Regions Of Epicotyl Research Articles

Phenotyping for Waterlogging Tolerance as a Proxy for Phytophthora medicaginis Resistance in Chickpea

Phytophthora root rot (PRR) caused by the soilborne oomycete Phytophthora medicaginis is a significant constraint to chickpea (Cicer arietinum) production across the northern grains region of Australia. In flooded soil, which is conducive to PRR disease development, up to 70% yield loss can occur in the most resistant Australian cultivars. Incorporating waterlogging tolerance in soybean (Glycine max) has been shown to improve quantitative resistance to Phytophthora sojae. Root growth of three chickpea genotypes was assessed at the seedling stage under waterlogging, PRR, and the combination of these abiotic and biotic constraints. Levels of waterlogging tolerance in chickpea are inherently low, yet selected genotypes displayed variability in root traits linked to improved waterlogging tolerance. The PRR moderately susceptible chickpea cultivar Yorker and PRR very susceptible Rupali demonstrated an eightfold increase in early adventitious root growth from the epicotyl region under waterlogging stress, compared with the PRR resistant interspecific backcross genotype 04067-81-2-1-1 (C. echinospermum × C. arietinum*2). Selection for primary root depth, which was significantly greater in 04067-81-2-1-1 under waterlogging, appears to improve PRR resistance compared with root replacement traits. Soilborne Phytophthora spp. are reportedly attracted to branch sites and leached exudates. We propose that compromised root barriers at emergence sites of adventitious roots under waterlogging conditions hasten hyphal entry, potentially increasing susceptibility to PRR. Hence, screening for root depth and absence of adventitious root development under waterlogged conditions may offer a novel proxy phenotyping method for PRR resistance traits at early stages of chickpea breeding.

Dehydrocostus lactone, a naturally occurring polar auxin transport inhibitor, inhibits epicotyl growth by interacting with auxin in etiolated Pisum sativum seedlings

We have isolated germacranolide-type sesquiterpene lactones with an α-methylene-γ-lactone moiety, dehydrocostus lactone (DHCL), costunolide, santamarine, and a novel compound denoted artabolide [3-hydroxy-4,6,7(H)-germacra-1(10),11(13)-dien-6,12-olide] from oriental medicinal Asteraceae plants as novel naturally occurring inhibitors of polar auxin transport detected by the radish hypocotyl bioassay. To investigate the mode of action of natural sesquiterpene lactones on the inhibition of polar auxin transport as well as its relation to the growth of seedlings, the function of DHCL on growth and auxin dynamics in etiolated pea seedlings was studied intensively. DHCL reduced polar auxin transport in a dose-dependent manner together with the inhibition of the accumulation of mRNA of <em>PsAUX1</em> and <em>PsPIN1</em> genes encoding influx and efflux carrier proteins of auxin, respectively. DHCL applied to the apical hook region as a lanolin paste substantially inhibited elongation growth in the subapical region of epicotyls in intact etiolated pea seedlings, coupled with a significant reduction of endogenous levels of indole-3-acetic acid (IAA). DHCL also revealed the inhibition of IAA-induced cell elongation in etiolated pea epicotyl segments by affecting IAA-induced changes in the mechanical properties of cell walls. These facts suggest that germacranolide-type sesquiterpene lactones with an α-methylene-γ-lactone moiety affect the expression of <em>PsAUX1</em> and <em>PsPINs</em> genes, and then inhibit polar auxin transport and reduce endogenous levels of IAA necessary for stem growth in etiolated pea seedlings. These compounds are also suggested to show the inhibitory effects on auxin action in pea stem growth.

Regulation of asymmetric polar auxin transport by PsPIN1 in endodermal tissues of etiolated Pisum sativum epicotyls: focus on immunohistochemical analyses.

This manuscript reportsthe production of specific polyclonal antibodies for PsPIN1, a putative auxin efflux carrier in Alaska pea (Pisum sativum L.) plants, and the cellular immunolocalization of PsPIN1. When pea seeds were set with the seed axis horizontal to the upper surface of a rockwool block, and allowed to germinate and grow for 3days in the dark, the epicotyl grew upward. On the other hand, the application of 2,3,5-triiodobenzoic acid (TIBA) inhibited graviresponse. In the subapical epicotyl regions, PsPIN1 has been found to localize in the basal side of the plasma membrane of cells in endodermal tissues. Asymmetric PsPIN1 localization between the proximal and distal sides of the epicotyl was observed, the total amounts of PsPIN1 being more abundant in the proximal side. The asymmetric PsPIN1 distribution between the proximal and distal sides of the epicotyl was well correlated with unequal polar auxin transport as well as asymmetric accumulation of mRNA of PsPIN1 (Ueda et al. in Biol Sci Space 26:32-41, 2012; Ueda et al. in Plant Biol 16(suppl 1):43-49, 2014). In the proximal side of an apical hook, PsPIN1 localized in the basal side of the plasma membrane of cells in endodermal tissues, whereas in the distal side, the abundant distribution of PsPIN1 localized in the basal-lower (endodermal) side of the basal plasma membrane, suggesting possible lateral auxin movement from the distal side to the proximal side in this region. The application of TIBA significantly reduced the amount of PsPIN1 in the proximal side of epicotyls, but little in the distal side. These results suggest that unequal auxin transport in epicotyls during the early growth stage of etiolated pea seedlings is derived from asymmetric PsPIN1 localization in the apical hook and subapical region of epicotyls, and that asymmetric transport between the proximal and distal sides of epicotyls is required for the graviresponse of epicotyls.

Changes in the transcript levels of microtubule-associated protein MAP65-1 during reorientation of cortical microtubules in azuki bean epicotyls

The relation between orientation of cortical microtubules and the transcript levels of a 65 kDa microtubule-associated protein (VaMAP65-1) was investigated along epicotyls of azuki bean (Vigna angularis) seedlings. In addition, the effects of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, and hypergravity, a gravitational force exceeding 1g, on the relation were examined. The percentage of cells with longitudinal microtubule increased, whereas that with transverse microtubules decreased from the apical to the basal regions of epicotyls. The transcript levels of VaMAP65-1 decreased toward the basal region. ACC induced reorientation of cortical microtubules from transverse to longitudinal directions and down-regulation of VaMAP65-1 expression. Hypergravity also induced reorientation of cortical microtubules and down-regulation of the expression. Strong correlations were observed between the percentage of cells with longitudinal or transverse microtubules and the transcript levels of VaMAP65-1. These results suggest that down-regulation of VaMAP65-1 expression is at least partly involved in the regulation of the orientation of cortical microtubules in azuki bean epicotyls.

Cell wall-bound peroxidase activity and lignin formation in azuki bean epicotyls grown under hypergravity conditions

The effects of accelerated gravity stimuli on the cell wall-bound peroxidase activity and the lignin content were investigated along epicotyls of azuki bean (Vigna angularis) seedlings. The endogenous growth occurred primarily in the upper regions of the epicotyl, but no growth was detected in the middle or basal regions. Hypergravity treatment at 300g for 6h suppressed elongation growth and stimulated lateral expansion of the upper regions. The content of acetyl bromide-soluble lignin increased gradually from the apical to the basal regions of epicotyls. Hypergravity treatment stimulated the increase in the lignin content in epicotyls, particularly in the middle and basal regions. The peroxidase activity in the protein fraction extracted with a high ionic strength buffer from the cell wall preparation also increased gradually toward the basal region, and hypergravity treatment increased the activity in all epicotyl regions. There was a close correlation between the lignin content and the enzyme activity. These results suggest that hypergravity increases the activity of cell wall-bound peroxidase followed by increases of the lignin formation in epicotyl cell walls, which may contribute to increasing the rigidity of cell walls against the gravitational force.

Germination and conversion of somatic embryos derived from mature Quercus robur trees: the effects of cold storage and thidiazuron

Somatic embryos of Quercus robur L. belonging to lines derived from mature trees were germinated in experiments designed to determine the effects of pregermination chilling and the temporary inclusion of thidiazuron (TDZ) in the germination medium. Germination response was evaluated in terms of the percentages of embryos exhibiting root-only elongation, shoot-only elongation, or both (conversion to plantlets). Conversion rates depended on genotype and were lower than those of oak zygotic embryonic axes germinated in vitro. Two months’ storage of somatic embryos at 4°C between maturation and germination treatments did not influence their conversion frequency, although root-only germination was promoted in the three embryogenic lines investigated. Addition of 0.01–5.0 µM TDZ to the germination medium for 7 or 18 days induced multiple shoot formation in the epicotyl region of germinating embryos, although the 18-day treatment was detrimental with TDZ concentrations of 0.5–5.0 µM. Exposure to 0.05–0.1 µM TDZ for 7 days increased the frequency of shoot-only elongation and decreased that of root-only elongation in embryogenic lines with both high and low conversion ability, but whereas the conversion rate of the hard-to-convert line Sainza was significantly increased, that of the readily converted line B-17 was negatively affected. As a result, with 0.1 µM TDZ the total potential plant recovery of Sainza (the conversion rate plus the percentage of embryos exhibiting shoot-only germination) was 64%, approximately the same as that of B-17. Shoots excised from shoot-only germinating embryos can be elongated and rooted by proven micropropagation procedures. An additional result was the finding that adventitious bud regeneration was induced on the cotyledons of TDZ treated embryos (69% with 0.5 µM TDZ), which may prove useful for genetic transformation. This study is the first to have shown that TDZ can increase the germination efficiency of oak somatic embryos.

Modifications of xyloglucan metabolism in azuki bean epicotyls under hypergravity conditions

We examined the effects of hypergravity on both the synthesis and the depolymerization processes of xyloglucan metabolisms. Azuki bean cuttings were incubated with [6- 3H]- l-fucose, and the levels and the molecular size of synthesized xyloglucans were investigated in the upper growing regions of epicotyls. The amounts of radioactivity incorporated into xyloglucan fraction increased during incubation, irrespective of the gravity conditions. 3H-Xyloglucans were eluted in the higher molecular mass regions than xyloglucans already present in the cell wall. Hypergravity at 300 g did not influence the molecular size of newly deposited xyloglucans. Thus, the process of xyloglucan synthesis was not modified by the gravitational force. On the other hand, 3H-xyloglucans obtained from epicotyls grown at 1 g were shifted to the low molecular mass regions during the incubation period, which was inhibited by hypergravity. These results indicate that xyloglucans were deposited into the cell wall as large molecules but they were then depolymerized in the cell wall, and that the inhibition of the depolymerization is mainly responsible for the increase in the molecular size of xyloglucans under hypergravity conditions.

Organogênese in vitro a partir de diferentes regiões do epicótilo de Citrus sp

O estabelecimento de protocolos para regeneração de plantas in vitro é essencial para o uso de técnicas de transformação genética no melhoramento de citros. Visando à obtenção de um protocolo eficiente de regeneração in vitro para laranja-azeda (Citrus aurantium), laranjas 'Natal' e 'Pêra' (C. sinensis), limão 'Volkameriano' (C. volkameriana) e citrange 'Carrizo' (Poncirus trifoliata x C. sinensis), avaliou-se a resposta morfogênica de diferentes regiões do epicótilo (basal, mediana e apical) em relação a distância do nó cotiledonar, na presença (1,0 mg/L-1) ou ausência de 6-BAP, em meio de cultura MT. Após 60 dias, avaliaram-se a porcentagem de explantes responsivos e o número de gemas adventícias por explante. A resposta morfogênica em função da região do epicótilo e da presença ou ausência da citocinina (6-BAP) foi influenciada pelo genótipo. A presença de 6-BAP no meio de cultura promoveu aumento na porcentagem de explantes responsivos para citrange 'Carrizo'. A suplementação do meio de cultura com a citocinina 6-BAP proporcionou aumento no número de brotos por explante para citrange 'Carrizo', laranja 'Natal' e limão 'Volkameriano'.

Isolation of PsPIN2 and PsAUX1 from etiolated pea epicotyls and their expression on a three-dimensional clinostat

We isolated novel cDNAs containing the complete open reading frames of a putative auxin influx carrier, PsAUX1, and a putative auxin efflux carrier, PsPIN2, from etiolated pea epicotyls. High levels of homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 (Accession No. AY222857) and AtPINs. Phylogenetic analyses based on deduced amino acid sequences revealed that PsPIN2 belonged to a subclade including AtPIN3, AtPIN4 and AtPIN7, while PsPIN1 belonged to the same clade as AtPIN1. The results were similar for PsAUX1 and AtAUX1, where PsAUX1 belongs to the same subclade as AtAUX1 and CS-AUX1. Expression of PsPIN1, PsPIN2 and PsAUX1 in pea epicotyl segments was promoted upon incubation of the segments with auxin (indole-3-acetic acid). In 3.5-d-old etiolated pea seedlings, relatively high expression of PsPIN1 and PsAUX1 was observed in the hook region, growing epicotyls and root tips as compared with those in mature regions of epicotyls and roots. Expression of PsPIN2 in roots was less than that in shoots. Simulated microgravity conditions on a three-dimensional clinostat remarkably increased gene expression of PsPIN1 and PsAUX1 in the hook and the internodes of pea epicotyls, but the increase in PsPIN2 was less. In contrast, polar auxin transport of pea epicotyls was substantially suppressed under simulated microgravity conditions on a 3D clinostat, similar to data from a space experiment on STS-95. These results suggest that PsPINs and PsAUX1 are auxin-inducible genes, and that the expression of PsPINs and PsAUX1 genes is sensitive to gravistimulation.

Photoregulation of Diamine Oxidase from Pea Seedlings

Summary Diamine oxidase (DAO) of pea (Pisum sativum L.) seedlings is sensitive to white light exposure. In light-grown seedlings the enzyme activity decreased in terminal bud and upper and middle regions of epicotyl but increased in the lower region with period of growth. The light-mediated response in both tissues was reversed upon transfer of seedlings to darkness. The light response was found to be phytochromemediated, causing a decrease in terminal bud and an increase in the upper region of epicotyl on single exposure and a decrease on multiple exposures. Middle and lower regions of epicotyl were insensitive to phytochrome. The phytochrome-mediated increase in epicotyl was due to increased synthesis of the enzyme. The phytochrome-mediated response in both of the tissues was reversed by the Ca chelator EGTA and Ca channel blockers, i.e. Lanthanum trichloride and diltiazem, suggesting that Ca-ions may be required as second messenger.

Interaction of gibberellins and phytochrome in the control of cowpea epicotyl elongation

The physiological response of cowpea (Vigna sinensis L.) epicotyl explants to far‐red light (FR) and its interaction with gibberellins (GAs) have been investigated. The effect of FR and GA1 varied with the age of the seedlings from which the explants were made: for FR, it decreased progressively with age (though the sensitivity of the epicotyls to FR did not change significantly until at least day 11), whereas it remained essentially constant for applied GA1 between days 5 and 9 after sowing. This indicates that the loss of response to FR may be due to a decrease in endogenous GA levels in the epicotyl. For a range of GA1 and GA20 (0.01–1 µg explant−1), both hormones were more active in FR than in R irradiated epicotyls, suggesting that phytochrome may affect GA sensitivity besides GA metabolism. The location of the epicotyl region most sensitive to FR (between 5 and 20 mm below the apex) was different from that to GAs (the upper 10 mm). Nevertheless, FR extended the region responsive to applied GAs, even in paclobutrazol‐treated epicotyls where elongation was due entirely to exogenous GAs. This means that modulation of epicotyl elongation by phytochrome, that occurs in a zone different from though overlapping with the GA‐sensitive subapical zone, is also mediated by GAs. Growth in the most FR‐sensitive region of the epicotyl stimulated by FR or GA1 was due to cell elongation, and in the most GA‐sensitive region to both cell division and elongation. The effect of FR and GA1 was negated by colchicine, indicating that microtubules may be involved in the response to both factors.

Role of genetic background in somatic embryogenesis in Medicago

Seventy-six cultivars of alfalfa (Medicago sativa L., M. falcata L. and M. varia Martyn) were tested in vitro for their capacity to produce callus and somatic embryos. A three-step media protocol was used to survey the response of the cotyledons and hypocotyl of each genotype while the epicotyl region was conserved in order to recover highly responding genotypes. The best regeneration response was observed in creepingrooted cultivars which contained a strong genetic contribution of two landrace germplasm sources, defined as M. falcata and Ladak, in their ancestry. The callus and embryogenesis responses showed a high degree of variation both between cultivars and among the plants of many of the 76 cultivars tested. A higher number of plants produced somatic embryos in the high regenerating cultivars compared to the low regenerating cultivars regardless of the media protocol or explant.

Roles of auxin and gibberellic acid in growth and maturation of epicotyls of Vigna angularis: Cell wall changes

The effects of auxin and gibberellic acid on cell wall composition in various regions of epicotyls of azuki bean (Vigna angularis Ohwi and Ohashi cv. Takara) were investigated with the following results. (1) Young segments excised from apical regions of the epicotyl elongated in response to added 10−4M indole‐3‐acetic acid (IAA). When the segments were supplied with 50 mM sucrose, the IAA‐induced segment growth was accompanied by enhanced overall synthesis of cell wall polysaccharides, such as xyloglucans, polyuronides and cellulose. This IAA effect on the cell wall synthesis is a consequence of extension growth induced by IAA. Gibberellic acid (GA) at 10−4M synergistically enhanced the IAA‐induced cell wall synthesis as well as IAA‐induced extension growth, although GA by itself neither stimulated the cell wall synthesis nor extension growth. In the absence of sucrose, cell wall synthesis was not induced by IAA or GA. (2) In mature segments excised from basal regions of the epicotyl, no extension growth was induced by IAA or GA. GA enhanced the synthesis of xylans and cellulose when the segments were supplied with 50 mM sucrose. IAA had no effect on the cell wall synthesis. These findings indicate that synthesis of polyuronides, xyloglucans and cellulose, which occurs during extension growth of the apical region of the epicotyl, is regulated chiefly by auxin whereas synthesis of xylans and cellulose during cell maturation in the basal region of the epicotyl is regulated by GA.

Die Wirkung des Cytostaticum ?Proresid? auf das Wachstum und die Blaulicht-induzierte Chloroplastendifferenzierung isolierter und normaler Keimlingswurzeln von Pisum sativum

“Proresid” (podophyllic acid ethylhydrazide; SP-1, Sandoz), a cytostatic drug with rapid but reversible effects on mitosis, inhibits growth, fresh weight increase and chlorophyll synthesis in excised roots of pea seedlings (var. Alaska) cultured in blue light (350–550 nm). Upon transferal of the treated fragments to normal culture medium the activity of these processes is fully restored within a few days. The reactions of intact seedlings on exposure of only the root tip to Proresid indicate that the drug exerts its action indirectly by blocking certain activities of the apical meristem. The important role of the latter in supporting or inhibiting various processes in the root is demonstrated by the observation that removal of the root tip from excised roots reduced the chloroplast differentiation in blue light to a very low yield, but induced cell divisions and lateral root formation. Moreover, there is good evidence that the drug is not actively transported in the root when supplied to the root tip only. Exposure of the whole root of a seedling to Proresid for longer periods does not only prevent its growth and differentiation but also gives rise to the formation of adventitious roots in the epicotyl region of the shoot.

Sugar levels in the pea epicotyl: Regulation by invertase and sucrose synthetase

In meristematic regions of the pea epicotyl (plumule and hook), fructose was the predominant sugar (about 1 per cent fr. wt.), sucrose was also present (0·4 per cent) but glucose was barely detectable. In adjacent regions of internode where cell expansion occurred, glucose became the main sugar (1 per cent) and levels of fructose and sucrose declined. None of these sugars was absorbed or utilized for metabolism preferentially by excised sections of any one region of the epicotyl. The distribution of various glucosidase, exo-glucanase and sugar phosphatase activities bore no clear relationship to actual hexose concentrations. However, the distribution of invertase activity was parallel to that of glucose, and sucrose synthetase was most active in regions where fructose and sucrose were concentrated. It is suggested, therefore, that the principal factors controlling sugar levels in the pea epicotyl are location and relative activities of these two enzymes, both utilizing translocated sucrose as substrate.