Evidence that Auxin-induced Growth of Soybean Hypocotyls Involves Proton Excretion

The role of H⁺ excretion in auxin-induced growth of soybean hypocotyl tissues has been investigated, using tissues whose cuticle was rendered permeable to protons or buffers by scarification (scrubbing). Indoleacetic acid induces both elongation and H⁺ excretion after a lag of 10 to 12 minutes. Cycloheximide inhibits growth and causes the tissues to remove protons from the medium. Neutral buffers (pH 7.0) inhibit auxin-induced growth of scrubbed but not intact sections; the inhibition increases as the buffer strength is increased. Both live and frozen-thawed sections, in the absence of auxin, extend in response to exogenously supplied protons. Fusicoccin induces both elongation and H⁺ excretion at rates greater than does auxin. These results indicate that H⁺ excretion is involved in the initiation of auxin-induced elongation in soybean hypocotyl tissue.     

Metabolic Regulation during Glyceollin Biosynthesis in Green Soybean Hypocotyls

The accumulation of the isoflavonoid phytoalexin, glyceollin, occurs in hypocotyls of green soybean seedlings (Glycine max L. Merr. cv Harosoy 63) in response to the injection of a glucan elicitor isolated from the mycelial walls of the fungus, Phytophthora megasperma f. sp. glycinea. This accumulation, which levels off after 24 hours, is preceded by a dramatic, transient rise in extractable activities of two early enzymes in the biosynthetic pathway, phenylalanine ammonia-lyase (PAL) and p-coumaryl CoA ligase (pCL). The maximum amount of extractable activity occurs 12 to 16 hours after elicitor treatment and is coincident with the most rapid period of glyceollin accumulation. These results suggest a regulatory role for these early enzymes in the biosynthesis of this secondary metabolite. High performance liquid chromatography analysis of the early intermediates in the pathway further corroborates this hypothesis. The relative pool size and rate of turnover of p-coumaric acid, an early intermediate in glyceollin production, increase during the period of rapid increases in enzyme activities. Removal of cotyledons from elicitor-treated seedlings reduces glyceollin accumulation approximately 70%. This limitation of phytoalexin accumulation by cotyledon removal is correlated with a similar cotyledon effect on reduction of extractable activities of both PAL and pCL as well as a decrease in the flux of carbon through the p-coumaric acid pool. This research further supports the hypothesis that early enzymic steps in a biosynthetic pathway diverting carbon from primary to secondary metabolites function as regulatory control points.     

Isoaccepting Transfer Ribonucleic Acids during Chilling Stress in Soybean Seedling Hypocotyls

Total aminoacylation of glycine and leucine transfer RNAs was compared between chilled and nonchilled hypocotyls of 7-day-old soybean seedlings. Total charging was greater for both specific transfer RNAs from nonchilled sources. Isoaccepting transfer RNA species for glycine and leucine were fractionated using reverse phase column chromatography. Leucyltransfer RNAs were fractionated into six distinct fractions with relatively small shifts appearing in specific fractions between chilled and nonchilled sources. Glycyl-transfer RNAs were fractionated into two distinct fractions with major shifts appearing for both fractions between chilled and nonchilled sources.     

Pectin May Hinder the Unfolding of Xyloglucan Chains during Cell Deformation： Implications of the Mechanical Performance of Arabidopsis Hypocotyls with Pectin Alterations

Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on tour1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants （Col-0） and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels （-tensile strength） of the hypocotyls of the mutants with pectin alterations （mur1, qua2） were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for tour1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.     

Selective Inhibition of Auxin-Stimulated NADH Oxidase Activity and Elongation Growth of Soybean Hypocotyls by Thiol Reagents

The NADH oxidase activity of isolated vesicles of soybean (Glycine max cv Williams 82) plasma membranes and elongation growth of 1-cm-long hypocotyl segments were stimulated by auxins (indole-3-acetic acid or 2,4-dichlorophenoxyacetic acid [2,4-D]). The auxin-induced stimulations of both NADH oxidase and growth were prevented by the thiol reagents N-ethylmaleimide, p-chloromercuribenzoate, 5,5[prime]-dithiobis(2-nitrophenylbenzoic acid), dithiothreitol, and reduced glutathione. These same reagents largely were without effect on or stimulated slightly the basal levels of NADH oxidase and growth when assayed in the absence of auxins. In the presence of dithiothreitol or reduced glutathione, both 2,4-D and indole-3-acetic acid either failed to stimulate or inhibited the NADH oxidase activity. The rapidity of the response at a given concentration of thiol reagent and the degree of inhibition of the 2,4-D-induced NADH oxidase activity were dependent on order of reagent addition. If the thiol reagents were added first, auxin stimulations were prevented. If auxins were added first, the inhibitions by the thiol reagents were delayed or higher concentrations of thiol reagents were required to achieve inhibition. The results demonstrate a fundamental difference between the auxin-stimulated and the constitutive NADH oxidase activities of soybean plasma membranes that suggest an involvement of active-site thiols in the auxin-stimulated but not in the constitutive activity.     

红光抑制绿豆下胚轴伸长时富含羟脯氨酸蛋白质的含量变化

红光能增加细胞壁的Hyp含量和细胞各部分的Hyp／Pro,绿豆下胚轴切段伸长被红光抑制的程度与两者是正相关。环己酰亚胺和放线菌素D明显降低细胞壁的Hyp含量,提高WSFC和CWRSE中的Hyp／Pro,抑制切段伸长。香豆素不仅显著减少细胞壁的Hyp含量,而且降低WSFC和CWRSE中的Hyp／Pro,促进下胚轴伸长。α,α．二吡啶降低细胞壁的Hyp含量和WSFC中的Hyp／Pro,解除红光对切段伸长的抑制。EGTA、verapamil、La3 和A23187均可解除红光抑制切段伸长的作用,但对细胞壁的Hyp含量和细胞三部分蛋白质中Hyp／Pro影响较小。     

Autophagy-Related Proteins Are Required for Degradation of Peroxisomes in Arabidopsis Hypocotyls during Seedling Growth

Plant peroxisomes play a pivotal role during postgerminative growth by breaking down fatty acids to provide fixed carbons for seedlings before the onset of photosynthesis. The enzyme composition of peroxisomes changes during the transition of the seedling from a heterotrophic to an autotrophic state; however, the mechanisms for the degradation of obsolete peroxisomal proteins remain elusive. One candidate mechanism is autophagy, a bulk degradation pathway targeting cytoplasmic constituents to the lytic vacuole. We present evidence supporting the autophagy of peroxisomes in Arabidopsis thaliana hypocotyls during seedling growth. Mutants defective in autophagy appeared to accumulate excess peroxisomes in hypocotyl cells. When degradation in the vacuole was pharmacologically compromised, both autophagic bodies and peroxisomal markers were detected in the wild-type vacuole but not in that of the autophagy-incompetent mutants. On the basis of the genetic and cell biological data we obtained, we propose that autophagy is important for the maintenance of peroxisome number and cell remodeling in Arabidopsis hypocotyls.     

The Existence of Expansin and Its Properties in the Hypocotyls of Soybean Seedlings

Expansins, a newly discovered class of cell wall proteins, were the only proteins that, to date, have been shown to have the ability to restore the "acid growth" response of the heat-inactivated cell wall in an in vitro assay. In order to characterize these proteins, an automatic extensometer had been previously constructed by modification of an equal-arm mechanical balance with a linear variable differential transformer (LVDT) and with some easily available laboratory equipment. The objective of this study was to confirm and complement the work on expansin in cucumber ( Cucumis sativus L. ) seedlings carried out in the expansin-discoverers' laboratory and in addition, to further examination of the extensometer built in the authors' laboratory. It was reported that, firstly, expansin activity was maximal in cell wall from the growing region of soybean (Glycine max L. ) hypocotyls but was negligible or lacking in that from mature, basal regions and cotyledons. Corre- spondingly, walls from the growing tissue had a strong susceptibility to the action of expansin, whereas the nongrowing tissues became insensitive to the expansin action. It was concluded that the growth of soybean hypocotyl was associated with an increase in both expansin activity and wall susceptibility to the expansin action. Secondly, the heat-inactivated wall extension could be induced by cross reconstitution with crude expansin extract between soybean and cucumber species. Thirdly, once the heat-inactivated wall has been pretreated with the exogenous expansin, the reconstituted wall required no further expansin for extension indicating that exogenous expansin could specifically bind to cell wall and be enough to repeatedly exert its action without releasing from the cell wall into the external solution, i.e., a single expansin molecule could gradually break a series of load-bearing bonds one by one while moving along the cell wall, and thereby permitting the wall to extend. Fourthly, reconstitution of the wall extension activity was evidently dependent on the expansin concentration and the pH of the bathing solution, which was consistent with the catalytic characteristics of classical enzymes. Finally, endogenous and reconstituted wall extension could be significantly induced in 50 mmoL/L sodium acetate at pH 4.5 and completely inhibited in 50 mmol/L Hepes at pH 6.8, especially these phenomena could continuously be caused by switching incubation buffer from one to the other alternately, suggesting that change in pH of bathing solution could only affect the conformation of expansin (thus leading to denaturation or renaturation of it) but not the affinity of it for cell wall. In summary, these observations lend further support to the fact that expansin could mediate the acid-induced extension of the isolated wall, probably through a biochemical or enzymatic process exerting directly to the cell wall. This protein may play an essential role in the control of plant cell growth in vivo.     

Changes in Amino Acid Composition during Germination of Soybean

As a fundamental approach to the problem of amino acid metabolism in soybean, changes in content of twenty-three free amino acids, two acidic peptides, ammonia, ethanolamine, urea and seventeen total amino acids of cotyledon, hypocotyl and root of soybean during germination were determined with an amino acid analyzer. Glycine Max M. var. T201 (non-nodule-forming) and Glycine Max M. var. T202 (nodule-forming) were used for this experiment. The content and composition of free and total amino acids of cotyledon in both T201 and T202 differ from those of other tissues in any stage of germination. However, no significant difference between these two varieties of soybean has been recognized in patterns of free and total amino acids changes during germination.

In dry bean and initial stage of germination a relatively large unknown peak appeared and disappeared thereafter when the change in free amino acid content during germination of soybean was analyzed with amino acid analyzer. From various tests on the unknown peak, it became obvious that the peak was consisted of two peptides, γ-glutamyl-tyrosine and γ-glutamylphenylalanine, which were discovered in soybean by Thompson et al. in 1962. The content of these peptides did not change during the first 20 hours of germination, but they decreased rapidly thereafter and disappeared after 70 hours.     

Changes in the Lipid Composition of Rhizoctonia solani Infected Groundnut Hypocotyls

Alterations in the total and individual lipids in healthy and Rhizoctonia solani infected groundnut hypocotyl tissues were determined. Infection resulted in an increase in the total as well as individual lipid components at various stages of disease development.     

Changes in Amino Acid Composition during Germination of Soybean

Malonogalactan, a malonylated polysaccharide (—74° (c=1.6, H₂O)) produced by Penicillium citrinum, consisted of d-galactose and malonic acid in the approximate molar ratio of 3:1. Molecular weight of the demalonylated galactan (-99° (c=4.6, H₂O)) was about 40,000. From the data regarding optical rotation, nuclear magnetic resonance spectrum, infrared spectrum, glycosidase susceptibility, periodate oxidation, Smith degradation, methylation and acid hydrolysis, the possible structure of the Penicillium malonogalactan is deduced as follows: A galactan, 1,5-β-galactofuranoside polymer esterified with malonic acid at the position of 2 or 3.     

Changes in Amino Acid Composition during Germination of Soybean

α- and γ-Glutamylaspartic acids were detected in acidic fraction of soybean seedling. R_F values of both peptides were consistent with those of authentic samples with several solvent systems.     

Differential Expression of ADC mRNA during Development and upon Acid Stress in Soybean (Glycine max) Hypocotyls

Arginine decarboxylase (ADC) is one of the key enzymes in thebiosynthesis of putrescine in plants. The regulation of itsactivity depends on the physiological condition, developmentalstage, and type of tissue. We have cloned ADC cDNA from soybean(Glycine max) hypocotyls to understand the regulation mechanismsof this enzyme activity. Using the cDNA clone, we examined therelationship between changes in the ADC activity and the levelof ADC mRNA during development, in different tissues, and uponacid stress. The ADC activity began to increase 2 d after initiationof germination, reached a peak at the 5th d, and then declined.This change in the enzyme activity was preceded by similar changesin the level of the mRNA. The ADC activity was expressed tissue-specifically;this expression was well corelated with the mRNA content ofthe respective tissues. Incubation of the 5-d-old hypocotylsin pH 3 potassium phosphate solution caused a rapid increasein ADC activity. Within 2 h of acid treatment, the ADC activityincreased more than threefold. This increase was also precededby a corresponding increase in the mRNA content and was alsoregulated tissue-specifically. These results suggest that thechange in the content of ADC mRNA has an important role in theregulation of the enzyme activity during early development,in different tissues, and upon acid stress. (Received April 2, 1997; Accepted August 18, 1997)     

Association of Phytochrome with Rough-surfaced Endoplasmic Reticulum Fractions from Soybean Hypocotyls

Distribution of phytochrome (as Pfr) among membranes from soybean hypocotyls (Glycine max L. cv. Wayne) was determined by the combined techniques of cell fractionation, difference spectrometry, and electron microscopic morphometry. More than 90% of the phytochrome was found in the soluble fraction. With homogenates prepared in the presence or absence of Mg²⁺, the portion associated with membrane was only 6.5% and 1%, respectively. In the presence of Mg²⁺, the content of particulate phytochrome correlated with the amount of endoplasmic reticulum with attached ribosomes in the fractions but not with mitochondria or other membranes (including endoplasmic reticulum membranes from which the ribosomes may have been lost during cell fractionation). In the absence of Mg²⁺, phytochrome was associated with a “heavy” plasma membrane fraction. The phytochrome content was sufficiently low to be accounted for by a contamination of less than 10% by rough-surfaced fragments of endoplasmic reticulum. The findings show association of phytochrome with a particulate fraction enriched in rough-surfaced fragments of endoplasmic reticulum but do not rule out cosedimentation of some unknown or unspecific phytochrome aggregate with this fraction.     

Roles of Pectin Methylesterases in Pollen-Tube Growth

Elongation of the pollen tube in pistil is essential for delivering sperms into the female gametophyte in sexual plant reproduction. Recently, a group of cell wall enzymes, pectin methylesterases （PMEs）, have been identified as playing an important role in this process. This article reviews the new understanding of the roles of PMEs in regulating pollen tube growth.     

Changes in the Enzymes for Fatty Acid Synthesis and Desaturation during Acclimation of Developing Soybean Seeds to Altered Growth Temperature

Temperature-induced changes in the enzymes for fatty acid synthesis and desaturation were studied in developing soybean seeds (Glycine max L. var Williams 82). Changes were induced by culture of the seed pods for 20 hours in liquid media at 20, 25, or 35°C. Linoleoyl and oleoyl desaturases were 94 and 10 times as active, respectively, in seeds cultured at 20°C as those cultured at 25°C. Both desaturases had negligible activity in seeds cultured at 35°C compared to seeds cultured at 20°C. Though less dramatic, other enzymes also showed differences in activity after 20 hours in culture at 20, 25, or 35°C. Stearoyl-acyl carrier protein (ACP) desaturase and CDP-choline:diacylglycerol phosphorylcholine transferase were most active in preparations from 20°C cultures. Activities were twofold lower at 25°C and a further threefold lower in 35°C cultures. Cultures from 25 and 35°C had 60 and 40%, respectively, of the phosphorylcholine:CTP cytidylyl transferase activity present in cultures grown at 20°C. Fatty acid synthetase, malonyl-coenzyme A:ACP transacylase, palmitoyl-ACP elongation, and choline kinase were not significantly altered by culture temperature. These data suggest that the enzymes for fatty acid desaturation and phosphatidylcholine synthesis can be rapidly modulated in response to altered growth temperatures, while the enzymes for fatty acid synthesis and elongation are not.     

大豆下胚轴质膜H+-ATPase质子转运的测定

以大豆下胚轴为材料,采用改进的匀浆介质,通过两相法制得具有质子转运活力的高纯度质膜微囊.并且发现冻融处理可以促进质膜微囊的翻转而提高荧光猝灭效率.质子载体和质子转运特性分析表明,由Mg^2＋-ATP引发的荧光猝灭可以被质子载体CCCP恢复,并被质子通道抑制剂DCCD抑制;并且发现质膜H^＋-ATPase专一抑制剂钒酸钠可以完全抑制荧光猝灭,同时发现荧光猝灭依赖于Mg^2＋,并受K^＋刺激,最适pH为6.5.以上证明所测荧光猝灭是由质膜H^＋-ATPase所进行的质子转运引起的.结果同时表明,维持H^＋-ATPase合适构象和提高质膜微囊封闭性是制备具有H^＋转运活力质膜微囊的两个关键因素.     

Precocious Germination during In Vitro Growth of Soybean Seeds

Immature Glycine max (L.) Merrill seeds were grown and matured in liquid medium at 25°C under fluorescent light. In standard medium containing minerals, 146 millimolar sucrose and 62.5 millimolar glutamine (osmolality 0.24), precocious germination seldom occurred with a starting seed size of less than 300 milligrams fresh weight. Frequency of precocious germination increased with increased starting seed size. Sucrose concentration strongly affected precocious germination while glutamine concentration had no effect. Starting with 300 to 350 milligrams fresh weight seeds, treatments which reduced the sucrose concentration or lowered the osmolality of the culture medium stimulated precocious germination, and increased the fresh weight growth but not the dry weight growth of seeds. Increasing the osmolality to 0.38 with sucrose or mannitol prevented precocious germination without reducing dry weight accumulation in seeds. In medium with initially low osmolality, precocious germination was inhibited by addition of 1 to 100 micromolar abscisic acid to the medium without a reduction in seed growth. During growth and maturation of large soybean seeds in vitro, precocious germination and other abnormal tissue growth can be prevented by high sucrose or mannitol concentrations in the medium or by addition of abscisic acid.     

Cell Enlargement during Gravicurvature of Sunflower Hypocotyls

Three independent methods have shown that cells on the uppermostside of a gravistimulated sunflower hypocotyl stop enlarging;they do not merely switch from elongation to lateral growth.Upper and lower cell diameters have been compared as have freshweight changes of upper and lower cells. The radius of the hypocotylon the upper side of a horizontal hypocotyl has also been monitoredwith a linear displacement transducer and shows no significantincrease. Key words: Gravitropism, Hypocotyl, Epidermis, Growth     

Greater Length of Ribonucleic Acid Synthesized by Chromatin-bound Polymerase from Auxin-treated Soybean Hypocotyls

An investigation has been made of the RNA synthesized by chromatin-bound RNA polymerase from soybean hypocotyls (Glycine max var. Wayne). Polymerase activity is 4- to 5-fold higher with chromatin from tissue treated with 2,4-dichlorophenoxyacetic acid, a synthetic auxin, compared to untreated tissue. Thin layer chromatography of the RNA hydrolysis products and acrylamide gel electrophoresis of the RNA synthesized by the chromatin show that increased activity induced by 2,4-dichlorophenoxyacetic acid is due primarily to the production of longer RNA chains, with only 20 to 50% increase in the number of RNA chains. The observation that 2,4-dichlorophenoxyacetic acid treatment leads to greater rates of RNA synthesis, producing longer chains in unit time, suggests that one manifestation of auxin activity is in activation of RNA polymerase I (ribosomal RNA polymerase).