Jeong-Il Kim

PIL5, a Phytochrome-Interacting Basic Helix-Loop-Helix Protein, Is a Key Negative Regulator of Seed Germination in Arabidopsis thaliana

The first decision made by an angiosperm seed, whether to germinate or not, is based on integration of various environmental signals such as water and light. The phytochromes (Phys) act as red and far-red light (Pfr) photoreceptors to mediate light signaling through yet uncharacterized pathways. We report here that the PIF3-like 5 (PIL5) protein, a basic helix-loop-helix transcription factor, is a key negative regulator of phytochrome-mediated seed germination. PIL5 preferentially interacts with the Pfr forms of Phytochrome A (PhyA) and Phytochrome B (PhyB). Analyses of a pil5 mutant in conjunction with phyA and phyB mutants, a pif3 pil5 double mutant, and PIL5 overexpression lines indicate that PIL5 is a negative factor in Phy-mediated promotion of seed germination, inhibition of hypocotyl negative gravitropism, and inhibition of hypocotyl elongation. Our data identify PIL5 as the first Phy-interacting protein that regulates seed germination.

Light and Brassinosteroid Signals Are Integrated via a Dark-Induced Small G Protein in Etiolated Seedling Growth

Plant growth and development are regulated through coordinated interactions between light and phytohormones. Here, we demonstrate that a dark-induced small G protein, pea Pra2, regulates a variant cytochrome P450 that catalyzes C-2 hydroxylation in brassinosteroid biosynthesis. The cytochrome P450 is dark-induced and predominantly expressed in the rapidly elongating zone of etiolated pea epicotyls, where Pra2 is also most abundant. Transgenic plants with reduced Pra2 exhibit a dark-specific dwarfism, which is completely rescued by exogenous brassinolide. Overexpression of the cytochrome P450 results in enhanced hypocotyl growth even in the light, which phenocopies the etiolated hypocotyls. We therefore propose that Pra2 and its orthologs are molecular mediators for the cross-talk between light and brassinosteroids in the etiolation process in plants.

Phytochrome-Specific Type 5 Phosphatase Controls Light Signal Flux by Enhancing Phytochrome Stability and Affinity for a Signal Transducer

Environmental light information such as quality, intensity, and duration in red (approximately 660 nm) and far-red (approximately 730 nm) wavelengths is perceived by phytochrome photoreceptors in plants, critically influencing almost all developmental strategies from germination to flowering. Phytochromes interconvert between red light-absorbing Pr and biologically functional far-red light-absorbing Pfr forms. To ensure optimal photoresponses in plants, the flux of light signal from Pfr-phytochromes should be tightly controlled. Phytochromes are phosphorylated at specific serine residues. We found that a type 5 protein phosphatase (PAPP5) specifically dephosphorylates biologically active Pfr-phytochromes and enhances phytochrome-mediated photoresponses. Depending on the specific serine residues dephosphorylated by PAPP5, phytochrome stability and affinity for a downstream signal transducer, NDPK2, were enhanced. Thus, phytochrome photoreceptors have developed an elaborate biochemical tuning mechanism for modulating the flux of light signal, employing variable phosphorylation states controlled by phosphorylation and PAPP5-mediated dephosphorylation as a mean to control phytochrome stability and affinity for downstream transducers.

Phytochrome Phosphorylation Modulates Light Signaling by Influencing the Protein–Protein Interaction

Plant photoreceptor phytochromes are phosphoproteins, but the question as to the functional role of phytochrome phosphorylation has remained to be elucidated. We investigated the functional role of phytochrome phosphorylation in plant light signaling using a Pfr-specific phosphorylation site mutant, Ser598Ala of oat (Avena sativa) phytochrome A (phyA). The transgenic Arabidopsis thaliana (phyA-201 background) plants with this mutant phyA showed hypersensitivity to light, suggesting that phytochrome phosphorylation at Serine-598 (Ser598) in the hinge region is involved in an inhibitory mechanism. The phosphorylation at Ser598 prevented its interaction with putative signal transducers, Nucleoside Diphosphate Kinase-2 and Phytochrome-Interacting Factor-3. These results suggest that phosphorylation in the hinge region of phytochromes serves as a signal-modulating site through the protein–protein interaction between phytochrome and its putative signal transducer proteins.

Overexpression of Arabidopsis translationally controlled tumor protein gene AtTCTP enhances drought tolerance with rapid ABA-induced stomatal closure

Translationally controlled tumor protein (TCTP), also termed P23 in human, belongs to a family of calcium- and tubulin-binding proteins, and it is generally regarded as a growth-regulating protein. Recently, Arabidopsis TCTP (AtTCTP) has been reported to function as an important growth regulator in plants. On the other hand, plant TCTP has been suggested to be involved in abiotic stress signaling such as aluminum, salt, and water deficit by a number of microarray or proteomic analyses. In this study, the biological functions of AtTCTP were investigated by using transgenic Arabidopsis plants overexpressing AtTCTP. Interestingly, AtTCTP overexpression enhanced drought tolerance in plants. The expression analysis showed that AtTCTP was expressed in guard cells as well as in actively growing tissues. Physiological studies of the overexpression lines showed increased ABA- and calcium-induced stomatal closure ratios and faster stomatal closing responses to ABA. Furthermore, in vitro protein-protein interaction analysis confirmed the interaction between AtTCTP and microtubules, and microtubule cosedimentation assays revealed that the microtubule binding of AtTCTP increased after calcium treatment. These results demonstrate that the overexpression of AtTCTP confers drought tolerance to plants by rapid ABA-mediated stomatal closure via the interaction with microtubules in which calcium binding enhances the interaction. Collectively, the present results suggest that the plant TCTP has molecular properties similar to animal TCTPs, such as tubulin- and calcium-binding, and that it functions in ABA-mediated stomatal movement, in addition to regulating the growth of plants.

Environmental risk assessment of genetically engineered herbicide-tolerant Zoysia japonica.

Herbicide-tolerant Zoysia grass (Zoysia japonica Steud.) has been generated previously through Agrobacterium tumefaciens-mediated transformation. The genetically modified (GM) Zoysia grass survived Basta spraying and grew to maturity normally while the wild-type (WT) grass stopped growing and died. GM Zoysia grass will permit more efficient weed control for various turf grass plantings such as home lawns, golf courses, and parks. We examined the environmental/biodiversity risks of herbicide-tolerant GM Zoysia before applying to regulatory agencies for approval for commercial release. The GM and WT Zoysia grass substantial trait equivalence, ability to cross-pollinate, and gene flow in confined and unconfined test fields were selectively analyzed for environmental/biodiversity effects. No difference between GM and WT Zoysia grass in substantial traits was found. To assess the potential for cross-pollination and gene flow, a non-selective herbicide, Basta, was used. Results showed that unintended cross-pollination with and gene flow from GM Zoysia grass were not detected in neighboring weed species examined, but were observed in WT Zoysia grass (on average, 6% at proximity, 1.2% at a distance of 0.5 m and 0.12% at a radius of 3 m, and 0% at distances over 3 m). On the basis of these initial studies, we conclude that the GM Zoysia grass generated in our laboratory and tested in the Nam Jeju County field does not appear to pose a significant risk when cultivated outside of test fields.

Production of purple-colored creeping bentgrass using maize transcription factor genes Pl and Lc through Agrobacterium-mediated transformation

Purple-colored transgenic creeping bentgrass (Agrostis stolonifera L.) plants were developed for ornamental purpose by means of Agrobacterium-mediated transformation. Embryogenic creeping bentgrass calli were transformed with the pCAMBIA 3301 vector harboring maize (Zea mays) flavonoid/anthocyanin biosynthetic pathway transcription factor genes, Lc (Leaf color) and Pl (Purple leaf), individually and in combination, and three types of putative transgenic plants (Lc, Pl, and Lcxa0+xa0Pl) were generated. Genomic integration and expression of the transgenes were confirmed by Southern and northern blot analyses, respectively. The transgenic creeping bentgrass plants expressing both Lc and Pl genes were entirely purple, whereas those expressing Pl alone had purple stems and those expressing Lc alone lacked purple pigmentation in adult plants. The anthocyanin content was estimated in all the three types of transgenic plant and correlated well with the degree of purple coloration observed. These results suggest that both Lc and Pl genes are necessary and sufficient to confer purple coloration to creeping bentgrass.

A novel protein phosphatase indirectly regulates phytochrome-interacting factor 3 via phytochrome.

Light signal transduction in plants involves an intricate series of pathways which is finely regulated by interactions between specific signalling proteins, as well as by protein modifications such as phosphorylation and ubiquitination. The identification of novel phytochrome-interacting proteins and the precise signalling mechanisms that they mediate is still ongoing. In our present study, we show that the newly identified putative phytochrome-associated protein, PAPP2C (phytochrome-associated protein phosphatase type 2C), interacts in the nucleus with phyA (phytochrome A) and phyB, both in vitro and in vivo. Moreover, the phosphatase activity of PAPP2C and its association with phytochromes were found to be enhanced by red light, indicating that it plays a role in mediating phytochrome signalling. In particular, PAPP2C specifically binds to the N-terminal PHY domain of the phytochromes. We thus speculate that this interaction reflects a unique regulatory function of this phosphatase toward established phytochrome-associated proteins. We also show that PAPP2C effectively dephosphorylates phytochromes in vitro. Interestingly, PAPP2C indirectly mediates the dephosphorylation of PIF3 (phytochrome-interacting factor 3) in vitro. Taken together, we suggest that PAPP2C functions as a regulator of PIF3 by dephosphorylating phytochromes in the nucleus.

Functional Characterization of Phytochrome Autophosphorylation in Plant Light Signaling

Plant phytochromes, molecular light switches that regulate various aspects of plant growth and development, are phosphoproteins that are also known to be autophosphorylating serine/threonine kinases. Although a few protein phosphatases that directly interact with and dephosphorylate phytochromes have been identified, no protein kinase that acts on phytochromes has been reported thus far, and the exact site of phytochrome autophosphorylation has not been identified. In this study, we investigated the functional role of phytochrome autophosphorylation. We first mapped precisely the autophosphorylation sites of oat phytochrome A (phyA), and identified Ser8 and Ser18 in the 65 amino acid N-terminal extension (NTE) region as being the autophosphorylation sites. The in vivo functional roles of phytochrome autophosphorylation were examined by introducing autophosphorylation site mutants into phyA-deficient Arabidopsis thaliana. We found that all the transgenic plants expressing the autophosphorylation site mutants exhibited hypersensitive light responses, indicating an increase in phyA activity. Further analysis showed that these phyA mutant proteins were degraded at a significantly slower rate than wild-type phyA under light conditions, which suggests that the increased phyA activity of the mutants is related to their increased protein stability. In addition, protoplast transfection analyses with green fluorescent protein (GFP)-fused phyA constructs showed that the autophosphorylation site mutants formed sequestered areas of phytochrome (SAPs) in the cytosol much more slowly than did wild-type phyA. These results suggest that the autophosphorylation of phyA plays an important role in the regulation of plant phytochrome signaling through the control of phyA protein stability.

Photoactivation and inactivation of Arabidopsis cryptochrome 2

Turning off the blue-light response In plants, blue light is perceived by cryptochromes, which, once activated, set off signaling events that regulate gene expression, circadian rhythms, and photomorphogenesis. Wang et al. now show that in the model plant Arabidopsis, one of the functions of activated cryptochromes, which are dimers or oligomers when active, is to activate production of the protein BIC1 (blue-light inhibitor of cryptochromes 1) (see the Perspective by Fankhauser and Ulm). BIC1 then favors monomerization and thus inactivation of the cryptochromes. This feedback loop resets the system so that blue-light responses can be turned off as well as turned on. Science, this issue p. 343; see also p. 282 A feedback loop ensures against a runaway response to light in plants. Cryptochromes are blue-light receptors that regulate development and the circadian clock in plants and animals. We found that Arabidopsis cryptochrome 2 (CRY2) undergoes blue light–dependent homodimerization to become physiologically active. We identified BIC1 (blue-light inhibitor of cryptochromes 1) as an inhibitor of plant cryptochromes that binds to CRY2 to suppress the blue light–dependent dimerization, photobody formation, phosphorylation, degradation, and physiological activities of CRY2. We hypothesize that regulated dimerization governs homeostasis of the active cryptochromes in plants and other evolutionary lineages.