Nitric Oxide-Induced Dormancy Removal of Apple Embryos Is Linked to Alterations in Expression of Genes Encoding ABA and JA Biosynthetic or Transduction Pathways and RNA Nitration.

Short-term (3 h) treatment of embryos isolated from dormant apple (Malus domestica Borkh.) seeds with NO donors stimulates their transition from dormancy to germination. Seed dormancy is maintained by ABA, while germination is controlled mainly by gibberellins (GAs) and jasmonic acid (JA). NO-induced dormancy removal correlates with low ABA concentration in embryonic axes and reduced embryo sensitivity to ABA. We analyzed the expression of genes encoding key enzymes of ABA degradation (CYP707A1, CYP707A2), biosynthesis (NCED3, NCED9), and elements of the ABA transduction pathway (PYL1, PYL2, RCAR1, RCAR3, PP2CA, ABI1, ABI2, SNRK2, ABI5, AREB3, ABF). A role for JA in the regulation of germination led us to investigate the expression of genes encoding enzymes of JA biosynthesis (AOS1, JMT, JAR1) and the transduction pathway (COI1, MYC2, JAZ3, JAZ12). The expression profiles of the genes were estimated in embryonic axes isolated from dormant or NO fumigated apple embryos. The analyzed genes were differentially regulated during dormancy alleviation, the main modifications in the transcription level were detected for NCED3, NCED9, CYP707A2, RCAR1, ABF, AOS1, JMT, JAR1 and JAZ3. A regulatory role of NO in the removal of seed dormancy is associated with the stimulation of expression of genes related to ABA degradation, down-regulation of genes responsible for ABA synthesis, an increase of expression level of genes engaged in JA synthesis and modification of the expression of genes engaged in signaling pathways of the hormones. To confirm a signaling role of NO during dormancy breakage, an increased RNA nitration level in embryonic axes was demonstrated.

Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level.

BACKGROUND: The amount and structure of genetic diversity in dessert apple germplasm conserved at a European level is mostly unknown, since all diversity studies conducted in Europe until now have been performed on regional or national collections. Here, we applied a common set of 16 SSR markers to genotype more than 2,400 accessions across 14 collections representing three broad European geographic regions (North + East, West and South) with the aim to analyze the extent, distribution and structure of variation in the apple genetic resources in Europe. RESULTS: A Bayesian model-based clustering approach showed that diversity was organized in three groups, although these were only moderately differentiated (FST = 0.031). A nested Bayesian clustering approach allowed identification of subgroups which revealed internal patterns of substructure within the groups, allowing a finer delineation of the variation into eight subgroups (FST = 0.044). The first level of stratification revealed an asymmetric division of the germplasm among the three groups, and a clear association was found with the geographical regions of origin of the cultivars. The substructure revealed clear partitioning of genetic groups among countries, but also interesting associations between subgroups and breeding purposes of recent cultivars or particular usage such as cider production. Additional parentage analyses allowed us to identify both putative parents of more than 40 old and/or local cultivars giving interesting insights in the pedigree of some emblematic cultivars. CONCLUSIONS: The variation found at group and subgroup levels may reflect a combination of historical processes of migration/selection and adaptive factors to diverse agricultural environments that, together with genetic drift, have resulted in extensive genetic variation but limited population structure. The European dessert apple germplasm represents an important source of genetic diversity with a strong historical and patrimonial value. The present work thus constitutes a decisive step in the field of conservation genetics. Moreover, the obtained data can be used for defining a European apple core collection useful for further identification of genomic regions associated with commercially important horticultural traits in apple through genome-wide association studies.

Modification of the endogenous NO level influences apple embryos dormancy by alterations of nitrated and biotinylated protein patterns.

MAIN CONCLUSION: NO donors and Arg remove dormancy of apple embryos and stimulate germination. Compounds lowering NO level (cPTIO, L -NAME, CAN) strengthen dormancy. Embryo transition from dormancy state to germination is linked to increased nitric oxide synthase (NOS)-like activity. Germination of embryos is associated with declined level of biotin containing proteins and nitrated proteins in soluble protein fraction of root axis. Pattern of nitrated proteins suggest that storage proteins are putative targets of nitration. Nitric oxide (NO) acts as a key regulatory factor in removal of seed dormancy and is a signal necessary for seed transition from dormant state into germination. Modulation of NO concentration in apple (Malus domestica Borkh.) embryos by NO fumigation, treatment with NO donor (S-nitroso-N-acetyl-D,L-penicillamine, SNAP), application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), N ω-nitro-L-arginine methyl ester (L-NAME), canavanine (CAN) or arginine (Arg) allowed us to investigate the NO impact on seed dormancy status. Arg analogs and NO scavenger strengthened embryo dormancy by lowering reactive nitrogen species level in embryonic axes. This effect was accompanied by strong inhibition of NOS-like activity, without significant influence on tissue NO2 (-) concentration. Germination sensu stricto of apple embryos initiated by dormancy breakage via short term NO treatment or Arg supplementation were linked to a reduced level of biotinylated proteins in root axis. Decrease of total soluble nitrated proteins was observed at the termination of germination sensu stricto. Also modulation of NO tissue status leads to modification in nitrated protein pattern. Among protein bands that correspond to molecular mass of approximately 95 kDa, storage proteins (legumin A-like and seed biotin-containing protein) were identified, and can be considered as good markers for seed dormancy status. Moreover, pattern of nitrated proteins suggest that biotin containing proteins are also targets of nitration.

Differential expression and modification of proteins during ontogenesis in Malus domestica.

Many morphological and physiological changes have been widely reported during ontogeny in higher plants. In order for the better understanding of the proteomic differences between ontogenetic phases, protein compositions between leaves of juvenile, adult vegetative and reproductive phases were compared in an apple (Malus domestica Borkh., Jonathan × Golden Delicious) seedling. Totally, 122 differentially expressed or modified protein spots were separated by DIGE. Of the 122 protein spots, 44, 17 and 29 were abundant in the leaf samples from the juvenile, adult vegetative and reproductive phases, respectively, two spots showed a lower level in the adult vegetative tissue, while the amount of protein increased in 21 spots during ontogeny and declined in nine spots. One hundred and fifteen spots were successfully picked and 95 spots were identified by MALDI-TOF-TOF high-resolution tandem mass spectrometry. Twenty-three juvenile phase abundant or down-regulated spots were photosynthesis-associated proteins, implying a juvenile phase-related photosynthesis enhancement. The expression of 10 enzymes and coenzymes involved in protein synthesis and catabolism was elevated in the adult reproductive phase or up-regulated during ontogeny, contributing a phase change-related activation in protein metabolism. Six proteins generated 30 differential gel spots via post-translational modifications. The differential expression of NADP-dependent D-sorbitol-6-phosphate dehydrogenase was confirmed by Western blotting in six seedlings derived from two hybrid populations. The results of semi-quantitative PCR indicate that some but not all of these proteomic changes were transcriptionally regulated.

Dormancy removal in apple embryos by nitric oxide or cyanide involves modifications in ethylene biosynthetic pathway.

The connection between classical phytohormone-ethylene and two signaling molecules, nitric oxide (NO) and hydrogen cyanide (HCN), was investigated in dormancy removal and germination "sensu stricto" of apple (Malus domestica Borkh.) embryos. Deep dormancy of apple embryos was removed by short-term (3-6 h) pre-treatment with NO or HCN. NO- or HCN-mediated stimulation of germination was associated with enhanced emission of ethylene by the embryos, coupled with transient increase in ROS concentration in embryos. Ethylene vapors stimulated germination of dormant apple embryos and eliminated morphological anomalies characteristic for young seedlings developed from dormant embryos. Inhibitors of ethylene receptors completely impeded beneficial effect of NO and HCN on embryo germination. NO- and HCN-induced ethylene emission by apple embryo was only slightly reduced by inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity during first 4 days of germination. Short-term pre-treatment of the embryos with NO and HCN modified activity of both key enzymes of ethylene biosynthetic pathway: ACC synthase and ACC oxidase. Activity of ACC synthase declined during first 4 days of germination, while activity of ACC oxidase increased markedly at that time. Additional experiments point to non-enzymatic conversion of ACC to ethylene in the presence of ROS (H(2)O(2)). The results indicate that NO and HCN may alleviate dormancy of apple embryos "via" transient accumulation of ROS, leading to enhanced ethylene emission which is required to terminate germination "sensu stricto". Therefore, ethylene seems to be a trigger factor in control of apple embryo dormancy removal and germination.

Comparative study of ultrasonic pretreatment and ultrasound assisted three phase partitioning for extraction of custard apple seed oil.

Custard apple seed oil is a marketable product extracted from custard apple seed which is a potential agriculture waste. The present work aims to elucidate simultaneous extraction of the custard apple seed oil and proteins using three phase partitioning (TPP). The efficient oil extraction was executed by optimization of parameters including time, slurry ratio, salt concentration, and slurry to t-butanol ratio. Additionally, the application of ultrasound as process intensification tool for TPP was studied that reduces the time of conventional TPP and increases the yield by 2.53%. The work also comprises a comparative study of two modes of ultrasound application, namely ultrasound pre-treatment and simultaneous ultrasound assisted TPP. This work proves ultrasonic pre-treatment followed by TPP as a superior mode of ultrasound application that attributes 33.6 ±â€¯0.56% (w/w) oil extraction with optimized pre-treatment time of 150 s, 30 W ultrasound power and 75% duty cycle. Extraction kinetics studied for conventional, Ultrasound assisted Three Phase Partitioning (UTPP), and Ultrasound Pre-treatment assisted Three Phase Partitioning (UPTPP) were observed to fit Peleg's model.

Arabinogalactan proteins mediate intercellular crosstalk in the ovule of apple flowers.

KEY MESSAGE: AGP-rich glycoproteins mediate pollen-ovule interactions and cell patterning in the embryo sac of apple before and after fertilization. Glycoproteins are significant players in the dialog that takes place between growing pollen tubes and the stigma and style in the angiosperms. Yet, information is scarce on their possible involvement in the ovule, a sporophytic organ that hosts the female gametophyte. Apple flowers have a prolonged lapse of time between pollination and fertilization, offering a great system to study the developmental basis of glycoprotein secretion and their putative role during the last stages of the progamic phase and early seed initiation. For this purpose, the sequential pollen tube elongation within the ovary was examined in relation to changes in arabinogalactan proteins (AGPs) in the tissues of the ovule before and after fertilization. To evaluate what of these changes are developmentally regulated, unpollinated and pollinated flowers were compared. AGPs paved the pollen tube pathway in the ovules along the micropylar canal, and the nucellus entrance toward the synergids, which also developmentally accumulated AGPs at the filiform apparatus. Glycoproteins vanished from all these tissues following pollen tube passage, strongly suggesting a role in pollen-ovule interaction. In addition, AGPs marked the primary cell walls of the haploid cells of the female gametophyte, and they further built up in the cell walls of the embryo sac and developing embryo, layering the interactive walls of the three generations hosted in the ovule, the maternal sporophytic tissues, the female gametophyte, and the developing embryo.

Dormancy alleviation by NO or HCN leading to decline of protein carbonylation levels in apple (Malus domestica Borkh.) embryos.

Deep dormancy of apple (Malus domestica Borkh.) embryos can be overcome by short-term pre-treatment with nitric oxide (NO) or hydrogen cyanide (HCN). Dormancy alleviation of embryos modulated by NO or HCN and the first step of germination depend on temporary increased production of reactive oxygen species (ROS). Direct oxidative attack on some amino acid residues or secondary reactions via reactive carbohydrates and lipids can lead to the formation of protein carbonyl derivatives. Protein carbonylation is a widely accepted covalent and irreversible modification resulting in inhibition or alteration of enzyme/protein activities. It also increases the susceptibility of proteins to proteolytic degradation. The aim of this work was to investigate protein carbonylation in germinating apple embryos, the dormancy of which was removed by pre-treatment with NO or HCN donors. It was performed using a quantitative spectrophotometric method, while patterns of carbonylated protein in embryo axes were analyzed by immunochemical techniques. The highest concentration of protein carbonyl groups was observed in dormant embryos. It declined in germinating embryos pre-treated with NO or HCN, suggesting elevated degradation of modified proteins during seedling formation. A decrease in the concentration of carbonylated proteins was accompanied by modification in proteolytic activity in germinating apple embryos. A strict correlation between the level of protein carbonyl groups and cotyledon growth and greening was detected. Moreover, direct in vitro carbonylation of BSA treated with NO or HCN donors was analyzed, showing action of both signaling molecules as protein oxidation agents.

Tissue-specific expression of SORBITOL DEHYDROGENASE in apple fruit during early development.

Sorbitol, the primary photosynthate and translocated carbohydrate in apple (Malusxdomestica Borkh.), is converted to fructose by sorbitol dehydrogenase (SDH; EC 1.1.1.14) which is active in apple fruit throughout development. In the apple genome, nine SDH genes have been isolated and their sequences characterized, but their individual expression patterns during apple fruit set and development have not been determined. The objective of this work was to ascertain if SDH genes are differentially expressed and how their patterns of expression may relate to SDH activity in apple seed and cortex during early fruit development. Seed SDH activity was found to be much higher than cortex SDH activity per mg and g fresh weight (FW), and seed SDH activity contributed significantly to whole fruit SDH activity during weeks 2-5 after bloom. Five of the nine SDH genes present in the apple genome were expressed in apple fruit. Two SDH genes, SDH1 and SDH3, were expressed in both seed and cortex tissues. SDH2 expression was limited to cortex, while SDH6 and SDH9 were expressed in seed tissues only. SDH isomeric proteins of different pI values were detected in apple fruit. SDH isomers with pI values of 4.2, 4.8, 5.5, and 6.3 were found in seeds, and SDH isomers with pI values of 5.5, 6.3, 7.3, and 8.3 were found in cortex. The present work is the first to show that SDH is highly active in apple seed and that SDH genes are differentially expressed in seed and cortex during early development.

In vitro androgenesis in apple--improvement of the induction phase.

Based on a protocol for microspore culture in apple ( Malus domestica Borkh.), the embryo induction phase has been improved with regard to pretreatment of microspores for initiation of microspore embryogenesis, the concentration of carbon source in the induction medium and the microspore density in the suspension. Furthermore, the effect of the genotype was studied. To determine the efficiency of in vitro androgenesis, both methods, via anther and microspore culture, were investigated using the same bud material. A comparison of the efficiency of embryo induction in anther and microspore cultures showed that microspore culture resulted in an increase up to 10 times, depending on the genotype. The regeneration route in microspore culture is similar to that of androgenic embryos via anther culture and showed adventitious shoot formation in most cases after a long period of secondary embryogenesis.