Plant Physiol. (1977) 59, 1034-1038

Interaction of Boron with Components of Nucleic AcidMetabolism in Cotton Ovules Cultured in vitro

Received for publication July 27, 1976 and in revised form January 17, 1977

ELLIOTr H. BIRNBAUM,1 W. MACK DUGGER, AND BUD C. A. BEASLEYDepartments of Biology and Plant Sciences, University of California, Riverside, California 92502

ABSTRACT

Cotton (Gossypium hirsutum L.) ovules grown in a defined nutrientmedium undergo normal morphogenesis, induding fiber production. Inidentical medium lacking boron, ovules callus and accumulate brownsubstances. Boron deficiency-like symptoms were induced by 6-azauraciland 6-azauridine in ovules growing in boron-sufficient media. Othernudeoside base analogs either reduced or had no effect on over-allgrowth, but did not cause typical boron-deficient callus growth of cottonovules. Orotic acid and uracl countered the effects of 6-azauracil.Actinomycin D, fluorodeoxyundine, and ethidium bromide reduced notonly fiber production on ovules growing in boron-sufficient media butalso callusing of ovules in boron-deficient media.

Similarties between symptoms of boron deficiency and 6-azauracilinjury, and the ability of uracil to suppress both, suggest that borondefciency symptoms are related to reduced activity in the pyrimidinebiosynthetic pathway. Growth inhibition by most nudeoside base ana-logs tested, actinomycin D, fluorodeoxyuridine, and ethidium bromide,as compared to callusing brought on by boron deficiency and 6-azauracil,indicates that boron deficiency symptoms are not related to a reductionin nudeic acid biosynthesis. Based on this information, a discussion ofthe possibility that boron deficiency causes reduced synthesis of UDP-glucose is presented.

Research on the role(s) of boron in higher plants suggests itsinvolvement not only with translocation and nucleic acid metab-olism, but also with responses to plant growth regulators, pheno-lic acid biosynthesis, cell wall metabolism, cell maturation anddivision, and certain enzyme-mediated reactions (11). Directinvolvement of boron in only one or a few metabolic processesmight have many secondary effects on plant growth; thus, any ofthe above might be influenced only indirectly by the sufficiencyor deficiency of the element.Changes in nucleic acid levels have been correlated with boron

deficiency. Some workers have reported that RNA levels in-crease during boron deficiency (8-10) while others have ob-served a decrease in nucleic acid levels (5, 7). Chapman andJackson (7) found that an early sign of boron deficiency inPhaseolus aureus root tip segments was increased incorporationof radioactive precursors into RNA. Only later, concomitantwith increased ribonuclease activity and cessation of elongation,did a reduction in RNA levels occur. They point out that reportsof boron deficiency-related reductions in RNA were in grosslydeficient tissues (1, 18) and that a much earlier and possiblymore direct effect of boron deficiency was increased RNA syn-thesis (8, 9).

1 Present address: Research and Development Authority. Ben-Gur-ion University of the Negev, P.O. Box 1025, Beer-Sheva, 84110, Israel.

Shkol'nik and Soloviyova (30) reported that deleterious ef-fects of boron deficiency might be eliminated by addition ofRNA to the growth medium. Johnson and Albert (19) tested theeffects of various nitrogen bases on root growth of tomato plantscultured in boron-sufficient and -deficient liquid media. Theirresults suggested that thymine, guanine, and cytosine suppresseddevelopment of boron deficiency symptoms as reflected in re-duced root elongation, lowered RNA content, loss of fluores-cence, and browning. On the other hand, 6-AU2 and barbituricacid induced these aberrations in root tips grown in the presenceof boron.

Unfertilized cotton (Gossypium hirsutum L.) ovules culturedon completely defined liquid growth medium will produce fiberand mimic in vivo growth of fertilized ovules (although embryosare not produced) if the medium is supplemented with 5 AMIAA, 0.5 ,UM GA3, and 0.05 AM Kin (3). If boron is omittedfrom this medium, growth is quite aberrant; little or no fiber isproduced, and the inundated and peripheral surfaces of theovules callus profusely, obliterating any semblance of normalmorphology (4). The morphological and physiological processesof cotton fiber growth involve elongation and maturation ofsingle epidermal cells. Ovular callus proliferation, on the otherhand, involves cell division that leads to abnormal structure.We have reported that development of boron deficiency

symptoms in cultured cotton ovules is determined in part by thephytohormones included in the basal growth medium (4). Pro-fuse callusing in the absence of boron occurs only in the presenceof GA3. Phytohormones have been shown to affect nucleic acidmetabolism (20). Thus, one possible cause of callus productionin boron deficiency is altered nucleic acid metabolism. In thispaper, we report the effects of nucleoside bases, nucleotides andtheir analogs, and inhibitors of nucleic acid biosynthesis ongrowth of unfertilized cotton ovules in boron-sufficient and-deficient media.

MATERIALS AND METHODS

Procedures for flower production, boron-sufficient and boron-deficient ovule culture, and measurement of fiber and ovulegrowth have been published previously (2-4). Fiber productionis expressed in terms of TFU, as determined by the stain-destainmethod (2), and total mass is expressed as dry wt (mg/ovule).3Replicated treatments consisted of two to six flasks, each con-taining 28 to 32 ovules from a single boll (ovary). Experimentswere terminated after 14 days of culture. Twenty ovules repre-

2 Abbreviations: 6-AU: 6-azauracil; Kin: kinetin; 5-FU: 5-fluoroura-cil; TFU: total fiber units; 2-TU 2-thiouracil; 5-AU: 5-azauracil; 6-AUR: 6-azauridine; 8-AG: 8-azaguanine; OMP: orotidine monophos-phate; FUdR: fluorodeoxyuridine; TdR: thymidine.

3 Callus tissue adsorbs a small amount of the stain used to determineTFU. Low TFU values are therefore observed for fiberless ovules whichhave undergone callusing. Thus, treatments which reduce callusing alsoresult in slightly lower TFU values.

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BORON DEFICIENCY IN COTTON OVULES

sentative of the treatment were selected from each flask for TFUdetermination. Dry wt was determined by pooling all sets of 20ovules utilized in any treatment.Composition of the culture medium was as previously re-

ported (4) and included IAA, GA3, and Kin (5, 0.5, and 0.05,UM, respectively). Phytohormones and other supplements wereadded to the autoclaved basal medium by filter sterilization.

RESULTS

Effects of Intermediates in Nucleic Acid Biosynthesis. In pre-liminary experiments, the effects of 0.1 mm adenine, guanine,thymine, cytosine, and uracil were determined on cotton ovulescultured in media without boren. Only uracil appeared to par-tially alleviate boron deficiency symptoms. Concentration stud-ies with uracil showed that 1 mm was most effective in enhancingovule and fiber development in boron-deficient medium (Fig. 1).In experiments where the medium contained 1 aM boron, ade-nine, thymine, and cytosine at 1 mm had no effect; uracil, UMP,and perhaps orotic acid and uridine did enhance cotton fibergrowth. Guanine was insoluble and not tested (Table I).

Uracil was analyzed for boron contamination by flame spec-trography and found to be boron-free. There was variability inresponse to uracil between experiments, caused perhaps by thevariable level of endogenous boron in the ovules, and related tothe variable light conditions during growth of the parent cottonplant. There are indications in the literature that light qualityand duration influence the responses of plants to boron or thelack of it (14, 17). Although the effect of uracil was variable,inhibition was always recorded at concentrations greater than 1mM.The measurements reported in Figure 2 were made on experi-

ments conducted over a shorter period of time and with lessvariability in total irradiance received by the plants from whichthe ovaries were harvested. In this series of experiments, theuracil effect is apparent only when there is a small amount ofboron in the culture media (>1 ,UM and <10 UM). In cultureswhere no exogenous boron was added to the flask, there was noenhancement of cotton fiber production by 1 mm uracil; wherethere was 10 ,UM or more boron added to the media there was nosignificant response brought about by the inclusion of uracil.

FIG. 1. Unfertilized cotton ovules grown for 14 days in (A) mediumcontaining 100 ,UM boron; (B) boron-free medium; (C) boron-freemedium supplemented with 1 mM uracil.

TABLE I. Effects of nitrogenous bases, uridine, orotic acid and UIP, at1I., on TFU production of unfertilized cotton ovules culturedfor 14 days in boron deficient(1) nutrient media

TREATMENT

B+ B- Be(10008) (1aM) ( 1 )control control AM3) T C OA UtMP UR U

TFU 5.42 1.69 1.85 1.71 1.61 2.55 3.01 2.58 3.15

+2xSE(2) +0.52 +0.46 +0.33 +0.16 +0.49 +0.51 +0.39 *0.59 +0.55

1 aM H3B032 Twiee the standard error3 Abbreviations: A. adenine; T, thysine. C, cytosine; OA, orotic acid;

UMP, uridine monophosphate; uR, uridine; U. uracil.

0£ -Urocil-0-Im 80E00-70-

-J0

60 ----.--pU.)

FIG. 2.Efcso rclo"hF odyw aio fufriie

o

40V

coto ov0e grw for 14dy ncutr hrebrnwsiiig

F-~~~~ 0~ -

FIG.II.Efcts of urailon the T o o of unfertilized

Cottou Ovules for 14 Da" in oron-ffcient (lO0 uN) lediu

Conce-tration of Apolnq,hB(-)

Analog 3 0.1 1 10 10m Control'iM

6-Azauracil TFUa 100 90.6 108.9 18.9 Brown shrunken 26.0DWa 100 36.4 86.0 77.5 7.56 134.3

6-Azauridine TFU 100 133.4 94.9 59.7 2.53 15.8DW 100 95.7 92.6 89.4 57.0 164.3

aData are expressed as percent of control (100 tM boron).TFU-total fiber units; DLI-Dry weight, me/ovule.

Effects of Purine and Pyrimidine Analogs. The influence ofthe following purine and pyrimidine analogs on cotton ovulegrowth in boron-sufficient media was determined at concentra-tions between 0.1 and 100 UM: 5-FU, 2-TU, 5-AU, 6-AU, 6-AUR, and 8-AG. Only with 5-AU was there no inhibition infiber growth and dry wt production at any of the concentrationsused. At certain concentrations, the others inhibited both TFUand dry wt, e.g. 100 MM 5-FU reduced TFU and dry wt, respec-tively, to 45.5% and 65% of the boron-sufficient control. Thesame concentration of 2-TU caused ovules to become brown-shrunken and reduced dry wt to 15.1 % of the B(+) control. At1 LM 8-AG, TFU were reduced to 62.7% and dry wt to 81.5%of the B(+) control. Ten ,UM 8-AG resulted in 15.5% of theB(+) control TFU and 41.1% of the B(+) control dry wt. Incontrast to these effects, ovules in boron-free media developedto 19% the TFU of B(+) ovules but dry wt increased as aconsequence of callusing to 178.2 %. Reduction of TFU and drywt by these compounds may indicate that toxicity is caused bytheir incorporation into nucleic acids (6, 16). In cotton ovules,there is no similarity between boron deficiency symptoms andthose of 8-AG poisoning, as was suggested by Shkol'nik andSmirnov (29).

In contrast to the other compounds tested, 6-AU and 6-AURinduced callusing and reduced TFU (Table II), thereby causingthe appearance of boron deficiency-like growth. Fiber produc-tion was reduced about 40% by 10 ,UM 6-AUR, and about 97%

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BIRNBAUM, DUGGER, AND BEASLEY

by 100 AM. Dry wt accumulation, however, was inhibited only10% by 10 AM 6-AUR and 43% by 100 AM 6-AUR. Evengreater inhibition of fiber production was caused by 6-AU; at 10AM it reduced TFU to the level of boron deficiency, i.e. to 19%of the control. As with 6-AUR, the effect of 6-AU on dry wtproduction was much less than on TFU development. The ef-fects of 1 to 10 AM 6-AU on TFU production and dry wt areshown in Figure 3.An interesting contrast exists between the effects of 6-AU on

ovules growing in boron-sufficient (but excess) and boron-defi-cient media. During ovule growth at 1 and 10 mm B, 6.4 jM 6-AU reduced TFU production about 40% (Table III) whilehaving a lesser effect on dry wt. However, in the absence ofexogeneous boron, there was not a significant effect on TFU, butthe dry wt was reduced by 43 %. This, in effect, produced ovuleswhich were normal in appearance although reduced in size. Thissuggests that 6-AU inhibits growth of the most rapidly develop-ing cells -fiber in the case of ovules in boron-sufficient mediumand callus in boron-deficient medium.

Interactions of 6-Azauracil and Uracil. Uracil enters the py-rimidine pathway (Fig. 4) by a salvage mechanism through whichUMP is synthesized directly from uracil or with uridine as anintermediate. Thus, it might be expected that uracil would over-come 6-AU interference with cotton fiber growth by serving asan alternate precursor in pyrimidine biosynthesis. In Table IV itis shown that 1 mm uracil did partially overcome 6-AU inhibi-tion of TFU production in boron-sufficient medium. The factthat uracil did not reverse 6-AU inhibition of growth to a greaterextent might be related to Ross' observation (26) that incorpora-tion of uracil into UDP-glucose was reduced by 6-AU, perhapsbecause 6-AU increased the rate of uracil catabolism.

Effects of 6-Azauracil and Orotic Acid. Synthesis of UMP byOMP decarboxylase is inhibited by 6-AU (26). Orotic acid,

6

5

Cl)

z

wGo-

0I-

5

4

3

2

4

20

8 ?PE

6 1I0

w4 :'r

2 0

00 2 4 6 8 lo

MiM 6-AZAURACIL

FIG. 3. Effects of 6-AU on TFU and dry wt production of unfertil-ized cotton ovules cultured in boron-sufficient (100 ;iM) nutrient mediafor 14 days.

Table III. Effects of 6-Azauracil on TFU and Dry Weight Production ofUnfertilized Cotton Ovules Cultured for 14 Days In Nutrient Media

Containing No Boron, laM or lDaM Levels of Boron

Treatmentif H3B03 0 1 10oIl 6-AU 3 6.4 0 6.4 0 6.4

TFU 0.54 0.69 5.59 3.30 3.09 1.81±2 x SE(a) ±0.23 ±0.34 ±1.01 ±0.25 ±0.67 ±0.19DW 6.4 3.6 4.3 3.7 3.4 2.6mg/ovuleaTwice the standard error

U ( salvage6- AU k pathway)

OROTIC ACID--OMP -4-X UMP -UDP--- UTPPRNA

UDP-sugars UDP CTPand cellulose glucose

glucose I-P

Pentose T FUdR 7|Phosphate (salvage FCycle pothwoy) TdR 'dCTR

'"TMP C\TPhenols DNA

Noncellulosic Cell Wall Components TTPNucleic Acid Precursors

FIG. 4. Pyrimidine biosynthetic and salvage pathways, and sites ofinteraction with the pentose phosphate cycle and nucleoside diphosphatesugar synthesis.

Table IV. Effects oT 6-Azauracil ad Uracil Plus -muraciln0Tf sad DryV*ilt Productiom of Unfertililad Cotton OvalA. Cultured for 14

Days in Boroa-ufficliet (100 A) Medium

Treatment6-AU 0 6uM 6iM

U 0 0 lmlt

TFU +2 x SF(a) 5.90 + 0.67 1.64 ± 0.27 3.25 t 0.43

DW, mg/ovule 4.4 4.1 3.9

Twice the standard error

Table V. Effects of 6-Azauracil and 6-Azauracil-Orotic Acid Combinations onTFU and Dry Weight Production of Unfertilized Cotton Ovules Grown14 Days In Nutrient Media Containing either IM, GA3 and Kin or

only IAA

Phytohormones IAA, GA3, Kin(a) IAA(b)

Treatment

H3103 6-AU OA TFU Dry wt TFU -y wt±2xSE(c) ±2xSE

IM mg/ovule mg/ov.e

0 0 0 0.36 7.5 0.57 2.4±0.03 ±0.14

100 0 0 5.02 5.4 2.16 2.7t0.25 ±0.42

100 6 0 1.76 4.9 0.18 1.4±0.50 ±0.07

100 6 0.1 3.65 4.8 2.19 2.4±0.65 ±0.16

100 6 10 3.39 1.9 0.50 1.1±0.49 +0.13

aIM, 5uM; GA3, 0.5 uM; Kin, 0.05 uHbLUS, 5UMcTwice the standard error

which is the precursor of OMP (the substrate of this reaction),had little effect on boron-deficient growth (Table I). Whenovules were cultured in boron-sufficient medium with 6 AM 6-AU, 10 mm orotic acid restored TFU to 68% of the control level(Table V). At the same time, dry wt was only 36% that ofcontrol ovules, resulting in small ovules of normal appearance. Ifonly IAA was included in the growth medium rather than theusual combination of IAA, GA3, and Kin, 6 gM 6-AU inhibitednormal growth. Orotic acid (0.1 mM) completely prevented thisinhibition, but greater amounts of orotic acid were inhibitory(Table V). These data support information that the effect of 6-AU is via competition with substrates in the pyrimidine biosyn-thetic pathway. Orotic acid has the ability to prevent 6-AUinhibition of growth and to reach inhibitory levels at lowerconcentrations in ovules cultured in the presence of only IAAthan in ovules cultured with both IAA and GA3. This suggests,as do the differences in morphology in boron-deficient culture,that boron might normally promote IAA-mediated processes,whereas its absence might allow GA3-mediated processes toproceed.

Effects of Actinomycin D. With the exceptions of 6-AU and6-AUR, the analogs heretofore discussed are incorporated into

DRY WEIGHT, _

BC-) CONTROL

1\ AAAlA A '* A

TOTAL FIBER UNITS, -B(-) CONTROL

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BORON DEFICIENCY IN COTTON OVULES

RNA (6, 27) and probably limit growth by producing nonfunc-tional or aberrant RNAs. Both 6-AU and 6-AUR interfere withthe pyrimidine biosynthetic pathway and caused an effect incotton ovules closely resembling boron deficiency. It was ofinterest to determine whether actinomycin D, an inhibitor ofRNA synthesis, would limit growth as did most of the otheranalogs, or whether it might cause aberrant (boron deficiency-like) growth.

Actinomycin D, 2 and 20 ,Mg/ml, was tested on ovules growingin boron-sufficient medium. At 2 ug/ml, the TFU level wasabout 4% that of the control. With 20 ,ug/ml, ovules enlargedslightly, produced no fiber, and remained white except for asmall area on the inner concave surface which browned (data notshown). Ovules were transferred from boron-sufficient or -defi-cient media into identical media containing 2 ,ug/mi actinomycinD on various days after initiation of culture (Fig. 5). Whentransferred on day 5, ovules from boron-sufficient medium pro-duced 37 % as much fiber as the control; when transferred onday 2, fiber production was 30% of the control. In boron-deficient medium, ovules transferred to actinomycin D mediumon day 5 produced about 29% of the B(-) control TFU, and51 % of the dry wt at the end of 2 weeks in culture. Thosetransferred on day 2 produced 23% of the B(-) control TFU,and only 29% of the dry wt. Thus, RNA synthesis appears to benecessary for fiber development and normal morphogenesis, aswell as for callus growth induced when boron was absent. RNAsynthesis appears to occur at all stages of the 14-day cultureperiod.

Inhibitors of DNA Synthesis. In order to gain insight intopossible differences in DNA composition of normal and boron-deficient tissues, two inhibitors of DNA synthesis, FUdR andethidium bromide, were tested.FUdR. FUdR interferes with all DNA synthesis by function-

ing as an alternate substrate for TMP synthetase (ref. 6 and Fig.4). In the presence of 100 gM boron, 1 and 10 Mm FUdRreduced TFU production to the level of boron deficiency (TableVI), however, unlike boron-deficient growth, FUdR reduceddry wt as well. Thus, it appears that both fiber development andovular growth require DNA synthesis. When ovules were grownin boron-deficient medium, 1 and 10 gM FUdR had a lesser

7

0

0

J3

° 4

effect on fiber production than when the ovules were grown inboron-sufficient medium. In both cases, dry wt was decreased byFUdR (Table VI). Such ovules resembled B(+) ovules grownwith FUdR, as well as B(-) ovules cultured with IAA but noGA3. In other words, inundated surfaces neither callused norproduced fiber, and the upper surfaces were covered by sparsefiber growth.Thymine had no effect on boron-deficient cotton ovule growth

(Table I). When 10 uM TdR was added to media in which fiberdevelopment (in the presence of boron [Table VI and Fig. 6]) orcallus development (in the absence of boron [Table VI]) wouldhave been prevented by 1 ,UM FUdR, growth occurred as thoughFUdR had not been included. Thymine and TdR are precursorsof TMP via a salvage pathway which bypasses TMP synthetase(ref. 6 and Fig. 4). Reversal of the FUdR effect by TdR issupportive of FUdR's proposed site of action. (Inasmuch asuptake studies were not made, the possibility of competitionbetween FUdR and TdR for transport into cells cannot be ruledout as at least a partial cause of the reversal.) FUdR preventedcallusing in a boron-deficient medium as well as fiber develop-ment in boron-sufficient medium. This indicates that callusing asan alternate growth form is not a result of insufficient precursormaterial for pyrimidine biosynthesis, and indeed is as dependenton pyrimidine and DNA synthesis as is normal growth.

Effects of Ethidium Bromide. In mammalian cells, ethidiumbromide inhibits mitochondrial associated RNA synthesis bybinding to DNA, and at low concentrations it does not inhibitnuclear DNA synthesis (23). The effects of ethidium bromide oncotton ovule morphogenesis were similar to those of FUdR. In

Table VI. Effects of Fluorodeoxyuridine (FUdR) Concentration on Fiber (TFU)and Dry Weight Production of Unfertilized Cotton Ovules Cultured

in Boron-Sufficient and Deficient Nutrient Media for 14 Days

B(+) (a) B(-) (b)FUdR,pIM 0 0.1 1 10 0 0.1 1 10

TFU 3.98 3.27 0.98 0.54 0.62 0.37 0.25 0.30

t2xSE(c) ±0.41 ±0.15±0.25 ±0.12 ±0.15 ±0.14 ±0.11 ±0.16

Dry vtmg/ovule 3.4 4.4 1.3 1.4 5.7 4.1 1.5 1.3

a100 u?4 113B030 H3BO3

Twice the standard error

7

64

5 o

E

3223-

2>.

cc

14 5 2 0 14 5 2 0

DAYS OF CULTURE PRIOR TO TRANSFER TO MEDIACONTAINING 2 Ag ACTINOMYCIN D/mI

FIG. 5. Effects of actinomycin D (2 Atg/ml) on TFU and dry wtproduction of unfertilized cotton ovules in boron-sufficient (100 ;LM)and -deficient nutrient media. Ovules were cultured 14, 5, 2, or 0 days inboron-sufficient or -deficient media prior to transfer to identical mediacontaining actinomycin D for the remainder of the 14-day culture pe-

riod.

5

UtI-

z

w

Ui.

0

4

3

2

,SM FUdR;tM TdR

0 lO 0 0.1 10

5

4

0

3'0

E

3

2>,

0C

FIG. 6. Effects of fluorodeoxyuridine (FUdR) and thymidine (TdR)combinations on TFU and dry wt production of unfertilized cottonovules cultured for 14 days in boron-sufficient (100 gM) nutrient media.

IOOj.tm H3B03 B8(-)

A~ ~~~~

A

A

A

A

AA

A

Plant Physiol. Vol. 59, 1977 1037

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1038 BIRNBAUM, DUGGER, AND BEASLEY

Table VII. Effects of Ethidium Bromide on TFU and Dry Weight Production ofUnfertilized Cotton Ovules in Boron-Sufficient Media, and on Dry

Weight (a) Production in Boron-Deficient Nutrient Media for 14 Days

Ethidiumbromide

ot

0.1

10

10I

B(+) (b) B (-) (c)TFUJ Dry *yt Drv wt±2xSE(d)

mg/ovule mg/ovule

5.04 4.4 8.7+0.533.72 3.5 10.9+0.190.62 1.1 1.5

±+.180.12 0.8 1.1

50.030.04 0. 5 0.9+0.01

No TFU data obtained

bDOM H3Bo3H3Bo3

Twice the standard error

the presence of boron (Table VII), ethidium bromide reducedfiber and dry wt production; in the absence of boron, it pre-vented callusing. In both cases, 1 ,tM ethidium bromide causedovules to be reduced in size with fiber on the upper surface butnone on the inundated surface. Both 10 and 100 uM ethidiumbromide reduced ovular growth still further and essentially elim-inated fiber production.

DISCUSSION

The over-all reduction in growth of boron-deficient ovulesaffected by inhibitors of RNA and DNA synthesis shows that forcallusing to occur, nucleic acid biosynthesis is necessary. Both 6-AU and 6-AUR are converted to 6-AUR monophosphate whichinteracts with OMP-decarboxylase (27, 32), thereby reducingsynthesis of UMP and, consequently, synthesis of nucleic acidpyrimidines and/or UDP-glucose (refs. 25, 26, and Fig. 4).Callus growth on cotton ovules in media containing 6-AU, 6-AUR, and in boron-deficient medium indicated that their effectsare not due to insufficient nucleic acid biosynthesis. One possiblehypothesis to explain the results of this study is that during borondeficiency a reduction in UDP-glucose pyrophosphorylase activ-ity and subsequent synthesis of UDP-glucose occurs. UDP-glu-cose is a likely precursor of cellulose and a precursor of otherUDP-sugars involved in cell wall composition (e.g. in elongatingand maturing fibers). UDP-glucose pyrophosphorylase appearsto play a key role in morphogenesis of the slime mold, Dictyoste-lium discoideum, increasing in activity during the period offruiting body formation by aggregated amoeboid cells (15, 24).With respect to higher plants, evidence in the literature lendssupport to the hypothesis that a critical function of boron is inthe activity of UDPG pryophosphorylase. In cocklebur leaf discsincubated with '4C-uracil and 6-AU, the most significant effectof 6-AU was reduction of incorporation of label into UDP-glucose by more than 50% (26). Similarly, in bean roots, borondeficiency resulted in accumulation of UTP and decline in thelevel of UDP-glucose (ref. 31 and personal communications withthe author). Dugger and Humphreys (12) earlier reported thatin vitro activity of UDP-glucose pyrophosphorylase was stimu-lated when boron was added to the reaction mixture.A number of reports connect boron to various phases of

carbohydrate and nucleotide metabolism (11). Lee and Aronoff(21) found that phenol biosynthesis is limited by a borate-6-P-gluconate complex which inhibits 6-P-gluconate dehydrogenaseactivity. In the absence of boron, greater enzyme activity resultsin elevated phenol biosynthesis. Phosphoglucomutase activity(22) and starch biosynthesis (13, 28) have also been reported tobe inhibited by boron. Regulation of any of these reactions byboron might influence glucose-i-P levels and, thereby, UDPGsynthesis and the direction of UTP metabolism.

Although there is evidence of roles for boron in activities ofseveral plant enzymes (11, 12, 14), we favor the hypothesis thatboron plays a major role in the development of cotton ovules by

Plant Physiol. Vol. 59, 1977

regulating UDPG synthesis. If this is so, glucose-i-P wouldaccumulate during boron deficiency and be available for metab-olism by these alternate routes. It is thus possible that UDP-glucose pyrophosphorylase plays a key role in the morphoge-netic development of organisms with cell walls in general, by its:(a) involvement in sugar nucleotide biosynthesis and, the subse-quent synthesis of cell wall material; and (b) peripheral effectson the availability of substrates for other reactions.

Acknowledgments - -The authors are grateful to G. Bradford for the flame spectrographicanalysis of uracil and to F. Foster for technical assistance. E. H. B. wishes to express particularappreciation to W. Belser for advice and encouragement.

LITERATURE CITED

1. Albert LS 1965 Ribonucleic acid content, boron deficiency symptoms and elongation oftomato root tips. Plant Physiol 40: 649-652

2. Beasley CA, EH Birnbaum, WM Dugger, IP Ting 1974 A quantitative procedure for

estimating cotton fiber growth. Stain Technol 49: 85-923. Beasley CA, IP Ting 1974 Effects of plant growth substances on in vitro fiber development

from unfertilized cotton ovules. Am J Bot 61: 188-1944. Birnbaum EH, CA Beasley, WM Dugger 1974 Boron deficiency in unfertilized cotton

(Gossypium hirsutum) ovules grown in vitro. Plant Physiol 54: 931-9355. Bozhenko VP, VN Belyaeva, MY Shkol'nik 1973 Nucleotide composition of RNA and its

individual fractions in the apical cone and roots of sunflowers in conditions of boron

deficiency and high temperatures. Fiziol Biokhim Kul't Rast 5: 125-130 (Russ, fromChem Abstr)

6. Brockman RW, EP Anderson 1963 Pyrimidine analogues. In RM Hochster, JH Quastel,eds, Metabolic Inhibitors Vol 1. Academic Press, New York pp 239-285

7. Chapman KSR, JF Jackson 1974 Increased RNA labeling in boron-deficient root-tipsegments. Phytochemistry 13: 1311-1318

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