Epidermis integrity and epicotyl growth in azuki bean

In order to verify if epidermis integrity played a determinant role in epicotyl elongation induced by fusicoccin (FC), buffers at different pH's, and indoleacetic acid (IAA), we studied the short-term kinetics of elongation growth, the increase of fresh weight in long-term treatment, and the H⁺ excretion in intact, abraded, and peeled azuki bean epicotyl sections. We demonstrated that the epidermis is more sensitive to IAA, whereas the cortex is highly responsive to protons. Our data are consistent with the acid growth theory. In addition, our studies support the idea that the epidermis may be the tissue target for auxin, but its integrity is necessary for IAA-induced elongation.     

Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by fucose-binding lectins from Tetragonolobus purpureus Moench and Ulex europaeus L. These lectins also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. Auxin caused a decrease in molecular mass of xyloglucans extracted with 24% KOH from the cell walls. The lectins inhibited auxin-induced changes in molecular mass of the xyloglucans. The autolytic release of xylose-containing products from the pectinase-treated cell walls was also suppressed by the lectins. Fucose-binding lectins pretreated with fucose exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosning, or breakdown of xyloglucans. These results support the view that the breakdown of xyloglucans is involved in the cell wall loosening responsible for auxin-induced elongation in dicotyledons.     

Role of xyloglucan breakdown in epidermal cell walls for auxininduced elongation of azuki bean epicotyl segments

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi et Ohashi cv. Takara) was suppressed by a fucose-binding lectin from Tetragonolobus purpureas Moench and by polyclonal antibodies raised against xyloglucan heptasaccharide (Xyl₃Glc₄) when the cuticle present in the outer surface of epicotyls was abraded. In contrast, elongation of non-abraded segments was not influenced by the lectin or the antibodies. Epicotyl segments, from which the epidermal and the outer cortical cells had been removed, elongated rapidly for 2 h and than only slowly. Auxin slightly stimulated elongation of the inner tissue segments in the phase of slow growth. Neither in the presence nor in the absence of auxin did the lectin or the antibodies affect elongation of the inner tissue segments. The split portions of outer surface-abraded epicotyl segments incubated in buffer extended outward, and auxininhibited this outward bending. The lectin and the antibodies reversed the effect of auxin on bending. The fucose-binding lectin pretreated with fucose or the immunoglobulin fraction obtained from preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation of abraded or split segments. These results support the view that a breakdown of xyloglucans in the epidermal cell walls plays an essential role in auxin-induced elongation in dicotyledons.     

Role of xyloglucan in gravitropic bending of azuki bean epicotyl

The mechanism of the gravitropic bending was studied in azuki bean epicotyls. The cell wall extensibility of the lower side became higher than that of the upper side in the epicotyl bending upward. The contents of matrix polysaccharides of the cell wall (pectin and xyloglucan in hemicellulose-II) in the lower side became smaller than those in the upper side. The molecular mass of xyloglucans in the lower side decreased. After an epicotyl was fixed to a metal rod to prevent the bending, gravistimulation was applied. Fundamentally the same results were obtained with respect to rheological and chemical characteristics of the cell wall as those of epicotyls showing gravitropic bending. The present results suggested that the initial gravitropic bending was caused by the increase in extensibility of the lower side and the decrease in extensibility of the upper side via the change of the cell wall matrix, especially xyloglucans.     

Effect of lectins on auxin-induced elongation and wall loosening in oat coleoptile and azuki bean epicotyl segments

A marine unicellular aerobic nitrogen-fixing cyanobacterium Synechococcus sp. strain Miarni BG 043511 was pretreated with different light and dark regimes in order to induce higher growth synchrony. A pretreatment of two dark and light cycles of 16 h each yielded good synchrony for 3 cell division cycles. Longer dark treatments decreased the degree of synchrony and shorter dark treatments caused irregular cell division. Once synchronous culture was established, distinct phases of cellular carbohydrate accumulation and cellular carbohydrate degradation were observed even under continuous illumination. Changes in carbohydrate content were repeated in a cyclic manner with approximately 20 h intervals, the same as the cell division cycle. This change in carbohydrate metabolism provided a good index of growth synchrony under nitrogen-fixing conditions.
Photosynthetic oxygen evolution and nitrogen fixation capabilities and their activities in near, in situ, culture conditions were measured in well synchronized cultures of this strain under continuous illumination. Distinct oscillations of both photosynthetic oxygen evolution and nitrogen fixation capabilities with ca 20-h intervals, similar to the interval of the cell division cycle, were observed for three cycles. However, the activities of photosynthetic oxygen evolution were inversely correlated with those of nitrogen fixation. During the nitrogen fixation period, net oxygen consumption was observed even in the light under conditions approximating in situ culture conditions. The phase of temporal appearance of nitrogenase activity during the cell division cycle coincided with the phase of carbohydrate net degradation. These data indicate that this unicellular cyanobacterium can grow diazotrophically under conditions of continuous illumination by the segregation of photosynthesis and nitrogen fixation within a cell division cycle.     

The fine structure of the epidermal cell wall in azuki bean epicotyl

Journal of Plant Research - The arrangement of microfibrils in the wall of epidermal cells in the epicotyl of the azuki bean plant has been observed. The outer and inner tangential walls have a...     

Modifications of cell wall polysaccharides during auxin-induced growth in azuki bean epicotyl segments

Changes in sugar compositions and the distribution pattern ofthe molecular weight of cell wall polysaccharides during indole-3-aceticacid (IAA)-induced cell elongation were investigated. Differentialextraction of the cell wall and gel permeation chroma-tographyof wall polysaccharides indicated that galactans, polyuronides,xylans, glucans and cellulose were present in the azuki beanepicotyl cell wall. When segments were incubated in the absence of sucrose, IAAenhanced the degradation of galactans in both the pectin andhemicellulose fractions, whereas to some extent it enhancedthe polymerization of xylans and glucans in the hemicellulosefraction and an increase in the amounts of polyuronides in thepectin fraction and of -cellulose. In the presence of 50 mMsucrose, IAA caused large increases in the amounts of all thewall polysaccharides, and enhanced the polymerization of galactans,xylans and glucans in the hemicellulose fraction. These results and an important role of galactan turnover incell wall extension are discussed. (Received December 11, 1979; )     

Possible involvement of 65 kda MAP in elongation growth of azuki bean epicotyls

Although regulation of the dynamics of plant microtubules (MTs) by microtubule-associated proteins (MAPs) has been suggested, the mechanism has not yet been elucidated. As one candidate, a MAP composed of a 65 kDa polypeptide (65 kDa MAP) has been isolated from tobacco cultured cells [Jiang and Sonobe (1993), J. Cell Sci 105: 8911. To investigate the physiological role of the 65 kDa MAP in situ, we analyzed the changes in content and colocalization of this MAP with cortical MTs in relation to elongation growth, using azuki bean epicotyls (Vigna angularis Ohwi et Ohashi). All apical, intermediate, and basal segments prepared from 6 d seedlings showed high growth activity. In 12 d seedlings, growth activity of intermediate and basal segments was low, although that of apical segments was high. The relationship between the growth activity and the orientation of cortical MTs in the epidermal cells was analyzed. Cells could be classified into four types with respect to orientation of cortical MTs: transverse (T), oblique (O), longitudinal (L) to the vertical axis of cells, and random (R). In rapidly growing segments, three types of cells, T, O, L, were observed at similar ratios. In such segments, significant amounts of the 65 kDa MAP were expressed, and it colocalized well with cortical MTs. In segments showing low growth activity, most of the cells showed oblique and longitudinal orientation of cortical MTs. In such segments, the content of the 65 kDa MAP was low. These results suggested involvement of this 65 kDa MAP in regulation of the elongation growth of this epicotyl.     

Transformation of azuki bean by Agrobacterium tumefaciens

Stable transformation and regeneration was developed for a grain legume, azuki bean (Vigna angularis Willd. Ohwi & Ohashi). Two constructs containing the neomycin phosphotransferase II gene (nptII) and either the -glucuronidase (GUS) gene or the modified green fluorescent protein [sGFP(S65T)] gene were introduced independently via Agrobacterium tumefaciens-mediated transformation. After 2 days of co-cultivation on MS medium supplemented with 100 M acetosyringone and 10 mg l^–1 6-benzyladenine, seedling epicotyl explants were placed on regeneration medium containing 100 mg l^–1 kanamycin. Adventitious shoots developing from explant calli were excised onto rooting medium containing 100 mg l^–1 kanamycin. Rooted shoots were excised and repeatedly selected on the same medium containing kanamycin. Surviving plants were transferred to soil and grown in a green house to produce viable seeds. This process took 5 to 7 months after co-cultivation. Molecular analysis confirmed the stable integration and expression of foreign genes.     

The chloroplast genomes of azuki bean and its close relatives: a deletion mutation found in weed azuki bean

A physical map of the azuki bean (Vigna angularis cv. Erimo-shozu) chloroplast DNA (cpDNA) was constructed by localising the cleavage sites of PstI, SalI, SmaI, SacI, KpnI, PvuII and XhoI. The resulting map is more similar to the cpDNA-maps of two Vigna species (mung bean, V. radiata, and V. nakashimae) than to common bean (Phaseolus vulgaris) cpDNA map. Azuki bean was originally classified in the genus Phaseolus, and the inclusion of this taxon in the genus Vigna is a recent taxonomic decision. Our result is thus in favour of the taxonomic placement of azuki bean in the same genus as V. nakashimae and mung bean. We also found that a weed-form accession of azuki bean has a 96-bp deletion relative to the cultivar Erimo-shozu. The 96-bp deletion is located between the trnS-UGA and psbC genes in the large single-copy region of the chloroplast genome. This deletion is flanked by imperfect 9-bp direct repeats, suggesting that the deletion was a result of intra-molecular recombination mediated by these direct repeats.     

Xyloglucan antibodies inhibit auxin-induced elongation and cell wall loosening of azuki bean epicotyls but not of oat coleoptiles

Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl₁Glc₁), xyloglucan heptasaccharides (Xyl₃Glc₄), and octasaccharides (Gal₁Xyl₃Glc₄). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin-induced elongation or cell wall loosening of oat coleoptiles.     

The pH profile for acid-induced elongation of coleoptile and epicotyl sections is consistent with the acid-growth theory

The acid-growth theory predicts that a solution with a pH identical to that of the apoplast of auxintreated tissues (4.5–5.0) should induce elongation at a rate comparable to that of auxin. Different pH profiles for elongation have been obtained, however, depending on the type of pretreatment between harvest of the sections and the start of the pH-incubations. To determine the acid sensitivity under in vivo conditions, oat (Avena sativa L.) coleoptile, maize (Zea mays L.) coleoptile and pea (Pisum sativum L.) epicotyl sections were abraded so that exogenous buffers could penetrate the free space, and placed in buffered solutions of pH 3.5–6.5 without any preincubation. The extension, without auxin, was measured over the first 3 h. Experiments conducted in three laboratories produced similar results. For all three species, sections placed in buffer without pretreatment elongated at least threefold faster at pH 5.0 than at 6.0 or 6.5, and the rate elongation at pH 5.0 was comparable to that induced by auxin. Pretreatment of abraded sections with pH-6.5 buffer or distilled water adjusted to pH 6.5 or above gave similar results. We conclude that the pH present in the apoplast of auxin-treated coleoptile and stems is sufficiently low to account for the initial growth response to auxin.Abbreviations FS free space - IAA indole-3-acetic acid This research was supported by a grant from the National Adonautics and space Administration (NASA), NAGW 1394 to R.E.C., NASA grant NAGW-297 to M.L.E., and NASA grant NAG 1849 to D.L.R.     

Two distinct blue-light responses regulate epicotyl elongation in pea

Blue light induces a long-term suppression of epicotyl elongation in red-light-grown pea (Pisum sativum L.) seedlings. The fluence-response characteristics are bell-shaped, indicating the possibility of two different blue-light responses: a lower fluence response causing suppression and a higher fluence response alleviating the suppression. To determine if two responses are in effect, we have grown pea seedlings under dark conditions hoping to eliminate one or the other response. Under these growth conditions, only the lower fluence portion of the response (suppression of elongation) is apparent. The kinetics of suppression are similar to those observed for the lower fluence response of red-light-grown seedlings. The response to blue light in the dark-grown seedlings is not due to the excitation of phytochrome because a pulse of far-red light large enough to negate phytochrome-induced suppression has no effect on the blue-light-induced suppression. Furthermore, treatment of the dark-grown seedlings with red light immediately prior to treatment with high fluence blue light does not elicit the higher fluence response, indicating that the role of red light in the blue high fluence response is to allow the plant to achieve a specific developmental state in which it is competent to respond to the higher fluences of blue light.     

Physiology of gibberellin-induced elongation of epicotyl explants from Vigna sinensis

The physiological characteristics of the response of excised cowpea (Vigna sinensis cv Blackeye pea No. 5) epicotyls to gibberellins (GAs) were studied. Epicotyl explants, retaining the petioles and a 2-cm portion of hypocotyl, were placed upright in small vials containing water. Plant growth substances were injected into the subapical tissues as ethanol solutions.Epicotyl elongation resulting from treatment with 0.5 g of GA ranged between 5 and 13 times that of the control, depending on the GA applied. With GA₁, no differences were obtained with explants prepared from 5 to 9-day-old seedlings. The increase in elongation could be detected within 6 h of treatment, and the stimulus of a single application lasted at least 4 days. Final elongation was proportional to the logarithm of the amount of GA, applied, 0.01 to lug. The response to GA treatment was limited to the upper part, the most sensitive zone being located between 2 to 4 mm below the apex of the epicotyl; this effect was entirely due to cell elongation.The induction of epicotyl elongation by GAs seems to be specific and independent of the effect of auxin. IAA had no effect on elongation and 4-chloro-phenoxyisobutyric acid (PCIB) did not affect the response to GA₁ Abbreviations ABA abscisic acid - GA gibberellin - IAA Indole-3-acetic acid - TIBA 2,3,5-triiodobenzoic acid - PCIB 4-chloro-phenoxyisobutyric acid     

Mechano-sensitive orientation of cortical microtubules during gravitropism in azuki bean epicotyls

The orientation of cortical microtubules (cMT) during gravitropism was studied in epidermal cells of azuki epicotyls. The relative proportion of cells with longitudinal cMT increased in the upper epidermis, and those with transverse cMT increased in the lower epidermis. When epicotyls were kept straight during gravistimulation, no change in cMT orientation occurred in either the upper and lower epidermis. When epicotyls were forced to bend downward, cells with transverse cMT increased in the upper epidermis, and those with longitudinal cMT increased in the lower epidermis. When epicotyls were loaded with naphthylphthalamic acid, an inhibitor of auxin transport, both gravitropic bending and change in cMT orientation were inhibited. However, when a change in cMT orientation was induced by forced downward bending, cells with longitudinal cMT increased in the compressed (lower) side and those with transverse cMT increased in the extended (upper) side. It was suggested that cMT orientation was controlled by the bending of the epicotyl and not by a gravity signal per se. Loading with Gd³⁺, an inhibitor of the stretch-activated channel, did not inhibit gravitropic bending. However, it inhibited cMT reorientation induced by gravitropic bending and by forced bending. Involvement of the stretch-activated channel in mechano-sensitive orientation of cMT was suggested.     

Cellular basis of growth suppression by submergence in azuki bean epicotyls

Background and Aims

Complete submergence severely reduces growth rate and productivity of terrestrial plants, but much remains to be elucidated regarding the mechanisms involved. The aim of this study was to clarify the cellular basis of growth suppression by submergence in stems.

Methods

The effects of submergence on the viscoelastic extensibility of the cell wall and the cellular osmotic concentration were studied in azuki bean epicotyls. Modifications by submergence to chemical properties of the cell wall; levels of osmotic solutes and their translocation from the seed to epicotyls; and apoplastic pH and levels of ATP and ethanol were also examined. These cellular events underwater were compared in etiolated and in light-grown seedlings.

Key Results

Under submergence, the osmotic concentration of the cell sap was substantially decreased via decreased concentrations of organic compounds including sugars and amino acids. In contrast, the viscoelastic extensibility of the cell wall was kept high. Submergence also decreased ATP and increased the pH of the apoplastic solution. Alcoholic fermentation was stimulated underwater, but the resulting accumulated ethanol was not directly involved in growth suppression. Light partially relieved the inhibitory effects of submergence on growth, osmoregulation and sugar translocation.

Conclusions

A decrease in the levels of osmotic solutes is a main cause of underwater growth suppression in azuki bean epicotyls. This may be brought about by suppression of solute uptake via breakdown of the H⁺ gradient across the plasma membrane due to a decrease in ATP. The involvement of cell wall properties in underwater growth suppression remains to be fully elucidated.Key words: Apoplastic pH, cell wall extensibility, growth suppression, osmoregulation, osmotic concentration, submergence, sugar translocation, Vigna angularis     

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.     

Gibberellin-induced ethylene production and its effect on cowpea epicotyl elongation

The effect of gibberellin A₁ (GA₁) on production of ethylene by cowpea (Vigna sinensis cv Blackeye pea no. 5) epicotyl explants and its relationship to epicotyl elongation was investigated. The explants were placed upright in water and incubated in sealed culture tubes or in large jars. GA, and IAA in ethanol solution were injected into the subapical tissues of the decapitated epicotyls. Cowpea epicotyl explants elongated after GA but not after IAA treatment, and they were very sensitive to exogenous ethylene. As little as 0.14 1/1 ethylene reduced significantly GA₁-induced epicotyl elongation.Treatment with GA₁ induced the production of ethylene which began 10 h after GA application, showed a peak at about 22 h and then declined. The yield of ethylene was proportional to the amount of GA, injected. The inhibition of epicotyl elongation in closed tubes was avoided by absorbing ethylene released with Hg(Cl0₄)₂ , or by adding AVG to the incubation solution to inhibit ethylene production. Treatment with IAA elicited a rapid production of ethylene which ceased about 10 h after application. The effects of IAA and GA₁ on ethylene production were additive.Abbreviations AVG aminoethoxyvinylglycine 2-amino-4-(2-aminoethoxy)-trans-3butenoic acid - ACC 1-aminocyclopropane-1-carboxylic acid - GA gibberellin - IAA indole-3-acetic acid     

Effects of hypergravity on expression of XTH genes in azuki bean epicotyls

Hypergravity produced by centrifugation caused inhibition of elongation growth and a decrease in the cell wall extensibility in azuki bean epicotyls ( Vigna angularis Ohwi et Ohashi). Also, hypergravity increased the molecular mass of xyloglucans, whereas it decreased xyloglucan-degrading activity in epicotyls. When the expression profiles of three xyloglucan endotransglucosylase/hydrolase ( XTH ) genes, VaXTHS4 , VaXTH1 and VaXTH2 , were analyzed under hypergravity conditions, the expression of VaXTHS4 , which shows only hydrolase activity, was downregulated in proportion to the logarithm of the magnitude of gravity (R = −0.94). However, the gene expression of VaXTH1 or VaXTH2 , which shows only transglucosylase activity, was not affected by gravitational conditions. When the seedlings that had been grown at 1  g were transferred to hypergravity conditions at 300  g , the downregulation of VaXTHS4 expression was detected within 1 h. By removal of hypergravity stimulus, VaXTHS4 expression was increased within 1 h. These results suggest that azuki bean epicotyls promptly regulate the expression level of only VaXTHS4 in response to gravity stimuli. The regulation of xyloglucan-hydrolyzing activity as a result of changes in VaXTHS4 expression may be involved in the regulation by gravity of molecular mass of xyloglucans, leading to modifications of cell wall mechanical properties and cell elongation. Lanthanum and gadolinium, potential blockers of mechanosensitive calcium ion permeable channels (mechanoreceptors), nullified the suppression of VaXTHS4 expression, suggesting that mechanoreceptors are responsible for inhibition by hypergravity of VaXTHS4 expression.