Gibberellin Signaling through RGA Suppresses GCN5 Effects on Arabidopsis Developmental Stages.

Histone acetyltransferases (HATs) modify the amino-terminal tails of the core histone proteins via acetylation, regulating chromatin structure and transcription. GENERAL CONTROL NON-DEREPRESSIBLE 5 (GCN5) is a HAT that specifically acetylates H3K14 residues. GCN5 has been associated with cell division and differentiation, meristem function, root, stem, foliar, and floral development, and plant environmental response. The flowers of gcn5 plants display a reduced stamen length and exhibit male sterility relative to the wild-type plants. We show that these effects may arise from gibberellin (GA)-signaling defects. The signaling pathway of bioactive GAs depends on the proteolysis of their repressors, DELLA proteins. The repressor GA (RGA) DELLA protein represses plant growth, inflorescence, and flower and seed development. Our molecular data indicate that GCN5 is required for the activation and H3K14 acetylation of genes involved in the late stages of GA biosynthesis and catabolism. We studied the genetic interaction of the RGA and GCN5; the RGA can partially suppress GCN5 action during the whole plant life cycle. The reduced elongation of the stamen filament of gcn5-6 mutants is reversed in the rga-t2;gcn5-6 double mutants. RGAs suppress the GCN5 effect on the gene expression and histone acetylation of GA catabolism and GA signaling. Interestingly, the RGA and RGL2 do not suppress ADA2b function, suggesting that ADA2b acts downstream of GA signaling and is distinct from GCN5 activity. In conclusion, we propose that the action of GCN5 on stamen elongation is partially mediated by RGA and GA signaling.

Silencing of a Nicotiana benthamiana ascorbate oxidase gene reveals its involvement in resistance against cucumber mosaic virus.

MAIN CONCLUSION: Silencing of an ascorbate oxidase (AO) gene in N. benthamiana enhanced disease severity from cucumber mosaic virus (CMV), showing higher accumulation and expansion of the spreading area of CMV. A Nicotiana benthamiana ascorbate oxidase (NbAO) gene was found to be induced upon cucumber mosaic virus (CMV) infection. Virus-induced gene silencing (VIGS) was employed to elucidate the function of AO in N. benthamiana. The tobacco rattle virus (TRV)-mediated VIGS resulted in an efficient silencing of the NbAO gene, i.e., 97.5% and 78.8% in relative quantification as compared to the control groups (TRV::eGFP- and the mock-inoculated plants), respectively. In addition, AO enzymatic activity decreased in the TRV::NtAO-silenced plants as compared to control. TRV::NtAO-mediated NbAO silencing induced a greater reduction in plant height by 15.2% upon CMV infection. CMV titer at 3 dpi was increased in the systemic leaves of NbAO-silenced plants (a 35-fold change difference as compared to the TRV::eGFP-treated group). Interestingly, CMV and TRV titers vary in different parts of systemically infected N. benthamiana leaves. In TRV::eGFP-treated plants, CMV accumulated only at the top half of the leaf, whereas the bottom half of the leaf was "occupied" by TRV. In contrast, in the NbAO-silenced plants, CMV accumulated in both the top and the bottom half of the leaf, suggesting that the silencing of the NbAO gene resulted in the expansion of the spreading area of CMV. Our data suggest that the AO gene might function as a resistant factor against CMV infection in N. benthamiana.

Efficient artificial microRNA-mediated resistance against zucchini yellow mosaic virus in zucchini via agroinfiltration.

Zucchini yellow mosaic virus (ZYMV) infects cucurbits causing yellow mosaic in leaves, malformations in fruits, and degradation of the product quality. RNA interference (RNAi) is a cellular mechanism in eukaryotes and it is exploited to protect them against viruses. The artificial micro RNA (amiRNA) mediated approach was employed to develop resistance against ZYMV. Four amiRNAs, amiZYMV_HC-115s and amiZYMV_HC-1162s (sense), amiZYMV_HC-182as and amiZYMV_HC-196as (antisense), were computationally designed and introduced into the AtMIR390a backbone. At four days post agroinfiltration (dpa) of zucchini cotyledons the corresponding pre- and the mature amiRNAs were identified in local tissue. Upon ZYMV inoculation of zucchini, ZYMV titer was significantly lower where amiZYMV_HCs were applied in relation to control starting at two days post inoculation (dpi). Control zucchini plants exhibited symptoms at 5-8 dpi, whereas the amiZYMV_HC-treated zucchini had symptoms at 14 dpi; at 21 dpi treated zucchini exhibited a 16 %, 19 %, 32 %, and 42.5 % protection, respectively. For luffa, we observed a lower protection (0 %, 17 %, 22.5 %, and 31 % at 21 dpi). Nicotiana benthamiana DCL4 knock-down mutants were infected by ZYMV, whereas when the amiZYMV_HC-196as was agroinfiltrated ZYMV was not detected by RT-PCR. These results indicate that amiRNA-mediated resistance could be applied against ZYMV in zucchini.

RNA-Based Vaccination of Plants for Control of Viruses.

Plant viruses cause nearly half of the emerging plant diseases worldwide, contributing to 10-15% of crop yield losses. Control of plant viral diseases is mainly accomplished by extensive chemical applications targeting the vectors (i.e., insects, nematodes, fungi) transmitting these viruses. However, these chemicals have a significant negative effect on human health and the environment. RNA interference is an endogenous, cellular, sequence-specific RNA degradation mechanism in eukaryotes induced by double-stranded RNA molecules that has been exploited as an antiviral strategy through transgenesis. Because genetically modified crop plants are not accepted for cultivation in several countries globally, there is an urgent demand for alternative strategies. This has boosted research on exogenous application of the RNA-based biopesticides that are shown to exhibit significant protective effect against viral infections. Such environment-friendly and efficacious antiviral agents for crop protection will contribute to global food security, without adverse effects on human health.

Metabolomic and Genomic Approach to Study Defense Induction by Nesidiocoris tenuis against Tuta absoluta and Tetranychus urticae in Tomato Plants.

The phytophagy of the predator Nesidiocoris tenuis (Hemiptera: Miridae) can trigger defense responses in tomato plants against pests, such as two spotted spider mite Tetranychus urticae (Acari: Tetranychidae) and South American leaf miner Tuta absoluta (Lepidoptera: Gelechiidae). The expression of genes governing Jasmonic Acid (JA) biosynthesis pathway and fluctuations in the levels of underlying metabolites have been rarely studied in mirid-infested plants. In the present study, fifteen 3rd instar nymphs of N.tenuis were caged on each top and lower leaf of tomato plants for 4 d to induce plant defense; after this period the predators were removed. With regard to T. absoluta, oviposition preference; larval period; and pupal weight were significantly reduced in N. tenuis-punctured plants. T. urticae adults exhibited a significantly higher escape tendency and reduced survival on punctured plants. Metabolomics confirmed such observations revealing substantial differences between N. tenuis-punctured and unpunctured (control) plants. Metabolites directly associated with the activation of the JA defense pathway, such as the precursor α-linolenic acid, had increased concentrations. The expression of the defense-related genes PI-II, MYC2, VSP2, and HEL was increased in the top leaves and only VSP2 and MBP2 in the lower leaves; interestingly, in the middle (unpunctured) leaves VSP2, HEL, and MBP2 were also upregulated, indicating systemic signaling. Collectively, phytophagy of N. tenuis caused adverse effects on T. absoluta and T. urticae, whereas the multi-omics approach (phenomics, metabolomics, and genomics) offered valuable insights into the nature of the plant defense responses and provided useful evidence for future applications in integrated pest management, plausibly resulting in the reduction in the required pesticide volumes.

The Citrus yellow mosaic badnavirus ORFI functions as a RNA-silencing suppressor.

Citrus yellow mosaic badnavirus (CMBV) causes mosaic disease in all economically important citrus cultivars of India, with losses reaching up to 70%. CMBV belongs to the genus Badnavirus, family Caulimoviridae, possessing a circular double-stranded (ds) DNA genome with six open reading frames (ORFs I to VI), whose functions are yet to be deciphered. The RNA-silencing suppressor (RSS) activity has not been assigned to any CMBV ORF as yet. In the present study, it was found that ORFI exhibited RSS activity among all the six CMBV ORFs tested. Studies were done by employing the well-established Agrobacterium-mediated transient assay based on the transgenic Nicotiana benthamiana 16c plant line expressing the green fluorescent protein (GFP). The RSS activity of ORFI was confirmed by the analysis of the GFP visual expression in the agroinfiltrated leaves, further supported by quantification of GFP expression by RT-PCR. Based on the GFP visual expression, the CMBV ORFI was a weak RSS when compared to the p19 protein of tomato bushy stunt virus. In contrast, the ORFII, ORFIV, ORFV, ORFVI, and CP gene did not exhibit any RSS activity. Hence, ORFI is the first ORF of CMBV to be identified with RNA-silencing suppression activity.

dsRNA Molecules From the Tobacco Mosaic Virus p126 Gene Counteract TMV-Induced Proteome Changes at an Early Stage of Infection.

Exogenous application of double-stranded RNA (dsRNA) in the tobacco-Tobacco mosaic virus (TMV) pathosystem was shown previously to induce resistance against TMV providing an alternative approach to transgenesis. In the present study, we employed proteomics technology to elucidate the effect of TMV on tobacco as well as the effect of exogenous application of TMV p126 dsRNA molecules (dsRNAp126) at an early stage of the tobacco-TMV interaction. The proteome of tobacco leaf at 15 min post inoculation (mpi) in the presence or absence of dsRNAp126 molecules was studied. Thirty-six tobacco proteins were differentially accumulated in TMV-infected vs. healthy tobacco leaf tissue. The identified main differential TMV-responsive proteins were found to be involved in photosynthesis, energy metabolism, stress, and defense responses. Most of the virus-induced changes in the tobacco leaf proteome were not observed in the leaves treated with dsRNAp126 + TMV. The results indicated that the protein changes induced by TMV infection were counteracted by the exogenous application of dsRNAp126 molecules. Moreover, using small RNA sequencing, we showed that the exogenously applied dsRNAp126 was efficiently processed in tobacco as early as 15 min post application (mpa) to produce small interfering RNAs (siRNAs); the dicing pattern was not affected by the presence of TMV. The presence of dsRNAp126 reduced TMV p126 RNA abundance suggesting virus titer reduction via a sequence-specific mechanism, since a non-homologous dsRNA did not protect from TMV infection nor affect TMV accumulation.

dsRNA-Mediated Pest Management of Tuta absoluta Is Compatible with Its Biological Control Agent Nesidiocoris tenuis.

RNAi-mediated insect pest management has recently shown promising results against the most serious pest of tomato, the tomato leafminer, Tuta absoluta. This study aimed to investigate whether dsRNA (dsTa-αCOP) designed to target the T. absoluta-αCOP gene could cause adverse effects to its biocontrol agent, the mirid predator, Nesidiocoris tenuis. Oral exposure of N. tenuis to dsRNA (dsNt-αCOP) designed to target N. tenuis-αCOP resulted in a 61%, 67% and 55% reduction in its transcript level in comparison to the sucrose, dsGFP and dsTa-αCOP treatments, respectively. In addition, significantly higher mortality of 57% was recorded in dsNt-αCOP-treated N. tenuis when compared to the sucrose (7%), dsGFP (10%) and dsTa-αCOP (10%) treatments. Moreover, the predation rate of ~33-39 Ephestia kuehniella eggs per N. tenuis adult dramatically reduced to almost half in the surviving dsNt-αCOP-treated N. tenuis. This worst-case exposure scenario confirmed for the first time that the RNAi machinery is functional in this species and that the risk of exposure through the oral route is possible. In contrast, dsTa-αCOP did not cause any sub-lethal effects to N. tenuis upon oral exposure. Oral exposure of T. absoluta to dsTa-αCOP resulted in 50% mortality. In the context of a biosafety risk assessment of RNAi-mediated insect management, investigating the effects on non-target organisms is essential in order to include this method as part of an integrated pest management strategy. Based on our laboratory assays, RNAi-mediated control is compatible with the biological control of T. absoluta by its natural enemy N. tenuis, adding the RNAi approach in the armoire of integrated pest management of T. absoluta.

DsRNA-mediated protection against two isolates of Papaya ringspot virus through topical application of dsRNA in papaya.

Papaya ringspot virus (PRSV) infections in papaya result in heavy yield losses, severely affecting the papaya industry worldwide, and hence warranting for effective control measures. In the past, transgenic papaya cultivars were developed that overexpressed parts of the PRSV genome and exhibited high levels of virus resistance. In the present study, a non-transgenic approach was employed, in which in vitro produced dsRNA molecules derived from a PRSV isolate from South India (PRSV-Tirupati) was tested for dsRNA-mediated protection against two isolates of PRSV through topical application of the dsRNA on papaya. The results showed that the dsRNA molecules from both the coat protein (CP) and helper component-proteinase (HC-Pro) genes of the PRSV-Tirupati isolate conferred 100 % resistance against PRSV-Tirupati infection. Further, the same dsRNA molecules were highly effective against the PRSV-Delhi isolate on the papaya cv. Pusa Nanha, conferring a resistance of 94 % and 81 %, respectively. Systemic papaya leaves of the dsRNA-treated plants were virus-free at 14 days post-inoculation, confirming the robustness of this non-transgenic virus control strategy. In contrast, the control TMV dsRNA did not protect against the PRSV infection. This study on the topical application of dsRNA opened up a new avenue for the control of papaya ringspot disease worldwide.

The histone acetyltransferase GCN5 and the transcriptional coactivator ADA2b affect leaf development and trichome morphogenesis in Arabidopsis.

MAIN CONCLUSION: The histone acetyltransferase GCN5 and associated transcriptional coactivator ADA2b are required to couple endoreduplication and trichome branching. Mutation of ADA2b also disrupts the relationship between ploidy and leaf cell size. Dynamic chromatin structure has been established as a general mechanism by which gene function is temporally and spatially regulated, but specific chromatin modifier function is less well understood. To address this question, we have investigated the role of the histone acetyltransferase GCN5 and the associated coactivator ADA2b in developmental events in Arabidopsis thaliana. Arabidopsis plants with T-DNA insertions in GCN5 (also known as HAG1) or ADA2b (also known as PROPORZ1) display pleiotropic phenotypes including dwarfism and floral defects affecting fertility. We undertook a detailed characterization of gcn5 and ada2b phenotypic effects in rosette leaves and trichomes to establish a role for epigenetic control in these developmental processes. ADA2b and GCN5 play specific roles in leaf tissue, affecting cell growth and division in rosette leaves often in complex and even opposite directions. Leaves of gcn5 plants display overall reduced ploidy levels, while ada2b-1 leaves show increased ploidy. Endoreduplication leading to increased ploidy is also known to contribute to normal trichome morphogenesis. We demonstrate that gcn5 and ada2b mutants display alterations in the number and patterning of trichome branches, with ada2b-1 and gcn5-1 trichomes being significantly less branched, while gcn5-6 trichomes show increased branching. Elongation of the trichome stalk and branches also vary in different mutant backgrounds, with stalk length having an inverse relationship with branch number. Taken together, our data indicate that, in Arabidopsis, leaves and trichomes ADA2b and GCN5 are required to couple nuclear content with cell growth and morphogenesis.

Exogenously applied dsRNA molecules deriving from the Zucchini yellow mosaic virus (ZYMV) genome move systemically and protect cucurbits against ZYMV.

Zucchini yellow mosaic virus (ZYMV) causes serious damage in a large number of cucurbits, and control measures are necessary. Transgenic cucurbits expressing parts of the ZYMV genome have been shown to be resistant to the cognate virus. A non-transgenic approach involving the exogenous application of double-stranded RNA (dsRNA) has also been shown to induce resistance in tobacco against Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV). In the present study, dsRNA molecules derived from the helper component-proteinase (HC-Pro) and coat protein (CP) genes of the ZYMV_DE_2014 isolate were produced in vitro. On exogenous dsRNA application in cucumber, watermelon and squash plants, dsRNA HC-Pro conferred resistance of 82%, 50% and 18%, and dsRNA CP molecules of 70%, 43% and 16%, respectively. On deep sequencing analysis of ZYMV-infected watermelon, hot-spot regions for viral small interfering RNAs (vsiRNAs) in the genome of ZYMV were identified. Stem-loop reverse transcription-polymerase chain reaction (RT-PCR) detection of selected 21-nucleotide-long vsiRNAs in plants that received only dsRNA molecules suggested that the dsRNAs exogenously applied onto plants were successfully diced, thus initiating RNA silencing. dsRNA molecules were found to be progressively degraded in planta, and strongly detected by semi-quantitative RT-PCR for at least 9 days after exogenous application. Moreover, dsRNA molecules were detected in systemic tissue of watermelon and squash, showing that dsRNA is transported long distances in these plants.

Plant insects and mites uptake double-stranded RNA upon its exogenous application on tomato leaves.

MAIN CONCLUSION: Exogenously applied double-stranded RNA (dsRNA) molecules onto tomato leaves, moved rapidly from local to systemic leaves and were uptaken by agricultural pests namely aphids, whiteflies and mites. Four small interfering RNAs, deriving from the applied dsRNA, were molecularly detected in plants, aphids and mites but not in whiteflies. Double-stranded RNA (dsRNA) acts as the elicitor molecule of the RNA silencing (RNA interference, RNAi), the endogenous and evolutionary conserved surveillance system present in all eukaryotes. DsRNAs and their subsequent degradation products, namely the small interfering RNAs (siRNAs), act in a sequence-specific manner to control gene expression. Exogenous application of dsRNAs onto plants elicits resistance against plant viruses. In the present work, exogenously applied dsRNA molecules, derived from Zucchini yellow mosaic virus (ZYMV) HC-Pro region, onto tomato plants were detected in aphids (Myzus persicae), whiteflies (Trialeurodes vaporariorum) and mites (Tetranychus urticae) that were fed on treated as well as systemic tomato leaves. Furthermore, four siRNAs, deriving from the dsRNA applied, were detected in tomato and the agricultural pests fed on treated tomato plants. More specifically, dsRNA was detected in agricultural pests at 3 and 10 dpt (days post treatment) in dsRNA-treated leaves and at 14 dpt in systemic leaves. In addition, using stem-loop RT-PCR, siRNAs were detected in agricultural pests at 3 and 10 dpt in aphids and mites. Surprisingly, in whiteflies carrying the applied dsRNA, siRNAs were not molecularly detected. Our results showed that, upon exogenous application of dsRNAs molecules, these moved rapidly within tomato and were uptaken by agricultural pests fed on treated tomato. As a result, this non-transgenic method has the potential to control important crop pests via RNA silencing of vital genes of the respective pests.

Exogenous application of double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco.

MAIN CONCLUSION: External application of dsRNA molecules from Tobacco mosaic virus (TMV) p126 and CP genes confers significant resistance against TMV infection. Exogenously applied dsRNA exhibits a rapid systemic trafficking in planta , and it is processed successfully by DICER-like proteins producing small interfering RNAs. RNA interference (RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism, induced by double-stranded RNA (dsRNA), which protects eukaryotic cells against invasive nucleic acids like viruses and transposons. In the present study, we used a non-transgenic strategy to induce RNAi in Nicotiana tabacum cv. Xanthi plants against TMV. DsRNA molecules for the p126 (TMV silencing suppressor) and coat protein (CP) genes were produced by a two-step PCR approach followed by in vitro transcription. The application of TMV p126 dsRNA onto tobacco plants induced greater resistance against TMV infection as compared to CP dsRNA (65 vs. 50 %). This study also reported the fast systemic spread of TMV p126 dsRNA from the treated (local) to non-treated (systemic) leaves beginning from 1 h post-application, confirmed by both conventional and real-time RT-PCR. Furthermore, we employed a stem-loop RT-PCR and confirmed the presence of a putative viral siRNA for up to 9 days in local leaves and up to 6 days in systemic leaves post-application. The approach employed could represent a simple and environmentally safe way for the control of plant viruses in future agriculture.

The role of transcriptional coactivator ADA2b in Arabidopsis abiotic stress responses.

Plant growth and crop production can be greatly affected by common environmental stresses such as drought, high salinity and low temperatures. Gene expression is affected by several abiotic stresses. Stress-inducible genes are regulated by transcription factors and epigenetic mechanisms such as histone modifications. In this Mini-Review, we have explored the role of transcriptional adaptor ADA2b in Arabidopsis responses to abiotic stress. ADA2b is required for the expression of genes involved in abiotic stress either by controlling H3 and H4 acetylation in the case of salt stress or affecting nucleosome occupancy in low temperatures response.

Arabidopsis thaliana transcriptional co-activators ADA2b and SGF29a are implicated in salt stress responses.

The transcriptional co-activator ADA2b is a component of GCN5-containing complexes in eukaryotes. In Arabidopsis, ada2b mutants result in pleiotropic developmental defects and altered responses to low-temperature stress. SGF29 has recently been identified as another component of GCN5-containing complexes. In the Arabidopsis genome there are two orthologs of yeast SGF29, designated as SGF29a and SGF29b. We hypothesized that, in Arabidopsis, one or both SGF29 proteins may work in concert with ADA2b to regulate genes in response to abiotic stress, and we set out to explore the role of SGF29a and ADA2b in salt stress responses. In root growth and seed germination assays, sgf29a-1 mutants were more resistant to salt stress than their wild-type counterparts, whereas ada2b-1 mutant was hypersensitive. The sgf29a;ada2b double mutant displayed similar phenotypes to ada2b-1 mutant with reduced salt sensitivity. The expression of several abiotic stress-responsive genes was reduced in ada2b-1 mutants after 3 h of salt stress in comparison with sgf29a-1 and wild-type plants. In the sgf29a-1;ada2b-1 double mutant, the salt-induced gene expression was affected similarly to ada2b-1. These results suggest that under salt stress the function of SGF29a was masked by ADA2b and perhaps SGF29a could play an auxiliary role to ADA2b action. In chromatin immunoprecipitation assays, reduced levels of histone H3 and H4 acetylation in the promoter and coding region of COR6.6, RAB18, and RD29b genes were observed in ada2b-1 mutants relative to wild-type plants. In conclusion, ADA2b positively regulates salt-induced gene expression by maintaining the locus-specific acetylation of histones H4 and H3.

The histone acetyltransferase GCN5 affects the inflorescence meristem and stamen development in Arabidopsis.

A central question in biology is to understand how gene expression is precisely regulated to give rise to a variety of forms during the process of development. Epigenetic effects such as DNA methylation or histone modification have been increasingly shown to play a critical role in regulation of genome function. GCN5 is a prototypical histone acetyltransferase that participates in regulating developmental gene expression in several metazoan species. In Arabidopsis thaliana, plants with T-DNA insertions in GCN5 (also known as HAG1) display a variety of pleiotropic effects including dwarfism, loss of apical dominance, and floral defects affecting fertility. We sought to determine when during early development floral abnormalities first arise. Using scanning electron microscopy, we demonstrate that gcn5-1/hag1-1 and gcn5-5/hag1-5 mutants display overproliferation of young buds and development of abnormal structures around the inflorescence meristem. gcn5 mutants also display defects in stamen number and arrangement at later stages. This analysis provides temporal and spatial information to aid in the identification of GCN5 target genes in the developing flower. Preliminary studies of putative targets using reverse transcriptase PCR suggest that the floral meristem identity gene LEAFY is among factors upregulated in gcn5-1 mutants.

Two Arabidopsis orthologs of the transcriptional coactivator ADA2 have distinct biological functions.

Histone acetylation is an example of covalent modification of chromatin structure that has the potential to regulate gene expression. Gcn5 is a prototypical histone acetyltransferase that associates with the transcriptional coactivator Ada2. In Arabidopsis, two genes encode proteins that resemble yeast ADA2 and share approximately 45% amino acid sequence identity. We previously reported that plants harboring a T-DNA insertion in the ADA2b gene display a dwarf phenotype with developmental defects in several organs. Here we describe T-DNA insertion alleles in the ADA2a gene, which result in no dramatic growth or developmental phenotype. Both ADA2a and ADA2b are expressed in a variety of plant tissues; moreover, expression of ADA2a from a constitutive promoter fails to complement the ada2b-1 mutant phenotype, consistent with the hypothesis that the two proteins have distinct biochemical roles. To further probe the cellular roles of ADA2a and ADA2b, we studied the response of the transcriptional coactivator mutants to abiotic stress. Although ada2b seedlings display hypersensitivity to salt and abscisic acid and altered responses to low temperature stress, the responses of ada2a seedlings to abiotic stress generally parallel those of wildtype plants. Intriguingly, ada2a;ada2b double mutant plants display an intermediate, gcn5-like phenotype, suggesting that ADA2a and ADA2b each work independently with GCN5 to affect genome function in Arabidopsis.

Synergy between the light-induced acute response and the circadian cycle: a new mechanism for the synchronization of the Phaseolus vulgaris clock to light.

PvLHY and Lhcb expression has been studied in primary bean leaves after exposure of etiolated leaves to two or three white light-pulses and under different photoperiods. Under the tested photoperiods, the steady-state mRNA levels exhibit diurnal oscillations with zenith in the morning between ZT21 and 4 for PvLHY and between ZT4 and 6 for Lhcb. Nadir is in the evening between ZT12 and 18 for PvLHY and ZT18 and 24 for Lhcb. Light-pulses to etiolated seedlings induce a differentiated acute response that is reciprocally correlated with the amplitude of the following circadian cycle. In addition, the clock modulates the duration of the acute response (descending part of the curve included), which according to the phase of the rhythm at light application extends from 7 to 18 h. This constitutes the response dynamics of the Phaseolus clock to light. Similarly, the waveform of PvLHY and Lhcb expression during the day of different photoperiods resembles in induction capability (accomplishment of peak after lights-on) and duration (from lights-on phase to trough) the phase-dependent progression of acute response in etiolated seedlings. Consequently, the peak of Lhcb (all tested photoperiods) and PvLHY (in LD 18:6) attained in the photophase corresponds to the acute response peak, while the peak of PvLHY during the scotophase (in LD 12:12 and 6:18) corresponds to the circadian peak. Thus, the effect of the response dynamics in the photoperiod determines the coincidence of the peak with the photo- or scotophase, respectively. This represents a new model mechanism for the adaptation of the Phaseolus clock to light.

Light and circadian regulation in the expression of LHY and Lhcb genes in Phaseolus vulgaris.

In order to understand some aspects of the circadian clock function in Phaseolus vulgaris, we analyzed the temporal transcript profile of Lhcb genes, typical clock reporters in plants, and that of PvLHY, an orthologue of Arabidopsis thaliana LHY which is a putative transcription factor of Lhcb genes. Under different light regimes, Lhcb and PvLHY exhibit a clear circadian pattern of expression. Moreover, the rhythm of Lhcb genes appears to be tightly coupled to that of PvLHY with the latter having a slightly earlier phase. This supports the idea that the oscillating capacity of PvLHY may be one of the causes of the rhythmic expression of Lhcb genes in bean. In addition to their circadian regulation, Lhcb and PvLHY are induced by light with similar and relatively slow induction kinetics. Moreover, this light induction is gated by the circadian oscillator: minimal responses occur at times around peaks of the pre-existing rhythm, while maximal ones occur at troughs of the pre-existing rhythm. This pattern of gating is opposite to that observed in Arabidopsis. The failure to block the light induction pathways at pre-existing troughs appears to have a detrimental effect to the subsequent circadian rhythmicity. Briefly, the overall regulation of PvLHY and Lhcb genes by light and the circadian clock reveals different strategies between Phaseolus and Arabidopsis in the adaptation to photoperiodic conditions.