GIBBERELLIN PERCEPTION SENSOR 2 reveals genesis and role of cellular GA dynamics in light-regulated hypocotyl growth.

The phytohormone gibberellic acid (GA) is critical for environmentally sensitive plant development including germination, skotomorphogenesis, and flowering. The Förster resonance energy transfer biosensor GIBBERELLIN PERCEPTION SENSOR1, which permits single-cell GA measurements in vivo, has been used to observe a GA gradient correlated with cell length in dark-grown, but not light-grown, hypocotyls. We sought to understand how light signaling integrates into cellular GA regulation. Here, we show how the E3 ligase CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) and transcription factor ELONGATED HYPOCOTYL 5 (HY5) play central roles in directing cellular GA distribution in skoto- and photomorphogenic hypocotyls, respectively. We demonstrate that the expression pattern of the GA biosynthetic enzyme gene GA20ox1 is the key determinant of the GA gradient in dark-grown hypocotyls and is a target of COP1 signaling. We engineered a second generation GPS2 biosensor with improved orthogonality and reversibility. GPS2 revealed a previously undetectable cellular pattern of GA depletion during the transition to growth in the light. This GA depletion partly explains the resetting of hypocotyl growth dynamics during photomorphogenesis. Achieving cell-level resolution has revealed how GA distributions link environmental conditions with morphology and morphological plasticity. The GPS2 biosensor is an ideal tool for GA studies in many conditions, organs, and plant species.

Cell wall integrity modulates HOOKLESS1 and PHYTOCHROME INTERACTING FACTOR4 expression controlling apical hook formation.

Formation of the apical hook in etiolated dicot seedlings results from differential growth in the hypocotyl apex and is tightly controlled by environmental cues and hormones, among which auxin and gibberellins (GAs) play an important role. Cell expansion is tightly regulated by the cell wall, but whether and how feedback from this structure contributes to hook development are still unclear. Here, we show that etiolated seedlings of the Arabidopsis (Arabidopsis thaliana) quasimodo2-1 (qua2) mutant, defective in pectin biosynthesis, display severe defects in apical hook formation and maintenance, accompanied by loss of asymmetric auxin maxima and differential cell expansion. Moreover, qua2 seedlings show reduced expression of HOOKLESS1 (HLS1) and PHYTOCHROME INTERACTING FACTOR4 (PIF4), which are positive regulators of hook formation. Treatment of wild-type seedlings with the cellulose inhibitor isoxaben (isx) also prevents hook development and represses HLS1 and PIF4 expression. Exogenous GAs, loss of DELLA proteins, or HLS1 overexpression partially restore hook development in qua2 and isx-treated seedlings. Interestingly, increased agar concentration in the medium restores, both in qua2 and isx-treated seedlings, hook formation, asymmetric auxin maxima, and PIF4 and HLS1 expression. Analyses of plants expressing a Förster resonance energy transfer-based GA sensor indicate that isx reduces accumulation of GAs in the apical hook region in a turgor-dependent manner. Lack of the cell wall integrity sensor THESEUS 1, which modulates turgor loss point, restores hook formation in qua2 and isx-treated seedlings. We propose that turgor-dependent signals link changes in cell wall integrity to the PIF4-HLS1 signaling module to control differential cell elongation during hook formation.

Brassinosteroid-regulated bHLH transcription factor CESTA induces the gibberellin 2-oxidase GA2ox7.

Brassinosteroids (BRs) are plant steroids that have growth-promoting capacities, which are partly enabled by an ability to induce biosynthesis of gibberellins (GAs), a second class of plant hormones. In addition, BRs can also activate GA catabolism; here we show that in Arabidopsis (Arabidopsis thaliana) the basic helix-loop-helix transcription factor CESTA (CES) and its homologues BRASSINOSTEROID-ENHANCED EXPRESSION (BEE) 1 and 3 contribute to this activity. CES and the BEEs are BR-regulated at the transcriptional and posttranslational level and participate in different physiological processes, including vegetative and reproduction development, shade avoidance, and cold stress responses. We show that CES/BEEs can induce the expression of the class III GA 2-oxidase GA2ox7 and that this activity is increased by BRs. In BR signaling - and CES/BEE-deficient mutants, GA2ox7 expression decreased, yielding reduced levels of GA110, a product of GA2ox7 activity. In plants that over-express CES, GA2ox7 expression is hyper-responsive to BR, GA110 levels are elevated and amounts of bioactive GA are reduced. We provide evidence that CES directly binds to the GA2ox7 promoter and is activated by BRs, but can also act by BR-independent means. Based on these results, we propose a model for CES activity in GA catabolism where CES can be recruited for GA2ox7 induction not only by BR, but also by other factors.

DNA sequence properties that predict susceptibility to epiallelic switching.

Transgenerationally heritable epialleles are defined by the stable propagation of alternative transcriptional states through mitotic and meiotic cell cycles. Given that the propagation of DNA methylation at CpG sites, mediated in Arabidopsis by MET1, plays a central role in epigenetic inheritance, we examined genomewide DNA methylation in partial and complete loss-of-function met1 mutants. We interpreted the data in relation to transgenerational epiallelic stability, which allowed us to classify chromosomal targets of epigenetic regulation into (i) single copy and methylated exclusively at CpGs, readily forming epialleles, and (ii) transposon-derived, methylated at all cytosines, which may or may not form epialleles. We provide evidence that DNA sequence features such as density of CpGs and genomic repetitiveness of the loci predispose their susceptibility to epiallelic switching. The importance and predictive power of these genetic features were confirmed by analyses of common epialleles in natural Arabidopsis accessions, epigenetic recombinant inbred lines (epiRILs) and also verified in rice.

HIV+ children and their families in the UK: results from a family-clinic based 'look-back' exercise in London.

Advances in antiretroviral treatment have resulted in a growing number of HIV+ children surviving into adolescence and adulthood. However, HIV remains a chronic condition compounded by additional psychosocial stressors associated with living with HIV. The gold standard for treatment of HIV+ children and young people is within a family context. This 'look-back' exercise was conducted within an HIV family clinic context in London. HIV+ children's clinical notes were examined with the aim of describing the medical and social context of the families attending the clinic. Results showed that families are living with many psychosocial challenges that may have an impact on their ability to respond effectively to the challenge of living with HIV disease.

HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 is required for circadian periodicity through the promotion of nucleo-cytoplasmic mRNA export in Arabidopsis.

Cold acclimation has been shown to be attenuated by the degradation of the INDUCER OF CBF EXPRESSION1 protein by the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1). However, recent work has suggested that HOS1 may have a wider range of roles in plants than previously appreciated. Here, we show that hos1 mutants are affected in circadian clock function, exhibiting a long-period phenotype in a wide range of temperature and light environments. We demonstrate that hos1 mutants accumulate polyadenylated mRNA in the nucleus and that the circadian defect in hos1 is shared by multiple mutants with aberrant mRNA export, but not in a mutant attenuated in nucleo-cytoplasmic transport of microRNAs. As revealed by RNA sequencing, hos1 exhibits gross changes to the transcriptome with genes in multiple functional categories being affected. In addition, we show that hos1 and other previously described mutants with altered mRNA export affect cold signaling in a similar manner. Our data support a model in which altered mRNA export is important for the manifestation of hos1 circadian clock defects and suggest that HOS1 may indirectly affect cold signaling through disruption of the circadian clock.

Network balance via CRY signalling controls the Arabidopsis circadian clock over ambient temperatures.

Circadian clocks exhibit 'temperature compensation', meaning that they show only small changes in period over a broad temperature range. Several clock genes have been implicated in the temperature-dependent control of period in Arabidopsis. We show that blue light is essential for this, suggesting that the effects of light and temperature interact or converge upon common targets in the circadian clock. Our data demonstrate that two cryptochrome photoreceptors differentially control circadian period and sustain rhythmicity across the physiological temperature range. In order to test the hypothesis that the targets of light regulation are sufficient to mediate temperature compensation, we constructed a temperature-compensated clock model by adding passive temperature effects into only the light-sensitive processes in the model. Remarkably, this model was not only capable of full temperature compensation and consistent with mRNA profiles across a temperature range, but also predicted the temperature-dependent change in the level of LATE ELONGATED HYPOCOTYL, a key clock protein. Our analysis provides a systems-level understanding of period control in the plant circadian oscillator.

Mathematical modeling elucidates the role of transcriptional feedback in gibberellin signaling.

The hormone gibberellin (GA) is a key regulator of plant growth. Many of the components of the gibberellin signal transduction [e.g., GIBBERELLIN INSENSITIVE DWARF 1 (GID1) and DELLA], biosynthesis [e.g., GA 20-oxidase (GA20ox) and GA3ox], and deactivation pathways have been identified. Gibberellin binds its receptor, GID1, to form a complex that mediates the degradation of DELLA proteins. In this way, gibberellin relieves DELLA-dependent growth repression. However, gibberellin regulates expression of GID1, GA20ox, and GA3ox, and there is also evidence that it regulates DELLA expression. In this paper, we use integrated mathematical modeling and experiments to understand how these feedback loops interact to control gibberellin signaling. Model simulations are in good agreement with in vitro data on the signal transduction and biosynthesis pathways and in vivo data on the expression levels of gibberellin-responsive genes. We find that GA-GID1 interactions are characterized by two timescales (because of a lid on GID1 that can open and close slowly relative to GA-GID1 binding and dissociation). Furthermore, the model accurately predicts the response to exogenous gibberellin after a number of chemical and genetic perturbations. Finally, we investigate the role of the various feedback loops in gibberellin signaling. We find that regulation of GA20ox transcription plays a significant role in both modulating the level of endogenous gibberellin and generating overshoots after the removal of exogenous gibberellin. Moreover, although the contribution of other individual feedback loops seems relatively small, GID1 and DELLA transcriptional regulation acts synergistically with GA20ox feedback.

Plant development: light exposure directs meristem fate.

Leaf initiation was previously thought to be self-regulated and not reliant on environmental cues. However, a recent study has revealed that light redirects meristem fate from maintenance to lateral organ initiation, through the regulation of the plant hormones auxin and cytokinin.

ALTERED MERISTEM PROGRAM 1 is involved in development of seed dormancy in Arabidopsis.

Mutants in the rice PLASTOCHRON 3 and maize VIVIPAROUS 8 genes have been shown to have reduced dormancy and ABA levels. In this study we used several mutants in the orthologous gene ALTERED MERISTEM PROGRAM 1 (AMP1) to determine its role in seed dormancy in Arabidopsis. Here we report that there are accession-specific effects of mutations in AMP1. In one accession, amp1 mutants produce seeds with higher dormancy, while those in two other accessions produce seeds of lower dormancy. These accession-specific effects of mutating AMP1 were shown to extend to ABA levels. We assayed global gene transcription differences in seeds of wild-type and mutant from two accessions demonstrating opposing phenotypes. The transcript changes observed indicate that the amp1 mutation shifts the seed transcriptome from a dormant into an after-ripened state. Specific changes in gene expression in the mutants give insight into the direct and indirect effects that may be contributing to the opposing dormancy phenotypes observed, and reveal a role for AMP1 in the acquisition and/or maintenance of seed dormancy in Arabidopsis.

"I look forward. I feel insecure but I am ok with it". The experience of young HIV+ people attending transition preparation events: a qualitative investigation.

Transition programmes which prepare young people with HIV to manage the medical, social and psychological consequences of the condition can provide clinical benefits for both young people and their families. The London-based Looking Forward Project (LFP) is embedded within a National Health Service HIV family clinic. The project uses a group work approach and aims to equip HIV+ young people over the age of 12 years who know their status with the emotional, psychological and behavioural skills necessary to face the challenges of living with HIV. This small scale qualitative study investigated the experience of attendance, explored factors which facilitated participation in the groups and investigated the impact on their lives as a result of participation. Participants reported that the LFP events were educational but different to school-like activities, being with other young people reduced isolation and that receiving a voucher was an incentive to attend. Participation was facilitated through family support. Attendance at the LFP facilitated a positive attitude towards medication and hope for the future.

Gene expression profiling identifies two regulatory genes controlling dormancy and ABA sensitivity in Arabidopsis seeds.

Seed dormancy is a very important trait that maximizes the survival of seed in nature, the control of which can have important repercussions on the yield of many crop species. We have used gene expression profiling to identify genes that are involved in dormancy regulation in Arabidopsis thaliana. RNA was isolated from imbibed dormant (D) and after-ripened (AR) ecotype C24 seeds, and then screened by quantitative RT-PCR (qRT-PCR) for differentially expressed transcription factors (TFs) and other regulatory genes. Out of 2207 genes screened, we have identified 39 that were differentially expressed during the first few hours of imbibition. After analyzing T-DNA insertion mutants for 22 of these genes, two displayed altered dormancy compared with the wild type. These mutants are affected in genes that encode a RING finger and an HDZip protein. The first, named DESPIERTO, is involved in ABA sensitivity during seed development, regulates the expression of ABI3, and produces a complete loss of dormancy when mutated. The second, the HDZip (ATHB20), is expressed during seed germination in the micropylar endosperm and in the root cap, and increases ABA sensitivity and seed dormancy when mutated.

Genetic analysis reveals that C19-GA 2-oxidation is a major gibberellin inactivation pathway in Arabidopsis.

Bioactive hormone concentrations are regulated both at the level of hormone synthesis and through controlled inactivation. Based on the ubiquitous presence of 2beta-hydroxylated gibberellins (GAs), a major inactivating pathway for the plant hormone GA seems to be via GA 2-oxidation. In this study, we used various approaches to determine the role of C(19)-GA 2-oxidation in regulating GA concentration and GA-responsive plant growth and development. We show that Arabidopsis thaliana has five C(19)-GA 2-oxidases, transcripts for one or more of which are present in all organs and at all stages of development examined. Expression of four of the five genes is subject to feed-forward regulation. By knocking out all five Arabidopsis C(19)-GA 2-oxidases, we show that C(19)-GA 2-oxidation limits bioactive GA content and regulates plant development at various stages during the plant life cycle: C(19)-GA 2-oxidases prevent seed germination in the absence of light and cold stimuli, delay the vegetative and floral phase transitions, limit the number of flowers produced per inflorescence, and suppress elongation of the pistil prior to fertilization. Under GA-limited conditions, further roles are revealed, such as limiting elongation of the main stem and side shoots. We conclude that C(19)-GA 2-oxidation is a major GA inactivation pathway regulating development in Arabidopsis.

The gibberellin biosynthetic genes AtGA20ox1 and AtGA20ox2 act, partially redundantly, to promote growth and development throughout the Arabidopsis life cycle.

The activity of the gibberellin (GA) biosynthetic enzymes GA 20-oxidases (GA20ox) is of particular importance in determining GA concentration in many plant species. In Arabidopsis these enzymes are encoded by a family of five genes: AtGA20ox1-AtGA20ox5. Transcript analysis indicated that they have different expression patterns and may thus participate differentially in GA-regulated developmental processes. We have used reverse genetics to determine the physiological roles of AtGA20ox1 and AtGA20ox2, the most highly expressed GA20ox genes during vegetative and early reproductive development. AtGA20ox1 and AtGA20ox2 act redundantly to promote hypocotyl and internode elongation, flowering time, elongation of anther filaments, the number of seeds that develop per silique and elongation of siliques, with AtGA20ox1 making the greater contribution to internode and filament elongation, and AtGA20ox2 making the greater contribution to flowering time and silique length. Pollination of the double mutant with wild-type pollen indicated that the GA promoting silique elongation is of maternal origin. The ga20ox2 phenotype revealed that GA promotes the number of stem internodes that elongate upon bolting, and does so independently of its effect on internode elongation. Comparison of the phenotype of the double mutant with that of the highly GA-deficient ga1-3 mutant indicates that other GA20ox genes contribute to all the developmental processes examined, and, in some cases such as root growth and leaf expansion, make major contributions, as these processes were unaffected in the double mutant. In addition, the effects of the mutations are mitigated by the homeostatic mechanism that acts on expression of other GA dioxygenase and GID1 receptor genes.

Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis.

We investigated the physiological function of three Arabidopsis thaliana homologs of the gibberellin (GA) receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) by determining the developmental consequences of GID1 inactivation in insertion mutants. Although single mutants developed normally, gid1a gid1c and gid1a gid1b displayed reduced stem height and lower male fertility, respectively, indicating some functional specificity. The triple mutant displayed a dwarf phenotype more severe than that of the extreme GA-deficient mutant ga1-3. Flower formation occurred in long days but was delayed, with severe defects in floral organ development. The triple mutant did not respond to applied GA. All three GID1 homologs were expressed in most tissues throughout development but differed in expression level. GA treatment reduced transcript abundance for all three GID1 genes, suggesting feedback regulation. The DELLA protein REPRESSOR OF ga1-3 (RGA) accumulated in the triple mutant, whose phenotype could be partially rescued by loss of RGA function. Yeast two-hybrid and in vitro pull-down assays confirmed that GA enhances the interaction between GID1 and DELLA proteins. In addition, the N-terminal sequence containing the DELLA domain is necessary for GID1 binding. Furthermore, yeast three-hybrid assays showed that the GA-GID1 complex promotes the interaction between RGA and the F-box protein SLY1, a component of the SCF(SLY1) E3 ubiquitin ligase that targets the DELLA protein for degradation.