KLU/CYP78A5, a Cytochrome P450 Monooxygenase Identified via Fox Hunting, Contributes to Cuticle Biosynthesis and Improves Various Abiotic Stress Tolerances.

Acquired osmotolerance after salt stress is widespread among Arabidopsis thaliana (Arabidopsis) accessions. Most salt-tolerant accessions exhibit acquired osmotolerance, whereas Col-0 does not. To identify genes that can confer acquired osmotolerance to Col-0 plants, we performed full-length cDNA overexpression (FOX) hunting using full-length cDNAs of halophyte Eutrema salsugineum, a close relative of Arabidopsis. We identified EsCYP78A5 as a gene that can confer acquired osmotolerance to Col-0 wild-type (WT) plants. EsCYP78A5 encodes a cytochrome P450 monooxygenase and the Arabidopsis ortholog is known as KLU. We also demonstrated that transgenic Col-0 plants overexpressing AtKLU (AtKLUox) exhibited acquired osmotolerance. Interestingly, KLU overexpression improved not only acquired osmotolerance but also osmo-shock, salt-shock, oxidative, and heat-stress tolerances. Under normal conditions, the AtKLUox plants showed growth retardation with shiny green leaves. The AtKLUox plants also accumulated higher anthocyanin levels and developed denser cuticular wax than WT plants. Compared to WT plants, the AtKLUox plants accumulated significantly higher levels of cutin monomers and very-long-chain fatty acids, which play an important role in the development of cuticular wax and membrane lipids. Endoplasmic reticulum (ER) stress induced by osmotic or heat stress was reduced in AtKLUox plants compared to WT plants. These findings suggest that KLU is involved in the cuticle biosynthesis, accumulation of cuticular wax, and reduction of ER stress induced by abiotic stresses, leading to the observed abiotic stress tolerances.

ECERIFERUM 10 Encoding an Enoyl-CoA Reductase Plays a Crucial Role in Osmotolerance and Cuticular Wax Loading in Arabidopsis.

Acquired osmotolerance induced after salt stress is widespread across Arabidopsis thaliana (Arabidopsis) accessions (e.g., Bu-5). However, it remains unclear how this osmotolerance is established. Here, we isolated a mutant showing an acquired osmotolerance-defective phenotype (aod2) from an ion-beam-mutagenized M2 population of Bu-5. aod2 was impaired not only in acquired osmotolerance but also in osmo-shock, salt-shock, and long-term heat tolerances compared with Bu-5, and it displayed abnormal morphology, including small, wrinkled leaves, and zigzag-shaped stems. Genetic analyses of aod2 revealed that a 439-kbp region of chromosome 4 was translocated to chromosome 3 at the causal locus for the osmosensitive phenotype. The causal gene of the aod2 phenotype was identical to ECERIFERUM 10 (CER10), which encodes an enoyl-coenzyme A reductase that is involved in the elongation reactions of very-long-chain fatty acids (VLCFAs) for subsequent derivatization into cuticular waxes, storage lipids, and sphingolipids. The major components of the cuticular wax were accumulated in response to osmotic stress in both Bu-5 WT and aod2. However, less fatty acids, primary alcohols, and aldehydes with chain length ≥ C30 were accumulated in aod2. In addition, aod2 exhibited a dramatic reduction in the number of epicuticular wax crystals on its stems. Endoplasmic reticulum stress mediated by bZIP60 was increased in aod2 under osmotic stress. The only cer10 showed the most pronounced loss of epidermal cuticular wax and most osmosensitive phenotype among four Col-0-background cuticular wax-related mutants. Together, the present findings suggest that CER10/AOD2 plays a crucial role in Arabidopsis osmotolerance through VLCFA metabolism involved in cuticular wax formation and endocytic membrane trafficking.

Ectopic expression of AtNF-YA6-VP16 in petals results in a novel petal phenotype in Torenia fournieri.

MAIN CONCLUSION: A novel Torenia phenotype having separate petals was obtained by the combination of NF-YA6-VP16 with a floral organ-specific promoter. Genetic engineering techniques helped in obtaining novel flower colors and shapes, in particular, by introducing functionally modified transcription factors (TFs) to ornamental flower species. Herein, we used functionally modified Arabidopsis TFs fused with the repression domain SRDX and the activation domain VP16 to screen for novel floral traits in Torenia fournieri Lind (torenia). We avoided undesired phenotypes unrelated to flowers by expressing these TFs through a floral organ-specific promoter belonging to the class-B genes, GLOBOSA (TfGLO). Fourteen constructs were produced to express functionally modified Arabidopsis TFs in which each of SRDX and VP16 was fused into 7 TFs that were used for the collective transformation of Torenia plants. Among the obtained transgenic plants, phenotypes with novel floral traits reflected in separate petals within normally gamopetalous flower lines. Sequencing analysis revealed that the transgenic plants contained nuclear factor-YA6 (NF-YA6) fused with the VP16. In the margin between the lips of the petals and tube in the TfGLOp:NF-YA6-VP16 plants, staminoid organs have been developed to separate petals. In the petals of the TfGLOp:NF-YA6-VP16 plants, the expression of a Torenia class C gene, PLENA (TfPLE), was found to be ectopically increased. Moreover, expression of TfPLE-VP16 under the control of the TfGLO promoter brought a similar staminoid phenotype observed in the TfGLOp:NF-YA6-VP16 plants. These results suggest that the introduction of the TfGLOp:NF-YA6-VP16 induced TfPLE expression, resulting in the formation of staminoid petals and separation of them.

Two types of bHLH transcription factor determine the competence of the pericycle for lateral root initiation.

The molecular basis of the competence of the pericycle cell to initiate lateral root primordium formation is totally unknown. Here, we report that in Arabidopsis, two types of basic helix-loop-helix (bHLH) transcription factors, named PERICYCLE FACTOR TYPE-A (PFA) proteins and PERICYCLE FACTOR TYPE-B (PFB) proteins, govern the competence of pericycle cells to initiate lateral root primordium formation. Overexpression of PFA genes confers hallmark pericycle characteristics, including specific marker gene expression and auxin-induced cell division, and multiple loss-of-function mutations in PFA genes or the repression of PFB target genes results in the loss of this specific pericycle function. PFA and PFB proteins physically interact and are under mutual- and self-regulation, forming a positive feedback loop. This study unveils the transcriptional regulatory system that determines pericycle participation in lateral root initiation.

Transcriptional profiling reveals signatures of latent developmental potential in Arabidopsis stomatal lineage ground cells.

In many developmental contexts, cell lineages have variable or flexible potency to self-renew. What drives a cell to exit from a proliferative state and begin differentiation, or to retain the capacity to divide days or years later is not clear. Here we exploit the mixed potential of the stomatal lineage ground cell (SLGC) in the Arabidopsis leaf epidermis as a model to explore how cells might balance potential to differentiate with a reentry into proliferation. By generating transcriptomes of fluorescence-activated cell sorting-isolated populations that combinatorically define SLGCs and integrating these data with other stomatal lineage datasets, we find that SLGCs appear poised between proliferation and endoreduplication. Furthermore, we found the RNA polymerase II-related mediator complex interactor DEK and the transcription factor MYB16 accumulate differentially in the stomatal lineage and influence the extent of cell proliferation during leaf development. These findings suggest that SLGC latent potential is maintained by poising of the cell cycle machinery, as well as general and site-specific gene-expression regulators.

Efficient transient gene expression system using buckwheat hypocotyl protoplasts for large-scale experiments.

Buckwheat (Fagopyrum esculentum) is cultivated worldwide and its flour is used in a variety of food products. Although functional analyses of genes in buckwheat are highly desired, reliable methods to do it have yet to be developed. In this study we established a simple and efficient transient gene expression system using buckwheat protoplasts isolated from young hypocotyls using 96-well plates as a high-throughput platform. The transformation efficiency was comparable with that of similar systems, such as Arabidopsis mesophyll protoplasts. Stable results were obtained in a typical example of the experiment to examine transcription factor activity. This system shows potential for the large-scale analysis of gene function using protoplast isolated from fewer and younger plants than the conventional system and may provide novel information for efficient buckwheat breeding.

Buckwheat R2R3 MYB transcription factor FeMYBF1 regulates flavonol biosynthesis.

Buckwheat (Fagopyrum esculentum) contains high amounts of flavonoids, especially flavonols (e.g., rutin), which are thought to be highly beneficial for human health. Little is known, however, about the regulation of flavonol synthesis in buckwheat. We identified a buckwheat gene encoding an R2R3 MYB transcription factor, and named this gene FeMYBF1. Analysis of the deduced amino acid sequence and phylogenetic analysis suggested that FeMYBF1 encodes an ortholog of the Arabidopsis flavonol regulators AtMYB11, AtMYB12 and AtMYB111. Expression of FeMYBF1 in a flavonol-deficient Arabidopsis triple mutant (myb11 myb12 myb111) restored flavonol synthesis. Constitutive expression of FeMYBF1 driven by the CaMV 35S promoter in Arabidopsis resulted in over-accumulation of flavonol glycosides and upregulation of the expression of AtFLS1. Transient expression assays showed that FeMYBF1 activated the promoter of the Arabidopsis gene encoding AtFLS1, and the promoters of buckwheat genes related to anthocyanin and proanthocyanidin synthesis such as dihydroflavonol 4-reductase (DFR) and leucoanthocyanidin dioxygenase (LDOX) in addition to genes encoding FLS. The results indicate that FeMYBF1 regulates flavonol synthesis and may have a role in synthesis of other flavonoid compounds, and also that buckwheat may have alternative pathway of flavonol synthesis through DFR and LDOX.

Dissecting promoter of InMYB1 gene showing petal-specific expression.

We had previously reported that the InMYB1 promoter, the 1023 bp upstream region of InMYB1, works petal-specifically in various dicot plants by recognizing petal identity at a cellular level. To determine the petal-specific region in the InMYB1 promoter, Arabidopsis plants harboring InMYB1_1023b::GUS (ß-glucuronidase), InMYB1_713b::GUS, InMYB1_506b::GUS, InMYB1_403b::GUS, InMYB1_332b::GUS, InMYB1_200b::GUS and InMYB1_140b::GUS were produced and confirmed a shortest region, which has the petal-specific promoter activity by using histochemical GUS assay. Petal-specific GUS staining was not observed in the Arabidopsis plants transformed with InMYB1_200b::GUS and InMYB1_140b::GUS, but observed in transgenic Arabidopsis plants harboring from InMYB1_1023b::GUS to InMYB1_332b::GUS. cDNA sequence of InMYB1 shows that 120 bp upstream region of InMYB1 is 5' untranslated region, suggesting that the 332-121 bp upstream region of InMYB1 contains an important element for petal-specific gene expression. In the Arabidopsis harboring the InMYB1_332-121b×3_TATA_Ω::GUS, petal-specific GUS staining was observed and the staining was stronger than in the Arabidopsis harboring InMYB1_1023b::GUS. This result shows that the 332-121 bp region is enough and essential for the petal specificity and the InMYB1_332-121b×3_TATA_Ω could be used for the molecular breeding of floricultural crops.

Efficient production of glycosylated Cypridina luciferase using plant cells.

Cypridina noctiluca luciferase has been utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays. Some of these applications require a large amount of purified luciferase. However, conventional protein expression systems are not capable of producing sufficient quantities of protein with a high quality and purity without laborious and costly purification processes. To improve the productivity and expand the breadth of possibilities for Cypridina luciferase applications, we employed a variety of secretion expression systems, including yeast, mammalian cells, and silk worms. In this study, we established a simple production procedure using plant cell cultures. The plant cell culture BY-2 efficiently secreted luciferase, which was easily purified using a simple one-step ion-exchange chromatography method. The production yield was 20-30 mg of luciferase per liter of culture medium, and its Km for the luciferin (0.45 µM) was similar to that of the native protein. Additionally, we characterized its glycosylation pattern and confirmed that the two potential N-glycosylation sites were modified with plant-type oligosaccharide chains. Interestingly, the oligosaccharide chains could be trimmed without any detectable decrease in recombinant protein activity. Therefore, the results of our study indicate that this method offers a more cost-effective production method for Cypridina luciferase than conventional methods.

The Petal-Specific InMYB1 Promoter Functions by Recognizing Petaloid Cells.

The InMYB1 gene in Japanese morning glory (Ipomoea nil) is a member of the MYB transcription factor family. The promoter of InMYB1 has been reported to induce petal-specific gene expression in Arabidopsis and Eustoma, and has the same function in several other dicotyledonous plants. Most flowers consist of sepals, petals, stamens and a carpel, whose identity establishment is explained by the ABC model. The establishment of the identity of petals is determined by the expression of class A and B genes in whorl 2. The aim of this study was to clarify whether the InMYB1 promoter functions by recognizing whorl position or petal identity by examining its activity in various mutant and transgenic Arabidopsis thaliana plants in which genes related to the ABC model have been modified. In plants defective in class C gene function, the InMYB1 promoter functioned not only in petals generated in whorl 2 but also in petaloid organs generated in whorl 3; while in the plants defective in class B gene function, the InMYB1 promoter did not function in the sepaloid organs generated in whorl 2. Plants overexpressing class A, B and E genes set flowers with petaloid sepals in whorl 1, i.e. the lateral parts were white and looked like petals, while the central parts were green and looked like sepals. The InMYB1 promoter functioned in the lateral white parts but not in the central green parts. These results show that the InMYB1 promoter functions by recognizing petal identity at the cellular level rather than the whorl position. The petal-specific function of the InMYB1 promoter could be used as a marker to identify petaloid cells.

Wood reinforcement of poplar by rice NAC transcription factor.

Lignocellulose, composed of cellulose, hemicellulose, and lignin, in the secondary cell wall constitutes wood and is the most abundant form of biomass on Earth. Enhancement of wood accumulation may be an effective strategy to increase biomass as well as wood strength, but currently only limited research has been undertaken. Here, we demonstrated that OsSWN1, the orthologue of the rice NAC Secondary-wall Thickening factor (NST) transcription factor, effectively enhanced secondary cell wall formation in the Arabidopsis inflorescence stem and poplar (Populus tremula×Populus tremuloides) stem when expressed by the Arabidopsis NST3 promoter. Interestingly, in transgenic Arabidopsis and poplar, ectopic secondary cell wall deposition in the pith area was observed in addition to densification of the secondary cell wall in fiber cells. The cell wall content or density of the stem increased on average by up to 38% and 39% in Arabidopsis and poplar, respectively, without causing growth inhibition. As a result, physical strength of the stem increased by up to 57% in poplar. Collectively, these data suggest that the reinforcement of wood by NST3pro:OsSWN1 is a promising strategy to enhance wood-biomass production in dicotyledonous plant species.

A petal-specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops.

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal-specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal-specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal-specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical ß-glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal-specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA-like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal-specific promoter in molecular breeding of floricultural crops.

A step towards understanding plant responses to multiple environmental stresses: a genome-wide study.

In natural habitats, especially in arid areas, plants are often simultaneously exposed to multiple abiotic stresses, such as salt, osmotic and heat stresses. However, most analyses of gene expression in stress responses examine individual stresses. In this report, we compare gene expression in individual and combined stresses. We show that combined stress treatments with salt, mannitol and heat induce a unique pattern of gene expression that is not a simple merge of the individual stress responses. Under multiple stress conditions, expression of most heat and salt stress-responsive genes increased to levels similar to or higher than those measured in single stress conditions, but osmotic stress-responsive genes increased to lower levels. Genes up-regulated to higher levels under multiple stress condition than single stress conditions include genes for heat shock proteins, heat shock regulators and late embryogenesis abundant proteins (LEAs), which protect other proteins from damage caused by stresses, suggesting their importance in multiple stress condition. Based on this analysis, we identify candidate genes for engineering crop plants tolerant to multiple stresses.

The MIXTA-like transcription factor MYB16 is a major regulator of cuticle formation in vegetative organs.

Cuticle secreted on the surface of the epidermis of aerial organs protects plants from the external environment. We recently found that Arabidopsis MIXTA-like R2R3-MYB family members MYB16 and MYB106 regulate cuticle formation in reproductive organs and trichomes. However, the artificial miRNA (amiRNA)-mediated knockdown plants showed no clear phenotypic abnormality in vegetative tissues. In this study, we used RNA interference (RNAi) targeting MYB16 to produce plants with reduced expression of both MYB16 and MYB106. The rosette leaves of RNAi plants showed more severe permeable cuticle phenotypes than the myb106 mutants expressing the MYB16 amiRNA in the previous study. The RNAi plants also showed reduced expression of cuticle biosynthesis genes LACERATA and ECERIFERUM1. By contrast, expression of a gain-of-function MYB16 construct induced over-accumulation of waxy substances on leaves. These results suggest that MYB16 functions as a major regulator of cuticle formation in vegetative organs, in addition to its effect in reproductive organs and trichomes.

Multi-petal cyclamen flowers produced by AGAMOUS chimeric repressor expression.

Cyclamen persicum (cyclamen) is a commercially valuable, winter-blooming perennial plant. We cloned two cyclamen orthologues of AGAMOUS (AG), CpAG1 and CpAG2, which are mainly expressed in the stamen and carpel, respectively. Cyclamen flowers have 5 petals, but expression of a chimeric repressor of CpAG1 (CpAG1-SRDX) caused stamens to convert into petals, resulting in a flower with 10 petals. By contrast, CpAG2-SRDX only caused incomplete formation of stamens and carpels. Expression in Arabidopsis thaliana showed similar effects on flower organ specification. Simultaneous expression of CpAG1-SRDX and CpAG2-SRDX in cyclamen induced rose-like, multi-petal flowers, a potentially valuable trait in commercial ornamental varieties. Expression of CpAG2-SRDX in a cyclamen mutant lacking expression of CpAG1 more effectively produced multi-petal flowers. Here, we controlled the number of petals in cyclamen by simple genetic engineering with a chimeric repressor. This strategy may be applicable useful for other ornamental plants with two distinct AG orthologues.

MIXTA-like transcription factors and WAX INDUCER1/SHINE1 coordinately regulate cuticle development in Arabidopsis and Torenia fournieri.

The waxy plant cuticle protects cells from dehydration, repels pathogen attack, and prevents organ fusion during development. The transcription factor WAX INDUCER1/SHINE1 (WIN1/SHN1) regulates the biosynthesis of waxy substances in Arabidopsis thaliana. Here, we show that the MIXTA-like MYB transcription factors MYB106 and MYB16, which regulate epidermal cell morphology, also regulate cuticle development coordinately with WIN1/SHN1 in Arabidopsis and Torenia fournieri. Expression of a MYB106 chimeric repressor fusion (35S:MYB106-SRDX) and knockout/down of MYB106 and MYB16 induced cuticle deficiencies characterized by organ adhesion and reduction of epicuticular wax crystals and cutin nanoridges. A similar organ fusion phenotype was produced by expression of a WIN1/SHN1 chimeric repressor. Conversely, the dominant active form of MYB106 (35S:MYB106-VP16) induced ectopic production of cutin nanoridges and increased expression of WIN1/SHN1 and wax biosynthetic genes. Microarray experiments revealed that MYB106 and WIN1/SHN1 regulate similar sets of genes, predominantly those involved in wax and cutin biosynthesis. Furthermore, WIN1/SHN1 expression was induced by MYB106-VP16 and repressed by MYB106-SRDX. These results indicate that the regulatory cascade of MIXTA-like proteins and WIN1/SHN1 coordinately regulate cutin biosynthesis and wax accumulation. This study reveals an additional key aspect of MIXTA-like protein function and suggests a unique relationship between cuticle development and epidermal cell differentiation.

Double-dose pravastatin versus add-on ezetimibe with low-dose pravastatin - effects on LDL cholesterol, cholesterol absorption, and cholesterol synthesis in Japanese patients with hypercholesterolemia (PEAS study).

AIM: This study compared the effect of doubling the dose of pravastatin with that of adding ezetimibe to low-dose pravastatin on the LDL cholesterol (LDL-C) level and on cholesterol absorption and synthesis markers. The tolerability of the 2 regimens was also compared. METHODS: This was a multicenter, open-label, parallel-group trial. Subjects were aged from 20 to 74 years and had an LDL-C ≥ 120 mg/dL despite pravastatin therapy at 5-10 mg/day. They were randomly allocated to receive either add-on ezetimibe (10 mg/day) or double-dose pravastatin, and follow-up was performed for 12 weeks. The primary endpoints were the changes of LDL-C and apolipoprotein (apo) B levels after 12 weeks of treatment. Cholesterol absorption and synthesis markers were also determined. RESULTS: LDL-C and apo B decreased by 16% and 14% in the ezetimibe add-on group versus 5.9% and 4.4%, respectively, in the pravastatin double-dose group. The between-group differences of these decreases were highly significant. Cholesterol absorption markers (sitosterol, campesterol, and cholestanol) were reduced by 48%, 36%, and 10%, respectively, in the ezetimibe add-on group, and were increased by 17%, 14%, and 6%, respectively, in the pravastatin double-dose group. Lathosterol (a cholesterol synthesis marker) increased by 76% in the ezetimibe add-on group and by 24% in the pravastatin double-dose group. The difference was statistically significant. No serious adverse effect was observed in either group. CONCLUSIONS: Adding ezetimibe to low-dose pravastatin achieves greater decreases in LDL-C, apo B, and cholesterol absorption markers than doubling the dose of pravastatin.

CRES-T, an effective gene silencing system utilizing chimeric repressors.

Chimeric REpressor gene Silencing Technology (CRES-T) is a useful tool for functional analysis of plant transcription factors. In this system, a chimeric repressor that is produced by fusion of a transcription factor to the plant-specific EAR-motif repression domain (SRDX) suppresses target genes of a transcription factor dominantly over the activity of endogenous and functionally redundant transcription factors. As a result, the transgenic plants that express a chimeric repressor exhibit phenotypes similar to loss-of-function of the alleles of the gene encoding the transcription factor. This system is simple and effective and can be used as a powerful tool not only for functional analysis of redundant transcription factors but also for the manipulation of plant traits by active suppression of the gene expression. Strategies for construction of the chimeric repressors and their expression in transgenic plants are described. Transient effector-reporter assays for functional analysis of transcription factors and detection of protein-protein interactions using the trans-repressive activity of SRDX repression domain are also described.

Plant catalase is imported into peroxisomes by Pex5p but is distinct from typical PTS1 import.

We have previously demonstrated that the targeting signal of pumpkin catalase, Cat1, is an internal PTS1 (peroxisomal targeting signal 1)-like sequence, QKL, located at -13 to -11 from the C-terminus, which is different from the typical PTS1 SKL motif located in the C-terminus. Here we show that Cat1 import into peroxisome is dependent on the cytosolic PTS receptor, Pex5p, in Arabidopsis, similar to typical PTS1 import, and that other components for transport of peroxisomal matrix proteins such as Pex14p, Pex13p, Pex12p and Pex10p also contribute to the import of Cat1. Interestingly, however, we found that Cat1 interacts with the N-terminal domain of Pex5p, but not the C-terminal domain for interaction with the typical PTS1, revealing that Pex5p recognizes Cat1 in a manner distinct from typical PTS1.