What regulates the shapes and sizes of organisms is one of the most intriguing unanswered questions of developmental biology. For plants, this is an especially challenging problem because most plant organs do not arise until after the seed germinates, and organ size and shape are optimized to suit the local environment. Because they are photosynthetic and fixed in space, plants need to be especially plastic in response to their light environment. Further, light signals do not act autonomously, but must be integrated with seasonal and diurnal changes in temperature, as well as with intrinsic developmental programs, to specify correct spatial and temporal regulation of gene expression, organelle development, and cellular differentiation. Our lab studies the mechanisms by which plants respond to changes in their environment. We use Arabidopsis thaliana, an ideal organism for genetic and molecular investigations of signaling networks.
Our efforts probe the mechanisms of developmental plasticity in plants by answering three interrelated questions: What are the initial events of photoreceptor signaling, and how do light and temperature changes alter the development of plants? How do the photoreceptor action pathways interact with endogenous hormone programs to establish the body plan of the emerging seedling and determine the final size of the plant? What are the mechanisms by which the expression of nuclear light-regulated genes is coordinated with chloroplast development?
Light Control of Gene Expression and Development
Plants have evolved sophisticated systems to sense incident light through the combined action of a suite of photoreceptors. Among these, the red/far-red–absorbing receptors called phytochromes (PHYA–PHYE) are the best characterized. Phytochromes are light-regulated kinases that translocate from the cytosol to discrete sites in the nucleus after light excitation, where they regulate expression of hundreds to thousands of genes, depending on the response. Our early studies identified a number of phytochrome mutants and defined roles for individual phytochromes in Arabidopsis development. Currently, we focus on early events in phytochrome signaling, with an emphasis on determinants that control phytochromes' subcellular locations. Ongoing genetic and proteomic studies have revealed new proteins associated with phytochrome trafficking and suggested models for the mechanisms by which phytochromes cause major developmental transitions.
One important physiological role for phytochromes is to sense the proximity of neighboring plants. Plants under a canopy or grown in dense agricultural plots are "starved" for red light and initiate a complex program called the shade-avoidance syndrome. Shade avoidance is the ability of plants to reach for light in dense canopies and is a key trait in aboveground competition between plants. We have performed genetic screens for Arabidopsis plants that are unable to sense shade light. Our studies have identified PFT, a component of the mediator complex, as playing a specific role in a light-quality flowering time pathway downstream from phytochrome b. Other genes identified in the mutant screen indicate that rapid new synthesis and transport of the plant hormone auxin is necessary for shade avoidance.
Plants not only sense quality of light, but also measure its duration, as well as the coincidence of light with other environmental factors. Specifically, the relationship between diurnal cycles of light and temperature reflects time of year and location, providing a plant with essential developmental cues. Diurnal cycles of ambient temperature are an important, yet poorly understood, developmental signal. To isolate the specific effects of temperature cycles on diurnal gene expression, we monitored the Arabidopsis transcriptome with microarrays under different regimens of temperature cycles, photoperiods, or constant conditions. A model-based, pattern-matching algorithm was developed to identify genes that are diurnally regulated, revealing that temperature and light/dark cycles diurnally regulate almost the entire Arabidopsis transcriptome. The phase of peak expression for most diurnally regulated genes depends on the specific regimens of temperature cycles or photoperiod, supporting a distinct role for each environmental cue. We developed a method to identify regulatory elements in the promoters of coordinately expressed genes. These results provide insights into how unknown, as well as known, light and circadian elements interact combinatorially to establish phase-specific diurnal expression.
Steroid Hormones and Light-Regulated Seedling Development
Earlier studies in my laboratory provided evidence that the action of a steroid hormone, brassinolide (BL), may be involved in light-regulated gene expression and cell elongation responses in plants. Steroid hormones are crucial for animal development, differentiation, and homeostasis; however, little was known about the biosynthesis, function, or mechanism of perception of plant steroids. We have shown that BL-deficient plants display many defects throughout development. In the dark, these mutants develop as light-grown plants and inappropriately express light-regulated genes. In the light, BL-deficient mutants are dwarfs, have reduced male fertility, and display a significant delay in the senescence program.
Over the past 15 years, most components of the BL signaling pathway have been identified (many in our lab), and signaling mechanisms from the cell surface to changes in gene expression are now known. We have shown that BLs are perceived by a plasma membrane receptor dual-specificity kinase, called BRI1. BRI1's extracellular domain contains 25 tandem leucine-rich repeats (LRRs) interrupted by a 70–amino acid domain between LRR 21 and 22. The 70–amino acid insertion is required for BRI1's function and binds BL directly. We solved the three-dimensional structures of BRI1's extracellular domain in the presence and absence of ligand (Figure 1). The BRI ectodomain structure at 2.5 angstrom resolution reveals a superhelix of 25 highly twisted LRRs, an architecture that is strikingly different from the LRR domains of animal Toll-like receptors. The small 70–amino acid domain between LRR 21 and 22 folds back into the interior of the helix. The steroid hormone brassinolide binds to a surface pocket formed by the island domain and the underlying LRRs 21–25. Several known loss- and gain-of-function alleles closely map to the hormone-binding site. We propose that steroid binding to BRI1 generates a docking platform for its coreceptor that is necessary for receptor activation. Our work has novel mechanistic implications for diverse hormone, developmental, and innate immunity signaling pathways in plants that employ similar membrane receptors.
BRI1 functions in proximity with BAK1, a second LRR kinase. BRI1 is kept in a basal state by BKI1, a negative regulator whose function is to prevent the interaction of BRI1 with BAK1 in the absence of ligand. Downstream from BRI1 and BAK1, BIN2, a member of the glycogen synthase kinase-3 family, negatively regulates BL signaling by phosphorylating members of a plant-specific family of transcriptional regulators, defined by the BES1 and BZR1genes. In the presence of BL, BIN2 is inhibited by an unknown mechanism, leading to the dephosphorylation of BES1 and BZR1 by a nuclear serine/threonine/tyrosine phosphatase. Dephosphorylated BES1 and BZR1 then homodimerize or cooperate with other transcription factors, which allows DNA binding and regulation of hundreds of brassinosteroid (BR)-responsive genes.
The size of an organism is genetically determined, yet how a plant or animal achieves its final size is largely unknown. The shoot of higher plants has a simple conserved body plan composed of three major tissue systems: the epidermis (L1) and the inner ground (L2) and vascular (L3) tissues. Which tissue system drives or restricts growth has been a subject of debate for more than a century. We used dwarf BR biosynthesis and response mutants in conjunction with tissue-specific expression of these components as tools to examine the role of the epidermis in shoot growth. We have shown that expression of the BR receptor or a BR biosynthetic enzyme in the epidermis, but not in the vasculature, of null mutants is sufficient to rescue their dwarf phenotypes. BR signaling from the epidermis is not sufficient to establish normal vascular organization. Moreover, shoot growth is restricted when BRs are depleted from the epidermis and BRs act locally within a leaf. Our studies indicate that the epidermis both promotes and restricts shoot growth by providing a nonautonomous signal to the ground tissues.
Retrograde Signaling from the Chloroplast Regulates the Expression of Nuclear Light-Responsive Genes
Plant cells coordinately regulate the expression of nuclear and plastid genes that encode components of the photosynthetic apparatus. Nuclear genes that regulate chloroplast development and chloroplast gene expression provide part of this control, but information also flows in the opposite direction, from chloroplasts to the nucleus. Plastid-to-nucleus retrograde signaling coordinates nuclear gene expression with chloroplast function and is essential for the photoautotrophic lifestyle of plants. We have performed four different genetic screens for retrograde-pathway signaling mutants, and all four of the screens have implicated the chloroplast-localized tetrapyrrole biosynthetic pathway that gives rise to both heme and chlorophyll (Figure 2). Our data are most consistent with a model in which a specific pool of heme functions as a positive signal of chloroplast function. A second, negative signal may also be produced from the tetrapyrrole biosynthetic pathway when plants are stressed. This leads to the accumulation of heme/chlorophyll precursors, such as protoporphyrin IX. These highly reactive metabolites are generators of singlet oxygen, which is necessary for the response. GUN1 (a chloroplast nucleoid-associated pentatricopeptide-repeat protein) and ABI4 (a nuclear-localized transcription factor) are required for multiple retrograde signaling pathways. ABI4 binds the promoter of a retrograde-regulated gene through a conserved motif found in proximity to a light-regulatory element. We are testing a model in which multiple indicators of aberrant plastid function are integrated upstream of GUN1 within plastids, leading to ABI4-mediated repression of nuclear-encoded genes.
Grants from the National Institutes of Health and the Department of Energy provided partial support for these projects.
As of April 11, 2016