Cloning of a Zoysia ZjLsL and its overexpression to induce axillary meristem initiation and tiller formation in Arabidopsis and bentgrass.

Zoysia grass and creeping bentgrass are important turf grasses used in parks, gardens and playing fields. Development of grasses with increased tiller formation will enhance their commercial cultivation. To investigate the regulatory mechanism of tiller formation, we cloned the Zoysia japonica Lateral suppressor-like (ZjLsL) gene. The Lateral suppressor (Ls) gene encodes a transcriptional regulator belonging to the plant-specific GRAS protein family of putative transcription factors, and regulates axillary meristem initiation. A full-length DNA of the ZjLsL gene was isolated by 5'/3' DNA walking. Phylogenetic analysis showed that ZjLsL is closely related to Ls genes. Southern blot analysis revealed that zoysia grass has two copies of the ZjLsL gene. ZjLsL expression was detected in all organs of zoysia grass but was most highly expressed in culms. Overexpression of ZjLsL in creeping bentgrass and Arabidopsis plants promoted axillary bud formation. These results suggest that ZjLsL plays an important role in axillary meristem initiation and tiller formation.

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.

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.

REP1, a basic helix-loop-helix protein, is required for a branch pathway of phytochrome A signaling in arabidopsis.

Phytochromes are primary photoreceptors mediating diverse responses ranging from induction of germination to floral induction in higher plants. We have isolated novel recessive rep1 (reduced phytochrome signaling 1) mutants, which exhibit a long-hypocotyl phenotype only under far-red light but not under red light. Physiological characterization showed that rep1 mutations greatly reduced a subset of phytochrome A-regulated responses, including the inhibition of hypocotyl elongation, cotyledon expansion, modulation of gravitropic growth of hypocotyl, and induction of the CAB (encoding chlorophyll a/b binding protein) gene, without affecting the accumulation of anthocyanin, far-red-preconditioned blocking of greening, induction of germination, and induction of CHS (encoding chalcone synthase) and FNR (encoding ferredoxin-NADP(+) oxidoreductase) genes. These results suggest that REP1 is a positive signaling component, functioning in a branch of the phytochrome A signaling pathway. Molecular cloning and characterization of the REP1 gene revealed that it encodes a light-inducible, putative transcription factor containing the basic helix-loop-helix motif.

Crystallization and preliminary X-ray crystallographic studies of response regulator for cyanobacterial phytochrome, Rcp1.

The key response-regulator gene of light regulation, rcp1, from Synechocystis sp. has been overexpressed, purified and subsequently crystallized using ammonium sulfate as a precipitant in forms suitable for X-ray crystallographic studies. A native data set was collected to a resolution of 2.5 A at cryogenic temperature. The crystals belong to the hexagonal space group P6(3), with unit-cell parameters a = b = 89.04 (5), c = 60.29 (3) A. The Matthews parameter suggests that Rcp1 crystallizes with two molecules per asymmetric unit.

A second photochromic bacteriophytochrome from Synechocystis sp. PCC 6803: spectral analysis and down-regulation by light.

It now appears that photosynthetic prokaryotes and lower eukaryotes possess higher plant phytochrome-like proteins. In this work, a second phytochrome-like gene was isolated, in addition to the recently identified Cph1 phytochrome, from the Synechocystis sp. PCC 6803, and its gene product was characterized photochemically. The open reading frame sll0821 (designated cph2 in this work) has structural characteristics similar to those of the plant phytochromes and the Synechocystis Cph1 with high amino acid sequence homology in the N-terminal chromophore binding domain. The predicted Cph2 protein consists of 1276 amino acids with a calculated molecular mass of 145 kDa. Interestingly, the Cph2 protein has two putative chromophore binding domains, one around Cys-129 and the other around Cys-1022. The Cph2 was overexpressed in E. coli as an Intein/CBD (chitin binding domain) fusion and in vitro reconstituted with phycocyanobilin (PCB) or phytochromobilin (PPhiB). Both the Cph2-PCB and Cph2-PPhiB adducts showed the typical photochromic reversibility with the difference spectral maxima at 643/690 and 655/701 nm, respectively. The Cys-129 was confirmed to be the chromophore binding residue by in vitro mutagenesis and Zn(2+) fluorescence. The microenvironment of the chromophore in Cph2 seems to be similar to that in plant phytochromes. The cph2 gene expression was dark-induced and down-regulated to a basal level by light, like the cph1 gene. These observations suggest that Synechocystis species have multiple photosensory proteins, probably with distinct roles, as in higher plants.

Chromophore-apoprotein interactions in Synechocystis sp. PCC6803 phytochrome Cph1.

The secondary, tertiary, and quaternary structures of the Synechocystis Cph1 phytochrome were investigated by absorption and circular dichroism spectroscopy, size exclusion chromatography, and limited proteolysis. The Cph1 protein was coexpressed with a bacterial thioredoxin in Escherichia coli, reconstituted in vitro with tetrapyrrole chromophores, and purified by chitin affinity chromatography. The resultant Cph1 holoproteins were essentially pure and had the specific absorbance ratio (SAR) of 0.8-0.9. Circular dichroism spectroscopy and limited proteolysis showed that the chromophore binding induced marked conformational changes in the Cph1 protein. The alpha-helical content increased to 42-44% in the holoproteins from 37% in the apoprotein. However, no significant difference in the secondary structure was detected between the Pr and Pfr forms. The tertiary structure of the Cph1 apoprotein appeared to be relatively flexible but became more compact and resistant to tryptic digestion upon chromophore binding. Interestingly, a small chromopeptide of about 30 kDa was still predominant even after longer tryptic digestion. The N-terminal location of this chromopeptide was confirmed by expression in E. coli and in vitro reconstitution with chromophores of the 32.5 kDa N-terminal fragment of the Cph1 protein. This chromopeptide was fully photoreversible with the spectral characteristic similar to that of the full-size Cph1 protein. The Cph1 protein forms dimers through the C-terminal region. These results suggest that the prokaryotic Cph1 phytochrome shares the structural and conformational characteristics of plant phytochromes, such as the two-domain structure consisting of the relatively compact N-terminal and the relatively flexible C-terminal regions, in addition to the chromophore-induced conformational changes.

Inter-domain crosstalk in the phytochrome molecules.

Phytochromes are bifunctional photoreceptors with a two-domain structure, consisting of the N-terminal photosensory domain and the C-terminal regulatory domain. The photo-induced Pr <--> Pfr phototransformation accompanies subtle conformational changes, primarily triggered by the apoprotein-chromophore interactions in the N-terminal domain. The conformational signals are subsequently transmitted to the C-terminal domain through various inter-domain crosstalks, resulting in the interaction of the activated C-terminal domain with phytochrome interacting factors. Thus the inter-domain crosstalks play critical roles in the photoactivation of the phytochromes. Protein phosphorylation, such as that of Ser-598, is implicated in this process by inducing conformational changes and by modulating inter-domain signaling.

Phytochrome signalling is mediated through nucleoside diphosphate kinase 2.

Because plants are sessile, they have developed intricate strategies to adapt to changing environmental variables, including light. Their growth and development, from germination to flowering, is critically influenced by light, particularly at red (660 nm) and far-red (730 nm) wavelengths. Higher plants perceive red and far-red light by means of specific light sensors called phytochromes(A-E). However, very little is known about how light signals are transduced to elicit responses in plants. Here we report that nucleoside diphosphate kinase 2 (NDPK2) is an upstream component in the phytochrome signalling pathway in the plant Arabidopsis thaliana. In animal and human cells, NDPK acts as a tumour suppressor. We show that recombinant NDPK2 in Arabidopsis preferentially binds to the red-light-activated form of phytochrome in vitro and that this interaction increases the activity of recombinant NDPK2. Furthermore, a mutant lacking NDPK2 showed a partial defect in responses to both red and farred light, including cotyledon opening and greening. These results indicate that NDPK2 is a positive signalling component of the phytochrome-mediated light-signal-transduction pathway in Arabidopsis.

Photomovement of the gliding cyanobacterium Synechocystis sp. PCC 6803.

Using a computerized videomicroscope motion analysis system, we investigated the photomovements of two Synechocystis sp. (PCC 6803 and ATCC 27184). Synechocystis sp. PCC 6803 displays a relatively slow gliding motion. The phototactic and photokinetic speeds of this cyanobacterium in liquid media were 5 microns/min and 15.8 microns/min, respectively, at 3 mumol/m2/s of stimulant white light. Synechocystis sp. PCC 6803 senses light direction rather than intensity for phototaxis. Synechocystis sp. ATCC 27184 showed a weak photokinesis but no phototaxis. Analysis of Synechocystis sp. ATCC 27184 suggests that the loss of phototaxis results from spontaneous mutation during several years of subculture. When directional irradiation was applied, the cell population of Synechocystis sp. PCC 6803 began to deviate from random movement and reached maximum orientation at 5 min after the onset of stimulant white light. Synechocystis sp. PCC 6803 showed high sensitivity to the stimulant white light of fluence rates as low as 0.002 mumol/m2/s. Neither 1,3-dichlorophenyldimethyl urea nor cyanide affected phototactic orientation, whereas cyanide inhibited gliding speed. This result suggests that the phototaxis of Synechocystis sp. PCC 6803 is independent of photosynthetic phosphorylation and that its gliding movement is primarily powered by oxidative phosphorylation. In the visible wavelength region, 560 nm, 660 nm and even 760 nm caused positive phototaxis. However, 360 nm light induced strikingly negative phototaxis. Therefore, at least two independent photoreceptors may exist to control phototaxis. The photoreceptor for positive phototaxis appears likely to be a phytochrome-like tetrapyrrole rather than chlorophyll a.

Mass spectrometric characterization of oat phytochrome A: isoforms and posttranslational modifications.

At least four mRNAs for oat phytochrome A (phyA) are present in etiolated oat tissue. The complete amino acid sequences of two phyA isoforms (A3 and A4) and the N-terminal amino acid sequence of a third isoform (A5) were deduced from cDNA sequencing (Hershey et al., 1985). In the present study, heterogeneity of phyA on a protein level was studied by tryptic mapping using electrospray ionization mass-spectrometry (ESIMS). The total tryptic digest of iodoacetamide-modified phyA was fractionated by gel filtration chromatography followed by reversed-phase high-performance liquid chromatography. ESIMS was used to identify peptides. Amino acid sequences of the peptides were confirmed or determined by collision-induced dissociation mass spectrometry (CID MS), MS/MS, or by subdigestion of the tryptic peptides followed by ESIMS analysis. More than 97% of the phyA3 sequence (1,128 amino acid residues) was determined in the present study. Mass-spectrometric analysis of peptides unique to each form showed that phyA purified from etiolated oat seedling is represented by three isoforms A5, A3, and A4, with ratio 3.4:2.3:1.0. Possible light-induced changes in phytochrome in vivo phosphorylation site at Ser7 (Lapko VN et al., 1997, Biochemistry 36:10595-10599) as well at Ser17 and Ser598 (known as in vitro phosphorylation sites) were also analyzed. The extent of phosphorylation at Ser7 appears to be the same for phyA isolated from dark-grown and red-light illuminated seedlings. In addition to Ser7, Ser598 was identified as an in vivo phosphorylation site in oat phyA. Ser598 phosphorylation was found only in phyA from the red light-treated seedlings, suggesting that the protein phosphorylation plays a functional role in the phytochrome A-mediated light-signal transduction.

Surface topography of phytochrome A deduced from specific chemical modification with iodoacetamide.

Phytochromes are a photoreversible photochromic light switch for photomorphogenesis in plants. The molecular structure and functional mechanism of phytochromes are not fully understood. On the basis of complete mapping of total tryptic digest of the iodoacetamide-modified oat phytochrome A (phyA), the molecular surface topography of phyA was probed by specific chemical modification of cysteine residues with [14C]iodoacetamide. Under native conditions, only two cysteines (Cys-158 and Cys-311) of eleven half-cystines of the N-terminal chromophore binding domain were modified to a significant extent. In the C-terminal domain, six cysteine residues (Cys-715, Cys-774, Cys-809, Cys-869, Cys-961, Cys-995) were readily accessible to iodoacetamide. Among the reactive cysteine residues, only cysteine-311 displayed reactivity that was dependent on the photochromic form (Pr left arrow over right arrow Pfr) of the photoreceptor. Surprisingly, the modification of Cys-311 in the vicinity of the chromophore attachment site (Cys-321) did not have any detectable effect on spectral properties of phyA. Most of the cysteines of the N-terminal domain (Cys-83, Cys-175, Cys-291, Cys-370, Cys-386, Cys-445, Cys-506) are deeply buried in the core of the chromophore binding domain, as they can be modified only after denaturation of the chromoprotein. In the C-terminal domain, modification of only one cysteine residue (Cys-939) required protein denaturation. Since all 22 half-cystines can be modified with iodoacetamide without reduction of the chromoprotein, it follows that oat phyA does not have any disulfide bonds. We found that Cys-311, Cys-774, Cys-961, and Cys-995 could be easily partially oxidized under the conditions used for phytochrome isolation. The surface topography/conformation of oat phyA and its role in protein-protein recognition in phytochrome-mediated signal transduction are discussed in terms of the relative reactivity of cysteine residues.

Posttranslational modification of oat phytochrome A: phosphorylation of a specific serine in a multiple serine cluster.

Phytochrome A (phyA) is a photoreceptor of higher plants which mediates a variety of biochemical and physiological processes in response to red/far-red light. By detailed structural analysis of the peptides of the total tryptic digest of oat phyA, we found that the photoreceptor isolated from red light irradiated seedlings contains only one site of phosphate attachment, in the N-terminal Ser-rich region. The N-terminal tryptic phosphopeptide (residues 1-12) contains eight serine residues, any of which may be phosphorylated. Direct fast atom bombardment mass spectrometry (FAB MS/MS) analysis of the phosphorylated peptide as well as of its phosphate-containing fragment (residues 1-9) was not successful due to their hydrophilic nature and instability of the phosphate bond. beta-Elimination of the phosphorylated tryptic peptide in the presence of ethanethiol converted the phosphoserine residue to S-ethylcysteine that is stable under FAB MS/MS. FAB MS/MS analysis of the modified peptide clearly showed that the phosphate group was attached to Ser7. The in vivo phosphorylation site at Ser7 in oat phyA is discussed for its possible regulatory role in phyA function.

Dynamics of the N-terminal alpha-helix unfolding in the photoreversion reaction of phytochrome A.

Time-resolved circular dichroism spectroscopy in the far-UV spectral region was used to examine the intermediates of the phytochrome photoreversion reaction (Pfr --> Pr). Three intermediates, lumi-F (tau = 320 ns), meta-Fa (tau = 265 micros) and meta-Fb (tau = 5.5 ms), have been identified in a simple sequential kinetic photoreversion mechanism by absorption spectroscopy [Linschitz, H., Kasche, V., Butler, W. L., & Siegelman, H. W. (1966) J. Biol. Chem. 241, 3395-3403; Pratt, L. H., & Butler, W. L. (1968) Photochem. Photobiol. 8, 477-485; Burke, M., Pratt, D. C., & Moscowitz, A. (1972) Biochemistry 11, 4025-4031; Spruit, C. J. P., Kendrick, R. E., & Cooke, R. J. (1975) Planta (Berlin) 127, 121-132; Eilfeld, P., & Rüdiger, W. (1985) Z. Naturforsch. 40c, 109-114; Chen, E., Lapko, V. N., Lewis, J. W., Song, P.-S., & Kliger, D. S. (1996) Biochemistry 35, 843-850]. In order to correlate the unfolding of the N-terminal alpha-helical segment with one or more of the intermediate species, time-resolved methods were coupled with the structurally sensitive probe of CD in the far-UV spectral region. Analysis of the TRCD data associates the decrease in alpha-helical content that occurs upon formation of Pr with decay of the meta-Fa intermediate. This unfolding process occurs with a time constant of 310 +/- 125 micros, which is consistent with the 265-micros lifetime for meta-Fa.

A possible tyrosine phosphorylation of phytochrome.

Red/far-red light signal transduction by the phytochrome family of photoreceptors regulates plant growth and development. We investigated the possibility that tyrosine kinases and/or phosphatases are involved in phytochrome-mediated signal transduction using crude extracts of oat seedlings that are grown in the dark. We found that a 124 kDa protein was tyrosine-phosphorylated as determined by Western blotting with a phosphotyrosine-specific monoclonal antibody. The 124 kDa protein was recognized by the anti-phosphotyrosine antibody in anti-phytochrome A immunoprecipitates. The level of anti-phosphotyrosine antibody binding to the 124 kDa protein(s) in phytochrome immunoprecipitates that had been treated with red light prior to immunoprecipitation decreased relative to dark controls. These results suggest that either phytochrome from dark-grown seedlings is tyrosine phosphorylated or that it co-immunoprecipitates with a phosphotyrosine-containing protein of the same molecular weight. The implications of these results in the regulation of (a) the putative Ser/Thr kinase activity of the photoreceptor and (b) the binding of signaling molecules, such as phospholipase C to phytochrome, are discussed.

Protein kinase A-catalyzed phosphorylation and its effect on conformation in phytochrome A.

Phytochromes are ubiquitous red/far-red wavelength-sensitive photoreceptors in plants. Oat phytochrome A is a phosphoprotein. Phytochrome A (phyA) possesses two spatially different sites for phosphorylation with cAMP-dependent protein kinase (PKA) [McMichael & Lagarias (1990) Biochemistry 29, 3872-3878]. To assess the modulation of protein conformation by phosphorylation/dephosphorylation and its possible implication in phytochrome-mediated signal transduction, the conformations of phytochrome have been probed by PKA catalyzed phosphorylation. The phosphorylated species were purified and analyzed, along with untreated phytochrome, by limited proteolysis, circular dichroism (CD) and fluorescence quenching measurements. No significant changes in secondary structure of the phyA molecule after its phosphorylation were observed by CD. However, a subtle topographic and/or electrostatic effect of the phytochrome phosphorylation was detected by the time-resolved fluorescence quenching of Trp residues with Cs+ ions. N-Terminal phosphorylation at Ser17 was unique to the Pr form, but both Pr and Pfr phytochromes were phosphorylated at the hinge region to some extent. Phosphorylation at the hinge region resulted in noticeable changes in the proteolytic patterns, inhibiting cleavage near the phosphorylation site and favoring tryptic digestion of the Lys536-Asn537 peptide bond. Phosphorylation at the N-terminus did not cause observable changes in the helical structure of this region, but had an inhibitory effect on proteinase V8 accessibility at a site near the chromophore attachment. The functional relevance of protein phosphorylation of phyA is also discussed.

Light signal transduction mediated by phytochromes: preliminary studies and possible approaches.

Phytochromes mediate a variety of developmental and growth processes involved in the photomorphogenesis of plants. In this article, we review the current understanding of the structure and function of the photoreceptor, discuss some very preliminary results, and offer speculations and even conjectures that may elicit future studies into the molecular mechanisms of the phytochrome-mediated light signal transduction in plants.

Mechanism of native oat phytochrome photoreversion: a time-resolved absorption investigation.

The regulation of plant photomorphogenesis is mediated by the thermal reactions that follow light absorption by the phytochrome photoreceptor. Phytochromes are tetrapyrrolic chromoproteins that exist in two photochromically interconvertible forms, a red light absorbing species, Pr, and a far-red light absorbing form, Pfr. Upon irradiation with 670 nm light, the inactive, red light sensing Pr form is converted to the active Pfr form. Although the forward phototransformation has been studied extensively by several groups using various techniques, the Pfr-->Pr photoreversion reaction that occurs upon irradiation with 730 nm light is not as thoroughly characterized. In this study, time-resolved absorption (TROD) spectroscopy is used to examine the intermediate species involved in the phytochrome photoreversion mechanism at 10 degrees C. Analysis of the TROD data identifies three species with lifetimes of 320 ns, 265 microseconds, and 5.5 ms. TROD results are described in terms of the simplest parallel and sequential kinetic models. Comparison of intermediate spectra from these mechanisms with those of previously reported species from flash photoreversion and low-temperature studies indicates that Pfr photoreversion follows a sequential pathway that does not share any intermediates with the Pr phototransformation pathway.

Initial spectroscopic characterization of the ciliate photoreceptor stentorin.

Stentorin serves as the primary photosensor in the single cell ciliate, Stentor coeruleus, for its photophobic and phototactic response to light of visible wavelengths. We separated two subunits, stentorin-2A and -2B, from the previous stentorin complex ('stentorin-2') of greater than half a million molecular mass isolated from the photoreceptor organelle (pigment granule). Stentorin-2B bears the chromophore covalently linked to an approx. 50 kDa apoprotein, as determined by SDS-urea-PAGE. Partial amino acid sequences were obtained from this 50 kDa subunit. Its visible and CD spectra were found to be similar to those of stentorin-2. The steady-state and time-resolved fluorescence spectra of stentorin-2B, in H2O and D2O buffers, were also similar to those of stentorin-2. This suggests that the 50 kDa subunit retains the spectral integrity and primary photoreactivity of the stentorin-complex. The picosecond time-resolved fluorescence study revealed that the short picosecond emission component (tau F approximately equal to 8-10 ps) was the predominant emitting species in stentorin-2B and -2, followed by longer decaying species. No deuterium solvent effect was seen in this fast-decaying species. The possible mechanism for the primary photoreaction appears to involve electron transfer coupled with proton transfer.

A simple and improved method of isolation and purification for native oat phytochrome.

A simple procedure for the isolation and purification of 124 kDa phytochrome (phyA) form etiolated Avena seedlings has been developed employing ammonium sulfate back-extraction. After solubilization of the ammonium sulfate precipitate (250 g/L) an additional ammonium sulfate fractionation with 17 g per 100 mL rather than column chromatography was performed. After several steps of the "washing-out" procedure with 100 mM phosphate buffer, phytochrome was solubilized in 10 mM phosphate buffer. The resulting phytochrome had a specific absorbance ratio (SAR = A666/ A280) ranging from 0.60 to 0.85. These values are equivalent to those of phytochrome preparations after hydroxylapatite chromatography-ammonium sulfate back-extraction. The total isolation-purification time was 8 h and yield of the chromoprotein was 50% higher than the yield using conventional techniques. The phytochrome preparation, after application to a Toyopearl HW-65S gel filtration column, produced very pure 124 kDa phyA with a specific absorbance ratio greater than 1.00. The spectral characteristics are identical to those described for the best of the highly purified native chromoprotein preparations.