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.

Differential Chloroplast Proteomics of Temperature Adaptation in Apple (Malus x domestica Borkh.) Microshoots.

Temperature stress is one of the most common external factors that plants have to adapt to. Accordingly, plants have developed several adaptation mechanisms to deal with temperature stress. Chloroplasts are one of the organelles that are responsible for the sensing of the temperature signal and triggering a response. Here, chloroplasts are purified from low temperature (4° C), control (22° C) and high temperature (30° C) grown Malus x domestica microshoots. The purity of the chloroplast fractions is evaluated by marker proteins, as well as by using in silico subcellular localization predictions. The proteins are digested using filter-aided sample processing and analyzed using nano-LC MS/MS. 733 proteins are observed corresponding to published Malus x domestica gene models and 16 chloroplast genome -encoded proteins in the chloroplast preparates. In ANOVA, 56 proteins are found to be significantly differentially abundant (p < 0.01) between chloroplasts isolated from plants grown in different conditions. The differentially abundant proteins are involved in protein digestion, cytoskeleton structure, cellular redox state and photosynthesis, or have protective functions. Additionally, a putative chloroplastic aquaporin is observed. Data are available via ProteomeXchange with identifier PXD014212.

Patterns of low temperature induced accumulation of dehydrins in Rosaceae crops-Evidence for post-translational modification in apple.

Important crop plants of Rosaceae family are often damaged during winter due to the lack of acclimation and cold hardiness. One of the cellular responses of plants to cold stress is the accumulation of dehydrin proteins. We studied the expression of dehydrins in several Rosaceae species during low temperature treatment in vitro. Microshoots of Pyrus communis, Malus×domestica, Fragaria vesca, Fragaria×ananassa, Prunus cerasus and Prunus avium cultivars were grown in low temperature conditions. Genotype -specific accumulation of dehydrins was detected by immunoblot analysis of the extracted proteins. Untargeted difference gel electrophoresis of Malus x domestica microshoots revealed an extensive accumulation of three dehydrins. In a protein phosphatase assay, MdDHN2 and MdDHN4, but not MdDHN6 proteins were found to be extensively phosphorylated. In terms of the amount of protein synthesized, dehydrins are a major protein-level adaptation mechanism to low temperature in M. x domestica. In addition to dehydrins, the induction of proteins involved in the response for oxidative stress were observed. Additionally, a Xero2 -like dehydrin of F. vesca was detected by difference gel electrophoresis and identified by nano LC-MS/MS.