Transcriptome analysis of Pinus halepensis under drought stress and during recovery
. Tree Physiology 2018
, 423-441. Publisher's VersionAbstract
Forest trees use various strategies to cope with drought stress and these strategies involve complex molecular mechanisms. Pinus halepensis Miller (Aleppo pine) is found throughout the Mediterranean basin and is one of the most drought-tolerant pine species. In order to decipher the molecular mechanisms that P. halepensis uses to withstand drought, we performed large-scale physiological and transcriptome analyses. We selected a mature tree from a semi-arid area with suboptimal growth conditions for clonal propagation through cuttings. We then used a high-throughput experimental system to continuously monitor whole-plant transpiration rates, stomatal conductance and the vapor pressure deficit. The transcriptomes of plants were examined at six physiological stages: pre-stomatal response, partial stomatal closure, minimum transpiration, post-irrigation, partial recovery and full recovery. At each stage, data from plants exposed to the drought treatment were compared with data collected from well-irrigated control plants. A drought-stressed P. halepensis transcriptome was created using paired-end RNA-seq. In total, ∼6000 differentially expressed, non-redundant transcripts were identified between drought-treated and control trees. Cluster analysis has revealed stress-induced down-regulation of transcripts related to photosynthesis, reactive oxygen species (ROS)-scavenging through the ascorbic acid (AsA)-glutathione cycle, fatty acid and cell wall biosynthesis, stomatal activity, and the biosynthesis of flavonoids and terpenoids. Up-regulated processes included chlorophyll degradation, ROS-scavenging through AsA-independent thiol-mediated pathways, abscisic acid response and accumulation of heat shock proteins, thaumatin and exordium. Recovery from drought induced strong transcription of retrotransposons, especially the retrovirus-related transposon Tnt1-94. The drought-related transcriptome illustrates this species' dynamic response to drought and recovery and unravels novel mechanisms. © The Author 2017.
Quantitative Trait Loci Analysis Identifies a Prominent Gene Involved in the Production of Fatty Acid-Derived Flavor Volatiles in Tomato
. Molecular Plant 2018
, 1147-1165. Publisher's VersionAbstract
To gain insight into the genetic regulation of lipid metabolism in tomato, we conducted metabolic trait loci (mQTL) analysis following the lipidomic profiling of fruit pericarp and leaf tissue of the Solanum pennellii introgression lines (IL). To enhance mapping resolution for selected fruit-specific mQTL, we profiled the lipids in a subset of independently derived S. pennellii backcross inbred lines, as well as in a near-isogenic sub-IL population. We identified a putative lecithin:cholesterol acyltransferase that controls the levels of several lipids, and two members of the class III lipase family, LIP1 and LIP2, that were associated with decreased levels of diacylglycerols (DAGs) and triacylglycerols (TAGs). Lipases of this class cleave fatty acids from the glycerol backbone of acylglycerols. The released fatty acids serve as precursors of flavor volatiles. We show that LIP1 expression correlates with fatty acid-derived volatile levels. We further confirm the function of LIP1 in TAG and DAG breakdown and volatile synthesis using transgenic plants. Taken together, our study extensively characterized the genetic architecture of lipophilic compounds in tomato and demonstrated at molecular level that release of free fatty acids from the glycerol backbone can have a major impact on downstream volatile synthesis. In this work, we conducted large-scale lipid profiling of fruit pericarp and leaf materials of a population of S. pennellii introgression line to gain insight into the genetic regulation of lipid metabolism in tomato. By combination of QTL mapping, metabolic and transcriptomic data, we identified and cloned novel lipid–genes that have a major effect on production of multiple fatty acid-derived flavor volatiles. These metabolites are positively correlated with consumer liking and are crucial for key agronomical traits in tomato. © 2018 The Author
The effect of irrigation level in the kernel dry matter accumulation period on almond yield, kernel dry weight, fruit count, and canopy size
. Irrigation Science 2018
. Publisher's VersionAbstract
The effect of irrigation regime in the kernel filling stage of almond was examined in a field experiment. The experiment was conducted on 9-year-old local variety (Um-El-Fahem) grafted on GF677 rootstock in Israel. Five irrigation treatments were applied during the main kernel dry matter accumulation. Irrigation rates in June varied from 1 to 8 mm/day and midday stem water potentials varied from −2.6 to −1.3 MPa. Seasonal irrigation varied from 394 to 801 mm. Kernel yield increased in the high irrigation treatments in the first four seasons where it decreased in the three lower irrigation treatments. Similarly, the four-season trunk cross-sectional area accumulation increased with increasing irrigation. Kernel yield increased with both midday stem water potential and irrigation rate. Kernel dry weight decreased with increasing fruit count where higher kernel weights were found in the higher irrigation treatments at similar fruit count. Kernel relative growth rates of all treatments were similar along the dry matter accumulation stage except for around 1 June where the lowest irrigation rate had significantly lower growth rate. Spurs survival analysis showed that the number of fruits per spur, fruiting spurs, and alive spurs increased with increasing irrigation. © 2017, Springer-Verlag GmbH Germany.
Water availability dynamics have long-term effects on mature stem structure in Vitis vinifera
. American Journal of Botany 2018
, 1443-1452. Publisher's VersionAbstract
Premise of the Study: The stem of Vitis vinifera, a climbing vine of global economic importance, is characterized by both wide and narrow vessels and high specific hydraulic conductivity. While the effect of drought stress has been studied in 1- and 2-yr-old stems, there are few data documenting effects of drought stress on the anatomical structure of the mature, woody stem near the base of the vine. Here we describe mature wood anatomical responses to two irrigation regimes on wood anatomy and specific hydraulic conductivity in Vitis vinifera Merlot vines. Methods: For 4 years, irrigation was applied constantly at low, medium, or high levels, or at alternating levels at two different periods during the growing season, either early spring or late summer, resulting in late season or early spring deficits, respectively. The following variables were measured: trunk diameter, annual ring width and area, vessel diameter, specific hydraulic conductivity and stem water potential. Key Results: High water availability early in the season (late deficit) resulted in vigorous vegetative growth (greater trunk diameter, ring width and area), wider vessels and increased specific hydraulic conductivity. High water availability early in the season caused a shift of the vessel population towards the wider frequency classes. These late deficit vines showed more negative water potential values late in the season than vines that received low but relatively constant irrigation. Conclusions: We concluded that high water availability during vegetative growth period of Vitis increases vessels diameter and hydraulic conductivity and causes the vines to be more vulnerable to drought stress late in the season. © 2018 Botanical Society of America
Controlling the seed bank of the invasive plant Acacia saligna: comparison of the efficacy of prescribed burning, soil solarization, and their combination
. Biological Invasions 2018
, 2875-2887. Publisher's VersionAbstract
Reducing the seed bank of invasive plants is a prerequisite for successful restoration of invaded ecosystems. The Australian legume tree Acacia saligna is one of the worst invasive plants in Mediterranean climate regions. This fire-adapted species possesses a large persistent seed bank characterized by physical dormant seeds. The present research was aimed to compare the efficacy of prescribed burning, soil solarization by solar heating of moist soil, and their use in combination on A. saligna seed bank reduction, using the buried seed method, and on seedling emergence from the natural seed bank, as a model of controlling fire-adapted plants. The data obtained show that the direct effect of prescribed burning on the buried seed dynamics was highly variable, and it only reduced seed viability from 98% to about 56%. Soil solarization, particularly in combination with prescribed burning, was much more effective than prescribed burning alone, reducing seed viability to about 29 and 4%, respectively. These results were confirmed by recording seedling emergence from the natural seed bank during two successive germination years following the treatments. Only a relatively very small number of seedlings emerged in the soil solarization treatment and none in the combined treatment. Based on the above data, it is recommended to apply prescribed burning as a pretreatment for soil solarization, or to utilize wild fires followed by soil solarization to reduce the seed bank of invasive fire-adapted plants. In situations in which fire cannot be used as a pretreatment, soil solarization alone is reasonably effective. © 2018, Springer International Publishing AG, part of Springer Nature.
Rooting of cuttings of selected Diospyros virginiana clonal rootstocks and bud growth in rooted cuttings
. Scientia Horticulturae 2018
, 13-21. Publisher's VersionAbstract
Diospyros virginiana is a common rootstock in several countries growing commercial plantations of persimmon. However, trees grafted on this rootstock, which is exclusively propagated from seeds, are not uniform in both vegetative and reproductive growth. The aim of the present research was to study the effect of various factors on the rooting of D. virginiana cuttings, in order to develop an efficient rooting procedure for selected clonal rootstocks. The clone genotype had a significant effect on the rooting rate, which ranged from zero to close to 100% in the various clones examined. Cuttings collected from different positions on the mother plant shoots did not differ significantly in their rooting rate, suggesting that juvenility does not play an important role in rooting of D. virginiana cuttings. Collecting cuttings at the early stage of the growing season yielded the highest rooting rate, which gradually decreased as the growing season progressed. Using three to 4-node cuttings and treatment with 6000 mg L−1 indole-3-butyric acid (IBA) by quick-dip in solution led to a relatively high rooting rate. Anatomical analysis demonstrated that adventitious root primordia were initiated at the phloem side of the fascicular cambium, with no relationship to the callus tissue that was formed on the cut surface of the cutting base. Out of the examined clones, rooted cuttings of only one clone exhibited a high rate of bud growth, a phenomenon which was also found to be genotype-dependent. Increasing the applied IBA concentration to cuttings of a clone with a high bud growth rate inhibited bud growth, whereas increasing the length of cuttings of clones with a low bud growth rate promoted bud growth. These observations suggest that the IBA applied to promote rooting is transported acropetally and inhibits bud growth. No cutting mortality was observed during rooting and above 90% of the rotted cuttings which exhibited bud growth survived after transplanting into the growing pots. © 2017 Elsevier B.V.
The Tomato Hybrid Proline-rich Protein regulates the abscission zone competence to respond to ethylene signals
. Horticulture Research 2018
. Publisher's VersionAbstract
The Tomato Hybrid Proline-rich Protein (THyPRP) gene was specifically expressed in the tomato (Solanum lycopersicum) flower abscission zone (FAZ), and its stable antisense silencing under the control of an abscission zone (AZ)-specific promoter, Tomato Abscission Polygalacturonase4, significantly inhibited tomato pedicel abscission following flower removal. For understanding the THyPRP role in regulating pedicel abscission, a transcriptomic analysis of the FAZ of THyPRP-silenced plants was performed, using a newly developed AZ-specific tomato microarray chip. Decreased expression of THyPRP in the silenced plants was already observed before abscission induction, resulting in FAZ-specific altered gene expression of transcription factors, epigenetic modifiers, post-translational regulators, and transporters. Our data demonstrate that the effect of THyPRP silencing on pedicel abscission was not mediated by its effect on auxin balance, but by decreased ethylene biosynthesis and response. Additionally, THyPRP silencing revealed new players, which were demonstrated for the first time to be involved in regulating pedicel abscission processes. These include: gibberellin perception, Ca2+-Calmodulin signaling, Serpins and Small Ubiquitin-related Modifier proteins involved in post-translational modifications, Synthaxin and SNARE-like proteins, which participate in exocytosis, a process necessary for cell separation. These changes, occurring in the silenced plants early after flower removal, inhibited and/or delayed the acquisition of the competence of the FAZ cells to respond to ethylene signaling. Our results suggest that THyPRP acts as a master regulator of flower abscission in tomato, predominantly by playing a role in the regulation of the FAZ cell competence to respond to ethylene signals. © 2018 The Author(s).
Onions and allied crops: Volume I: Botany, physiology, and genetics
; Onions and Allied Crops: Volume I: Botany, Physiology, and Genetics; 2018; pp. 1-273. Publisher's VersionAbstract
First Published in 2018. Routledge is an imprint of Taylor & Francis, an Informa company. © 1990 by CRC Press, LLC.
Physiology of flowering
; Onions and Allied Crops: Volume I: Botany, Physiology, and Genetics; 2018; pp. 113-134. Publisher's VersionAbstract
Flowering of onions is of great economical importance during the two growing seasons. When grown for bulbs, bolting plants - those which flower during the first season - are of very poor quality. The flower stalk of the onion, as in all Alliums, is an apical extension of the stem, but without histological differentiation into nodes and intern odes. Following induction and initiation, the differentiated central axis starts protruding and the scape is formed. The flower stalks of most commercially available cultivars reach a final length of 1 to 2 m, but flowering may also occur, though very rarely, within the fleshy scales of the bulb. The buds are grouped in small cymes of five to ten flowers each; the umbel is entirely enveloped by a spathe. This thin sheathing bract splits open when flowering begins. Temperature, day length and a level of nitrogen fertilizer seem to be the important environmental factors affecting flowering of the bulb onion. © 1990 by CRC Press, LLC.
; Onions and Allied Crops: Volume I: Botany, Physiology, and Genetics; 2018; pp. 151-159. Publisher's VersionAbstract
Onion seed set and development are markedly affected by a number of factors, the most important ones being growth conditions, availability of pollinating insects, and the genetic make up of the individual plant. Cross-pollination is predominant in onions and leeks, in chives and most probably also in other alliums. However, onions are self-compatible, and regardless of earlier accounts, protandry does not prevent their self-pollination. In onions, pollen shed begins at anthesis and continues at irregular intervals for the next 24 to 36 h and may last up to 2 d. High temperatures and air humidities below 70% accelerate the process. Twelve to twenty four hours after pollination, the first pollen tubes with two sperm-nuclei, enter the micropyle. Onion seeds are produced in a wide range of environments, and weather conditions may change considerably during seed growth and ripening on the mother plant. These influence seed composition and subsequent performance. © 1990 by CRC Press, LLC.
Temporal and spatial effect of low pre-planting temperatures on plant architecture and flowering in bolting garlic
. Scientia Horticulturae 2018
, 69-75. Publisher's VersionAbstract
Garlic flowering and bulbing are prone to photo-thermal regulation during its annual cycle. As in many other geophytes, these pathways are parallel but competitive, and can be manipulated by the environment. Both flowering and bulbing are of paramount physiological value, and of great horticultural interest. We argue that, in bolting garlic, differential regulation of only one of the two pathways by pre-planting vernalization is unfeasible, and that garlic's response to cold shows an optimal curve. Within limits, long vernalization treatments have resulted in rapid development of ‘Reproductive and Bulbing Phenotype’ with fast leaf elongation, early transition of the apical meristem to the reproductive state, development of axillary buds, flowering and bulbing. Low temperatures trigger primary signaling components, thus modulating organogenesis even under a relatively short photoperiod. We therefore propose that under a suboptimal photoperiod, favorable temperatures could substitute the plant's requirements for photoperiod and signal for the meristem transition, flowering and bulbing. The optimum response of the studied genotype was obtained after vernalization of four weeks at 4 °C. No transition of the apical meristem was evident in plants exposed to only short or no vernalization, namely the apex remained vegetative. These plants continuously produced foliage leaves, thus forming a “Leafy Phenotype” which did not branch, bulb or flower. Comprehension of the plant's response to environment is expected to facilitate physiological manipulations on the production of either bulbs or true seeds in garlic. © 2018
Evaluation of resistance to Blumeria graminis (DC.) f. sp. avenae, in Avena murphyi and A. magna genotypes
. Crop Protection 2018
, 177-181. Publisher's VersionAbstract
Powdery mildew is one of the main factors reducing oat yield quality and quantity. New sources of resistance are needed in many breeding programs. The most effective sources of resistance to fungal diseases in cereals have often been found in lower-ploidy species. Thus, tetraploid species could be valuable source of resistance to powdery mildew. The aim of the present study was to identify resistance among tetraploid genotypes that could be used in breeding programmes to increase the level of resistance to powdery mildew in oat. Sixty two Avena magna and 17 A. murphyi accessions were tested and all showed high level of resistance to oat powdery mildew. None of the accessions were completely susceptible to the tested pathogen isolates. Twelve A. magna and six A. murphyi accessions were resistant to all five isolates of the powdery mildew pathogen tested. The rest of the accessions showed a resistant or an intermediate response. The results of the present study show that there are several unexplored resistance sources among A. magna and A. murphyi. Because of the genomic similarity of tetraploid species to hexaploid oats the introduction of the new resistance sources in cultivated oats is promising. © 2017 Elsevier Ltd
Asexual reproduction of weeds
; Weed Physiology: Volume I: Reproduction and Ecophysiology; 2018; pp. 65-100. Publisher's VersionAbstract
Weeds are plant species that adapted to man-made habitats, 1 and are usually innocuous wild plants in their geographical center of origin. 2 They are unintentionally spread and perpetuated by the activities of man and his domesticated animals, including those that have unintentionally become domesticated. Most weeds are herbaceus, but some shrubs and trees, such as Prosopis spp., Acacia spp., and Rubus spp., and cacti, such as Opuntia spp., are extremely noxious weeds in certain habitats. © 1985 by CRC Press, Inc.
Cattle foraging in Mediterranean oak woodlands – Effects of management practices on the woody vegetation
. Forest Ecology and Management 2018
, 160-169. Publisher's VersionAbstract
Vegetation structure and composition of woodlands in the Mediterranean Basin have experienced extensive land-use change during recent decades. Decline in traditional foraging by goats is leading to more closed and spatially homogeneous woody vegetation, reduced plant diversity, and increased fire risk because of accumulation of inflammable material. We studied the use of cattle foraging as an alternative to goat foraging in Mediterranean oak woodlands. Our main goal was to provide basic information on the responses of woody vegetation to cattle foraging intensity, and on the factors affecting spatial patterns of woodland utilization. We conducted the study in the Western Galilee, Israel, in oak woodland dominated by Palestine oak (Quercus calliprinos Webb.) interspersed with patches of shrubs and herbaceous vegetation. Effects of two animal population densities, moderate (0.33 cow⋅ha−1) and high (0.55 cow⋅ha−1), on the structure, composition, and regeneration potential of dense and of open woody formations were examined. Four consecutive annual seasons of cattle foraging resulted in relatively large amounts of woody vegetation removal, especially under high animal density, but had no negative effects on woody species richness or regeneration potential from saplings. The type of vegetation formation and initial state of the woody vegetation were important factors affecting the degree of change. Woody biomass removal by cattle, as shown in this study, can reduce fire hazards and increase vegetation heterogeneity and plant diversity. These findings support the use of cattle as an efficient alternative tool for multi-purpose, sustainable management of Mediterranean oak woodlands. © 2018 Elsevier B.V.
Development and ecophysiology of amaranths
; Amaranth Biology, Chemistry, and Technology; 2018; pp. 39-73. Publisher's VersionAbstract
Amaranthus (Amaranthaceae) is a genus of tropical origin but widely distributed all over the world, including temperate regions. 1,2About 50 species are native to the Americas and another 15 can be found in Europe, Asia, Africa, and Australia. Most amaranth species are pioneer nitrophilous annuals of open habitats and produce many small dormant seeds. This extensive seed production is associated with prolonged seed-dormancy and with prompt germination after soil disturbance and seed exposure to light. This assemblage of reproductive traits enables amaranths to survive by continuous colonization of new disturbed sites with full sunlight and little competition from other plants. With such strategy of reproduction it is not surprising that several species of amaranth were preadapted for invasion of habitats drastically modified by human activities. Thus, today, amaranths are best known as opportunistic weeds, associated with soil cultivation (e.g., A. hybridus L., A. powellii S. Watt., A. retroflexus L., A. spinosus L., etc.). However, other amaranth species have been domesticated in tropical and subtropical regions and are cultivated for their grain (A. hypochondriacus L., A. cruentus L., A. caudatus L. = A. edulis Spegazzini) or for leaf consumption (A. tricolor L. = A. gangeticus L., A. viridis L. = A. gracilis Desf., A. blitum L. = A. lividus L.). © 1994 by CRC Press, Inc.
; Handbook of Flowering: Volume I; 2018; pp. 427-433. Publisher's VersionAbstract
Amaranthus is a genus of tropical origin that belongs to the Amaranthaceae. Today it is widely distributed all over the world, with species reaching temperate regions even before man converted some of them into cosmopolitan weeds or domesticates (crops or ornamentals).15 About 60 species are native to the Americas, and about 15 others to Europe, Asia, Africa, and Australia. Most of them are pioneer annuals of naturally open habitats, producing abundant seed. They thrive by constant colonization of sites of disturbed soil, with full sun and little competition. Long natural selection for such a way of life preadaptated certain of the species for success in habitats drastically disturbed by man. Thus, today Amaranthus spp. are best known as noxious weeds (e.g., A. hybridus L., A. retroflexus L., A. spinosus L., A. powellii S. Wats.). However, in tropical and subtropical countries, certain species are grown for grain (A. hypocondriacus L., A. cruentus L., A. caudatus L.), whereas the young plants of others are eaten as pot-herbs (A. dubius Mart., A. tricolor L.). Other species are cultivated as ornamentals for their highly colored inflorescence (A. caudatus L.) or leaves (A. tricolor L.). © 1995 by CRC Press, Inc.
Application Timing and Degradation Rate of Sulfosulfuron in Soil Co-affect Control Efficacy of Egyptian broomrape (Phelipanche aegyptiaca) in Tomato
. Weed Science 2018
, 780-788. Publisher's VersionAbstract
Egyptian broomrape (Phelipanche aegyptiaca Pers.) is a root-parasitic weed that severely damages many crops worldwide, including tomato (Solanum lycopersicum L.). In Israel, the management protocol used for P. aegyptiaca in open-field tomato includes PPI sulfosulfuron at 37.5 g ai ha-1 to the top 10-cm soil layer. The objective of this study was to investigate the co-effect of sulfosulfuron application timing and variable degradation rate in soil on the control efficacy of P. aegyptiaca in tomato. Degradation of sulfosulfuron (80ng g-1 soil) at a temperature of 15C, measured in soil samples from three farms using liquid chromatography-tandem mass spectrometry, followed a first-order kinetics with variable degradation rate constant among sites (0.008 to 0.012 d-1). Incubation at 25 C increased sulfosulfuron degradation rate constant by a factor of 2 to 2.7 in soils from the different sites, with a similar degradation rate order among soils. A higher degradation rate in the soil resulted in a shorter period of residual activity, measured using a sorghum [Sorghum bicolor (L.) Moench.] bioassay. Phelipanche aegyptiaca management in open-field tomatoes was investigated in five independent field experiments. Sulfosulfuron soil concentration throughout the growing season (following preplant incorporation of 37.5 g ha-1) was calculated from laboratory-measured degradation rates, which were corrected to represent the effect of recorded temperatures at each field. At the end of the tomato growing season, control efficacy of P. aegyptiaca varied among experiments (70.4% to 100%) and positively correlated with predicted sulfosulfuron concentration at the critical period for seedling control (R2=0.67). The current study confirms that sulfosulfuron is degraded in soil to nonphytotoxic metabolites and that rapid degradation rates would result in reduced injury to P. aegyptiaca seedling and, consequently, lower control efficacy. © Weed Science Society of America, 2018.
An evolutionary platform for alternate bearing in fruit trees
. Acta Horticulturae 2018
, 1-7. Publisher's VersionAbstract
Absract Alternate bearing has been and still is a major problem in fruit trees. From the point of view of a horticulturist it is a tree productivity syndrome which must be addressed by agro-technical means in order to eliminate or, at least, mitigate the alternation cycle. ‘Masting’ is a widespread forest tree irregular sexual reproduction pattern that has attracted ecologists who wonder about the survival-advantage of this behavior. From an evolutionary perspective alternate bearing and masting seem to be related and may share a common bio-evolutionary mechanism, responsible for ‘yield alternation’. A recently developed resource budget model, based on nonlinear chaos dynamics, seems to account for a broad spectrum of yield alternation patterns. From an evolutionary standpoint, one may envision a domestication-evolutionary continuum from extreme masting, through intermediate alternate bearing, down to regular yield of fully-managed fruit trees. Domestication records of pecan (Carya illinoiensis) support this hypothesis. The alternate bearing of fruit trees appears to be mediated by a ‘fruit overload’ signal but resource depletion still plays a critical role in the alternate bearing of numerous tree crops. © 2018 International Society for Horticultural Science. All Rights Reserved.
Isolation of Intact Vacuoles from Petunia Petals and Extraction of Sequestered Glycosylated Phenylpropanoid Compounds
. Bio-protocol 2018
e2912. Publisher's VersionAbstract
Plant vacuoles are the largest compartment in plant cells, occupying more than 80% of the cell volume. A variety of proteins, sugars, pigments and other metabolites are stored in these organelles (Paris et al., 1996; Olbrich et al., 2007). Flowers produce a variety of specialized metabolites, some of which are unique to this organ, such as components of pollination syndromes, i.e., scent volatiles and flavonoids (Hoballah et al., 2007; Cna'ani et al., 2015). To study the compounds stored in floral vacuoles, this compartment must be separated from the rest of the cell. To enable isolation of vacuoles, protoplasts were first generated by incubating pierced corollas with cellulase and macrozyme enzymes. After filtering and several centrifugation steps, protoplasts were separated from the debris and damaged/burst protoplasts, as revealed by microscopic observation. Concentrated protoplasts were lysed, and vacuoles were extracted by Ficoll-gradient centrifugation. Vacuoles were used for quantitative GC-MS analyses of sequestered metabolites. This method allowed us to identify vacuoles as the subcellular accumulation site of glycosylated volatile phenylpropanoids and to hypothesize that conjugated scent compounds are sequestered in the vacuoles en route to the headspace (Cna'ani et al., 2017).
Expression and Immunogenicity of M2e Peptide of Avian Influenza Virus H5N1 Fused to Ricin Toxin B Chain Produced in Duckweed Plants
. Frontiers in Chemistry 2018
22. Publisher's VersionAbstract
The amino acid sequence of the extracellular domain of the virus-encoded M2 matrix protein (peptide M2e) is conserved among all subtypes of influenza A strains, enabling the development of a broad-range vaccine against them. We expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005 (H5N1) in nuclear-transformed duckweed plants for further development of an avian influenza vaccine. The 30-amino acid N-terminal fragment of M2, including M2e (denoted M130), was selected for expression. The M2e DNA sequence fused in-frame to the 3′ end of ricin toxin B chain (RTB) was cloned under control of the CaMV 35S promoter into pBI121. The resulting plasmid was used for duckweed transformation, and 23 independent transgenic duckweed lines were obtained. Asialofetuin-binding ELISA of protein samples from the transgenic plants using polyclonal anti-RTB antibodies confirmed the expression of the RTB–M130 fusion protein in 20 lines. Quantitative ELISA of crude protein extracts from these lines showed RTB–M130 accumulation ranging from 0.25–2.5 μg/g fresh weight (0.0006–0.01% of total soluble protein). Affinity chromatography with immobilized asialofetuin and western blot analysis of protein samples from the transgenic plants showed expression of fusion protein RTB–M130 in the aggregate form with a molecular mass of about 70 kDa. Mice were immunized orally with a preparation of total soluble protein from transgenic plants, receiving four doses of 7 μg duckweed-derived RTB–M130 each, with no additional adjuvant. Specific IgG against M2e was detected in immunized mice, and the endpoint titer of nti-M2e IgG was 1,024. It was confirmed that oral immunization with RTB-M130 induces production of specific antibodies against peptide M2e, one of the most conserved antigens of the influenza virus. These results may provide further information for the development of a duckweed-based expression system to produce a broad-range edible vaccine against avian influenza.