chemical affecting anthocyanin formation morphogenesis ... · impatiens balsamina' allan...

Chemical Factors Affecting Anthocyanin Formation andMorphogenesis in Cultured Hypocotyl Segments of

Impatiens Balsamina '

Allan W. Arnold2' 3and Luke S. AlbertDepartment of Botany, University of Rhode Island, Kingston

IntroductionThe effects of various chemicals on anthocyanin

synthesis and morphogenesis in the Impatiens hypo-cotyl were investigated to determine possible relation-ships between a specific biochemical process (antho-cyanin synthesis) and morphogenetic events (theformation of roots and epidermal hairs). Culturedsegments of balsam (Impatiens balsamina L.) pro-vide excellent material for the study of anthocyaninformation as an indicator of metabolic conditionsaccompanying differentiation (3). A similar ap-proach to studying morphogenesis has been takenby Klein and Hagen (7) using cultured balsam flowerpetals.

The axis of the etiolated hypocotyl presents agradient of tissue differentiation from an activelydividing meristem in the region of the hypocotyl archto highly specialized tissue zones toward the base(2, 8). Correspondingly, different segments of thebalsam hypocotyl axis differ in their capacity toform roots, epidermal hairs, and anthocyanin pigmentswhen cultured on an agar medium (3).

Methods and MaterialsVaughn Seed Company's Scarlet Balsam (Impa-

tiens balsamina L.) was used in all experiments. Theseeds were germinated under sterile conditions intotal darkness for 7 days as previously described (3).

The hypocotyls (about 50 mm in length) wereexcised from the etiolated seedlings and cut into 10equal segments which were transferred to a petri dishcontaining an agar medium. The segments werearranged in a radial fashion on the agar, the seg-ment proximal to the cotyledons being designated as1 and the more distal as 2 through 10. Sterile condi-tions were maintained throughout.

White's medium (14) was used in all experimentsand it was supplemented in various experiments withthe following constituents: compounds reported toaffect growth; naphthalene acetic acid (NAA, 5.0 x10-6 and 5.0 X 10-8 M), 2, 3, 5-triiodobenzoic acid(TIBA, 5.0 X 10-5 M), gibberellic acid (GA, 2.7 x

1 Received Aug. 2, 1963.2 This work is a portion of a thesis submitted in partial

fulfillment for the Ph.D. degree. Allan W. Arnold wassupported by a National Defense Education Act Fellow-ship.

3 Present address: Department of Botany, Brown Uni-versity, Providence, Rhode Island.

10-5 M) and compounds reported to affect anthocyaninsynthesis; benzimidazole (3.0 X 10-3M), 8-azagua-nine (3.0 X 10-5 and 3.0 x 10-6 M), and riboflavin(3.0 x 10-4 and 1.0 x 104M). The constituentswere used singly or in combinations in some experi-ments.

Each experiment, comprised of 35 cultures, wascarried out in darkness or under 450 ft-c of light fromcool white fluorescent tubes at 250. In "dark" ex-periments transfers were made under dim green light.During the experimental period of 10 days the "dark"cultures received brief periodic exposures to lightwhich were necessary for the collection of data.Periodic observations for the presence or absence ofanthocyanin pigments, epidermal hairs, and the num-ber of roots were recorded for each of the 10 hypo-cotyl segments in each culture. These data represent35 replicates of each of the 10 segments along theaxis of a hypocotyl.

Quantitative estimates of total anthocyaninformed in the hypocotyl segments at the end of the 10-day experimental period were determined as before(3).

Results

The segments of the hypocotyl which present agradient of tissue differentiation often responded dif-ferently to treatment depending on their position alongthe hypocotyl axis. Physiological gradients were alsoindicated and were found to shift in response to sometreatments. Table I records the results obtained withthe various compounds tested at the indicated con-centrations. Measurements were taken on the inci-dence of epidermal hairs and anthocyanin pigment,number of roots formed, and anthocyanin content perpigmented hypocotyl segment. When NAA wasabsent from the culture medium only segment 1formed epidermal hairs. Since NAA added to themedium induced hair formation along the hypocotylaxis, a concentration of 5 X 10-8 M NAA was rou-tinely used in the control medium except where indi-cated at a higher or lower concentration.

Compounds Affecting Morphogenesis. NAAadded to White's medium caused an increase in theformation of hairs and in the number of roots formed(table I). NAA at 5 x 10-6 M caused somewhatless than a twofold increase in root formation over thecontrol except in segments 1,9, and 10, in which theincrease was over 250 %. In the absence of NAA,

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Table IEffect of Chemlical Factors ont Anthocyanin Synthesis and Growth in Cultured Hypocotyl Segmnents

Hypocotyl segmentCompound M Observation*

1 2 3 4 5 6 7 8 9 10

Light Pigment: incidence 35 35 35 35 35 35 35 35 35 35control** Rel. amt./seg. 2.94 3.89 4.34 4.66 4.57 4.09 5.00 3.88 4.23 4.00

Roots 137 138 143 134 136 132 138 126 130 114Hairs 24 13 3 1 0 0 1 1 0 0

NAA 0 Pigment: incidence 35 35 35 35 35 35 35 35 35 34Rel. amt./seg. 4.86 4.69 4.29 4.11 3.71 3.66 3.91 3.60 3.37 2.66

Roots 139 136 123 121 110 111 114 101 102 62Hairs 15 0 0 0 0 0 0 0 0 0

NAA 5.0 X 10-6 Pigment: incidence 34 35 35 35 35 35 34 33 35 35Rel. amt./seg. 4.17 3.37 3.40 4.28 5.00 4.77 5.82 6.88 6.34 6.03

Roots 372 208 222 237 224 230 241 212 259 314Hairs 29 20 11 5 5 0 0 0 0 0

TIBA 5.0 x 10- Pigment: incidence 35 35 35 35 34 35 35 34 35 32Rel. amt./seg. 4.32 3.92 3.63 3.72 3.47 3.34 3.23 3.23 2.40 2.41

Roots 36 10 0 7 10 23 20 24 19 57Hairs 4 0 0 0 0 0 0 0 0 0

TIBA 5.0 x 10-5 Pigment: incidence 28 29 35 33 29 27 29 28 32 33NAA 5.0 X 10-6 Rel. amt./seg. 1.50 2.00 1.94 2.03 2.55 3.26 3.24 3.18 3.16 2.73

Roots 60 103 103 87 66 68 65 49 43 58Hairs 20 7 3 1 0 1 0 0 0 0

GA 2.7 X I0- Pigment: incidence 35 35 35 35 35 35 35 35 35 35Rel. amt./se-. 1.86 3.34 2.77 3.06 2.94 2.95 2.92 2.94 2.89 2.32

Roots 105 138 133 118 120 121 135 137 131 112Hairs 14 24 9 11 3 2 3 2 1 0

Aza*** 3.0 x 1')-6 Pigment: incidence 35 35 35 34 35 34 35 35 32 30Rel. amt./seg. 3.03 1.17 1.03 1.29 1.00 1.65 1.43 1.03 1.06 0.73

Roots 58 68 80 71 62 86 61 41 40 32Hairs 2 0 0 0 0 0 0 0 0 0

Bz 3.0 x 10-3 Pigment: incidenice 34 32 31 31 31 28 28 31 29 30Rel. amt./seg. 0.71 0.72 0.68 0.71 0.74 1.00 0.96 0.92 0.72 0.97

Roots 110 7 2 2 4 6 3 2 0 7Hairs 4 0 0 0 0 0 0 0 0 0

Bz 3.0 X 103 Pigmenit: incidence 4 5 3 4 1 10 21 18 23 26NAA 5.0 x 10-6 Rel. amt./seg. 0 0.40 0.67 0.25 0 0.40 0.10 0.28 0.26 0.31

Roots 36 10 0 7 10 23 20 24 19 57Hairs 30 31 18 11 7 7 8 3 1 1

Bz 3.0 X 10-; Pigment: incidence 31 0 0 0 0 0 0 0 0 0Rb 1.0 x 10-1 Rel. amt./seg. 0.23 ... ... ... ... ... ...

Roots 4 0 0 0 0 0 0 0°0 0Hairs 0 0 0 0 0 0 0 0 0 0

Dark Pigment: incidence 7 34 35 35 34 35 34 35 35 35control** Rel. amt./se-. 0.14 0.32 0.46 0.37 0.35 0.26 0.41 0.34 0.31 0.34

Roots 87 137 123 129 121 118 120 119 110 124Hairs 35 35 27 18 10 8 12 11 11 0

Rb (dark) Pigment: incidence 8 35 35 35 35 34 34 34 34 353.0 x 10-4 Rel. amt./seg. 0.25 0.09 0.23 0.23 0.23 0.15 0.32 0.23 0.18 0

Roots 64 137 137 130 133 130 135 137 128 93Hairs 11 34 31 26 16 12 6 4 5 7

* Pigment: incidence - number of replicate segments showing visible anthocyanin. Rel. amt./seg. = relativeamount of anthocyanin expressed as absorbency of extract X 103/pigmented segment. Roots = total number ofroots found on 35 replicate segments. Hairs = number of replicate segments having epidermal hairs.

** Control medium contained 5.0 x 10-8A NAA, which is present in all other experiments except where NAAis indicated at a higher or lower level.AAza, 8-azaguaninle; Bz, benzimidazole; Rb, riboflavin.

308 PLANT PHYSIOLOGY

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ARNOLD AND ALBERT-ANTHOCYANIN FORMATION AND MORPHOGENESIS

only segments 3 to 10 formed fewershowing nearly a 50 % decrease.

During the course of the experhairs appeared earlier in the NAAThe appearance of anthocyanin piglow this pattern. Figure 1 shows I

40

30

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(Dw

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w

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z

.CDIL

o

0

40

30

20

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TIME (HOUFIG. 1. Influence of NAA on the

of anthocyanin pigments in segmentspocotyl. The control cultures cot

NAA. Pigmented segments = the i

segments having visible pigment, thebeing 35.

NAA in the culture medium causedset of anythocyanin synthesis in <which are typical examples. In s

the total amount of anthocyanin foiments of the hypocotyl increased wNAA and decreased when NAA w

I). NAA affected the total anthcthe entire hypocotyl in the same

influenced the quantitative distribalong the axis of the hypocotyl.cyanin content occurred in segmenof NAA, in segments 4 and 5 ofcontained 5.0 X 10-8 M NAA, and5.0 x 10-6 M NAA (table I). Thto NAA was somewhat similar in tments were more responsive. Thesion of root formation in segments

roots, segment 10 sence of NAA, but segment 1 formed the most rootsof any segment at the 5.0 x 10-6 M level (table I).

*iments, roots and TIBA (5.0 x 10-5 M) was found to have no ef-t treated cultures. fect on the number of segments forming pigment, but,ments did not fol- root and hair formation was strongly inhibited (tablethat increments of I). TIBA also depressed the amount of anthocyanin

formed in all the segments except segment 1, whichshowed about a 50 % increase over the control. The

I quantitative distribution of anthocyanin along theaxis of the hypocotyl was significantly changed byTIBA. Segment 1 contained the most anthocyaninwith gradually decreasing amounts toward the baseof the hypocotyl, while segments 4 and 5 were maxi-

x 10-6M NAA mum in the control. While being generally lower inamount, the quantitative distribution of anythocyaninin the presence of TIBA was very similar to that forthe segments cultured in the absence of NAA. Whenthe same level of TIBA (5.0 X 10-5 M) was chal-lenged with a higher level of NAA (5.0 X 10-6 M)than was in the control, the point of maximum antho-cyanin synthesis was again shifted toward the base ofthe hypocotyl, occurring in segments 6 and 7. NAAalso partially eliminated the inhibitory action ofTIBA on root and hair formation. Clearly an auxin-antiauxin relationship is active in the control of an-thocyanin synthesis as well as root and hair forma-tion.

GA had its greatest effects on segment 1 (table106M NAA I). The formation of roots and hairs, and the total

anthocyanin formed were depressed. Segment 1 wasobserved to elongate markedly in the presence of GA,but the remaining hypocotyl segments did not elon-gate noticeably. In segments 2 and 9 hair formation

Iwas enhanced, rooting was only slightly affected and160 240 the total anthocyanin content was generally inhibited.

All segments of the hypocotyl, with only a few iso-JRS) lated exceptions, formed visible amounts of pigmentsrate of appearance in the presence of NAA, TIBA, and GA. The most2 and 9 of the hy- striking results were obtained with NAA which gen-ntain 5.0 X 10-8 M erally enhanced growth and anthocyanin formation innumber of replicate the hypocotyl segments. Besides increasing the totalmaximum number anthocyanin formed, the addition of NAA caused a

shift in the point of maximum synthesis toward thebase of the hypocotyl. In the absence of NAA seg-

a delay in the on- ment I formed the most anthocyanin and the totalsegments 2 and 9, formedl by the entire hypocotyl was diminished. Dif-;pite of this delay, ferent parts of the hypocotyl were found to respondrmed by most seg- quite differently to the same treatment. For instance,rith the addition of NAA inhibited anthocyanin synthesis in the upper 3ras withheld (table segments, in segment 4 the content is about the same)cyanin formed by in the presence and absence of NAA, and in segmentsway and strongly 5 through 10 an increase in NAA resulted in an in-ution of pigment creased anthocyanin content (table I).Maximum antho- Compounds Affecting An thocyaniii Synthesis.

It 1 in the absence The data for the experiments using 8-azaguanine,the control which benzimidazole, and riboflavin are presented in table I.in segment 8 with Both purine analogs, 8-azaguanine and benzimida-

.e rooting response zole, effectively inhibited hair production in all seg-that the lower segments of the hypocotyl. Root formation was severely!re was no depres- inhibited by 8-azaguanine in all segments at both1 anid 2 in the ab- levels used. Benzimidazole, on the other hand, had

I ISEGMENT 2

NO NA'A /-CONTROL

- 0 /

-I /5.0

1/ /

SEGMENT 9NO NAA-zo00o -

7 CONTROL

-o 0 Au

309



PLANT PHYSIOLOGY

little effect on segment 1, while reducing the numberof roots formed on segments 2 to 10 negligible values.The relative ineffectiveness of benzimidazole as com-pared with 8-azaguanine on segment 1, while bothcompounds inhibited rooting in the rest of the hypo-cotyl indicates the unique response of segment 1.Both compounds generally inhibited anthocyanin syn-thesis, but 8-azaguanine was a much more potent in-hibitor of anythocyanin synthesis than benzimidazole.In segments 2 to 10, 8-azaguanine at 3.0 X 10-5 Mwas about equally as effective as 3.0 X 10-3 M benzi-midazole. Again, segment 1 is of particular interest,since 8-azaguanine had relatively little effect on an-thocyanin synthesis in segment 1, whereas benzimida-zole inhibited pigment formation in segment 1 as wellas the other segments. The anthocyanin formed insegment 1 with 3.0 X 10-6 AI 8-azaguanine is equalto the control, while the remaining segments formedonly about 20 to 30 % of the total anthocyanin foundin the control.

The combination of benzimidazole and NAA re-duced both the incidence of and the total anthocyaninformed per segment well below the values for eachcompound used alone (table I). The suppression ofthe incidence of pigment was very pronounced in seg-ments 1 to 5.

Hair formation was effected in nearly an oppositemanner to anthocyanin formation by the benzimida-zole-NAA interaction (table I). Benzimidazole usedalone almost completely eliminated hair productionwhile NAA alone favored hair production. Moresegments were found to have epidermal hairs on thebenzimidazole-NAA medium than with either sub-stance used alone. Similar opposing responses werealso noted in the number of roots formed on the vari-ous segments. Benzimidazole alone severely inhibitedrooting in all segments except segment 1. NAA en-hanced rooting in all segments of the hypocotyl, espe-cially segment 1. When benzimidazole and NAAwere used together, rooting in segments 2 to 10 wasabout equal to or slightly greater than that in a me-dium with benzimidazole alone. Segment 1, how-ever, formed less than 40 % of the number of roots onthe benzimidazole-NAA medium than it did on thebenzimidazole medium. The results indicate that acompound such as benzimidazole may act as a NAAantagonist for a response such as anthocyanin syn-thesis while acting synergistically with NAA in thesame tissue to cause the production of epidermal hairs.Furthermore, NAA counteracted the inhibition ofrooting by benzimidazole in the more highly differen-tiated tissue of segments 6 to 10, while NAA actedsynergistically with benzimidazole to cause a mini-mum number of roots formed in segment 1 wvhich con-tains a large amount of meristematic tissue (2, 8).

DiscussionThe objective of this research was to slhow inter-

relationships between the development of morphologi-cal structures and a biochemical process. Definiterelationships have been shown to occur. Anatomi-

cally, the axis of the hypocotyl of Impatiens balsanzinaL. has been shown to present a gradient of tissue typesfrom meristematic at its upper end to highly differen-tiated and specialized types toward its basal end(3, 8). Therefore the various physiological re-sponses, as well as the morphogenetical manifesta-tions observed in cultured hypocotyl segments, can beinterpreted with reference to the basic structure andlevel of differentiation of the tissue.

The most interesting results were obtained fromexperiments using NAA. Suggestions made by Al-ston (1) concerning the mode of gene control of an-thocyanin synthesis are supported by these results.In Impatiens, a series of genes is known to govern thedistribution of anthocyanin pigments fromii the firstnode upward in the stem axis and even to the midribsof the leaves (1). The hypocotyls used for the pres-ent studies when grown on a medium containiing noNAA were shown to have a specific quanititative dis-tribution of anthocyanin in which segment 1 formsthe most with gradually decreasing aimiouihts in eachsubsequent segment toward the base of the hy1pocotyl.The rates of anthocyanin synthesis in the v-arious seg-ments do not appear to differ greatly, silnce pigmentappears first in segment 1 and proceeds stepwise to-ward the base (3). A similar appearalnce and dis-tribution of anthocv-anins are found in the intactseedling grown in the light. This pattern of antlho-cyanin formation is under genetic control (1). NAAcaused a general increase in anthocyanin content, butmore important, NAA treatment resulted in a shiftin the point of maximum anthocyanin syntlhesis to-ward the base of the hypocotyl. NAA also retardedthe appearance of anthocyanin in the upper segmentsduring the course of the experiments, while the lowersegments were affected only slightly (fig 1). Ratesof anthocyanin synthesis in the lower segmenits wereapparently greatly increased since segment 9 formednearly twice the amount of pigment of segmilent 2 inthe same amount of time on the mediumii with 5.0 X10-6 M, NAA (table I). The hypocotyls grown on the5.0 x 10-6 Al, 5.0 X 10-8 al and the zero level ofNAA may be looked upon as 3 hypothetical mutantssimilar to the previously mentioned Impatiens plantsin which the quantitative distribution of anthocyaninsalong the stem and petioles is controlled by an allelicseries. The hypothetical mutant hypocotyls differ inthe rates of anthocyanin synthesis, the times of ap-pearance of anthocyanins and the quantitative dis-tribution of anthocyanins along the axis of the hypo-cotyl, which suggests the shift of a physiologicalgradient favoring anthocyanin synthesis, towN-ard thebase of the hypocotyl. The control of anthocyaninsynthesis then could be accomplishedI by controllinglevels of auxins in the hypocotyl. This concept issupported by the results of experiments using the anti-auxin TIBA, which has been shown to effectivelvlower endogenous auxin levels (4, 5, 6). TIBA at alevel of 5.0 X 10-5 Al completely overcamlie the levelof NAA (5.0 X 10-8 Mr) in the control to cause aquantitative distribution of pigmenit along the axis of

310



ARNOLD AND ALBERT-ANTHOCYANIN FORMATION AND MORPHOGENESIS

the hypocotyl which was nearly identical to the seg-ments where no NAA was added to the medium.When the same concentration of TIBA was inter-acted with the level of NAA which caused the great-est shift in the pattern of anthocyanin formation(5.0 X 10-6 M) results intermediate between noNAA and the highest level of NAA were obtained.Along with the anthocyanin responses, NAA en-hanced both root and hair formation by the hypocotylsegments and these effects were likewise counteractedby TIBA. Thus, it is quite possible that such appar-ently unrelated processes as anthocyanin synthesis,root formation, and hair formation may be controlledsimultaneously by factors (genetic or environmental)which influence the endogenous levels of auxins in theImpatiens hypocotyl.

GA caused a marked increase in elongation of seg-ment 1 in the light which was similar to its responsewhen cultured in the dark without GA. The meri-stematic tissues of segment 1 are responsible for itsability to elongate (2, 8). Elongation appears to beunfavorable for the formation of both roots and an-thocyanins. Root formation and anthocyanin syn-thesis in segment 1 was inhibited with respect to theother segments of the hypocotyl in the dark withoutGA and in the light in the presence of GA (table I).

Thimann and Radner (12) have reported thatanthocyanin formation in Spirodela is directly de-pendent upon riboflavin synthesis. In the Impatienshypoctoyl riboflavin was found to actually inhibitanthocyanin formation in the dark and also inhibitroot and hair formation in segment 1 (table I).When used in combination with benzimidazole in thelight, riboflavin severely inhibited all activities of thehypocotyl and was lethal to most of the lower seg-ments. In agreement with our results, Straus (9)has shown that riboflavin inhibited anthocyanin syn-thesis in cultured corn endosperm tissue.

In contrast to the other compounds tested, thepurine analogs, benzimidazole and 8-azaguanine, usedindividually, and benzimidazole used in combinationwith NAA effectively separated the growth responsesof root and hair formation from each other, and fromanthocyanin synthesis. These responses were un-coupled only through the use of metabolic inhibitorsbenzimidazole and 8-azaguanine and not by com-pounds such as sugars, auxins, or other chemicalswhich would be expected to act as factors known tooccur naturally in plant tissues. Both benzimidazoleand 8-azaguanine effectively inhibited growth and an-thocyanin synthesis in all segments of the hypocotylexcept segment 1 (table I). The results of the inhibi-tion of anthocyanin formation in segments 2 to 10 bythe purine analogs agree well with the report of Thi-mann and Radner (13), but in segment 1 only benzi-midazole was effective in preventing anthocyaninformation while 8-azaguanine had relatively little ef-fect. However, 8-azaguanine did strongly inhibit rootformation while benzimidazole was relatively ineffec-tive. The relationship between the level of differen-tiation of a tissue and the sensitivity of anthocyanin

synthesis to 8-azaguanine was further supported bythe observation that the anthocyanin was formed wellup into the hypocotyl arch (meristematic region)when 8-azaguanine was present in the medium.Straus has reported that 8-azaguanine caused a slightenhancement of anthocyanin formation in culturedcorn endosperm tissue (9). The corn endospermseems to approximate more nearly the condition ofthe meristematic tissue of segment 1 than the matureSpirodela plants used by Thimann et al (10-13).The fact that 8-azaguanine acts as a potent inhibitorof anthocyanin synthesis in the mature tissues of thehypocotyl and has little effect on the meristematic areaof segment 1, offers a likely explanation for the con-flicting results of Straus (9) and Thimann and Rad-ner (13). The mature Spirodela plants which Thi-mann and Radner used were severely inhibited by 8-azaguanine as was anthocyanin synthesis in the ma-ture segments of the hypocotyl, while 8-azaguaninedid not affect anthocyanin synthesis in the undifferen-tiated corn endosperm tissue and segment 1 of theImpatiens hypocotyl. The effectiveness of 8-azagua-nine in inhibiting anthocyanin synthesis appears de-pendent on the state of differentiation of the tissue.

SummaryEtiolated 5-day-old hypocotyls of Impatiens balsa-

mnina L. were cut into 10 equal segments and cul-tured under sterile conditions on White's agar me-dium in petri dishes. Segment 1 was at the cotyledonend of the hypocotyl and the other segments werenumbered 2 to 10 towards the base of the hypocotyl.Measurements were made on growth and anthocyaninformation in each of the segments during and at theend of a 10-day experimental period.

Naphthalene acetic acid caused a general increasein both growth and anthocyanin formation. Also ashift in the point of maximum anthocyanin synthesisfrom segment 1 to segment 8 was caused by increas-ing naphthalene acetic acid in the medium. Theseeffects were reversed by triiodobenzoic acid.

The medium was also supplemented with gib-berellic acid, 8-azaguanine, benzimidazole, and ribo-flavin in various experiments. Responses to thesechemical factors which indicate interrelationshipsbetween anthocyanin synthesis and morphogenesis inthe hypocotyl segments are described.

Literature Cited1. ALSTON, R. E. 1959. Physiology and the inheri-

tance of anthocyanin pattern. Genetica 30: 261-77.2. ARNOLD, A. W. 1962. Factors affecting anthocya-

nin formation and morphogenesis in the hypocotylImpatiens balsaminia L. Ph.D. Thesis. Univ. ofRhode Island.

3. ARNOLD, A. AND R. E. ALSTON. 1961. Certainproperties of hypocotyl of Impatiens balsamina re-flecting physiological complexity. Plant Physiol.36: 650-56.

4. AUDUs, L. J. 1959. Plant Growth Substances.Leonard Hill (Books) Ltd., London.

5. AUDUs, L. J. AND R. THRESH. 1956. The effects

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of synthetic growth regulator treatmenits on thelevel of free endogenous growth substances inplants. Ann. Botany N.S. 20: 439-59.

6. AUDUS, L. J. AND R. THRESH. 1956. The effectsof synthetic growth substances in the level ofendogenous auxins in planits. In: The Chemistryand Mode of Action of Plant Growth Substances.Wain and Wightman, ed. Butterworths, London.p 248-52.

7. KLEIN, A. 0. AND C. Wi. HAGEN, JR. 1961. Antlio-cyanin production in detached petals of I1iipatienisbalsauinina. L. Plant Physiol. 36: 1-9.

8. NOEL, R. 1951. Contribution a l'etud de la rhizo-genesis. Neoformation des racines dans les frag-ments de hypocotyles d'Im patiens balsamiiina L.cultives in vitro. Arch. Inst. Bot., IUniv. Liege,Belgique 21: 1-164.

9. STRAUSS, J. 1960. Anthocyanin synthesis in cornendosperm tissue cultures. II. Effect of certain

inhibitory and stimulatory agenits. Plant Phvsiol.35: 645-50.

10. THIMANN, K. AND Y. EDMUNDSON. 1951. Thebiogenesis of the anthocyanins. III. The roleof sugars in anthocyanin formation. Arch. Bio-chem. Biophys. 34: 305-23.

11. THIMANN, K. AND Y. EDMUNDSON. 1949. Thebiogenesis of the anthocyanins. I. General nutri-tional conditions leading to anthocyanin formation.Arch. Biochem. 22: 33-53.

12. THIMANN, K. AND B. RADNER. 1958. The bio-genesis of anthocyanin. VI. The role oi ribo-flavin. Arch Biochem. Biophys. 74: 209-23.

13. THIMANN, K. AND B. RADNER. 1955. The bio-genesis of anthocyanin. V. Evidenice of themediation of pyrimidines in anthocyanin svnthesis.Arch. Biochem. Biophys. 59: 511-25.

14. WHITE, P. R. 1943. A Handbook of Plant TissueCulture. Cattell Press, Lancaster, Pa.

Metabolic Processes in Cytoplasmic Particles of theAvocado Fruit VI. Controlled Oxidations

and Coupled Phosphorylations 1, 2, 3J. T. Wiskich4, Roy E. Young, and Jacob B. Biale

Department of Botany and Plant Biochemistry,University of California, Los Angeles

AIn explanation of the climacteric rise of the res-piration rate of ripening fruits has been sought bystudies of subcellular particles isolated fronm the fruit.It is known that in most plant tissues the rate of 0Ouptake can be increased by treating the tissue withuncoupling agents, suggesting that the rate of 03 up-take is limited by the rate of oxidative phosphoryla-tion. If during the process of ripening the restric-tion on oxidation exerted by phosphorylation wasrelieved, a stimulation of the rate of 03 uptake wouldbe expected. Thus, Millerd et al. (24) establishedthat whereas the respiration of avocado tissue slicesof preclimacteric fruit could be stimulated by applica-tion of DNP5, slices from the climacteric peak fruit

1 Received Aug. 6, 1963.2 A preliminary report of this work has appeared (31).3 This research was supported by Grant RG-8224

from the United States Public Health Service and by agrant from the Cancer Research Coordinating Committeeof the University of California.

4 Present address: Department of Botany, Universityof Adelaide, South Australia.

5 Abbreviations: CCP, carbonyl cyanide m-chloro-phenylhydrazone; DNP, 2,4-dinitrophenol; TPP, thiaminepyrophosphate.

were not stimulated by such treatment. From studiesof the isolated mitochondria and of the other fractionsthese workers concluded that there wvas a natuiral un-coupler present in the fruit. Several criticisnms havebeen offered against such an hypothesis (5), andPearson and Robertson (25) suggested that the cli-macteric rise was due to a higher turnover of thephosphorylation cycle. These explanations of the in-creased 0. uptake, involving changes in the control-ling influence of oxidative phosphorylation on themitochonclrial oxidations, are possible, but it is im-probable that the study of the isolated mitochonidriawill reveal the causes of the changes. Nevertheless,much valuable information has been obtained fromthese studies, and in particular, the properties of avo-cado particles have been thoroughly examine(I anddocumented (1, 5, 6, 24, 26).

Ronmani and Biale (26) observed that mitochon-dria isolated from ripe avocado fruit appeared to havean oxidative phosphorylation mechanism which wasinsensitive to DNP. Mitochondria isolated fronm un-ripe avocado fruit were sensitive to DNP, but theuncoupling effect observedl with these particle, was

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