Cold and drought are two of the most severe threats affecting the growth and productivity of the tea plant, limiting its global spread. Both stresses cause osmotic changes in the cells of the tea plant by decreasing their water potential. To develop cultivars that are tolerant to both stresses, it is essential to understand the genetic responses of tea plant to these two stresses, particularly in terms of the genes involved. In this study, we combined literature data with interspecific transcriptomic analyses (using Arabidopsis thaliana and Solanum lycopersicum) to choose genes related to cold tolerance. We identified 45 stress-inducible candidate genes associated with cold and drought responses in tea plants based on a comprehensive homologous detection method. Of these, nine were newly characterized by us, and 36 had previously been reported. The gene network analysis revealed upregulated expression in ICE1-related cluster of bHLH factors, HSP70/BAM5 connected genes (hexokinases, galactinol synthases, SnRK complex, etc.) indicating their possible co-expression. Using qRT-PCR we revealed that 10 genes were significantly upregulated in response to both cold and drought in tea plant: HSP70, GST, SUS1, DHN1, BMY5, bHLH102, GR-RBP3, ICE1, GOLS1, and GOLS3. SnRK1.2, HXK1/2, bHLH7/43/79/93 were specifically upregulated in cold, while RHL41, CAU1, Hydrolase22 were specifically upregulated in drought. Interestingly, the expression of CIP was higher in the recovery stage of both stresses, indicating its potentially important role in plant recovery after stress. In addition, some genes, such as DHN3, bHLH79, PEI54, SnRK1.2, SnRK1.3, and Hydrolase22, were significantly positively correlated between the cold and drought responses. CBF1, GOLS1, HXK2, and HXK3, by contrast, showed significantly negative correlations between the cold and drought responses. Our results provide valuable information and robust candidate genes for future functional analyses intended to improve the stress tolerance of the tea plant and other species.

Background Cold and frost are two serious factors limiting the yield of many crops worldwide, including the tea plant (Camellia sinensis (L.) Kuntze). The acclimatization of tea plant from tropical to temperate climate regions resulted in unique germplasm in the North–Western Caucasus with extremely frost-tolerant genotypes. Methods The aim of the current research was to evaluate the physiological, biochemical and genetic responses of tolerant and sensitive tea cultivars exposed to cold (0 to +2 °C for 7 days) and frost (−6 to −8 °C for 5 days). Relative water content, cell membranes integrity, pH of the cell sap, water soluble protein, cations, sugars, amino acids were measured under cold and frost. Comparative expression of the following genes ICE1, CBF1, WRKY2, DHN1, DHN2, DHN3, NAC17, NAC26, NAC30, SnRK1.1, SnRK1.2, SnRK1.3, bHLH7, bHLH43, P5CS, LOX1, LOX6, LOX7 were analyzed. Results We found elevated protein (by 3–4 times) and cations (potassium, calcium and magnesium) contents in the leaves of both cultivars under cold and frost treatments. Meanwhile, Leu, Met, Val, Thr, Ser were increased under cold and frost, however tolerant cv. Gruzinskii7 showed earlier accumulation of these amino acids. Out of 18 studied genes, 11 were expressed at greater level in the frost- tolerant cultivar comparing with frost-sensitive one: ICE1, CBF1, WRKY2, DHN2, NAC17, NAC26, SnRK1.1, SnRK1.3, bHLH43, P5CS and LOX6. Positive correlations between certain amino acids namely, Met, Thr, Leu and Ser and studied genes were found. Taken together, the revealed cold responses in Caucasian tea cultivars help better understanding of tea tolerance to low temperature stress and role of revealed metabolites need to be further evaluated in different tea genotypes.

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Osmotic stress is a major factor reducing the growth and yield of many horticultural crops worldwide. To reveal reliable markers of tolerant genotypes, we need a comprehensive understanding of the responsive mechanisms in crops. In vitro stress induction can be an efficient tool to study the mechanisms of responses in plants to help gain a better understanding of the physiological and genetic responses of plant tissues against each stress factor. In the present study, the osmotic stress was induced by addition of mannitol into the culture media to reveal biochemical and genetic responses of tea microplants. The contents of proline, threonine, epigallocatechin, and epigallocatechin gallate were increased in leaves during mannitol treatment. The expression level of several genes, namely DHN2, LOX1, LOX6, BAM, SUS1, TPS11, RS1, RS2, and SnRK1.3, was elevated by 2–10 times under mannitol-induced osmotic stress, while the expression of many other stress-related genes was not changed significantly. Surprisingly, down-regulation of the following genes, viz. bHLH12, bHLH7, bHLH21, bHLH43, CBF1, WRKY2, SWEET1, SWEET2, SWEET3, INV5, and LOX7, was observed. During this study, two major groups of highly correlated genes were observed. The first group included seven genes, namely CBF1, DHN3, HXK2,SnRK1.1, SPS, SWEET3, and SWEET1. The second group comprised eight genes, viz. DHN2, SnRK1.3, HXK3, RS1, RS2,LOX6, SUS4, and BAM5. A high level of correlation indicates the high strength connection of the genes which can be co-expressed or can be linked to the joint regulons. The present study demonstrates that tea plants develop several adaptations to cope under osmotic stress in vitro; however, some important stress-related genes were silent or downregulated in microplants.

The effects of application of calcium-containing natural fertilizer on the functional state of the tea plant were studied. Application of calcium resulted weakening of the negative effects of high temperatures and water deficiency due to an increase in its heat resistance (on average by 30 - 40%), an increase in catalase activity (on average 5 - 10 %), as well as adaptive rearrangement of pigment ratio to increasing the content of carotenoids, chlorophylls and the their functional activity. In general, the more efficient functioning of the signaling intracellular network due to the calcium application provided better adaptability of plants to extreme conditions and more efficient recovery after subsequent rehydration, which in a whole contributed to an increase in shoot growth activity (on average 20%) and an increase in yield by an average of 27 - 33%.

The tea collection of the FRC SSC RAS (Sochi, Maykop in Russia) represents one of the northernmost germplasm comprising a number of locally derived cultivars and ɣ-irradiation mutants. The latter are often characterized by larger genome size, which may lead to better adaptation to biotic and abiotic stress. Such genotypes may be a valuable genetic resource for better adaptability to extreme environmental conditions, which could enable tea cultivation outside global growing regions. Microsatellite markers are often the best choice for genetic diversity analysis in genebank collections. However, their use in polyploid species is questionable because simple sequence repeat (SSR) allele dosage cannot be readily determined. Therefore, the efficiency of SSR and start codon targeted (SCoT) markers was investigated using 43 selected cultivars from the Russian genebank collection derived from mutant breeding and clonal selection. Previously, the increase in genome size was confirmed in 18 mutants within this collection. Despite the presence of polyploid tea genotypes, our study revealed higher efficiency of SSR markers than SCoT markers. Subsequent SSR analysis of the 106 genotypes in the Russian genebank collection revealed three distinct genetic clusters after STRUCTURE analysis. Greater genetic variation was observed within genetic clusters than between clusters, indicating low genetic variation between collections. Nevertheless, the northernmost tea collection exhibited a greater genetic distance from the other two clusters than they did from each other. Close genetic relationships were found between many cultivars with particularly large leaves and mutant forms. Pearson’s correlation analysis revealed a significant, moderate correlation between genome size and leaf area size. Our study shows that microsatellite fingerprinting is useful to estimate the genetic diversity and genetic background of tea germplasm in Russia despite polyploid tea accessions. Thus, the results of our study contribute to the development of future tea germplasm conservation strategies and modern tea breeding programs.

К важным факторам, определяющим продуктивность растений, относится их устойчивость к стрессовым воздействиям, в том числе к засухе, гипотермии, минеральной недостаточности и засолению. Решению этих проблем, актуальных в связи со всеобщей аридизацией климата, посвящена серия исследований на различных сельскохозяйственных культурах (J.K. Zhu, 2016; E. Fleta-Soriano, S. Munné-Bosch, 2016), в том числе на чае (Camellia sinensis L.) (T.K. Maritim с соавт., 2015; Л.С. Самарина с соавт., 2019). При достаточно детальном изучении физиолого-биохимических и молекулярных механизмов устойчивости чая к засухе практически не охвачена тема их экзогенной регуляции на основе использования химических и биологических веществ. При этом на многих культурах показана важная роль ионов кальция (Ca 2+) в распознавании клеткой внешнего стрессорного воздействия и запуске системы трансдукции ответного сигнала (M.C. Kim, 2009; Е.Г Рихванов с соавт., 2014). При исследовании этих аспектов достаточно часто в качестве «моделей засухи» используют агаризованные питательные среды с добавлением в них осмотически активных веществ (R.M. Pérez-Clemente с соавт., 2012; M.K. Rai с соавт., 2011) и модельные биосистемы (микропобеги и ткани in vitro), позволяющие раскрыть клеточные механизмы адаптации. Однако в отношении растений чая число подобных исследований невелико (Л.С. Самарина с соавт., 2018; М.В. Гвасалия с соавт., 2019), и они направлены на расшифровку биохимических и молекулярных ответов растений на стрессы. В настоящем сообщении мы впервые на основе отечественных методик получения микропобегов чая в культуре in vitro (М.В. Гвасалия, 2013) и протоколов моделирования осмотического стресса исследовали роль кальция в адаптации растений к стрессовым условиям, вызванным длительным культивированием и осмотическим стрессом, а также продемонстрировали перспективу изучения роли экзогенных индукторов в повышении устойчивости растений на такого рода «моделях засухи». Целью работы было выявление особенностей функционального состояния культивируемых in vitro микропобегов чая, выращиваемых в оптимальных условиях и при моделировании слабого осмотического стресса, обусловленного действием маннита, на фоне разных концентраций кальция (Ca 2+) в питательной среде. Оценивали морфофизиологическое состояние листьев, их оводненность, проницаемость мембран растительных клеток, содержание малонового диальдегида, пролина и фотосинтетических пигментов. Установлено, что при повышении концентрации Ca 2+ в питательной среде (с 440 до 880 мг/л) при длительном культивировании микропобегов чая in vitro (4 мес) происходит замедление формирования и развития их листьев, а также достоверное снижение содержания малонового диальдегида и проницаемости мембран растительных клеток (в среднем на 50 %, р  0,05), свидетельствующее о менее выраженном развитии процессов липопероксидации. Добавление в питательную среду маннита (40 г/л) снижало оводненность побегов (в среднем на 2 %, р  0,05), формируя тем самым незначительный осмотический стресс, что приводило к накоплению пролина (увеличение...

In vitro plant propagation is a developed biotechnology, however until now there are no effective protocols for many perennials, especially for trees. High contamination of mature explants during tissue culture initiation, low multiplication and rooting during following passages are the main challenges. Aseptic culture of explants is associated with stress due to tissue damage and exposition to aggressive disinfectants and antibiotics during initiation. These could be the reasons of virulence of endophytes in following propagation. Plant-associated microorganisms were until recently seen as a problem for micropropagation, leading to contamination of in vitro explants. However recent studies showed that colonization of endophytes often play crucial role for increasing viability of in vitro and ex vitro plants. Most endophytes affect positively plant growth, providing nutrients and exhibiting antagonism to pathogens, as well as decreasing stress effects on plants. Beneficial effects were obtained in using Beauveria bassiana (