A novel basic helix-loop-helix transcription factor, ZjICE2 from Zoysia japonica confers abiotic stress tolerance to transgenic plants via activating the DREB/CBF regulon and enhancing ROS scavenging.

KEY MESSAGE: ZjICE2 works as a positive regulator in abiotic stress responses and ZjICE2 is a valuable genetic resource to improve abiotic stress tolerance in the molecular breeding program of Zoysia japonica. The basic helix-loop-helix (bHLH) family transcription factors (TFs) play an important role in response to biotic or abiotic stresses in plants. However, the functions of bHLH TFs in Zoysia japonica, one of the warm-season turfgrasses, remain poorly understood. Here, we identified ZjICE2 from Z. japonica, a novel MYC-type bHLH transcription factor that was closely related to ICE homologs in the phylogenetic tree, and its expression was regulated by various abiotic stresses. Transient expression of ZjICE2-GFP in onion epidermal cells revealed that ZjICE2 was a nuclear-localized protein. Also, ZjICE2 bound the MYC cis-element in the promoter of dehydration responsive element binding 1 of Z. japonica (ZjDREB1) using yeast one-hybrid assay. A phenotypic analysis showed that overexpression of the ZjICE2 in Arabidopsis enhanced tolerance to cold, drought, and salt stresses. The transgenic Arabidopsis and Z. japonica accumulated more transcripts of cold-responsive DREB/CBFs and their downstream genes than the wild type (WT) after cold treatment. Furthermore, the transgenic plants exhibited an enhanced Reactive oxygen species (ROS) scavenging ability, which resulted in an efficient maintenance of oxidant-antioxidant homeostasis. In addition, overexpression of the ZjICE2 in Z. japonica displayed intensive cold tolerance with increases in chlorophyll contents and photosynthetic efficiency. Our study suggests that ZjICE2 works as a positive regulator in abiotic stress responses and the ICE-DREB/CBFs response pathway involved in cold stress tolerance is also conserved in Z. japonica. These results provide a valuable genetic resource for the molecular breeding program especially for warm-season grasses as well as other leaf crop plants.

Zoysia japonica MYC type transcription factor ZjICE1 regulates cold tolerance in transgenic Arabidopsis.

ICE1 (Inducer of CBF Expression 1) is a regulator of cold-induced transcriptome, which plays an important role in plant cold response pathway. To enhance the cold tolerance of Zoysia japonica, one of the warm-season turfgrasses, it is helpful to understand the cold response mechanism in Zoysia japonica. We identified stress-responsive ZjICE1 from Zoysia japonica and characterized its function in cold stress. Our results showed that ZjICE1 shared the typical feature of ICE homolog proteins belonging to a nucleic protein. Transactivation activity assay revealed that ZjICE1 bound to the MYC cis-element in the ZjDREB1's promotor. The ZjICE1 overexpressed transgenic Arabidopsis showed enhanced tolerance to cold stress with an increases in SOD, POD, and free proline content and reduction in MDA content. They also induced the transcripts abundance of cold-responsive genes (CBF1, CBF2, CBF3, COR47A, KIN1, and RD29A) after cold treatment. These results suggest that ZjICE1 is a positive regulator in Zoysia japonica plant during cold stress and can be a useful gene for the molecular breeding program to develop the cold tolerant zoysiagrass. Furthermore, the ZjICE1 also conferred resistance to salt and drought stresses, providing the better understanding of the basic helix-loop-helix (bHLH) gene family in abiotic stress responses.

Development of transgenic crops based on photo-biotechnology.

The phenotypes associated with plant photomorphogenesis such as the suppressed shade avoidance response and de-etiolation offer the potential for significant enhancement of crop yields. Of many light signal transducers and transcription factors involved in the photomorphogenic responses of plants, this review focuses on the transgenic overexpression of the photoreceptor genes at the uppermost stream of the signalling events, particularly phytochromes, crytochromes and phototropins as the transgenes for the genetic engineering of crops with improved harvest yields. In promoting the harvest yields of crops, the photoreceptors mediate the light regulation of photosynthetically important genes, and the improved yields often come with the tolerance to abiotic stresses such as drought, salinity and heavy metal ions. As a genetic engineering approach, the term photo-biotechnology has been coined to convey the idea that the greater the photosynthetic efficiency that crop plants can be engineered to possess, the stronger the resistance to biotic and abiotic stresses. Development of GM crops based on photoreceptor transgenes (mainly phytochromes, crytochromes and phototropins) is reviewed with the proposal of photo-biotechnology that the photoreceptors mediate the light regulation of photosynthetically important genes, and the improved yields often come with the added benefits of crops' tolerance to environmental stresses.

Evidence that phytochrome functions as a protein kinase in plant light signalling.

It has been suggested that plant phytochromes are autophosphorylating serine/threonine kinases. However, the biochemical properties and functional roles of putative phytochrome kinase activity in plant light signalling are largely unknown. Here, we describe the biochemical and functional characterization of Avena sativa phytochrome A (AsphyA) as a potential protein kinase. We provide evidence that phytochrome-interacting factors (PIFs) are phosphorylated by phytochromes in vitro. Domain mapping of AsphyA shows that the photosensory core region consisting of PAS-GAF-PHY domains in the N-terminal is required for the observed kinase activity. Moreover, we demonstrate that transgenic plants expressing mutant versions of AsphyA, which display reduced activity in in vitro kinase assays, show hyposensitive responses to far-red light. Further analysis reveals that far-red light-induced phosphorylation and degradation of PIF3 are significantly reduced in these transgenic plants. Collectively, these results suggest a positive relationship between phytochrome kinase activity and photoresponses in plants.

In Vivo Assessment of Cold Tolerance through Chlorophyll-a Fluorescence in Transgenic Zoysiagrass Expressing Mutant Phytochrome A.

Chlorophyll-a fluorescence analysis provides relevant information about the physiology of plants growing under abiotic stress. In this study, we evaluated the influence of cold stress on the photosynthetic machinery of transgenic turfgrass, Zoysia japonica, expressing oat phytochrome A (PhyA) or a hyperactive mutant phytochrome A (S599A) with post-translational phosphorylation blocked. Biochemical analysis of zoysiagrass subjected to cold stress revealed reduced levels of hydrogen peroxide, increased proline accumulation, and enhanced specific activities of antioxidant enzymes compared to those of control plants. Detailed analyses of the chlorophyll-a fluorescence data through the so-called OJIP test exhibited a marked difference in the physiological status among transgenic and control plants. Overall, these findings suggest an enhanced level of cold tolerance in S599A zoysiagrass cultivars as reflected in the biochemical and physiological analyses. Further, we propose that chlorophyll-a fluorescence analysis using OJIP test is an efficient tool in determining the physiological status of plants under cold stress conditions.

Photo-biotechnology as a tool to improve agronomic traits in crops.

Phytochromes are photosensory phosphoproteins with crucial roles in plant developmental responses to light. Functional studies of individual phytochromes have revealed their distinct roles in the plant's life cycle. Given the importance of phytochromes in key plant developmental processes, genetically manipulating phytochrome expression offers a promising approach to crop improvement. Photo-biotechnology refers to the transgenic expression of phytochrome transgenes or variants of such transgenes. Several studies have indicated that crop cultivars can be improved by modulating the expression of phytochrome genes. The improved traits include enhanced yield, improved grass quality, shade-tolerance, and stress resistance. In this review, we discuss the transgenic expression of phytochrome A and its hyperactive mutant (Ser599Ala-PhyA) in selected crops, such as Zoysia japonica (Japanese lawn grass), Agrostis stolonifera (creeping bentgrass), Oryza sativa (rice), Solanum tuberosum (potato), and Ipomea batatas (sweet potato). The transgenic expression of PhyA and its mutant in various plant species imparts biotechnologically useful traits. Here, we highlight recent advances in the field of photo-biotechnology and review the results of studies in which phytochromes or variants of phytochromes were transgenically expressed in various plant species. We conclude that photo-biotechnology offers an excellent platform for developing crops with improved properties.

Expression of recombinant full-length plant phytochromes assembled with phytochromobilin in Pichia pastoris.

We have successfully developed a system to produce full-length plant phytochrome assembled with phytochromobilin in Pichia pastoris by co-expressing apophytochromes and chromophore biosynthetic genes, heme oxygenase (HY1) and phytochromobilin synthase (HY2) from Arabidopsis. Affinity-purified phytochrome proteins from Pichia cells displayed zinc fluorescence indicating chromophore attachment. Spectroscopic analyses showed absorbance maximum peaks identical to in vitro reconstituted phytochromobilin-assembled phytochromes, suggesting that the co-expression system is effective to generate holo-phytochromes. Moreover, mitochondria localization of the phytochromobilin biosynthetic genes increased the efficiency of holophytochrome biosynthesis. Therefore, this system provides an excellent source of holophytochromes, including oat phytochrome A and Arabidopsis phytochrome B.

Overexpression of phytochrome A and its hyperactive mutant improves shade tolerance and turf quality in creeping bentgrass and zoysiagrass.

Phytochrome A (phyA) in higher plants is known to function as a far-red/shade light-sensing photoreceptor in suppressing shade avoidance responses (SARs) to shade stress. In this paper, the Avena PHYA gene was introduced into creeping bentgrass (Agrostis stolonifera L.) and zoysiagrass (Zoysia japonica Steud.) to improve turf quality by suppressing the SARs. In addition to wild-type PHYA, a hyperactive mutant gene (S599A-PHYA), in which a phosphorylation site involved in light-signal attenuation was removed, was also transformed into the turfgrasses. Phenotypic traits of the transgenic plants were compared to assess the suppression of SARs under a simulated shade condition and outdoor field conditions after three growth seasons. Under the shade condition, the S599A-PhyA transgenic creeping bentgrass plants showed shade avoidance-suppressing phenotypes with a 45 % shorter leaf lengths, 24 % shorter internode lengths, and twofold increases in chlorophyll concentrations when compared with control plants. Transgenic zoysiagrass plants overexpressing S599A-PHYA also showed shade-tolerant phenotypes under the shade condition with reductions in leaf length (15 %), internode length (30 %), leaf length/width ratio (19 %) and leaf area (22 %), as well as increases in chlorophyll contents (19 %) and runner lengths (30 %) compared to control plants. The phenotypes of transgenic zoysiagrass were also investigated in dense field habitats, and the transgenic turfgrass exhibited shade-tolerant phenotypes similar to those observed under laboratory shade conditions. Therefore, the present study suggests that the hyperactive phyA is effective for the development of shade-tolerant plants, and that the shade tolerance nature is sustained under field conditions.

Photochemical characterization of phytochrome missense mutants.

Phytochromes are photoreceptors that regulate many aspects of plant growth and development in response to red/far-red light signals from the environment. In this study, we analyzed chromophore ligation and photochromism of missense phytochrome mutants in the Per-Arnt-Sim (PAS)-related domain (PRD). Among the 14 mutants analyzed, the Gly768Asp mutant of Avena phytochrome A showed aberrant photochromism and dark reversion, suggesting that amino acid residues in the C-terminal domain affect the photochemical properties of the photosensory N-terminal domain.

A small GTPase activator protein interacts with cytoplasmic phytochromes in regulating root development.

Phytochromes enable plants to sense light information and regulate developmental responses. Phytochromes interact with partner proteins to transmit light signals to downstream components for plant development. PIRF1 (phytochrome-interacting ROP guanine-nucleotide exchange factor (RopGEF 1)) functions as a light-signaling switch regulating root development through the activation of ROPs (Rho-like GTPase of plant) in the cytoplasm. In vitro pulldown and yeast two-hybrid assays confirmed the interaction between PIRF1 and phytochromes. PIRF1 interacted with the N-terminal domain of phytochromes through its conserved PRONE (plant-specific ROP nucleotide exchanger) region. PIRF1 also interacted with ROPs and activated them in a phytochrome-dependent manner. The Pr form of phytochrome A enhanced the RopGEF activity of PIRF1, whereas the Pfr form inhibited it. A bimolecular fluorescence complementation analysis demonstrated that PIRF1 was localized in the cytoplasm and bound to the phytochromes in darkness but not in light. PIRF1 loss of function mutants (pirf1) of Arabidopsis thaliana showed a longer root phenotype in the dark. In addition, both PIRF1 overexpression mutants (PIRF1-OX) and phytochrome-null mutants (phyA-211 and phyB-9) showed retarded root elongation and irregular root hair formation, suggesting that PIRF1 is a negative regulator of phytochrome-mediated primary root development. We propose that phytochrome and ROP signaling are interconnected through PIRF1 in regulating the root growth and development in Arabidopsis.

Autophosphorylation desensitizes phytochrome signal transduction.

Plant red/far-red photoreceptor phytochromes are known as autophosphorylating serine/threonine kinases. However, the functional roles of autophosphorylation and kinase activity of phytochromes are largely unknown. We recently reported that the autophosphorylation of phytochrome A (phyA) plays an important role in regulating plant phytochrome signaling by controlling phyA protein stability. Two serine residues in the N-terminal extension (NTE) region were identified as autophosphorylation sites, and phyA mutant proteins with serine-to-alanine mutations were degraded in plants at a significantly slower rate than the wild-type under light conditions, resulting in transgenic plants with hypersensitive light responses. In addition, the autophosphorylation site phyA mutants had normal protein kinase activities. Collectively, our results suggest that phytochrome autophosphorylation provides a mechanism for signal desensitization in phytochrome-mediated light signaling by accelerating the degradation of phytochrome A.

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.

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 Lc + Pl) 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.

Arabidopsis nucleoside diphosphate kinase-2 as a plant GTPase activating protein.

Nucleoside diphosphate kinase (NDPK) is involved in multiple signaling pathways in mammalian systems, including G-protein signaling. Arabidopsis NDPK2, like its mammalian counterparts, is multifunctional despite its initial discovery phytochrome-interacting protein. This similarity raises the possibility that NDPK2 may play a role in G-protein signaling in plants. In the present study, we explore the potential relationship between NDPK2 and the small G proteins, Pra2 and Pra3, as well as the heterotrimeric G protein, GPA1. We report a physical interaction between NDPK2 and these small G proteins, and demonstrate that NDPK2 can stimulate their GTPase activities. Our results suggest that NDPK2 acts as a GTPase-activating protein for small G proteins in plants. We propose that NDPK2 might be a missing link between the phytochromemediated light signaling and G protein-mediated signaling.

Differential interactions of phytochrome A (Pr vs. Pfr) with monoclonal antibodies probed by a surface plasmon resonance technique.

Phytochromes are red- and far-red light-reversible photoreceptors for photomorphogenesis in plants. Phytochrome A is a dimeric chromopeptide that mediates very low fluence and high irradiance responses. To analyze the surface properties of phytochrome A (phyA), the epitopes of 21 anti-phyA monoclonal antibodies were determined by variously engineered recombinant phyA proteins and the dissociation constants of seven anti-phyA monoclonal antibodies with phyA were measured using a surface plasmon resonance (SPR)-based resonant mirror biosensor (IAsys). Purified oat phyA was immobilized on the sensor surface using a carboxymethyl dextran cuvette in advance, and the interactions of each chosen monoclonal antibody against phyA in either red light absorbing form (Pr) or far-red light absorbing form (Pfr) at different concentrations were monitored. The binding profiles were analyzed using the FAST Fit program of IAsys. The resultant values of dissociation constants clearly demonstrated the differential affinities between the phyA epitopes and the monoclonal antibodies dependent upon Pr vs. Pfr conformations. Monoclonal antibody mAP20 preferentially recognized the epitope at amino acids 653-731 in the Pr form, whereas mAA02, mAP21 and mAR07/mAR08 displayed preferential affinities for the Pfr's surfaces at epitopes 494-601 (the hinge region between the N- and C-terminal domains), 601-653 (hinge in PASI domain), and 772-1128 (C-terminal domain), respectively. The N-terminal extension (1-74) was not recognized by mAP09 and mAP15, suggesting that the N-terminal extreme is not exposed in the native conformation of phyA. On the other hand, the C-terminal domain becomes apparently exposed on Pr-to-Pfr phototransformation, suggesting an inter-domain cross-talk. The use of surface plasmon resonance spectroscopy offers a new approach to study the surface properties of phytochromes associated with the photoreversible structural changes, as well as for the study of protein-protein interactions of phytochromes with their interacting proteins involved in light signaling events in plants.

Time-resolved detection of conformational changes in oat phytochrome A: time-dependent diffusion.

Conformational changes in oat phytochrome A (phy) in solution after photoexcitation of the red-absorbing form (Pr) were studied in time-domain by the pulsed laser-induced transient grating technique. It was found that the diffusion coefficient (D) of far-red-absorbing form (Pfr) of large phy (1.3 x 10(-11) m(2) s(-1)) is markedly reduced compared with that of Pr (5.8 x 10(-11) m(2) s(-1)). This large reduction indicates that the conformation of Pfr is significantly changed from that of Pr, so that the intermolecular interaction with water molecules increases. This change completes within 1 ms after the photoexcitation. On the other hand, D of Pr of intact phy (4.1 x 10(-11) m(2) s(-1)) first decreases upon photoexcitation to 0.89 x 10(-11) m(2) s(-1) within 1 ms and then gradually increases with a time constant of 100 ms to the value of Pfr, 1.7 x 10(-11) m(2) s(-1). This slower phase suggests that the conformation of the N-terminal region changes with 100 ms to decrease the intermolecular interaction with water after a global change in the large phy region. The increase of D was interpreted in terms of alpha-helix formation in the Pfr form from the random coil structure in the Pr form.

Autophosphorylation of Arabidopsis nucleoside diphosphate kinase 2 occurs only on its active histidine residue.

Arabidopsis nucleoside diphosphate kinase 2 (NDPK2) is a component in the phytochrome-mediated light signaling. In the present study, its autophosphorylation was investigated. Acid-stable and alkali-stable phosphorylated residues were analyzed under two different conditions. Results revealed that NDPK2 is phosphorylated only on its active histidine residue His197 and the presence of serine/threonine phosphorylation is an experimental artifact due to the harsh condition applied in the treatment of the phosphorylated protein sample. To resolve the controversy of whether serine/threonine phosphorylation of NDPK occurs as has been suggested by other NDPK studies, NDPK2 putative phosphorylation site mutants were generated and examined. No serine/threonine phosphorylation was identified in NDPK2 or implicated in its enzymatic activity. Further studies indicated that the low enzymatic activity and autophosphorylation level of NDPK2 mutant S199A are shown to be due to a damaged H-bonding with the active histidine residue His197 in the nucleotide-binding pocket. In addition, NDPK2 Kpn loop mutant T182A was found to possess an extremely low enzymatic activity and almost no autophosphorylation, suggesting the importance of the oligomeric states of NDPK2 in NDPK2 functioning.

Phytochrome phosphorylation in plant light signaling.

Reversible protein phosphorylation is a switching mechanism used in eukaryotes to regulate various cellular signalings. In plant light signaling, sophisticated photosensory receptor systems operate to modulate growth and development. The photoreceptors include phytochromes, cryptochromes and phototropins. Despite considerable progresses in defining the photosensory roles of these photoreceptors, the primary biochemical mechanisms by which the photoreceptor molecules transduce the perceived light signals into cellular responses remain to be elucidated. The signal-transducing photoreceptors in plants are all phosphoproteins and/or protein kinases, suggesting that light-dependent protein phosphorylation and dephosphorylation play important roles in the function of the photoreceptors. This review focuses on the role of phytochromes' reversible phosphorylation involved in the light signal transduction in plants.

A possible role for NDPK2 in the regulation of auxin-mediated responses for plant growth and development.

Auxin plays many crucial roles in the course of plant growth and development, such as hook opening, leaf expansion and inhibition of mesocotyl elongation. Although its mechanism of action has not been clarified at the molecular level, recent studies have indicated that auxin triggers the induction of a number of genes known as primary auxin-responsive genes. Hence, the identification of the regulatory components in auxin-mediated cellular responses would help to elucidate the mechanism of the action of this hormone in plant growth and development. NDPK2 encodes a nucleoside diphosphate kinase 2 (NDPK2) in Arabidopsis. We aim to elucidate the possible role of NDPK2 in auxin-related cellular processes, in view of the finding that a ndpk2 mutant displays developmental defects associated with auxin. Interestingly, the ndpk2 mutant exhibits defects in cotyledon development and increased sensitivity to an inhibitor of polar auxin transport (naphthylphthalamic acid; NPA). Consistent with this phenotype, the transcript levels of specific auxin-responsive genes were reduced in the ndpk2 mutant plants treated with auxin. The amount of auxin transported from the shoot apex to the shoot/root transition zone of ndpk2 mutant plants was increased, compared with that in the wild-type plants. These results collectively suggest that NDPK2 appears to participate in auxin-regulated processes, partly through the modulation of auxin transport.