Rajnish Khanna

A Novel Molecular Recognition Motif Necessary for Targeting Photoactivated Phytochrome Signaling to Specific Basic Helix-Loop-Helix Transcription Factors

The phytochrome (phy) family of sensory photoreceptors (phyA to phyE) in Arabidopsis thaliana control plant developmental transitions in response to informational light signals throughout the life cycle. The photoactivated conformer of the photoreceptor Pfr has been shown to translocate into the nucleus where it induces changes in gene expression by an unknown mechanism. Here, we have identified two basic helix-loop-helix (bHLH) transcription factors, designated PHYTOCHROME-INTERACTING FACTOR5 (PIF5) and PIF6, which interact specifically with the Pfr form of phyB. These two factors cluster tightly with PIF3 and two other phy-interacting bHLH proteins in a phylogenetic subfamily within the large Arabidopsis bHLH (AtbHLH) family. We have identified a novel sequence motif (designated the active phytochrome binding [APB] motif) that is conserved in these phy-interacting AtbHLHs but not in other noninteractors. Using the isolated domain and site-directed mutagenesis, we have shown that this motif is both necessary and sufficient for binding to phyB. Transgenic expression of the native APB-containing AtbHLH protein, PIF4, in a pif4 null mutant, rescued the photoresponse defect in this mutant, whereas mutated PIF4 constructs with site-directed substitutions in conserved APB residues did not. These data indicate that the APB motif is necessary for PIF4 function in light-regulated seedling development and suggest that conformer-specific binding of phyB to PIF4 via the APB motif is necessary for this function in vivo. Binding assays with the isolated APB domain detected interaction with phyB, but none of the other four Arabidopsis phys. Collectively, the data suggest that the APB domain provides a phyB-specific recognition module within the AtbHLH family, thereby conferring photoreceptor target specificity on a subset of these transcription factors and, thus, the potential for selective signal channeling to segments of the transcriptional network.

Phytochrome Induces Rapid PIF5 Phosphorylation and Degradation in Response to Red-Light Activation

The phytochrome (phy) family of sensory photoreceptors (phyA–phyE in Arabidopsis thaliana) induces changes in target-gene expression upon light-induced translocation to the nucleus, where certain members interact with selected members of the constitutively nuclear basic helix-loop-helix transcription factor family, such as PHYTOCHROME-INTERACTING FACTOR3 (PIF3). Previous evidence indicates that the binding of the photoactivated photoreceptor molecule to PIF3 induces rapid phosphorylation of the transcription factor in the cell prior to its degradation via the ubiqitin-proteosome system. To investigate whether this apparent primary signaling mechanism can be generalized to other phy-interacting partners, we have examined the molecular behavior of a second related phy-interacting member of the basic helix-loop-helix family, PIF5, during early deetiolation, immediately following initial exposure of dark-grown seedlings to light. The data show that red light induces very rapid phosphorylation and subsequent degradation (t1/2 < 5 min) of PIF5 via the proteosome system upon irradiation. Photobiological and genetic evidence indicates that the photoactivated phy molecule acts within 60 s to induce this phosphorylation of PIF5, and that phyA and phyB redundantly dominate this process, with phyD playing an apparently minor role. Collectively, the data support the proposal that the rapid phy-induced phosphorylation of PIF3 and PIF5 may represent the biochemical mechanism of primary signal transfer from photoactivated photoreceptor to binding partner, and that phyA and phyB (and possibly phyD) may signal to multiple, shared partners utilizing this common mechanism.

The Arabidopsis B-Box Zinc Finger Family

The Arabidopsis thaliana genome encodes >1500 transcription factors, and ∼45% of these belong to families specific to plants ([Riechmann et al., 2000][1]). Comparison of the entire complement of transcription factors of Arabidopsis , Drosophila melanogaster , Caenorhabditis elegans , and

The Basic Helix-Loop-Helix Transcription Factor PIF5 Acts on Ethylene Biosynthesis and Phytochrome Signaling by Distinct Mechanisms

PHYTOCHROME-INTERACTING FACTOR5 (PIF5), a basic helix-loop-helix transcription factor, interacts specifically with the photoactivated form of phytochrome B (phyB). Here, we report that dark-grown Arabidopsis thaliana seedlings overexpressing PIF5 (PIF5-OX) exhibit exaggerated apical hooks and short hypocotyls, reminiscent of the triple response induced by elevated ethylene levels, whereas pif5 mutants fail to maintain tight hooks like those of wild-type seedlings. Silver ions, an ethylene receptor blocker, rescued the triple-response phenotype, and we show that PIF5-OX seedlings express enhanced levels of key ethylene biosynthesis enzymes and produce elevated ethylene levels. Exposure of PIF5-OX seedlings to prolonged continuous red light (Rc) promotes hypocotyl elongation relative to dark controls, the reciprocal of the Rc-imposed hypocotyl inhibition displayed by wild-type seedlings. In contrast with this PIF5-OX hyposensitivity to Rc, pif5 mutant seedlings are hypersensitive relative to wild-type seedlings. We show that this contrast is due to reciprocal changes in phyB protein levels in prolonged Rc. Compared with wild-type seedlings, PIF5-OX seedlings have reduced, whereas pif5 mutants have increased, phyB (and phyC) levels in Rc. The phyB degradation in the overexpressors depends on a functional phyB binding motif in PIF5 and involves the 26S proteasome pathway. Our data thus indicate that overexpressed PIF5 causes altered ethylene levels, which promote the triple response in darkness, whereas in the light, the interaction of photoactivated phyB with PIF5 causes degradation of the photoreceptor protein. The evidence suggests that endogenous PIF5 negatively regulates phyB-imposed hypocotyl inhibition in prolonged Rc by reducing photoreceptor abundance, and thereby photosensory capacity, rather than functioning as a signaling intermediate.

Functional Profiling Reveals That Only a Small Number of Phytochrome-Regulated Early-Response Genes in Arabidopsis Are Necessary for Optimal Deetiolation

In previous time-resolved microarray-based expression profiling, we identified 32 genes encoding putative transcription factors, signaling components, and unknown proteins that are rapidly and robustly induced by phytochrome (phy)-mediated light signals. Postulating that they are the most likely to be direct targets of phy signaling and to function in the primary phy regulatory circuitry, we examined the impact of targeted mutations in these genes on the phy-induced seedling deetiolation process in Arabidopsis thaliana. Using light-imposed concomitant inhibition of hypocotyl and stimulation of cotyledon growth as diagnostic criteria for normal deetiolation, we identified three major mutant response categories. Seven (22%) lines displayed statistically significant, reciprocal, aberrant photoresponsiveness in the two organs, suggesting disruption of normal deetiolation; 13 (41%) lines displayed significant defects either unidirectionally in both organs or in hypocotyls only, suggesting global effects not directly related to photomorphogenic signaling; and 12 (37%) lines displayed no significant difference in photoresponsiveness from the wild type. Potential reasons for the high proportion of rapidly light-responsive genes apparently unnecessary for the deetiolation phenotype are discussed. One of the seven disrupted genes displaying a significant mutant phenotype, the basic helix-loop-helix factor–encoding PHYTOCHROME-INTERACTING FACTOR3-LIKE1 gene, was found to be necessary for rapid light-induced expression of the photomorphogenesis- and circadian-related PSEUDO-RESPONSE REGULATOR9 gene, indicating a regulatory function in the early phy-induced transcriptional network.

EARLY FLOWERING 4 Functions in Phytochrome B-Regulated Seedling De-Etiolation

To define the functions of genes previously identified by expression profiling as being rapidly light induced under phytochrome (phy) control, we are investigating the seedling de-etiolation phenotypes of mutants carrying T-DNA insertional disruptions at these loci. Mutants at one such locus displayed reduced responsiveness to continuous red, but not continuous far-red light, suggesting a role in phyB signaling but not phyA signaling. Consistent with such a role, expression of this gene is induced by continuous red light in wild-type seedlings, but the level of induction is strongly reduced in phyB-null mutants. The locus encodes a novel protein that we show localizes to the nucleus, thus suggesting a function in light-regulated gene expression. Recently, this locus was identified as EARLY FLOWERING 4, a gene implicated in floral induction and regulating the expression of the gene CIRCADIAN CLOCK-ASSOCIATED 1. Together with these previous data, our findings suggest that EARLY FLOWERING 4 functions as a signaling intermediate in phy-regulated gene expression involved in promotion of seedling de-etiolation, circadian clock function, and photoperiod perception.

Expression of the Arabidopsis thaliana BBX32 Gene in Soybean Increases Grain Yield

Crop yield is a highly complex quantitative trait. Historically, successful breeding for improved grain yield has led to crop plants with improved source capacity, altered plant architecture, and increased resistance to abiotic and biotic stresses. To date, transgenic approaches towards improving crop grain yield have primarily focused on protecting plants from herbicide, insects, or disease. In contrast, we have focused on identifying genes that, when expressed in soybean, improve the intrinsic ability of the plant to yield more. Through the large scale screening of candidate genes in transgenic soybean, we identified an Arabidopsis thaliana B-box domain gene (AtBBX32) that significantly increases soybean grain yield year after year in multiple transgenic events in multi-location field trials. In order to understand the underlying physiological changes that are associated with increased yield in transgenic soybean, we examined phenotypic differences in two AtBBX32-expressing lines and found increases in plant height and node, flower, pod, and seed number. We propose that these phenotypic changes are likely the result of changes in the timing of reproductive development in transgenic soybean that lead to the increased duration of the pod and seed development period. Consistent with the role of BBX32 in A. thaliana in regulating light signaling, we show that the constitutive expression of AtBBX32 in soybean alters the abundance of a subset of gene transcripts in the early morning hours. In particular, AtBBX32 alters transcript levels of the soybean clock genes GmTOC1 and LHY-CCA1-like2 (GmLCL2). We propose that through the expression of AtBBX32 and modulation of the abundance of circadian clock genes during the transition from dark to light, the timing of critical phases of reproductive development are altered. These findings demonstrate a specific role for AtBBX32 in modulating soybean development, and demonstrate the validity of expressing single genes in crops to deliver increased agricultural productivity.

BBX32, an Arabidopsis B-Box Protein, Functions in Light Signaling by Suppressing HY5-Regulated Gene Expression and Interacting with STH2/BBX21

A B-box zinc finger protein, B-BOX32 (BBX32), was identified as playing a role in determining hypocotyl length during a large-scale functional genomics study in Arabidopsis (Arabidopsis thaliana). Further analysis revealed that seedlings overexpressing BBX32 display elongated hypocotyls in red, far-red, and blue light, along with reduced cotyledon expansion in red light. Through comparative analysis of mutant and overexpression line phenotypes, including global expression profiling and growth curve studies, we demonstrate that BBX32 acts antagonistically to ELONGATED HYPOCOTYL5 (HY5). We further show that BBX32 interacts with SALT TOLERANCE HOMOLOG2/BBX21, another B-box protein previously shown to interact with HY5. Based on these data, we propose that BBX32 functions downstream of multiple photoreceptors as a modulator of light responses. As such, BBX32 potentially has a native role in mediating gene repression to maintain dark adaptation.

Ubiquitin ligase switch in plant photomorphogenesis: A hypothesis.

The E3 ubiquitin ligase COP1 (CONSTITUTIVE PHOTOMORPHOGENIC1) plays a key role in the repression of the plant photomorphogenic development in darkness. In the presence of light, COP1 is inactivated by a mechanism which is not completely understood. This leads to accumulation of COP1’s target transcription factors, which initiates photomorphogenesis, resulting in dramatic changes of the seedling’s physiology. Here we use a mathematical model to explore the possible mechanism of COP1 modulation upon dark/light transition in Arabidopsis thaliana based upon data for two COP1 target proteins: HY5 and HFR1, which play critical roles in photomorphogenesis. The main reactions in our model are the inactivation of COP1 by a proposed photoreceptor-related inhibitor I and interactions between COP1 and a CUL4 (CULLIN4)-based ligase. For building and verification of the model, we used the available published and our new data on the kinetics of HY5 and HFR1 together with the data on COP1 abundance. HY5 has been shown to accumulate at a slower rate than HFR1. To describe the observed differences in the timecourses of the “slow” target HY5 and the “fast” target HFR1, we hypothesize a switch between the activities of COP1 and CUL4 ligases upon dark/light transition, with COP1 being active mostly in darkness and CUL4 in light. The model predicts a bi-phasic kinetics of COP1 activity upon the exposure of plants to light, with its restoration after the initial decline and the following slow depletion of the total COP1 content. CUL4 activity is predicted to increase in the presence of light. We propose that the ubiquitin ligase switch is important for the complex regulation of multiple transcription factors during plants development. In addition, this provides a new mechanism for sensing the duration of light period, which is important for seasonal changes in plant development.