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.

Expression of the protective antigen for PEDV in transgenic duckweed, Lemna minor.

Duckweeds are small, floating aquatic plants with a number of useful characteristics, including edibility, fast-growing, and a clonal proliferation. Duckweed is also fed to animals as a diet complement because of its high nutritional value. Porcine epidemic diarrhea virus (PEDV) is a major causative agent of fatal diarrhea in piglets and is a serious problem in the hog-raising industry. In this study, we assessed the feasibility of producing a protective antigen for the PEDV spike protein 1 using duckweed, Lemna minor. Stably transformed Lemna were obtained by co-cultivation with A. tumefaciens EHA105 harboring the PEDV spike protein gene. Transgene integration and expression of the PEDV spike protein 1 gene were confirmed by genomic PCR and RT-PCR and western blot analysis of transgenic Lemna, respectively. This is the first report of the expression of a vaccine antigen against an animal infectious disease in duckweed.

Phototropins and chloroplast activity in plant blue light signaling.

In plants, phototropins 1 (phot1) and 2 (phot2) mediate chloroplast movement to blue light (BL). A recent report showed that phototropins (phot) are required for the expression of chloroplast genes in rice. The light-induced responses of phot1a rice mutants result in H(2)O(2)-mediated damage to chloroplast photosystems, indicating that phot-regulated responses might be associated with the other photoreceptor, such as cryptochrome (cry) BL receptor. This suggests diversification and specialization of photoreceptor signaling in plants.

Rice phot1a mutation reduces plant growth by affecting photosynthetic responses to light during early seedling growth.

The aim of this work was to characterize the phot1 mutant of rice during early seedling growth in various light conditions. We isolated the rice T-DNA insertion mutant phot1a-1 and compared it to the Tos17 insertion mutant phot1a-2. When phot1a mutants were grown under WL (100) and BL (40 miccromol m(-2) s(-1)), they demonstrated a considerable reduction in photosynthetic capacity, which included decreased leaf CO(2) uptake and plant growth. Pigment analysis showed no significant difference between wild-type and mutants in the Chl a:b ratios, whereas in the latter, total concentration was reduced (a 2-fold decrease). Carotenoid contents of the mutants were also decreased considerably, implying the involvement of phot1a in pigment degradation. Deletion of phot1a showed higher contents of H(2)O(2) in leaves. Chloroplastic APX and SOD activities were lower in the mutants whereas the activities of cytosolic enzymes were increased. Immunoblotting indicated reduced accumulation of photosystem proteins (D1, D2, CP43, Lhca2, and PsaC) relative to the other light-harvesting complexes in the mutant. We conclude that the defect of Os Phot1a affects degradation of chlorophylls and carotenoids, and under photosynthetically active photon fluxes, mutation of phot1a results in loss of photosynthetic capacity owing to the damage of photosystems caused by elevated H(2)O(2) accumulation, leading to a reduction in plant growth.

Loss of cytosolic fructose-1,6-bisphosphatase limits photosynthetic sucrose synthesis and causes severe growth retardations in rice (Oryza sativa).

During photosynthesis, triose-phosphates (trioseP) exported from the chloroplast to the cytosol are converted to sucrose via cytosolic fructose-1,6-bisphosphatase (cFBPase). Expression analysis in rice suggests that OscFBP1 plays a major role in the cytosolic conversion of trioseP to sucrose in leaves during the day. The isolated OscFBP1 mutants exhibited markedly decreased photosynthetic rates and severe growth retardation with reduced chlorophyll content, which results in plant death. Analysis of primary carbon metabolites revealed both significantly reduced levels of sucrose, glucose, fructose and starch in leaves of these mutants, and a high accumulation of sucrose to starch in leaves of rice plants. In the oscfbp1 mutants, products of glycolysis and the TCA cycle were significantly increased. A partitioning experiment of (14)C-labelled photoassimilates revealed altered carbon distributions including a slight increase in the insoluble fraction representing transitory starch, a significant decrease in the neutral fraction corresponding to soluble sugars and a high accumulation of phosphorylated intermediates and carboxylic acid fractions in the oscfbp1 mutants. These results indicate that the impaired synthesis of sucrose in rice cannot be sufficiently compensated for by the transitory starch-mediated pathways that have been found to facilitate plant growth in the equivalent Arabidopsis mutants.

Overexpression of Arabidopsis ZEP enhances tolerance to osmotic stress.

Zeaxanthin epoxidase (ZEP) is an enzyme important in ABA biosynthesis and in the xanthophyll cycle. ABA, a plant hormone, is a key molecule that regulates plant responses to abiotic stress, such as drought and salinity, and is required for stress tolerance. To investigate the biological roles of the Arabidopsis thaliana ZEP gene (AtZEP) in stress response, we generated transgenic plants overexpressing the AtZEP gene and analyzed their responses to salt and drought stresses. AtZEP-overexpressing plants exhibited more vigorous growth under high salt and drought treatments than wild-type plants. In addition to enhanced de novo ABA biosynthesis, AtZEP-overexpressing plants also exhibited much higher expression of the endogenous stress-responsive genes RD29A and Rab18 than wild-type plants under salt stress. Moreover, the stomatal aperture of the AtZEP-overexpressing plants was smaller than wild-type plants after exposure to light. Our results therefore indicated that AtZEP plays important roles in response to osmotic stress.

Glycine-rich RNA-binding protein 7 affects abiotic stress responses by regulating stomata opening and closing in Arabidopsis thaliana.

Despite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, their physiological functions and mechanisms of action in stress responses remain largely unknown. Here, we assessed the functional roles of GRP7, one of the eight GRP family members in Arabidopsis thaliana, on seed germination, seedling growth, and stress tolerance under high salinity, drought, or cold stress conditions. The transgenic Arabidopsis plants overexpressing GRP7 under the control of the cauliflower mosaic virus 35S promoter displayed retarded germination and poorer seedling growth compared with the wild-type plants and T-DNA insertional mutant lines under high salinity or dehydration stress conditions. By contrast, GRP7 overexpression conferred freezing tolerance in Arabidopsis plants. GRP7 is expressed abundantly in the guard cells, and has been shown to influence the opening and closing of the stomata, in accordance with the prevailing stress conditions. GRP7 is localized to both the nucleus and the cytoplasm, and is involved in the export of mRNAs from the nucleus to the cytoplasm under cold stress conditions. Collectively, these results provide compelling evidence that GRP7 affects the growth and stress tolerance of Arabidopsis plants under high salt and dehydration stress conditions, and also confers freezing tolerance, particularly via the regulation of stomatal opening and closing in the guard cells.

Functional characterization of a glycine-rich RNA-binding protein 2 in Arabidopsis thaliana under abiotic stress conditions.

Although glycine-rich RNA-binding protein 2 (GRP2) has been implicated in plant responses to environmental stresses, the function and importance of GRP2 in stress responses are largely unknown. Here, we examined the functional roles of GRP2 in Arabidopsis thaliana under high-salinity, cold or osmotic stress. GRP2 affects seed germination of Arabidopsis plants under salt stress, but does not influence seed germination and seedling growth of Arabidopsis plants under osmotic stress. GRP2 accelerates seed germination and seedling growth in Arabidopsis plants under cold stress, and contributes to enhancement of cold and freezing tolerance in Arabidopsis plants. No differences in germination between the wild-type and transgenic plants were observed following addition of abscisic acid (ABA) or glucose, implying that GRP2 affects germination through an ABA-independent pathway. GRP2 complements the cold sensitivity of an Escherichia coli BX04 mutant and exhibits transcription anti-termination activity, suggesting that it has an RNA chaperone activity during the cold adaptation process. Mitochondrial respiration and catalase and peroxidase activities were affected by expression of mitochondrial-localized GRP2 in Arabidopsis plants under cold stress. Proteome analysis revealed that expression of several mitochondrial-encoded genes was modulated by GRP2 under cold stress. These results provide new evidence indicating that GRP2 plays important roles in seed germination, seedling growth and freezing tolerance of Arabidopsis under stress conditions, and that GRP2 exerts its function by modulating the expression and activity of various classes of genes.

Arabidopsis cyp51 mutant shows postembryonic seedling lethality associated with lack of membrane integrity.

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14alpha-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14alpha-methyl-delta8-sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14alpha-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.

Mitochondria provide the main source of cytosolic ATP for activation of outward-rectifying K+ channels in mesophyll protoplast of chlorophyll-deficient mutant rice (OsCHLH) seedlings.

The role of mitochondria in providing intracellular ATP that controls the activity of plasma membrane outward-rectifying K+ channels was evaluated. The OsCHLH rice mutant, which lacks chlorophyll in the thylakoids, was isolated by T-DNA gene trapping (Jung, K.-H., Hur, J., Ryu, C.-H., Choi, Y., Chung, Y.-Y., Miyao, A., Hirochika, H., and An, G. (2003) Plant Cell Physiol. 44, 463-472). The OsCHLH mutant is unable to fix CO2 and exhibits reduced growth. Wild type and mutant plants exhibit similar rates of respiratory O2 uptake in the dark, whereas the rate of photosynthetic O2 evolution by the mutant was negligible during illumination. During dark respiration the wild type and mutant exhibited similar levels of cytoplasmic ATP. In the mutant oligomycin treatment (an inhibitor of mitochondrial F1F0-ATPase) drastically reduced ATP production. The fact that this was reversed by the addition of glucose suggested that the mutant produced ATP exclusively from mitochondria but not from chloroplasts. In whole cell patch clamp experiments, the activity of outward-rectifying K+ channels of rice mesophyll cells showed ATP-dependent currents, which were 1.5-fold greater in wild type than in mutant cells. Channels in both wild type and mutant cells were deactivated by the removal of cytosolic ATP, whereas in the presence of ATP the channels remained active. We conclude that mesophyll cells in the OsCHLH rice mutant derive ATP from mitochondrial respiration, and that this is critical for the normal function of plasma membrane outward-rectifying K+ channels.

The Arabidopsis dynamin-like proteins ADL1C and ADL1E play a critical role in mitochondrial morphogenesis.

Dynamin-related proteins are high molecular weight GTP binding proteins and have been implicated in various biological processes. Here, we report the functional characterization of two dynamin homologs in Arabidopsis, Arabidopsis dynamin-like 1C (ADL1C) and Arabidopsis dynamin-like 1E (ADL1E). ADL1C and ADL1E show a high degree of amino acid sequence similarity with members of the dynamin family. However, both proteins lack the C-terminal Pro-rich domain and the pleckstrin homology domain. Expression of the dominant-negative mutant ADL1C[K48E] in protoplasts obtained from leaf cells caused abnormal mitochondrial elongation. Also, a T-DNA insertion mutation at the ADL1E gene caused abnormal mitochondrial elongation that was rescued by the transient expression of ADL1C and ADL1E in protoplasts. In immunohistochemistry and in vivo targeting experiments in Arabidopsis protoplasts, ADL1C and ADL1E appeared as numerous speckles and the two proteins colocalized. These speckles were partially colocalized with F1-ATPase-gamma:RFP, a mitochondrial marker, and ADL2b localized at the tip of mitochondria. These results suggest that ADL1C and ADL1E may play a critical role in mitochondrial fission in plant cells.

Stress memory in plants: a negative regulation of stomatal response and transient induction of rd22 gene to light in abscisic acid-entrained Arabidopsis plants.

All organisms, including plants, perceive environmental stress, and they use this information to modify their behavior or development. Here, we demonstrate that Arabidopsis plants have memory functions related to repeated exposure to stressful concentrations of the phytohormone abscisic acid (ABA), which acts as a chemical signal. Repeated exposure of plants to ABA (40 micro m for 2 h) impaired light-induced stomatal opening or inhibited the response to a light stimulus after ABA-entrainment under both dark/light cycle and continuous light. Moreover, there were transient expressions of the rd22 gene during the same periods under both the growing conditions. Such acquired information in ABA-entrained plants produced a long-term sensitization. When the time of light application was changed, a transient induction of the rd22 gene in plants after ABA-entrainment indicated that these were light-associated responses. These transient effects were also observed in kin1, rab18, and rd29B. The transient expression of AtNCED3, causing the accumulation of endogenous ABA, indicated a possible regulation by ABA-dependent pathways in ABA-entrained plants. An ABA immunoassay supported this hypothesis: ABA-entrained plants showed a transient increase in endogenous ABA level from 220 to 250 pmol g-1 fresh mass at 1-2 h of the training period, whereas ABA-deficient (aba2) mutants did not. Taking into account these results, we propose that plants have the ability to memorize stressful environmental experiences, and discuss the molecular events in ABA-entrained plants.

Parallel recordings of photosynthetic electron transport and K+-channel activity in single guard cells.

Stomata open in response to red and blue light. Red light-induced stomatal movement is mediated by guard cell chloroplasts and related to K+-uptake into these motor cells. We have combined a new type of microchlorophyll fluorometer with the patch-clamp technique for parallel studies of the photosynthetic electron transport and activity of plasma membrane K+ channels in single guard cell protoplast. In the whole-cell configuration and presence of ATP in the patch-pipette, the activity of the K+-uptake channels remained constant throughout the course of an experiment (up to 30 min) while photosynthetic activity declined to about 50%. In the absence of ATP inward K+ currents declined in a time-dependent manner. Under these ATP-free conditions, photosynthetic electron transport was completely blocked within 8 min. ADP together with orthophosphate was able to prevent inhibition of photosynthetic electron transport and run-down of K+-channel activity. The results demonstrate that the combination of these two techniques is suited to directly study cytosolic factors as common regulators of photosynthesis and plasma membrane transport within a single-cell.

Extended leaf longevity in the ore4-1 mutant of Arabidopsis with a reduced expression of a plastid ribosomal protein gene.

The longevity of plant leaf organs is genetically determined. However, the molecular mechanisms underlying the control of longevity are still largely unknown. Here, we describe a T-DNA-insertional mutation of Arabidopsis thaliana that confers extended leaf longevity. The mutation, termed ore4-1, delays a broad spectrum of age-dependent leaf senescence, but has little effect on leaf senescence artificially induced by darkness, abscisic acid (ABA), methyl jasmonate (MeJA), or ethylene. The T-DNA was inserted within the promoter region of the plastid ribosomal small subunit protein 17 (PRPS17) gene, and this insertion dramatically reduced PRPS17 mRNA expression. In the ore4-1 mutant, the leaf growth rate is decreased, while the maturation timing is similar to that of wild-type. In addition, the activity of the photosystem I (PSI) is significantly reduced in the ore4-1 mutant, as compared to wild-type. Thus, the ore4-1 mutation results in a deficiency in various chloroplast functions, including photosynthesis, which may decrease leaf growth. Our results suggest a possible link between reduced metabolism and extended longevity of the leaf organs in the ore4-1 mutation.