Effect of Benzyladenine on DNA, RNA, Protein, and Chlorophyll Contents in Intact Bean Leaves: Differential Responses to Benzyladenine According to Leaf Age

Benzyladenine (BA) was applied to intact bean (Phaseolus vulgaris) leaves at different stages of their growth. Changes in the amounts of cellular constituents resulting from the different treatments were followed and compared. RNA, protein, and chlorophyll contents, dry weight, fresh weight, and leaf area per single leaf continued increasing when leaves were treated with BA from an early stage, whereas in untreated leaves all these values levelled off or declined with advancing age. Besides these changes, BA treatment induced an increase in the DNA content. Changes in RNA content was more remarkable in response to application or deprival of BA treatment than the corresponding ones in protein and chlorophyll contents. The pattern of response to BA varied greatly according to the age at which the leaf received the treatment. As leaves aged, they lost the ability to increase their area and fresh weight in response to BA. However, continuous treatment with BA from an early stage kept the leaves young and able to respond.     

CHANGES IN THE RESPONSE OF CARROT ROOT CALLUSES TO THE ROOT EXTRACT OCCURRING DURING THEIR SUCCESSIVE CULTURES

1. Alcohol extract of carrot root promoted the growth of the carrotroot callus which had been succesively cultured for more than18 months (CCL) on the medium containing WHITE'S inorganic salts,sucrose, yeast extract and 2, 4-D, but only a weak promotionwas observed for the growth of the carrot root callus whichhad been cultured for less than 14 months (CCS).
2. The activesubstances were fractionated by Amberlite IR-120and AmberliteIRA-400 into four fractions; C, D, E, and F. Eachfraction seemedto act synergistically to produce the effectof the whole carrotroot extract on the growth of CCL.
3. Fraction F of the carrotroot extract, which was adsorbed byAmberlite IRA-400 but notby Amberlite IR-120, promoted thegrowth of CCL in the presenceof other fractions, but had noeffect on the growth of CCS.So the different responses to thealcohol extract of the carrotroot calluses having differentlengths of successive cultureperiod seemed to depend mainlyon the ability of respondingto fraction F.
4. Using four strains of carrot root callusesof different origin,it was ascertained that different responsesof carrot root callusesto fraction F depended on the lengthof their culture and noton their strain-specific characters.
5. The substances active for the growth of CCL in the carrotrootextract passed through a dialysis membrane. These substanceswere little affected by autoclaving and remained in the aqueouslayer when shaken with several organic solvents: n-butanol,ethyl acetate, chloroform, benzene, ethyl ether and carbon tetrachloride.
6. Alcohol extract of carrot root also promoted the growth ofcarrotroot explant, tobacco stem callus and sunflower crowngall tissue.

(Received December 24, 1964; )     

CHANGES IN RESPONSE TO PURINE AND PYRIMIDINE DERIVATIVES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURING

1. The growth of the carrot root callus which had been subculturedfor a long period (CCL) was promoted by the addition of 5l0^–8and 5l0^–7 M kinetin, whereas in the callus subculturedfor a short period (CCS) no growth promotion was observed atany concentrations of kinetin tested.
2. CCL showed an increasedgrowth in response to the applicationof kinetin, guanine, adenine,hypoxanthine, uracil, thymine,and cytosine in the presenceof fractions A and C of carrotroot extract, whereas no suchresponse was observed in CCS.CCL required fraction C to respondto uracil and probably purineand pyrimidine derivatives ingeneral.
3. The growth of CCL was promoted by kinetin, guanine,adenine,or hypoxanthine in the medium containing inositol andaminoacids mixture. In this case the growth-promoting actionof guanine,adenine, or hypoxanthine was nullified by kinetin.

(Received December 24, 1964; )     

The Effect of Benzyladenine on Changes in Nuclease and Protease Activities in Intact Bean Leaves during Ageing

Primary leaves of intact bean plants (Phaseolus vulgaris L.) were treated with benzyladenine (BA) at different stages during growth. Changes in DNase, RNase, and proteas activities in the leaves were followed. Unlike the case of various excised tissues, cytokinin raised the activities of these hydrolases in intact bean leaves. Because BA elevated the levels of DNA, RNA, and protein in intact leaves, it may stimulate both synthesis and decomposition of these cellular constituents. The hydrolase activities showed differential responses to BA according to the age at which the leaf received the hormone treatment.     

Longitudinal changes in δ13C of riffle macroinvertebrates from mountain to lowland sections of a gravel‐bed river

Summary 1. To understand longitudinal changes in the trophic base of benthic macroinvertebrates from mountain to lowland river sections, we investigated carbon stable isotopic compositions (δ¹³C) of macroinvertebrates and their food resources in riffles for four seasons at 14 sites along the main stem of the Toyo River, Japan. 2. At each site, δ¹³C was usually highest or nearly highest for periphyton (epilithic biofilm) and was lowest for transported leaf materials. Among macroinvertebrate groups, grazers usually had higher δ¹³C values than filterers or predators. 3. During all seasons, δ¹³C of periphyton and all macroinvertebrate groups increased downstream from mountain to upland sections, but decreased downstream from upland to lowland sections. In addition, the difference between grazer δ¹³C and filterer δ¹³C decreased from mountain to upland sections, but increased from upland to lowland sections. 4. The observed changes in δ¹³C of periphyton and macroinvertebrates from mountain to upland sections agree with previous reports: the δ¹³C of periphyton and consumers increased with stream size and productivity. The decrease in δ¹³C of periphyton and macroinvertebrates from upland to lowland sections has not been reported previously, and this may have resulted from an increased importance of terrestrial detritus relative to periphyton production in the lowland section, where riffles were infrequent and pools dominated the reach. 5. A simple mixing model of δ¹³C showed that grazers rely mostly on periphyton at all sites, whereas the importance of periphyton for filterers changed longitudinally increasing from mountain to upland sections and decreasing from upland to lowland sections. This longitudinal trend for filterers is possibly associated with the changes in the availability or quality of terrestrial detritus in transported particulate organic matter. 6. Longitudinal changes in the relative importance of autochthonous production and allochthonous detritus appear to be reflected in δ¹³C of riffle benthic communities. The longitudinal changes were not monotonic, and specific reach characteristics may be responsible for the greater importance of allochthonous detritus in mountain and lowland sections.     

Effects of temperature on the cytokinin requirement of tobacco calluses

A cytokinin-nonrequiring strain (T22) was isolated from a cytokinin-requiringcallus strain T2 of tobacco (Nicotiana tabacum var. Bright Yellow). Strain T22 grew rapidly on the medium without added kinetinat 26?C. But its growth was completely suppressed at 16?C. Thisgrowth suppression at 16?C was partially recoverable by supplyingkinetin. Benzyladenine, geranylaminopurine and 2-methyl-8-benzylamino-s-triazolo[l,5-a]pyrazinewere also effective in removing growth suppression at 16?C.Adenine, which was unable to remove growth suppression of T22at 16?C, promoted the growth of T2 at 26?C, but not at 16?C.Physiological differences between cytokinin-requiring and -nonrequiringcalluses are discussed. ¹Part II in the series "Studies, on Plant Tissue Cultures";for Part I, See Plant & Cell Physiol. 9: 103–114 (1968). (Received May 29, 1970; )     

Studies on plant tissue cultures I. Relationship between inocula sizes and growth of calluses in liquid culture

1. 1. It was observed that lag of growth was longer in small inoculathan in large inocula using tobacco callus in liquid culture.
2. 2. These different growth responses between small and largeinocula were dependent on the ratio of inoculum to culture medium.
3. 3. The same result was obtained in a strain of carrot rootcallus.But the growth lag was very short in the carrot callus,whichwas subcultured for the shortest period among the 4 strainsused, even in small inocula. On the other hand, both small andlarge inocula of the strain, which were subcultured for thelongest period among the 4 strains, did not grow at all duringthe culture period; the longer the period of subculturing, thelonger the lag of growth.
4. 4. The longer lag of small inoculain tobacco callus was recoveredby gibberellin A₃ in the presenceof the acidic fraction ofcarrot root extract or vitamins suchas pyridoxine and thiamine.

(Received December 11, 1967; )     

ENZYMIC FORMATION OF GLUTAMINE IN RICE-PLANT SEEDLINGS

1. Purified preparation from rice-plant seedling catalyses a stoichiometricreaction between ATP, glutamate, and NH₂OH in the presence ofMg⁺⁺ to form glutamyl hydroxamate, ADP and inorganic phosphate.
2. The method of purification and some of the properties of theenzyme are described. Co⁺⁺ can be substituted for Mg⁺⁺. Mn⁺⁺,NaF, and PCMB inhibit the enzyme strongly.
3. Inorganic orthophosphateis liberated from ATP by the additionof or cysteine in the presence of glutamate, Mg⁺⁺ andthis preparation. Glutamine was detectedin the reaction productsby paperchromatography.
4. The same preparation catalyses a reactionbetween gluta mineand NH₂OH in the presence of Mg⁺⁺ or Mn⁺⁺,ADP and inorganicphosphate, to form glutamyl hydroxamate.

¹ Present address: The Department of Chemistry, Faculty of Science,Kanazawa University, Kanazawa. (Received October 31, 1960; )     

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURES

1. The longer the period of stock culture, the more remarkableis the growth inhibition by 8-azaguanine in callus.
2. Chloramphenicol,5-methyltryptophane and mitomycin C exert greaterinhibitionon growth in CCL than in CCS.
3. Bud formation is inhibited bysome concentrations of chloramphenicolwithout accompanyinginhibition of the growth.
4. Cell size and the contents of RNA,DNA, protein and lipid percell of CCL are greater than thoseof CCS, respectively. Thecontents per cell of RNA and lipidin "mitochondrial fraction"are higher in CCL than in CCS.
5. Incorporationof guanine-8-¹⁴C into RNA of CCS occurs rapidlyin the first12 hr and slows down thereafter, but that in CCL-RNAincreasessteadily for 16 hr. This difference in rate of theincorporationafter 12 hr between CCS and CCL is principallydue to the differencein rate of the incorporation into RNAof nuclear, mitochondrialand soluble fractions.

1. The rate of RNA breakdown in CCL wasnot so great as the rateof synthesis.
2. 8-azaguanine (10^–3and 10^–4M) inhibits incorporationof guanine-8.¹⁴C intoRNA of both CCS and CCL during 14 hr,but thereafter (up to25 hr) it inhibits the incorporation intoCCL-RNA alone leavingthat into CCS-RNA unaffected.

1. In CCL 510^–5M 8.azaguaninedoes not affect total radioactivityincorporated into bulk RNA,but inhibits incorporation intoRNA of "mitochondrial fraction".

(Received December 23, 1964; )