Phytocyanin-encoding genes confer enhanced ozone tolerance in Arabidopsis thaliana.

Ozone is a phytotoxic air pollutant that has various damaging effects on plants, including chlorosis and growth inhibition. Although various physiological and genetic studies have elucidated some of the mechanisms underlying plant ozone sensitivity and lesion development, our understanding of plant response to this gas remains incomplete. Here, we show evidence for the involvement of certain apoplastic proteins called phytocyanins, such as AtUC5, that protect against ozone damage. Two representative ozone-inducible responses, chlorosis and stomatal closure, were suppressed in AtUC5-overexpressing plants. Analysis of transgenic plants expressing a chimeric protein composed of AtUC5 fused to green fluorescent protein indicated that this fusion protein localises to the apoplast of plant cells where it appears to suppress early responses to ozone damage such as generation or signalling of reactive oxygen species. Moreover, yeast two-hybrid analyses suggest that AtUC5 may physically interact with stress-related proteins such as copper amine oxidase and late embryogenesis abundant protein-like protein. In addition to AtUC5, other examined phytocyanins such as AtUC6 and AtSC3 could confer ozone tolerance to plants when overexpressed in A. thaliana, suggesting that these proteins act together to protect plants against oxidative stress factors.

Quantitative evaluation of glycan-binding specificity of recombinant concanavalin A produced in lettuce (Lactuca sativa).

Concanavalin A (ConA), a mannose (Man)-specific leguminous lectin isolated from the jack bean (Canavalia ensiformis) seed extracts, was discovered over a century ago. Although ConA has been extensively applied in various life science research, recombinant mature ConA expression has not been fully established. Here, we aimed to produce recombinant ConA (rConA) in lettuce (Lactuca sativa) using an Agrobacterium tumefaciens-mediated transient expression system. rConA could be produced as a fully active form from soluble fractions of lettuce leaves and purified by affinity chromatography. From 12 g wet weight of lettuce leaves, 0.9 mg rConA could be purified. The glycan-binding properties of rConA were then compared with that of the native ConA isolated from jack bean using glycoconjugate microarray and frontal affinity chromatography. rConA demonstrated a glycan-binding specificity similar to nConA. Both molecules bound to N-glycans containing a terminal Man residue. Consistent with previous reports, terminal Manα1-6Man was found to be an essential unit for the high-affinity binding of rConA and nConA, while bisecting GlcNAc diminished the binding of rConA and nConA to Manα1-6Man-terminated N-glycans. These results demonstrate that the fully active rConA could be produced using the A. tumefaciens-mediated transient expression system and used as a recombinant substitute for nConA.

CRISPR/Cas9-mediated mutagenesis of the EPHEMERAL1 locus that regulates petal senescence in Japanese morning glory.

Flower longevity is one of the most important traits in ornamental plants. In Japanese morning glory (Ipomoea nil), EPHEMERAL1 (EPH1), a NAC transcription factor, is reportedly a key regulator of petal senescence. CRISPR/Cas9-mediated targeted mutagenesis is a powerful tool for crop breeding as well as for biological research. Here we report the application of CRISPR/Cas9 technology to targeted mutagenesis of the EPH1 gene in I. nil. Three regions within the EPH1 gene were simultaneously targeted by a single binary vector containing three single-guide RNA cassettes. We selected eight T0 transgenic plants containing the transferred DNA (T-DNA). Cleaved amplified polymorphic sequence (CAPS) analysis revealed that mutations occurred at single or multiple target sites in all eight plants. These plants harbored various mutations consisting of single base insertions and/or deletions of a single or more than two bases at the target sites. Several mutations generated at target sites were inherited in the T1 progeny with or without T-DNA insertions. Mutant plants in the T1 generations exhibited a clear delay in petal senescence. These results confirm that CRISPR/Cas9 technology can efficiently induce mutations in a target I. nil gene and that EPH1 plays a crucial role in the regulation of petal senescence. The eph1 mutants obtained in this study will be a useful tool for the elucidation of regulatory mechanisms in petal senescence.

Alteration of flower colour in Ipomoea nil through CRISPR/Cas9-mediated mutagenesis of carotenoid cleavage dioxygenase 4.

Japanese morning glory, Ipomoea nil, exhibits a variety of flower colours, except yellow, reflecting the accumulation of only trace amounts of carotenoids in the petals. In a previous study, we attributed this effect to the low expression levels of carotenogenic genes in the petals, but there may be other contributing factors. In the present study, we investigated the possible involvement of carotenoid cleavage dioxygenase (CCD), which cleaves specific double bonds of the polyene chains of carotenoids, in the regulation of carotenoid accumulation in the petals of I. nil. Using bioinformatics analysis, seven InCCD genes were identified in the I. nil genome. Sequencing and expression analyses indicated potential involvement of InCCD4 in carotenoid degradation in the petals. Successful knockout of InCCD4 using the CRISPR/Cas9 system in the white-flowered cultivar I. nil cv. AK77 caused the white petals to turn pale yellow. The total amount of carotenoids in the petals of ccd4 plants was increased 20-fold relative to non-transgenic plants. This result indicates that in the petals of I. nil, not only low carotenogenic gene expression but also carotenoid degradation leads to extremely low levels of carotenoids.

CRISPR/Cas9-mediated mutagenesis of the dihydroflavonol-4-reductase-B (DFR-B) locus in the Japanese morning glory Ipomoea (Pharbitis) nil.

CRISPR/Cas9 technology is a versatile tool for targeted mutagenesis in many organisms, including plants. However, this technique has not been applied to the Japanese morning glory (Ipomoea [Pharbitis] nil), a traditional garden plant chosen for the National BioResource Project in Japan. We selected dihydroflavonol-4-reductase-B (DFR-B) of I. nil, encoding an anthocyanin biosynthesis enzyme, as the target gene, and changes in the stem colour were observed during the early stages of plant tissue culture by Rhizobium [Agrobacterium]-mediated transformation. Twenty-four of the 32 (75%) transgenic plants bore anthocyanin-less white flowers with bi-allelic mutations at the Cas9 cleavage site in DFR-B, exhibiting a single base insertion or deletions of more than two bases. Thus, these results demonstrate that CRISPR/Cas9 technology enables the exploration of gene functions in this model horticultural plant. To our knowledge, this report is the first concerning flower colour changes in higher plants using CRISPR/Cas9 technology.

Overexpression of carotenogenic genes in the Japanese morning glory Ipomoea (Pharbitis) nil.

Japanese morning glory, Ipomoea nil, has several coloured flowers except yellow, because it can accumulate only trace amounts of carotenoids in the petal. To make the petal yellow with carotenoids, we introduced five carotenogenic genes (geranylgeranyl pyrophosphate synthase, phytoene synthase, lycopene ß-cyclase and ß-ring hydroxylase from Ipomoea obscura var. lutea and bacterial phytoene desaturase from Pantoea ananatis) to white-flowered I. nil cv. AK77 with a petal-specific promoter by Rhizobium (Agrobacterium)-mediated transformation method. We succeeded to produce transgenic plants overexpressing carotenogenic genes. In the petal of the transgenic plants, mRNA levels of the carotenogenic genes were 10 to 1,000 times higher than those of non-transgenic control. The petal colour did not change visually; however, carotenoid concentration in the petal was increased up to about ten-fold relative to non-transgenic control. Moreover, the components of carotenoids in the petal were diversified, in particular, several ß-carotene derivatives, such as zeaxanthin and neoxanthin, were newly synthesized. This is the first report, to our knowledge, of changing the component and increasing the amount of carotenoid in petals that lack ability to biosynthesize carotenoids.

XTH20 and XTH19 regulated by ANAC071 under auxin flow are involved in cell proliferation in incised Arabidopsis inflorescence stems.

One week after partial incision of Arabidopsis inflorescence stems, the repair process in damaged tissue includes pith cell proliferation. Auxin is a key factor driving this process, and ANAC071, a transcription factor gene, is upregulated in the distal region of the incised stem. Here we show that XTH20 and the closely related XTH19, members of xyloglucan endotransglucosylase/hydrolases family catalyzing molecular grafting and/or hydrolysis of cell wall xyloglucans, were also upregulated in the distal part of the incised stem, similar to ANAC071. XTH19 was expressed in the proximal incision region after 3 days or after auxin application to the decapitated stem. Horizontal positioning of the plant with the incised side up resulted in decreased ProDR 5 :GUS, ANAC071, XTH20, and XTH19 expression and reduced pith cell proliferation. In incised stems of Pro35S :ANAC071-SRDX plants, expression of XTH20 and XTH19 was substantially and moderately decreased, respectively. XTH20 and XTH19 expression and pith cell proliferation were suppressed in anac071 plants and were increased in Pro35S :ANAC071 plants. Pith cell proliferation was also inhibited in the xth20xth19 double mutant. Furthermore, ANAC071 bound to the XTH20 and XTH19 promoters to induce their expression. This study revealed XTH20 and XTH19 induction by auxin via ANAC071 in the distal part of an incised stem and their involvement in cell proliferation in the tissue reunion process.

CLE6 expression recovers gibberellin deficiency to promote shoot growth in Arabidopsis.

Small peptides act as local signals during plant development, but few studies have examined their interaction with phytohormone signaling. Here, we show that application of gibberellin (GA) to Arabidopsis shoots induces substantial accumulation of transcripts encoded by CLE6, a member of the CLAVATA/ESR-RELATED (CLE) gene family, in the root stele, followed by promotion of organ growth by CLE6 in GA-deficient plants. The long-distance effect of GA4 was demonstrated by the observation that its application to the shoot apex of the GA-deficient mutant ga3ox1/ga3ox2 rescued the short-root phenotype. Microarray analysis was used to identify root-expressed genes that respond to systemic application of GA, and CLE6 was selected for further analysis. CLE6 was highly expressed in roots at the young seedling stage, and CLE6 promoter activity was strong in hypocotyls and roots, especially in root stele cells at branch points. Application of CLE6 peptide had no obvious effect on the growth and development of GA-deficient mutant plants. Nonetheless, the fact that ectopic over-expression of CLE6 in the GA-deficient mutant promoted root growth and branching, petiole elongation, bolting rate and stem length showed that CLE6 expression partially compensates for the GA deficiency. Reciprocal grafting of GA-deficient mutant plants to 35S::CLE6 transformants complemented the shoot phenotype associated with GA deficiency, demonstrating the systemic effect of CLE6 from root to shoot. These data suggest that root-expressed CLE6 is systemically involved in shoot growth under GA action in Arabidopsis.

Isolation and characterization of a carrot nucleolar protein with structural and sequence similarity to the vertebrate PESCADILLO protein.

The nuclear matrix is involved in many nuclear events, but its protein architecture in plants is still not fully understood. A cDNA clone was isolated by immunoscreening with a monoclonal antibody raised against nuclear matrix proteins of Daucus carota L. Its deduced amino acid sequence showed about 40% identity with the PESCADILLO protein of zebrafish and humans. Primary structure analysis of the protein revealed a Pescadillo N-terminus domain, a single breast cancer C-terminal domain, two nuclear localization signals, and a potential coiled-coil region as also found in animal PESCADILLO proteins. Therefore, we designated this gene DcPES1. Although DcPES1 mRNA was detected in all tissues examined, its levels were highest in tissues with proliferating cells. Immunofluorescence using specific antiserum against the recombinant protein revealed that DcPES1 localized exclusively in the nucleolus. Examination of fusion proteins with green fluorescent protein revealed that the N-terminal portion was important for localization to the nucleoli of tobacco and onion cells. Moreover, when the nuclear matrix of carrot cells was immunostained with an anti-DcPES1 serum, the signal was detected in the nucleolus. Therefore, the DcPES1 protein appears to be a component of or tightly bound to components of the nuclear matrix.

Generative cell-specific activation of the histone gH2A gene promoter of Lilium longiflorum in tobacco.

The Lilium longiflorum gH2A promoter is active exclusively in the generative cells of mature pollen in transgenic tobacco expressing the gH2A promoter::GUS (ß-glucuronidase) construct as a reporter gene. Temporal and spatial aspects of gH2A promoter activity examined during pollen development in transgenic tobacco reveal that GUS reporter activity was not detected until developing pollen entered the early bicellular developmental stage. Activity was first detected in generative cells at early-mid stages and gradually increased to maximum levels at mid-bicellular stages. The patterns of appearance and longevity of GUS activity in tobacco were very similar to those of gH2A mRNA during pollen development in Lilium. Exogenous treatment with colchicine, a well-known microtubule depolymerize, blocked microspore mitosis and inhibited generative cell differentiation. No GUS signal was detected in the resulting anomalous pollen, which lacked generative cell differentiation. These data strongly suggest that normal generative cell development is essential for activation of the gH2A promoter. Furthermore, these results indicate that common transcriptional activator(s) of the gH2A promoter may be present in both Lilium and Nicotiana, and that such putative factor(s) activates the gH2A promoter only when generative cells undergo normal development.

Constitutive expression of the GIGANTEA ortholog affects circadian rhythms and suppresses one-shot induction of flowering in Pharbitis nil, a typical short-day plant.

GIGANTEA (GI) is a key regulator of flowering time, which is closely related to the circadian clock function in Arabidopsis. Mutations in the GI gene cause photoperiod-insensitive flowering and altered circadian rhythms. We isolated the GI ortholog PnGI from Pharbitis (Ipomoea) nil, an absolute short-day (SD) plant. PnGI mRNA expression showed diurnal rhythms that peaked at dusk under SD and long-day (LD) conditions, and also showed robust circadian rhythms under continuous dark (DD) and continuous light (LL) conditions. Short irradiation with red light during the flower-inductive dark period did not change PnGI expression levels, suggesting that such a night break does not abolish flowering by affecting the expression of PnGI. In Pharbitis, although a single dusk signal is sufficient to induce expression of the ortholog of FLOWERING LOCUS T (PnFT1), PnGI mRNA expression was not reset by single lights-off signals. Constitutive expression of PnGI (PnGI-OX) in transgenic plants altered period length in leaf-movement rhythms under LL and affected circadian rhythms of PnFT mRNA expression under DD. PnGI-OX plants formed fewer flower buds than the wild type when one-shot darkness was given. In PnGI-OX plants, expression of PnFT1 was down-regulated, suggesting that PnGI functions as a suppressor of flowering, possibly in part through down-regulation of PnFT1.

ICE1 Ser403 is necessary for protein stabilization and regulation of cold signaling and tolerance.

ICE1, a MYC-type transcription factor, has an important role in the induction of CBF3/DREB1A for regulation of cold signaling and tolerance. Here we reveal that serine 403 of ICE1 is involved in regulating the transactivation and stability of the ICE1 protein. Substitution of serine 403 by alanine enhanced the transactivational activity of ICE1 in Arabidopsis protoplasts. Over-expression of ICE1(S403A) conferred more freezing tolerance than ICE1(WT) in Arabidopsis, and the expression of cold-regulated genes such as CBF3/DREB1A, COR47 and KIN1 was enhanced in plants over-expressing ICE1(S403A). Furthermore, the ICE1(S403A) protein level was not changed after cold treatment, whereas the ICE1(WT) protein level was reduced. Interestingly, polyubiquitylation of the ICE1(S403A) protein in vivo was apparently blocked. These results demonstrate that serine 403 of ICE1 has roles in both transactivation and cold-induced degradation of ICE1 via the ubiquitin/26S proteasome pathway, suggesting that serine 403 is a key residue for the attenuation of cold-stress responses by HOS1-mediated degradation of ICE1.

Historical and contemporary gene dispersal in wild carrot (Daucus carota ssp. carota) populations.

BACKGROUND AND AIMS: Wild carrot is the ancestor of cultivated carrot and is the most important gene pool for carrot breeding. Transgenic carrot may be released into the environment in the future. The aim of the present study was to determine how far a gene can disperse in wild carrot populations, facilitating risk assessment and management of transgene introgression from cultivated to wild carrots and helping to design sampling strategies for germplasm collections. METHODS: Wild carrots were sampled from Meijendel and Alkmaar in The Netherlands and genotyped with 12 microsatellite markers. Spatial autocorrelation analyses were used to detect spatial genetic structures (SGSs). Historical gene dispersal estimates were based on an isolation by distance model. Mating system and contemporary pollen dispersal were estimated using 437 offspring of 20 mothers with different spatial distances and a correlated paternity analysis in the Meijendel population. KEY RESULTS: Significant SGSs are found in both populations and they are not significantly different from each other. Combined SGS analysis indicated significant positive genetic correlations up to 27 m. Historical gene dispersal sigma(g) and neighbourhood size N(b) were estimated to be 4-12 m [95 % confidence interval (CI): 3-25] and 42-73 plants (95 % CI: 28-322) in Meijendel and 10-31 m (95 % CI: 7-infinity) and 57-198 plants (95 % CI: 28-infinity) in Alkmaar with longer gene dispersal in lower density populations. Contemporary pollen dispersal follows a fat-tailed exponential-power distribution, implying pollen of wild carrots could be dispersed by insects over long distance. The estimated outcrossing rate was 96 %. CONCLUSIONS: SGSs in wild carrots may be the result of high outcrossing, restricted seed dispersal and long-distance pollen dispersal. High outcrossing and long-distance pollen dispersal suggest high frequency of transgene flow might occur from cultivated to wild carrots and that they could easily spread within and between populations.

PnMADS1, encoding an StMADS11-clade protein, acts as a repressor of flowering in Pharbitis nil.

We previously isolated PnMADS1, a MADS-box transcription factor and member of the functionally diverse StMADS11 clade of the MADS-box family, from Pharbitis nil, which is a typical SD plant. However, its precise function remained unclear. To investigate the biological role of PnMADS1, and especially its involvement in flowering, we constructed transgenic P. nil plants that overexpresses or underexpresses PnMADS1. PnMADS1-RNAi transformants had an increased number of flower buds, whereas overexpression of PnMADS1 led to a decrease in the number of flower buds, although both transgenic plants maintained the photoperiodic responses of flowering. These results suggest that PnMADS1 negatively regulates floral evocation from the vegetative phase to the reproductive phase but it has no essential role in floral induction by photoperiodic signals. Results of yeast two-hybrid experiments revealed that PnMADS1 can interact with itself, suggesting that this protein functions in floral evocation as a homodimer. PnMADS1 also interacts with PnSAH3, an AP1-clade protein, suggesting that PnMADS1 has a functional role in flower formation as a heterodimer with other MADS-box protein(s).

Male gametic cell-specific histone gH2A gene of Lilium longiflorum: genomic structure and promoter activity in the generative cell.

A genomic clone containing the gH2A gene, a histone variant specifically expressed in male gametic cells within the pollen of Lilium longiflorum, was isolated. Sequence analysis revealed that the coding region of the gene is interrupted by one intron, as is the case with the somatic type of plant histone H2A genes, suggesting derivation from the same ancestral gene containing one intron. In addition, a 2.8-kbp fragment of the 5' upstream region of gH2A contained TATA and CAAT boxes, but neither a plant histone-specific regulatory DNA element nor vegetative cell-specific cis-elements were found. A histochemical study of stable transformants demonstrated that the 5' upstream region of the gene can drive gene expression specifically in the generative cell of pollen; no activity was detectable in the vegetative cell or in other reproductive and vegetative tissues of transgenic Nicotiana tabacum. These results strongly suggest that the generative cell can direct specific gene expression, that this expression may be regulated by a putative male gametic factor, and that the gH2A promoter may therefore serve as a useful male gametic cell fate marker in angiosperms.

Genomic structure of a novel Arabidopsis clock-controlled gene, AtC401, which encodes a pentatricopeptide repeat protein.

We isolated and characterized AtC401, a novel Arabidopsis clock-controlled gene that encodes a protein containing the pentatricopeptide repeat (PPR) motif. AtC401 was isolated as an Arabidopsis homolog of Pharbitis nil C401 (PnC401), a gene that encodes a leaf protein closely related to the photoperiodic induction of flowering and displays a circadian rhythm at the transcriptional level. The AtC401 gene spans 5.6 kb and contains 12 exons. Comparisons of the sequences and genomic organization of AtC401 and PnC401 revealed that each has two exons near the 3'-end, which encode a highly conserved domain consisting of 12 repeats of the PPR motif. Phylogenetic analysis of at least 450 Arabidopsis proteins containing PPR motifs revealed that AtC401 and related proteins form a distinct group. Moreover, the position of the intron between the two exons that encode the PPR domain has been conserved exactly in other C401-like genes. Using a reporter assay, we found a fragment (-174 to +73) of AtC401 that was sufficient to regulate circadian rhythmic expression. These results suggest that the conserved domain of AtC401 has a function similar to that of PnC401, and that the expression of C401 genes according to a circadian rhythm is important for protein function.

Characterization of transcriptional oscillation of an Arabidopsis homolog of PnC401 related to photoperiodic induction of flowering in Pharbitis nil.

AtC401 is an Arabidopsis homolog of PnC401 that is related to photoperiodic induction of flowering in Pharbitis nil. These genes show free-running rhythms. To study the free-running rhythm of AtC401, we fused a firefly luciferase reporter to the AtC401 promoter and transformed it into Arabidopsis plants. The observed bioluminescence oscillated under continuous light and continuous dark only with sucrose supplementation. The free-running period of bioluminescence was temperature-compensated between 22 degrees C and 30 degrees C. Light-pulse experiments under continuous darkness produced a phase-response curve typical of circadian rhythms. We conclude that rhythmic expression of AtC401 is controlled by a circadian oscillator.

Molecular characterization of a novel protein disulfide isomerase in carrot.

A protein disulfide isomerase (PDI) coding sequence was cloned from a cDNA library derived from carrot (Daucus carota L.) somatic embryos. The cDNA is 2060 bp in length and encodes for a protein of 581 amino acids and molecular weight of 64.4 kDa. Primary structure analysis of the deduced protein revealed two thioredoxin-like active sites and an endoplasmic reticulum-retention signal at its C-terminus, which is also found in PDIs in plants and animals. Although between the carrot protein and other plant PDIs there is only about 30% identity, the active site regions are almost identical. The corresponding mRNA was found in varying amounts, in all tissues investigated. A recombinant protein expressed from the carrot cDNA clone effectively catalyzed both glutathione-insulin transhydrogenation and the oxidative renaturation of denatured RNase A. These results suggest that the protein coded for by the carrot gene is a novel member of the PDI family in plants. We therefore designated this novel carrot gene PDIL1. The protein expressed by the PDIL1 cDNA sequence had a highly acidic stretch at its N-terminal region (no such domain exists in known plant PDIs), and was located far from known plant PDIs on a maximum likelihood tree. The PDIL1 gene, together with closely-related genes identified in Arabidopsis and tomato, was suggested to belong to a novel subfamily of PDIs.