Plant Physiol. (1975) 56, 429-433

Promotion of Radish Cotyledon Enlargement and ReducingSugar Content by Zeatin and Red Light1

Received for publication December 11, 1973 and in revised form August 5, 1974

ALBERT K. HuFF2 AND CLEON W. RossDepartment of Botany and Plant Pathology, Colorado State University, Fort Collins, Colorado 80521

ABSTRACT

Effects of zeatin on amino acid and sugar contents of de-tached radish (Raphanus saivus L) cotyledons were in-vestigated to determine if accumulation of these solutes con-tributes to cytokinin-enhanced growth. Protein and aminoacid levels were not sigificantly affected, but in cotyledonsincubated in light the hormone caused greater accumula-tions of reducing sugars than occurred in light controls. Con-tinuous fluorescent light or a few minutes of red light in-creased both the growth rate and the reducing sugar levelscompared to dark controls. A far red treatment following redlight overcame the promoting effect of the latter. Amountsof reducing sugars were closely associated with growth underthe above conditions. Activity of an unidentified amylase waselevated by continuous light or a red light treatment (nulli-fiable by far red), suggesting that reducing sugars were de-rived from starch. Zeatin-treated cotyledons exhibited lessamylase activity than did light controls, perhaps implicatingcytokinin-stimulated conversion of fats to sugars in light. Indarkness zeatin promoted cotyledon growth but did not in-crease sugar levels nor amylase activity, suggesting that en-hanced ion accumulation also contributes to the more rapidwater uptake leading to growth.

Cytokinins and red light stimulate growth of various planttissues. For example, red light and cytokinins both enhanceexpansion of discs from etiolated dicot leaves (14, 18, 21), andcertain portions of grass leaves expand faster after treatmentwith either of these stimuli, as evidenced by leaf unrolling (11,23). In light-sensitive lettuce seeds, cytokinins increase thegrowth rate of cotyledons (5), while red light primarily en-hances elongation of radicle cells (20). Cotyledons fromnumerous other dicots also grow faster in the presence ofexogenous cytokinins (2, 4, 9, 10, 16, 19), and this phenomenonhas been developed as a cytokinin bioassay by Esashi andLeopold (2), Letham (9, 10), and Narain and Laloraya (16).

Although many kinds of tissues do not exhibit faster ex-pansion when exposed to cytokinins or red light, the questionarises as to the driving force for growth in the examples noted.Auxins are well known to stimulate cell elongation by increas-

1This work was supported in part by National Science Founda-tion Grant GB-38625.

I Present address: Plant Science Department, South Dakota StateUniversity, Brookings, S. D. 57006.

ing wall plasticity, while red light-enhanced lettuce seedgermination seems to arise from an osmotic effect due to morerapid degradation of some macromolecule (15). We are notaware of data indicating that either red light or cytokinins in-crease wall plasticity, and our studies were to determinewhether increased cell expansion (10) of excised radish cot-yledons by cytokinins could be due to water uptake resultingfrom osmotic effects of enhanced solute accumulation. Wefound that both cytokinins (in the presence of light) and redlight increased the reducing sugar contents of the cotyledons,and these increases were highly correlated with cotyledongrowth.

MATRIAILS AND METHODS

Radish seeds (Raphanus sativus L. var. Early Scarlet Globe)were surface sterilized with 10% (v/v) Clorox and, afterthoroughly rinsing, were germinated in darkness for 48 hr onwet paper. The smaller cotyledon cut from each selectedseedling was used in all studies. Usually five cotyledons (20-35 mg fresh weight) were randomly selected for each sample,blotted gently, weighed as a group, and placed adaxial sidedown on Whatman No. 1 filter paper covering the bottom of a5.5-cm Petri dish. The filter paper was previously wetted with1 ml of 2 mm potassium phosphate buffer (pH 6.4) containingdesignated additives. The Petri dishes were placed on wetpaper towels in glass trays covered with clear plastic film, andcotyledons were incubated at 25 C. The trays were also coveredwith aluminum foil for incubation in darkness. After incuba-tion, cotyledons were blotted and reweighed.

Immediately after obtaining final weights, cotyledons ineach sample to be analyzed for protein and amino acids wereplaced in 10 ml of hot 80% (v/v) ethanol and boiled untilvolumes were reduced to about 2 ml. An additional 10 ml of80% ethanol was then added, and samples were homogenizedwith a Brinkmann Polytron and centrifuged for 15 min at17,300g at 4 C. Supernatant volumes were adjusted to 20 ml,and a-amino nitrogen was determined on 1-ml aliquots, asdescribed by Yemm and Cocking (24), using glutamine as astandard. Residues from centrifugation were prepared forprotein analysis by twice dissolving in 0.1 N KOH and pre-cipitating with 10% (w/v) trichloroacetic acid. The finalprecipitates were dissolved in 0.1 N KOH, and proteinanalyses were made on aliquots of these solutions using themethod of Lowry et al. (12). BSA was used as a standard.

Cotyledons analyzed for sugars were homogenized in 6 ml of70% (v/v) ethanol, centrifuged, and aliquots of the solublefraction were analyzed for reducing sugars, after removal ofethanol, by the method of Marais et al. (13) using D-glucoseas a standard. Sucrose was estimated by the increase in reduc-

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430 HUFF AND ROSS

ing sugars after treatment of ethanol-free aliquots with 1.5units of invertase (Sigma Chemical Co.) for 1 hr at pH 4.6.To measure extractable amylase activity, generally six

cotyledons were placed in 2 ml of ice-cold 10 mm tris-Cl buffer(pH 7), homogenized twice for 30 sec at top speed with thePolytron, and centrifuged at 4 C for 20 min at 23,500g.Supernatants were analyzed for amylase activity at 30 to 31C by adding 1.5-ml portions to 10 ml of 0.05% (w/v) solublepotato starch dissolved in 20 mm sodium acetate, pH 5.5. Atintervals, 2-ml aliquots of enzyme incubation medium were re-moved and each was added to a tube containing 5 ml of 0.05N HCl to stop the reaction. Starch remaining in these tubeswas subsequently measured by the iodine method of Jones andVarner (7). With most of our enzyme preparations, it wasnecessary to measure the absorbance of the starch-iodinecomplex immediately after the iodine reagent was added;otherwise a slow precipitation of an apparent complex betweenthe starch, iodine reagent, and protein significantly loweredthe spectrophotometer readings. Amylase activity was deter-mined as the rate of change in absorbance at 620 nm/i 00cotyledons.

500

400F

300F

I-

C,)

z 200nw

C,

z

e 300w

c.-

w

a. 200RESULTS

Lack of Zeatin Effects on Cotyledon Protein and AminoAcid Levels. Protein and amino acid contents of cotyledons

Table 1. ZeatiUi Effects onz Radish Cotyledoni Gro)twth anld Proteinanld Amino Acid Conitenlts

Cotyledons were incubated under continuous fluorescent lightof 250 ft-c for 4 days. Results are means of three samples of fivecotyledons each.

Zeatin Concn Fresh Wt Increase Protein a-Amino N

AM % Ag/cotyledon0 (Controls) 226 (20)1 516 (22) 5.92 (0.68)0.12 396 (18) 460 (17) 6.38 (0.34)2.2 467 (12) 538 (8) 5.94 (0.06)

22 539 (29) 532 (60) 6.19 (0.63)

Standard errors.

2

E

39-a

coI

zw

0

cl)

48 72TiME (hr)

FIG. 1. Sugar accumulation in detached radish cotyledons withtime. Cotyledons were incubated under continuous fluorescentlight of 250 ft-c. Controls (C); incubation media containing 9.8 ,uMzeatin (Z). Each data point represents the mean of four samples offive cotyledons each. Vertical bars indicate standard errors.

100o

A (1

Plant Physiol. Vol. 56, 1975

* III hr

riME STUDY) 72 hr *

ZEATIN

48 hr

III hr23 hr

48hr 72hr

23hr

CONTROLS

=, ~ ~ ~~~LZ

B (LIGHT EFFECTS)

ZEATIN

RZ/ LC

RFRZ *

/RC, CONTROLSRFRCDc

O0 0.1 0.2 0.3 0.4 0.5REDUCING SUGARS (pmoles/mg initial fresh wt.)

FIG. 2. Relationship between cotyledon growth and reducingsugar content. A: Conditions same as in Figure 1; B: cotyledonswere prepared under dim green safe light. Incubation time was 48hr. Red (maximum at 680 nm) and far red (maximum at 780 nm)light exposures of 2 min and 5 min, respectively, were repeated at8-hr intervals for 40 hr beginning with time zero. Controls (C);incubation media containing 2.5 am zeatin (Z); cotyledons in-cubated in darkness (D); cotyledons exposed to red light (R);cotyledons exposed to red light immediately followed by far-redlight (RFR); cotyledons incubated under continuous fluorescentlight of 250 ft-c (L). Each data point represents the mean of fivesamples of five cotyledons each. Vertical bars indicate standarderrors.

incubated in presence of different concentrations of zeatinare shown in Table I. The lowest zeatin concentration causeda small decrease in protein levels, but there was no correlationbetween protein degradation to amino acids and growth stimu-lation.

Effect of Zeatin on Cotyledon Sugar Levels. Cotyledons in-cubated under continuous fluorescent light of 250 ft-c ex-hibited a gradual accumulation of reducing sugars (Fig. 1),which was markedly increased after 23 hr by 9.8 ,uM zeatin.After 111 hr, zeatin-treated cotyledons exhibited a 430% in-crease in reducing sugars, compared to a 230% increase forcontrol cotyledons. Zeatin-treated cotyledons contained some-what lower quantities of sucrose than did control cotyledonsduring much of the time period investigated, but sucrose levelswere low in all cotyledons, and no pronounced changes oc-curred throughout the 11 1-hr incubation period.

Cotyledon enlargement was linearly related to reducingsugar content (Fig. 2) with zeatin-treated as well as controlcotyledons, although the former were somewhat larger thancontrol cotyledons with similar quantities of reducing sugars.When zeatin-induced cotyledon growth was inhibited by in-

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COTYLEDON GROWTH AND REDUCING SUGARS

Table II. Effect of0.25 Molal Mannitol on Cotyledon Enlargementand Reducing Sugar Content

Cotyledons were incubated 72 hr under fluorescent light of 125ft-c. Results are means of five samples of five cotyledons each.

Incubation Medium Fresh Wt Reducing SugarsIncrease

e% jmnote/mg Amolt/mginitial fresh wI final fresh wI

Controls 186 (12)1 0.16 (0.01) 0.055 (0.004)Zeatin (2.5 Mm) 330 (10) 0.28 (0.01) 0.063 (0.005)Zeatin + mannitol 208 (10) 0.28 (0.01) 0.091 (0.010)

I Standard errors.

cubating in the presence of 0.25 molal mannitol, reducingsugar accumulation still occurred (Table II). These resultsand similar results obtained with 9% (w/v) PEG-4000 (datanot shown) indicate that sugar production is not an effect ofgrowth.

Effect of Red and Far Red Light on Growth and ReducingSugar Levels. Letham (10) previously indicated that light in-creased detached radish cotyledon growth over that occurringin darkness. It is apparent from results presented in Figure 2Bthat red light stimulated reducing sugar production as well ascotyledon enlargement, and that far red light nullified both redlight effects. Furthermore, reducing sugar accumulation wasagain closely correlated with cotyledon expansion. Althoughthe maximum increase in reducing sugar was obtained fromzeatin plus light treatments, zeatin did not significantly affectreducing sugar levels in darkness (compare DC and DZvalues in Fig. 2B). This result has been verified in time coursestudies over 4-day incubation periods (data not shown). Zeatinin combination with red light does cause an increased sugarcontent (Fig. 2B).

Effects of Zeatin on Extractable Amylase Activity. Whendetached cotyledons were incubated under continuous light of125 ft-c, amylase activity increased rapidly during the first 24hr after excision (Fig. 3). There was little change in amylaseactivity subsequent to this initial rapid increase. Cotyledonsincubated in the presence of 24.9 FM zeatin also exhibited arapid initial increase in amylase activity, but this activity re-mained lower than that in control cotyledons in light. Increasesin amylase levels were also observed in cotyledons incubatedin darkness with or without zeatin, but these increases weresmaller than those occurring in light (Fig. 3). The period ofinitial increase in amylase levels was longer in dark-incubatedcotyledons than in those exposed to light. The effect of zeatinon amylase activity in dark-incubated cotyledons was variableamong experiments, but was always small and of questionablephysiological significance. These results suggest that enhancedsugar levels in cotyledons exposed to light arise from starchdegradation, but they reduce the possibility that zeatin furtherincreases the reducing sugar contents of light-treated cot-yledons by effects on amylase activity.Red and Far Red Light Effects on Extractable Amylase

Activity. If the light-induced rise in reducing sugars is effectedby stimulation of amylase activity, then red and far red lightshould exhibit an influence on amylase activity similar to thaton reducing sugars. In Table III results of experiments to testthe effects of red and far red light on amylase activity areshown. Red light stimulated extractable amylase activity com-

pared with dark-incubated control cotyledons, and far redlight nullified the red light stimulation.

Excision Effects on Amylase Activity. The early rise inamylase activity in dark-incubated cotyledons (Fig. 3) sug-

gested that a stimulus for this was introduced at the time ofexcision from the dark-grown seedlings. To determine if re-moval of cotyledons from the remainder of the seedlings alsopromoted amylase activity, detached cotyledons and intactseedlings were incubated for 24 hr prior to analysis of amylaseactivities in the smaller cotyledon. Results from one such ex-periment are presented in Table IV. Each part of the experi-ment (light or dark) was repeated at least twice and resultsfrom this experiment are representative of all data. Amylaseactivity in detached cotyledons incubated in darkness wasabout 40% higher than in cotyledons left intact on seedlings.Under the stimulus of fluorescent light differences were evenmore pronounced, with about 100% more activity extractedfrom detached cotyledons than from cotyledons of intact seed-lings. Although trauma may be a stimulus for increasedamylase activity, these results may indicate inhibition ofamylase activity in intact cotyledons by some other part of theseedling.

Properties of Radish Cotyledon Amylase. The enzyme wasfairly stable and crude extracts or fractions eluted from a

0

0

10

4.0

.E

E 3.0

0

2.0

I-_

E 1.0

n

1-J

Z4 46 hrzTIME (hr)

96

FIG. 3. Changes in extractable amylase activity in detachedradish cotyledons with time. Light-treated cotyledons were in-cubated under continuous fluorescent light of 125 ft-c. Zeatin con-centration was 24.9 AM. Data points for dark-incubated cotyledonsrepresent means of two samples of 12 cotyledons each. Data pointsfor light-treated cotyledons represent means of four values fromtwo experiments, one with 12 cotyledons for each sample andone with six cotyledons each. Bars represent standard errors.

Table III. Effects of Red anid Far Red Light ont Amylase ActivityCotyledons from 48-hr-old seedlings were treated with 10 min

of red light or 10 min of red light followed immediately with 15min of far red light, and incubated for 24 hr in darkness prior toamylase analyses. Results are means of four samples from twoexperiments with six cotyledons in each sample. Initial amylaseactivity was 0.56 4 0.10 -AA620/min-100 cotyledons.

Light Conditions Amylase Activity

-A 620/wini 100 cotyledons

Dark 1.38 (0.04)'10 min red 2.10 (0.08)10 min red + 15 min far red 1.24 (0.15)Continuous light (125 ft-c) 2.61 (0.20)

Standard errors.

Plant Physiol. Vol. 56, 1975 431

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Plant Physiol. Vol. 56, 1975

Table IV. Effect of Excision on Cotyledon Amylase ActivitiesCotyledons and intact seedlings with seed coats removed were

incubated 24 hr in darkness or under continuous fluorescent lightof 125 ft-c. Results are from a single experiment with duplicatesamples of six cotyledons each. Initial amylase activity was 0.35

1 0.05 -AA620/min .100 cotyledons.

Amylase Activity

Dark Light

-AA620/min-100 cotyledons

Intact 0.41 (0.04)1 1.03 (0.33)Detached 0.57 (0.03) 2.04 (0.61)

1 Standard errors.

0.6

E 0.50es 0.4

*T 0.3

0.2c4 5 6 7 8

pH

FIG. 4. pH dependency of starch-degrading activity. Samples of0.1 ml from an extract of 480 cotyledons in 40 ml of 1 mM CaCI2were incubated for 10 min in 2 ml of 0.08% soluble starch, bufferedeither with 20 mm sodium acetate, 20 mm tris-Cl, or with 20 mMpotassium phosphate. Cotyledons had been exposed to 24 hr offluorescent light of 125 ft-c. Homogenized cotyledons were centri-fuged at 3,020g for 10 min at 4 C, then the resulting supernatantwas recentrifuged at 23,500g for 20 min at 4 C.

Sephadex column could be left at least 24 hr at 4 C withoutapparent loss of activity, even though considerable precipita-tion of protein from the crude extract occurred. When effectsof pH on starch-degrading activity were studied over the pHrange 4 to 7.9, maximal activity was observed at about pH5.5 with sodium acetate-acetic acid buffer (Fig. 4). Thisoptimum compares with a maximum at pH 5.3 to 5.9 for peacotyledon amylase (22). There was no increase in activity ofcrude extracts when potassium phosphate was used as bufferinstead of sodium acetate, suggesting that little starch phos-phorylase was present and that starch-degrading activity was

therefore primarily due to an amylase.Gel filtration of an enzyme extract from cotyledons ex-

posed to 22 hr of fluorescent light of 125 ft-c resulted in a

single broad fraction (Fig. 5). Three analyses gave maximalactivity at estimated mol wt of 22,400, 23,500, and 26,600.BSA (mol wt 67,000), human a-globulin (160,000), and spermwhale myoglobin (17,800), all from Schwarz-Mann, were usedas protein standards. Contents of all tubes in each of the threeanalyses were assayed both with and without 2 mm CaCl,; thissalt did not affect starch degradation in any fraction.No attempts to purify and further characterize the enzymes

as a or ,B-amylase have been made, but preliminary experi-ments with crude 23,500g supernatants (after homogenizingcotyledons in 10 mm tris-Cl, pH 7) suggest that the enzymesmight be primarily /8 amylases. Chromatography of starchdegradation products revealed similar amounts of glucose andmaltose but none of the higher mol wt oligosaccharides ex-

pected from an a amylase. These reducing sugar products were

both obtained at time periods corresponding to degradationof 27% and 81% of the starch in a 0.05% solution at pH 5.5.Also, polarimetric analysis of the products formed, asdescribed by Swain and Dekker (22), gave results consistentwith a predominance of /8 amylase activity. Use of /8 limitdextrin, an a amylase specific substrate (1), gave significantrelease of soluble blue chromophore indicative of a amylaseactivity. Lack of a requirement for added Ca does not neces-sarily indicate that purified enzymes would not require Ca formaximal activity (22), and therefore does not eliminate thepossibility that a amylases were involved.

DISCUSSION

The results show that red light caused an increase in thecontent of reducing sugars in radish cotyledons. Zeatin furtherincreased the accumulation of these sugars in continuous lightor after brief red light treatments, but not in darkness. Theseincreases were well correlated with growth effects. This cor-relation apparently does not result from an effect of growthon sugar production, since the content of reducing sugars wasindependent of water uptake; this is evidenced by the in-hibitory effects of negative external osmotic potentials ongrowth, but not on sugar levels (Table II). Instead, the correla-tion probably results, at least in part, from the osmotic effectsof the sugars. If water uptake was not concomitant with therise in sugars, our calculations show that the osmotic potentialswould decrease as much as 10.5 bars. However, since sugarproduction is slow compared to water uptake, a nearly con-stant concentration of sugars should be maintained. Actually,the sugar concentrations are expected to rise slightly to counterdilution of solutes that do not increase, thus maintaining waterpotentials in the growing cotyledons in near equilibrium withthe external water potentials. We emphasize here the osmoticeffects of an increased sugar supply upon growth, yet werecognize that influences of sugars on the energy supply andon numerous metabolic reactions may also more indirectlylead to growth.

Although growth enhancement due to zeatin (with light)

Ve/Vo=2.24 /0.5 -

000.3

0.2-

0.1

2 4 6 8 10 12 14TUBE NO.

FiG. 5. Elution profile of amylases from a Sephadex G-150column. Sample was a 5-ml tris-Cl (pH 7) extract of 220 cotyledonsthat had been exposed to 22 hr of fluorescent light (125 ft-c).Sucrose was added to a final concentration of 5% (w/v) beforesample was added to the 2.7 x 20 cm column. After elution of22-ml void volumn, 15 fractions of 5 ml each were eluted with 20mm sodium acetate (pH 5.5) at 4 C. Amylase activity was measuredby mixing 0.3 ml from each fraction with 2 ml of 0.05% (w/v)starch buffered at pH 5.5 with 20 mm sodium acetate, then in-cubating 15 min at 30 C. Ve/Vo represents the ratio of sampleelution volume to void volume, determined with Blue Dextran2000 from Pharmacia.

* ACETATEO TRIS-CIPHOSPHATE

432 HUFF AND ROSS

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COTYLEDON GROWTH AND REDUCING SGUARS

seems to result in part from sugar accumulation, another factormust be involved in the cytokinin effect. Zeatin did not in-crease the level of reducing sugars when cotyledons werestimulated to grow in the dark (Fig. 2B), nor did it increasethese levels during the first 24 hr of incubation occurring incontinuous light even though it stimulated growth (Fig. 1).Zeatin-treated cotyledons incubated in light were larger thancontrol cotyledons with similar sugar contents (Fig. 2). Insunflower cotyledons, kinetin promotes both reducing sugarproduction (4) and K' uptake (6), so it is possible that part ofthe growth promotion due to zeatin in our studies results fromenhanced K' uptake from the potassium phosphate bufferused. An alternative possibility is that zeatin decreases theinternal pressure potential by somehow affecting the cell wall,but we are aware of no precedent for this.The accumulation of reducing sugars resulting from phyto-

chrome activation might result from increased amylase activ-ity. This phenomenon appears to be similar to light-enhancedstarch breakdown during unrolling of the first leaf in grassesafter light treatment (8). Starch degradation is probably notresponsible for the additional zeatin-promoted sugar increase,since extractable amylase activity was inhibited by zeatin rela-tive to control tissues in the light (Fig. 3). These results are thusdifferent from those of Gepstain and Ilan (3), who found anincrease of amylase activity in bean cotyledons by kinetin.Although our amylase measurements may not reflect the invivo situation, the results suggest an alternate source of sugars(perhaps fats) in cotyledons treated with the hormone. Pennerand Ashton (17) previously demonstrated that benzyladeninepromotes isocitrate lyase activity in squash cotyledons, andthis enzyme is important in the conversion of fatty acids tosugars in fat-rich seeds.

Acknowledgment-The capable technical assistance of Patricia Fox is grate-fully acknowledged.

LITERATURE CITED

1. BILDERBACK, D. E. 1973. A simple method to differentiate between a- and,8-amylase. Plant Physiol. 51: 594595.

2. EBAsHI, Y. AND A. C. LEOPOLD. 1969. Cotyledon expansion as a bioassay forcytokinins. Plant Physiol. 44: 618-20.

3. GEPSTAIN, S. AND I. ILAN. 1970. A promotive action of kinetin on amylaseactivity in cotyledons of Phaseolus vulgaris. Plant Cell Physiol. 11: 819-822.

4. GIAD, T., I. ILN, AND L. REINHOLD. 1970. The effect of kinetin and ofthe embryo axis on the level of reducing sugars in sunflower cotyledons.Isr. J. Bot. 19: 447450.

5. IxumA, H. ANm K. V. TMMUNN. 1963. Action of kinetin on photosensitivegermination of lettuce seeds as compared with that of gibberellic acid.Plant Cell Physiol. 4: 113-128.

6. ILAN, I., T. Gn.A , AND L. REINHOLD. 1971. Specific effects of kinetin onthe uptake of monovalent cations by sunflower cotyledons. Physiol. Plant.24: 337-341.

7. JoNEs, R. L. AND J. E. VAINER. 1967. The bioassay of gibberellins. Planta72: 155-161.

8. KLEIN, W. H., L. PRICE, AND K. MrrRAxos. 1963. Light-stimulated starchdegradation in plastids and leaf morphogenesis. Photochem. Photobiol.2: 233-240.

9. LETHAM, D. S. 1968. A new cytokinin bioassay and the naturally occurringcytokinin complex. In: F. Wightman and G. S. Setterfield, eds., Bio-chemistry and Physiology of Plant Growth Substances. Runge Press,Ottawa. pp. 3345.

10. LETHAM, D. S. 1971. Regulators of cell division in plant tissues Xm. Acytokinin bioassay using excised radish cotyledons. Physiol. Plant. 25: 391-396.

11. LovEYs, B. R. AN-D P. F. WAREING. 1971. The hormonal control of wheat leafunrolling. Planta 98: 117-127.

12. LowRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AN R. J. RANDAL. 1951.Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

13. MARAIs, J. P., J. L. DE WIT, AND G. V. QuiCxE. 1966. A critical examinationof the Nelson-Somogyi method for determination of reducing sugars.Anal. Biochem. 15: 373-381.

14. MILLER, C. 0. 1956. Similarity of some kinetin and red light effects. PlantPhysiol. 31: 318-319.

15. NABORS, M. W. AND A. LAN-G. 1971. The growth physics and water relationsof red light-induced germination in lettuce seeds. II. Embryos germinatingin water. Planta 101: 26-42.

16. NARAIN, A. AND M. M. LALORAYA. 1974. Cucumber cotyledon expansion asa bioassay for cytokinins. Z. Pflanzenphysiol. 71: 313-322.

17. PENNER, D. AND F. M. AsTroN. 1967. Hormonal control of isocitrate lyasesynthesis. Biochim. Biophys. Acta 148: 481485.

18. POWELL, R. D. AND M. GRIFFITH. 1960. Some anatomical effects of kinetinand red light on disks of bean leaves. Plant Physiol. 35: 273-275.

19. RIJVEN, A. H. G. C. AND V. PARKASH. 1970. Cytokinin-induced growthresponses by fenugreek cotyledons. Plant Physiol. 45: 638-40.

20. SCHEIBE, J. AND A. LANG. 1967. Lettuce seed germination: a phytochrome-mediated increase in the growth rate of lettuce seed radicles. Planta 72:348-354.

21. ScOTr, R. A. AND J. L. LIVERMAN. 1956. Promotion of leaf expansion bykinetin and benzylaminopurine. Plant Physiol. 31: 321-322.

22. SWAIN, R. R. AND E. E. DEEKER. 1966. Seed germination studies I. Purifica-tion and properties of an c-amylase from the cotyledons of germinatingpeas. Biochim. Biophys. Acta 122: 75-86.

23. VIRGIN, H. I. 1962. Light-induced unfolding of the grass leaf. Physiol.Plant. 15: 380-389.

24. YEMM, E. W. A" E. C. COcKING. 1955. The determination of amino acidswith ninhydrin. Analyst 80: 209-213.

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