Plant U-box E3 ligases PUB20 and PUB21 negatively regulate pattern-triggered immunity in Arabidopsis.

KEY MESSAGE: Plant U-box E3 ligases PUB20 and PUB21 are flg22-triggered signaling components and negatively regulate immune responses. Plant U-box proteins (PUBs) constitute a class of E3 ligases that are associated with various stress responses. Among the class IV PUBs featuring C-terminal Armadillo (ARM) repeats, PUB20 and PUB21 are closely related homologs. Here, we show that both PUB20 and PUB21 negatively regulate innate immunity in plants. Loss of PUB20 and PUB21 function leads to enhanced resistance to surface inoculation with the virulent bacterium Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). However, the resistance levels remain unaffected after infiltration inoculation, suggesting that PUB20 and PUB21 primarily function during the early defense stages. The enhanced resistance to Pst DC3000 in PUB mutant plants (pub20-1, pub21-1, and pub20-1/pub21-1) correlates with extensive flg22-triggered reactive oxygen production, strong MPK3 activation, and enhanced transcriptional activation of early immune response genes. Additionally, PUB mutant plants (except pub21-1) exhibit constitutive stomatal closure after Pst DC3000 inoculation, implying the significant role of PUB20 in stomatal immunity. Comparative analyses of flg22 responses between PUB mutants and wild-type plants reveals that the robust activation of the pattern-induced immune responses may enhance resistance against Pst DC3000. Notably, the hypersensitivity responses triggered by RPM1/avrRpm1 and RPS2/avrRpt2 are independent of PUB20 and PUB21. These results suggest that PUB20 and PUB21 knockout mutations affect bacterial invasion, likely during the early stages, acting as negative regulators of plant immunity.

Genome-Wide Association Study for Agronomic Traits in Gamma-Ray-Derived Mutant Kenaf (Hibiscus cannabinus L.).

Kenaf (Hibiscus cannabinus L.), in the Malvaceae family, is an important crop for not only fiber production, but also various other industrial materials. We performed phylogenetic analysis and a genome-wide association study (GWAS) of seven agronomic traits: days to flowering, plant height, fresh weight, dry weight, flower color, stem color, and leaf shape, using 96 kenaf genotypes, including gamma-irradiation-derived mutant lines. Genotypes were determined by genotyping-by-sequencing (GBS) and a total of 49,241 single-nucleotide polymorphisms (SNPs) were used in the analysis. Days to flowering, plant height, fresh weight, and dry weight were positively correlated with each other, and stem color was also correlated with fresh weight and dry weight. The phylogenetic analysis divided the 96 lines into nine related groups within two independent groups, and the GWAS analysis detected a total of 49 SNPs for days to flowering, plant height, fresh weight, dry weight, flower color, stem color, and leaf shape with -log10(P) ≥ 4, of which 22 were located in genic regions. The detected SNPs were located in genes with homology ranging from 45% to 96% to plants of the Malvaceae and Betulaceae, and these genes were found to be involved in plant growth and development via various pathways. Our identification of SNP markers related to agronomic traits is expected to help improve the quality of selective breeding programs for kenaf.

A GBS-based genetic linkage map and quantitative trait loci (QTL) associated with resistance to Xanthomonas campestris pv. campestris race 1 identified in Brassica oleracea.

The production of Brassica oleracea, an important vegetable crop, is severely affected by black rot disease caused by the bacterial pathogen Xanthomonas campestris pv. campestris. Resistance to race 1, the most virulent and widespread race in B. oleracea, is under quantitative control; therefore, identifying the genes and genetic markers associated with resistance is crucial for developing resistant cultivars. Quantitative trait locus (QTL) analysis of resistance in the F2 population developed by crossing the resistant parent BR155 with the susceptible parent SC31 was performed. Sequence GBS approach was used to develop a genetic linkage map. The map contained 7,940 single nucleotide polymorphism markers consisting of nine linkage groups spanning 675.64 cM with an average marker distance of 0.66 cM. The F2:3 population (N = 126) was evaluated for resistance to black rot disease in summer (2020), fall (2020), and spring (2021). QTL analysis, using a genetic map and phenotyping data, identified seven QTLs with LOD values between 2.10 and 4.27. The major QTL, qCaBR1, was an area of overlap between the two QTLs identified in the 2nd and 3rd trials located at C06. Among the genes located in the major QTL interval, 96 genes had annotation results, and eight were found to respond to biotic stimuli. We compared the expression patterns of eight candidate genes in susceptible (SC31) and resistant (BR155) lines using qRT-PCR and observed their early and transient increases or suppression in response to Xanthomonas campestris pv. campestris inoculation. These results support the involvement of the eight candidate genes in black rot resistance. The findings of this study will contribute towards marker-assisted selection, additionally the functional analysis of candidate genes may elucidate the molecular mechanisms underlying black rot resistance in B. oleracea.

RING-Type E3 Ubiquitin Ligases AtRDUF1 and AtRDUF2 Positively Regulate the Expression of PR1 Gene and Pattern-Triggered Immunity.

The importance of E3 ubiquitin ligases from different families for plant immune signaling has been confirmed. Plant RING-type E3 ubiquitin ligases are members of the E3 ligase superfamily and have been shown to play positive or negative roles during the regulation of various steps of plant immunity. Here, we present Arabidopsis RING-type E3 ubiquitin ligases AtRDUF1 and AtRDUF2 which act as positive regulators of flg22- and SA-mediated defense signaling. Expression of AtRDUF1 and AtRDUF2 is induced by pathogen-associated molecular patterns (PAMPs) and pathogens. The atrduf1 and atrduf2 mutants displayed weakened responses when triggered by PAMPs. Immune responses, including oxidative burst, mitogen-activated protein kinase (MAPK) activity, and transcriptional activation of marker genes, were attenuated in the atrduf1 and atrduf2 mutants. The suppressed activation of PTI responses also resulted in enhanced susceptibility to bacterial pathogens. Interestingly, atrduf1 and atrduf2 mutants showed defects in SA-mediated or pathogen-mediated PR1 expression; however, avirulent Pseudomonas syringae pv. tomato DC3000-induced cell death was unaffected. Our findings suggest that AtRDUF1 and AtRDUF2 are not just PTI-positive regulators but are also involved in SA-mediated PR1 gene expression, which is important for resistance to P. syringae.

Jasmonate regulates plant resistance to Pectobacterium brasiliense by inducing indole glucosinolate biosynthesis.

Pectobacterium brasiliense (P. brasiliense) is a necrotrophic bacterium that causes the soft rot disease in Brassica rapa. However, the mechanisms underlying plant immune responses against necrotrophic bacterial pathogens with a broad host range are still not well understood. Using a flg22-triggered seedling growth inhibition (SGI) assay with 455 Brassica rapa inbred lines, we selected six B. rapa flagellin-insensitive lines (Brfin2-7) and three B. rapa flagellin-sensitive lines (Brfs1-3). Brfin lines showed compromised flg22-induced immune responses (oxidative burst, mitogen-activated protein kinase (MAPK) activation, and seedling growth inhibition) compared to the control line R-o-18; nevertheless, they were resistant to P. brasiliense. To explain this, we analyzed the phytohormone content and found that most Brfin lines had higher P. brasiliense-induced jasmonic acid (JA) than Brfs lines. Moreover, MeJA pretreatment enhanced the resistance of B. rapa to P. brasiliense. To explain the correlation between the resistance of Brfin lines to P. brasiliense and activated JA signaling, we analyzed pathogen-induced glucosinolate (GS) content in B. rapa. Notably, in Brfin7, the neoglucobrassicin (NGBS) content among indole glucosinolates (IGS) was significantly higher than that in Brfs2 following P. brasiliense inoculation, and genes involved in IGSs biosynthesis were also highly expressed. Furthermore, almost all Brfin lines with high JA levels and resistance to P. brasiliense had higher P. brasiliense-induced NGBS levels than Brfs lines. Thus, our results show that activated JA-mediated signaling attenuates flg22-triggered immunity but enhances resistance to P. brasiliense by inducing indole glucosinolate biosynthesis in Brassica rapa. This study provides novel insights into the role of JA-mediated defense against necrotrophic bacterial pathogens within a broad host range.

Comparative Study on Phenolic Compounds and Antioxidant Activities of Hop (Humulus lupulus L.) Strobile Extracts.

In this study, we investigated the phenolic compounds in hop strobile extracts and evaluated their antioxidant property using DPPH and ABTS assay. The total phenolic compound (TPC) and total flavonoid compound (TFC) estimated in two different solvent extracts considerably varied depending on the extraction solvent. The most abundant phenolic compound in hop strobile was humulones (α-acid) with levels ranging from 50.44 to 193.25 µg/g. El Dorado accession revealed higher antioxidant activity in ethanol extracts (DPPH: IC50 124.3 µg/mL; ABTS: IC50 95.4 µg/mL) when compared with that of the other accessions. Correlations between DPPH (IC50) scavenging TFC in ethanol extract (TFC_E, -0.941), and TPC_E (-0.901), and between ABTS (IC50) scavenging TFC_E (-0.853), and TPC_E (-0.826), were statistically significant at p < 0.01 level, whereas no significant correlation was observed between antioxidant activities, TPC and TFC in water extract. This study is the first to report that variations in the level of phenolic contents and antioxidant activity of various hop cultivars depended on the type of extraction solvent used and the cultivation regions. These results could provide valuable information on developing hop products.

Mutagenic Effect of Proton Beams Characterized by Phenotypic Analysis and Whole Genome Sequencing in Arabidopsis.

Protons may have contributed to the evolution of plants as a major component of cosmic-rays and also have been used for mutagenesis in plants. Although the mutagenic effect of protons has been well-characterized in animals, no comprehensive phenotypic and genomic analyses has been reported in plants. Here, we investigated the phenotypes and whole genome sequences of Arabidopsis M2 lines derived by irradiation with proton beams and gamma-rays, to determine unique characteristics of proton beams in mutagenesis. We found that mutation frequency was dependent on the irradiation doses of both proton beams and gamma-rays. On the basis of the relationship between survival and mutation rates, we hypothesized that there may be a mutation rate threshold for survived individuals after irradiation. There were no significant differences between the total mutation rates in groups derived using proton beam or gamma-ray irradiation at doses that had similar impacts on survival rate. However, proton beam irradiation resulted in a broader mutant phenotype spectrum than gamma-ray irradiation, and proton beams generated more DNA structural variations (SVs) than gamma-rays. The most frequent SV was inversion. Most of the inversion junctions contained sequences with microhomology and were associated with the deletion of only a few nucleotides, which implies that preferential use of microhomology in non-homologous end joining was likely to be responsible for the SVs. These results show that protons, as particles with low linear energy transfer (LET), have unique characteristics in mutagenesis that partially overlap with those of low-LET gamma-rays and high-LET heavy ions in different respects.

Metabolite Profiling and Dipeptidyl Peptidase IV Inhibitory Activity of Coreopsis Cultivars in Different Mutations.

Coreopsis species have been developed to produce cultivars of various floral colors and sizes and are also used in traditional medicine. To identify and evaluate mutant cultivars of C. rosea and C. verticillata, their phytochemical profiles were systematically characterized using ultra-performance liquid chromatography time-of-flight mass spectrometry, and their anti-diabetic effects were evaluated using the dipeptidyl peptidase (DPP)-IV inhibitor screening assay. Forty compounds were tentatively identified. This study is the first to provide comprehensive chemical information on the anti-diabetic effect of C. rosea and C. verticillata. All 32 methanol extracts of Coreopsis cultivars inhibited DPP-IV activity in a concentration-dependent manner (IC50 values: 34.01-158.83 µg/mL). Thirteen compounds presented as potential markers for distinction among the 32 Coreopsis cultivars via principal component analysis and orthogonal partial least squares discriminant analysis. Therefore, these bio-chemometric models can be useful in distinguishing cultivars as potential dietary supplements for functional plants.

Suggested doses of proton ions and gamma-rays for mutation induction in 20 plant species.

PURPOSE: Proton ions are expected to be used as a discriminative radiation source to induce different kinds of mutations than those produced by γ-rays and carbon ions; however, there is little systemic information about radiosensitivity in plants. MATERIALS AND METHODS: We analyzed the LD30, LD50, and RD50 values in response to proton ions and γ-rays using 20 plant species. Plant seeds were irradiated, and growth responses were measured one month after planting, except for cymbidium, for which in vitro rhizomes were irradiated. The rhizomes were analyzed at six and nine months after subculturing. RESULTS: Resistance to proton ions and γ-rays was observed in Chinese cabbage, watermelon, and melon, while Japanese atractylodes, naked barley, and lentil were susceptible. Plants belonging to the Brassicaceae and Cucurbitaceae families were highly resistant to radiation, and plants belonging to the Compositae and Poaceae families were highly susceptible. In addition, plants with genome sizes greater than 8,000 Mbp were highly sensitive to radiation, but there was no clear relationship between radiosensitivity and genome size in plants with genomes smaller than 2,500 Mbp. CONCLUSIONS: The biological effectiveness of proton ions was greater than that of γ-rays in 16 plant species, indicating that they could be used as a discriminative radiation source to induce mutations compared with γ-rays.

A Comparison of the Transcriptomes of Cowpeas in Response to Two Different Ionizing Radiations.

In this study, gene expression changes in cowpea plants irradiated by two different types of radiation: proton-beams and gamma-rays were investigated. Seeds of the Okdang cultivar were exposed to 100, 200, and 300 Gy of gamma-rays and proton-beams. In transcriptome analysis, the 32, 75, and 69 differentially expressed genes (DEGs) at each dose of gamma-ray irradiation compared with that of the control were identified. A total of eight genes were commonly up-regulated for all gamma-ray doses. However, there were no down-regulated genes. In contrast, 168, 434, and 387 DEGs were identified for each dose of proton-beam irradiation compared with that of the control. A total of 61 DEGs were commonly up-regulated for all proton-beam doses. As a result of GO and KEGG analysis, the ranks of functional categories according to the number of DEGs were not the same in both treatments and were more diverse in terms of pathways in the proton-beam treatments than gamma-ray treatments. The number of genes related to defense, photosynthesis, reactive oxygen species (ROS), plant hormones, and transcription factors (TF) that were up-/down-regulated was higher in the proton beam treatment than that in gamma ray treatment. Proton-beam treatment had a distinct mutation spectrum and gene expression pattern compared to that of gamma-ray treatment. These results provide important information on the mechanism for gene regulation in response to two ionizing radiations in cowpeas.

Comparative Analysis of Volatile Compounds of Gamma-Irradiated Mutants of Rose (Rosa hybrida).

Roses are one of the most important floricultural crops, and their essential oils have long been used for cosmetics and aromatherapy. We investigated the volatile compound compositions of 12 flower-color mutant variants and their original cultivars. Twelve rose mutant genotypes were developed by treatment with 70 Gy of 60Co gamma irradiation of six commercial rose cultivars. Essential oils from the flowers of the 18 genotypes were analyzed by gas chromatography-mass spectrometry. Seventy-seven volatile compounds were detected, which were categorized into six classes: Aliphatic hydrocarbons, aliphatic alcohols, aliphatic ester, aromatic compounds, terpene alcohols, and others. Aliphatic (hydrocarbons, alcohols, and esters) compounds were abundant categories in all rose flowers. The CR-S2 mutant had the highest terpene alcohols and oil content. Three (CR-S1, CR-S3, and CR-S4) mutant genotypes showed higher ester contents than their original cultivar. Nonacosane, 2-methylhexacosane, and 2-methyltricosane were major volatile compounds among all genotypes. Hierarchical cluster analysis (HCA) of the rose genotypes gave four groups according to grouping among the 77 volatile compounds. In addition, the principal component analysis (PCA) model was successfully applied to distinguish most attractive rose lines. These findings will be useful for the selection of rose genotypes with improved volatile compounds.

Phenolic Compounds in Extracts of Hibiscus acetosella (Cranberry Hibiscus) and Their Antioxidant and Antibacterial Properties.

Hibiscus species are rich in phenolic compounds and have been traditionally used for improving human health through their bioactive activities. The present study investigated the phenolic compounds of leaf extracts from 18 different H. acetosella accessions and evaluated their biofunctional properties, focusing on antioxidant and antibacterial activity. The most abundant phenolic compound in H. acetosella was caffeic acid, with levels ranging from 14.95 to 42.93 mg/100 g. The antioxidant activity measured by the ABTS assay allowed the accessions to be classified into two groups: a high activity group with red leaf varieties (74.71-84.02%) and a relatively low activity group with green leaf varieties (57.47-65.94%). The antioxidant activity was significantly correlated with TAC (0.933), Dp3-Sam (0.932), Dp3-Glu (0.924), and Cy3-Sam (0.913) contents (p < 0.001). The H. acetosella phenolic extracts exhibited antibacterial activity against two bacteria, with zones of inhibition between 12.00 and 13.67 mm (Staphylococcus aureus), and 10.67 and 13.33 mm (Pseudomonas aeruginosa). All accessions exhibited a basal antibacterial activity level (12 mm) against the Gram-positive S. aureus, with PI500758 and PI500764 exhibiting increased antibacterial activity (13.67 mm), but they exhibited a more dynamic antibacterial activity level against the Gram-negative P. aeruginosa.

Upregulation of the MYB2 Transcription Factor Is Associated with Increased Accumulation of Anthocyanin in the Leaves of Dendrobium bigibbum.

Orchids with colorful leaves and flowers have significant ornamental value. Here, we used γ-irradiation-based mutagenesis to produce a Dendrobium bigibbum mutant that developed purple instead of the normal green leaves. RNA sequencing of the mutant plant identified 2513 differentially expressed genes, including 1870 up- and 706 downregulated genes. The purple leaf color of mutant leaves was associated with increased expression of genes that encoded key biosynthetic enzymes in the anthocyanin biosynthetic pathway. In addition, the mutant leaves also showed increased expression of several families of transcription factors including the MYB2 gene. Transient overexpression of D. biggibumMYB2 in Nicotiana benthamiana was associated with increased expression of endogenous anthocyanin biosynthesis genes. Interestingly, transient overexpression of orthologous MYB2 genes from other orchids did not upregulate expression of endogenous anthocyanin biosynthesis genes. Together, these results suggest that the purple coloration of D. biggibum leaves is at least associated with increased expression of the MYB2 gene, and the MYB2 orthologs from orchids likely function differently, regardless of their high level of similarity.

Composition and Antioxidant Activities of Volatile Organic Compounds in Radiation-Bred Coreopsis Cultivars.

Coreopsis is a flowering plant belonging to the Asteraceae family. It is an ornamental plant native to the Americas, Asia and Oceania and its flower is used as a raw material for tea and food manufacture in China. In this study, new cultivars of C. rosea ("golden ring") were developed via radiation-induced mutation of the original cultivar, "pumpkin pie". The chemical composition and antioxidant activities of flowers belonging to three different Coreopsis cultivars were evaluated: "golden ring", "pumpkin pie" and "snow chrysanthemum" (coreopsis tea; C. tinctoria). The volatile compounds were characterized via gas chromatography-mass spectrometry (GC-MS) and 50-59 oils representing 95.3-96.8% of the total volatile compounds in these flower materials were identified. "Golden ring" contained a high amount of fatty acids (38.13%), while "pumpkin pie" and "snow chrysanthemum" teas were rich in aliphatic amides (43.01%) and esters (67.22%), respectively. The antioxidant activities of the volatile oils of these cultivars were evaluated using 1,1-diphenyl-2-picrylhydraxyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assays. The volatile extract of "golden ring" showed higher antioxidant activities compared with the extracts of the other cultivars. Therefore, "golden ring" can be used for further development as a raw material for tea manufacture or as a dietary supplement.

Characterization of Gene Isoforms Related to Cellulose and Lignin Biosynthesis in Kenaf (Hibiscus cannabinus L.) Mutant.

Kenaf is a source of fiber and a bioenergy crop that is considered to be a third world crop. Recently, a new kenaf cultivar, "Jangdae," was developed by gamma irradiation. It exhibited distinguishable characteristics such as higher biomass, higher seed yield, and earlier flowering than the wild type. We sequenced and analyzed the transcriptome of apical leaf and stem using Pacific Biosciences single-molecule long-read isoform sequencing platform. De novo assembly yielded 26,822 full-length transcripts with a total length of 59 Mbp. Sequence similarity against protein sequence allowed the functional annotation of 11,370 unigenes. Among them, 10,100 unigenes were assigned gene ontology terms, the majority of which were associated with the metabolic and cellular process. The Kyoto encyclopedia of genes and genomes (KEGG) analysis mapped 8875 of the annotated unigenes to 149 metabolic pathways. We also identified the majority of putative genes involved in cellulose and lignin-biosynthesis. We further evaluated the expression pattern in eight gene families involved in lignin-biosynthesis at different growth stages. In this study, appropriate biotechnological approaches using the information obtained for these putative genes will help to modify the desirable content traits in mutants. The transcriptome data can be used as a reference dataset and provide a resource for molecular genetic studies in kenaf.

Dark/Light Treatments Followed by γ-Irradiation Increase the Frequency of Leaf-Color Mutants in Cymbidium.

Radiation randomly induces chromosomal mutations in plants. However, it was recently found that the frequency of flower-color mutants could be specifically increased by upregulating anthocyanin pathway gene expression before radiation treatments. The mechanisms of chlorophyll biosynthesis and degradation are active areas of plant study because chlorophyll metabolism is closely connected to photosynthesis. In this study, we determined the dark/light treatment conditions that resulted in upregulation of the expression levels of six chlorophyll pathway genes, uroporphyrinogen III synthase (HEMD), uroporphyrinogen III decarboxylase (HEME2), NADPH-protochlorophyllide oxidoreductase (POR) A (PORA), chlorophyll synthase (CHLG), chlorophyllase (CLH2), and red chlorophyll catabolite reductase (RCCR), and measured their effects on the γ-irradiation-induced frequencies of leaf-color mutants in two Cymbidium cultivars. To degrade chlorophyll in rhizomes, 60-75 days of dark treatment were required. To upregulate the expressions of chlorophyll pathway genes, 10 days of light treatment appeared to be optimal. Dark/light treatments followed by γ-irradiation increased chlorophyll-related leaf mutants by 1.4- to 2.0-fold compared with γ-ray treatment alone. Dark/light treatments combined with γ-irradiation increased the frequency of leaf-color mutants in Cymbidium, which supports the wider implementation of a plant breeding methodology that increases the mutation frequency of a target trait by controlling the expression of target trait-related genes.

Molecular Characterization and Identification of Calnexin 1 As a Radiation Biomarker from Tradescantia BNL4430.

Calnexin (CNX) is an integral membrane protein that functions as a chaperone in the endoplasmic reticulum for the correct folding of proteins under stress conditions, rendering organisms tolerant under adverse conditions. Studies have investigated the cytogenetic effects of gamma irradiation (Ɣ-IR) on plants, but information on the molecular response under Ɣ-IR remains limited. Previously, we constructed a cDNA library of an irradiation-sensitive bioindicator plant, Tradescantia BNL4430 (T-4430) under Ɣ-IR, in which the Calnexin-1 gene was highly upregulated at 50 mGy treatment. TrCNX1 encodes a 61.4 kDa protein with conserved signature motifs similar to already reported CNX1s. TrCNX1 expression was evaluated by semiquantitative reverse transcriptase PCR and quantitative real-time PCR and was ubiquitously expressed in various tissues and highly upregulated in flower petals under 50 mGy Ɣ-IR stress. The protective function of TrCNX1 was investigated by overexpression of TrCNX1 in an Escherichia coli BL21(DE3) heterologous system. Using plate assay, we showed that TrCNX1 increased the viability of E. coli transformants under both UV-B and Ɣ-IR compared with the control, demonstrating that TrCNX1 functions under irradiation stress. TrCNX1 may enhance irradiation stress tolerance in crops and act as a radio marker gene to monitor the effects of radiation.

Transcriptome analysis to identify candidate genes associated with the yellow-leaf phenotype of a Cymbidium mutant generated by γ-irradiation.

Leaf color is an important agronomic trait in flowering plants, including orchids. However, factors underlying leaf phenotypes in plants remain largely unclear. A mutant displaying yellow leaves was obtained by the γ-ray-based mutagenesis of a Cymbidium orchid and characterized using RNA sequencing. A total of 144,918 unigenes obtained from over 25 million reads were assigned to 22 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes database. In addition, gene ontology was used to classify the predicted functions of transcripts into 73 functional groups. The RNA sequencing analysis identified 2,267 differentially expressed genes between wild-type and mutant Cymbidium sp. Genes involved in the chlorophyll biosynthesis and degradation, as well as ion transport, were identified and assayed for their expression levels in wild-type and mutant plants using quantitative real-time profiling. No critical expression changes were detected in genes involved in chlorophyll biosynthesis. In contrast, seven genes involved in ion transport, including two metal ion transporters, were down-regulated, and chlorophyllase 2, associated with chlorophyll degradation, was up-regulated. Together, these results suggest that alterations in chlorophyll metabolism and/or ion transport might contribute to leaf color in Cymbidium orchids.

Mitotypes Based on Structural Variation of Mitochondrial Genomes Imply Relationships With Morphological Phenotypes and Cytoplasmic Male Sterility in Peppers.

Plant mitochondrial genomes characteristically contain extensive structural variation that can be used to define and classify cytoplasm types. We developed markers based on structural variation in the mitochondrial genomes of fertile and cytoplasmic male sterility (CMS) pepper lines and applied them to a panel of Capsicum accessions. We designed a total of 20 sequence characterized amplified region (SCAR) markers based on DNA rearrangement junctions or cytoplasm-specific segments that did not show high similarity to any nuclear mitochondrial DNA segments. We used those markers to classify the mitotypes of 96 C. annuum accessions into 15 groups. Precise genotyping of other Capsicum species (C. frutescens, C. chinense, and C. baccatum) was hampered because of various stoichiometric levels of marker amplicons. We developed a multiplex PCR system based on four of the markers that efficiently classified the C. annuum accessions into five mitotype groups. Close relationships between specific mitotypes and morphological phenotypes implied that diversification or domestication of C. annuum germplasm might have been accompanied by structural rearrangements of mitochondrial DNA or the selection of germplasms with specific mitotypes. Meanwhile, CMS lines shared the same amplification profile of markers with another mitotype. Further analysis using mitochondrial DNA (mtDNA) markers based on single-nucleotide polymorphisms (SNPs) or insertions and deletions (InDels) and CMS-specific open reading frames (orfs) provided new information about the origin of the CMS-specific mitotype and evaluation of candidates for CMS-associated genes, respectively.

Biological effects of three types of ionizing radiation on creeping bentgrass.

Purpose: Gamma-rays and carbon ions are frequently used for mutation breeding in diverse plant species, whereas proton ions have been rarely used for this purpose. This study assessed the potential of proton ions for plant mutation breeding. Materials and methods: We compared the effects of radiation on creeping bentgrass seeds with γ-rays, proton ions, and carbon ions on seed germination, plant growth parameters, and DNA fragmentation. Results and conclusions: The lethal dose 50 (LD50) doses based on seed germinability were 115.9 Gy (γ-rays), 225.1 Gy (proton ions), and 57.7 Gy (carbon ions). Threshold doses for survival were 150 Gy (γ-rays), 150 Gy (proton ions), and 25 Gy (carbon ions). Suppression of plant growth was displayed at 100 Gy (γ-rays), 25 Gy (proton ions), and 25 Gy (carbon ions). Similar patterns of decreasing head DNA percentage were observed for γ-rays and proton ions. Carbon ions induced the lowest frequency of DNA fragmentation. The biological effects of the ionizing radiation types on creeping bentgrass are summarizable as follows: germination, carbon ions (C)>γ-rays (G)>proton ions (P); survival, C > P = G; growth, C ≥ P > G; DNA fragmentation, G ≥ P > C. These results indicate that proton ions are useful as a physical mutagen in plant mutation breeding.