Ploidy's Role in Daylily Plant Resilience to Drought Stress Challenges.

This study aimed to understand the differences in the performance of diploid and tetraploid daylily cultivars under water deficit conditions, which are essential indicators of drought tolerance. This research revealed that tetraploid daylilies performed better than diploid varieties in arid conditions due to their enhanced adaptability and resilience to water deficit conditions. The analysis of the results highlighted the need to clarify the specific physiological and molecular mechanisms underlying the enhanced drought tolerance observed in tetraploid plants compared to diploids. This research offers valuable knowledge for improving crop resilience and sustainable floricultural practices in changing environmental conditions. The morphological and physiological parameters were analyzed in 19 diploid and 21 tetraploid daylily cultivars under controlled water deficit conditions, and three drought resistance groups were formed based on the clustering of these parameters. In a high drought resistance cluster, 93.3% tetraploid cultivars were exhibited. This study demonstrates the significance of ploidy in shaping plant responses to drought stress. It emphasizes the importance of studying plant responses to water deficit in landscape horticulture to develop drought-tolerant plants and ensure aspects of climate change.

One Step Forwards in Knowledge of Blossom Blight Brown Rot Disease: Monilinia spp. SSR Marker Database.

A freely available Monilinia spp. marker database was created, containing microsatellite (SSR) data of the three most essential European fungal pathogens: M. fructigena, M. laxa, and M. fructicola. These pathogens cause brown rot blossom blight. Microsatellites were identified using the bioinformatics tool Genome-wide Microsatellite Analyzing Toward Application (GMATA). The database provides information about SSR markers: forward and reverse sequences of the primers, fragment sizes, SSR motifs (and repeats), and the exact locations with the coordinates in the reference genome. This database currently contains information about 39,216 SSR motifs and 26,366 markers. In total, eight primers generated in silico were validated experimentally and they are marked in the database. All scientists can join this collaboration by adding their experimental data. This database is the initial start of organizing Monilinia spp. molecular data worldwide and, in the future, it could be extended by adding more molecular and genomic information.

Assessing the Genetic Diversity of Daylily Germplasm Using SSR Markers: Implications for Daylily Breeding.

This work aims to characterize the genetic diversity of species, early hybrids, and cultivars using microsatellite simple sequence repeat (SSR) markers, as well as analyze and identify the origin of Hemerocallis spp. early hybrids. For this research, samples were collected from different types of daylily species, early hybrids (known or hypothetically first-generation hybrids from Hemerocallis species), foreign, and Lithuanian varieties. An initial screening of SSR primers developed for Hemerocallis citrina was performed, and their suitability for testing other daylily species and hybrids was evaluated. The genetic diversity was assessed with the selected eight-primer set, and molecular SSR profiles were created. Primer SAU00097 is the most informative according to heterozygosity (0.95) and polymorphism information content (PIC) (0.17). The highest heterozygosity was observed in Lithuanian cultivars (0.713), the lowest in species (0.583). Genetic relationships between species show that only fulvous daylilies are separated into a different cluster. The highest variation among genotypes was observed in the species group (18%), while modern cultivars had the slightest variation among genotypes (1%). The putative origin of early hybrids was analyzed using a likelihood heatmap of all genotypes. Results show what species might be used in breeding for early hybrids. Several modern diploid and tetraploid daylily cultivars have triploid species as ancestors.

Fingerprinting of Plum (Prunus domestica) Genotypes in Lithuania Using SSR Markers.

This study's aim was to evaluate the genetic diversity of European plum (Prunus domestica) cultivars and hybrids in Lithuania using SSR markers. In total, 107 plum genotypes (including 68 European plum cultivars and 39 hybrids) from the genetic resources collection of the Institute of Horticulture of the Lithuanian Research Centre for Agriculture and Forestry (LRCAF IH) were evaluated using nine microsatellite markers (SSRs) previously published and suggested by the European Cooperative Programme for Plant Genetic Resources (ECPGR). Up to six alleles per locus with each primer pair were generated for some genotypes due to the hexaploidy of plums. The number of alleles in each primer ranged from 18 to 30, with an average of 24.33. The highest number of alleles was generated with the PacA33 primer pair (30). The most informative primer, according to the PIC value, was BPPCT007. Sixty-two unique alleles (representing 39.5% of all polymorphic alleles) have been detected in the plum germplasm developed in Lithuania. According to UPGMA cluster analysis, 58 European plum genotypes were separated into eight groups without any relation to fruit color or shape. By genetic diversity (UPGMA) and structure (Bayesian) analysis, European plum hybrids were grouped into clusters according to their pedigree.

Performance of Sweet Cherry Cultivars and Advanced Selections on Gisela 5 Rootstock in Young Orchards.

Six sweet cherry cultivars and two advanced selections of Gisela 5 rootstock were tested in 2015-2021 at the Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry. Fruit trees were planted at distances of 4.5 × 2.5 m and trained as spindles. Orchard floor management included frequently mown grass in alleyways with herbicide strips along tree rows. Cultivars 'Mindauge' and 'Irema BS' were the most vigorous at the end of the seventh leaf. Their trunk diameter achieved 11.6 cm. The 'Merchant' cultivar had the smallest trunk diameter-9.3 cm. The average yield in 2018-2021 ranged from 2.75 t/ha for 'Vega' to 8.73 t/ha for 'Regina'. Cultivars 'Regina', 'Sunburst', 'Irema BS' and 'Merchant' had the highest cumulative yield efficiency of 0.440-0.503 kg/cm2 with respect to the trunk cross-section area (TCSA). The least productive cultivar 'Vega' produced fruits of the highest average weight-9.9 g. Fruits of 'Regina' and 'Sunburst' were large as well-8.8-9.1 g. 'Irema BS' fruits had the highest soluble solids content (SSC)-20.2%. The lowest SSC was recorded in 'Merchant' and 'Sunburst' fruits-14.7-15.8%. The yield of advanced selection, No. 102, equaled to the yield of cv. 'Regina'. No. 102 had a high fruit weight, and fruits were distinguished by attractiveness and taste.

Analysis of R Genes Related to Blackcurrant Reversion Virus Resistance in the Comparative Transcriptome of Ribes nigrum cv. Aldoniai.

Blackcurrant reversion virus (BRV) is the most destructive mite-transmitted pathogen in blackcurrants. The understanding of the resistance to BRV is limited, hindering and delaying the selection process. To identify the resistance (R) gene for BRV resistance, a gene expression analysis based on de novo blackcurrant cv. Aldoniai comparative transcriptome analysis (mock- and BRV-inoculated samples at 2 and 4 days post-inoculation (dpi)) was performed. In this study, 111 annotated clusters associated with pathogenesis according to conservative R gene domains were identified. In virus-infected samples, only Cluster-12591.33361 showed significant expression at 4 dpi. The expression profiles of this cluster were significantly associated with the presence of BRV particles in plant tissues, making it a putative R gene in the dominant resistance strategy in the BRV-Ribes nigrum interaction. The newly identified gene R.nigrum_R belongs to the CC-NBS-LRR class and has 63.9% identity with RPM1 in Populus spp. This study provides new insights on dominant putative R genes related to resistance to BRV in R. nigrum, which could aid targeted research and genetic improvement in breeding programs of blackcurrants.

De Novo Transcriptome Analysis of R. nigrum cv. Aldoniai in Response to Blackcurrant Reversion Virus Infection.

The most damaging pathogen in blackcurrant plantations is mite-transmitted blackcurrant reversion virus (BRV). Some Ribes species have an encoded genetic resistance to BRV. We performed RNA sequencing analysis of BRV-resistant blackcurrant cv. Aldoniai to evaluate the molecular mechanisms related to the BRV infection response. The RNA of virus-inoculated and mock-inoculated microshoots was sequenced, and the transcriptional changes at 2- and 4-days post inoculation (dpi) were analyzed. The accumulation and expression of BRV RNA1 were detected in infected plants. In total, 159,701 transcripts were obtained and 30.7% were unigenes, annotated in 7 databases. More than 25,000 differentially expressed genes (DEGs) according to FPKM were upregulated or downregulated. We observed 221 and 850 DEGs at 2 and 4 dpi, respectively, in BRV-infected microshoots related to the stress response. The proportion of upregulated DEGs at 4 dpi was about 3.5 times higher than at 2 dpi. Pathways of the virus defense response were activated, and key candidate genes were identified. The phenylpropanoid and the cutin, suberine, and wax biosynthesis pathways were activated in infected plants. Our comparative de novo analysis of the R. nigrum transcriptome provides clues not only for understanding the molecular BRV resistance mechanisms but also for breeding BRV-tolerant genotypes.

An Effective Method of Ribes spp. Inoculation with Blackcurrant Reversion Virus under In Vitro Conditions.

Blackcurrant reversion virus (BRV) is the most destructive currant-infecting and mite-transmitted pathogen from the genus Nepovirus. In this work, BRV transmission in the system Ribes ex vitro-Ribes in vitro was applied for the first time. Triple infection of BRV identified in blackcurrant cv. Gojai was used for phylogenetic analysis and inoculation assay. Transmission of BRV was successful due to its stability in the inoculum for up to 8 days at 4 °C; all BRV isolates were infectious. Our suggested inoculation method through roots was applied in six Ribes spp. genotypes with 100.0% reliability, and the expression levels of defence-related gene PR1 to biotic stress was observed. The prevalence of the virus in microshoots after 2-14 days post-inoculation (dpi) was established by PCR. In resistant genotypes, the BRV was identified up to 8 dpi; meanwhile, infection remained constant in susceptible genotypes. We established that BRV transmission under controlled conditions depends on the inoculum quality, post-inoculation cultivation temperature, and host-plant susceptibility to pathogen. This in vitro inoculation method opens possibilities to reveal the resistance mechanisms or response pathways to BRV and can be used for the selection of resistant Ribes spp. in breeding programs.

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.

Putative Genes of Pathogenesis-Related Proteins and Coronatine-Insensitive Protein 1 in Ribes spp.

In response to pathogen attacks, plants activate a complex of defense mechanisms including an accumulation of the endogenous signaling compounds salicylic acid and jasmonic acid. The activity of pathogenesis-related genes (PRs) and coronatine-insensitive 1 (COI1) in defense-response pathways are established in plants. The aim of this study was to identify homologs of the PRs and COI1 in blackcurrants. Primers with degenerate nucleotides were designed based on the most conservative parts of PR1 and COI1 genes from other plants and applied for amplification of specific fragments of PRs and COI1 in Ribes spp. Seven heterogeneous sequences of PR with a diversity of 66.0-98.3% at nucleic acid level were found. The phylogenetic analysis revealed the dependence of R. nigrum PR homologs on the PR1 and PR6 families. Four heterogeneous sequences of R. nigrum COI1 with an identity of 95.9-98.8% at nucleic acid level were isolated. Specific primers for newly detected genes' homologs were designed in this study and could be useful for evaluating the defense response to pathogen attacks in blackcurrants.

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.

Anthocyanins: From the Field to the Antioxidants in the Body.

Anthocyanins are biologically active water-soluble plant pigments that are responsible for blue, purple, and red colors in various plant parts-especially in fruits and blooms. Anthocyanins have attracted attention as natural food colorants to be used in yogurts, juices, marmalades, and bakery products. Numerous studies have also indicated the beneficial health effects of anthocyanins and their metabolites on human or animal organisms, including free-radical scavenging and antioxidant activity. Thus, our aim was to review the current knowledge about anthocyanin occurrence in plants, their stability during processing, and also the bioavailability and protective effects related to the antioxidant activity of anthocyanins in human and animal brains, hearts, livers, and kidneys.

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.

Anthocyanins: From plant pigments to health benefits at mitochondrial level.

Anthocyanins are water-soluble pigments providing certain color for various plant parts, especially in edible berries. Earlier these compounds were only known as natural food colorants, the stability of which depended on pH, light, storage temperature and chemical structure. However, due to the increase of the in vitro, in vivo experimental data, as well as of the epidemiological studies, today anthocyanins and their metabolites are also regarded as potential pharmaceutical compounds providing various beneficial health effects on either human or animal cardiovascular system, brain, liver, pancreas and kidney. Many of these effects are shown to be related to the free-radical scavenging and antioxidant properties of anthocyanins, or to their ability to modulate the intracellular antioxidant systems. However, it is generally overlooked that instead of acting exclusively as antioxidants certain anthocyanins affect the activity of mitochondria that are the main source of energy in cells. Therefore, the aim of the present review is to summarize the major knowledge about the chemistry and regulation of biosynthesis of anthocyanins in plants, to overview the facts on bioavailability, and to discuss the most recent experimental findings related to the beneficial health effects emphasizing mitochondria.

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.

Expression and Anthocyanin Biosynthesis-Modulating Potential of Sweet Cherry (Prunus avium L.) MYB10 and bHLH Genes.

Anthocyanins are essential contributors to fruit coloration, an important quality feature and a breed determining trait of a sweet cherry fruit. It is well established that the biosynthesis of anthocyanins is regulated by an interplay of specific transcription factors belonging to MYB and bHLH families accompanied by a WD40 protein. In this study, we isolated and analyzed PaWD40, PabHLH3, PabHLH33, and several closely related MYB10 gene variants from different cultivars of sweet cherry, analyzed their expression in fruits with different anthocyanin levels at several developmental stages, and determined their capabilities to modulate anthocyanin synthesis in leaves of two Nicotiana species. Our results indicate that transcription level of variant PaMYB10.1-1 correlates with fruit coloration, but anthocyanin synthesis in Nicotiana was induced by another variant, PaMYB10.1-3, which is moderately expressed in fruits. The analysis of two fruit-expressed bHLH genes revealed that PabHLH3 enhances MYB-induced anthocyanin synthesis, whereas PabHLH33 has strong inhibitory properties.

Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families.

In this study, a protocol is described for rapid preparation of an enriched, reasonably pure fraction of nuclear proteins from the leaves of tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and apple (Malus domestica). The protocol gives reproducible results and can be carried out quickly in 2 hours. Tissue extracts clarified with filtration were treated with non-ionic detergent (Triton X-100) to lyse membranes of contaminating organelles. Nuclei were collected from a 60% Percoll layer of density gradient following low-speed centrifugation. Western blot analysis using antibodies to marker proteins of organelles indicated that the nuclear protein fractions were highly enriched and free or nearly free of proteins from the endoplasmic reticulum and chloroplasts.

Phenolics, antioxidative activity and characterization of anthocyanins in berries of blackcurrant interspecific hybrids.

The interest in phenols and anthocyanins has increased due to their antioxidant properties and to their potential usage as dietary antioxidants in human nutrition. Total phenols and anthocyanin content, composition and stability in berry extracts of blackcurrant interspecific hybrids, and antioxidative activity of extracts was evaluated. Berries of interspecific hybrids accumulated 530 to 614 mg 100 g(-1) FW of total phenolic compounds, while 621 mg 100 g(-1) FW of phenolics was established in berries of control Ribes nigrum cultivar 'Ben Tirran'. 'Ben Tirran' berries accumulated 444 mg 100 g(-1) FW of anthocyanins and higher amount was identified in berries of interspecific hybrids No. 11-13 (R. nigrum × R. petraeum) and No. 57 (R. nigrum × R. aureum), 522 and 498 mg100 g(-1) FW respectively. Berry extracts of hybrid No. 11-13 distinguished by the highest antioxidative activity (80%) and it was higher than antioxidant activity of 'Ben Tirran' (70%). Antioxidative activity of all tested berry extracts (70-80%) was twice higher compared to synthetic antioxidant BHT (39%). However correlation between phenolics or total anthocyanin content and antioxidative activity degree was not established. Amount of cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside in berries of hybrids No. 57, No. 11-13 and No. 8 ((R. nigrum × R. americanum) × (R. nigrum × R. americanum)) was higher than in berries of 'Ben Tirran'. It was established that cyanidins are more stable anthocyanins in all studied temperature and irradiation conditions. Therefore interspecific hybrids No. 57 and No. 11-13 were the most agronomically valuable hybrids.