Enhanced Carbonylation of Photosynthetic and Glycolytic Proteins in Antibiotic Timentin-Treated Tobacco In Vitro Shoot Culture.

Antibiotics are used in plant in vitro tissue culture to eliminate microbial contamination or for selection in genetic transformation. Antibiotic timentin has a relatively low cytotoxic effect on plant tissue culture; however, it could induce an enduring growth-inhibiting effect in tobacco in vitro shoot culture that persists after tissue transfer to a medium without antibiotic. The effect is associated with an increase in oxidative stress injury in plant tissues. In this study, we assessed changes of reactive oxygen species accumulation, protein expression, and oxidative protein modification response associated with enduring timentin treatment-induced growth suppression in tobacco (Nicotiana tabacum L.) in vitro shoot culture. The study revealed a gradual 1.7 and 1.9-fold increase in superoxide (O2•-) content at the later phase of the propagation cycle for treatment control (TC) and post-antibiotic treatment (PA) shoots; however, the O2•- accumulation pattern was different. For PA shoots, the increase in O2•- concentration occurred several days earlier, resulting in 1.2 to 1.4-fold higher O2•- concentration compared to TC during the period following the first week of cultivation. Although no protein expression differences were detectable between the TC and PA shoots by two-dimensional electrophoresis, the increase in O2•- concentration in PA shoots was associated with a 1.5-fold increase in protein carbonyl modification content after one week of cultivation, and protein carbonylation analysis revealed differential modification of 26 proteoforms involved in the biological processes of photosynthesis and glycolysis. The results imply that the timentin treatment-induced oxidative stress might be implicated in nontranslational cellular redox balance regulation, accelerates the development of senescence of the shoot culture, and contributes to the shoot growth-suppressing effect of antibiotic treatment.

Enduring Effect of Antibiotic Timentin Treatment on Tobacco In Vitro Shoot Growth and Microbiome Diversity.

Plant in vitro cultures initiated from surface-sterilized explants often harbor complex microbial communities. Antibiotics are commonly used to decontaminate plant tissue culture or during genetic transformation; however, the effect of antibiotic treatment on the diversity of indigenous microbial populations and the consequences on the performance of tissue culture is not completely understood. Therefore, the aim of this study was to assess the effect of antibiotic treatment on the growth and stress level of tobacco (Nicotiana tabacum L.) shoots in vitro as well as the composition of the plant-associated microbiome. The study revealed that shoot cultivation on a medium supplemented with 250 mg L-1 timentin resulted in 29 ± 4% reduced biomass accumulation and a 1.2-1.6-fold higher level of oxidative stress injury compared to the control samples. Moreover, the growth properties of shoots were only partially restored after transfer to a medium without the antibiotic. Microbiome analysis of the shoot samples using multivariable region-based 16S rRNA gene sequencing revealed a diverse microbial community in the control tobacco shoots, including 59 bacterial families; however, it was largely dominated by Mycobacteriaceae. Antibiotic treatment resulted in a decline in microbial diversity (the number of families was reduced 4.5-fold) and increased domination by the Mycobacteriaceae family. These results imply that the diversity of the plant-associated microbiome might represent a significant factor contributing to the efficient propagation of in vitro tissue culture.

Stimulation of Nicotiana tabacum L. In Vitro Shoot Growth by Endophytic Bacillus cereus Group Bacteria.

In vitro plant tissue cultures face various unfavorable conditions, such as mechanical damage, osmotic shock, and phytohormone imbalance, which can be detrimental to culture viability, growth efficiency, and genetic stability. Recent studies have revealed a presence of diverse endophytic bacteria, suggesting that engineering of the endophytic microbiome of in vitro plant tissues has the potential to improve their acclimatization and growth. Therefore, the aim of this study was to identify cultivated tobacco (Nicotiana tabacum L.) endophytic bacteria isolates that are capable of promoting the biomass accumulation of in vitro tobacco shoots. Forty-five endophytic bacteria isolates were obtained from greenhouse-grown tobacco plant leaves and were assigned to seven Bacillus spp. and one Pseudomonas sp. based on 16S rRNA or genome sequence data. To evaluate the bacterial effect on in vitro plant growth, tobacco shoots were inoculated with 22 isolates selected from distinct taxonomic groups. Four isolates of Bacillus cereus group species B. toyonensis, B. wiedmannii and B. mycoides promoted shoot growth by 11-21%. Furthermore, a contrasting effect on shoot growth was found among several isolates of the same species, suggesting the presence of strain-specific interaction with the plant host. Comparative analysis of genome assemblies was performed on the two closely related B. toyonensis isolates with contrasting plant growth-modulating properties. This revealed distinct structures of the genomic regions, including a putative enzyme cluster involved in the biosynthesis of linear azol(in)e-containing peptides and polysaccharides. However, the function of these clusters and their significance in plant-promoting activity remains elusive, and the observed contrasting effects on shoot growth are more likely to result from genomic sequence variations leading to differences in metabolic or gene expression activity. The Bacillus spp. isolates with shoot-growth-promoting properties have a potential application in improving the growth of plant tissue cultures in vitro.

Cold Plasma Treatment of Sunflower Seeds Modulates Plant-Associated Microbiome and Stimulates Root and Lateral Organ Growth.

Cold atmospheric pressure (CP) plasma irradiation of seeds has been shown to promote plant growth, but the molecular basis of this phenomenon is poorly understood. In our study, optimum irradiation of common sunflower seeds using a dielectric barrier discharge CP device stimulated growth of sunflower lateral organs and roots by 9-14% compared to the control. Metagenomic analysis revealed that the structure of plant-associated bacterial assembly was greatly modified upon CP treatment and could be attributed to the antimicrobial effect of CP-generated reactive species. The treatment resulted in the domination of spore forming Mycobacterium sp. in the above-ground tissues of the seedlings. While the overall bacterial diversity in the roots was barely affected, the CP-induced shift in microbial composition is the likely basis for the observed seedling root growth stimulation and the long-term effect on lateral organ growth and could be mediated by increase in water uptake and/or direct root signaling. Low amplitude protein abundance differences were detected in the roots of the emerging seedlings that are characteristic to low intensity stress stimuli response and could be linked to the changes in plant-associated microbiome upon CP treatment.

Treatment of Common Sunflower (Helianthus annus L.) Seeds with Radio-frequency Electromagnetic Field and Cold Plasma Induces Changes in Seed Phytohormone Balance, Seedling Development and Leaf Protein Expression.

Treatment of plant seeds with electromagnetic fields or non-thermal plasmas aims to take advantage of plant functional plasticity towards stimulation of plant agricultural performance. In this study, the effects of pre-sowing seed treatment using 200 Pa vacuum (7 min), 5.28 MHz radio-frequency cold plasma (CP -2, 5, and 7 min) and electromagnetic field (EMF -5, 10, 15 min) on seed germination kinetics, content of phytohormones, morphometric parameters of seedlings and leaf proteome were assessed. CP 7 min and EMF 15 min treatments caused 19-24% faster germination in vitro; germination in the substrate was accelerated by vacuum (9%) and EMF 15 min (17%). The stressors did not change the seed germination percentage, with exception of EMF 5 min treatment that caused a decrease by 7.5%. Meanwhile both CP 7 min and EMF 15 min treatments stimulated germination, but the EMF treatment resulted in higher weight of leaves. Stressor-specific changes in phytohormone balance were detected in seeds: vacuum treatment decreased zeatin amount by 39%; CP treatments substantially increased gibberellin content, but other effects strongly varied with the treatment duration; the abscisic acid content was reduced by 55-60% after the EMF treatment. Analysis of the proteome showed that short exposure of seeds to the EMF or CP induced a similar long-term effect on gene expression in leaves, mostly stimulating expression of proteins involved in photosynthetic processes and their regulation.

Endophytic Bacillus and Pseudomonas spp. Modulate Apple Shoot Growth, Cellular Redox Balance, and Protein Expression Under in Vitro Conditions.

Interactions between host plants and endophytic microorganisms play an important role in plant responses to pathogens and environmental stresses and have potential applications for plant stress management under in vitro conditions. We assessed the effect of endophytic bacteria on the growth and proliferation of domestic apple cv. Gala shoots in vitro. Further, a model apple cell suspension system was used to examine molecular events and protein expression patterns at an early stage of plant-endophyte interaction. Among the seven strains used in the study, Bacillus spp. strains Da_1, Da_4, and Da_5 and the Pseudomonas fluorescens strain Ga_1 promoted shoot growth and auxiliary shoot proliferation. In contrast, Bacillus sp. strain Oa_4, P. fluorescens strain Ga_3 and P. orientalis strain G_12 inhibited shoot development. In the cell suspension, the effects of the association between endophytic bacteria and plant cells were specific to each strain. Modulation of the cellular redox balance was monitored in the apple cells using a 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) probe, and strain-specific effects were observed that correlated with the in vitro shoot development results. Proteomic analysis revealed differences in protein expressions in apple cells co-cultivated with different Bacillus spp. strains that had contrasting effects on cellular redox balance and shoot development. The Bacillus sp. strain Da_4, which enhanced shoot development and oxidation of H2DCFDA, induced differential expression of proteins that are mainly involved in the defense response and regulation of oxidative stress. Meanwhile, treatment with Bacillus sp. strain Oa_4 led to strong upregulation of PLAT1, HSC70-1 and several other proteins involved in protein metabolism and cell development. Taken together, the results suggest that different cell signaling and response events at the early stage of the plant-endophyte interaction may be important for strain-dependent regulation of cellular redox balance and development of shoot phenotype.