Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation.

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.

TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts.

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.

Pedobacter endophyticus sp. nov., an endophytic bacterium isolated from Carex pumila.

An aerobic, Gram-stain-negative, weak-motile, short-rod-shaped bacterial strain, designated JBR3-12T, was isolated from halophyte Carex pumila plants, and its taxonomic position was investigated by using a polyphasic taxonomic approach. The strain produced a pink pigment on tryptic soy agar and grew optimally at 25 °C, pH 8 and in the presence of 3 % (w/v) NaCl. Results of phylogenetic analysis based on 16S rRNA gene sequences showed that strain JBR3-12T formed a lineage within the genus Pedobacter and was most closely related to Pedobacter sandarakinus DS-27T (98.0 %) and Pedobacter agri PB92T (97.6 %). The DNA G+C content of the genome was 41.3 mol%; the whole genome length was 5 426 070 bp. The major fatty acids of JBR3-12T were iso-C15 : 0, summed feature 3 (comprising C16 : 1 ω6c and/or C16 : 1 ω7c) and iso-C17 : 0 3-OH. The predominant polar lipid was phosphatidylethanolamine. The predominant quinone was menaquinone-7. Based on its phenotypic, phylogenetic and genotypic features, strain JBR3-12T is proposed to represent a novel species of the genus Pedobacter, for which the name is Pedobacter endophyticus sp. nov. The type strain is JBR3-12T (=KCTC 82363T=NBRC 114901T).

Development of a Rapid Selection System for Salt-Resistant Mutants of Nicotiana benthamiana through Protoplast Culture after Gamma Irradiation.

We aimed to develop a novel technology capable of rapidly selecting mutant plant cell lines. Salt resistance was chosen as a rapid selection trait that is easily applicable to protoplast-derived cell colonies. Mesophyll protoplasts were cultured in a medium supplemented with 0, 50, 100, 150, 200, 250, and 300 mM NaCl. At NaCl concentrations ≥ 100 mM, cell colony formation was strongly inhibited after 4 weeks of culture. Tobacco protoplasts irradiated with 0, 50, 100, 200, and 400 Gy were then cultured to investigate the effects of radiation intensity on cell division. The optimal radiation intensity was 50 Gy. To develop salt-resistant tobacco mutant plants, protoplasts irradiated with 50 Gy were cultured in a medium containing 100 mM NaCl. The efficiency of cell colony formation from these protoplasts was approximately 0.002%. A salt-resistant mutant callus was selected and proliferated in the same medium and then transferred to a shoot inducing medium for adventitious shoot formation. The obtained shoots were then cultured in a medium supplemented with 200 mM NaCl and developed into normal plantlets. This rapid selection technology for generating salt-resistant tobacco mutants will be useful for the development of crop varieties resistant to environmental stresses.

The Effect of Sodium Butyrate on Adventitious Shoot Formation Varies among the Plant Species and the Explant Types.

Histone acetylation plays an important role in plant growth and development. Here, we investigated the effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on adventitious shoot formation from protoplast-derived calli and cotyledon explants of tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum). The frequency of adventitious shoot formation from protoplast-derived calli was higher in shoot induction medium (SIM) containing NaB than in the control. However, the frequency of adventitious shoot formation from cotyledon explants of tobacco under the 0.1 mM NaB treatment was similar to that in the control, but it decreased with increasing NaB concentration. Unlike in tobacco, NaB decreased adventitious shoot formation in tomato explants in a concentration-dependent manner, but it did not have any effect on adventitious shoot formation in calli. NaB inhibited or delayed the expression of D-type cyclin (CYCD3-1) and shoot-regeneration regulatory gene WUSCHEL (WUS) in cotyledon explants of tobacco and tomato. However, compared to that in control SIM, the expression of WUS was promoted more rapidly in tobacco calli cultured in NaB-containing SIM, but the expression of CYCD3-1 was inhibited. In conclusion, the effect of NaB on adventitious shoot formation and expression of CYCD3-1 and WUS genes depended on the plant species and whether the effects were tested on explants or protoplast-derived calli.

Reclassification of genus Izhakiella into the family Erwiniaceae based on phylogenetic and genomic analyses.

The genus Izhakiella was established and designated as a member of the family Enterobacteriaceae in 2016. Although the taxonomical classification of most members in this family has been relatively resolved after two reclassifications in 2016 and 2017, the classification of the genus Izhakiella remains ambiguous. In this study, a polyphasic approach was used to provide evidence supporting the fact that the genus Izhakiella should no longer be considered a member of Enterobacteriaceae and proposes its reclassification into the family Erwiniaceae. The phylogenetic tree of type species in the families Enterobacteriaceae and Erwiniaceae based on the sequences of the 16S rRNA gene, rpoB housekeeping gene, and the whole-genome comprising the 92 core genes revealed that the genus Izhakiella forms a phylogenetic lineage within the family Erwiniaceae. The average nucleotide identity (ANI) value of the type species with genus Izhakiella was found to be higher for the family Erwiniaceae than that for the family Enterobacteriaceae. Notably, 12 conserved signature indels (CSIs) that are exclusively shared among the Erwiniaceae clade members were found in the type strains of the genus Izhakiella. Based on these analyses, this study suggests the reclassification of I. capsodis and I. australiensis into the family Erwiniaceae.

Saccharibacillus brassicae sp. nov., an endophytic bacterium isolated from kimchi cabbage (Brassica rapa subsp. pekinensis) seeds.

Strain ATSA2T was isolated from surface-sterilized kimchi cabbage (Brassica rapa subsp. pekinensis) seeds and represents a novel bacterium based on the polyphasic taxonomic approach. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain ATSA2T formed a lineage within genus Saccharibacillus and was most closely to Saccharibacillus deserti WLG055T (98.1%) and Saccharibacillus qing-shengii H6T (97.9%). The whole-genome of ATSA2T comprised a 5,619,468 bp of circular chromosome with 58.4% G + C content. The DNA-DNA relatedness values between strain ATSA2T and its closely related type strains S. deserti WLJ055 and S. qingshengii H6T were 26.0% and 24.0%, respectively. Multiple gene clusters associated with plant growth promotion activities (stress response, nitrogen and phosphorus metabolism, and auxin biosynthesis) were annotated in the genome. Strain ATSA2T was Gram-positive, endospore-forming, facultatively anaerobic, and rod-shaped It grew at 15-37°C (optimum 25°C), pH 6.0-10.0 (optimum pH 8.0), and in the presence of 0-5% (w/v) NaCl (optimum 1%). The major cellular fatty acids (> 10%) of strain ATSA2T were anteiso-C15:0 and C16:0. MK-7 was the major isoprenoid quinone. The major polar lipids present were diphosphatidylglycerol, phosphatidylglycerol, and three unknown glycolipids. Based on its phylogenetic, genomic, phenotypic, and chemotaxo-nomic features, strain ATSA2T is proposed to represent a novel species of genus Saccharibacillus, for which the name is Saccharibacillus brassicae sp. nov. The type strain is ATSA2T (KCTC 43072T = CCTCC AB 2019223T).

Neobacillus endophyticus sp. nov., an endophytic bacterium isolated from Selaginella involvens roots.

A Gram-stain-positive, facultatively anaerobic, rod-shaped, endospore-forming, oxidase-positive, and catalase-negative strain designated as BRMEA1T was isolated from the surface-sterilized Selaginella involvens roots. Growth of strain BRMEA1T was found to occur at pH 6.0-8.0 (optimum, pH 7.0), 15-50 °C (optimum, 25-30 °C) and in the absence of NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BRMEA1T formed a lineage within the genus Neobacillus (family Bacillaceae) and showed the highest sequence similarity to Neobacillus drentensis DSM 15600T (98.3 %) and Neobacillus fumarioli KCTC 13885T (98.2 %), and less than 98.2 % 16S rRNA gene sequence similarity to the other members of the genus Neobacillus. Whole-genome analysis of strain BRMEA1T comprised a circular chromosome (5 632 809 bp in size) with 38.5 mol% G+C content. Digital DNA-DNA hybridization analyses revealed that strain BRMEA1T showed 20.5 and 22.0% genomic DNA relatedness with the closest species, N. drentensis DSM 15600T and N. fumarioli KCTC 13885T, respectively. The whole-genome sequence of strain BRMEA1T showed the presence of 11 specific conserved signature indels for the genus Neobacillus. The major cellular fatty acids (>10 %) of strain BRMEA1T were found to be iso-C15 : 0 and anteiso-C15 : 0, while the major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Polyphasic analysis results revealed that BRMEA1T represents a novel species of the genus Neobacillus, with the proposed name Neobacillus endophyticus sp. nov. The type strain is BRMEA1T (=KCTC 43208T=CCTCC AB 2020071T).

An Alcohol Dehydrogenase Gene from Synechocystis sp. Confers Salt Tolerance in Transgenic Tobacco.

Synechocystis salt-responsive gene 1 (sysr1) was engineered for expression in higher plants, and gene construction was stably incorporated into tobacco plants. We investigated the role of Sysr1 [a member of the alcohol dehydrogenase (ADH) superfamily] by examining the salt tolerance of sysr1-overexpressing (sysr1-OX) tobacco plants using quantitative real-time polymerase chain reactions, gas chromatography-mass spectrometry, and bioassays. The sysr1-OX plants exhibited considerably increased ADH activity and tolerance to salt stress conditions. Additionally, the expression levels of several stress-responsive genes were upregulated. Moreover, airborne signals from salt-stressed sysr1-OX plants triggered salinity tolerance in neighboring wild-type (WT) plants. Therefore, Sysr1 enhanced the interconversion of aldehydes to alcohols, and this occurrence might affect the quality of green leaf volatiles (GLVs) in sysr1-OX plants. Actually, the Z-3-hexenol level was approximately twofold higher in sysr1-OX plants than in WT plants within 1-2 h of wounding. Furthermore, analyses of WT plants treated with vaporized GLVs indicated that Z-3-hexenol was a stronger inducer of stress-related gene expression and salt tolerance than E-2-hexenal. The results of the study suggested that increased C6 alcohol (Z-3-hexenol) induced the expression of resistance genes, thereby enhancing salt tolerance of transgenic plants. Our results revealed a role for ADH in salinity stress responses, and the results provided a genetic engineering strategy that could improve the salt tolerance of crops.

Regulation of the major vacuolar Ca²âº transporter genes, by intercellular Ca²âº concentration and abiotic stresses, in tip-burn resistant Brassica oleracea.

Calcium is an essential plant macronutrient that has unique structural and signaling roles related to tip-burn disorder in Brassica spp. crops. For two types of cabbage inbred lines, tip-burn susceptible and resistant, we measured and compared major macronutrient cations, including Ca(2+), in leaves. In both lines, Ca(2+), Mg(2+), Na(+), and K(+), accumulated more in leaf base than in leaf apex. Ca(2+) and K(+) were >2 times more abundant in the tip-burn resistant line, while Na(+) was higher in the susceptible line. Ca(2+) differences between the two lines resulted from differential accumulation of calcium into cell vacuoles. We profiled major vacuolar Ca(2+) transporters, in both cabbage lines, by growth time and intercellular Ca(2+) concentration. Expression pattern of several Ca(2+) transporter genes differed between tip-burn susceptible and resistant lines by growth time points. We also identified promoter regions of the major Ca(2+) vacuole transporter genes, CAX1, ACA4, and ACA11, which displayed hormonal, light and defense-related cis-acting regulatory elements. Finally, transporter genes in the two cabbage lines responded differently to abiotic stresses, demonstrating diversity in gene regulation among orthologous genes.