Growth and cell wall changes in azuki bean epicotyls I. Changes in wall polysaccharides during intact growth

Measurement of endogenous growth rates and the mechanical propertyof the cell wall in various regions of light-grown azuki beanepicotyls revealed diat the minimum stress-relaxation time (T_o)was the shortest in the upper region (0–30 mm below theapex) of the epicotyl, where vigorous endogenous growth tookplace, and became longer toward the basal region, which wasmature and not growing. In the upper region of the epicotyl, a lower percentage of a-celluloseand a higher percentage of pectic substances than in the lowerregion were found. The percentage of hemicellulose content wasalmost constant over the whole epicotyl. Major components ofnoncellulosic neutral sugars in the cell wall were galactoseand xylose. The percentage of the galactose content to the noncellulosicpolysaccharide was highest in the upper region and lowest inthe basal region of the epicotyl, and a clearly negative correlationbetween the galactose composition and the T_o value was obtained.On the contrary, the percentage of die xylose content was highestin the basal region and lowest in die upper region, and a clearlypositive correlation between die xylose composition and theT_o value was obtained. During die endogenous growth of die intactepicotyl, all die neutral sugars, particularly galactose, increasedin die upper region, whereas in die middle and basal regions,only xylose increased. Similar changes in sugar compositionswere observed during IAA-induced elongation of die segment excisedfrom various regions of die epicotyl. (Received July 27, 1978; )     

Growth and cell wall changes in azuki bean epicotyls II. Changes in wall polysaccharides during auxininduced growth of excised segments

Changes in cell wall polysaccharides and mechanical propertiesof the cell wall were examined during IAA-induced elongationgrowth of excised azuki bean epicotyl segments under differentgrowth conditions. Sucrose promoted IAA-induced cell elongation,but had very little effect on IAA-induced cell wall loosening.In the absence of sucrose, the amount of galactose in the cellwall decreased during the incubation period. IAA enhanced thedecrease in the galactose level. In the presence of sucrose,on the other hand, IAA induced increases in the amounts of cellulose,galactose and xylose in noncellulosic polysaccharides. TheseIAA-induced increases were not observed in the presence of mannitolat concentrations higher than 0.1 M, although cell wall looseningwas induced by IAA even in the presence of 0.2 M mannitol. (Received November 21, 1978; )     

Photosynthetic activities of a thermophilic blue-green alga

Photosynthetic activities of a thermophilic blue-green alga,a species of Synechococcus, were studied with special referenceto its growth at high temperatures. A rapid algal growth occurredin the temperature range between 50 and 60?C, showing the maximumrate, six doublings per day, at about 57?C. Photosynthetic oxygenevolution and methyl viologen photoreduction in the cells werealso active at high temperatures and the optimum temperaturesfor these activities agreed with that of the algal growth. Thegrowth and photosynthetic activities were very low at room temperatureand irreversibly inactivated at temperatures above 60?C. The thylakoid membranes isolated from the alga were also photochemicallyactive at high temperatures. The membranes mediated ferricyanidephotoreduction coupled with a stoichiometric oxygen evolutionat a rate comparable to that of photosynthetic oxygen evolutionin the cells. The optimum temperature for the reaction was ashigh as 50?C. The membranes also showed a photosystem I-mediatedreaction at high temperatures. These observations indicate thatthe thylakoid membranes are intrinsically thermophilic in thisorganism. Thus the growth of the alga at high temperatures canbe well correlated to thermophilic properties of the photosyntheticapparatus. (Received February 20, 1978; )     

Effects of dibromothymoquinone and bathophenanthroline on flash-induced cytochrome f oxidation in spinach chloroplasts

The effects of several electron transport inhibitors on themagnitude and kinetics of cytochrome f oxidation induced byflash illumination were studied in the - and -band regions.On the flash excitation only a fraction of cytochrome f presentin the chloroplasts was oxidized with a half time of 0.1 to0.3 msec and then reduced with a half time of 10 to 25 msec. Dibromothymoquinone (DBMIB) at concentrations which severelysuppressed the reduction of cytochrome f approximately doubledthe magnitude of cytochrome f oxidation caused by a flash, mainlyby inducing an additional slow oxidation of cytochrome f witha half time longer than 1 msec. Enhancement of the cytochromef oxidation was also observed in the presence of bathophenanthroline.Such enhanced oxidation in duced by the two inhibitors was largelydiminished with the addition of reduced 2,6-dichlorophenolindophenolwhich accelerated cytochrome f reduction. In contrast, the inhibitionof cytochrome f reduction by 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) was not associated with an increase in the magnitudeof cytochrome f oxidation. However, addition of DBMIB to theDCMU-poisoned chloroplasts enhanced cytochrome f oxidation,suggesting that this is related to a block of the electron transportbetween plastoquinone and cytochrome f. The results are explainedby assuming the occurrence of an electron carrier between plastoquinoneand cytochrome f. (Received May 10, 1978; )     

Scanning electron microscopy of giemsa-stained chromosomes and surface-spread chromosomes

Giemsa-stained chromosomes as prepared for light microscopy, and including G-banded, C-banded, and FPG-stained chromosomes, were examined by scanning electron microscopy. Although suitable for light microscopy, these chromosomes were too flat for a close examination of their fine structure by scanning electron microscopy. The surface of Giemsa-positive regions was rough and bright, whereas that of unstained or poorly stained regions was smoother and less bright. Giemsa-staining, therefore, seems to produce the bulkiness of the chromosomes. On topographical examination by scanning electron microscopy, the transparent chromosomes as observed with the light microscope proved to be footprints. Stereographical examinations of surface-spread chromosomes showed that minimally stretched chromosomes were composed of a mass of nodular and twisted looping fibers with an average diameter of about 300 Å. The substructure of these chromosome fibers was not determined. The kinetochore region was discernible as a constriction in the mass of the chromosome fibers, and was distinguishable from gaps by the presence of several chromosome fibers parallel to the axis of the chromatid. The organization of the chromosome fibers, however, was disordered rather than regular.     

Insulin binds to type V collagen with retention of mitogenic activity

The abilities of eight extracellular matrix proteins, fibronectin, vitronectin, laminin, and collagen types I, II, III, IV, and V to bind insulin were examined by binding studies with insulin conjugated with peroxidase. At a physiological pH and ionic strength, type V collagen bound to insulin most strongly. The other types of collagen, laminin, and vitronectin also bound insulin with affinity lower than that of type V collagen. The insulin-binding site of type V collagen was in a 30-kDa CNBr fragment of the alpha 1 (V) chain. Analysis of the amino acid sequence showed that this 30-kDa fragment was identical to the heparin-binding fragment of type V collagen. The insulin-binding sites of laminin and vitronectin were located in the A chain and in the heparin-binding domain, respectively. Insulin bound to type V collagen stimulated the synthesis of DNA by mouse mammary tumor MTD cells, indicating that bound insulin retained mitogenic activity.     

Auxin-induced changes in the cell wall structure: Changes in the sugar compositions, intrinsic viscosity and molecular weight distributions of matrix polysaccharides of the epicotyl cell wall of Vigna angularis

Rapid effects of indole-3-acetic acid (IAA) on the mechanical properties of cell wall, and sugar compositions, intrinsic viscosity and molecular weight distribution of cell wall polysaccharides were investigated with excised epicotyl segments of Vigna angularis Ohwi et Ohashi cv. Takara.

• 1 IAA caused cell wall loosening as studied by stress-relaxation analysis within 15 min after the IAA application.
• 2 IAA stimulated the decrease in the content of arabinose and galactose in the hemicellulose 1 h after its application. The amounts of other component sugars in the cell wall polysaccharides remained constant during the IAA-induced segment growth.
• 3 The intrinsic viscocity of the pectin increased as early as 30 min after the IAA application. This effect was not prevented when elongation growth of the segment was osmotically suppressed by 0.15 M mannitol.
• 4 Gel permeation chromatography of the pectin on a Sepharose 4 B column demonstrated that IAA caused increase in the mass-average molecular weight of the pectin. Analysis of the sugar compositions of the pectin eluted from the Sepharose 4 B column indicated that IAA increased the molecular weight of the polysaccharides composed of uronic acid, galactose, rhamnose and arabinose. This effect became apparent within 30 min after the IAA application. Furthermore, IAA increased the molecular weight of the pectin when elongation growth of the epicotyl segments was osmotically suppressed by 0.15 M mannitol.
• 5 Hemicellulose of the cell wall chromatographed on a Sepharose CL-4 B column. Analysis of the neutral sugar compositions and the iodine staining property (specific for xyloglucans) of the polysaccharide solution eluted from the column indicated that the hemicellulose consisted of xyloglucans, arabinogalactans and polysaccharides composed of xylose and/or mannose. IAA caused a decrease in the arabinogalactan content and depolymerization of xyloglucans. These IAA effects became apparent within 30 min after the IAA application. These changes occurred even when elongation growth of the epicotyl segments was osmotically suppressed by 0.15 M mannitol.

Polymerization of the pectin, degradation of arabinogalactans and depolymerization of xyloglucans appear to be involved in the mechanism by which IAA induces cell wall loosening and therefore extension growth of cells.     

A Study on the Mechanism of Nodding Initiation of the Flower Stalk in a Poppy, Papaver Rhoeas L.

A study was made to find whether the nodding of the flower stalkin a poppy, Papaver Rhoeas L., immediately after its formationwas triggered by the weight of its flower bud or by positivegeoreaction and the following results were obtained.

1. The direction of the nodding was mostly toward the inclinedside of the stalk, which was opposite the leaf, for apical flowerbuds.
2. If the weight of the flower bud at stage 1 was cancelledbyapplying a load equivalent to the bud weight, the noddingofthe stalk was not initiated.
3. The stalk at stages 1 and2 and the upper part of the stalk(bending zone), as comparedwith the basal part, at later stageswere highly deformableaccording to measurements by the bendingmethod.
4. The cellwall of highly deformable stalks was rich in hemicellulosesand that of the basal part was abundant in pectic substances.

From these results, we concluded that the initiation of thenodding in the flower stalk was caused by the weight of theflower bud and positive geotropic reaction was probably notinvolved. (Received December 22, 1980; Accepted January 26, 1981)     

Polymerization of deoxyhemoglobin CHarlem (β6 Glu → Val, β73 Asp → Asn): The effect of β73 asparagine on the gelation and crystallization of hemoglobin

The kinetics of aggregation and the solubility of deoxy Hb2 C_Harlem (α₂β₂ 6 Val, 73 Asn) in concentrated phosphate buffers were studied in comparison with those of deoxy Hb S and deoxy Hb A. Deoxy Hb C_Harlem aggregated with a clear exhibition of a delay time. The length of the delay and aggregation times and the degree of the aggregation depended upon the initial hemoglobin concentration.The initial hemoglobin concentration required for the aggregation of deoxy Hb C_Harlem was approximately 200% of its solubility, a value much higher than that required for the aggregation of deoxy Hb S (120%). With the same hemoglobin concentration, the delay time for the aggregation of deoxy Hb C_Harlem was approximately 100 times longer than that of deoxy Hb S. The logarithmic plotting of the delay time versus hemoglobin concentration in 1.8 m-phosphate buffer (pH 7.4) showed linear lines with a slope (n) of 4.0 for deoxy Hb C_Harlem. In contrast to the results for the aggregation of deoxy Hb S, n values for deoxy Hb C_Harlem were unchanged with phosphate concentrations varying from 1.2 m to 2.0 m. The solubilities of deoxy Hb S and deoxy Hb C_Harlem were increased exponentially by lowering the pH of the medium, with the increase being more conspicuous for Hb C_Harlem. The gels (or aggregates) of Hb C_Harlem were converted to crystals at a rate much faster than were those of Hb A and Hb S. The kinetics for gelation and crystallization of deoxy Hb C_Harlem can be explained by the following scheme, where nuclei G and nuclei C are formed before gelation and crystallization, respectively. Monomenc deoxy Hb

The hemoglobin concentration required for the crystallization of deoxy Hb C_Harlem was about ten times lower than that required for deoxy Hb A. The solubility of deoxy Hb C_Harlem after aggregation was about twice that of deoxy Hb S, suggesting that the substitution of Asn for Asp at the β73 residue inhibits the formation of nuclei G and accelerates the formation of nuclei C.