Effect of light on ethylene production and hypocotyl growth of soybean seedlings

The apical 1-cm hypocotyl of dark-grown `Clark' soybean (Glycine max [L.] Merr.) seedlings produced ethylene at rates of 7 to 11 nanoliters per hour per gram when attached to the cotyledons. Such physiologically active rates occurred prior to the deceleration of hypocotyl elongation caused by the temperature of 25 C.

Daily exposure of the etiolated seedlings to red light promoted hypocotyl elongation and prevented its lateral swelling. Red light treatment also caused a 45% decrease in ethylene production. Far red irradiation following the red treatment reversed the red effects, suggesting that the ethylene intervenes as a regulator in the phytochrome control of `Clark' soybean hypocotyl growth at 25 C.

     

Influence of ethylene and Ag+ on hypocotyl growth in etiolated lupin seedlings. Effects on cell growth and division

Lupin seeds treated with 1-amino-cyclopropane-1-carboxylic acid (ACC) or2-chloroethylphosphonic acid (CEPA) produced hypocotyls showing a typicalethylene growth response (reduced elongation and increased thickness), whichcould be efficiently counteracted by the presence of silver thiosulfate (STS).The fact that ACC and CEPA stimulated the ethylene produced in different zonesalong the hypocotyls suggests that these compounds, which are stored in theseeds during treatment, were transported to and along the hypocotyl. The same istrue in hypocotyls from STS-treated seeds, which indicates that stress ethyleneis induced by metal toxicity. CEPA was more effective than ACC in both producingethylene and influencing growth due to the high capacity of the hypocotyl toconjugate ACC. At the same time that CEPA inhibited hypocotyl elongation, thehypocotyl diameter increased and ethylene production exceeded the maximum valueof the control. The subsequent recovery of hypocotyl elongation coincided with adecrease in ethylene production and involved cell elongation. The final celllength was similar (in ACC-) or higher (in CEPA-treated plants) than in thecontrol, although the hypocotyls were shorter in both cases, while the number ofcells per column was reduced to half that observed in the control. Thisinhibition of cell division caused by ethylene was selective since the number ofcell layers did not change. The variations in cell diameter in the epidermisand, especially, in the cortex and pith were correlated with the variations inhypocotyl diameter produced by ACC, CEPA and STS. The results show that theethylene-induced hypocotyl thickening was irreversible and mainly due to anincrease in cell diameter, while the inhibition of hypocotyl elongation wasreversible and involved irreversible inhibition of cell division and,paradoxically, stimulation of cell elongation to produce cells longer than thoseof the control.     

Effect of the defoliant thidiazuron on ethylene evolution from mung bean hypocotyl segments

The effect of the defoliant thidiazuron (N-phenyl-N′1,2,3-thiadiazol-5-ylurea) on ethylene evolution from etiolated mung bean hypocotyl segments was examined. Treatment of hypocotyl segments with concentrations of thidiazuron equal to or greater than 30 nanomolar stimulated ethylene evolution. Increased rates of ethylene evolution from thidiazuron-treated tissues could be detected within 90 minutes of treatment and persisted up to 30 hours after treatment. Radioactive methionine was readily taken up by thidiazuron-treated tissues and was converted to ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) and an acidic conjugate of ACC. Aminoethoxyvinylglycine, aminooxyacetic acid, cobalt chloride, and α-aminoisobutyric acid reduced ethylene evolution from treated tissues. An increase in the endogenous content of free ACC coincided with the increase in ethylene evolution following thidiazuron treatment. Uptake and conversion of exogenous ACC to ethylene were not affected by thidiazuron treatment. No increases in the extractable activities of ACC synthase were detected following thidiazuron treatment.     

Influence of ethylene on adventitious root formation in mung bean hypocotyl cuttings

The relationship between ethylene and adventitious root formation in mung bean hypocotyl cuttings was studied.Ethephon, an ethylene-releasing compound, at 5 x 10 -5 M increased root number and root dry weight on hypo-cotyl cuttings. When ethephon was applied to hypocotyl at different times after excision, there were two effectivetimes for root production i.e. between 06 h and 18-24 h. These two time periods correspond to the induction phase and the late initiation phase of root development, respectively. After excision, three peaks of ethylene productionwere observed. The first peak commencing at 6 h started the sequence of reactions leading root formation, the second peak appearing at 12 h coincided with the beginning of the increase of the IAA level during primordia initiation, and the third peak showing at 48 h played a role in root differentiation and growth. Ethylene stimulated rooting by enhancing the increase in auxins. Thus it appears that the IAA-induced ethylene production may be a factor involved in the stimulation of adventitious root formation.     

Cobalt and plant development: interactions with ethylene in hypocotyl growth

Co²⁺ promoted elongation of hypocotyl segments of light-grown cucumber (Cucumis sativus) seedlings. Time course and dose response data are presented and interactions with IAA, gibberellin, cyclohexanol, and cotyledons described. Segments without cotyledons responded to Co²⁺ only if grown in gas-tight vessels with IAA added. When bases of cotyledons were ringed with an inhibitor of auxin transport, Co²⁺ caused no growth promotion in the hypocotyl. Co²⁺ prevented lateral swelling of hypocotyls treated with supraoptimal IAA. Removal of ethylene from the atmosphere reduced the Co²⁺ response, but Co²⁺ did not counteract the inhibitory effect of increased ethylene levels. These results are consistent with the hypothesis that Co²⁺ promotes hypocotyl elongation by inhibiting ethylene production. The hypothesis was confirmed by a direct demonstration that Co²⁺, at growth-promoting concentrations, powerfully inhibited ethylene production in the cucumber hypocotyl.     

Influence of auxin and incubation on the relative level of polyribosomes in excised soybean hypocotyl

The influence of incubation and auxin (2,4-D) on polyribosome level in soybean hypocotyl was studied.     

Lithium,aminoethoxyvinylglycine and cobalt reversal of the cotyledonary pricking-induced growth inhibition in the hypocotyl of Bidens pilosus in relation to ethylene and peroxidases

Pretreatment of young Bidens pilosus plants with lithium (Li), aminoethoxyvinylglycine (AVG) or cobalt (Co) prevents the cotyledonary pricking-induced growth inhibition of the hypocotyl. The effect is correlated with parallel prevention of the pricking-induced enhancement of peroxidase and ethylene production in the hypocotyl. Only Co prevents the increased capacity of hypocotyl segments or microsomes to convert aminocyclopropane carboxylic acid to ethylene. Li, AVG, and Co do not interfere with peroxidase and ethylene metabolism in pricked cotyledons. Although Li, AVG as well as Co are known to interfere with ethylene biosynthesis and action, they could well remove the wounding effect by different modes of action.     

The interaction of microgravity and ethylene on the ultrastructure cell and Ca2+ localization in soybean hook hypocotyl

Calcium ions are secondary messenger in numerous cellular processes of plant grown at 1 g. Ca2+ are connected with oxygen atoms, of pectin carboxy groups and/or with H(+)-groups of protein (Roux and Slocum, 1982; Hepler and Wayne, 1985). The influence of altered gravity on the calcium balance in some cells is established. The increased synthesis of ethylene in plant grown in microgravity caused the change of the structural-functional organization of cell (Hensel and Iversen, 1980; Hilaire et al., 1996). Available data put the new question: how do high ethylene level and microgravity influence on the redistribution of Ca2+ in cell of seedling in early stage of growth? Therefore, the goal of our data was the comparable study of the cell ulltrastructure and localization of Ca2+ in hook hypocotyl of soybean seedling under interaction of microgravity and ethylene.     

Isolation and culture of soybean hypocotyl protoplasts

Summary Protoplasts were isolated seedling hypocotyls of soybean (Glycine max), and cultured in both liquid and agarose-solidified, modified K8P medium. Nuclear staining revealed that only 2% of protoplasts lacked a nucleus, 93% contained a single nucleus, and 5% contained more than one. Maximum protoplast yields and subsequent division frequencies, in liquid medium, were obtained from 5 days-old seedlings. Maximum division frequencies (54%) were obtained from hypocotyl protoplasts plated at a density of 5×10⁴ ml⁻¹. Using different osmolality reduction régimes for liquid cultures, hypocotyl protoplasts developed into green, nodular callus, similar to that which has previously given rise to shoot buds in perennialGlycine species. This tissue, however, did not produce shoot buds in soybean. N. H. was supported by a SERC CASE studentship and a postdoctoral fellowship from Shell Research Ltd., Sittingbourne, Kent, UK.     

Auxin, ethylene and brassinosteroids: tripartite control of growth in the Arabidopsis hypocotyl

Dark-grown Arabidopsis seedlings develop an apical hook by differential cell elongation and division, a process driven by cross-talk between multiple hormones. Auxins, ethylene and gibberellins interact in the formation of the apical hook. In the light, a similar complexity of hormonal regulation has been revealed at the level of hypocotyl elongation. Here, we describe the involvement of brassinosteroids (BRs) in auxin- and ethylene-controlled processes in the hypocotyls of both light- and dark-grown seedlings. We show that BR biosynthesis is necessary for the formation of an exaggerated apical hook and that either application of BRs or disruption of BR synthesis alters auxin response, presumably by affecting auxin transport, eventually resulting in the disappearance of the apical hook. Furthermore, we demonstrate that ethylene-stimulated hypocotyl elongation in the light is largely controlled by the same mechanisms as those governing formation of the apical hook in darkness. However, in the light, BRs appear to compensate for the insensitivity to ethylene in hls mutants, supporting a downstream action of BRs. Hence, our results indicate that HLS1, SUR1/HLS3/RTY1/ALF1 and AMP1/HPT/COP2/HLS2/PT act on the auxin-ethylene interaction, rather than at the level of BRs. A model for the tripartite hormone interactions is presented.     

Organogenic regeneration of soybean from hypocotyl explants

Summary Thirteen soybean genotypes representing maturity groups IV−VI were compared for organogenic responses on three media cultured under two lighting conditions with hypocotyl sections excised from 7-d-old seedlings. All soybean lines responsed by producing adventitious shoots on the acropetal end of the hypocotyl explants, confirming genotype-independence of shoot initiation. Media containing 6-benzyladenine (BA; 5.0–10 μM) induced the greatest numbers of shoots. Histological studies confirmed the adventitious nature of arising shoots by indicative formation of meristematic zones and shoot primordia from parenchymatous tissues of central pith and plumular trace regions of the hypocotyl. Incompletely excised cotyledonary buds also contributed to shoot initiation. Degrees of responses were media-dependent and varied with regard to genotype. Centennial, Epps, and Lyon gave the greatest individual responses. Between cultivars (across all treatments), the regeneration potential (percentage of explants producing meristem-like structures or shoot primordia) 4 wk after initiation ranged from 47 to 75%. Four wk later, regenerative ability (number of shoots produced per responding explant) and regeneration efficiency (number of shoots produced per explant plated) yielded 1.4–7.1 and 1.0–5.0 shoots, respectively. The optimized protocol included initiation on a medium containing 5.0 μM BA for 4 wk, then transfer onto a shoot elongation medium (0.36 μM BA) for 4 wk. For 11 genotypes tested, 66–100% of excised shoots produced roots after 4 wk on media containing 12.5–29.2 μM indole-3-butyric acid. Of 109 regenerants transplanted to soil, 94% survived and no sterility has been observed on those mature enough to flower.     

Effect of hypobaric conditions on ethylene evolution and growth of lettuce and wheat

Elevated levels of ethylene occur in enclosed crop production systems and in spaceflight environments, leading to adverse plant growth and sterility. There are engineering advantages in growing plants at hypobaric (reduced atmospheric pressure) conditions in biomass production for extraterrestrial base or spaceflight environments. Objectives of this research were to characterize the influence of hypobaria on growth and ethylene evolution of lettuce (Lactuca sativa) and wheat (Triticum aestivum). Plants were grown under variable total gas pressures [from 30 to 101 kPa (ambient)]. In one study, lettuce and wheat were direct seeded, germinated and grown in the same chambers for 28 d at 50 or 101 kPa. Hypobaria increased plant growth and did not alter germination rate. During a 10-day study, 28-day-old lettuce and 40-day-old wheat seedlings were transplanted together in the same low and ambient pressure chambers; ethylene accumulated in the chambers, but the rate of production by both lettuce and wheat was reduced more than 65% under 30 kPa compared with ambient pressure (101 kPa). Low O2 concentrations [partial pressure of O2 (pO2) = 6.2 kPa] inhibited ethylene production by lettuce under both low (30 kPa) and ambient pressure, whereas ethylene production by wheat was inhibited at low pressure but not low O2 concentration. There was a negative linear correlation between increasing ethylene concentration and decreasing chlorophyll content of lettuce and wheat. Lettuce had higher production of ethylene and showed greater sensitivity to ethylene than wheat. The hypobaric effect on reduced ethylene production was greater than that of just hypoxia (low oxygen).     

Characterization of post-flooding recovery-responsive enzymes in soybean root and hypocotyl

Soybean exhibits markedly reduced growth and yields under flooding stress. To determine the functional roles of four soybean proteins in post-flooding recovery, the organ/stress specificity and time-dependency of their enzymatic activities were analyzed. Peroxidase activity decreased in root and hypocotyl exposed to flooding and cold stresses, but increased during the post-stress recovery period. In contrast, its activity increased in both root and hypocotyl under drought stress. Acid phosphatase activity was suppressed in root treated with flooding and cold stresses, and slightly increased during the recovery period; however, the opposite profile was observed in hypocotyl. In response to drought stress, it did not change in root, but was decreased in hypocotyl. Beta-ketoacyl reductase activity did not change in root under flooding conditions, but was decreased in hypocotyl, although the activity increased slightly during the recovery period. In addition, it was decreased in both organs under drought and cold stresses, but again increased during the recovery period. Nucleotidylyl transferase activity was increased in root under flooding and drought stresses, but was decreased in hypocotyl. It was decreased in response to cold stress, but exhibited a slight increase during the recovery period. Furthermore, the treatment with jasmonate and salicylate suppressed the activities of peroxidase and acid phosphatase in root and hypocotyl under flooding stress; however, the activity of acid phosphatase increased during the recovery period. Nucleotidylyl transferase activity in root was also elevated by treatment with jasmonate, but gradually decreased during the recovery period. These results suggest that jasmonate-induced changes in nucleotidylyl transferase activity may facilitate soybean root recovery after flooding stress.     

Proteome analysis of soybean hypocotyl and root under salt stress

To evaluate the response of soybean to salt stress, the related changes in protein expression were investigated using the proteomic approach. Soybean plants were exposed to 0, 50, 100, and 200 mM NaCl. Especially at 200 mM, the length and fresh weight of the hypocotyl and root reduced under salt stress, while the proline content increased. Proteins from the hypocotyl and root treated with 100 mM NaCl were extracted and separated by two-dimensional polyacrylamide gel electrophoresis; 321 protein spots were detected. In response to salt stress, seven proteins were reproducibly found to be up- or down-regulated by two to sevenfold: late embryogenesis-abundant protein, beta-conglycinin, elicitor peptide three precursor, and basic/helix-loop-helix protein were up-regulated, while protease inhibitor, lectin, and stem 31-kDa glycoprotein precursor were down-regulated. These results indicate that salinity can change the expression level of some special proteins in the hypocotyl and root of soybean that may in turn play a role in the adaptation to saline conditions.     

Adenylate cyclase activity not found in soybean hypocotyl and onion meristem

Tissue, homogenates, and purified cell fractions prepared from hypocotyls of a dicot, soybean (Glycine max), and meristematic tissue of a monocot, onion (Allium cepa), were examined critically for evidence of adenylate cyclase activity. Three assay methods were used: chemical analysis, isotope dilution analysis, and enzyme cytochemistry. In both crude extracts or whole tissue, as well as purified membranes, with or without auxin, no adenylate cyclase was detected by any of the three methods.     

Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl

We have previously shown that endoreduplication levels in hypocotyls of Arabidopsis thaliana (L.) Heynh. are under negative control of phytochromes. In this study, the hormonal regulation of this process was analysed using a collection of A. thaliana mutants. The results show that two hormones in particular, gibberellin (GA) and ethylene, play distinct roles. Hypocotyl cells of the GA-deficient mutant ga1-11 grown in the dark did not elongate and showed a greatly reduced endoreduplication. Normal endoreduplication could be restored by supplying 10⁻⁹ M of the gibberellin GA₄₊₇, whereas the restoration of normal cell growth required 100-fold higher concentrations. The GA-insensitive mutant gai showed reduced cell elongation but normal ploidy levels. We conclude that (i) GA₄₊₇ has a global positive effect on endoreduplication and (ii) that endoreduplication is more sensitive to GA₄₊₇ than cell elongation. Ethylene had a completely different effect. It induced an extra round of endoreduplication both in light- and dark-grown seedlings and acted mainly on discrete steps rather than having a global effect on endoreduplication. The genes EIN2 and CTR1, components of the ethylene signal transduction pathway were both involved in this process. Received: 27 February 1999 / Accepted: 21 May 1999