How the Ethylene Biosynthesis Pathway of Semi-Halophytes Is Modified with Prolonged Salinity Stress Occurrence?

The mechanism of ethylene (ET)-regulated salinity stress response remains largely unexplained, especially for semi-halophytes and halophytes. Here, we present the results of the multifaceted analysis of the model semi-halophyte Mesembryanthemum crystallinum L. (common ice plant) ET biosynthesis pathway key components' response to prolonged (14 days) salinity stress. Transcriptomic analysis revealed that the expression of 3280 ice plant genes was altered during 14-day long salinity (0.4 M NaCl) stress. A thorough analysis of differentially expressed genes (DEGs) showed that the expression of genes involved in ET biosynthesis and perception (ET receptors), the abscisic acid (ABA) catabolic process, and photosynthetic apparatus was significantly modified with prolonged stressor presence. To some point this result was supported with the expression analysis of the transcript amount (qPCR) of key ET biosynthesis pathway genes, namely ACS6 (1-aminocyclopropane-1-carboxylate synthase) and ACO1 (1-aminocyclopropane-1-carboxylate oxidase) orthologs. However, the pronounced circadian rhythm observed in the expression of both genes in unaffected (control) plants was distorted and an evident downregulation of both orthologs' was induced with prolonged salinity stress. The UPLC-MS analysis of the ET biosynthesis pathway rate-limiting semi-product, namely of 1-aminocyclopropane-1-carboxylic acid (ACC) content, confirmed the results assessed with molecular tools. The circadian rhythm of the ACC production of NaCl-treated semi-halophytes remained largely unaffected by the prolonged salinity stress episode. We speculate that the obtained results represent an image of the steady state established over the past 14 days, while during the first hours of the salinity stress response, the view could be completely different.

Transcriptomic and metabolic studies on the role of inorganic and organic iodine compounds in lettuce plants.

Iodine (I) is considered a beneficial element or even micronutrient for plants. The aim of this study was to determine the molecular and physiological processes of uptake, transport, and metabolism of I applied to lettuce plants. KIO3, KIO3 + salicylic acid, 5-iodosalicylic acid and 3,5-diiodosalicylic acid were applied. RNA-sequencing was executed using 18 cDNA libraries constructed separately for leaves and roots from KIO3, SA and control plants. De novo transcriptome assembly generated 1937.76 million sequence reads resulting in 27,163 transcripts with N50 of 1638 bp. 329 differentially expressed genes (DEGs) in roots were detected after application of KIO3, out of which 252 genes were up-regulated, and 77 were down-regulated. In leaves, 9 genes revealed differential expression pattern. DEGs analysis indicated its involvement in such metabolic pathways and processes as: chloride transmembrane transport, phenylpropanoid metabolism, positive regulation of defense response and leaf abscission, and also ubiquinone and other terpenoid-quinone biosynthesis, protein processing in endoplasmic reticulum, circadian rhythm including flowering induction as well as a putative PDTHA (i.e. Plant Derived Thyroid Hormone Analogs) metabolic pathway. qRT-PCR of selected genes suggested their participation in the transport and metabolism of iodine compounds, biosynthesis of primary and secondary metabolites, PDTHA pathway and flowering induction.

Transcriptome Dynamics Underlying Planticine®-Induced Defense Responses of Tomato (Solanum lycopersicum L.) to Biotic Stresses.

The induction of natural defense mechanisms in plants is considered to be one of the most important strategies used in integrated pest management (IPM). Plant immune inducers could reduce the use of chemicals for plant protection and their harmful impacts on the environment. Planticine® is a natural plant defense biostimulant based on oligomers of α(1→4)-linked D-galacturonic acids, which are biodegradable and nontoxic. The aim of this study was to define the molecular basis of Planticine's biological activity and the efficacy of its use as a natural plant resistance inducer in greenhouse conditions. Three independent experiments with foliar application of Planticine® were carried out. The first experiment in a climatic chamber (control environment, no pest pressure) subjected the leaves to RNA-seq analysis, and the second and third experiments in greenhouse conditions focused on efficacy after a pest infestation. The result was the RNA sequencing of six transcriptome libraries of tomatoes treated with Planticine® and untreated plants; a total of 3089 genes were found to be differentially expressed genes (DEGs); among them, 1760 and 1329 were up-regulated and down-regulated, respectively. DEG analysis indicated its involvement in such metabolic pathways and processes as plant-pathogen interaction, plant hormone signal transduction, MAPK signaling pathway, photosynthesis, and regulation of transcription. We detected up-regulated gene-encoded elicitor and effector recognition receptors (ELRR and ERR), mitogen-activated protein kinase (MAPKs) genes, and transcription factors (TFs), i.e., WRKY, ERF, MYB, NAC, bZIP, pathogenesis-related proteins (PRPs), and resistance-related metabolite (RRMs) genes. In the greenhouse trials, foliar application of Planticine® proved to be effective in reducing the infestation of tomato leaves by the biotrophic pathogen powdery mildew and in reducing feeding by thrips, which are insect herbivores. Prophylactic and intervention use of Planticine® at low infestation levels allows the activation of plant defense mechanisms.

Proteomic Studies of Roots in Hypoxia-Sensitive and -Tolerant Tomato Accessions Reveal Candidate Proteins Associated with Stress Priming.

Tomato (Solanum lycopersicum L.) is a vegetable frequently exposed to hypoxia stress induced either by being submerged, flooded or provided with limited oxygen in hydroponic cultivation systems. The purpose of the study was to establish the metabolic mechanisms responsible for overcoming hypoxia in two tomato accessions with different tolerance to this stress, selected based on morphological and physiological parameters. For this purpose, 3-week-old plants (plants at the juvenile stage) of waterlogging-tolerant (WL-T), i.e., POL 7/15, and waterlogging-sensitive (WL-S), i.e., PZ 215, accessions were exposed to hypoxia stress (waterlogging) for 7 days, then the plants were allowed to recover for 14 days, after which another 7 days of hypoxia treatment was applied. Root samples were collected at the end of each time-point and 2D-DIGE with MALDI TOF/TOF, and expression analyses of gene and protein-encoded alcohol dehydrogenase (ADH2) and immunolabelling of ADH were conducted. After collating the obtained results, the different responses to hypoxia stress in the selected tomato accessions were observed. Both the WL-S and WL-T tomato accessions revealed a high amount of ADH2, which indicates an intensive alcohol fermentation pathway during the first exposure to hypoxia. In comparison to the tolerant one, the expression of the adh2 gene was about two times higher for the sensitive tomato. Immunohistochemical analysis confirmed the presence of ADH in the parenchyma cells of the cortex and vascular tissue. During the second hypoxia stress, the sensitive accession showed a decreased accumulation of ADH protein and similar expression of the adh2 gene in comparison to the tolerant accession. Additionally, the proteome showed a greater protein abundance of glyceraldehyde-3-phosphate dehydrogenase in primed WL-S tomato. This could suggest that the sensitive tomato overcomes the oxygen limitation and adapts by reducing alcohol fermentation, which is toxic to plants because of the production of ethanol, and by enhancing glycolysis. Proteins detected in abundance in the sensitive accession are proposed as crucial factors for hypoxia stress priming and their function in hypoxia tolerance is discussed.

Waterlogging-Stress-Responsive LncRNAs, Their Regulatory Relationships with miRNAs and Target Genes in Cucumber (Cucumis sativus L.).

Low oxygen level is a phenomenon often occurring during the cucumber cultivation period. Genes involved in adaptations to stress can be regulated by non-coding RNA. The aim was the identification of long non-coding RNAs (lncRNAs) involved in the response to long-term waterlogging stress in two cucumber haploid lines, i.e., DH2 (waterlogging tolerant-WL-T) and DH4 (waterlogging sensitive-WL-S). Plants, at the juvenile stage, were waterlogged for 7 days (non-primed, 1xH), and after a 14-day recovery period, plants were stressed again for another 7 days (primed, 2xH). Roots were collected for high-throughput RNA sequencing. Implementation of the bioinformatic pipeline made it possible to determine specific lncRNAs for non-primed and primed plants of both accessions, highlighting differential responses to hypoxia stress. In total, 3738 lncRNA molecules were identified. The highest number (1476) of unique lncRNAs was determined for non-primed WL-S plants. Seventy-one lncRNAs were depicted as potentially being involved in acquiring tolerance to hypoxia in cucumber. Understanding the mechanism of gene regulation under long-term waterlogging by lncRNAs and their interactions with miRNAs provides sufficient information in terms of adaptation to the oxygen deprivation in cucumber. To the best of our knowledge, this is the first report concerning the role of lncRNAs in the regulation of long-term waterlogging tolerance by priming application in cucumber.

New Aspects of Uptake and Metabolism of Non-organic and Organic Iodine Compounds-The Role of Vanadium and Plant-Derived Thyroid Hormone Analogs in Lettuce.

The process of uptake and translocation of non-organic iodine (I) ions, I- and IO3 -, has been relatively well-described in literature. The situation is different for low-molecular-weight organic aromatic I compounds, as data on their uptake or metabolic pathway is only fragmentary. The aim of this study was to determine the process of uptake, transport, and metabolism of I applied to lettuce plants by fertigation as KIO3, KIO3 + salicylic acid (KIO3+SA), and iodosalicylates, 5-iodosalicylic acid (5-ISA) and 3,5-diiodosalicylic acid (3,5-diISA), depending on whether additional fertilization with vanadium (V) was used. Each I compound was applied at a dose of 10 µM, SA at a dose of 10 µM, and V at a dose of 0.1 µM. Three independent 2-year-long experiments were carried out with lettuce; two with pot systems using a peat substrate and mineral soil and one with hydroponic lettuce. The effectiveness of I uptake and translocation from the roots to leaves was as follows: 5-ISA > 3,5-diISA > KIO3. Iodosalicylates, 5-ISA and 3,5-diISA, were naturally synthesized in plants, similarly to other organic iodine metabolites, i.e., iodotyrosine, as well as plant-derived thyroid hormone analogs (PDTHA), triiodothyronine (T3) and thyroxine (T4). T3 and T4 were synthesized in roots with the participation of endogenous and exogenous 5-ISA and 3,5-diISA and then transported to leaves. The level of plant enrichment in I was safe for consumers. Several genes were shown to perform physiological functions, i.e., per64-like, samdmt, msams5, and cipk6.

Protuberances are organized distinct regions of long-term callus: histological and transcriptomic analyses in kiwifruit.

KEY MESSAGE: Macroscopic, ultrastructural, and molecular features-like a ball shape, the presence of starch granules, and the up-regulation of genes involved in carbohydrate metabolism and secondary metabolite biosynthesis-distinguish PT regions within a callus. The modification of the mass of pluripotent cells into de novo shoot bud regeneration is highly relevant to developmental biology and for agriculture and biotechnology. This study deals with protuberances (PT), structures that appear during the organogenic long-term culturing of callus (OC) in kiwifruit. These ball-shaped regions of callus might be considered the first morphological sign of the subsequent shoot bud development. Sections of PT show the regular arrangement of some cells, especially on the surface, in contrast to the regions of OC beyond the PT. The cells of OC possess chloroplasts; however, starch granules were observed only in PTs' plastids. Transcriptomic data revealed unique gene expression for each kind of sample: OC, PT, and PT with visible shoot buds (PT-SH). Higher expression of the gene involved in lipid (glycerol-3-phosphate acyltransferase 5 [GPAT5]), carbohydrate (granule-bound starch synthase 1 [GBSS1]), and secondary metabolite (beta-glucosidase 45 [BGL45]) pathways were detected in PT and could be proposed as the markers of these structures. The up-regulation of the regulatory associated protein of TOR (RAPTOR1) was found in PT-SH. The highest expression of the actinidain gene in leaves from two-year-old regenerated plants suggests that the synthesis of this protein takes place in fully developed organs. The findings indicate that PT and PT-SH are specific structures within OC but have more features in common with callus tissue than with organs.

Long-Term Waterlogging as Factor Contributing to Hypoxia Stress Tolerance Enhancement in Cucumber: Comparative Transcriptome Analysis of Waterlogging Sensitive and Tolerant Accessions.

Waterlogging (WL), excess water in the soil, is a phenomenon often occurring during plant cultivation causing low oxygen levels (hypoxia) in the soil. The aim of this study was to identify candidate genes involved in long-term waterlogging tolerance in cucumber using RNA sequencing. Here, we also determined how waterlogging pre-treatment (priming) influenced long-term memory in WL tolerant (WL-T) and WL sensitive (WL-S) i.e., DH2 and DH4 accessions, respectively. This work uncovered various differentially expressed genes (DEGs) activated in the long-term recovery in both accessions. De novo assembly generated 36,712 transcripts with an average length of 2236 bp. The results revealed that long-term waterlogging had divergent impacts on gene expression in WL-T DH2 and WL-S DH4 cucumber accessions: after 7 days of waterlogging, more DEGs in comparison to control conditions were identified in WL-S DH4 (8927) than in WL-T DH2 (5957). Additionally, 11,619 and 5007 DEGs were identified after a second waterlogging treatment in the WL-S and WL-T accessions, respectively. We identified genes associated with WL in cucumber that were especially related to enhanced glycolysis, adventitious roots development, and amino acid metabolism. qRT-PCR assay for hypoxia marker genes i.e., alcohol dehydrogenase (adh), 1-aminocyclopropane-1-carboxylate oxidase (aco) and long chain acyl-CoA synthetase 6 (lacs6) confirmed differences in response to waterlogging stress between sensitive and tolerant cucumbers and effectiveness of priming to enhance stress tolerance.

Iodine biofortification through expression of HMT, SAMT and S3H genes in Solanum lycopersicum L.

The uptake process and physiological reaction of plants to aromatic iodine compounds have not yet been documented. The aim of this research was to compare uptake by tomato plants of KI and KIO3, as well as of organic iodine compounds - 5-ISA (5-iodosalicylic acid), 3,5-diISA (3,5-diiodosalicylic acid), 2-IBeA (2-iodobenzoic acid), 4-IBeA (4-iodobenzoic acid) and 2,3,5-triIBeA (2,3,5-triiodobenzoic acid). Only 2,3,5-triIBeA had a negative influence on plant development. All organic iodine compounds were taken up by roots and transported to leaves and fruits. Among all the compounds applied, the most efficiently transferred iodine was 2-IBeA - to fruits, and 4-IBeA - to leaves. The order of iodine accumulation in fruit cell compartments was as follows: organelles > cell walls > soluble portions of cells; for leaf and root cells, it was: organelles > cell walls or soluble portions, depending on the compound applied. The compounds studied influence iodine metabolism through expression of the HMT gene which encodes halide ion methyltransferase in leaves and roots. Also, their influence on modification of the activity of the SAMT and S3H genes that encode salicylic acid carboxyl methyltransferase and salicylic acid 3-hydroxylase was established. It was discovered that exogenously applied 5-ISA, 3,5-diISA, 2-IBeA and 4-IBeA are genuinely (endogenously) synthesised in tomato plants; to date, this has not been described for the tomato, nor for any other species of higher plant.

Iodine and Selenium Biofortification with Additional Application of Salicylic Acid Affects Yield, Selected Molecular Parameters and Chemical Composition of Lettuce Plants (Lactuca sativa L. var. capitata).

Iodine (I) and selenium (Se) are included in the group of beneficial elements. They both play important roles in humans and other animals, particularly in the regulation of thyroid functioning. A substantial percentage of people around the world suffer from health disorders related to the deficiency of these elements in the diet. Salicylic acid (SA) is a compound similar to phytohormones and is known to improve the efficiency of I biofortification of plants. The influence of SA on Se enrichment of plants has not, however, been recognized together with its effect on simultaneous application of I and Se to plants. Two-year studies (2014-2015) were conducted in a greenhouse with hydroponic cultivation of lettuce in an NFT (nutrient film technique) system. They included the application of I (as KIO3), Se (as Na2SeO3) and SA into the nutrient solution. KIO3 was used at a dose of 5 mg I⋅dm-3 (i.e., 39.4 µM I), while Na2SeO3 was 0.5 mg Se⋅dm-3 (i.e., 6.3 µM Se). SA was introduced at three doses: 0.1, 1.0, and 10.0 mg⋅dm-3 nutrient solutions, equivalent to 0.724, 7.24, and 72.4 µM SA, respectively. The tested combinations were as follows: (1) control, (2) I + Se, (3) I + Se + 0.1 mg SA⋅dm-3, (4) I + Se + 1.0 mg SA⋅dm-3 and (5) I + Se + 10.0 mg SA⋅dm-3. The applied treatments had no significant impact on lettuce biomass (leaves and roots). Depending on the dose, a diverse influence of SA was noted with respect to the efficiency of I and Se biofortification; chemical composition of leaves; and mineral nutrition of lettuce plants, including the content of macro- and microelements and selenocysteine methyltransferase (SMT) gene expression. SA application at all tested doses comparably increased the level of selenomethionine (SeMet) and decreased the content of SA in leaves.

Genetic diversity of F1 and F2 interspecific hybrids between dwarf birch (Betula nana L.) and Himalayan birch (B. utilis var. jacquemontii (Spach) Winkl. 'Doorenbos') using RAPD-PCR markers and ploidy analysis.

Crosses between Betula nana and B. utilis 'Doorenbos' were undertaken in order to obtain interspecific hybrids which could be characterized by wide spreading stems, strong branching habit, decorative clear white bark and an interesting shape of purple leaves. The research purpose was to examine genetic diversity of the 16 F1 and F2 putative progenies by using the RAPD-PCR method and the ploidy analysis. A total of 242 RAPD markers were scored with 24 primers and 220 (90.9%) polymorphic bands were found. In the NJ dendrogram, cluster I consisted of the female parent--B. nana and 12 hybrids and cluster II grouped the male parent--B. utilis 'Doorenbos' with 4 hybrids (F2/2, F1/8, F1/7 and F2/1). The 2-D scaling by PCoA was in agreement with the similarity index, i.e. two hybrids (F1/8, F2/2) grouped with the male parent while others with female parent. Classification of the hybrid plants by chromosome counting demonstrated that 13 hybrids were confirmed with accurate chromosome counts as being diploid (2n=2x=28) and 3 plants (F1/7, F1/8, F2/2) as triploid with 42 chromosomes.

The complete genome of Blastobotrys (Arxula) adeninivorans LS3 - a yeast of biotechnological interest.

BACKGROUND: The industrially important yeast Blastobotrys (Arxula) adeninivorans is an asexual hemiascomycete phylogenetically very distant from Saccharomyces cerevisiae. Its unusual metabolic flexibility allows it to use a wide range of carbon and nitrogen sources, while being thermotolerant, xerotolerant and osmotolerant. RESULTS: The sequencing of strain LS3 revealed that the nuclear genome of A. adeninivorans is 11.8 Mb long and consists of four chromosomes with regional centromeres. Its closest sequenced relative is Yarrowia lipolytica, although mean conservation of orthologs is low. With 914 introns within 6116 genes, A. adeninivorans is one of the most intron-rich hemiascomycetes sequenced to date. Several large species-specific families appear to result from multiple rounds of segmental duplications of tandem gene arrays, a novel mechanism not yet described in yeasts. An analysis of the genome and its transcriptome revealed enzymes with biotechnological potential, such as two extracellular tannases (Atan1p and Atan2p) of the tannic-acid catabolic route, and a new pathway for the assimilation of n-butanol via butyric aldehyde and butyric acid. CONCLUSIONS: The high-quality genome of this species that diverged early in Saccharomycotina will allow further fundamental studies on comparative genomics, evolution and phylogenetics. Protein components of different pathways for carbon and nitrogen source utilization were identified, which so far has remained unexplored in yeast, offering clues for further biotechnological developments. In the course of identifying alternative microorganisms for biotechnological interest, A. adeninivorans has already proved its strengthened competitiveness as a promising cell factory for many more applications.

Paternity determination of interspecific rhododendron hybrids by genomic in situ hybridization (GISH).

Genomic in situ hybridization (GISH) has been proved to be the most effective and accurate technique for confirmation of hybrid character. The objective of our study was to adapt and optimize a GISH protocol for identification of donor chromatin in hybrids obtained by interspecific crosses between five Rhododendron taxa (R. aureum, R. brachycarpum, R. catawbiense 'Catharine van Tol', R. catawbiense 'Nova Zembla', and R. yakushimanum 'Koichiro Wada'). Positive results were obtained only when we used mitotic chromosome spreads prepared from anthers. The best differentiation of maternal and paternal chromosomes in hybrid genomes was obtained when 50 ng of probe was applied together with blocking DNA at a concentration of 3.0 microg/microL. The results demonstrate that GISH is a practical tool for detection of alien genomes and analysis of the constitution of the chromosomes in rhododendron hybrids.