To investigate the gene function of radish (Raphanus sativus L.), several attempts have been made to generate genetically transformed radish. However, no efficient and relatively simple method for ...the genetic transformation of radish has been developed to date. In this study, we established an Agrobacterium-mediated genetic transformation method using the hypocotyl-derived explants of radish cultivar “Pirabikku”. Primarily based on the Brassica transformation procedure, we optimized it for radish transformation. Using this system, the transformation efficiency of radish hypocotyl explants by Agrobacterium tumefaciens strain GV3101 harboring pIG121-Hm was 13.3%. The copy number of transfer DNA integrated into the genome was either one or two in the four independent transgenic plants. Two of the four plants exhibited male sterility and did not produce self-pollinated seeds. Examination of the expression of the β-glucuronidase (GUS) gene in T1 plants from fertile T0 plants showed that the GUS genes were inherited. The improvement in the genetic transformation in this study might pave the way for accelerated molecular breeding and genetic analysis of radish.
Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as ...hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the mossPhyscomitrella patenswith reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated “ARK” (for “ABA and abiotic stress-responsive Raf-like kinase”) plays an essential role in the activation of SnRK2. ARK encoded by a single gene inP. patensbelongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms.
Fusarium wilt race 1, caused by
Fusarium oxysporum
f. sp.
lactucae
, is one of the main diseases of lettuce in tropical and subtropical regions, including under summer conditions in Japan. Resistant ...lettuce cultivars have shown symptoms on occasion, which have led breeding programs to focus on the improvement of resistance phenotypes to race 1. Disease assays using Fusarium wilt race 1 were performed, and the lettuce cultivar ‘VI185’ was categorized as highly resistant, which was validated by subsequent population analyses. F
2
individuals obtained from a cross between ‘VI185’ (highly resistant) and ‘ShinanoGreen’ (partially resistant) were analyzed, and an F
3
population was subjected to an infection assay using the race 1 pathogen. ddRAD-seq analysis revealed two significant QTLs on LG7 (
qFOL7.1
) and LG8 (
qFOL8.1
) that accounted for the race 1 resistance phenotype. Furthermore, the loci that conferred elevated resistance originated from ‘VI185’ at both QTLs. Our results provide a foundation for understanding the QTLs for resistance to Fusarium wilt race 1 and provide evidence that genetic diversity is available for enhancing the current resistance in existing lettuce cultivars.
Understanding the processes that regulate plant sink formation and development at the molecular level will contribute to the areas of crop breeding, food production and plant evolutionary studies. We ...report the annotation and analysis of the draft genome sequence of the radish Raphanus sativus var. hortensis (long and thick root radish) and transcriptome analysis during root development. Based on the hybrid assembly approach of next-generation sequencing, a total of 383 Mb (N50 scaffold: 138.17 kb) of sequences of the radish genome was constructed containing 54,357 genes. Syntenic and phylogenetic analyses indicated that divergence between Raphanus and Brassica coincide with the time of whole genome triplication (WGT), suggesting that WGT triggered diversification of Brassiceae crop plants. Further transcriptome analysis showed that the gene functions and pathways related to carbohydrate metabolism were prominently activated in thickening roots, particularly in cell proliferating tissues. Notably, the expression levels of sucrose synthase 1 (SUS1) were correlated with root thickening rates. We also identified the genes involved in pungency synthesis and their transcription factors.
Abscisic acid (ABA) controls seed dormancy and stomatal closure through binding to the intracellular receptor Pyrabactin resistance1 (Pyr1)/Pyr1-like/regulatory components of ABA receptors ...(PYR/PYL/RCAR) in angiosperms. Genes encoding PYR/PYL/RCAR are thought to have arisen in the ancestor of embryophytes, but the roles of the genes in nonvascular plants have not been determined. In the liverwort
, ABA reduces growth and enhances desiccation tolerance through increasing accumulation of intracellular sugars and various transcripts such as those of
(
)-like genes. In this study, we analyzed a gene designated Mp
which is closely related to
/
/
of angiosperms, in transgenic liverworts. Transgenic lines overexpressing Mp
-
showed ABA-hypersensitive growth with enhanced desiccation tolerance, whereas Mp
generated by CRISPR-Cas9-mediated genome editing showed ABA-insensitive growth with reduced desiccation tolerance. Transcriptome analysis indicated that Mp
is a major regulator of abiotic stress-associated genes, including all 35 ABA-induced
-like genes. Furthermore, these transgenic plants showed altered responses to extracellular Suc, suggesting that ABA and PYR/PYL/RCAR function in sugar responses. The results presented here reveal an important role of PYR/PYL/RCAR in the ABA response, which was likely acquired in the common ancestor of land plants. The results also indicate the archetypal role of ABA and its receptor in sugar response and accumulation processes for vegetative desiccation tolerance in bryophytes.
Abscisic acid (ABA) is not a plant-specific compound but one found in organisms across kingdoms from bacteria to animals, suggesting that it is a ubiquitous and versatile substance that can modulate ...physiological functions of various organisms. Recent studies have shown that plants developed an elegant system for ABA sensing and early signal transduction mechanisms to modulate responses to environmental stresses for survival in terrestrial conditions. ABA-induced increase in stress tolerance has been reported not only in vascular plants but also in non-vascular bryophytes. Since bryophytes are the key group of organisms in the context of plant evolution, clarification of their ABA-dependent processes is important for understanding evolutionary adaptation of land plants. Molecular approaches using
Physcomitrella patens
have revealed that ABA plays a role in dehydration stress tolerance in mosses, which comprise a major group of bryophytes. Furthermore, we recently reported that signaling machinery for ABA responses is also conserved in liverworts, representing the most basal members of extant land plant lineage. Conservation of the mechanism for ABA sensing and responses in angiosperms and basal land plants suggests that acquisition of this mechanism for stress tolerance in vegetative tissues was one of the critical evolutionary events for adaptation to the land. This review describes the role of ABA in basal land plants as well as non-land plant organisms and further elaborates on recent progress in molecular studies of model bryophytes by comparative and functional genomic approaches.
The SNF1-related protein kinase 2 (SnRK2) family includes key regulators of osmostress and abscisic acid (ABA) responses in angiosperms and can be classified into three subclasses. Subclass III ...SnRK2s act in the ABA response while ABA-nonresponsive subclass I SnRK2s are regulated through osmostress. Here we report that an ancient subclass III SnRK2-based signalling module including ABA and an upstream Raf-like kinase (ARK) exclusively protects the moss
from drought. Subclass III SnRK2s from both Arabidopsis and from the semiterrestrial alga
, which contains all the components of ABA signalling except ABA receptors, complement
mutants, whereas Arabidopsis subclass I SnRK2 cannot. We propose that the earliest land plants developed the ABA/ARK/subclass III SnRK2 signalling module by recruiting ABA to regulate a pre-existing dehydration response and that subsequently a novel subclass I SnRK2 system evolved in vascular plants conferring osmostress protection independently from the ancient system.
Vegetative desiccation tolerance is common in bryophytes, although this character has been lost in most vascular plants. The moss Physcomitrella patens survives complete desiccation if treated with ...abscisic acid (ABA). Group A protein phosphatases type 2C (PP2C) are negative regulators of abscisic acid signalling. Here we show that the elimination of Group A PP2C is sufficient to ensure P. patens survival to full desiccation, without ABA treatment, although its growth is severely hindered. Microarray analysis shows that the Group A PP2C-regulated genes exclusively overlap with genes exhibiting a high level of ABA induction. Group A PP2C disruption weakly affects ABA-activated kinase activity, indicating Group A PP2C action downstream of these kinases in the moss. We propose that Group A PP2C emerged in land plants to repress desiccation tolerance mechanisms, possibly facilitating plants propagation on land, whereas ABA releases the intrinsic desiccation tolerance from Group A PP2C regulation.
To identify genes associated with genic male sterility (GMS) that are useful for hybrid breeding in
Brassica
crops, we analyzed the “ms77B” strain, which is a spontaneous turnip GMS mutant derived ...from a cv. 77B population. Previously, we reported that the GMS phenotype of ms77B is caused by a single gene with sterility as the recessive trait. To identify the causative gene of GMS in ms77B, we performed bulked segregant and transcriptome analyses with high-throughput sequencing and identified
BrEMS1
as the most likely candidate.
BrEMS1
is a single-copy gene homologous to
Arabidopsis thaliana EMS1/EXS
, which is essential for anther development. The
BrEMS1
gene of ms77B has a long insertion (1460 bp) in the coding sequence, and RNA sequencing revealed that the gene is not transcribed beyond the insertion site. The region that is not transcribed because of the insertion contains sequences that encode multiple leucine-rich repeats and a catalytic kinase domain. Furthermore, genotyping of an F2 population revealed a complete correlation between the insertion and the male sterility phenotype. Together, our results indicate that the male sterility phenotype of ms77B is due to a null mutation of
BrEMS1
, which is essential for anther development.
Objective
To determine the safety and efficacy of the one‐surgeon basketing technique for stone extraction during flexible ureteroscopy when carried out by novice surgeons under instructor guidance.
...Methods
We retrospectively compared perioperative results, complications and the “stone‐free” rate (defined as ≤2 mm fragments on kidney–ureter–bladder imaging 1 month after flexible ureteroscopy) between experienced (group A, n = 50) and novice (group B, n = 50) surgeons using the one‐surgeon basketing technique.
Results
Baseline patients’ characteristics were similar between the two groups. There were no significant differences between groups A and B in operative time (mean 76 min vs 85 min, P = 0.46), stone‐free rate (98% vs 92%, P = 0.36), postoperative fever events (4% vs 4%, P = 1.00) and postoperative hospital stay (24 h vs 24 h, P = 1.00). Clavien–Dindo grade IIIa complications (ureter injury) were only observed in two cases (4%) in group B.
Conclusions
The one‐surgeon basketing technique for the extraction of stone fragments during flexible ureteroscopy might be safely and effectively carried out by surgeons with no prior experience under proper guidance.
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