RELATIONS AMONG CELL GROWTH, DNA SYNTHESIS, AND GIBBERELLIN ACTION*

Anton Lang and Joseph Nitsan MSUIAEC Plant Research Laboratory,

Michigan State University, East Lansing, Mich.

Introduction

Plant cells pass through three developmental stages in their life: cell division, cell enlargement, or, if the size increase in one direction is greatly in excess of the others, cell elongation, and cell differentiation.

Each of these three stages may be affected by any of the three well-established plant growth hormones: the auxins, gibberellins, and cytokinins.

In recent years tremendous advances have been made in the understanding of some of the most fundamental processes of cell metabolism, namely, the control of synthesis of proteins by specific ribonucleic acids (RNA) which in turn are under the control of specific sections of deoxyribonucleic acid (DNA) carrying the appropriate genetic information. These advances have provided us with a new avenue for approaching the problem of the mode of action of plant growth hor- mones in the control of cell growth and development.

The first author to demonstrate a connection between those processes of cell metabolism, and plant hormone action was Masuda who, in 1959, found that treatment of a plant tissue (oat coleoptile sections) with ribonuclease reduced, at least temporarily, its ability to respond to auxin. Some years later, NoodCn and Thimann (1963) and Key ( 1964) showed, by means of inhibitors of these processes, that protein and RNA synthesis were required for optimal plant cell growth both in the absence and in the presence of auxin.

No similar information was available with respect to gibberellin (GA) , and the experiments to be reviewed in this paper were initially undertaken to obtain this information. The very first experiments gave results quite analogous to those of NoodCn and Thimann, and of Key; several inhibitors of protein and RNA synthesis (chloramphenicol, puromycin, actinomycin D) reduced growth of plant cells both in the presence and absence of exogenous GA (Nitsan & Lang, 1965). However, in our experiments, we also included some inhibitors of DNA synthesis, particularly 5-fluorodeoxyuridine (FUDR) , which, like the RNA inhibitors, proved highly effective in reducing both GA-enhanced and GA-free growth. Since we knew that at least a large part of the growth of the tissue used was based on cell elongation, this result raised the possibility that cell elongation, perhaps par- ticularly cell elongation regulated by GA, was dependent not only on protein and RNA synthesis, but also on DNA synthesis. The following experiments were therefore focused upon this question.

Methods and Materials

In order to be useful for our proposed work, a plant tissue had to meet three conditions: (1) It had to grow exclusively, or at least to a major extent, by cell elongation; (2) it had to be highly responsive to GA; and (3) preferably, it had

* This work was started at the California Institute of Technology, with support from the National Science Foundation (grant GB-3056), and continued at the MSU/AEC Plant Re- search Laboratory under U. S. Atomic Energy Commission contract no. (11-1)-1338.

180

Lang & Nitsan: Cell Growth, DNA, and Gibberellin 181 to be capable of growing submerged in the test solutions to avoid complications which may be caused by translocation phenomena following localized application of chemicals. The earlier experiments were done with seedlings of lettuce (Lactuca mtiva L., cultivar Grand Rapids). The gibherellin sensitivity of this material is well known; lettuce seedlings are used for bioassays of gibberellins (Frankland & Wareing, 1960). We found that the seedlings grew very well when shaken in a simple basal medium (0.1 Hoagland’s nutrient solution). The efficiency of this procedure is attested by the fact that the sensitivity to G A was increased by two orders of magnitude, a significant growth promotion being obtained at mg/l of gibberellic acid (GA,) as compared to 10-1 mg/l in seedlings grown on filter paper with only the root in contact with the test solution. The seedlings were used approximately 30 hr after soaking and were incubated for a period of 44 hr. Experiments were done in both light and darkness, with quite similar results.

Later, we switched to another material, epicotyls of lentils (Lens culinaris Medik = L . esculenta Moench, cv. Persian), because it was found that this tissue, at least under the conditions of our experiments, grew by cell elongation, with at best only a negligible amount of cell division. The lentil seeds were germinated for ca. 40 hr; they were then decoated and both the root and the shoot tip were removed, the former 2 mm below the cotyledons, the latter at the first node. The operated seedlings were incubated for 24 or 3 8 hr in the dark, using 0.1 Hoagland plus 1000 units of penicillin per milliliter as basal medium. For determinations of nucleic acids or nucleic-acid synthesis, the seedlings were grown intact, but the analyses were made only on the epicotyl.

Other plant materials used were embryos or coleoptiles of seedlings of wheat (Triricum aestivum L. = T . vulgare Vill,, cv. Genesee) and oat (Avena sativa L., cv. Torch) raised in the dark, and the uppermost 18 mm of the epicotyl of dark- grown cucumber (Cucumis .rativus L., cv. National Pickling) seedlings. The experiments with wheat seedlings were done by Mr. Edwin C. Liu, those with cucumber epicotyl section by Dr. Richard H. Groves.

Nucleic acids were extracted according to Smillie and Krotkov (1960) , with slight modifications; DNA and R N A were determined as phosphorus according to King ( 1932) and by optical density, D N A also as deoxyribose by the diphenyl- amine method according to Burton ( 1956) ; the correlations between determina- tions as P and by the other two methods were 0.96 and 0.98, respectively. Syn- thesis of DNA and R N A were studied by means of incorporation of 14C- or 3H-labeled thymidine and uridine, respectively; these experiments were carried out under aseptic conditions. For fractionation, the labeled nucleic acids were extracted according to Ingle et al. (1965) and fractionated on a methylated- albumin-kieselguhr ( M A K ) column according to Mandell and Hershey (1960) .

GA (GA,,) was used at optimal concentrations, namely, 10 mg/l with lettuce and cucumber hypocotyls, 100 mg/l with lentils epicotyls, and 1-10 mg/l with oat and wheat coleoptiles.

Results

The effect of F U D R on the growth of lentil epicotyls is shown in FIGURE 1, on that of lettuce hypocotyls in FIGURE 2. In either case, growth was markedly in- hibited, the effect increasing with the concentration of the antimetabolite; the effect was equally present in tissue incubated with and without GA; it was fully reversible by simultaneous application of thymidine but not affected by applica- tion of uridine.* * In lettuce seedlings, the effects of a number of other substances

** The inhibition by 10-4 M FUDR was only partially reversed by thymidine. At this high level, FUDR may also inhibit RNA synthesis, or may exert unspecific inhibitory effects.

182

E E v

-I > I- 0 0 n W

L L 0 I !- c3 z W -I

Annals New York Academy of Sciences

\- lo - CONTROL

A- - -A + I O - ~ M THYMIDINE

0-- + I O - ~ M URIDINE

I

7 6 5

5- FLUORODEOXYURIDINE (-log M I FIGURE 1. Effects of FUDR, thymidine, and uridine on the growth of lentil epicotyls in

the dark, in presence and absence of G A (10 mg/l). From Nitsan and Lang, 1965; repro- duced by permission, Developmental Biology, Academic Press, Inc., N. Y.

which have been reported to inhibit DNA synthesis (amethopterin, mitomycin C, phenethyl alcohol) were also studied. These substances also resulted in strong growth inhibitions both in the presence and absence of GA; the inhibition by amethopterin, which, like FUDR, creates a deficiency in endogenous thymidine, was partially reversible by thymidine but not by uridine. The results are thus in line with the FUDR data. However, because of the complete and specific reversi- bility of the FUDR inhibition with thymidine, these latter data are the most con- clusive to date.

TABLE 1 summarizes determinations of cell numbers in the two tissues, made either by counting the numbers of cells in longitudinal cell files in the cortex, or by the hemocytometric method of Brown and Rickless (1949). Both cell multi- plication and cell elongation contributed to the growth of lettuce hypocotyls. GA promoted, while FUDR inhibited, both processes. However, the reduction in cell number accounted for only a relatively small fraction of the reduction in length of the whole organ. Thus, the FUDR inhibition clearly extended also to cell e1ongation.f

Nevertheless, it may be argued that the inhibition of cell elongation in the lettuce hypocotyls was not due to inhibition of DNA synthesis by FUDR in the

t The contribution of inhibition of cell elongation to the inhibition of elongation of the hypocotyl as a whole is particularly evident from a comparison of control hypocotyls (no GA, no FUDR) and hypocotyls treated with both G A and FUDR. The cell numbers are not sig- nificantly different, but the FUDR-treated hypocotyls are considerably shorter than the controls.

Lang & Nitsan: Cell Growth, DNA, and Gibberellin 183

E E

I c (3 Z w _J 1,-+-

4 - - C H L O R A M P H E N I ~ L

- --ACTINOMYCIN D

F--T PUROMYCIN

I I

6 5 4 3

CONCENTRATION OF INHIBITOR ( - log M )

-4 ACTINOMYCIN D

F--T PUROMYCIN

I I

6 5 4 3

CONCENTRATION OF INHIBITOR ( - log M )

t , FIGURE 2. Effect of FUDR, thymidine, and uridine on the growth of lettuce hypocotyls

in the dark, in presence and absence of GA (10 mg/l). From Nitsan and Lang, 1965; repro- duced by permission, Developmental Biology, Academic Press, Inc., N. Y.

TABLE 1

BEFORE AND AFTER INCUBATION* CELL NUMBERS IN LE~TUCE HYPOCOTYLS AND LENTIL EPICOTYLS

Lentil Edcotyls Lettuce Hymcotvls . . _. .

GA** FUDR No. Cells Length No. Cells (1WM) Length pc$$!l Length in Cortex

(mm) Cell Filet (mm) E p i & 4 t t (mm) Cell File+

Before incubation - - 5.4 12.4 5.9 199 1.8 43

After incubation( - 16.4 139 19.0 172 8.2 73

+ - 30.0 140 27.6 170 19.1 119 - + 10.9 144 10.9 180 3.6 59 + + 14.6 126 16.9 167 5.5 68

-

* (After data in Nitsan & Lang, 1965,1966).

t Microscopic determination.

9 Lentils, 48 hr; lettuce, 44 hr; in dark.

** Lentil experiments, 100 mg/l; lettuce experiments, 10 mgll.

tt Hemocytometric determination; X 103; standard error, f 9.

184 Annals New York Academy of Sciences elongating cells themselves, but to a secondary effect of the inhibition of this process in the dividing cells of the hypocotyl.

In the lentil epicotyls, however, the differences in cell numbers are negligible, at least for the incubation period of 48 hr. The data with this tissue, therefore, show that inhibition of DNA synthesis results in a direct inhibition of cell elon- gation or, conversely, that DNA synthesis is needed for this growth process of plant cells, at least in certain tissues.$

If cell elongation, both that in the absence of added GA and that enhanced by exogenous GA, depends on DNA synthesis, it ought to be possible to detect DNA synthesis in the elongating cells. Furthermore, exogenous GA ought to promote this process, and FUDR to inhibit both the DNA synthesis occurring in the absence of added GA and the increased synthesis in the presence of GA. The experiments reviewed in the following paragraphs were undertaken to test the validity of these assumptions. In order to reduce any possible interference by cell division they were all done on lentil epicotyls and have been published elsewhere in more detail (Nitsan & Lang, 1966).

TABLE 2 shows the results of DNA and RNA determination in epicotyls before and after 48 hr of incubation. TABLE 3 shows measurements of DNA synthesis by means of labeled thymidine, and TABLE 4 shows the same for RNA and uridine. Finally, TABLE 5 and FIGURE 3 show the results of experiments in which feeding with labeled thymidine was combined with fractionation of the nucleic acids of the tissue on an MAK column. The following points emerge:

(a) The DNA content of the epicotyl tissue increases during incubation. The increase is greater in the presence than in the absence of exogenous GA. FUDR reduces the increase, in the case of GA-free incubation, keeping the DNA con- tent at the initial level.

(b) During the incubation, there is active synthesis of DNA in the tissue. This is increased in the presence of added GA. A simple calculation which need not be repeated here (see Nitsan & Lang, 1966) showed that most of the DNA in- crease evident in TABLE 2 is based on the DNA synthesis evident in TABLE 3, although some turnover of DNA also takes place.

TABLE 2

DNA AND RNA CONTENT OF LENTIL EPICOTYLS AFTER INCUBATION WITH AND WITHOUT GA, IN THE PRESENCE AND ABSENCE OF FUDR*

DNA RNA Fresh Weight la per w g per Length

Ulm mg &%:. ?!er Epicot. Cell

Initial state 5.5 5.0 2.45 12.3 13.2 66.5

Control 19.0 14.9 2.80 16.3 11.9 69.2 GA. 100 mg/f 27.6 29.4 3.14 22.0 15.4 90.9 FUDR, 10-5 M 10.9 12.0 2.20 12.2 11.2 62.1 GA + FUDR 16.9 20.0 2.96 17.7 11.9 71.3

S. E. t0.5 t0.4 t0.15 f0.6

After 48 hr incubation

* (After Nitsan & Lang, 1966). $ The hemocytometric determinations in the lentil epicotyls indicate actually a decrease in

cell numbers. This may be caused by differentiation of vascular tissue, particularly of xylem vessels, which is in progress in the organ and is associated with loss of integrity of certain cells.

Lang & Nitsan: Cell Growth, DNA, and Gibberellin 185

TABLE 3

INCUBATED WITH AND WITHOUT GA, I N THE PRESENCE AND ABSENCE OF FUDR*

Fresh Thymidine Thymidine Incorporation

INCORPORATION OF THYMIDINE-I4C INTO DNA AND RNA OF LENTIL EPICOTYLS

Treatment Weight Uptake (cpm per Epicotyl ) mp. cum per mg DNA RNA

~~~ ~

24hr** 48hr** 24 hr 48 hr 24hr 48 hr 24 hr 48 hr

Control 14.4 21.8 65 114 436 851 75 105 GA,, 100 mg/l 19.0 31.0 77 110 925 1081 67 75 FUDR, M 13.4 19.8 100 123 716 1107 73 69 GA, + FUDR 16.4 40.6 117 147 1102 1890 73 128

* (After Nitsan & Lang, 1966). All incubation media contained 104 M thymidine. * * Incubation period.

TABLE 4

INCUBATED WITH AND WITHOUT GA* INCORPORATION OF uRIDlNE-’4c INTO RNA AND DNA OF LENTIL EPICOTYLS

Fresh Weight Uridine Uptake Uridine Incorporation kpm)

RNA DNA (mg) (cpm)

Control 22.6 236 2177 315

GA,, 100 mg/l 33.6 215 2966 437

* (After Nitsan & Lang, 1966). All data are per epicotyl. Incubation media contained lo* M uridine. Incubation time, 48 hr.

(c ) Presence of exogenous GA also results in an increase of the RNA content and an enhancement of RNA synthesis in the epicotyl tissue. These increases concern ribosomal RNA and are suppressed by FUDR, indicating that they are dependent on the DNA synthesis taking place in the tissue.

The results of these experiments support the assumptions stated at the outset of this section. In addition, they prove that there is an enhancement of synthesis of ribosomal RNA under the influence of GA and show the dependence of this en- hanced RNA synthesis on the DNA synthesis occurring in the cells.

A few minor but still significant points should be discussed here: (1) In the experiments in TABLES 3 and 4, some thymidine label appeared also in RNA, and some uridine label in the DNA. This may have been because of contamination of the preparations with the “opposite” nucleic acid, or because of some inter- conversion of thymidine and uridine, or both. However, the extent of these incor- porations was so small as not to affect the essential results. (2) T h e incorporation of thymidine into DNA in the presence of FUDR was increased. However, this is expected since FUDR blocks thymidine synthesis, thereby decreasing the pool of endogenous thymidine and increasing utilization of exogenous precursor. ( 3 ) The amount of label taken up by the tissues was not affected by the presence or absence of GA and of FUDR in the incubation media, certainly not to an extent which would account for the differences in incorporation into DNA or RNA. This is an important point. In similar experiments with 32P it was noted that uptake of the precursor was very significantly modified by applied GA; the meaning of different levels of incorporation is in such a case equivocal.

186 Annals New York Academy of Sciences TABLE 5

INCORPORATION OF THYMIDINE-JH INTO THE MAK COLUMN DNA FRACTION OF LENTIL EPICOTYLS INCUBATED WITH AND WITHOUT GA*

Fresh Thy- Thy- DNA Specific Ribc- Weight midine midine Content Activity soma1

Uptake Incorpo- RNA ration Content

into DNA cpm X

1V mg/ cpm/ O.D./ per O.D./

Epicot. cpm/mg Eplcot. Epicot. O.D. Epicot.

Control 12.9 1126 2371 0.039 60.8 0.135 GA,, 100 mg/l 19.2 1073 4001 4.049 81.6 0.191

* (After Nitsan & Lana. 1966). Incubation media contained lo4 M thymidine and 10-5 M FUDR.

0.4

0.3

0.2

0.1

a 0 (D hl b

a 0.5

0.4

0.3

0.2

0. I

0 20 40 60 80 I00 120 140

TUBE NO

FIGURE 3. MAK column chromatographic pattern of nucleic acids from lentil epicotyls after 24 hr incubation with thymidineJH in presence or absence of 100 mg GA,. The incu- bation media contained lo-* M thymidine carrier and 10-5 M FUDR. From Nitsan and Lang, 1966; reproduced by permission, Planf Physiology, American Society of Plant Physiologists.

Lang & Nitsan: Cell Growth, DNA, and Gibberellin 187

The results summarized so far permit us to make the following points: (a) The cells of the lentil hypocotyl-a tissue growing almost exclusively by cell elon- gation, at least during the time interval and under the conditions used in our experiments-are synthesizing DNA while they elongate; (b) Presence of exoge- nous GA, which enhances elongation, also enhances DNA synthesis during this process. DNA synthesis also takes place during “GA-less” elongation; however, the existence of endogenous gibberellins in dark-grown pea seedlings (Kende & Lang, 1964; R. L. Jones, unpublished data) suggests that gibberellins also par- ticipate in the regulation of the “endogenous” growth of the lentil epicotyl; and (c ) FUDR inhibits both DNA synthesis and elongation in this tissue.

Synthesis of DNA in the nuclei of nondividing cells has been demonstrated, mostly by radioautography, in both plant and animal cells, in the latter some- times limited to a few specific regions of the chromosomes (Ficq & Pavan, 1957; Rudkin & Corlette, 1957; Pelc & LaCour, 1959; Woodard et al., 1961; Pelc, 1964; Wessels, 1964). A metabolic turnover of DNA in some plant tissues has also been reported (Hotta et al., 1965). However, this was DNA of cytoplasmic organelles. We have found (unpublished data) that most, if not all, of the thymi- dine label in the elongating lentil cells was localized in the nuclei.

Our results pose the question of the significance of DNA synthesis for cell elongation. Two major possibilities may be envisaged. Firstly, it may be assumed that the process of DNA synthesis itself is not essential and that it is only its result, that is, increased DNA content in the cells, which is important. Increases of the DNA content in growing and differentiating plant cells have long been known, as the phenomena of endopolyploidy and polyteny. However, there is no evidence to suggest that these phenomena are essential for growth and differentia- tion of the cells (for a brief discussion, see, e.g., Buvat, 1965, pp. 107-109). The possibility remains, of course, that only a small fraction of the new DNA is essen- tial for cell elongation and that only the increase in this specific fraction is important.

Secondly, it may be assumed that some genes have to replicate or “turn over” in order to produce their characteristic RNA which would be the factor required for cell elongation. In this connection, it may be of interest that in the elongating lentil epicotyls RNA synthesis was proceeding along with DNA synthesis, and was promoted by exogenous GA by 3 1 percent by net determination (TABLE 2) and 36 percent by uridine incorporation (TABLE 4) . This GA-induced increase could be fully accounted for by an increase in ribosomal RNA (41 percent above the minus-CIA ControI-TABLE 5) and was inhibited by FUDR, that is, was evidently dependent on the concurrent DNA synthesis. While at present this is clearly not more than a conjecture, it appears possible that active DNA syn- thesis is required for the production of ribosomal RNA, and that the latter is needed for elongation growth of certain plant cells.

The mechanism by which GA causes an increase of DNA synthesis is entirely unknown, and to propose speculative possibilities would be of doubtful use at this time.

In the introduction, it was pointed out that several authors (Masuda; NoodCn & Thimann: Key) had established a relationship among elongation growth of plant cells, auxin action, and RNA (and protein) synthesis. Our experiments estab- lish a connection among cell elongation, gibberellin action, and DNA synthesis. The question arises whether these results, similar in principle but divergent at a very important point, reflect a deeper difference in auxin- and gibberellin-regu- lated elongation growth.

188 Annals New York Academy of Sciences In the “classical” tissue for auxin assays, the three-day-old -Avena coleoptile,

the response of which to GA is either very small or nonexistent, no effect of FUDR on 3-indoleacetic acid (IAA) -promoted growth was detected (TABLE 6) . It appeared desirable to pursue the problem in tissues in which growth can be promoted by both auxin and GA. Two such tissues, coleoptiles of etiolated wheat seedlings and sections from etiolated cucumber seedlings, were selected.

For etiolated wheat coleoptiles, Wright (1961, 1966) had reported that three distinct growth phases may be distinguished: (1) an early phase of cell elonga- tion; (2) a phase of cell division; and (3) a second, late phase of cell elongation. The three phases exhibited considerable relative differences in their sensitivity to exogenous plant growth hormones. In the first phase, maximal growth promo- tion was obtained with GA; in the second, with kinetin; in the third, with IAA. Liu (unpublished data; see TABLE 7) found that growth in the first phase was inhibited by FUDR and the inhibition reversed by thymidine, whereas the third

TABLE 6

EFFECT OF FUDR ON THE ELONQATION OF AVENA COLEOFTILE SECTIONS m PRESENCE AND AESENCE OF IAA AND GAI*

OAo (mu) n 1 in

Control FUDR Control FUDR Control FUDR

No IAA 5.95 5.95 6.20 6.40 6.80 6.42

IAA 10 mg/l 9.72 10.10 9.98 10.60 9.52 9.00

* FUDR, 10-5 M. Values represent h a 1 length in mm.

TABLE I

EFFEC~ OF GA, IAA, FUDR AND THYMIDINE ON GROWTH OF WHEAT AND OAT COLEOPTILES m DIFFERENT STAQES OF DEVELOPMENT*

Initial Final Increase Increase Final Increase Increase LcnHh Length over Length over

Control Control mm mm % % m m % %

63 hr old (initial length, 10 mm) Two hr old

~~

GA, (1W M) 1.11 2.62 136 85 15.3 53 GA, + FUDR (~WJ M) 1.04 1.89 81 30 14.8 48 GA, + FUDR

4- thymidine (l(r M ) 1.18 2.50 112 61 14.1 41 Control 1.18 1.78 51 - 15.7 57

IAA + FUDR IAA (1W M ) 1.04 1.18 13 22.0 120 57

0.97 1.19 22 22.3 123 60 IAA + FUDR

-t thymidine 1.12 1.28 17 21.5 115 52 GA, Control

1.01 2.89 153 121 0.94 1.23 32 - 16.3 63 -

* Two hr old, whole embryos incubated, coleoptile measured; 63 hr old, 10-mm coleoptile sections incubated, Incubation period in all cases, 24 hr. Germination and incubation in dark, 25’.

Lang & Nitsan: Cell Growth, DNA, and Gibberellin 189 TABLE 8

TO GA, IAA, FUDR, AND THYMIDINE* RESPONSE OF 18-MM-LONG, APICAL SECTIONS FROM ETIOLATED CUCUMBER HYPOCOTYLS

Hormones Antimeta-

Metabolite 10mg/l Img/l + 10mg/l l m g / l + bolite,, None GA IAA GA None GA IAA GA

(M) IAA IAA

With cotyledons and apex Without cotyledons and apex None 32.5 41.7 37.4 45.0 22.7 23.9 28.7 30.7 FUDR (10-5) 31.9 32.5 37.6 39.4 22.8 23.2 29.0 30.2 FUDR -I- 33.3 40.7 38.8 45.7 21.5 23.6 29.8 30.8

thymidine ( 1 0 9

* The figures are the final lengths in nun; incubation period, three days.

phase was quite FUDR-insensitive. It should be mentioned that, thus far, we have not done cell counts in this tissue. For cucumber seedlings, Katsumi et al. (1965) had reported that growth can

be equally promoted by applied GA and IAA, the effects being much less than additive. Using the uppermost 18 mm of the hypocotyls of three-day-old, dark- grown cucumber seedlings, Groves (unpublished data) found a material in which the effects of GA and IAA were clearly additive. The GA-promoted growth was suppressed by FUDR and was restored by thymidine; the IAA-promoted growth was insensitive to FUDR (TABLE 8).

While much further work is needed, the results suggest the intriguing possi- bility that, for gibberellin-regulated elongation growth of plant cells, synthesis of DNA is essential, whereas auxin-promoted growth is dependent on the syn- thesis of RNA and protein, but not on that of DNA.

Acknowledgments

FUDR was generously supplied by Hoffman-La Roche; actinomycin D by Merck, Sharp & Dohme; “Persian” lentils by Dr. V. E. Youngman, Washington State University; and Grand Rapids lettuce seed by the Pieters-Wheeler Seed Company. Mr. Elliot N. Light has ably assisted throughout the course of the work. To all these persons, institutions and companies we wish to express our grateful appreciation.

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