(9):370-375.
Haemoglobins are found ubiquitously in eukaryotes and many bacteria. In plants, haemoglobins were first identified in species, which can fix nitrogen via symbiosis with bacteria. Recent findings suggest that another class of haemoglobins termed as nonsymbiotic haemoglobins are present through out the plant kingdom and are expressed differentially during plant development. Limited data available suggests that non-symbiotic haemoglobins are involved in hypoxic stress and oversupply of nutrients. Due to lack of information on structurally conserved, functionally important residues in non-symbiotic haemoglobins, further studies to elucidate the molecular mechanisms underlying the biological role are hampered. To determine functionally important residues in non-symbiotic haemoglobins, I have analyzed a number of sequences from plant haemoglobin family, in the context of the known crystal structures of plant by evolutionary trace method. Results indicate that the, evolutionary trace method like conventional phylogentic analysis, could resolve phylogentic relationships between plant haemoglobin family. Evolutionary trace analysis has identified candidate functional (trace) residues that uniquely characterize the heme-binding pocket, dimer interface and possible novel functional surfaces. Such residues from specific three-dimensional clusters might be of functional importance in nonsymbiotic haemoglobins. These data, together with our improved knowledge of possible functional residues, can be used in future structure-function analysis experiments.
PMCID: PMC1891720  PMID: 17597924
evolution; haemoglobin; site directed mutagenesis; phylogeny
16.  Ethylene and the regulation of plant development 
BMC Biology  2012;10:9.
Often considered an 'aging' hormone due to its role in accelerating such developmental processes as ripening, senescence, and abscission, the plant hormone ethylene also regulates many aspects of growth and development throughout the life cycle of the plant. Multiple mechanisms have been identified by which transcriptional output from the ethylene signaling pathway can be tailored to meet the needs of particular developmental pathways. Of special interest is the report by Lumba et al. in BMC Biology on how vegetative transitions are regulated through the effect of the transcription factor FUSCA3 on ethylene-controlled gene expression, providing an elegant example of how hormonal control can be integrated into a developmental pathway.
See research article http://www.biomedcentral.com/1741-7007/10/8
Commentary
One of the amazing qualities of plants is their phenotypic plasticity. Consider, for example, how a pine tree will grow to a towering hundreds of feet in height in Yosemite Valley, but to only a gnarled few feet in height up near the timberline. This diversity of form, though originating from the same genotype, points to the degree to which plant growth and development can be modulated. Much of this control is mediated by a small group of plant hormones that include auxin, cytokinin, gibberellin, abscisic acid, brassinosteroid, jasmonic acid, and ethylene [1]. These are often considered 'classical' plant hormones because they were discovered decades ago; indeed, the presence of some was inferred over a century ago. Their early discovery is no doubt due in part to their general function throughout the life cycle of the plant. More recently, and in the remarkably short period of time since the advent of Arabidopsis as a genetic model, key elements in the primary signaling pathways of these plant hormones have been uncovered. The important question is no longer simply how are these hormones perceived, but how are the hormonal signals integrated into the control of particular developmental pathways? In pursuing such a question, Lumba et al. [2] have now uncovered a role for the plant hormone ethylene in regulating the conversion of juvenile to adult leaves. These new data, in combination with prior research implicating the plant hormones abscisic acid and gibberellin in this transition [3], form an important step in defining how a hormonal network regulates a key developmental process.
doi:10.1186/1741-7007-10-9
PMCID: PMC3282650  PMID: 22348804
17.  ESTs Analysis Reveals Putative Genes Involved in Symbiotic Seed Germination in Dendrobium officinale 
PLoS ONE  2013;8(8):e72705.
Dendrobiumofficinale (Orchidaceae) is one of the world’s most endangered plants with great medicinal value. In nature, D. officinale seeds must establish symbiotic relationships with fungi to germinate. However, the molecular events involved in the interaction between fungus and plant during this process are poorly understood. To isolate the genes involved in symbiotic germination, a suppression subtractive hybridization (SSH) cDNA library of symbiotically germinated D. officinale seeds was constructed. From this library, 1437 expressed sequence tags (ESTs) were clustered to 1074 Unigenes (including 902 singletons and 172 contigs), which were searched against the NCBI non-redundant (NR) protein database (E-value cutoff, e-5). Based on sequence similarity with known proteins, 579 differentially expressed genes in D. officinale were identified and classified into different functional categories by Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression levels of 15 selected genes emblematic of symbiotic germination were confirmed via real-time quantitative PCR. These genes were classified into various categories, including defense and stress response, metabolism, transcriptional regulation, transport process and signal transduction pathways. All transcripts were upregulated in the symbiotically germinated seeds (SGS). The functions of these genes in symbiotic germination were predicted. Furthermore, two fungus-induced calcium-dependent protein kinases (CDPKs), which were upregulated 6.76- and 26.69-fold in SGS compared with un-germinated seeds (UGS), were cloned from D. officinale and characterized for the first time. This study provides the first global overview of genes putatively involved in D. officinale symbiotic seed germination and provides a foundation for further functional research regarding symbiotic relationships in orchids.
doi:10.1371/journal.pone.0072705
PMCID: PMC3742586  PMID: 23967335
18.  NO homeostasis is a key regulator of early nitrate perception and root elongation in maize*  
Journal of Experimental Botany  2013;65(1):185-200.
Nitrate reductase produces nitric oxide (NO) as an early response to nitrate, and the coordinated induction of ns-haemoglobins finely modulates NO level. The control of NO homeostasis regulates root elongation and represents a novel key component of nitrate signaling in maize
Crop plant development is strongly dependent on nitrogen availability in the soil and on the efficiency of its recruitment by roots. For this reason, the understanding of the molecular events underlying root adaptation to nitrogen fluctuations is a primary goal to develop biotechnological tools for sustainable agriculture. However, knowledge about molecular responses to nitrogen availability is derived mainly from the study of model species. Nitric oxide (NO) has been recently proposed to be implicated in plant responses to environmental stresses, but its exact role in the response of plants to nutritional stress is still under evaluation. In this work, the role of NO production by maize roots after nitrate perception was investigated by focusing on the regulation of transcription of genes involved in NO homeostasis and by measuring NO production in roots. Moreover, its involvement in the root growth response to nitrate was also investigated. The results provide evidence that NO is produced by nitrate reductase as an early response to nitrate supply and that the coordinated induction of non-symbiotic haemoglobins (nsHbs) could finely regulate the NO steady state. This mechanism seems to be implicated on the modulation of the root elongation in response to nitrate perception. Moreover, an improved agar-plate system for growing maize seedlings was developed. This system, which allows localized treatments to be performed on specific root portions, gave the opportunity to discern between localized and systemic effects of nitrate supply to roots.
doi:10.1093/jxb/ert358
PMCID: PMC3883287  PMID: 24220653
Maize; nitrate; nitrate reductase; nitric oxide; non-symbiotic haemoglobin; root; transition zone.
19.  Genome-wide analysis of the GH3 family in apple (Malus × domestica) 
BMC Genomics  2013;14:297.
Background
Auxin plays important roles in hormone crosstalk and the plant’s stress response. The auxin-responsive Gretchen Hagen3 (GH3) gene family maintains hormonal homeostasis by conjugating excess indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acids (JAs) to amino acids during hormone- and stress-related signaling pathways. With the sequencing of the apple (Malus × domestica) genome completed, it is possible to carry out genomic studies on GH3 genes to indentify candidates with roles in abiotic/biotic stress responses.
Results
Malus sieversii Roem., an apple rootstock with strong drought tolerance and the ancestral species of cultivated apple species, was used as the experimental material. Following genome-wide computational and experimental identification of MdGH3 genes, we showed that MdGH3s were differentially expressed in the leaves and roots of M. sieversii and that some of these genes were significantly induced after various phytohormone and abiotic stress treatments. Given the role of GH3 in the negative feedback regulation of free IAA concentration, we examined whether phytohormones and abiotic stresses could alter the endogenous auxin level. By analyzing the GUS activity of DR5::GUS-transformed Arabidopsis seedlings, we showed that ABA, SA, salt, and cold treatments suppressed the auxin response. These findings suggest that other phytohormones and abiotic stress factors might alter endogenous auxin levels.
Conclusion
Previous studies showed that GH3 genes regulate hormonal homeostasis. Our study indicated that some GH3 genes were significantly induced in M. sieversii after various phytohormone and abiotic stress treatments, and that ABA, SA, salt, and cold treatments reduce the endogenous level of axuin. Taken together, this study provides evidence that GH3 genes play important roles in the crosstalk between auxin, other phytohormones, and the abiotic stress response by maintaining auxin homeostasis.
doi:10.1186/1471-2164-14-297
PMCID: PMC3653799  PMID: 23638690
Malus sieversii Roem; Phytohormone; Biotic stress; GH3; DR5; GUS
20.  Genome-wide identification and transcriptional profiling analysis of auxin response-related gene families in cucumber 
BMC Research Notes  2014;7:218.
Background
Auxin signaling has a vital function in the regulation of plant growth and development, both which are known to be mediated by auxin-responsive genes. So far, significant progress has been made toward the identification and characterization of auxin-response genes in several model plants, while no systematic analysis for these families was reported in cucumber (Cucumis sativus L.), a reference species for Cucurbitaceae crops. The comprehensive analyses will help design experiments for functional validation of their precise roles in plant development and stress responses.
Results
A genome-wide search for auxin-response gene homologues identified 16 auxin-response factors (ARFs), 27 auxin/indole acetic acids (Aux/IAAs), 10 Gretchen Hagen 3 (GH3s), 61 small auxin-up mRNAs (SAURs), and 39 lateral organ boundaries (LBDs) in cucumber. Sequence analysis together with the organization of putative motifs indicated the potential diverse functions of these five auxin-related family members. The distribution and density of auxin response-related genes on chromosomes were not uniform. Evolutionary analysis showed that the chromosomal segment duplications mainly contributed to the expansion of the CsARF, CsIAA, CsGH3, and CsLBD gene families. Quantitative real-time RT-PCR analysis demonstrated that many ARFs, AUX/IAAs, GH3s, SAURs, and LBD genes were expressed in diverse patterns within different organs/tissues and during different development stages. They were also implicated in IAA, methyl jasmonic acid, or salicylic acid response, which is consistent with the finding that a great number of diverse cis-elements are present in their promoter regions involving a variety of signaling transduction pathways.
Conclusion
Genome-wide comparative analysis of auxin response-related family genes and their expression analysis provide new evidence for the potential role of auxin in development and hormone response of plants. Our data imply that the auxin response genes may be involved in various vegetative and reproductive developmental processes. Furthermore, they will be involved in different signal pathways and may mediate the crosstalk between various hormone responses.
doi:10.1186/1756-0500-7-218
PMCID: PMC4108051  PMID: 24708619
21.  Auxin Response Factor2 (ARF2) and Its Regulated Homeodomain Gene HB33 Mediate Abscisic Acid Response in Arabidopsis 
PLoS Genetics  2011;7(7):e1002172.
The phytohormone abscisic acid (ABA) is an important regulator of plant development and response to environmental stresses. In this study, we identified two ABA overly sensitive mutant alleles in a gene encoding Auxin Response Factor2 (ARF2). The expression of ARF2 was induced by ABA treatment. The arf2 mutants showed enhanced ABA sensitivity in seed germination and primary root growth. In contrast, the primary root growth and seed germination of transgenic plants over-expressing ARF2 are less inhibited by ABA than that of the wild type. ARF2 negatively regulates the expression of a homeodomain gene HB33, the expression of which is reduced by ABA. Transgenic plants over-expressing HB33 are more sensitive, while transgenic plants reducing HB33 by RNAi are more resistant to ABA in the seed germination and primary root growth than the wild type. ABA treatment altered auxin distribution in the primary root tips and made the relative, but not absolute, auxin accumulation or auxin signal around quiescent centre cells and their surrounding columella stem cells to other cells stronger in arf2-101 than in the wild type. These results indicate that ARF2 and HB33 are novel regulators in the ABA signal pathway, which has crosstalk with auxin signal pathway in regulating plant growth.
Author Summary
Abscisic acid is a phytohormone that regulates many aspects in plant growth and development and response to different biotic and abiotic stresses. Research on ABA inhibiting seed germination, controlling stomatal movement, and regulating gene expression has been widely performed. However, the molecular mechanism for ABA regulating root growth is not well known. We have set up a genetic screen by using ABA inhibiting root growth to identify ABA related mutants and to dissect the molecular mechanism of ABA regulating root growth. In this study, we identified two new mutant alleles that are defective in ARF2 gene. ARF2 is a transcriptional suppressor that has been found to be involved in ethylene, auxin, and brassinosteroid pathway to control plant growth and development. Our study indicates that ARF2 is an ABA responsive regulator that functions in both seed germination and primary root growth. ARF2 directly regulates the expression of a homeodomain gene HB33. We demonstrate that ABA treatment reduces the cell division and alters auxin distribution more in arf2 mutant than in the wild type, suggesting an important mechanism in ABA inhibiting the primary root growth through mediating cell division in root tips.
doi:10.1371/journal.pgen.1002172
PMCID: PMC3136439  PMID: 21779177
22.  Boolean modeling of transcriptome data reveals novel modes of heterotrimeric G-protein action 
Classical mechanisms of heterotrimeric G-protein signaling are observed to function in regulation of the transcriptome. Conversely, many theoretical regulatory modes of the G-protein are not manifested in the transcriptomes we investigate.A new mechanism of G-protein signaling is revealed, in which the β subunit regulates gene expression identically in the presence or absence of the α subunit.We find evidence of cross-talk between G-protein-mediated and hormone-mediated transcriptional regulation.We find evidence of system specificity in G-protein signaling.
Heterotrimeric G-proteins, composed of α, β, and γ subunits, participate in a wide range of signaling pathways in eukaryotes (Morris and Malbon, 1999). According to the typical, mammalian paradigm, in its inactive state, the G-protein exists as an associated heterotrimer. G-protein signaling begins with ligand binding that results in a conformational change in a G-protein-coupled receptor (GPCR). Once activated by the GPCR, the Gα separates from the associated Gβγ dimer and the freed Gα and Gβγ proteins can then interact with downstream effector molecules, alone or in combination, to transduce the signal. Subsequent to signal propagation, Gα re-associates with the Gβγ dimer to reform the G-protein complex.
There are several classical routes for signal propagation through heterotrimeric G-proteins that have been categorized in mammalian systems (Marrari et al, 2007; Dupre et al, 2009). One route, which we designate classical I, requires the presence of both subunits, and can invoke one of two distinct mechanisms. In one mechanism, on GPCR activation, freed Gα and Gβγ each interact with downstream effectors to elicit the downstream response. In a related mechanism, Gα but not Gβγ interacts with downstream effectors, but the Gβγ dimer is nevertheless required to facilitate coupling of Gα with the relevant GPCR (Marrari et al, 2007). In a second route, which we designate classical II, it is solely the Gβγ dimer that interacts with downstream effectors; in this case, sequestration of Gβγ within the heterotrimer prevents signal propagation. In addition, a few non-classical G-protein regulatory modes have also been implicated in some systems, for example signaling by the intact heterotrimer in yeast (Klein et al, 2000; Frank et al, 2005). Observations such as these lead to a fundamental question, namely, which of all the theoretical regulatory modes of G-protein signaling are realized biologically. Our study answers this question in the context of the model plant Arabidopsis thaliana, and in addition analyzes the manner in which G-protein signaling couples with signaling by the plant hormone abscisic acid. The Arabidopsis genome encodes only one canonical Gα subunit, GPA1, and one canonical Gβ subunit, AGB1, and knockout mutants are available for each of these, allowing clear dissection of Gα- and Gβ-related phenotypes.
Abscisic acid (ABA) is a major plant hormone, which inhibits growth and promotes tolerance of abiotic stresses such as drought, salinity, and cold. ABA signaling is known to interact with heterotrimeric G-protein signaling in both developmental and stress responses in a complex manner, causing, for example, ABA hyposensitivity of guard cell stomatal opening in gpa1 and agb1 single mutants as well as agb1 gpa1 double mutants (Fan et al, 2008), but ABA hypersensitivity of the inhibition of seed germination and post-germination seedling development in the same mutants (Pandey et al, 2006). These experimental observations implicate G-proteins as one of the components of ABA signaling, but to date no systematic study has been conducted in either plant or metazoan systems to define the co-regulatory modes of a G-protein and a hormone.
In this study, we conduct genome-wide gene expression profiling in G-protein subunit mutants of A. thaliana guard cells and leaves, with or without treatment with ABA. By introducing one or more mediators acting downstream of the G-protein and ABA to control transcript levels, we propose nine G-protein/ABA signaling pathways including ABA-independent G-protein signaling pathways, G-protein-independent ABA signaling pathways, and seven distinct ABA–G-protein-coupled signaling pathways (Figure 1). We develop a Boolean modeling framework to systematically enumerate 14 possible theoretical regulatory modes of the G-protein and 142 co-regulatory modes of the G-protein and ABA, and then use a pattern matching approach to associate target genes with theoretical regulatory modes.
Our analysis shows that the G-protein regulatory mode that requires the presence of both Gα and Gβγ subunits (consistent with classical I mechanisms), is well represented in both guard cells and leaves. The G-protein regulatory mode that requires a freed Gβγ subunit (classical II G-protein regulatory mechanism) is well supported in guard cells and somewhat less so in leaves. In addition, a G-protein regulatory mode representing a non-classical regulatory mechanism is prevalent in guard cells but less so in leaves (Figure 5). In this regulatory mode, signaling by Gβ(γ) occurs, and this signaling is not regulated in any way by Gα.
By relating the target genes with the nine proposed G-protein/ABA signaling pathways, we are able to gauge the plausibility of regulatory modes of the G-protein and ABA at the pathway level. We find that G-protein-independent ABA signaling pathways are prevalent in both guard cells and leaves. The existence of an ABA-independent regulatory activity of the G-protein is well supported in guard cells, but not supported in leaves. Additive regulation by G-protein signaling plus G-protein-independent ABA signaling is rare in both guard cells and leaves. In addition, combinatorial cross-talk between G-protein signaling and ABA signaling and additive cross-talk between ABA–G-protein signaling and G-protein-independent ABA signaling are observed in both guard cells and leaves. Our transcriptome analysis indicates that in some cases, ABA definitely does not influence G-protein signaling, though it may do so in some other cases.
To investigate whether previously observed hypersensitivity or hyposensitivity of developmental and dynamic transient responses to ABA in G-protein mutants is recapitulated at the level of transcriptional regulation, we compare gene regulation by ABA in guard cells and leaves of the G-protein mutants versus wild type. We find that in guard cells, equal ABA hyposensitivity of all mutants combined is significant, although hyposensitivity in individual mutants is not. There is also a separate group of genes in guard cells that show ABA hypersensitivity in the gpa1 mutant, suggesting complex interactions between ABA and G-protein signaling in gene regulation in this cell type. In leaves, ABA hyposensitivity of gene expression in the three individual mutants and equal hyposensitivity in all mutants are strongly supported. In addition, several of the functional categories identified by our analysis of G-protein regulatory modes have been implicated in previous physiological analyses of G-protein mutants, providing validation to the biological interpretation of our results.
In summary, by conducting a genome-wide gene expression profiling study in G-protein subunit mutants of A. thaliana guard cells and leaves and developing a Boolean modeling framework, we systematically evaluate the biological utilization of mechanisms of G-protein regulatory action and reveal novel regulatory modes of the G-protein. The results generate empirical evidence and insights regarding molecular events of G-protein signaling and response at the physiological level in both plants and mammals.
Heterotrimeric G-proteins mediate crucial and diverse signaling pathways in eukaryotes. Here, we generate and analyze microarray data from guard cells and leaves of G-protein subunit mutants of the model plant Arabidopsis thaliana, with or without treatment with the stress hormone, abscisic acid. Although G-protein control of the transcriptome has received little attention to date in any system, transcriptome analysis allows us to search for potentially uncommon yet significant signaling mechanisms. We describe the theoretical Boolean mechanisms of G-protein × hormone regulation, and then apply a pattern matching approach to associate gene expression profiles with Boolean models. We find that (1) classical mechanisms of G-protein signaling are well represented. Conversely, some theoretical regulatory modes of the G-protein are not supported; (2) a new mechanism of G-protein signaling is revealed, in which Gβ regulates gene expression identically in the presence or absence of Gα; (3) guard cells and leaves favor different G-protein modes in transcriptome regulation, supporting system specificity of G-protein signaling. Our method holds significant promise for analyzing analogous ‘switch-like' signal transduction events in any organism.
doi:10.1038/msb.2010.28
PMCID: PMC2913393  PMID: 20531402
abscisic acid; Arabidopsis thaliana; Boolean modeling; heterotrimeric G-protein; transcriptome
23.  Prediction of transcriptional regulatory elements for plant hormone responses based on microarray data 
BMC Plant Biology  2011;11:39.
Background
Phytohormones organize plant development and environmental adaptation through cell-to-cell signal transduction, and their action involves transcriptional activation. Recent international efforts to establish and maintain public databases of Arabidopsis microarray data have enabled the utilization of this data in the analysis of various phytohormone responses, providing genome-wide identification of promoters targeted by phytohormones.
Results
We utilized such microarray data for prediction of cis-regulatory elements with an octamer-based approach. Our test prediction of a drought-responsive RD29A promoter with the aid of microarray data for response to drought, ABA and overexpression of DREB1A, a key regulator of cold and drought response, provided reasonable results that fit with the experimentally identified regulatory elements. With this succession, we expanded the prediction to various phytohormone responses, including those for abscisic acid, auxin, cytokinin, ethylene, brassinosteroid, jasmonic acid, and salicylic acid, as well as for hydrogen peroxide, drought and DREB1A overexpression. Totally 622 promoters that are activated by phytohormones were subjected to the prediction. In addition, we have assigned putative functions to 53 octamers of the Regulatory Element Group (REG) that have been extracted as position-dependent cis-regulatory elements with the aid of their feature of preferential appearance in the promoter region.
Conclusions
Our prediction of Arabidopsis cis-regulatory elements for phytohormone responses provides guidance for experimental analysis of promoters to reveal the basis of the transcriptional network of phytohormone responses.
doi:10.1186/1471-2229-11-39
PMCID: PMC3058078  PMID: 21349196
24.  The CRE1 Cytokinin Pathway Is Differentially Recruited Depending on Medicago truncatula Root Environments and Negatively Regulates Resistance to a Pathogen 
PLoS ONE  2015;10(1):e0116819.
Cytokinins are phytohormones that regulate many developmental and environmental responses. The Medicago truncatula cytokinin receptor MtCRE1 (Cytokinin Response 1) is required for the nitrogen-fixing symbiosis with rhizobia. As several cytokinin signaling genes are modulated in roots depending on different biotic and abiotic conditions, we assessed potential involvement of this pathway in various root environmental responses. Phenotyping of cre1 mutant roots infected by the Gigaspora margarita arbuscular mycorrhizal (AM) symbiotic fungus, the Aphanomyces euteiches root oomycete, or subjected to an abiotic stress (salt), were carried out. Detailed histological analysis and quantification of cre1 mycorrhized roots did not reveal any detrimental phenotype, suggesting that MtCRE1 does not belong to the ancestral common symbiotic pathway shared by rhizobial and AM symbioses. cre1 mutants formed an increased number of emerged lateral roots compared to wild-type plants, a phenotype which was also observed under non-stressed conditions. In response to A. euteiches, cre1 mutants showed reduced disease symptoms and an increased plant survival rate, correlated to an enhanced formation of lateral roots, a feature previously linked to Aphanomyces resistance. Overall, we showed that the cytokinin CRE1 pathway is not only required for symbiotic nodule organogenesis but also affects both root development and resistance to abiotic and biotic environmental stresses.
doi:10.1371/journal.pone.0116819
PMCID: PMC4285552  PMID: 25562779
25.  Transient Hypermutagenesis Accelerates the Evolution of Legume Endosymbionts following Horizontal Gene Transfer 
PLoS Biology  2014;12(9):e1001942.
Stress-responsive error-prone DNA polymerase genes transferred along with key symbiotic genes ease the evolution of a soil bacterium into a legume endosymbiont by accelerating adaptation of the recipient bacterial genome to its new plant host.
Horizontal gene transfer (HGT) is an important mode of adaptation and diversification of prokaryotes and eukaryotes and a major event underlying the emergence of bacterial pathogens and mutualists. Yet it remains unclear how complex phenotypic traits such as the ability to fix nitrogen with legumes have successfully spread over large phylogenetic distances. Here we show, using experimental evolution coupled with whole genome sequencing, that co-transfer of imuABC error-prone DNA polymerase genes with key symbiotic genes accelerates the evolution of a soil bacterium into a legume symbiont. Following introduction of the symbiotic plasmid of Cupriavidus taiwanensis, the Mimosa symbiont, into pathogenic Ralstonia solanacearum we challenged transconjugants to become Mimosa symbionts through serial plant-bacteria co-cultures. We demonstrate that a mutagenesis imuABC cassette encoded on the C. taiwanensis symbiotic plasmid triggered a transient hypermutability stage in R. solanacearum transconjugants that occurred before the cells entered the plant. The generated burst in genetic diversity accelerated symbiotic adaptation of the recipient genome under plant selection pressure, presumably by improving the exploration of the fitness landscape. Finally, we show that plasmid imuABC cassettes are over-represented in rhizobial lineages harboring symbiotic plasmids. Our findings shed light on a mechanism that may have facilitated the dissemination of symbiotic competency among α- and β-proteobacteria in natura and provide evidence for the positive role of environment-induced mutagenesis in the acquisition of a complex lifestyle trait. We speculate that co-transfer of complex phenotypic traits with mutagenesis determinants might frequently enhance the ecological success of HGT.
Author Summary
Horizontal gene transfer has an extraordinary impact on microbe evolution and diversification, by allowing exploration of new niches such as higher organisms. This is the case for rhizobia, a group of phylogenetically diverse bacteria that form a nitrogen-fixing symbiotic relationship with most leguminous plants. While these arose through horizontal transfer of symbiotic plasmids, this in itself is usually unproductive, and full expression of the acquired traits needs subsequent remodeling of the genome to ensure the ecological success of the transfer. Here we uncover a mechanism that accelerates the evolution of a soil bacterium into a legume symbiont. We show that key symbiotic genes are co-transferred with genes encoding stress-responsive error-prone DNA polymerases that transiently elevate the mutation rate in the recipient genome. This burst in genetic diversity accelerates the symbiotic evolution process under selection pressure from the host plant. A more widespread involvement of plasmid mutagenesis cassettes in rhizobium evolution is supported by their overrepresentation in rhizobia-containing lineages. Our findings provide evidence for the role of environment-induced mutagenesis in the acquisition of a complex lifestyle trait and predict that co-transfer of complex phenotypic traits with mutagenesis determinants might help successful horizontal gene transfer.
doi:10.1371/journal.pbio.1001942
PMCID: PMC4151985  PMID: 25181317

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