Identification of six CPC-like genes and their differential expression in leaves of tea plant, Camellia sinensis.

Tea is one of the most consumed beverages worldwide, and trichome formation in tea plant leaves impairs their commercial value. In Arabidopsis thaliana leaves, trichome formation is negatively regulated by the CPC family genes, which encode R3-type MYB transcription factors. Here, we identified six CPC-like genes in a tea plant (Camellia sinensis var. sinensis) for the first time. Simulated three-dimensional structure of the MYB domains of all the six CPC-like proteins exhibited negative charge on the surface, as observed on that of the Arabidopsis CPC protein that does not bind to DNA, indicating their similarity with regard to molecular interaction. We further found that the six CPC-like genes were differentially expressed in different developmental stages of tea leaves, and four out of the six genes were upregulated in the youngest 1st leaves, which formed more trichomes than other older leaves. Although it does not establish a causal link, the correlation between differential expression of CPC-like genes and variable trichome formation suggests that the R3-type MYB transcription factors are potential precipitating factors in affecting the value of tea leaf.

Exogenous proline has favorable effects on growth and browning suppression in rice but not in tobacco.

Proline is one of the amino acids that compose proteins and has various roles under non-stress and stress conditions. In this study, we investigated the effect of proline on the growth and browning of two plants, tobacco and rice, by exogenous application and endogenous increase of proline. Exogenous proline had a different effect on the growth and browning between tobacco and rice: proline affected negatively the growth of tobacco seedlings and favorably that of rice seedlings. In addition, proline prevented browning only in rice cultured cells, consistent with the increase of proline contents, but not in tobacco BY-2 cells. These results might be due to the difference of exogenous proline uptake activity in these cells. From the Lineweaver-Burk plots, proline inhibited polyphenol oxidase activity in vitro, which is a major factor of enzymatic browning in plants, by affecting the enzyme-substrate complex. Proline could suppress the browning of the plant callus by inhibition of PPO activity.

High levels of expression of multiple enzymes in the Smirnoff-Wheeler pathway are important for high accumulation of ascorbic acid in acerola fruits.

Acerola fruits contain abundant ascorbic acid (AsA). The gene expression levels of three upstream enzymes in the primary AsA biosynthesis pathway were correlated with AsA contents in the fruits of two acerola cultivars. Multiple overexpression of the enzymes increased AsA contents, suggesting their high expression is important for high AsA accumulation in acerola fruits and the breeding of AsA-rich plants. Abbreviations: AsA: ascorbic acid; PMI: phosphomannose isomerase; PMM: phosphomannomutase; GMP: GDP-d-mannose pyrophosphorylase; GME: GDP-d-mannose 3',5'-epimerase; GGP: GDP-l-galactose phosphorylase; GPP: l-galactose-1-phosphate phosphatase; GDH: l-galactose dehydrogenase; GLDH: l-galactono-1,4-lactone dehydrogenase.

Effect of mutation of C-terminal and heme binding region of Arabidopsis catalase on the import to peroxisomes.

We evaluated the import of Arabidopsis catalase to peroxisomes under homogenous transient expression. The amino acids at -11 to -4 from the C-terminus are necessary for catalase import. The results are in agreement with the previous work under stable expression. We first demonstrate that heme-binding sites are important for peroxisomal import, suggesting the importance of catalase folding. Abbreviations: AtCat: Arabidopsis catalase; PTS: peroxisomal targeting signal; PEX: Peroxin.

Distribution of three isoforms of antimicrobial peptide, chrysophsin-1, -2 and -3, in the red sea bream, Pagrus (Chrysophrys) major.

We report here a liquid chromatography/electrospray ionization-tandem mass spectrometry assay for the quantification of three isoforms of antimicrobial peptide (AMP), chrysophsin-1, -2 and -3, in the red sea bream, Pagrus (Chrysophrys) major. Chrysophsin-1 was mainly distributed in the pyloric caeca and gills, followed by intestine and stomach. Chrysophsin-2 was detected in the gills and stomach, but chrysophsin-3 was only in the gills. The present procedure is valuable as a general method for simultaneous determination of the level of multiple AMP isoforms in fish tissues, and the data give important information for understanding the significance of each AMP isoform in host defense.

Amino acid substitutions in CPC-LIKE MYB reveal residues important for protein stability in Arabidopsis roots.

TRYPTICHON (TRY) and ENHANCER OF TRY AND CPC2 (ETC2) encode R3-type MYB transcription factors that are involved in epidermal cell differentiation in Arabidopsis thaliana. TRY and ETC2 belong to the CPC-like MYB gene family, which includes seven homolog genes. Previously, we showed that among the CPC family members, TRY and ETC2 are characterized by rapid proteolysis compared with that of other members, and we demonstrated that this proteolysis is mediated by the proteasome-dependent pathway. In this study, we compared the functions of the wild-type TRY and ETC2 proteins and their amino acid-substituted versions. Our results showed that the substitution of amino acids in the C-terminal of TRY and ETC2 conferred them the ability to induce root hair formation. Furthermore, we confirmed that these mutations enhanced the stability of the TRY and ETC2 proteins. These results revealed that the amino acids, which are important for the functions of TRY and ETC2, mediate morphological pattern formation and can be useful in understanding the pathway determining the fate of root hair cells.

Two G-box-like elements essential to high gene expression of SlAKR4B in tomato leaves.

Aldo-keto reductases (AKRs) play important roles in aldehyde detoxification as well as primary and secondary metabolism in plants. We previously reported inducible expression of a Solanum lycopersicum AKR4B (SlAKR4B) in tomato leaves treated with salicylic acid and jasmonic acid, and high promoter activity of SlAKR4B in tomato leaf protoplasts. In this study, we investigated the expression response of SlAKR4B in the tomato leaves with infiltration treatment and the cis-element(s) involved in high promoter activity. Gene expression analysis in tomato leaf protoplasts and buffer-infiltrated tomato leaves suggested that cell damage caused the increased expression of SlAKR4B. Promoter activity of SlAKR4B was significantly reduced by mutation of two G-box like elements. It is suggested that the two G-box like elements are responsible for the high promoter activity.

A novel regulatory element responsible for high transcriptional expression of acerola GDP-d-mannose pyrophosphorylase gene.

GDP-d-mannose pyrophosphorylase (GMP) is one of the enzymes that highly expressed in acerola plants. A promoter assay suggests the presence of a new cis-element in the -1087 to -1083 bp sequence of the MgGMP promoter. Moreover, cis-elements, present in the -1080 to -600 bp sequence of the MgGMP promoter, function as enhancers of MgGMP expression.

Gene expression and promoter analysis of a novel tomato aldo-keto reductase in response to environmental stresses.

The functional role of an uncharacterized tomato (Solanum lycopersicum) aldo-keto reductase 4B, denoted as SlAKR4B, was investigated. The gene expression of tomato SlAKR4B was detected at a high level in the senescent leaves and the ripening fruits of tomato. Although d-galacturonic acid reductase activities tended to be higher in tomato SlAKR4B-overexpressing transgenic tobacco BY-2 cell lines than those in control cell lines, SlAKR4B gene expression was not well correlated with l-ascorbic acid content among the cell lines. The analysis of the transgenic cell lines showed that tomato SlAKR4B has enzyme activities toward d-galacturonic acid as well as glyceraldehyde and glyoxal, suggesting that the SlAKR4B gene encodes a functional enzyme in tomato. Gene expression of SlAKR4B was induced by NaCl, H2O2, and plant hormones such as salicylic acid and jasmonic acid, suggesting that SlAKR4B is involved in the stress response. The transient expression assay using protoplasts showed the promoter activity of the SlAKR4B gene was as high as that of the cauliflower mosaic virus 35S promoter. Also, the promoter region of the SlAKR4B gene was suggested to contain cis-element(s) for abiotic stress-inducible expression.

Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format.

BACKGROUND: The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. RESULTS: A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. CONCLUSIONS: A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome.

Analysis of ascorbic acid biosynthesis using a simple transient gene expression system in tomato fruit protoplasts.

We established a new method of transient expression using tomato fruit protoplasts. The method showed that L-ascorbic acid (AsA) content in tomato protoplasts was increased by transient expression of the L-galactose-1-phosphate phosphatase gene. This system provides a means of rapid analysis to clarify the function of AsA biosynthetic enzymes and AsA roles in tomato fruit.

Molecular cloning and characterization of L-galactose-1-phosphate phosphatase from tobacco (Nicotiana tabacum).

L-Galactose-1-phosphate phosphatase (GPPase) is an enzyme involved in ascorbate biosynthesis in higher plants. We isolated a cDNA encoding GPPase from tobacco, and named it NtGPPase. The putative amino acid sequence of NtGPPase contained inositol monophosphatase motifs and metal binding sites. Recombinant NtGPPase hydrolyzed not only L-galactose-1-phosphate, but also myo-inositol-1-phosphate. The optimum pH for the GPPase activity of NtGPPase was 7.5. Its enzyme activity required Mg2+, and was inhibited by Li+ and Ca2+. Its fluorescence, fused with green fluorescence protein in onion cells and protoplasts of tobacco BY-2 cells, was observed in both the cytosol and nucleus. The expression of NtGPPase mRNA and protein was clearly correlated with L-ascorbic acid (AsA) contents of BY-2 cells during culture. The AsA contents of NtGPPase over expression lines were higher than those of empty lines at 13 d after subculture. This suggests that NtGPPase contributes slightly to AsA biosynthesis.

Characterization of secretory phospholipase A2 with phospholipase A1 activity in tobacco, Nicotiana tabacum (L.).

A cDNA encoding protein with homology to plant secretory phospholipase A2 (sPLA2), denoted as Nt1 PLA2, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA2 has 12 cysteines, Ca²âº binding loop and catalytic site domain that are commonly conserved in plant sPLA2s. The recombinant Nt1 PLA2 was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA2 could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²âº essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA2 mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.

Ontogeny of gene expression of group IB phospholipase A2 isoforms in the red sea bream, Pagrus (Chrysophrys) major.

The red sea bream (Pagrus major) was previously found to express mRNAs for two group IB phospholipase A(2) (PLA(2)) isoforms, DE-1 and DE-2, in the digestive organs, including the hepatopancreas, pyloric caeca, and intestine. To characterize the ontogeny of the digestive function of these PLA(2)s, the present study investigated the localization and expression of DE-1 and DE-2 PLA(2) genes in red sea bream larvae/juveniles and immature adults, by in situ hybridization. In the adults, DE-1 PLA(2) mRNA was expressed in pancreatic acinar cells. By contrast, DE-2 PLA(2) mRNA was detected not only in digestive tissues, such as pancreatic acinar cells, gastric glands of the stomach, epithelial cells of the pyloric caeca, and intestinal epithelial cells, but also in non-digestive ones, including cardiac and lateral muscle fibers and the cytoplasm of the oocytes. In the larvae, both DE-1 and DE-2 PLA(2) mRNAs first appeared in pancreatic tissues at 3 days post-hatching (dph) and in intestinal tissue at 1 dph, and expression levels for both gradually increased after this point. In the juvenile stage at 32 dph, DE-1 PLA(2) mRNA was highly expressed in pancreatic tissue, and DE-2 PLA(2) mRNA was detected in almost all digestive tissues, including pancreatic tissue, gastric glands, pyloric caeca, and intestine, including the myomere of the lateral muscles. In conclusion, both DE-1 and DE-2 PLA(2) mRNAs are already expressed in the digestive organs of red sea bream larvae before first feeding, and larvae will synthesize both DE-1 and DE-2 PLA(2) proteins.

Gene expression of monodehydroascorbate reductase and dehydroascorbate reductase during fruit ripening and in response to environmental stresses in acerola (Malpighia glabra).

Acerola (Malpighia glabra) is an exotic fruit cultivated primarily for its abundant ascorbic acid (AsA) content. The molecular mechanisms that regulate the metabolism of AsA in acerola have yet to be defined. Monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) are key enzymes of the ascorbate-glutathione cycle that maintain reduced pools of ascorbic acid and serve as important antioxidants. cDNAs encoding MDHAR and DHAR were isolated from acerola using RT-PCR and RACE. Phylogenetic trees associated acerola MDHAR and DHAR with other plant cytosolic MDHARs and DHARs. Expressions of the two genes correlated with their enzymatic activities and were differentially regulated during fruit ripening. Interestingly, MDHAR expression was only detected in overripe fruits, whereas the transcript level of DHAR was highest at the intermediate stage of fruit ripening. Under dark conditions, there was a sharp and significant decline in the total and reduced ascorbate contents, accompanied by a decrease in the level of transcripts and enzyme activities of the two genes in acerola leaves. MDHAR and DHAR transcripts and enzyme activities were significantly up-regulated in the leaves of acerola under cold and salt stress conditions, indicating that expression of both genes are transcriptionally regulated under these stresses.

Light regulation of ascorbic acid biosynthesis in rice via light responsive cis-elements in genes encoding ascorbic acid biosynthetic enzymes.

The ascorbic acid (AsA) content and the mRNA levels of L-galactose-1-phosphate phosphatase (GPPase), and L-galactono-1,4-lactone dehydrogenase (GalLDH) increased by intense light, and decreased in the dark. Moreover, the promoter regions of GPPase and GalLDH contained light responsible cis-elements. These results suggest that in rice, AsA synthesis is regulated by light.

Luminescence detection of SNARE-SNARE interaction in Arabidopsis protoplasts.

Membrane associated proteins SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) provide the minimal fusion machinery necessary for cellular vesicles to fuse to target organelle membranes in eukaryotic cells. Despite the conserved nature of the fusion machinery in all eukaryotes, it still remains challenging to identify functional SNARE pairs in higher plants. We developed a method based on a split-luciferase complementation assay for detecting changes in SNARE-SNARE interaction by luminescence within Arabidopsis protoplasts that express recombinant proteins at physiological levels in 96-well plates. The reliability of the assay was confirmed by three experiments. First, reduction of the SNARE-SNARE interaction caused by a single amino acid substitution adjacent to the SNARE motif in endosome-localized AtVAM3/SYP22 (syntaxin of plant 22) was detected by a reduction of luminescence. Second, reduction of the interaction between plasma-membrane localized SYP121 and VAMP722 in response to sodium azide was detected in real-time. Third, the results of 21 SNARE pairs investigated by this method largely agreed with the results from previously reported co-immunoprecipitation assays. Using the method, we newly identified the interaction between SYP121 and VAMP722 was significantly increased when the protoplasts were incubated in the light. Microscopic observation of transgenic Arabidopsis expressing GFP-SYP121 (green fluorescent protein tagged SYP121) from its own promoter suggested that the plasma-membrane localization of GFP-SYP121 is maintained by light. These suggested that the vesicle trafficking pathway mediated by SYP121 might be regulated by light in Arabidopsis. In general, this article demonstrated the method that can generate new biological insight of the SNARE protein interactions in plant cells.

Increase in ascorbate content of transgenic tobacco plants overexpressing the acerola (Malpighia glabra) phosphomannomutase gene.

Phosphomannomutase (PMM; EC 5.4.2.8) catalyzes the interconversion of mannose-6-phosphate to mannose-1-phosphate in the Smirnoff-Wheeler pathway for the biosynthesis of l-ascorbic acid (AsA). We have cloned the PMM cDNA from acerola (Malpighia glabra), a plant containing an enormous amount of AsA. The AsA contents correlate with the PMM gene expression of the ripening fruits and leaves. The PMM activities in the leaves of acerola, tomato and Arabidopsis correlate with their respective AsA contents. Transgenic tobacco plants overexpressing the acerola PMM gene showed about a 2-fold increase in AsA contents compared with the wild type, with a corresponding correlation with the PMM transcript levels and activities.

Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola (Malpighia glabra).

The Smirnoff-Wheeler (SW) pathway has been proven to be the only significant source of l-ascorbic acid (AsA; vitamin C) in the seedlings of the model plant Arabidopsis thaliana. It is yet uncertain whether the same pathway holds for all other plants and their various organs as AsA may also be synthesized through alternative pathways. In this study, we have cloned some of the genes involved in the SW-pathway from acerola (Malpighia glabra), a plant containing enormous amount of AsA, and examined the expression patterns of these genes in the plant. The AsA contents of acerola leaves were about 8-fold more than that of Arabidopsis with 5-700-fold higher mRNA abundance in AsA-biosynthesizing genes. The unripe fruits have the highest AsA content but the accumulation was substantially repressed as the fruit transitions to maturation. The mRNAs encoding these genes showed correlation in their expression with the AsA contents of the fruits. Although very little AsA was recorded in the seeds the mRNAs encoding all the genes, with the exception of the mitochondrially located L-galactono-1,4-lactone dehydrogenase, were clearly detected in the seeds of the unripe fruits. In young leaves of acerola, the expression of most genes were repressed by the dark and induced by light. However, the expression of GDP-D-mannose pyrophosphorylase similar to that encoded by A. thaliana VTC1 was induced in the dark. The expressions of all the genes surged after 24h following wounding stress on the young leaves. These findings will advance the investigation into the molecular factors regulating the biosynthesis of abundant AsA in acerola.

RNAi-mediated knockdown of the XIP-type endoxylanase inhibitor gene, OsXIP, has no effect on grain development and germination in rice.

OsXIP (Oryza sativa xylanase inhibitor protein) is a XIP-type xylanase inhibitor which was identified as a protein encoded by a wound stress-responsive gene in rice. Although the OsXIP gene was specifically expressed in mature grains under basal conditions, recombinant OsXIP had no effect on rice endogenous xylanases, and OsXIP-suppressed transgenic rice plants did not exhibit any change in grain development and germination, suggesting that rice development may be independent of OsXIP. Analysis using an OsXIP-specific antibody revealed that OsXIP is markedly accumulated in apoplast in rice root cells by wounding. These results reinforced the possibility that OsXIP is involved in plant defense mechanisms against phytopathogens.