Auxin-Induced Changes in the Molecular Weight Distribution of Cell Wall Xyloglucans in Avena Coleoptiles

The effect of auxin on the molecular weight (Mw) distributionof cell wall xyloglucans was investigated by gel permeationchromatography using coleoptile segments of Avena sativa L.cv. Victory, and the following results were obtained.

1. The water-insoluble hemicellulose (HC-A) mainly consisted ofxyloglucans. Iodine staining method revealed that relativelylarge amounts of xyloglucans were present in the water-solublehemicellulose (HC-B) and water-soluble polysaccharide (WS) fractions.
2. IAA did not cause remarkable changes in xyloglucan contentsin the hemicellulose, but significantly increased the xyloglucancontent in the WS fraction.
3. IAA substantially decreased theweight-average Mw of HC-A. Thiseffect became apparent within30 min of the incubation period,and was not affected by the0.15 M mannitol or 2% sucrose applied.Hydrogen ions also causeda decrease in the weight-average Mwof HC-A; its effect beingreversible.
4. Neither IAA nor hydrogen ions caused any remarkablechangesin the weightaverage Mw of water-soluble xyloglucansin theHC-B.

These results suggest that cell wall xyloglucans have an importantrole in auxininduced cell wall loosening in oat coleoptile cells. (Received May 10, 1984; Accepted August 20, 1984)     

Changes in cell wall polysaccharides and monosaccharides during development and ripening of Japanese pear fruit

1. Changes in polysaccharide and monosaccharide components in thecell wall were studied during cell division, cell enlargmementand softening in Japanese pear fruit. Wall polysaccharides werefractionated into water soluble carbohydrate, NaClO₂ solublecarbohydrate, EDTA soluble carbohydrate, acid soluble hemicellulose,alkali soluble hemicellulose and cellulose. These polysaccharideswere composed of glucose, uronic acid, xylose, arabinose, galactose,rhamnose, mannose and fucose.
2. The total polysaccharide contentof the cell wall per cell (DNAcontent basis) remained constantduring the cell division period(S1). But during the pre-enlargementperiod (S2) it began toincrease rapidly in spite of the slightnessof cell enlargement.Thereafter, during the enlargement period(S3) the polysaccharidesremained almost constant although thefruits enlarged dramatically,and the polysaccharides increasedsomewhat with ripening. Thequality of the polysaccharides,however, seemed to change activelyat each stage. This suggestedthat the extensive fruit enlargementdid not require an increasein polysaccharide content, and wasrather accompanied by thepartial breakdown or partial interconversionof polysaccharidecomponents already present.
3. The loss of arabinose and galactosein acid soluble hemicellulosewas prominant in fruit softeningoccurring in the ripening stage.The cellulose component decreasedwith overripening. Water solublepectin increased parallel tothe increase in total pectin withripening. On the other hand,xylose and non-cellulosic glucoseresidues did not alter withripening or overripening. Non-cellulosicglucose continued toaccumulate during cell enlargement.

¹ This paper is Contribution A-88, Fruit Tree Research Station. (Received August 4, 1978; )     

Studies on the Cell Wall of Chlorella IV. Incorporation of Glucose-Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

Uniformly ¹⁴C-labeIled glucose was fed to synchronously growingChlorella cells in the dark or in light. The rate of ¹⁴C-incorporationinto hemicellulose showed two maxima one in the growth phaseand one in the reproductive phase. Significant ¹⁴Cincorporationinto a "rigid wall" was found only in the reproductive phase. (Received April 14, 1983; Accepted June 15, 1983)     

Effects of hypergravity on growth and cell wall properties of cress hypocotyls

Elongation growth of etiolated hypocotyls of cress (Lepidiumsativum L.) was suppressed when they were exposed to basipetalhypergravity at 35 g and above. Acceleration at 135 g causeda decrease in the mechanical extensibility and an increase inthe minimum stress-relaxation time of the cell wall. Such changesin the mechanical properties of the cell wall were prominentin the lower regions of hypocotyls. The amounts of cell wallpolysaccharides per unit length of hypocotyls increased underthe hypergravity condition and, in particular, the increasein the amount of cellulose in the lower regions was conspicuous.Hypergravity did not influence the neutral sugar compositionof either the pectin or the hemicellulose fraction. The amountof lignin was also increased by hypergravity treatment, althoughthe level was low. The data suggest that hypergravity modifiesthe metabolism of cell wall components and thus makes the cellwall thick and rigid, thereby inhibiting elongation growth ofcress hypocotyls. These changes may contribute to the plants'ability to sustain their structures against hypergravity. Key words: Cell wall extensibility, cellulose, hypergravity, Lepidium sativum L., lignin     

Studies on the Cell Wall of Chlorella III. Incorporation of Photosynthetically Fixed Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

The carbon metabolism in cell walls of Chlorella ellipsoideawas studied by following ¹⁴C incorporation into cell wall constituentsin photosynthesizing, synchronously growing cells. The rateof incorporation was higher at an early growth phase of thecell cycle. The ¹⁴C was incorporated into both the major cellwall constituents, hemicellulose and ‘rigid wall’,and the radioactivity in the latter was distributed into itstwo components, glucosamine and amino acids. In pulse-chaseexperiments, the ¹⁴C fixed photosynthetically in the precedingcell cycle was rapidly transferred into the cell wall constituentsat the early growth phase of the ongoing cell cycle, and thereafterwas gradually released from the cell walls, although the totalamount of ¹⁴C in the cells remained constant. It was concludedthat the cell wall constituents are turned over during the growthphase of the algal cell cycle, and that the cell wall metabolismin the ongoing cell cycle is closely connected with the carbonmetabolism in the preceding cell cycle. (Received February 3, 1982; Accepted June 21, 1982)     

Stipe elongation during basidiocarp maturation in Coprinus macrorhizus: Mechanical properties of stipe cell wall

Stipe elongation during basidiocarp maturation in the wild-type,#5026+5132, and the elongationless mutant, NG0398, of Coprinusmacrorhizus was studied, and the following results were obtained.

1. In the wild-type the middle zone of the stipe elongated 8.4times in 15 hr during maturation, while in the mutant it elongatedoaiy 2.2 times.
2. Component cells of the stipe elongated inparallel with thestipe elongation in both the wild-type andthe mutant. The widthof stipe cells was almost constant duringelongation in thewild-type, while it increased 2 times in themutant. Cell volumeincreased ca. 8 times in both stocks.
3. Theosmotic value of stipe cells was almost constant (0.45–0.50M) throughout elongation of both the wild-type and the elongationlessstipes.
4. Mechanical properties of the cell wall were examinedby measuringshrinkage, extensibility and minimum stress-relaxationtime(To) of the stipe during maturation. These parameters weredirectlyproportional to the elongation rate to follow.
5. Whenthe wild-type stipes were incubated in various concentrationsof mannitol solution and then in plain buffer solution, theextensibility of the stipe after the incubation in mannitolsolutions changed proportionally with the stipe length afterthe mannitol treatment, and To with the elongation capacityin plain buffer solution.

(Received March 3, 1977; )     

Regulation of lettuce hypocotyl elongation by gibberellic acid. Correlation between cell elongation, stress-relaxation properties of the cell wall and wall polysaccharide content

Lettuce hypocotyl elongation caused by gibberellic acid wasstrongly inhibited by coumarin and dichlobenil, known inhibitorsof cellulose biosyndiesis. Stress-relaxation analysis of thecell wall revealed that gibberellic acid induces a decreasein both minimum relaxation time (To) and relaxation rate (b)and an increase in maximum relaxation time (Tm), when gibberellicacid stimulates hypocotyl elongation. Both coumarin and dichlobenilnullified the effect of gibberellic acid on changes in To, Tmand b values. The content of pectic, hemicellulosic and cellulosic substancesin the cell wall increased per hypocotyl but decreased per unithypocotyl length, in response to gibberellic acid treatment.Particularly, gibberellic acid caused a substantial increasein cellulose content per hypocotyl but a decrease per unit length.A good correlation existed between the decrease in To and thedecrease in hemicellulose content per unit lengdi of the cellwall. The increase in Tm was correlated with the decrease incellulose content per unit length of the cell wall. The decreasein b was correlated with the decrease in the content of bothcellulose and hemicellulose per unit length. Based on these results, we discuss the role of polysaccharidemetabolism of the cell wall in gibberellic acid-induced lettucehypocotyl elongation and the nature of gibberellic acid-inducedbiochemical modifications of the cell wall, which are representedby changes in stress-relaxation properties of the cell wall. ¹Present address: Department of Anatomy, Aichi Medical University,Nagakutecho, Aichigun, Aichi 480-11, Japan. (Received September 22, 1975; )     

WATER PERMEABILITY OF THE CELL WALL IN NITELLA

1. A method has been developed to measure the hydraulic conductivityof the wall of the internodal cell of Nitella flexilis.
2. Therate of water penetration through the cell wall varies linearlywith the hydrostatic pressure difference between the two sidesof the wall, showing that water permeability of the cell wallremains independent of the pressure difference applied.
3. Waterpermeability of the cell wall is inversely proportionalto itsthickness It is 30µµmin^–3{dot}atm^–3when the thickness of the wall is 10 µ.
4. Water permeabilityof the cell wall is the same for inward andoutward water flow.The polar water permeability of the entiremembrane system (walland protoplasmic part) of the living celldemonstrated by KAMIYAand TAZAWA (1) is, therefore, due tothe living protoplasmicpart.
5. The ratio of the inward to outward permeability constantsofthe protoplasmic layer alone is higher than that of the entiremembrane system composed of protoplasmic layer and cell wall.

¹ Dedicated to Prof. H. TAMIYA on the occasion of his 60th birthday.The present work was supported in part by a Grant-in-Aid forFundamental Scientific Research from the Ministry of Education. ² Present address: Sh?in Women's College, Kobe. (Received July 21, 1962; )     

Effects of gibberellic acid and indole-3-acetic acid on stress-relaxation properties of pea hook cell wall

The effects of GA, IAA and PCIB on the cell wall propertiesof Alaska pea hooks were examined using stress-relaxation analysis.The results were:

1. GA caused a decrease in the stress-relaxation parameter To ofplumular hook sections after the first 30 min of incubation,long before it induced elongation.
2. PCIB increased To, andIAA tended to negate the PCIB effecton To in GA-treated sectionsafter 90 min of incubation, whenthe effect of PCIB and IAAon the elongation was not yet found.In this case, IAA couldnot be substituted by an extra amountof GA.
3. GA decreasedTo in the middle part of the sections after 24hr of incubation,and then stimulated elongation.
4. In any case, the effect ofGA, IAA or PCIB on To was recognizedin both epidermis and innertissue of plumular hook sections.
5. The stress-relaxation parameterTo appears to represent thecapacity of the cell wall to extend;we thus concluded thatboth gibberellin and auxin increase theextensibility of thecell wall, when they stimulate the elongationof plumular hooksections.

(Received October 4, 1974; )     

Studies on nucleic acids in chloroplasts isolated from Chlorella protothecoides

1. 1. Subcellular fractions of Chlorella protothecoides were separatedby fractional centrifugation of the algal cell homogenate inmixtures of cyclohexane and CCl₄. The base composition, meltingprofiles and IRC-50 column chromatographic patterns of DNA preparationsfrom the chloroplast and non-chloroplast fractions were examined.It was shown that the algal chloroplast contains at least oneDNA species which is different from the nuclear DNA.
2. 2. RNApreparations from the subcellular fractions were subjectedtoMAK column chromatography, sucrose density gradient centrifugationand analysis for base composition. It was demonstrated thatthe chloroplast contains ribosomal RNA and soluble RNA. Twocomponents of the chloroplast ribosomal RNA were found to havethe same patterns as those of the E. coli ribosomal RNA in MAKcolumn chromatography and zone centrifugation. The major componentof the chloroplast ribosomal RNA was distinctly different fromthat of the non-chloroplastic (cytoplasmic) ribosomal RNA inall properties examined.

¹This work was partly reported at the Symposium on Mitochondriaand Chloroplasts as Self-duplicating Units sponsored by theBotanical Society of Japan in August, 1966, and at the Symposiumon Biogenesis of Subcellular Particles, the 7th Internatl. Congressof Biochemistry, Tokyo, 1967.     

Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Turnover of cell wall components was examined in two growth phases of a batch suspension culture of Vinca rosea L. Three-day-cultured cells (cell division phase) and 5-day-cultured cells (cell expansion phase) were incubated with d -[U-¹⁴C]glucose. After various periods of incubation, extra-cellular polysaccharides (ECP) and cell walls were isolated, and then the cell walls were fractionated to pectic substance, hemicellulose, and cellulose fractions. The results of the measurement of radioactivities and amounts of total carbohydrate in the ECP and cell wall fractions indicated that synthesis of pectic substance was more active in the cell division phase than in the cell expansion phase. From the results of the pulse-chase experiments, in which cells prelabelled by incubation with d -[U-¹⁴C]glucose for 3 h were incubated in a medium containing unlabelled glucose for various periods, the gross degradation, net synthesis, and gross synthesis of cell wall components were estimated. Active degradation and synthesis were observed in the hemicellulose fraction, indicating that active turnover occurred in the hemicellulose fraction, while little degradation was found in the pectic substance and cellulose fractions.     

Cell elongation and metabolic turnover of the cell wall as affected by auxin and cell wall degrading enzymes

A cell wall fraction (pectic substances) of oat coleoptile segmentsfed with ¹⁴C-glucose contained more radioactivity under theeffect of auxin than did the control. When labeled segmentswere grown for 6 hr in auxin or glucanase solution the labelin the hemicellulose fraction decreased as growth increased.ß-1,3-Glucanase prepared from the culture of a fungus,Sclerotinia libertiana, induces elongation of segments of thepea stem and the oat coleoptile. Traces of cellulase and pectinmethylesterase contaminating the enzyme preparation are notresponsible for the stimulatory effect. Cellulase seemed tobe rather inhibitory and pectin methylesterase showed only aslight effect on coleoptile elongation. A possible relationshipbetween the metabolic turnover of hemicellulosic polysaccharideand cell wall extension is suggested. (Received February 5, 1968; )     

Studies on the cell wall of Chlorella II. Mode of increase of glucosamine in the cell wall during the synchronous growth of Chlorella ellipsoidea

The dry weight and glucosamine content of the alkali-insoluble"rigid wall" of Chlorella ellipsoidea were measured in synchronouslygrowing and dividing cells. The content of glucosamine, the major component of the rigidwall, was measured by the Elson-Morgan and the ninhydrin reactions.The results revealed, in agreement with previous observations,that the amount of the rigid wall, measured in terms of wholedry weight or glucosamine content, remains almost constant duringthe growing phase and increases only in the reproduction phase. (Received February 19, 1979; )     

"GIGANTISM" OF CHLORELLA VULGARIS I. MECHANISM OF INDUCTION OF CIGANTISM

1. Based on the microscopic observations, two stages, "giant cellstage" and the subsequent "palmelloid body stage", were distinguishedin the process of formation of giant Chlorella induced by theaddition of sugars. The "giant cell" is much larger in sizethan the control cell, but the other morphological featuresare the same as those of the latter. The "palmelloid body" isa form composed of many conjoined autospores.
2. When a highconcentration of glucose was maintained in the medium,gigantismwas also maintained. Under this condition, the algashows acyclic transformation between "giant cell" and "palmelloidbody"without returning to the small single cells.
3. Large amountsof carbohydrate composed of hexose were foundto be accumulatedin the giant algal cells, and it was inferredthat this carbohydrateaccumulation causes greater enlargementof cell volume as comparedwith control cells.
4. Uronic acids, which were found to be absentin the control cells,were formed and lost in the cells culturedin the glucose mediumin parallel with the appearance and disappearanceof gigantism.
5. Pectic substances, from which uronic acids areconsidered tobe derived during the extraction procedure, werefound to bepresent only in giant Chlorella.
6. The conjoinedautospores in giant Chlorella (at the palmelloidbody stage)were separated to some extent by the addition ofEDTA, and theresulting cells were similar to control Chlorellacells.
7. Basedon these results it was inferred that inductive formationofthe pectic substances is causally related with the appearanceof "palmelloid body".

¹ Present address: Department of Chemistry, College of GeneralEducation, Osaka University, Toyonaka, Osaka.     

Auxin-induced changes in the molecular weight of hemicellulosic polysaccharides of the Avena coleoptile cell wall

The average molecular weight of the water soluble hemicelluloses(hemicellulose B) of the Avena coleoptile cell wall was determinedby gel permeation chromatography (GPC) and viscometry. Analysisof the neutral sugar composition of henucellulose B eluted froma GPC column (Sepharose 4B) indicated that it consists of ß-glucanwith a high molecular weight and arabinoxylan with a low molecularweight. A kinetic study of the effect of auxin on the moleculardistribution of henucellulose B demonstrated that auxin decreasedthe ß-glucan content of the hemicellulose as earlyas the first hour incubation, but not the arabinoxylan content,when it stimulated the extension of the coleoptile segments.Calculation of the weight-average molecular weight from thechromatograms suggested that auxin decreased the molecular weightof hemicellulose B; this was also confirmed by viscometry. Thus,auxin may cause cell wall loosening, leading to cell extension,through its effect on ß-glucan degradation or throughthe decrease in the molecular weight of hemicellulose B. (Received July 16, 1979; )     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

SYNCHRONOUS CULTURE OF CHLORELLA: II. CHANGES IN CONTENT OF VARIOUS VITAMINS DURING THE COURSE OF THE ALGAL LIFE CYCLE

1. Chlorella ellipsoidea was grown synchronously and the changesin content of various vitamins during the algal life cycle werefollowed either by chemical or microbiological assay methods.
2. In terms of µg per gram of cell dry weight, the contentof some vitamins (niacin, biotin, inositol and choline) remainedalmost constant throughout the algal life cycle, while thatof others (vitamin B₆-complex, pantothenic acid, folic acid,thiamine and riboflavin) was found to decrease more or lessmarkedly during the "growing phase" and increase at later phasesof "ripening". The content of p-aminobenzoic acid increasedonly at an early stage of "ripening", and that of ascorbic acidincreased only at the stages in which photosynthesis occurredmost actively.
3. These results were discussed in an attemptto interprete theirrelationship with the previously reportedobservations pertainingto the physiological and biochemicalevents occurring in thelife cycle of the alga.

(Received November 7, 1959; )     

Presence of a xyloglucan in the cell wall of Phaseolus aureus hypocotyls

The non-cellulosic ß-glucan₁ in the cell wall of Phaseolusaureus hypocotyb was studied. Evidence that xyloglucan is presentin a hemicellulose fraction was obtained by its isolation fromcell wall preparations. This polysaccharide was homogeneouson zone electrophoresis and ultracentrifugation. On acid hydrolysis,it gave glucose, xylose, galactose, and fucose in the approximatemolar ratio of 10 : 7 : 2.5 : 1. Its solution gave a reddishviolet color with iodine-staining solution. The results of partialacid hydrolysis and cellulase treatment suggest a structurein which xylose, galactose, and fucose attached as side chainsto a sequenceof ß-l,4-linked glucose. The xyloglucanisolated accounted for 13.9% of the total non-cellulosic fractions. (Received May 10, 1976; )     

Polyphenoloxidase and the Respiration of Ivy Leaves

1. Polyphenoloxidase is present in ivy leaves but not in thoseof Aucuba or Euonymus.
2. Respiration of intact ivy leaves wasmarkedly stimulated bycatechol (R.Q. approximately=I), gallicacid, caffeic acid,and dihydroxyphenylalanine. The stimulationwas not relatedto injury as far as could be detected and wasnot followed byinhibition. The extra oxygen consumption representsa many timesrepeated oxidation of the amount of catechol supplied.
3. The effect of catechol on the respiration of Aucuba and Euonymusleaves was very small.
4. Cupferron and phenylthiourea, whichinhibit polyphenoloxidasein vitro, nevertheless increased respirationwhen administeredto leaves through the petiole. On the otherhand, when appliedby infiltration, cupferron did cause inhibition,but in Aucubaand Euonymus as much as in ivy.

     

Culture of chlorophyllous tobacco-cells not requiring any organic additives except sucrose in the medium I. Effects of light and temperature on the growth of the cells

1. In the dark, HNG (habituated Nicotiana glutinosa) and NG cellsscarcely grew at 15?C, and the difference between the growthrates of HNG and NG was small at both 15?C and 25?C.
2. The stimulatoryeffect of light (4000 lux) on the cell growthrate was higherat 25?C than at 15?C for both HNG and NG.
3. Light exerted muchmore effect on the growth rate of HNG thanof NG.
4. The thermaleffect was higher in the light than in the darkfor both HNGand NG, and was somewhat greater on NG than onHNG.
5. The synergisticeffect of light and temperature on cell growthwas greater onHNG than on NG.
6. HNG contained more chlorophyll than NG.
7. Inaddition, there was little difference between the friabilitiesof cell groups of HNG and NG.

(Received December 3, 1976; )