Heterotrophy in the Lemnaceae

J. Plant Physiol. Vol.

144. pp. 189-193 (1994)

Heterotrophy in the Lemnaceae1 HUGH FRICK Plant and Soil Sciences Department, University of Delaware, Newark, DE 19717-1303 , U.S.A. Received August 4, 1993 . Accepted January 28, 1994

Summary

Lemna minor L. and Wolffta brasiliensis Weddell can use sucrose to support heterotrophic growth in darkness and photomixotrophic growth in the light, but each is killed by galactose in the medium. Spirodela punctata (G. F. W. Meyer) Thompson growth on sucrose and galactose was indistinguishable. L. minor, Wolffia, and Spirodela plants could not utilize lactose or sorbitol; Callitriche stagnalis Scopoli could not utilize galactose or sorbitol and made only marginal use of sucrose to drive growth in light or darkness. L. minor callus and Callitriche callus, on the other hand, utilized galactose effectively, and L. minor callus also utilized sorbitol. The specific reducing sugar content was lowered in Lemna callus during heterotrophic growth on galactose, but not in Spirodela plants. The specific starch and sucrose contents were reduced in both when growing on galactose. Callitriche plants appeared to absorb galactose, but to not utilize it to drive growth, whereas the callus that did utilize galactose showed the Lemnacean pattern of diminished specific starch and sucrose pools. Catabolic sucrose synthase (EC 2.4.1.13) activity diminished during growth in darkness in all cases, consistent with an increase in the specific sucrose content. Invertase (EC 3.2.1.26) activity, alkaline and acidic, diminished in darkness (excepting heterotrophic Spirodela plants), consistent with the large increases in specific reducing sugar content being due to diminished alkaline invertase activity. L. minor callus not only showed lowered invertase and sucrose synthetase activities when growing on galactose or sorbitol, but also had lowered reducing sugar, sucrose, and starch contents. The enzymology of exogen?us lactose, galactose, and sorbitol utilization is open to study with respect to primary carbon partitionmg. Key words: Lemnaceae, Callitriche, heterotrophy, galactose metabolism.

Introduction

There are now many examples of gene expression differences, and polypeptide differences, between tissues of the same plant representing various stages of specialization, and between differentiated plants and de-differentiated callus/ cell cultures (Goldberg et aI., 1989; Kiyosue et aI., 1991; Nomura and Komamine, 1986; Ramagopal, 1989; Wink, 1989). Intact Lemna minor L. plants and their de-differentiated callus differ in the capability to utilize exogenous carbohydrates to support heterotrophic growth (Frick, 1991). Their I Published as miscellaneous paper 1500 of the Delaware Agricultural Experiment Station.

© 1994 by Gustav F ischer VerJag, Stuttgart

heterotrophic capabilities seem traceable to specific enzymology and may thereby permit a functional analysis of gene expression differences between the plant and its callus. There exist particular advantages in studying galactose-based heterotrophy in the Lemnaceae since preliminary evidence shows that Lemna minor callus can grow well on lactose, and can convert galactose to UDP glucose/glucose-1-phosphate using both Leloir and Isselbacher pathways (Frick and Morley, 1994). It is first necessary to establish the differences between sucrose- and galactose-based heterotrophy in terms of carbohydrate storage pools and soluble sugar pools so that enzyme activity differences can be interpreted. The Lemnaceae are a family of aquatic monocotyledons that show an apparent continuum of morphological simplifi-

190

HUGH FRICK

cation from Spirodela through Lemna to Wolffta and Wolf fiella (Arber, 1920; Hillman, 1961; Landolt, 1986; Sculthorpe, 1967). Lemna minor L. has found wide use in aseptic culture because of its steady-state vegetative growth and capacity to utilize sucrose to drive heterotrophic growth in darkness. In this report, comparative data for a yellow mutant of Lemna gibba L. (an obligate heterotroph) are included, as well as for an aquatic dicotyledon Callitriche stagnalis. The report focuses on the consequences of sucroseversus galactose-based heterotrophy to the specific contents of reducing sugars, sucrose, starch, and the specific activities of invertase and sucrose synthetase.

Materials and Methods

The Lemna minor L. clone was that previously employed (Frick, 1991); the yellow mutant of Lemna gibba L. was a gift of Janet Slovin (USDA-ARS, Beltsville, MD); Spirodela punctata (G. F. W. Meyer) C. H. Thompson, Wo/jfol brasiliensis Weddell and Calli· triche stagnalis Scopoli were isolated locally (Gleason and Cronquist, 1991; Landolt, 1986). Vouchers were deposited in DOV (Claude E. Phillips Herbarium, Delaware State University, Dover, DE). Plants were grown phototrophically in cool-white fluorescent + incandescent light (600 I1mol· m- 2 • S-1 PPF), on liquid basal salts F-medium (Frick, 1991) buffered at pH 5 with 20mM succinic acid, and growth was estimated as change in frond number (or frond weight), ..lFN, = (logFN, - 10gFNo) 1000, where FN is daily frond number; doubling time in this formulation is 301/..lFN" days. F-medium was supplemented with 2 % sucrose and 111M N 6(2-isopentenyl adenine) [2ip] for photomixotrophic growth in light or heterotrophic growth in darkness. Lemna minor callus was grown on F-medium + 2 % sucrose + an organic mixture (Frick, 1991) + 111m 2ip and 9 11m 2,4-dichlorophenoxy acetic acid [2,4-D]. Callitriche plants and callus were grown under identical conditions except for the absence of succinic acid from the medium and the use of NO) --nitrogen only. All carbohydrate supplements were at 2 %

Results

The utilization of exogenous carbohydrate supplements by photosynthetic Lemnaceae is unequivocal only when the initially green plants can use the carbohydrate to drive heterotrophic growth in darkness. Lemna minor and Woljfia brasiliensis are killed by galactose (Table 1 and Frick, 1991) but can use sucrose to support heterotrophic growth in darkness and photomixotrophic growth in the light (Table 1). Spirodela punctata growth on sucrose and galactose was indistinguishable in either light or darkness. L. minor, Woljfia, and Spirodela plants were unable to utilize lactose or sorbitol, and each plant was sensitive to sorbitol in the light (Table 1). The yellow mutant of Lemna gibba L. (Slovin and Cohen, 1988) and Callitriche stagnalis were also unable as intact plants to utilize galactose or sorbitol (Table 2). Callitriche made only marginal use of sucrose to drive growth in light or darkness, and neither plant was harmed by galactose. Whereas L. minor and Callitriche plants did not utilize galactose, their calli utilized it effectively (Table 2); L. minor callus also utilized sorbitol. (No callus yet exists of Woljfia or Spirodela, nor of the L. gibba mutant). The protein content of phototrophic plants of the Lemna· ceae did not differ strikingly from that of photomixotrophic Table 1: Phototrophic, heterotrophic, and mixotrophic growth of Lemnaceae plants.

Plant

Lemna minor

CWIV).

Plant and callus were hand-homogenized in cold 50 mM HEPES/ NaOH (PH 7.5), 1 mM N<4EDTA, 5 mM MgCb, 0.05 % (V IV) Triton X-100, 2 % polyvinylpolypyrollidone (PVPP), and 1 mM phenylmethylsulfonyl-fluoride (PMSF), and centrifuged to clarity. The pellet was digested with a-amylase + amyloglucosidase at pH 4.5 and 55 DC overnight, and reducing sugars were assayed colorimetrically after Lever (1972; p-hydroxybenzoic acid hydrazide) to estimate starch content. The supernatant was sequentially sampled in triplicate to estimate protein (Bradford, 1976), inorganic phosphate (Forbush, 1983) reducing sugars (Lever, 1972), sucrose (Monsigny et al., 1988) invertase (EC 3.2.1.26) activity at pH 8 and pH 4.5 in 35 mM Mcilvaine's buffer and 9.5 mM sucrose (Mcilvaine, 1921), and catabolic sucrose synthase (EC 2.4.1.13) activity in 28 mM HEPES (PH 6.6), 14 mM UDP and 11 mM sucrose (Sowokinos and Varns, 1992). Enzyme activities were measured in stopped assays at 37 DC for 15 or 30 min and reducing sugars estimated as glucose equivalents (Lever, 1972). Corrections were made where appropriate for endogenous reducing sugar content in the other assays. For each treatment to each plant, three replicates were harvested and triplicate samples from each homogenate assayed. Samples stored at -20 DC were assayed within 72 h of harvest. Tables 2, 3 and 4 present means ± S.D. for each estimate in a typical experiment. Logistics dictated that all plants could not be harvested, nor treatments administered, simultaneously; several experiments were performed three times, typical data are presented.

Wolffia brasiliensis

Growth Rate (6FN,)· DD LL Steady-state Steady-state slope slope

Relevant Medium Component b

(ill)

(%)

(ill)

(%)

basal sucrose galactose

14.4 43.7 28.2

lactose

10.0

sorbitol

8.9

33 - basal 100 ... sucrose 64 ... galactose sucrose 23'" lactose sucrose 20 ... sorbitol sucrose

103.8 137.7 78.9 145.6 150.9 176.8 112.7 171.3

75 100 57 106 110 128 82 124

basal sucrose galactose

46.6 91.3 3.2

lactose

15.9

83 117.2 141.5 lOa [dead] (dead] 86 122.5

sorbitol

39.0

51 - basal 100 ... sucrose 4 ... galactose sucrose 50 - lactose sucrose 43 ... sorbitol sucrose

Spirodela punctata

basal sucrose galactose

22.9 48.9 48.7

lactose

18.5

sorbitol

10.0

47'" basal 100'" sucrose 100 - galactose sucrose 38'" lactose sucrose 21 - sorbitol sucrose

126.4 158.5

89 112

111.9 97.8 124.6 118.2 106.1

114 100 127 121 108

85.9 116.3

88 119

• 6FN, - (log FN,.log FNo) 1000, where FN is daily frond number in con-

b

tinuous darkness, DD, or continuous light, LL. Twenty replicate cultures were incubated in darkness for 5 -7 days (with -10 sec!day exposure to 0.03I-1mol· m- 2 • S-l green light exposure during counting), then pruned to convenient numbers for a further 5 -7 day incubation in continuous light, 10 replicates with and 10 without a shift back to sucrose as carbohydrate supplement. Each slope, ill, was calculated during steady-state growth according to l>.FN. The arrow represents transfer from DD to LL. All carbohydrate supplements at 2 % (W IV).

191

Heterotrophy in the Lemnaceae Table 2: Growth, and protein and inorganic phosphate contents in Lemnaceae and Callitriche.

Lemna minor

plant

Lemna minor

Wo/jfta brasiliensis Spirodela punctata

Protein (g protein' kg fw-I) 18h:6h, L:D DD

Growth Rate (l'.FW,)a Ish : 6h, L:D DD

Relevant Medium Component

0 29 ±

11.2 ± 0.5 16.4 ± O.S

20.1 ± 1.3 16.6 ± 1.8

basal sucrose

57 ± 11 92 ± 9

callus

sucrose galactose sorbitol

30± 7 42 ± 7 24± 3

plant

basal sucrose

109± 3 113± 5

0 58± 2

13.3 ± 0 .2 12.7 ± 0.2

basal sucrose galactose

41 ± 13 113 ± 9 100 ± 16

0 57 ± 11 50± 6

12.6 ± 0 .1 14.9 ± 1.5 18.1 ± 0.6

9.0 ± 1.1 8.5± 1.7

62± 4

21 ±

9

20.0 ± 1.5

15.1 ± 0.7

0 27±20 9± 6

3.7 ± 0.6 4.5 ±0.6 4.7 ± 0 .6

1.2 ± 0.2 5.9 ±0.3 4.2±0.5

plant

Lemna gibba (yellow) plant

basal, (12)b sucrose, (105) galactose, (23) sorbitol, (8)

Callitriche stagnalis

plant

basal sucrose galactose

41 ± 16 60±25 48 ±29

Callitriche stagnalis

callus

sucrose galactose sorbitol

21 ± 2 22 ± 2 2± 1

2

13.3 ± 1.0 11.5 ± 0.7 14.3 ± 2.2 6.0 ± 0.3

4.1 ± 0.2 3.9±Ll

Inorganic phosphate (mmol Pi (kg protein)-I) ISh:6h,L:D DD 3308 ± 220 2307± 88 282± 64± 265 ±

35 9 51

383 ± 562 ±

67 63

2233 ± 268 2420± 82

1252 ±

82

1559 ± 71 1133± 193 1002± 75

2596± 166 2203 ± 216

1394±

1032 ±

32

7203 ± 1200 4909 ± 1012 4713 ± 249 599 ± 715 ±

76

4283 ± 1262 4645 ± 689 5832 ± 961

25 46

, l'.FW, = (log FW,-log FWo)1000, where FW is fresh weight, mg, in an 18h: 6h, light: dark regime, or in continuous darkness, DD, ± standard deviation of the mean for triplicate estimates of 3 samples. All calli were grown in the light. b Data in parentheses are the steady-state slopes for l'.FN, rather than the end-point l'.FW,. Table 3: Reducing sugars, starch, and sucrose contents of Lemnaceae and Callitriche. Reducing Sugars (g glc . kg protein -I) 18h : 6h, L:D DD

Lemna minor

plant

basal sucrose

100± 24 190 ± 17

Lemna minor

callus

sucrose galactose sorbitol

404± 34 160 ± 18 54± 2

Wol./fia brasiliensis

plant

basal sucrose

3± 4 219 ± 12

basal sucrose galactose

Lemma gibba (yellow) plant

sucrose

Callitriche stagnalis

plant

Callitriche stagnalis

callus

Spirodela punctata

plant

Starch (g glc . kg protein -I) 18h : 6h, L:D DD

88 ± 16 2922 ± 147

199 ± 3 199 ± 20

4276 ±370

784 ± 173 180 ± 18 255 ± 17 4135± 214

16 ± 12 126 ± 36

61 ± 7 27l± 26 256± 18 104 ±

Sucrose (g glc . kg protein -I) 18h:6h, L:D DD 234 ± 10 355± 20

7366± 960

1106± 96 297± 18 326± 46 2125± 127

128 ± 20 203 ± 9

5501 ± 847

6940±248 6376 ± 836

156 ± 12 337 ± 82 176 ± 16

5071 ± 238 4476± 82

207± 19 393 ± 49 304 ± 30

1489 ± 186 490± 46

7

3998 ± 421

109 ±

6

4497 ± 201

142 ±

8121 ± 1077

basal sucrose galactose

243 ± 24 2322 ± 289 3072 ± 312

137± 19 1578 ± 92 2323 ± 30

271 ± 107 559 ± 193 563 ± 37

180 ± 71 269 ± 63 293± 17

sucrose galactose

825 ± 64 926 ± 24

(18h:6h, light:dark regime) or heterotrophic (DD) plants (Table 2). Nor did calli of L. minor and Callitriche differ when grown on alternative carbohydrate supplements. The calli accumulated far less inorganic phosphate (Pi) than the corresponding intact plants, and L. minor callus using galactose accumulated very little Pi. Callitriche plants accumulated even more Pi than any Lemnaceae studied.

1263 ± 24 988 ± 2

3

265 ± 157 425 ± 73 260 ± 159

42± 3 554± 42 572± 176

3082 ± 522 1790 ± 287

The accumulation of reducing sugars in the light was modest compared to accumulation in darkness (Table 3). The specific reducing sugar content was lowered in Lemna callus during heterotrophic growth on galactose or sorbitol but not in Spirodela plants grown photomixotrophically on galactose. The specific reducing sugar content of Callitriche plants was 100times greater on sucrose or galactose supple-

192

HUGH FRICK

ment, and even though the plants did not utilize galactose to drive heterotrophic growth (Table 2) the reducing sugar content was increased in its presence (Table 3). The specific sucrose and starch contents of plants on sucrose were enhanced compared to these pools in phototrophic plants (Table 3). On galactose medium, however, both sucrose and starch pools were reduced during heterotrophic growth of L. minor callus and Spirodela plants. Thus, galactose could support heterotrophic growth in L. minor callus and Spirodela plants but did not support the standard sucrose or starch pool at the same time. In the case of Spiro· dela plants, the diminished sucrose and starch pools were not due to a diminished reducing sugar content, though that might serve as an explanation in L. minor callus (Table 3). The high levels of reducing sugars in Callitriche plants growing on galactose were associated with roughly equivalent amounts of sucrose and starch in light or darkness, but the callus pools of sucrose and starch were diminished during heterotrophic growth on galactose (Table3). Invertase activities, alkaline and acidic, diminished during growth in darkness, with the exception of acidic invertase of Spirodela plants growing heterotrophically on sucrose or galactose (Table 4). Sucrose synthase activity diminished in darkness in all cases. This is consistent, at least, with the observation that the specific sucrose content greatly increased in darkness, and consistent with the large increases in specific reducing sugar content in plants in darkness resulting from invertase activity, not sucrose synthase activity. Note that L. minor callus had greatly lowered invertase and sucrose synthase activities when growing on galactose or sorbitol, and also had lowered reducing sugar, sucrose, and starch contents (Table 4). Callitriche plants showed the same pattern of diminished alkaline invertase and sucrose synthase activities in darkness, but the acidic invertase activity did not diminish. The spe-

cific sucrose content of these plants did not greatly increase in darkness as it did in heterotrophic Lemnaceae, nor did the specific reducing sugar content increase in darkness in these plants. Note that in contrast to the Lemnaceae, Callitriche callus did not have lowered invertase and sucrose synthase activities when growing heterotrophically on galactose, nor did the callus have lowered reducing sugars - but did have lowered sucrose and starch contents. Discussion

The Lemnaceae appear to be a variable family with regard to the carbohydrate capable of supporting heterotrophic growth, with galactose utilization by intact plants distinguishing Spirodela and all other plants studied. Nevertheless, the plants that cannot utilize galactose retain the gene(s) to do so, demonstrated here for L. minor callus (and for Calli· triche call us). The pool sizes of reducing sugars, sucrose, and starch were much larger during Lemnacean heterotrophic growth in darkness, whatever the sugar source, but were not larger during the weakly heterotrophic growth of the dicot Callitriche stagnalis. Invertase and sucrose synthase activities decreased greatly in darkness, except for acid invertase in Spirodela plants. This could account for pool size increases of sucrose and starch, but not of reducing sugars. The same sort of decreases occurred in L. minor callus, but not during galactose heterotrophy in Callitriche callus. On the basis of these data, it appears that L. minor callus is able to grow as well on galactose or sorbitol as on sucrose, but only with diminished pool sizes of reducing sugars, sucrose, and starch, and lowered specific activities of acid and alkaline invertases, and sucrose synthase. This raises the

Table 4: Invertase and sucrose synthase activities in Lemnaceae and Callitriche. Invertase (g glucose· kg protein-I. h- I) pH 4.5 pH 8.0 DD 18h:6h, L:D DD 18h:6h, L:D

Sucrose Synthase (g glc· kg protein-I) 18h:6h, L:D

Lemna minor

plant

basal sucrose

Lemna minor

callus

sucrose galactose sorbitol

3320 ± 888 300 ± 64 1136 ± 168

556 ± 124 272 ± 48 320 ± 48

2032 ± 244 920 ± 48 1132 ± 112

Wolffza brasiliensis

plant

basal sucrose

1226± 144 1168 ± 40

692 ± 60 836 ± 68

1498 ± 266 1122 ± 34

Spirodela punctata

plant

basal sucrose galactose

2847± 34 3402 ±760 3476 ± 97

sucrose

3086± 120

basal sucrose galactose

2938 ± 186 4618 ± 278 3484 ± 248

sucrose galactose

712 ± 184 1032 ± 72

Lemna gibba (yellow) plant Callitriche stagnalis plant Callitriche stagnalis

callus

4363 ± 154 5712 ±219

304± 55 382± 170

7552± 1488 18132 ± 316 0 3588 ± 856 5084± 864

424 ± 35 480± 20

649± 10 950± 66 977± 52 518 ± 26 1420 ± 230 1056 ± 176 328 ± 298 2952 ±772 2688 ± 168

23 ± 11 0

63 ±56 42±55 0 81 ± 63 0 0

1336 ± 91 1217 ± 100

1053 ± 27 1305 ± 116 1133±120 1442 ± 26 3266± 376 3136 ± 234 2794 ± 580 2936 ± 360 3768 ± 560

DD 168 ± 57 90± 46

0 428 ± 182 796 ± 70 0 445± 72 1050 ± 230

Heterotrophy in the Lemnaceae

question of whether growth is possible on galactose or sorbitol only with exhaustion of carbohydrate storage pools. The metabolic disposition of galactose, for example, would include these pools through UDP galactose and/or glucoseI-phosphate. The following attempts to show that calli adapted to galactose or sorbitol are able to convert these carbohydrates to readily respirable substrates not including these other pools. Thirteen to 16% of Lemnacean sugars are galactose (Amado, 1980, cited in Landolt and Kandeler, 1987). DeKock et al. (1979) reported that Lemna gibba pectins were 22 % galactose, and hemicelluloses 11 %. Allowing for some inexactitude, this suggests that the soluble galactose pool could be relatively small in plants growing on galactose. Starting with estimates of reducing sugars plus sucrose presented here (Table 3) for calli grown on sucrose or galactose, an estimate can be reached about the quantity of galactose that must move to glycolysis in support of growth. The callus of L. minor is about 1.0 % ash (offresh weight), and its growth rate on sucrose is on the order of .lFW = 54, or 0.18d- l . Thus, 100mg of callus doubles in mass in 5.6d, and the new 100 mg mass, less 1 mg ash, would be associated with sucrose consumption, assuming no photosynthetic contribution. This is 9 mg dry weight at 90 % water content. If one takes the Growth Yield (Amthor, 1986; Thornley, 1977) to be 0.7 (i.e., 0.7 g dry weight gain per 1 g sucrose consumed), then 12.8 mg sucrose has to be removed from the growth medium for a fresh mass doubling. This is 2.28 mg per day to sustain the given growth rate, or 6.7llmol sucrose per day. Galactose-based growth must also consume this much sucrose for the growth rates to be equal (Table 2). Sucrose-grown L. minor callus contained 1510 g glc· (kg protein)-I (Table 3, reducing sugars + sucrose), and galactose-grown callus contained 457 gglc . (kg protein)-I. This is 8·4 and 2·5 mol glc· (kg protein)-I, respectively. Taking 12 g protein· (kg fw)-I as representative (Table 2), these specific glucose contents convert to 101 mol glc· (kg fw) -I and 30molglc·(kgfw)-1 for sucrose-grown and galactose-grown callus, respectively. Therefore, the pool sizes for reducing sugars + sucrose at any instant during growth which is consuming 6.7llmol sucrose· d -I appear to be adequate. The enzymology of galactose metabolism by callus growing on galactose must reflect this preferential flow of absorbed galactose in the glycolytic direction, as opposed to starch and sucrose (fructan?) pools, or cell wall and lipid components. The foregoing calculations imply a wide latitude on the part of callus in regulation of carbohydrate pool sizes during growth. The Lemnaceae system permits long-term heterotrophic growth in darkness, and mixotrophic growth in continuous light (Frick, 1991; Frick and Morley, 1994), and the direct comparison of plant and callus for metabolic capability. There are advantages to this model system in exploring the regulation of carbon partitioning at the cellular level, starting with perturbations by exogenous carbohydrate supplements. This work is focused on lactose, galactose, and sorbitol heterotrophy.

193

Acknowledgements The author thanks Elaine Eiker for the preparation of this manuscript, and Tom Pizzolato and John Frett for help in identifying Callitriche stagnalis.

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c.: