Plant Physiol. (1978) 62, 582-585

An Inborn Error of Potassium Metabolism in the Tomato,Lycopersicon esculentuml

Received for publication March 6, 1978 and in revised form June 8, 1978

EMANUEL EPSTEINDepartment of Land, Air and Water Resources, Soils and Plant Nutrition Program, University of California,Davis, California 95616

ABSTRACT

A mutant of the tomato (Lycopersico esculentum Mil) was obtainedthrough treatment of the seed with ethyl methane sulfonate. Its chiefdisuising characteristic is the requirement for an extraordinarily highK concentration in the medium (20 mM) if it is to develop withoutpronounced K deficiency symptoms, while the wild type grows without anysuch symptoms at 0.1 to 0.2 mM K. The lesion of the K-inefficient mutantdoes not lie in its lnability to absorb and translocate K.

The role of K in the metabolism of higher plants, like the roleof many other mineral nutrients, is investigated mainly by thefollowing two methods. (a) A deficiency of the element is broughtabout by supplying inadequate amounts of it, and the conse-quences of this deficiency are then followed in terms of growthand development, symptoms, and physiological and biochemicalabnormalities. Resupplying K to K-deficient plants, followed bysimilar kinds of observations, is often part of studies of this type.Limitations inherent in this method have been discussed by Ep-stein (1, pp. 296-297). (b) The effect of K in activating certainenzymes in vitro is studied; about 50 K-activated enzymes havebeen identified (2, 12).A third method, of a type common in investigations of metab-

olism in microorganisms, has not been brought to bear uponstudies of K metabolism in higher plants. It is the induction ofmutations by radiation or chemical mutagens, followed by selec-tion of individuals impaired or inefficient in K metabolism, andcomparison of plants having such genetically governed impair-ment with normal ones. The present paper is an account of thefirst results of an investigation in which K-inefficient mutants ofthe tomato, Lycopersicon esculentum, were obtained in this man-ner.

MATERIALS AND METHODS

Mutagenesis. Seeds of tomato, L. esculentum Mill., cv. VF 36,were soaked in a solution of 8 g/l of ethyl methane sulfonate for24 hr. The seeds were rinsed with water for 30 min and planted ina 1:1 mix of Yolo loam and peat moss. When the seedlings werelarge enough they were planted on field plot on the Davis Campus.There were 1,073 plants from ethyl methane sulfonate-treatedseed, and 40 from control (untreated) seed. The fruit was harvested

1 This research was supported by National Science Foundation GrantsBMS75-02285 and PCM75-02285 AOl. Some related experiments, thoughnone reported here, were done while the author was a Senior FulbrightResearch Fellow at the Department of Scientific and Industrial Research,Palmerston North, New Zealand.

in August, after the plants had grown in the field for 4 months.The pulp was squeezed from the fruit and kept for 4 days in the

greenhouse. The liquified pulp was then strained through a kitchenstrainer which retained the seeds. The seeds were dried on papertowels.

Solution Culture for Selection of Mutants. Seeds were germi-nated and plants grown by means of solution culture in thegreenhouse. There were four tanks made of sheet metal. Theywere painted inside and out with a gray paint which has provennontoxic (Amercoat, made by Ameron Corporation, Brea, Calif.).The inside dimensions of the tanks were 365.8 x 101.6 x 20.3 cm(height). Five straps spaced evenly across each tank served to holdsix plastic grids, each 100. 3 x 60.2 x 1.25 cm, on which the plantswere supported. With the solution just below the level of the grid,each tank held 700 liters.To germinate seedlings and grow plants, the tanks were filled

with nutrient solution, the grids were put in place on the straps,and cheesecloth was stretched over the grids. The level of thesolution was then raised so that it just made contact with thecheesecloth. The solution was aerated; there were six aerators/tank, or one for each of the plastic grids. Seeds were planteddirectly on the cheesecloth to germinate. When the seedlings hadreached a height of a few cm, the level of the solution was allowedto drop (by evaporation) until it was just below the bottoms of theplastic grids.

Solution Culture for Characterization of Plants. For a system-atic comparison of mutant and normal plants, each of four 700-liter tanks was used to grow two plants to maturity as describedbelow. Instead of six grids, six covers made of Plexiglas acrylicsheet were placed on the straps of the tanks. The sheets were 4.8mm thick and had the same length and width as the plastic grids.To keep them from warping they were reinforced by means ofPlexiglas bars fastened to their bottom sides using chloroform.Each cover had four holes 5.8 cm in diameter. The covers weresandpapered to roughen the surface and then painted above andbelow the gray Amercoat paint. To support seedlings on thecovers, the hypocotyl was carefully wrapped with some Dacronbatting, the seedling was then inserted into the central hole of acork impregnated with wax and the cork in turn inserted into oneof the holes of the covers. Unused holes were closed with corks.Aeration was by means of six aerating tubes.As the plants grew beyond the seedling stage, those in the low

K solutions (0.15 mm K) proceeded to withdraw K from thesolutions to an extent that their K concentrations, monitored byatomic absorption spectrometry, decreased measurably. The Klevels of the solutions were replenished by additions of KNO3stock solution. Eventually, it became impossible to maintain thesolutions at 0.15 mm K and a lower limit of 0.1 mm and an upperone of 0.2 mm were established for the low K solutions. Even withthis latitude K had to be analyzed for and replenished daily afterthe plants had grown for 2 months.The pH of both high K and low K solutions tended to drop

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AN INBORN ERROR OF POTASSIUM METABOLISM

from the initial value of approximately 5. To keep the pH frombecoming too low periodic additions were made of a suspensioncontaining 1 mol of Ca(OH)2/l.

After the plants had grown to an appreciable size (a height ofabout 60 cm of the normal VF 36 plants) the concentrations ofCa2+, Mg2+, N03-, H2PO4-, s042-, and micronutrients in some ofthe cultures were decreasing. Replenishments were made by ap-propriate additions of stock solutions. This was done twice, onApril 23 and on May 19, 1976, at which time the plants had grownin the solutions approximately 2 and 3 months, respectively.

EXPERIMENTS AND RESULTS

Selection. Seeds of fruit harvested from plants grown from ethylmethane sulfonate-treated seed as described above were plantedon two of the 700-liter tanks in the greenhouse. The nutrientsolution had the composition given in Table I. It is distinguishedby an extraordinarily high concentration of K, viz. 20 mm. Inorder not to have any one anion predominate in the solution, thestock K solution (1.0 M K) contained equivalent concentrations ofKNO3, KCI, and K2SO4. The pH of the solutions ranged from 4.9to 5.4. No adjustments ofthe solutions were made during the shortperiod the seedlings were grown in these solutions.When the seedlings had two to three true leaves the grids with

the plants were transferred to two other tanks containing the lowK nutrient solution whose composition is given in Table II.Thereafter, the plants were carefully monitored each day forsymptoms of K deficiency, which are highly characteristic andreadily identified by an experienced observer. Plants showing suchsymptoms were "rescued" by transfer back to high K nutrientsolutions (Table I). They were grown to maturity and seed washarvested as described under "Materials and Methods."

Characterization. Several mutants responding as describedabove were identified. The symptoms were similar in all of themthough varying in intensity. One such selection, mutant 42, waschosen for a systematic study involving two genotypes and twotreatments, as follows: the parent or wild type cv. VF 36, one setin high K solution, the other in low; mutant 42 in high K and lowK solutions. The procedure was as follows.About 100 seeds of each cv. VF 36 and mutant 42 were planted

on cheesecloth stretched over 3.5-liter polyethylene containersfilled with aerated high K nutrient solution (Table I). The seedsgerminated and all seedlings grew without developing K defi-ciency symptoms. After 18 days two seedlings of cv. VF 36 weretransferred onto a 700-liter tank with high K solution, being heldby corks as described above. Two VF 36 seedlings were transferredin like manner to a tank with low K solution. Two other tanks,

Table I. Composition of high K nutrient solution

Salt or element Concentration Concentration of individual nutrientsmM mM

Composite K 20 K, 20; No3, 6.7; Cl, 6.7; S04. 3.3

Ca(N03)2 4 Total No3, 14.7NH4H2PO4 2

MgSo4 1 Total S04, 4.3

Fe-EDTA 0.05Mn, Zn, Cu, Cl, B, Mo See Table 3-1 in ref. 1

Total Cl, 6.7

Table II. Composition of low K nutrient solution

Salt or element Concentration Concentration of individual nutrientsmM mM

KNO3 0.1 Total K, 0.15

Ca(N03)2 4 Total NO3, 8.1

NH4H2PO4 2

Mg1S04 1

Fe-EDTA 0.05Mn, Zn, Cu, C1, B, Mo See Table 3-1 in ref. 1

one with high K solution and the other with low K solution, eachreceived two seedlings of mutant 42.The plants were grown in these solutions from February 23

until June 1, 1976. The weather was bright for the most part, witha light intensity at plant level ranging from 26,000 lux in the earlypart of the experiment to about twice that value at the end.Daytime temperatures ranged from 25 to 35 C (higher on a fewoccasions) and night temperatures were approximately 18 C.The wild type VF 36 plants grew well at both low and high K

levels, without any abnormal symptoms. However, the yield ofvegetative matter (roots and shoots, dry weight basis) was some-what greater for the high K culture (1,855 g/plant compared with1,691 g for the low K one) and that of fruit (fresh weight basis)somewhat smaller (11,402 g/plant compared with 13,268 g for thelow K culture).The performance of the mutant 42 plants differed drastically

from that of the VF 36 ones, and the mutant 42 plants in the highand low K treatments differed even more dramatically. At both Klevels, the mutants developed more slowly than the VF 36 plants,the difference being greater under low K than high K conditions.The mutants produced less vegetative dry matter in low K culturethan at the high K level (748 and 1,076 g/plant, respectively) butsomewhat greater (fresh weight) yield of fruit (4,705 and 3,898 g/plant, respectively). The most dramatic difference, however, be-tween mutant plants grown at low and high K levels was that theformer developed typical K deficiency symptoms within a fewdays after transfer to the low K culture. With the progress of time,these symptoms became extremely severe. The mutants in the highK solution developed few relatively minor symptoms in only someof the oldest foliage.A more detailed account will now be given, with emphasis on

the most characteristic response of the plants observed in thecourse of the experiment, viz. the development (or lack of it) ofsymptoms like those ofK deficiency.Four days after the seedlings were planted onto the tanks, the

mutant 42 plants in the low K solution had necrotic spots andareas on the first two true leaves and near the tips of the cotyle-dons, while none of the other plants had any abnormal symptoms.The symptoms of the low K mutant plants became more severewith every passing day. Figure 1 shows a mutant plant on theeighth day, and Figure 2, a view of a mutant plant in the high Kculture, for comparison, taken the same day. This extremely rapidresponse of the mutant plants to low K conditions has beenconsistently observed in a number of experiments (7).By April 2, or 5 weeks after the start ofthe differential treatment,

the VF 36 plants on both low and high K solutions were of aboutthe same height (mean 57 cm) and similar in appearance, withoutabnormal symptoms. However, the plants on the high K cultureswere somewhat more vigorous as judged by over-all appearance,profusion of blossoms, and size of the leaflets of fully grownleaves.At that time, the mutant plants on the high and low K cultures

had a mean height of 40 and 30 cm, respectively. The mutants onthe low K culture looked severely abnormal; see Figure 3 which,like the subsequent figures, shows a typical leaflet at about mid-height of the plants, i.e. neither low and senescent nor high andnewly developed. Except for some of the youngest leaves near thetop of the plants, the leaves had severe necrotic lesions, had acurled and twisted appearance, and the color of the leaflets wasblue-green, most markedly so near the center. One of the twomutant plants had no open flowers at all, the only such plant inthe experiment. It did, however, have flower buds which began toopen 2 days later. Mutants on the high K culture had normallooking leaves (Fig. 4), except for occasional marginal spots on afew leaflets of old leaves.During the remainder of the experiment, April 2 to June 1, the

mutant plants somewhat narrowed the gap between them and thewild type ones, in terms of over-all growth. The disparity between

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the wild type VF 36 plants grown in high and low K solutionsremained small, as evidenced by both appearance and yield ofvegetative matter, while the mutant plants grown under the two

conditions differed greatly. The normal plants under low K con-

ditions produced 91% as much dry vegetative matter as they didunder high K conditions, while for the mutants, the correspondingvalue was 70%o (see yield data, above).

Except for variations in size, the wild type plants under bothconditions and the mutant ones in the high K culture lookednormal, while the mutant plants in low K culture looked obviouslyabnormal to even a casual observer. Severely affected leaves (Fig.5) were common, the degree of severity being least in the upper

and greatest in the lower part of the plants. The leaves of themutant grown in high K culture looked normal (Fig. 6), exceptfor occasional slight marginal spotting along some leaflets ofsomeof the oldest leaves.

Analyses of plant material for K, Ca, Mg, and P are shown inTable III. All samples were taken on the last day ofthe experiment

FIG. 1. Seedling of K-inefficient mutant of the tomato 8 days after

transfer from a nutrient solution with 20 mm K to one containing 0.15 mMK.

FIG. 2. Mutant of the same age as the plant shown in Figure 1, butkept on high K nutrient solution (20 mM).

FIG. 3. Leaflet of mutant 5 weeks after transfer to low K solution.FIG. 4. Leaflet of mutant of the same age as the one shown in Figure

3, but kept on high K nutrient solution.FIG. 5. Leaflet of mutant on low K solution, at the end of the experi-

ment.FIG. 6. Leaflet of mutant kept on high K solution, at the end of the

experiment.

(June 1). For leaf material, analysis of the petioles are reported, as

is customary for analysis of tomato leaf samples (8). Two suchvalues are given, A for the upper and B for the lower part of theplant. The laminae were also analyzed to determine whether inthe mutant there is a blockage of K movement from the petioleinto the lamina. The K concentrations in the laminae of bothgenotypes in the low K cultures were about half those reportedfor the petioles-still clearly in the "luxury consumption" range.

The lowest concentration ofK in any lamina sample in the entireexperiment was 2.6%; it came from a VF 36 plant in low K culturewhich grew without any abnormal symptoms. Analyses of rootsare included in Table III.

DISCUSSION

Both the high and the low K solutions supported normal growthand development of the VF 36 tomato plants. Although theconcentration ofK in the low K solutions was only 0.1 to 0.2 mm,this concentration was high enough to supply adequate, indeedluxury consumption levels of K to the plants, as shown by tissueanalysis (Table III). The concentrations of the anions of the Ksalts used to make up the high K solutions (NO3, Cl, and SO4)were greater in the high K solutions than in the low K ones, butthere was no indication that these levels were detrimental to theplants, except that the high NO3 concentration probably was

responsible for the smaller yield of fruit produced by both geno-

types in the high K cultures. For the wild type VF 36, at least,both solutions may therefore be said to constitute adequate nutri-tional substrates.The mutant plants, on the other hand, did not develop normally

in the low K culture solution but showed severe K deficiencysymptoms within days after transfer to the low K solution. Thiswas not so for the mutants in the high K solution, although theirdevelopment was somewhat retarded compared with that of theirVF 36 controls. We are dealing with a heritable character. Plantsgrown from seed obtained from several successive generations ofselfed progeny of the original variant have exhibited the same

phenotype described in the present report.The lesion afflicting the mutant plants does not lie in an

impairment of absorption and long distance transport of K. In thelow K cultures, the mutants had greater concentrations of K intheir tissues than did the wild type VF 36 plants, the differencebeing considerable in the lower leaves (leaf B in Table III) andthe roots. Under high K conditions, the roots of the mutants alsocontained appreciably greater concentrations ofK than did thoseof the normal plants, while in the shoots there were no largedifferences in K concentrations.

In terms of gross tissue analysis, the mutants are not deficientin K, yet they seem to suffer from a severe functional deficiencyof this element. The condition is specific for K; experiments notreported here showed high concentrations of Na in the solutionsto be ineffective in keeping mutants from developing the Kdeficiency syndrome. In the low K solutions the ratios of Ca/Kand Mg/K were quite high (26.7/1 and 6.7/1 on a molar basis,respectively). One might therefore suspect some form of antago-

Table III. Analyses of leaf and root tissue of wild type tomato plants and of a K-inefficient mutant

All values x dry weightK ~ ~ ~Ca Mg

High K culture Wild type Mutant Wild type Mutant Wild type Mutant Wild type Mutant

Leaf A 7.8 7.2 1.45 0.82 0.11 0.32 0.42 0.52Leaf B 8.6 7.8 2.09 0.89 0.28 0.31 0.43 0.73Roots 3.8 6.6 2.18 0.48 0.08 0.18 1.78 1.34

Low K culture

Leaf A 6.8 7.4 1.00 0.70 0.32 0.43 0.64 0.70

Leaf B 4.6 6.6 2.82 1.30 0.82 0.55 0.72 0.79Roots 2.6 3.8 1.36 0.45 0.42 0.20 1.58 1.72

A: leaves from the upper part of the shoot.B: leaves from the lower part of the shoot.

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Plant Physiol. Vol. 62, 1978 AN INBORN ERROR OF POTASSIUM METABOLISM 585

nism between one or the other of these two divalent cations andK. This idea is not only vague but specifically made unlikely bythe results of the Ca and Mg analyses of the plants grown in thelow K solutions (Table III). Despite the wide Ca/K ratios in thelow K solutions, the mutants contained lower concentrations ofCa than did the wild type plants, and except for the upper leaves(leaf A), the same was true for Mg. In fact, lower Ca concentrationsin the mutants than in the wild type plants are the most pro-nounced and consistent difference between the two genotypesrevealed by analysis, but this holds for both the high and low Kcultures, in which their performance differed profoundly.

Demonstration of genotypic differences in efficiency of K uti-lization in plants, and specifically in the tomato, is nothing new,though by no means common. As early as 1939 Harvey (6)experimented with a number of tomato genotypes under condi-tions of K deficiency and found them to differ in the degree towhich they suffered from the deficiency, in terms of total dryweight produced in comparison with the growth of K-sufficientcontrols. The most recent of the few reports on this subject arethose by Gabelman (4) and Gerloff (5).What is new in the present investigation is the deliberate

induction of mutations by means of a mutagen, followed byselection of K-inefficient genotypes. This approach has led to theisolation of a mutant of unprecedented inefficiency in K utiliza-tion. Only at a K concentration in the medium as high as 20 mmcould the development of pronounced K deficiency symptoms beprevented in this mutant-a concentration two orders of magni-tude higher than is needed for entirely normal development of thewild type VF 36 from which it was derived.

It is plain from these results that in the present instance, nocorrelation exists between K deficiency in terms of tissue analysis,on the one hand, and K deficiency in a functional sense, on theother. The former did not exist in any of the plants; on thecontrary, levels of K in all samples of all plants were in the rangeof luxury consumption. Yet functional K deficiency as manifestedby characteristic foliar symptoms appeared in the mutants soonafter their transfer to the low K solution. It is as though exposureto K concentrations on the order of 0.1 to 0.2 mm caused the rootsto send a signal, possibly hormonal, to the leaves, making themrespond as they do to K deficiency, perhaps by perturbations inamine metabolism (9, 1 1); or possibly in the mutant a K-activatedenzyme or enzymes require inordinately high K concentrationsfor activation.

Finally, an attractive hypothesis is that the leaf cells of themutant sequester K with unusual efficiency in the vacuoles, ren-dering the cytoplasm K-deficient except under conditions of anabnormally high K supply. Very efficient sequestration of ions invacuoles has been invoked as the mechanism whereby Na and Cl

contribute to the osmotic adjustment of leaf cells of halophyteswithout poisoning the metabolic machinery of the cytoplasm; seereference 3 for review. A similar instance involving the nitrate ionhas been discussed by Shaner and Boyer (10, and refs. quotedtherein). Nitrate sequestered in a large storage pool in leaf cells,presumably in the vacuoles, was ineffective in the induction ofnitrate reductase activity. The present findings parallel thoseconcerning salt and nitrate in suggesting such effective sequestra-tion of K in the vacuoles as to deplete the cytoplasm, unless anexceedingly high concentration of K in the medium causes anadequate flux of it from the xylem into the cytoplasm.Whatever the ultimate explanation, two conclusions clearly

emerge. First, the marked differences in the efficiency of Kutilization observed make it necessary to use caution in applyingtissue analysis of a nutrient as an index of nutritional adequacy.It is likely in view ofthe present and earlier results that conclusionsdrawn on the basis of work with one genotype of a species maynot be valid for another genotype of the same species. Second,induction of mutations by mutagens followed by appropriateselection can lead to the isolation of genotypes of higher plantswith inborn errors of mineral metabolism. Such genotypes maybecome useful tools for studying various aspects of mineral plantnutrition.

Acknowledgments-I thank D. L. Fredrickson and J. D. Norlyn for both assistance and helpfuldiscussion, and J. Quick and the staff of the Cooperative Extension Laboratory, Davis, forperforming analyses of culture solutions and tissues.

LITERATURE CITED

1. EPSTEIN E 1972 Mineral Nutrition of Plants: Principles and Perspectives. John Wiley & Sons,New York

2. EVANS HJ, RA WILDES 1971 Potassium and its role in enzyme activation. Proceedings of the8th Colloquium Intemational Potash Institute, Beme, pp 13-3913-39

3. FLOWERS TJ, PF TROKE, AR YEO The mechanism of salt tolerance in halophytes. Annu RevPlant Physiol 28: 89-121

4. GABELMAN WH 1977 Genetic potentials in nitrogen, phosphorus, and potassium efficiency. InMi Wright, ed, Proceedings of the Workshop on Plant Adaptation to Mineral Stress inProblem Soils. Comell University Press, Ithaca, pp 205-212

5. GERLOFF GC 1977 Plant efficiencies in the use of nitrogen, phosphorus, and potassium. In MiWright, ed, Proceedings of the Workshop on Plant Adaptation to Mineral Stress in ProblemSoils. Comell University Press, Ithaca, pp 161-173

6. HARVEY PH 1939 Hereditary variation in plant nutrition. Genetics 24: 437-4617. JOOSTE JH, E EPSTEIN 1977 A potassium-inefficient mutant of the tomato, Lycopersicon

esculentum. Plant Physiol 59: S-698. REISENAUER HM, ed 1976 Soil and Plant-Tissue Testing in Califomia. Division of Agricultural

Sciences, University of California Bull 18799. RICHARDS FJ, RG COLEMAN 1952 Occurrence of putrescine in potassium-deficient barley.

Nature 170: 46010. SHANER DL, JS Boyer 1976 Nitrate reductase activity in maize (Zea mays L.) leaves. I.

. Regulation by nitrate flux. Plant Physiol 58: 499-50411. SMITH TA 1970 The biosynthesis and metabolism of putrescine in higher plants. Ann NY

Acad Sci 171: 988-100112. SUELTER CH 1970 Enzymes activated by monovalent cations. Science 168: 789-795

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