Helen I. Rostovtseva

High-menthol mint varieties are traditionally grown in regions characterized by a continental climate, characterized by pronounced seasonality with cold winters and hot, usually dry summers. Climate change means that this plant is increasingly grown in extreme conditions, where primary and secondary soil salinization is the norm. However, by its origin, mint is a typical glycophyte, that is, a plant that is very sensitive to the effects of salt, suspending the gain of biomass under conditions of increasing salinity and limited available water. We demonstrate an attempt to desensitize and increase tolerance of peppermint to high-salinity conditions corresponding to 100 mM and 150 mM NaCl. To maintain productivity under these uncharacteristic conditions, it was necessary to modify plant metabolism in such a way as to effectively utilize the non-specific potential inherent in plant genetics for resistance to stress factors characteristic of other abiotic stresses, such as cold or drought, which cause oxidative and osmotic damage to metabolism and growth. By obtaining peppermint plants transgenic for Fe-SOD and codA genes, we intended to induce mild internal stress, inducing protective mechanisms, without damaging the systems that ensure resistance due to specific anti-stress systems. An in vitro experiment confirmed that with this strategy, despite a significant decrease in growth and biomass, a decrease in the pool of photosynthetic pigments, and changes in the morphology of epidermal cells, the parameters recording plant viability were significantly higher in transgenic lines than in the original peppermint genotype. We also confirmed the absence of significant growth inhibition in transgenic lines under optimal conditions. This method of inducing non-specific resistance will be quite effective, but only if the salt concentration is subsequently reduced, since it is difficult to achieve a fundamental shift, manifested in the complete removal of the effects of stress in such a highly sensitive glycophyte as mint.

The influence of crown illumination on leaf damage of horse chestnut species (Aesculus hippocastanum L., Aesculus glabra Willd, Aesculus flava Aiton, Aesculus pavia L., Aesculus × carnea Hayne, Aesculus parviflora Walter, Aesculus chinensis Bunge) affected by ohrid leaf miner (Cameraria ohridella Deschka & Dymić) was studied using some accessions from the arboretum botanical tree collection. A. hippocastanum, A. glabra, A. flava had the lowest chl a content in the foliage on the sunlit side of the crown, while in A. pavia, A. parviflora and A. chinensis this indicator was the highest. The chl a content in the leaves of A. hippocastanum and A. flava under shaded conditions was 1.3 and 2.4 times higher than in the sunlit part, while in A. pavia, A. parviflora and A. chinensis the chl a content on the shaded side was 1.2, 1.6 and 1.3 times lower. The quantitative content of chl b in the sunlit part of the crown in A. hippocastanum and A. flava was significantly higher than in the other species. Moreover, while A. flava and A. parviflora had the highest chl b content in the foliage of the shaded part of the crown, A. glabra and A. × carnea had the lowest. Similarly, differences in proline levels were found in the leaves of different horse chestnut species on the sunny side of the crown. Higher proline levels in less infested species were identified. Water content imbalances due to feeding by leaf miners were most characteristic of the severely affected species. Chlorophyll fluorescence determination revealed high photochemical activity with an effective defense system in resistant species, while non-resistant species exhibited weak defense mechanisms in both sunlight and shade. To assess horse chestnut species the hyperspectral analysis indices (DSWI and SIPI) were also successfully applied. Changes in chl a and chl b content, proline levels, and leaf water-holding properties can be used to assess the resistance of horse chestnut species using classical physiological and biochemical methods. Hyperspectral analysis indices (DSWI and SIPI) can also be successfully applied.

Main conclusion Moderate concentrations of Na⁺ ions in the growth medium have positive effects on growth and performance characteristics of the C₄ euhalophyte Suaeda altissima, optimizing functioning of both, stomata and photosynthetic apparatus. Abstract Effects of salinity on the morphology, ion relations, and gas exchange of the leaves in the euhalophyte Suaeda altissima (L.) Pall. were investigated with emphasis on the guard and epidermal cells. The presence of NaCl in the nutrient solution (NS) at both growth-stimulating (250 mM) and growth-inhibiting (750 mM) concentrations resulted in increased leaf succulence, net photosynthetic rate (P_n), and instantaneous water use efficiency (WUE_i), and reduced stomatal conductance (g_s), stomatal density on the leaf surface, and transpiration rate (E). X-ray microanalyses revealed Na and Cl accumulation in the guard and epidermal cells of leaves under salinity conditions. However, Na and Cl contents differed by not much in plants grown at 250 and 750 mM NaCl, indicating a mechanism preventing accumulation of Na⁺ and Cl⁻ ions in cells paving leaf surface at high NaCl concentrations. Examination of g_s and E as functions of CO₂ concentration in the leaf gas exchange chamber revealed better ability to regulate these parameters in 250 mM NaCl-grown plants than in 750 mM NaCl-grown or control plants. The study of P_n dependent on CO₂ concentration in leaf intercellular space revealed direct stimulating effect of NaCl on photosynthesis. We hypothesize that S. altissima, a species having anatomy and ultrastructure features of C₄ plants, improves its performance characteristics under saline conditions, optimizing not only the functioning of the stomata complex but also the process of CO₂ assimilation, including the C₄ fixation pathway.

Salt-tolerance improvement of tomatoes is largely a task of modern selection and plant molecular genetics because of cultivation on dry and irrigated lands under salt stress. To reveal the salt resistance gene, we need quantitative real-time polymerase chain reaction (qRT-PCR) normalization through reference genes analysis. Sometimes, housekeeping gene expression changes in response to various stress factors, especially salinity. In this manuscript, we evaluated expression changes of elongation factor 1α X53043.1 (EF1α), actin BT013707.1 (ACT), ubiquitin NM_001346406.1 (UBI), nuclear transcript factor XM_026030313.2 (NFT-Y), β-tubulin NM_001247878.2 (TUB), glyceraldehyde-3 phosphate dehydrogenase NM_001247874.2 (GAPDH), phosphatase 2A catalytic subunit NM_001247587.2 (PP2a), and phosphoglycerate kinase XM_004243920.4 (PGK) in salt-sensitive Solanum lycopersicum L. YaLF line and salt tolerance Rekordsmen cv. under 100 mM NaCl. We also suggested potential correlations between relative water content (RWC), ion accumulation, and reference gene expression in tomato genotypes with contrasting salinity tolerance. We used geNorm, NormFinder, BestKeeper, ∆Ct, and RefFinder algorithms to establish a set of the most reliable tomato candidate genes. The most stable genes for YaLF tomatoes were ACT, UBI, TUB, and PP2a. Despite differences in ranks, the NFT-Y was present in Rekordsmen’s stable set.

Cloning of the SaSLAH3 gene (OQ271227.1), an AtSLAH3 ortholog, member of the slow anion channels family (SLAC/SLAH), has been carried out from the euhalophyte Suaeda altissima. Under hydroponic conditions, these species related to Amaranthaceae is capable to complete life cycle on nutrient solutions containing up to 1 M NaCl. Cloning of SaSLAH3 was performed based on a putative SaSLAH3 similarity with SLAH3 homologs from the related Suaeda glauca and Suaeda fruticose through assembled transcriptomes previously. Membrane topology, analyzed by in silico approach, the 3-D structure, presence of conservative phenylalanine-bearing motives and as well as SaSLAH3 MEME-generated sequence motif patterns, all of these features were found typical for the SLAC/SLAH channels and confirmed predicted SaSLAH3 is a new member of this family. Phylogenetic relationship analysis demonstrated the evolutionary SLAC-like proteins divergence into three distinct groups with location in SLAH2/3 clade. Expression profile investigation by quantitative real-time PCR revealed roots and leaves SaSLAH3 transcript level increment upon NaCl elevation with sufficient nitrate supply and nitrate deficiency. Probably, SaSLAH3 plays an important role in chloride loading enhancing into root xylem and, thereafter in chloride delivery to shoot under salinity. Another SaSLAH3 physiological role may be cytoplasm nitrate concentration regulation to ensure proper S. altissima nitrogen nutrition to strong competition between nitrate and chloride ions.

Water Use Efficiency (WUE) regulation plays a key role in the plant response to global climate changes such as warming (New et al. in Philos Trans R Soc A Math Phys Eng Sci 369:6–19, 2011). Nowadays enhanced plant drought tolerance has become a priority trait in current crop improvement efforts. To gain insight into water requirement regulation of the drought-resistant halophyte Sueda altissima we determined WUE_i under various light conditions. RBL combination (50% red, 50% blue) contributed to twice stomatal conductance/transpiration decreases and a more pronounced response to green/far-red supplementation compared to glycophytic species. The most rapid WUE_i improvement was observed under far-red light conditions (0% R₆₆₀ + 100% FR). The critical role in halophyte growth and development played optimization of red-blue-green light balance in the spectrum. Besides, we also investigated moderate or strong (r > 0.5, r > 0.8) correlations between stomatal conductance (g _s ), transpiration (E), WUE_i and consequently SaSLAH3 expression. Moreover, this report identified 14- and 20-fold higher than control expression increases in responce to R/FR used together. These results suggested that SLAH3 channels have to be involved in phytochrome-controlled pathways of stomatal aperture regulation.

Plasma membrane-associated proteins of NAR2 family are involved in the uptake of nitrate by plant roots as partners of NRT2 family proteins, which carry out high- affinity nitrate transport across membranes of plant cells. In this study, the coding sequence of NAR2 family gene, SaNAR2.2, was cloned from the euhalophyte Suaeda altissima . This species is able to withstand high concentrations of NaCl, up to 750 mM, in the nutrient medium. The protein SaNAR2.2, of 196 amino acids in size, with a calculated molecular weight of 21.7 kDa, has been characterized in terms of structure and phylogeny. According to the prediction of cellular localization and protein topology, SaNAR2.2 is a resident of plasma membrane. It has one transmembrane segment at the C-terminal part and a small C-end located in cytoplasm. The large N-terminal part of SaNAR2.2 protein is located in the extracellular space. On the phylogenetic tree of NAR2 family proteins, the protein from S. altissima lies in a clade with the proteins of NAR2.2 subfamily from other plants of Amaranthaceae family. We detected the interaction between SaNAR2.2 and putative partner proteins SaNRT2.1 and SaNRT2.5, which are the members of high-affinity nitrate transporter family NRT2 from S. altissima , using the bimolecular fluorescence complementation (BiFC) assay in Nicotiana benthamiana leaves. The expression of SaNAR2.2 gene in organs of S. altissima plants grown at low (0.5 mM) or high (15 mM) nitrate concentrations in the nutrient medium, without NaCl or in the presence of increasing salt concentrations, was analyzed using RT-qPCR method. It has been found that SaNAR2.2 is expressed nearly exclusively in roots of S. altissima . As the concentrations of NaCl in the nutrient medium increased (up to 500 mM), the expression of SaNAR2.2 decreased at high external nitrate concentrations and significantly increased, up to ten-fold, at low nitrate concentrations; this points to the significant role of SaNAR2.2 protein under salinity conditions as a participant of high-affinity nitrate transport system. SaNAR2.2 is the first components of a high affinity nitrate transport system characterized in a terrestrial halophytic plant.

Two genes of nitrate transporters SaNRT2.1 and SaNRT2.5, putative orthologs of high-affinity nitrate transporter genes AtNRT2.1 and AtNRT2.5 from Arabidopsis thaliana, were cloned from the euhalophyte Suaeda altissima. Phylogenetic bioinformatic analysis demonstrated that the proteins SaNRT2.1 and SaNRT2.5 exhibited higher levels of homology to the corresponding proteins from the plants of family Amaranthaceae; the similarity of amino acid sequences between proteins SaNRT2.1 and SaNRT2.5 was lower (54%). Both SaNRT2.1 and SaNRT2.5 are integral membrane proteins forming 12 transmembrane helices as predicted by topological modeling. An attempt to demonstrate nitrate transporting activity of SaNRT2.1 or SaNRT2.5 by heterologous expression of the genes in the yeast Hansenula (Ogataea) polymorpha mutant strain Δynt1 lacking the only yeast nitrate transporter was not successful. The expression patterns of SaNRT2.1 and SaNRT2.5 were studied in S. altissima plants that were grown in hydroponics under either low (0.5 mM) or high (15 mM) nitrate and salinity from 0 to 750 mM NaCl. The growth of the plants was strongly inhibited by low nitrogen supply while stimulated by NaCl; it peaked at 250 mM NaCl for high nitrate and at 500 mM NaCl for low nitrate. Under low nitrate supply, nitrate contents in S. altissima roots, leaves and stems were reduced but increased in leaves and stems as salinity in the medium increased. Potassium contents remained stable under salinity treatment from 250 to 750 mM NaCl. Quantitative real-time PCR demonstrated that without salinity, SaNRT2.1 was expressed in all organs, its expression was not influenced by nitrate supply, while SaNRT2.5 was expressed exclusively in roots—its expression rose about 10-fold under low nitrate. Salinity increased expression of both SaNRT2.1 and SaNRT2.5 under low nitrate. SaNRT2.1 peaked in roots at 500 mM NaCl with 15-fold increase; SaNRT2.5 peaked in roots at 500 mM NaCl with 150-fold increase. It is suggested that SaNRT2.5 ensures effective nitrate uptake by roots and functions as an essential high-affinity nitrate transporter to support growth of adult S. altissima plants under nitrogen deficiency.

Cloning of the SaSLAH3 gene (OQ271227.1), an AtSLAH3 ortholog from the slow anion channels family (SLAC/SLAH), that was originally identified in Arabidopsis thaliana, has been carried out from the euhalophyte Suaeda altissima. Under hydroponic conditions, this species related to Amaranthaceae/Chenopodiaceae is capable to complete life cycle on nutrient solutions containing NaCl at high concentrations up to 1 M. Cloning of SaSLAH3 was performed based on a putative similarity of SaSLAH3 with the SLAH3 homologs from the related species Suaeda glauca and Suaeda fruticosa, using transcriptomes assembled previously. Membrane topology, analyzed by in silico approach, the 3-D structure, presence of conservative phenylalanine-bearing motives and as well as MEME-generated sequence motif patterns of SaSLAH3, all of these features were found typical for the SLAC/SLAH channels and had validated SaSLAH3 belonging to this family. Analysis of SLAC/SLAH phylogenetic relationship demonstrated the evolutionary divergence of SLAC-like proteins into three distinct groups characteristic of this family with location of SaSLAH3 in SLAH2/3 clade. Investigation of the expression profile by quantitative Real-Time PCR revealed increasing SaSLAH3 expression in roots and leaves upon elevation of NaCl concentration in nutrient solution under conditions of both sufficient nitrate supply and nitrate deficiency. SaSLAH3 is suggested to play an important role in enhancing chloride loading into root xylem and, thereafter in chloride delivery to shoot under salinity. Another physiological role of SaSLAH3 may be attributed to regulation of nitrate concentration in cytoplasm to ensure proper nitrogen nutrition of S. altissima plants under conditions of stiff competition of nitrate with chloride.

Transgenic tobacco plants overexpressing the choline oxidase gene from A. globiformis showed an increase in resistance at the level of primary and secondary biosynthesis of metabolites, removing the damage characteristic of salinity and stabilizing the condition of plants. We used 200 mM NaCl, which inhibits the growth of tobacco plants at all stages of development. Leaves of transgenic and wild-type (WT) plants Nicotiána tabácum were used for biochemical, cytological and molecular biological analysis. However, for transgenic lines cultivated under normal conditions (without salinity), we noted juvenile characteristics, delay in flowering, and slowing down of development, including the photosynthetic apparatus. This caused changes in the amount of chlorophyll, a delay in the plastid grana development with the preservation of prolamellar bodies. It also caused changes in the amount of sugars and indirectly downstream processes. A significant change in the activity of antioxidant enzymes and a change in metabolism is probably compensated by the regulation of a number of genes, the expression level of which was also changed. Thus, the tolerance of transgenic tobacco plants to salinity, which manifested itself as a result of the constitutive expression of codA, demonstrates an advantage over WT plants, but in the absence of salinity, transgenic plants did not have such advantages due to juvenilization.