Pollen tube growth is essential for the fertilization process in angiosperms. When pollen grains arrive on the stigma, they germinate, and the pollen tubes elongate through the styles of the pistils to deliver sperm cells into the ovules to produce the seeds. The relationship between the growth rate and style length remains unclear. In previous studies, we developed a liquid pollen germination medium for observing pollen tube growth. In this study, using this medium, we examined the pollen tube growth ability in Petunia axillaris subsp. axillaris, P. axillaris subsp. parodii, P. integrifolia, and P. occidentalis, which have different style lengths. Petunia occidentalis had the longest pollen tubes after 6 h of culture but had a relatively shorter style. Conversely, the pollination experiments revealed that P. axillaris subsp. parodii, which had the longest style, produced the longest pollen tubes in vivo. The results revealed no clear relationship between the style lengths and the growth rate of pollen tubes in vitro. Interspecific pollinations indicated that the styles affected pollen tube growth. We concluded that, in vitro, the pollen tubes grow without being affected by the styles, whereas, in vivo, the styles significantly affected pollen tube growth. Furthermore, interspecific pollination experiments implied that the pollen tube growth tended to be suppressed in the styles of self-incompatibility species. Finally, we discussed the pollen tube growth ability in relation to style lengths.
To introduce useful characteristics such as fragrance into Argyranthemum frutescens (L.) and to expand the variation, we conducted crosses using A. frutescens as the seed parent and Chamaemelum nobile (L.) All. as the pollen parent. All the tested cross combinations between the three strains of A. frutescens and one strain of C. nobile produced embryos, and healthy plants were obtained by ovule culture. The obtained plantlets had a white ray floret, and the leaf shape was intermediate to those of the parents. The individuals obtained from this cross were subjected to two methods to determine hybridity: flow cytometry analyses and cleaved amplified polymorphic sequence (CAPS) markers. For the CAPS marker, we selected the internal transcribed spacer (ITS) region, which is highly variable among the genera, as the region to be amplified. We selected restriction enzymes BmgT120 I and Afl II, which selectively cut common sequences in the genus Argyranthemum, based on the sequence analysis of one parent strain each of A. frutescens and C. nobile and alignment with known sequences of related species. Flow cytometry analyses and CAPS markers revealed that the individuals obtained from the cross between A. frutescens and C. nobile are intergeneric hybrids. In addition, these established methods were capable of quickly and reliably identifying hybrids between A. frutescens and C. nobile. This report shows for the first time that crossbreeding between A. frutescens (seed parent) and C. nobile (pollen parent) is possible, and further development of Argyranthemum breeding, such as the expansion of variation by intergeneric crosses, is expected.
Genome information has been accumulated for many species, and these genes and regulatory sequences are expected to be applied in plants by enhancing or creating new metabolic pathways. We hypothesized that manipulating a long array of repetitive sequences using tethered chromatin modulators would be effective for robust regulation of gene expression in close proximity to the arrays. This approach is based on a human artificial chromosome made of long synthetic repetitive DNA sequences in which we manipulated the chromatin by tethering the modifiers. However, a method for introducing long repetitive DNA sequences into plants has not yet been established. Therefore, we constructed a bacterial artificial chromosome-based binary vector in Escherichia coli cells to generate a construct in which a cassette of marker genes was inserted into 60-kb synthetic human centromeric repetitive DNA. The binary vector was then transferred to Agrobacterium cells and its stable maintenance confirmed. Next, using Agrobacterium-mediated genetic transformation, this construct was successfully introduced into the genome of cultured tobacco BY-2 cells to obtain a large number of stable one-copy strains. ChIP analysis of obtained BY-2 cell lines revealed that the introduced synthetic repetitive DNA has moderate chromatin modification levels with lower heterochromatin (H3K9me2) or euchromatin (H3K4me3) modifications compared to the host centromeric repetitive DNA or an active Tub6 gene, respectively. Such a synthetic DNA sequence with moderate chromatin modification levels is expected to facilitate manipulation of the chromatin structure to either open or closed.
Many microorganisms have been reported to produce compounds that promote plant growth and are thought to be involved in the establishment and maintenance of symbiotic relationships. 3-Phenyllactic acid (PLA) produced by lactic acid bacteria was previously shown to promote root growth in adzuki cuttings. However, the mode of action of PLA as a root-promoting substance had not been clarified. The present study therefore investigated the relationship between PLA and auxin. PLA was found to inhibit primary root elongation and to increase lateral root density in wild-type Arabidopsis, but not in an auxin signaling mutant. In addition, PLA induced IAA19 promoter fused β-glucuronidase gene expression, suggesting that PLA exhibits auxin-like activity. The inability of PLA to promote degradation of Auxin/Indole-3-Acetic Acid protein in a yeast heterologous reconstitution system indicated that PLA may not a ligand of auxin receptor. Using of a synthetic PLA labeled with stable isotope showed that exogenously applied PLA was converted to phenylacetic acid (PAA), an endogenous auxin, in both adzuki and Arabidopsis. Taken together, these results suggest that exogenous PLA promotes auxin signaling by conversion to PAA, thereby regulating root growth in plants.
In rice, the lysin motif (LysM) receptor-like kinase OsCERK1, originally identified as the essential molecule for chitin-triggered immunity, plays a key role in arbuscular mycorrhizal (AM) symbiosis. As we previously reported, although AM colonization was largely repressed at 2 weeks after inoculation (WAI), arbuscules were observed at 5 WAI in oscerk1 mutant. Conversely, most mutant plants that defect the common symbiosis signaling pathway exhibited no arbuscule formation. Concerning the reason for this characteristic phenotype of oscerk1, we speculated that OsRLK10, which is a putative paralog of OsCERK1, may have a redundant function in AM symbiosis. The protein sequences of these two genes are highly conserved and it is estimated that the gene duplication occurred 150 million years ago. Here we demonstrated that OsCERK2/OsRLK10 induced AM colonization and chitin-triggered reactive oxygen species production in oscerk1 knockout mutant as similar to OsCERK1. The oscerk2 mutant showed a slight but significant reduction of AM colonization at 5 WAI, indicating the contribution of OsCERK2 for AM symbiosis. However, the oscerk2;oscerk1 double-knockout mutant produced arbuscules at 5 WAI as similar to the oscerk1 mutant, indicating that the redundancy of OsCERK1 and OsCERK2 did not explain the mycorrhizal colonization in oscerk1 at 5 WAI. These results indicated that OsCERK2 has a potential to regulate both chitin-triggered immunity and AM symbiosis and at least partially contributes to AM symbiosis in rice though the contribution of OsCERK2 appears to be weaker than that of OsCERK1.
Endoplasmic reticulum (ER) stress activates unfolded protein responses (UPRs), such as promoting protein folding under the control of specific gene expression. Our previous study showed that ER stress induced by ER stress inducers such as tunicamycin (Tm), an inhibitor of N-linked glycan synthesis, causes ectopic lignin deposition in Arabidopsis roots, but the relationship between UPR and ectopic lignin deposition remains unclear. The receptor-like kinase THESEUS1 (THE1) has been shown to sense cell wall damage (CWD) induced in Arabidopsis by cellulose synthase inhibitors such as isoxaben (ISO) and to activate ectopic lignin deposition. In this study, we assessed the involvement of THE1 in ectopic lignin deposition caused by the ER stress inducer Tm. The loss-of-function mutation of THE1, the1-3, suppressed Tm-induced root growth inhibition and ectopic lignin deposition, revealing that THE1 is involved in root growth defects and ectopic lignin deposition caused by ER stress. Similarly, ISO treatment induced ectopic lignin deposition as well as the expression of the UPR marker genes binding protein 3 (BiP3) and ER-localized DnaJ 3b (ERdj3b). Conversely, in the the1-3 mutant, ISO-induced ectopic lignin deposition and the expression of BiP3 and ERdj3b were suppressed. These results showed that THE1 is involved in not only root growth inhibition and ectopic lignin deposition caused by ER stress but also CWD-induced UPR.
In transgenic experiments, we often face fundamental requirements such as overexpressing a certain gene, developing organelle markers, testing promoter activities, introducing large genomic fragments, and combinations of them. To fulfill these multiple requirements in rice, we developed simple binary vectors with or without maize ubiquitin (UBQ) promoter, Gateway cassette and fluorescent proteins. First, we compared stabilities of cauliflower mosaic virus 35S and maize UBQ promoters for constitutive gene expression in transgenic rice. We show that the 35S promoter was frequently silenced after shoot regeneration, whereas maize UBQ promoter achieved stable expression in various young tissues. Binary vectors with Gateway cassettes under the control of the UBQ promoter allowed us to develop stable organelle markers for nuclei, microtubules and P-bodies in rice. The maize UBQ promoter can be easily replaced with any promoters of interest as exemplified by reporters of mitotic cells and provascular bundles. Finally, by introducing two genomic fluorescent reporters, we showed utilities of the Gateway cassette and two selection markers in large DNA fragment transfer and sequential transformations, respectively. Thus, these binary vectors provide useful choices of transgenic experiments in rice.
An Arabidopsis NAC domain transcription factor VND-INTERACTING2 (VNI2) was originally isolated as an interacting protein with another NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), a master regulator of xylem vessel element differentiation. VNI2 inhibits transcriptional activation activity of VND7 by forming a protein complex. Here, to obtain insights into how VNI2 regulates VND7, we tried to identify the amino acid region of VNI2 required for inhibition of VND7. VNI2 has an amino acid sequence similar to the ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR (ERF)-associated amphiphilic repression (EAR) motif, conserved in transcriptional repressors, at the C-terminus. A transient expression assay showed that the EAR-like motif of VNI2 was not required for inhibition of VND7. The C-terminal deletion series of VNI2 revealed that 10 amino acid residues, highly conserved in the VNI2 orthologs contributed to effective repression of the transcriptional activation activity of VND7. Observation of transgenic plants ectopically expressing VNI2 showed that the identified 10 amino acid sequence strongly affected xylem vessel formation and plant growth. These data indicated that the 10 amino acid sequence of VNI2 has an important role in its transcriptional repression activity and negative regulation of xylem vessel formation.
Flowering locus T (FT) is known to promote flowering in response to photoperiodic conditions and has recently been shown to contribute to other phenomenon, such as diurnal stomatal movement. In legumes, FTs are classified into three subtypes, though the role of each subtype is not well defined. It has been reported that when FT of Lotus japonicus (LjFT) is heterologously expressed in Arabidopsis, LjFT functions as a mobile florigen to promote flowering, similar to Arabidopsis FT (AtFT). In this study, we expressed AtFT in L. japonicus using the SUC2 promoter and showed that heterologous expression of AtFT was able to promote flowering in the plant. We also showed that AtFT expression does not affect stomatal closing nor nyctinastic leaf movement. These findings contribute to our understanding of flower development and have potential application to breeding or plant biotechnology.
Blackberry is an economically important crop in Mexico, and its yield is substantially reduced by gray mold, a disease caused by Botrytis cinerea. One of the means to obtain B. cinerea-resistant plants is gamma irradiation. Shoot tips of in vitro-micropropagated blackberry plants (Rubus fruticosus ‘Tupy’) were irradiated with five doses of Cobalt-60 gamma radiation (0, 15, 30, 45, and 60 Gy) and cultured on Murashige and Skoog basal medium containing 1.0 mg l−1 benzylaminopurine and 0.06 mg l−1 indole-3-butyric acid (MSB medium). After 28 days of culture, survival was evaluated to determine mean lethal dose (LD50), and 200 shoots were further irradiated at the determined LD50 (30.8 Gy). After 28 days, the surviving shoots were micropropagated on MSB medium for 60 days. Non-irradiated shoots were screened for the in vitro selection of resistant B. cinerea, exposing them to different concentrations of sterile culture filtrate of B. cinerea (0, 2, 4, 6, 8, and 10 g l−1) for 28 days to determine mean lethal concentration (LC50), and the irradiated surviving shoots were further exposed to the determined LC50 (4.6 g l−1). Three surviving lines (rfgum5, rfgum6, and rfgum17) that did not present changes compared with the control shoots were micropropagated to obtain plantlets, which were further subjected to in vitro resistance assays using detached leaves inoculated with B. cinerea (1×103 spores ml−1). Plants of rfgum5 and rfgum6 mutant lines were highly resistant and presented similar growth to control plants. Therefore, this methodology is useful to obtain B. cinerea-resistant blackberry plants.
3-Phenyllactic acid (PLA) is a common secondary product of Lactobacillus sp. and promotes adventitious-root formation in Azuki beans (Vigna angularis). Root promotion activity of PLA is synergistically enhanced by tryptophan (Trp). In this study, stereoisomers of PLA and Trp amide conjugates and their alkyl esters were synthesized to investigate the structure–activity relationships on root-promotion activity. The rooting activity of D-PLA-L-Trp conjugate shows more than 40 times higher than that of the mixture of D-PLA and L-Trp. Modification of PLA-Trp with ethyl ester showed the highest activity at 3,400 times of a mixture of D-PLA and L-Trp. However, L-or D-PLA-D-Trp conjugate and the isopropyl ester of PLA-Trp conjugates, both lost the root promotion activity and implicated that a requirement for steric structure for PLA related root promotion mechanism. Unlike auxin substances, which are commonly used as rooting agents that displayed high activity in low concentrations, PLA-Trp ethyl ester exhibited far less phytotoxicity at high concentration of 1 mM, despite its high rooting activity. Innovation of PLA-Trp ethyl ester may be expected for agricultural aspects with low environmental impact.
Previously, we developed a method that uses temperature-controlled atmospheric-pressure plasma to induce protein uptake in plant cells. In the present work, we examined the mechanism underlying such uptake of a fluorescent-tagged protein in tobacco leaf cells. Intact leaf tissue was irradiated with N2 plasma generated by a multi-gas plasma jet and then exposed to the test protein (histidine-tagged superfolder green fluorescence protein fused to adenylate cyclase); fluorescence intensity was then monitored over time as an index of protein uptake. Confocal microscopy revealed that protein uptake potential was retained in the leaf tissue for at least 3 h after plasma treatment. Further examination indicated that the introduced protein reached a similar amount to that after overnight incubation at approximately 5 h after irradiation. Inhibitor experiments revealed that protein uptake was significantly suppressed compared with negative controls by pretreatment with sodium azide (inhibitor of adenosine triphosphate hydrolysis) or sucrose or brefeldin A (inhibitors of clathrin-mediated endocytosis) but not by pretreatment with genistein (inhibitor of caveolae/raft-mediated endocytosis) or cytochalasin D (inhibitor of micropinocytosis/phagocytosis), indicating that the N2 plasma treatment induced protein transportation across the plant plasma membrane via clathrin-mediated endocytosis.
The brassinosteroid (BR) phytohormone is an important regulator of plant growth. To identify novel transcription factors that regulate BR responses, we screened chimeric repressor gene silencing technology (CRES-T) plants, in which transcription factors were converted into chimeric repressors by the fusion of SRDX plant-specific repression domain, with brassinazole (Brz), an inhibitor of BR biosynthesis. We identified that a line that expressed the chimeric repressor for zinc finger homeobox transcription factor, BRASSINOSTEORID-RELATED-HOMEOBOX-2 (BHB2-sx), exhibited Brz-hypersensitive phenotype with shorter hypocotyl under dark, dwarf and round and dark green leaves similar to BR-deficient phenotype. Similar to BHB2-sx plants, bhb2 knockout mutant also exhibited Brz hypersensitive phenotype. In contrast, ectopic expression of BHB2 (BHB2-ox) showed hypocotyl elongation phenotype (BR excessive), showing decrease to Brz sensitivity. The expression of the DWF4 and CPD BR biosynthesis genes was repressed in BHB2-sx plants, whereas it was enhanced in BHB2-ox plants. The BR deficient-like phenotype of BHB2-sx plants was partially restored by treatment with brassinolide (BL), indicating that the BR deficient phenotype of BHB2-sx plant may be due to suppression of BR biosynthesis. Our results indicate that BHB2 is a positive regulator of BR response may be due to the promotion of BR biosynthesis genes.
Allene oxide synthase (AOS) is a key enzyme involved in the biosynthesis of 12-oxo-phytodienoic acid (OPDA) and jasmonic acid and plays an important role in plant defense against herbivore attacks. In the liverwort, Marchantia polymorpha, we previously identified cytosol-type MpAOS1 and chloroplast-type MpAOS2 that show AOS activities. However, there is no direct evidence to show the subcellular localization of MpAOSs and their contribution to plant defense via OPDA production in M. polymorpha. In this study, we generated M. polymorpha mutants, with the MpAOS1 and MpAOS2 genes disrupted via CRISPR/Cas9-mediated genome editing; the loss of OPDA production was analyzed in double-knockout mutants. On AOS mutants, the survival rate and oviposition of spider mites (Tetranychus urticae) increased relative to those on wild-type plants. Overall, these findings suggest that defense systems via OPDA-signaling pathways in response to spider mites have been established in M. polymorpha.
Potato, Solanum tuberosum L. is an important crop. However, it is difficult to breed potato cultivars by applying conventional crossing methods because potato has a tetraploid genome and is vegetatively propagated. Flower formation and tuber development occur simultaneously. Many potato cultivars hardly produce any fruits after crossing and fail to produce seeds. We report an improved procedure for obtaining progeny seeds by grafting potatoes onto tomatoes. The rate of fruit formation was more than 19% when the grafted potatoes were used for the crossing experiments, whereas crossing using the ungrafted plants showed a rate of 1.1%. This result suggests that our procedure results in the easy acquisition of null-segregant progenies by crossing mutant lines. It is also expected to improve conventional potato breeding.
L-3,4-dihydroxyphenylalanine (L-DOPA) is one of the important secondary metabolites of plants and has been used for various purposes, such as in clinical treatment for Parkinson’s disease and dopamine-responsive dystonia. In plants, L-DOPA is a precursor of many alkaloids, catecholamines, and melanin; the L-DOPA synthesis pathway is similar to that in mammals. L-DOPA acts as an allelochemical, has an important role in several biological processes, such as stress response and metabolism, in plants. L-DOPA is widely used in the clinical treatment as well as a dietary supplement or psychotropic drug, understanding of biosynthesis of L-DOPA in plant could lead to a stable supply of L-DOPA. This paper describes an improved method for simple and rapid quantification of L-DOPA content using liquid chromatography-tandem mass spectrometry. The standard quantitative methods for L-DOPA require multiple purification steps or relatively large amounts of plant material. In our improved method, quantification of L-DOPA was possible with extract of one–two pieces of cotyledon without any partitioning or column for purification. The endogenous L-DOPA (approximately 4,000 µg g−1 FW (fresh weight)) could be detected from the one pieces of cotyledon of the faba bean sprout using this method. This method was also effective for samples with low endogenous amounts of L-DOPA such as broccoli, Japanese white radish, pea, and red cabbage sprouts. Therefore, this improved method will allow to measurement of L-DOPA content easily and accurately from a small amount of plant tissue and contribute to understanding biosynthesis, catabolism, and transport of L-DOPA.
LEAFY (LFY), which encodes a plant-specific transcription factor, plays an important role in the transition from vegetative to reproductive development. Ectopic expression of LFY has been reported to induce dwarfism and early flowering in some model plants. In order to examine the possibility of using LFY for molecular breeding of ornamental plants, we produced and characterized transgenic plants ectopically expressing LFY from Arabidopsis thaliana (AtLFY) in the liliaceous ornamental plant Tricyrtis sp. Nine independent transgenic plants have been obtained, and all of them exhibited dwarf phenotypes compared with the vector control. These transgenic plants could be classified into three types according to the degree of dwarfism: one showed an extreamly dwarf phenotype with smaller leaves (Type I); two showed moderately dwarf phenotypes (Type II); and six showed slightly dwarf phenotypes (Type III). All of Type I, Type II and Type III transgenic plants produced flower buds 1–3 weeks earlier than the vector control. Vector control and Type III transgenic plants produced 1–4 apical flower buds, whereas Type I and Type II transgenic plants produced only a single apical flower bud. Type I and Type II transgenic plants often produced non-fully-opened flowers. Quantitative real-time reverse transcription-polymerase chain reaction analysis showed that the AtLFY expression level generally correlated with the degree of dwarfism. These results indicate that morphological alterations observed in the transgenic plants was induced by ectopic expression of AtLFY. Lower levels of ectopic expression of LFY may be valuable for producing dwarf and early flowering ornamental plants.
Brassinosteroid (BR) is a phytohormone that acts as important regulator of plant growth. To identify novel transcription factors that may be involved in unknown mechanisms of BR signaling, we screened the chimeric repressor expressing plants (CRES-T), in which transcription factors were converted into chimeric repressors by the fusion of SRDX plant-specific repression domain, to identify those that affect the expression of BR inducible genes. Here, we identified a homeobox-leucine zipper type transcription factor, BRASSINOSTEROID-RELATED-HOMEOBOX 3 (BHB3), of which a chimeric repressor expressing plants (BHB3-sx) significantly downregulated the expression of BAS1 and SAUR-AC1 that are BR inducible genes. Interestingly, ectopic expression of BHB3 (BHB3-ox) also repressed the BR inducible genes and shorten hypocotyl that would be similar to a BR-deficient phenotype. Interestingly, both BHB3-sx and BHB3-ox showed pale green phenotype, in which the expression of genes related photosynthesis and chlorophyll contents were significantly decreased. We found that BHB3 contains three motifs similar to the conserved EAR-repression domain, suggesting that BHB3 may act as a transcriptional repressor. These results indicate that BHB3 might play an important role not only to the BR signaling but also the regulation of greenings.