2012 Annual Report
1a.Objectives (from AD-416):
The long-term goal of this research is to define the molecular mechanisms by which the phytochrome (phy) family of photoreceptors perceive informational light signals from the environment and transduce them to photoresponsive nuclear genes, thereby controlling plant growth and development. Using genome-scale microarray-based expression profiling, we have begun to define the global transcriptional network regulated by these photoreceptors and have identified a set of rapidly light-induced or -repressed genes (primarily encoding various transcription factors) that are potential direct targets of the phytochrome signaling pathway. The specific objectives of this project plan are:
1. To identify which phy family members are responsible for signaling to each of
these genes, by examining light-induced expression profiles in null mutants of each phytochrome. [NP 301, C 4, PS 4a]
2. To define the cis elements in the promoters of these genes responsible for
coordinate light-regulated expression, using computational analysis coupled with
targeted mutagenesis and transgenic expression of promoter::reporter fusion
constructs. [NP 301, C 4, PS 4a]
3. To identify downstream targets of these genes in the light-regulated
transcriptional network, using microarray-based expression profiling in knockout
mutants null for each transcription factor, coupled with chromatin immuno- precipitation (ChIP) for target promoter identification and characterization. [NP 301, C 4, PS 4a]
1b.Approach (from AD-416):
The specific objectives of this proposal are: (a) to identify which phys are
responsible for signaling to these genes; (b) to define the cis elements in the
promoters of these genes responsible for coordinating light-regulated expression; (c) to identify downstream targets of these genes in the light-regulated transcriptional network. The experimental approaches will include: (a) microarray-based expression profiling of mutants null for each phy, both to define the phy-regulated transcriptional networks and to identify rapidly light-responsive genes for targeted reverse-genetic mutagenesis; (b) computational analysis of the promoters of these genes, coupled with targeted mutagenesis and transgenic expression of promoter::reporter fusion constructs; (c) expression profiling in knockout mutants null for each transcription factor, coupled with chromatin immunoprecipitation for identification of promoters that are direct targets of these factors.
Plants respond to light signals informing them of imposed or impending vegetative shade, via the phytochrome (phy) photoreceptor system, by adaptive changes in growth and development, collectively termed the shade avoidance syndrome (SAS). To examine the roles of the Phy-Interacting bHLH Factors, PIF1, 3, 4 and 5, in relaying this perceived information to the transcriptional network, we compared the genome-wide transcription profiles of wild-type and quadruple pif (pifq) mutants in response to shade. The data identify a subset of genes, enriched in transcription-factor-encoding loci, that respond rapidly (within 1 h), in a PIF-dependent manner, to the shade signal, and that contain promoter-located G-box-sequence motifs (CACGTG), known to be preferred PIF binding sites. These genes are thus potential direct targets of phy-PIF signaling that function in the primary transcriptional circuitry that controls downstream response elaboration. A second subset of PIF-dependent, early-response genes, lacking G-box motifs, are enriched for auxin-responsive loci, suggestive of being indirect targets of phy-PIF signaling involved in the rapid cell-expansion responses known to be induced by shade. A meta-analysis comparing deetiolation- and shade-responsive transcriptomes identifies a further subset of G-box-containing genes that reciprocally display rapid repression and induction in response to light and shade signals at the inception of deetiolation and shade-avoidance, respectively. These data define a core set of transcriptional and hormonal (auxin, cytokinin) processes that appear to be dynamically poised to react rapidly to changes in the light environment in response to perturbations in the mutually antagonistic regulatory activities of the phys and PIFs. Data from comparative analysis of the quadruple pifq and all triple pif-mutant combinations in response to light and shade, confirm that the PIF-quartet members act with overlapping redundancy on seedling morphogenesis and transcriptional regulation, but that the individual PIFs contribute differentially to these responses.
Seedling establishment. A critical factor in successful seedling establishment in crop plants is a developmental strategy that first enables postgerminative seedlings emerging from buried seed to grow vigorously upward in the subterranean darkness toward the soil surface (termed skotomorphogenic development), and then, upon initial exposure to light, undergo a process, termed deetiolation (the induction of photomorphogenesis), involving conversion to the fully-photosynthetically active state of green plants. Conversely, fully-established green plants respond to vegetative shade imposed by neighboring plants, by accelerating the vertical growth rate of stem tissue at the expense of reproductive and storage tissue deposition (e.g. seed or tuber production), a strategy partially resembling reversion toward the initial skotomorphogenic state. The underlying molecular processes that control these responses to the light environment were not well defined. ARS scientists in Albany, California, have discovered that a central component of the mechanism underlying these responses is the promotion, in darkness, by a family of transcription factors called PIFs (phytochrome-interacting factors), of the gene-expression network normally repressed upon exposure to light, and, conversely, the reversal of this light-imposed repression in a subset of these genes upon exposure to vegetative shade. The experiments show that light triggers this process initially by activating the phy photoreceptor molecules, which then bind to and induce rapid degradation of the PIF proteins in the nucleus, thereby initiating the normal gene expression pattern observed in green seedlings. But then, conversely, vegetative shade deactivates the phy molecules, permitting re-accumulation of the PIF proteins, which in turn then reactivate the expression of a subset of the genes initially highly expressed in the dark-grown seedlings.