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1.  Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions 
Journal of Experimental Botany  2010;62(1):155-166.
Homeodomain-leucine zipper type I (HD-Zip I) proteins are plant-specific transcription factors associated with the regulation of growth and development in response to changes in the environment. Nicotiana attenuata NaHD20 was identified as an HD-Zip I-coding gene whose expression was induced by multiple stress-associated stimuli including drought and wounding. To study the role of NaHD20 in the integration of stress responses with changes in growth and development, its expression was silenced by virus-induced gene silencing (VIGS), and control and silenced plants were metabolically and developmentally characterized. Phytohormone profiling showed that NaHD20 plays a positive role in abscisic acid (ABA) accumulation in leaves during water stress and in the expression of some dehydration-responsive genes including ABA biosynthetic genes. Moreover, consistent with the high levels of NaHD20 expression in corollas, the emission of benzylacetone from flowers was reduced in NaHD20-silenced plants. Additionally, bolting time and the opening of the inflorescence buds was decelerated in these plants in a specific developmental stage without affecting the total number of flowers produced. Water stress potentiated these effects; however, after plants recovered from this condition, the opening of the inflorescence buds was accelerated in NaHD20-silenced plants. In summary, NaHD20 plays multiple roles in N. attenuata and among these are the coordination of responses to dehydration and its integration with changes in flower transitions.
PMCID: PMC2993906  PMID: 20713465
ABA; benzylacetone; corolla; HD-Zip; Nicotiana
2.  The two α-dox genes of Nicotiana attenuata: overlapping but distinct functions in development and stress responses 
BMC Plant Biology  2010;10:171.
Plant fatty acid α-dioxygenases (α-DOX) are oxylipin-forming enzymes induced by biotic and abiotic stresses, which also participate in developmental processes. In Nicotiana attenuata, herbivory strongly induces the expression of an α-dox1 gene. To determine its role, we silenced its expression using Agrobacterium-mediated plant transformation with an inverted repeat construct. More than half of the transformed lines showed a severe dwarf growth phenotype that was very similar to the phenotype of tomato plants mutated at a second α-dox isoform. This led us to identify the corresponding α-dox2 gene in N. attenuata and examine the regulation of both α-dox genes as well as the consequences of their silencing in plant development and anti-herbivore defense.
The transformed lines exhibiting a dwarf growth phenotype are co-silenced for both α-dox genes resulting in a nearly complete suppression of α-DOX activity, which is associated with increases in ABA, JA and anthocyanin levels, all metabolic signatures of oxidative stress. The other lines, only silenced for α-dox1, developed similarly to wild-type plants, exhibited a 40% reduction of α-DOX activity resulting in a 50% reduction of its main product in planta (2-HOT) and showed no signs of oxidative stress. In contrast to α-dox1, the expression of α-dox2 gene is not induced by wounding or elicitors in the oral secretions of Manduca sexta. Instead, α-dox2 is expressed in roots and flowers which lack α-dox1 expression, but both genes are equally regulated during leaf maturation. We transiently silenced α-dox gene copies with gene-specific constructs using virus induced gene silencing and determined the consequences for plant development and phytohormone and 2-HOT levels. While individual silencing of α-dox1 or α-dox2 had no effects on plant growth, the co-suppression of both α-dox genes decreased plant growth. Plants transiently silenced for both α-dox genes had increased constitutive levels of JA and ABA but silencing α-dox1 alone resulted in lower M. sexta-induced levels of JA, 2-HOT and ABA.
Thus, both α-dox isoforms function in the development of N. attenuata. In leaf maturation, the two α-dox genes have overlapping functions, but only α-dox2 is involved in root and flower development and only α-dox1 functions in anti-herbivore defense.
PMCID: PMC3017789  PMID: 20701756
3.  Ethylene regulates phosphorus remobilization and expression of a phosphate transporter (PhPT1) during petunia corolla senescence 
Journal of Experimental Botany  2009;60(7):2179-2190.
The programmed degradation of macromolecules during petal senescence allows the plant to remobilize nutrients from dying to developing tissues. Ethylene is involved in regulating the timing of nucleic acid degradation in petunia, but it is not clear if ethylene has a role in the remobilization of phosphorus during petal senescence. To investigate ethylene's role in nutrient remobilization, the P content of petals (collectively called the corolla) during early development and senescence was compared in ethylene-sensitive wild type Petunia×hybrida ‘Mitchell Diploid’ (MD) and transgenic petunias with reduced sensitivity to ethylene (35S::etr1-1). When compared to the total P content of corollas on the day of flower opening (the early non-senescing stage), P in MD corollas had decreased 74% by the late stage of senescence (advanced wilting). By contrast, P levels were only reduced by an average of 32% during etr1-1 corolla (lines 44568 and Z00-35-10) senescence. A high-affinity phosphate transporter, PhPT1 (PhPht1;1), was cloned from senescing petunia corollas by RT-PCR. PhPT1 expression was up-regulated during MD corolla senescence and a much smaller increase was detected during the senescence of etr1-1 petunia corollas. PhPT1 mRNA levels showed a rapid increase in detached corollas (treated at 1 d after flower opening) following treatment with low levels of ethylene (0.1 μl l-1). Transcripts accumulated in the presence of the protein synthesis inhibitor, cycloheximide, indicating that PhPT1 is a primary ethylene response gene. PhPT1 is a putative phosphate transporter that may function in Pi translocation during senescence.
PMCID: PMC2682506  PMID: 19380421
Ethylene; flowers; high-affinity phosphate transporter; hormones; nitrogen; petal senescence
4.  RNA-sequencing reveals early, dynamic transcriptome changes in the corollas of pollinated petunias 
BMC Plant Biology  2014;14:307.
Pollination reduces flower longevity in many angiosperms by accelerating corolla senescence. This response requires hormone signaling between the floral organs and results in the degradation of macromolecules and organelles within the petals to allow for nutrient remobilization to developing seeds. To investigate early pollination-induced changes in petal gene expression, we utilized high-throughput sequencing to identify transcripts that were differentially expressed between corollas of pollinated Petunia × hybrida flowers and their unpollinated controls at 12, 18, and 24 hours after opening.
In total, close to 0.5 billion Illumina 101 bp reads were generated, de novo assembled, and annotated, resulting in an EST library of approximately 33 K genes. Over 4,700 unique, differentially expressed genes were identified using comparisons between the pollinated and unpollinated libraries followed by pairwise comparisons of pollinated libraries to unpollinated libraries from the same time point (i.e. 12-P/U, 18-P/U, and 24-P/U) in the Bioconductor R package DESeq2. Over 500 gene ontology terms were enriched. The response to auxin stimulus and response to 1-aminocyclopropane-1-carboxylic acid terms were enriched by 12 hours after pollination (hap). Using weighted gene correlation network analysis (WGCNA), three pollination-specific modules were identified. Module I had increased expression across pollinated corollas at 12, 18, and 24 h, and modules II and III had a peak of expression in pollinated corollas at 18 h. A total of 15 enriched KEGG pathways were identified. Many of the genes from these pathways were involved in metabolic processes or signaling. More than 300 differentially expressed transcription factors were identified.
Gene expression changes in corollas were detected within 12 hap, well before fertilization and corolla wilting or ethylene evolution. Significant changes in gene expression occurred at 18 hap, including the up-regulation of autophagy and down-regulation of ribosomal genes and genes involved in carbon fixation. This transcriptomic database will greatly expand the genetic resources available in petunia. Additionally, it will guide future research aimed at identifying the best targets for increasing flower longevity by delaying corolla senescence.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0307-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4245787  PMID: 25403317
RNA-seq; WGCNA; de novo assembly; String; KEGG; Trinity; Autophagy; Calcium signaling; Ethylene; Petal senescence
5.  A Jasmonate ZIM-Domain Protein NaJAZd Regulates Floral Jasmonic Acid Levels and Counteracts Flower Abscission in Nicotiana attenuata Plants 
PLoS ONE  2013;8(2):e57868.
Jasmonic acid is an important regulator of plant growth, development and defense. The jasmonate-ZIM domain (JAZ) proteins are key regulators in jasmonate signaling ubiquitously present in flowering plants but their functional annotation remains largely incomplete. Recently, we identified 12 putative JAZ proteins in native tobacco, Nicotiana attenuata, and initiated systematic functional characterization of these proteins by reverse genetic approaches. In this report, Nicotiana attenuata plants silenced in the expression of NaJAZd (irJAZd) by RNA interference were used to characterize NaJAZd function. Although NaJAZd transcripts were strongly and transiently up-regulated in the rosette leaves by simulated herbivory treatment, we did not observe strong defense-related phenotypes, such as altered herbivore performance or the constitutive accumulation of defense-related secondary metabolites in irJAZd plants compared to wild type plants, both in the glasshouse and the native habitat of Nicotiana attenuata in the Great Basin Desert, Utah, USA. Interestingly, irJAZd plants produced fewer seed capsules than did wild type plants as a result of increased flower abscission in later stages of flower development. The early- and mid-developmental stages of irJAZd flowers had reduced levels of jasmonic acid and jasmonoyl-L-isoleucine, while fully open flowers had normal levels, but these were impaired in NaMYB305 transcript accumulations. Previously, NaMYB305-silenced plants were shown to have strong flower abscission phenotypes and contained lower NECTARIN 1 transcript levels, phenotypes which are copied in irJAZd plants. We propose that the NaJAZd protein is required to counteract flower abscission, possibly by regulating jasmonic acid and jasmonoyl-L-isoleucine levels and/or expression of NaMYB305 gene in Nicotiana attenuata flowers. This novel insight into the function of JAZ proteins in flower and seed development highlights the diversity of functions played by jasmonates and JAZ proteins.
PMCID: PMC3585257  PMID: 23469091
6.  Effects of molybdenum on expression of cold-responsive genes in abscisic acid (ABA)-dependent and ABA-independent pathways in winter wheat under low-temperature stress 
Annals of Botany  2009;104(2):345-356.
Background and Aims
Molybdenum (Mo) is an essential trace element for higher plants. It has been shown that application of Mo enhances the cold resistance of winter wheat. In order to improve our understanding of the molecular mechanisms of cold resistance arising from application of Mo in winter wheat, investigations were made regarding the transcription of cold-responsive (COR) genes in abscisic acid (ABA)-dependent and ABA-independent pathways in winter wheat regulated by Mo application under low-temperature stress.
Two cultivars of winter wheat (Triticum aestivum), Mo-efficient cultivar ‘97003’ and Mo-inefficient cultivar ‘97014’, were grown in control (−Mo) and Mo fertilizer (+Mo) treatments for 40 d at 15/12 °C (day/night), and the temperature was then reduced to 5/2 °C (day/night) to create low-temperature stress. Aldehyde oxidase (AO) activities, ABA contents, the transcripts of basic leucine zipper (bZIP)-type transcription factor (TF) genes, ABA-dependent COR genes, CBF/DREB transcription factor genes and ABA-independent COR genes were investigated at 0, 3, 6 and 48 h post cold stress.
Key Results
Mo application significantly increased AO activity, ABA levels, and expression of bZIP-type TF genes (Wlip19 and Wabi5) and ABA-dependent COR genes (Wrab15, Wrab17, Wrab18 and Wrab19). Mo application increased expression levels of CBF/DREB transcription factor genes (TaCBF and Wcbf2-1) and ABA-independent COR genes (Wcs120, Wcs19, Wcor14 and Wcor15) after 3 and 6 h exposure to low temperature.
Mo might regulate the expression of ABA-dependent COR genes through the pathway: Mo → AO → ABA → bZIP → ABA-dependent COR genes in winter wheat. The response of the ABA-dependent pathway to Mo was prior to that of the ABA-independent pathway. Similarities and differences between the Mo-efficient and Mo-inefficient wheat cultivars in response to Mo under cold stress are discussed.
PMCID: PMC2710908  PMID: 19491090
Molybdenum; cold resistance; cold responsive gene; low-temperature stress; ABA-dependent pathway; ABA-independent pathway; aldehyde oxidase
7.  The regulatory network of ThbZIP1 in response to abscisic acid treatment 
Previously, a bZIP transcription factor from Tamarix hispida, ThbZIP1, was characterized: plants overexpressing ThbZIP1 displayed improved salt stress tolerance but were sensitive to abscisic acid (ABA). In the current study, we further characterized the regulatory network of ThbZIP1 and the mechanism of ABA sensitivity mediated by ThbZIP1. An ABF transcription factor from T. hispida, ThABF1, directly regulates the expression of ThbZIP1. Microarray analysis identified 1662 and 1609 genes that were respectively significantly upregulated or downregulated by ThbZIP1 when exposed to ABA. Gene ontology (GO) analysis showed that the processes including “response to stimulus,” “catalytic activity,” “binding function,” and “metabolic process” were highly altered in ThbZIP1 expressing plants exposed to ABA. The gene expression in ThbZIP1 transformed plants were compared between exposed to ABA and salt on the genome scale. Genes differentially regulated by both salt and ABA treatment only accounted for 9.75% of total differentially regulated genes. GO analysis showed that structural molecule activity, organelle part, membrane-enclosed lumen, reproduction, and reproductive process are enhanced by ABA but inhibited by salt stress. Conversely, immune system and multi-organism process were improved by salt but inhibited by ABA. Transcription regulator activity, enzyme regulator activity, and developmental process were significantly altered by ABA but were not affected by salt stress. Our study provides insights into how ThbZIP1 mediates ABA and salt stress response at the molecular level.
PMCID: PMC4322638  PMID: 25713576
bZIP transcription factor; cis-acting element; abiotic stress; yeast one hybrid; gene expression regulation; microarray analysis
8.  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.
PMCID: PMC2913393  PMID: 20531402
abscisic acid; Arabidopsis thaliana; Boolean modeling; heterotrimeric G-protein; transcriptome
9.  Diverting the Flux of the JA Pathway in Nicotiana attenuata Compromises the Plant's Defense Metabolism and Fitness in Nature and Glasshouse 
PLoS ONE  2011;6(10):e25925.
A plant's inducible defenses against herbivores as well as certain developmental processes are known to be controlled by the jasmonic acid (JA) pathway. We have previously shown that ectopically expressing Arabidopsis thaliana JA O-methyltransferase in Nicotiana attenuata (35S-jmt) strongly reduces the herbivory-elicited jasmonate bursts by acting as metabolic sink that redirects free JA towards methylation; here we examine the consequences of this metabolic sink on N. attenuata's secondary metabolism and performance in nature. In the glasshouse, 35S-jmt plants produced fewer seed capsules due to shorter floral styles, which could be restored to wild type (WT) levels after hand-pollination, and were more susceptible to Manduca sexta larvae attack. When transplanted into the Great Basin Desert in Utah, 35S-jmt plants grew as well as WT empty vector, but were highly attacked by native herbivores of different feeding guilds: leaf chewers, miners, and single cell feeders. This greater susceptibility was strongly associated with reduced emissions of volatile organic compounds (hexenylesters, monoterpenes and sesquiterpenes) and profound alterations in the production of direct defenses (trypsin proteinase inhibitors [TPI], nicotine, diterpene glycosides [DTGs] and phenylpropanoid-polyamine conjugates) as revealed by a combination of targeted and metabolomics analyses of field collected samples. Complementation experiments with JA-Ile, whose formation is outcompeted in 35S-jmt plants by the methylation reaction, restored the local TPI activation to WT levels and partially complemented nicotine and DTG levels in elicited but not systemic leaves. These findings demonstrate that MeJA, the major JA metabolite in 35S-jmt plants, is not an active signal in defense activation and highlights the value of creating JA sinks to disrupt JA signaling, without interrupting the complete octadecanoid pathway, in order to investigate the regulation of plants' defense metabolism in nature.
PMCID: PMC3189938  PMID: 22022469
10.  Do pollinator distributions underlie the evolution of pollination ecotypes in the Cape shrub Erica plukenetii? 
Annals of Botany  2013;113(2):301-316.
Background and Aims
According to the Grant–Stebbins model of pollinator-driven divergence, plants that disperse beyond the range of their specialized pollinator may adapt to a new pollination system. Although this model provides a compelling explanation for pollination ecotype formation, few studies have directly tested its validity in nature. Here we investigate the distribution and pollination biology of several subspecies of the shrub Erica plukenetii from the Cape Floristic Region in South Africa. We analyse these data in a phylogenetic context and combine these results with information on pollinator ranges to test whether the evolution of pollination ecotypes is consistent with the Grant–Stebbins model.
Methods and Key Results
Pollinator observations showed that the most common form of E. plukenetii with intermediate corolla length is pollinated by short-billed Orange-breasted sunbirds. Populations at the northern fringe of the distribution are characterized by long corollas, and are mainly pollinated by long-billed Malachite sunbirds. A population with short corollas in the centre of the range was mainly pollinated by insects, particularly short-tongued noctuid moths. Bird exclusion in this population did not have an effect on fruit set, while insect exclusion reduced fruit set. An analysis of floral scent across the range, using coupled gas chromatography–mass spectrometry, showed that the scent bouquets of flowers from moth-pollinated populations are characterized by a larger number of scent compounds and higher emission rates than those in bird-pollinated populations. This was also reflected in clear separation of moth- and bird-pollinated populations in a two-dimensional phenotype space based on non-metric multidimensional scaling analysis of scent data. Phylogenetic analyses of chloroplast and nuclear DNA sequences strongly supported monophyly of E. plukenetii, but not of all the subspecies. Reconstruction of ancestral character states suggests two shifts from traits associated with short-billed Orange-breasted sunbird pollination: one towards traits associated with moth pollination, and one towards traits associated with pollination by long-billed Malachite sunbirds. The latter shift coincided with the colonization of Namaqualand in which Orange-breasted sunbirds are absent.
Conclusions Erica plukenetii
is characterized by three pollination ecotypes, but only the evolutionary transition from short- to long-billed sunbird pollination can be clearly explained by the Grant–Stebbins model. Corolla length is a key character for both ecotype transitions, while floral scent emission was important for the transition from bird to moth pollination.
PMCID: PMC3890384  PMID: 24071499
Helicoverpa armigera; speciation; benzene propanoids; phylogeny; niche; floral scent; bird pollination; floral trait; ancestral character state; Erica plukenetii; pollination ecotype; Cape flora
11.  Identification and expression analysis of ERF transcription factor genes in petunia during flower senescence and in response to hormone treatments 
Journal of Experimental Botany  2010;62(2):825-840.
Ethylene-responsive element-binding factor (ERF) genes constitute one of the largest transcription factor gene families in plants. In Arabidopsis and rice, only a few ERF genes have been characterized so far. Flower senescence is associated with increased ethylene production in many flowers. However, the characterization of ERF genes in flower senescence has not been reported. In this study, 13 ERF cDNAs were cloned from petunia. Based on the sequence characterization, these PhERFs could be classified into four of the 12 known ERF families. Their predicted amino acid sequences exhibited similarities to ERFs from other plant species. Expression analyses of PhERF mRNAs were performed in corollas and gynoecia of petunia flower. The 13 PhERF genes displayed differential expression patterns and levels during natural flower senescence. Exogenous ethylene accelerates the transcription of the various PhERF genes, and silver thiosulphate (STS) decreased the transcription of several PhERF genes in corollas and gynoecia. PhERF genes of group VII showed a strong association with the rise in ethylene production in both petals and gynoecia, and might be associated particularly with flower senescence in petunia. The effect of sugar, methyl jasmonate, and the plant hormones abscisic acid, salicylic acid, and 6-benzyladenine in regulating the different PhERF transcripts was investigated. Functional nuclear localization signal analyses of two PhERF proteins (PhERF2 and PhERF3) were carried out using fluorescence microscopy. These results supported a role for petunia PhERF genes in transcriptional regulation of petunia flower senescence processes.
PMCID: PMC3003824  PMID: 20974735
ERF; ethylene; flower senescence; gene expression; petunia
12.  Cytoplasmic male sterility in Mimulus hybrids has pleiotropic effects on corolla and pistil traits 
Heredity  2011;106(5):886-893.
The mechanisms underlying genetic associations have important consequences for evolutionary outcomes, but distinguishing linkage from pleiotropy is often difficult. Here, we use a fine mapping approach to determine the genetic basis of association between cytonuclear male sterility and other floral traits in Mimulus hybrids. Previous work has shown that male sterility in hybrids between Mimulus guttatus and Mimulus nasutus is due to interactions between a mitochondrial gene from M. guttatus and two tightly linked nuclear restorer alleles on Linkage Group 7, and that male sterility is associated with reduced corolla size. In the present study, we generated a set of nearly isogenic lines segregating for the restorer region and male sterility, but with unique flanking introgressions. Male-sterile flowers had significantly smaller corollas, longer styles and greater stigmatic exsertion than fertile flowers. Because these effects were significant regardless of the genotypic composition of introgressions flanking the restorer region, they suggest that these floral differences are a direct byproduct of the genetic incompatibility causing anther abortion. In addition, we found a non-significant but intriguing trend for male-sterile plants to produce more seeds per flower than fertile siblings after supplemental pollination. Such pleiotropic effects may underlie the corolla dimorphism frequently observed in gynodioecious taxa and may affect selection on cytoplasmic male sterility genes when they initially arise.
PMCID: PMC3186224  PMID: 21245895
gynodioecy; sexual dimorphism; CMS; genetic correlation
13.  Rapid modification of the insect elicitor N-linolenoyl-glutamate via a lipoxygenase-mediated mechanism on Nicotiana attenuata leaves 
BMC Plant Biology  2010;10:164.
Some plants distinguish mechanical wounding from herbivore attack by recognizing specific constituents of larval oral secretions (OS) which are introduced into plant wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of Manduca sexta OS and strong elicitors of herbivore-induced defense responses in Nicotiana attenuata plants.
The metabolism of one of the major FACs in M. sexta OS, N-linolenoyl-glutamic acid (18:3-Glu), was analyzed on N. attenuata wounded leaf surfaces. Between 50 to 70% of the 18:3-Glu in the OS or of synthetic 18:3-Glu were metabolized within 30 seconds of application to leaf wounds. This heat-labile process did not result in free α-linolenic acid (18:3) and glutamate but in the biogenesis of metabolites both more and less polar than 18:3-Glu. Identification of the major modified forms of this FAC showed that they corresponded to 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu. The formation of these metabolites occurred on the wounded leaf surface and it was dependent on lipoxygenase (LOX) activity; plants silenced in the expression of NaLOX2 and NaLOX3 genes showed more than 50% reduced rates of 18:3-Glu conversion and accumulated smaller amounts of the oxygenated derivatives compared to wild-type plants. Similar to 18:3-Glu, 13-oxo-13:2-Glu activated the enhanced accumulation of jasmonic acid (JA) in N. attenuata leaves whereas 13-hydroxy-18:3-Glu did not. Moreover, compared to 18:3-Glu elicitation, 13-oxo-13:2-Glu induced the differential emission of two monoterpene volatiles (β-pinene and an unidentified monoterpene) in irlox2 plants.
The metabolism of one of the major elicitors of herbivore-specific responses in N. attenuata plants, 18:3-Glu, results in the formation of oxidized forms of this FAC by a LOX-dependent mechanism. One of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of JA biosynthesis and differential monoterpene emission.
PMCID: PMC3095298  PMID: 20696061
14.  Differential transcriptome analysis reveals insight into monosymmetric corolla development of the crucifer Iberis amara 
BMC Plant Biology  2014;14:285.
In the co-evolution between insects and plants, the establishment of floral monosymmetry was an important step in angiosperm development as it facilitated the interaction with insect pollinators and, by that, likely enhanced angiosperm diversification. In Antirrhinum majus, the TCP transcription factor CYCLOIDEA is the molecular key regulator driving the formation of floral monosymmetry. Although most Brassicaceae form a polysymmetric corolla, six genera develop monosymmetric flowers with two petal pairs of unequal size. In the monosymmetric crucifer Iberis amara, formation of the different petal pairs coincides with a stronger expression of the CYC-homolog IaTCP1 in the small, adaxial petals.
In this study, RNA-Seq was employed to reconstruct the petal transcriptome of the non-model species Iberis amara. About 9 Gb of sequence data was generated, processed and re-assembled into 18,139 likely Iberis unigenes, from which 15,983 showed high sequence homology to Arabidopsis proteins. The transcriptome gives detailed insight into the molecular mechanisms governing late petal development. In addition, it was used as a scaffold to detect genes differentially expressed between the small, adaxial and the large, abaxial petals in order to understand the molecular mechanisms driving unequal petal growth. Far more genes are expressed in adaxial compared to abaxial petals implying that IaTCP1 activates more genes than it represses. Amongst all genes upregulated in adaxial petals, a significantly enhanced proportion is associated with cell wall modification and cell-cell signalling processes. Furthermore, microarrays were used to detect and compare quantitative differences in TCP target genes in transgenic Arabidopsis plants ectopically expressing different TCP transcription factors.
The increased occurrences of genes implicated in cell wall modification and signalling implies that unequal petal growth is achieved through an earlier stop of the cell proliferation phase in the small, adaxial petals, followed by the onset of cell expansion. This process, which forms the monosymmetric corolla of Iberis amara, is likely driven by the enhanced activity of IaTCP1 in adaxial petals.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0285-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4245847  PMID: 25407089
Brassicaceae; Monosymmetry; CYC; TCP1; RNA-Seq; Microarray
15.  Marcescent corollas as functional structures: effects on the fecundity of two insect-pollinated plants 
Annals of Botany  2010;106(4):659-662.
Background and aims
Persistence of withered corollas after anthesis (‘corolla marcescence’) is widespread in angiosperms, yet its functional significance does not seem to have been explored for any species. This note reports the results of experiments assessing the fecundity effects of marcescent corollas in two southern Spanish insect-pollinated plants, Lavandula latifolia (Lamiaceae) and Viola cazorlensis (Violaceae).
The effect of marcescent corollas on seed production was evaluated experimentally on wild-growing plants. Newly open flowers were randomly assigned to either control or treatment groups in experimental plants. After anthesis, withered corollas of treatment flowers were removed and those in control flowers were left in place. Fruits produced by treatment and control flowers were collected shortly before dehiscence and the number of seeds counted.
Key Results
In V. cazorlensis, removal of withered corollas had no effect on percentage of fruit set, but mean seeds per fruit increased from 9·5 to 11·4. In L. latifolia, corolla removal had no effect on the number of seeds per fruit, but reduced the proportion of flowers ripening fruit from 60 % to 40 %. The detrimental effect of corolla removal on L. latifolia fecundity resulted from the drastic increase in fruit infestation by seed-predatory cecidomyiid larvae, which occurred in 4 % and 34 % of control and treatment fruits, respectively.
Because of their potential effects on plant fecundity, marcescent corollas should not be dismissed a priori as biologically irrelevant leftovers from past floral functions. The simplicity of the experimental layout required to test for short-term fecundity effects of corolla marcescence should help to achieve a better understanding of the ecological and evolutionary correlates of this widespread but poorly understood trait.
PMCID: PMC2944983  PMID: 20870656
Corolla marcescence; fecundity; Lavandula latifolia (Lamiaceae); seed predation; Viola cazorlensis (Violaceae)
16.  Genetic association mapping identifies single nucleotide polymorphisms in genes that affect abscisic acid levels in maize floral tissues during drought 
Journal of Experimental Botany  2010;62(2):701-716.
In maize, water stress at flowering causes loss of kernel set and productivity. While changes in the levels of sugars and abscisic acid (ABA) are thought to play a role in this stress response, the mechanistic basis and genes involved are not known. A candidate gene approach was used with association mapping to identify loci involved in accumulation of carbohydrates and ABA metabolites during stress. A panel of single nucleotide polymorphisms (SNPs) in genes from these metabolic pathways and in genes for reproductive development and stress response was used to genotype 350 tropical and subtropical maize inbred lines that were well watered or water stressed at flowering. Pre-pollination ears, silks, and leaves were analysed for sugars, starch, proline, ABA, ABA-glucose ester, and phaseic acid. ABA and sugar levels in silks and ears were negatively correlated with their growth. Association mapping with 1229 SNPs in 540 candidate genes identified an SNP in the maize homologue of the Arabidopsis MADS-box gene, PISTILLATA, which was significantly associated with phaseic acid in ears of well-watered plants, and an SNP in pyruvate dehydrogenase kinase, a key regulator of carbon flux into respiration, that was associated with silk sugar concentration. An SNP in an aldehyde oxidase gene was significantly associated with ABA levels in silks of water-stressed plants. Given the short range over which decay of linkage disequilibrium occurs in maize, the results indicate that allelic variation in these genes affects ABA and carbohydrate metabolism in floral tissues during drought.
PMCID: PMC3003815  PMID: 21084430
ASI; abscisic acid; association mapping; drought; flower set; kernel set
17.  Ethylene-Induced Inhibition of Root Growth Requires Abscisic Acid Function in Rice (Oryza sativa L.) Seedlings 
PLoS Genetics  2014;10(10):e1004701.
Ethylene and abscisic acid (ABA) have a complicated interplay in many developmental processes. Their interaction in rice is largely unclear. Here, we characterized a rice ethylene-response mutant mhz4, which exhibited reduced ethylene-response in roots but enhanced ethylene-response in coleoptiles of etiolated seedlings. MHZ4 was identified through map-based cloning and encoded a chloroplast-localized membrane protein homologous to Arabidopsis thaliana (Arabidopsis) ABA4, which is responsible for a branch of ABA biosynthesis. MHZ4 mutation reduced ABA level, but promoted ethylene production. Ethylene induced MHZ4 expression and promoted ABA accumulation in roots. MHZ4 overexpression resulted in enhanced and reduced ethylene response in roots and coleoptiles, respectively. In root, MHZ4-dependent ABA pathway acts at or downstream of ethylene receptors and positively regulates root ethylene response. This ethylene-ABA interaction mode is different from that reported in Arabidopsis, where ethylene-mediated root inhibition is independent of ABA function. In coleoptile, MHZ4-dependent ABA pathway acts at or upstream of OsEIN2 to negatively regulate coleoptile ethylene response, possibly by affecting OsEIN2 expression. At mature stage, mhz4 mutation affects branching and adventitious root formation on stem nodes of higher positions, as well as yield-related traits. Together, our findings reveal a novel mode of interplay between ethylene and ABA in control of rice growth and development.
Author Summary
Rice is a monocotyledonous plant that is distinct from the dicotyledonous model plant Arabidopsis in many aspects. In Arabidopsis, ethylene-induced root inhibition is independent of ABA action. In rice, however, we report here that ethylene inhibition of root growth requires ABA function. We identified MHZ4, a rice homolog of Arabidopsis ABA4 that is involved in ABA biosynthesis. The mhz4 mutant displayed reduced ABA level and exhibited ethylene-hyposensitive root, but -hypersensitive coleoptile phenotypes in etiolated seedlings. Exogenous application of ABA largely recovered the defective ethylene responses. Overexpression of MHZ4 resulted in enhanced and reduced ethylene response in the roots and coleoptiles, respectively. In root, MHZ4-dependent ABA pathway genetically acts at or downstream of ethylene receptors and positively regulates root ethylene response. Moreover, ethylene treatment stimulated ABA production in roots at least through transcriptional activation of MHZ4. The results indicate that ethylene-induced root inhibition in rice is largely mediated through MHZ4-dependent ABA function. In coleoptile, MHZ4-dependent ABA pathway acts at or upstream of OsEIN2 and negatively regulates coleoptile ethylene response, possibly via transcriptional suppression of OsEIN2. Together, our findings reveal a novel mode of ethylene-ABA interaction which is fundamentally different from that in Arabidopsis.
PMCID: PMC4199509  PMID: 25330236
18.  Endogenous Abscisic Acid Promotes Hypocotyl Growth and Affects Endoreduplication during Dark-Induced Growth in Tomato (Solanum lycopersicum L.) 
PLoS ONE  2015;10(2):e0117793.
Dark-induced growth (skotomorphogenesis) is primarily characterized by rapid elongation of the hypocotyl. We have studied the role of abscisic acid (ABA) during the development of young tomato (Solanum lycopersicum L.) seedlings. We observed that ABA deficiency caused a reduction in hypocotyl growth at the level of cell elongation and that the growth in ABA-deficient plants could be improved by treatment with exogenous ABA, through which the plants show a concentration dependent response. In addition, ABA accumulated in dark-grown tomato seedlings that grew rapidly, whereas seedlings grown under blue light exhibited low growth rates and accumulated less ABA. We demonstrated that ABA promotes DNA endoreduplication by enhancing the expression of the genes encoding inhibitors of cyclin-dependent kinases SlKRP1 and SlKRP3 and by reducing cytokinin levels. These data were supported by the expression analysis of the genes which encode enzymes involved in ABA and CK metabolism. Our results show that ABA is essential for the process of hypocotyl elongation and that appropriate control of the endogenous level of ABA is required in order to drive the growth of etiolated seedlings.
PMCID: PMC4334974  PMID: 25695830
19.  Pectin methylesterase NaPME1 contributes to the emission of methanol during insect herbivory and to the elicitation of defence responses in Nicotiana attenuata 
Journal of Experimental Botany  2009;60(9):2631-2640.
Pectin methylesterases (PMEs) catalyse the demethylation of pectin within plant cell walls, releasing methanol (MeOH) in the process. Thus far, PMEs have been found to be involved in diverse processes such as plant growth and development and defence responses against pathogens. Herbivore attack increases PME expression and activity and MeOH emissions in several plant species. To gain further insights into the role of PMEs in defence responses against herbivores, the expression of a Manduca sexta oral secretion (OS)-inducible PME in Nicotiana attenuata (NaPME1) was silenced by RNA interference (RNAi)-mediated gene silencing. Silenced lines (ir-pme) showed 50% reduced PME activity in leaves and 70% reduced MeOH emissions after OS elicitation compared with the wild type (WT), demonstrating that the herbivore-induced MeOH emissions originate from the demethylation of pectin by PME. In the initial phase of the OS-induced jasmonic acid (JA) burst (first 30 min), ir-pme lines produced WT levels of this hormone and of jasmonyl-isoleucine (JA-Ile); however, these levels were significantly reduced in the later phase (60–120 min) of the burst. Similarly, suppressed levels of the salicylic acid (SA) burst induced by OS elicitation were observed in ir-pme lines even though wounded ir-pme leaves contained slightly increased amounts of SA. This genotype also presented reduced levels of OS-induced trypsin proteinase inhibitor activity in leaves and consistently increased M. sexta larvae performance compared with WT plants. These latter responses could not be recovered by application of exogenous MeOH. Together, these results indicated that PME contributes, probably indirectly by affecting cell wall properties, to the induction of anti-herbivore responses.
PMCID: PMC2692009  PMID: 19380422
Defence; herbivory; jasmonic acid; Manduca; methanol; Nicotiana; pectin methylesterase; proteinase inhibitor
20.  Ectopic TPS expression enhances sesquiterpene emission in Nicotiana attenuata without altering defense or development of transgenic plants or neighbors 
Plant physiology  2014;166(2):779-797.
Sesquiterpenoids, with ca. 5000 structures, are the most diverse class of plant volatiles, with manifold hypothesized functions in defense, stress tolerance, and signaling between and within plants. These hypotheses have often been tested by transforming plants with sesquiterpene synthases (STPS’s) expressed behind the constitutively active 35S promoter, which may have physiological costs, measured as inhibited growth and reduced reproduction, or require augmentation of substrate pools to achieve enhanced emission, complicating the interpretation of data from affected transgenic lines. Here, we expressed Zea mays TPSlO (ZmTPSl0), which produces (E)-α-bergamotene and (E)-β-farnesene, or a point mutant, ZmTPSlOM, producing primarily (E)-β-farnesene, under control of the 35S promoter in the ecological model plant Nicotiana attenuata. Transgenic N. attenuata plants had specifically enhanced emission of target sesquiterpene(s) with no changes detected in their emission of any other volatiles. Treatment with herbivore or jasmonate elicitors induces emission of (E)-α-bergamotene in wild-type (WT) plants, and also tended to increase emission of (E)-α-bergamotene and (E)-β-farnesene in transgenics. However, transgenics did not differ from WT in defense signaling or chemistry, and did not alter defense chemistry in neighboring WT plants. These data are inconsistent with within- or between-plant signaling functions of (E)-β-farnesene and (E)-α-bergamotene in N. attenuata. Ectopic sesquiterpene emission was apparently not costly for transgenics, which were similar to WT plants in their growth and reproduction even when forced to compete for common resources. These transgenics would be well suited for field experiments to investigate indirect, ecological effects of sesquiterpenes for a wild plant in its native habitat.
PMCID: PMC4190577  PMID: 25187528
21.  Multiple interactions of NaHER1 protein with abscisic acid signaling in Nicotiana attenuata plants 
Plant Signaling & Behavior  2013;8(11):e26365.
Previously, we identified a novel herbivore elicitor-regulated protein in Nicotiana attenuata (NaHER1) that is required to suppress abscisic acid (ABA) catabolism during herbivore attack and activate a full defense response against herbivores. ABA, in addition to its newly defined role in defense activation, mainly controls seed germination and stomatal function of land plants. Here we show that N. attenuata seeds silenced in the expression of NaHER1 by RNA interference (irHER1) accumulated less ABA during germination, and germinated faster on ABA-containing media compared to WT. Curiously, epidermal cells of irHER1 plants were wrinkled, possibly due to the previously demonstrated increase in transpiration of irHER1 plants that may affect turgor and cause wrinkling of the cells. We conclude that NaHER1 is a highly pleiotropic regulator of ABA responses in N. attenuata plants.
PMCID: PMC4091387  PMID: 24022276
Nicotiana attenuata; abscisic acid (ABA); ABA metabolism; germination
22.  A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity 
PLoS Pathogens  2011;7(7):e1002148.
Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H2O2 and O2−, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H2O2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses.
Author Summary
This study provides an explanation for the strong resistance to B. cinerea observed in wounded plants or plants with cuticular defects. We have observed that a production of ROS and a permeable cuticle is common to all these situations. ROS, that include hydrogen peroxide, are known inducers of resistance and can also act directly against the invading fungus. Degradation of the cuticle by exposure to cutinase also results in the production of ROS and resistance. These observations lead to a model where the cuticle plays a central role as a barrier against water-soluble elicitors from the surface. Under normal circumstances, the cuticle does not allow the passage of elicitors and no responses are induced. Under conditions where the cuticular barrier is broken, ROS and resistance are induced. This illustrates why plants that are in fact permanently exposed to potential elicitors do not constantly induce immune responses: this only takes place once the cuticle has been permeabilized, for example after an infection with a pathogen. This study also demonstrates how a cuticle-degrading pathogen avoids the generation of ROS by producing an effector that interferes with ROS production. Removal of this effector restores both ROS and resistance.
PMCID: PMC3145797  PMID: 21829351
23.  Effects of abscisic acid on ethylene biosynthesis and perception in Hibiscus rosa-sinensis L. flower development 
Journal of Experimental Botany  2011;62(15):5437-5452.
The effect of the complex relationship between ethylene and abscisic acid (ABA) on flower development and senescence in Hibiscus rosa-sinensis L. was investigated. Ethylene biosynthetic (HrsACS and HrsACO) and receptor (HrsETR and HrsERS) genes were isolated and their expression evaluated in three different floral tissues (petals, style–stigma plus stamens, and ovaries) of detached buds and open flowers. This was achieved through treatment with 0.1 mM 1-aminocyclopropane-1-carboxylic acid (ACC) solution, 500 nl l−1 methylcyclopropene (1-MCP), and 0.1 mM ABA solution. Treatment with ACC and 1-MCP confirmed that flower senescence in hibiscus is ethylene dependent, and treatment with exogenous ABA suggested that ABA may play a role in this process. The 1-MCP impeded petal in-rolling and decreased ABA content in detached open flowers after 9 h. This was preceded by an earlier and sequential increase in ABA content in 1-MCP-treated petals and style–stigma plus stamens between 1 h and 6 h. ACC treatment markedly accelerated flower senescence and increased ethylene production after 6 h and 9 h, particularly in style–stigma plus stamens. Ethylene evolution was positively correlated in these floral tissues with the induction of the gene expression of ethylene biosynthetic and receptor genes. Finally, ABA negatively affected the ethylene biosynthetic pathway and tissue sensitivity in all flower tissues. Transcript abundance of HrsACS, HrsACO, HrsETR, and HrsERS was reduced by exogenous ABA treatment. This research underlines the regulatory effect of ABA on the ethylene biosynthetic and perception machinery at a physiological and molecular level when inhibitors or promoters of senescence are exogenously applied.
PMCID: PMC3223042  PMID: 21841180
Abscisic acid content; ACO; ACS; ERS; ethylene production; ETR; floral tissue; flower senescence
24.  Common and unique elements of the ABA-regulated transcriptome of Arabidopsis guard cells 
BMC Genomics  2011;12:216.
In the presence of drought and other desiccating stresses, plants synthesize and redistribute the phytohormone abscisic acid (ABA). ABA promotes plant water conservation by acting on specialized cells in the leaf epidermis, guard cells, which border and regulate the apertures of stomatal pores through which transpirational water loss occurs. Following ABA exposure, solute uptake into guard cells is rapidly inhibited and solute loss is promoted, resulting in inhibition of stomatal opening and promotion of stomatal closure, with consequent plant water conservation. There is a wealth of information on the guard cell signaling mechanisms underlying these rapid ABA responses. To investigate ABA regulation of gene expression in guard cells in a systematic genome-wide manner, we analyzed data from global transcriptomes of guard cells generated with Affymetrix ATH1 microarrays, and compared these results to ABA regulation of gene expression in leaves and other tissues.
The 1173 ABA-regulated genes of guard cells identified by our study share significant overlap with ABA-regulated genes of other tissues, and are associated with well-defined ABA-related promoter motifs such as ABREs and DREs. However, we also computationally identified a unique cis-acting motif, GTCGG, associated with ABA-induction of gene expression specifically in guard cells. In addition, approximately 300 genes showing ABA-regulation unique to this cell type were newly uncovered by our study. Within the ABA-regulated gene set of guard cells, we found that many of the genes known to encode ion transporters associated with stomatal opening are down-regulated by ABA, providing one mechanism for long-term maintenance of stomatal closure during drought. We also found examples of both negative and positive feedback in the transcriptional regulation by ABA of known ABA-signaling genes, particularly with regard to the PYR/PYL/RCAR class of soluble ABA receptors and their downstream targets, the type 2C protein phosphatases. Our data also provide evidence for cross-talk at the transcriptional level between ABA and another hormonal inhibitor of stomatal opening, methyl jasmonate.
Our results engender new insights into the basic cell biology of guard cells, reveal common and unique elements of ABA-regulation of gene expression in guard cells, and set the stage for targeted biotechnological manipulations to improve plant water use efficiency.
PMCID: PMC3115880  PMID: 21554708
25.  Physiological and molecular responses to drought in Petunia: the importance of stress severity 
Journal of Experimental Botany  2012;63(18):6335-6345.
Plant responses to drought stress vary depending on the severity of stress and the stage of drought progression. To improve the understanding of such responses, the leaf physiology, abscisic acid (ABA) concentration, and expression of genes associated with ABA metabolism and signalling were investigated in Petunia × hybrida. Plants were exposed to different specific substrate water contents (θ = 0.10, 0.20, 0.30, or 0.40 m3·m–3) to induce varying levels of drought stress. Plant responses were investigated both during the drying period (θ decreased to the θ thresholds) and while those threshold θ were maintained. Stomatal conductance (gs) and net photosynthesis (A) decreased with decreasing midday leaf water potential (Ψleaf). Leaf ABA concentration increased with decreasing midday Ψleaf and was negatively correlated with gs (r = –0.92). Despite the increase in leaf ABA concentration under drought, no significant effects on the expression of ABA biosynthesis genes were observed. However, the ABA catabolism-related gene CYP707A2 was downregulated, primarily in plants under severe drought (θ = 0.10 m3∙m–3), suggesting a decrease in ABA catabolism under severe drought. Expression of phospholipase Dα (PLDα), involved in regulating stomatal responses to ABA, was enhanced under drought during the drying phase, but there was no relationship between PLDα expression and midday Ψleaf after the θ thresholds had been reached. The results show that drought response of plants depends on the severity of drought stress and the phase of drought progression.
PMCID: PMC3504489  PMID: 23077204
abscisic acid; acclimation; automated irrigation; soil moisture sensor; stomatal conductance; substrate water content

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