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.

Potential Markers for Selecting Self-Eliminating Apple Genotypes.

The heavy blooming of apple trees results in the inefficient usage of energy and nutritional material, and additional expenditure on fruitlet thinning is required to maintain fruit quality. A possible solution for controlling the fruit load on trees is the development of new cultivars that self-eliminate excess fruitlets, thus controlling yield. The aim of our study was to identify biological differences in apple cultivars in terms of blooming intensity and fruitlet load self-regulation. In total, 19 apple cultivars were studied in the years 2015-2017. The dynamics of fruitlet self-elimination, seed development in fruitlets and fruits, photosynthetic parameters, carbohydrates, and plant hormones were evaluated. We established that apple cultivars self-eliminating a small number of fruitlets need a lower number of well-developed seeds in fruit, and their number of leaves and area per fruit on a bearing branch are larger, compared to cultivars, self-eliminating large numbers of fruitlets. A higher carbohydrate amount in the leaves may be related to smaller fruitlet self-elimination. The amount of auxin and a high indole-3-acetic acid/zeatin ratio between leaves of cultivar groups with heavy blooming were higher than in cultivars with moderate blooming. A lower amount of abscisic acid was found in heavy-blooming cultivars during drought stress. All these parameters may be used as markers for the selection of different apple genotypes that self-eliminate fruitlets.

Characteristics of Fragaria vesca Yield Parameters and Anthocyanin Accumulation under Water Deficit Stress.

Plants exposed to drought stress conditions often increase the synthesis of anthocyanins-natural plant pigments and antioxidants. However, water deficit (WD) often causes significant yield loss. The aim of our study was to evaluate the productivity as well as the anthocyanin content and composition of berries from cultivated Fragaria vesca "Rojan" and hybrid No. 17 plants (seedlings) grown under WD. The plants were grown in an unheated greenhouse and fully irrigated (control) or irrigated at 50% and 25%. The number of berries per plant and the berry weight were evaluated every 4 days. The anthocyanin content and composition of berries were evaluated with the same periodicity using HPLC. The effect of WD on the yield parameters of two evaluated F. vesca genotypes differed depending on the harvest time. The cumulative yield of plants under WD was not less than that of the control plants for 20-24 days after the start of the experiment. Additionally, berries accumulated 36-56% (1.5-2.3 times, depending on the harvest time) more anthocyanins compared with fully irrigated plants. Our data show that slight or moderate WD at a stable air temperature of about 20 °C positively affected the biosynthesis of anthocyanins and the yield of F. vesca berries.

Isolation and Analysis of Anthocyanin Pathway Genes from Ribes Genus Reveals MYB Gene with Potent Anthocyanin-Inducing Capabilities.

Horticultural crops of the Ribes genus are valued for their anthocyanin-rich fruits, but until now, there were no data about the genes and regulation of their flavonoid pathway. In this study, the coding sequences of flavonoid pathway enzymes and their putative regulators MYB10, bHLH3 and WD40 were isolated, and their expression analyzed in fruits with varying anthocyanin levels from different cultivars of four species belonging to the Ribes genus. Transcription levels of anthocyanin synthesis enzymes and the regulatory gene RrMYB10 correlated with fruit coloration and anthocyanin quantities of different Ribes cultivars. Regulatory genes were tested for the ability to modulate anthocyanin biosynthesis during transient expression in the leaves of two Nicotiana species and to activate Prunus avium promoters of late anthocyanin biosynthesis genes in N. tabacum. Functional tests showed a strong capability of RrMyb10 to induce anthocyanin synthesis in a heterologous system, even without the concurrent expression of any heterologous bHLH, whereas RrbHLH3 enhanced MYB-induced anthocyanin synthesis. Data obtained in this work facilitate further analysis of the anthocyanin synthesis pathway in key Ribes species, and potent anthocyanin inducer RrMyb10 can be used to manipulate anthocyanin expression in heterologous systems.

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.

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.