Planta

Planta (1992)187:83-88

9 Springer-Verlag1992

Subcellular localization of luteolin glucuronides and related enzymes in rye mesophyll* Stephan Anhalt and Gottfried Weissenb6ck** Botanisches Institut der Universit~itzu K61n, Gyrhofstrasse 15, W-5000 K61n 41, Federal Republic of Germany Received 30 July; accepted 3 December 1991

Abstract. Vacuoles were isolated by osmotic rupture of mesophyll protoplasts from the primary leaves of 4-dand 7-d-old plants of rye (Secale cereale L.). Their content of two flavones, luteolin 7-O-[13-o-glucuronosyl(1 ~2)13-D-glucuronide] (R2) and luteolin 7-O-[13-D-glucuronosyl (1 ~2)13-D-glucuronide]-4'-O-13-D-glucuronide ( R a ) , a s well as that of three specific flavone-glucuronosyltransferases involved in their biosynthesis and of a specific ~-glucuronidase was determined in comparison to the parent protoplasts. The two flavonoids were found to be entirely located in the vacuolar fraction, together with 70% of the activity of UDP-glucuronate: luteolin 7-O-diglucuronide-4'-O-glucuronosyl-transferase (LDT; EC 2.4.1.), the third enzyme of the sequence of three transferases in the anabolic pathway. The activities of the first and second anabolic enzymes, UDP-glucuronate: luteolin 7-O-glucuronosyltransferase (LGT; EC 2.4.1.) and UDP-glucuronate: luteolin 7-O-glucuronide-glucuronosyltransferase (LMT; EC 2.4.1.) could not be found in the vacuolar fraction in appreciable amounts. The specific 13-glucuronidase (EC 3.2.1.), catalyzing the deglucuronidation of luteolin triglucuronide to luteolin diglucuronide, was present with 90% of its activity in the digestion medium after isolation of mesophyll protoplasts, indicating an apoplastic localization of this enzyme. The data presented indicate a directed anabolic and subsequent catabolic pathway for the luteolin glucuronides in the mesophyll cells of rye primary leaves. This includes two cytosolic and a last vacuolar step of glucuronidation of luteolin, and the vacuolar storage of the luteolin triglucuronide. We propose the transport of the latter into the cell wall, after which the

* Dedicated to Professor Ludwig Bergmann, Botanisches Institut der Universit~itzu K61n, on the occasion of his 65th birthday ** To whom correspondence should be addressed Abbreviations: LDT=UDP-glucuronate: luteolin 7-O-di-glucuronide-4'-O-glucuronosyltransferase;LGT = UDP-glucuronate: luteolin 7-O-glucuronosyltransferase;LMT=UDP-glucuronate: luteolin 7-O-glucuronide-glucuronosyltransferase

triglucuronide is deglucuronidated, this being the first step for further turnover. Key words: [3-Glucuronidase - Flavone-glucuronosyltransferase - Luteolin glucuronide (localization) - Secale (enzyme localization) - Vacuole

Introduction Primary leaves of rye (Secale cereale L. cv. Kustro) display a strict compartmentation of ftavonoids in different tissues and a close correlation of leaf development and flavonoid metabolism. While two C-glucosyl-apigeninO-glycosides, isovitexin 2"-O-arabinoside (R3) and isovitexin 2"-O-galactoside (R4) (Dellamonica et al. 1983) are accumulated in both epidermal tissues, two anthocyanins, cyanidin 3-O-glucoside (R0 and cyanidin 3-0gentiobioside (Ru) (Strack et al. 1982; Busch et al. 1986), and two luteolin glucuronides, luteolin 7-O-diglucuronyl-4'-O-glucuronide (R1) and luteolin 7-O-diglucuronide (R/) (Schulz et al. 1985), are located in the mesophyll (Schulz and Weissenb6ck 1986). The synthesis of the two luteolin glucuronides starting from luteolin is sequentially catalyzed by three specific UDPglucuronate: flavone-glucuronosyltransferases (Schulz and Weissenb6ck 1988b) and is assumed to be channeled (as intermediates do not appear) in the cytosol of mesophyll cells. This leads to a maximum accumulation of the luteolin triglucuronide (R1) at the fifth day of primary-leaf development; the subsequent rapid decline of this compound during further leaf development (Strack et al. 1982) is due to a specific [3-glucuronidase (Schulz and Weissenb6ck 1987), and the product is luteolin 7-O-diglucuronide (R2). As the small amount of accumulation of R2 does not corrrespond to the rapid decline of R1, this deglycosylation is supposedly only the first step of a turnover of this flavonoid, although no soluble degradation products could be detected. Assuming a vacuolar localization of both flav0noids R1 and R2, as shown for other natural plant products

84

S. Anhalt and G. Weissenbrck: Luteolin glucuronide metabolism in rye mesophyll

( H o p p et al. 1985; M a t e r n et al. 1986), a n d a cytosolic g l y c o s i d a t i o n as s u g g e s t e d b y M a t i l e (1984), we e x p e c t e d the 13-glucuronidase to be v a c u o l a r like o t h e r glyc o s i d a s e s (Boller a n d K e n d e 1979; B o u d e t et al. 1981) a n d the low a c c u m u l a t i o n o f luteolin 7 - O - d i g l u c u r o n i d e t h e r e f o r e to be o f c a t a b o l i c origin.

Material and methods Chemicals. Luteolin was purchased from Roth, Karlsruhe, FRG. Luteolin 7-O-glucuronide, luteolin 7-O-diglucuronide and luteolin 7-O-diglucuronyl-4'-O-glucuronide were isolated from suitable plants and purified to 95-99% purity (HPLC) as described by Schulz and Weissenbrck (1987). Glucose-6-phosphate, 4-nitrophenol-ct-D-mannopyranoside, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), 2-(N-morpholino)ethanesulfonic acid (Mes), diethylaminoethyl (DEAE)-Dextran and Dextran sulfate were from Sigma, Deisenhofen, FRG. NADH, NADP, UDPglucuronate, cytochrome-c and Tris were from Boehringer, Mannheim, FRG. Dowex 1 • 2 (CI-), Polyclar AT, 2-mercaptoethanol, bovine serum albumin (BSA) (fraction 5) came from Serva, Heidelberg, FRG. Ficoll-400 was purchased from Pharmacia, Freiburg, FRG, and Rohament CT and Rohament P were from R6hm, Darmstadt, FRG. Methanol was purchased from Schindler, Krln, FRG, tetrahydrofurane and acetonitrile from Baker, Deventer, Ethylendiaminetetraacetate (EDTA), sorbitol and all other solvents, acids and buffer salts were purchased from Merck, Darmstadt, FRG. Media. The following buffers were used: A) 5 mM Mes-KOH pH 5.8, 0.6 M sorbitol; B) buffer A containing 1.5% (w/v) Rohament CT (cellulase) and 0.5% (w/v) Rohament P (pectinase); C) 5 mM Mes-KOH pH 5.8, 0.3 M sorbitol, 5 mM EDTA; D) 0.1 M KPi pH 7.0, 10 mM 2-mercaptoethanol. Plant material. Caryopses of Secale cereale L. cv. Kustro were purchased from F. von Lochow Petkus, Bergen, FRG, and seedlings were grown in a phytotron as previously described (Strack et al. 1982). Primary leaves were harvested without coleoptiles according to previously determined standard lengths, 2 h after the beginning of the light period. For the determination of enzyme activities of the glucuronosyltransferases 4-d-old leaves (7.0-7.5 cm; enzyme activities at or near maxima) were collected and for investigations of the 13-glucuronidase the leaves were 7 d old (14.5-15.0cm; enzyme activity has reached its maximum). Preparation of mesophyll protoplasts. Preparation of mesophyll protoplasts was similar to the method described by Schulz and Weissenb6ck (1988a), but with a different cellulase and pectinase. After manual peeling of the lower epidermal tissue of defined sections (4-d-old primary leaves: from 1 cm to 5 cm below tip; 7-d-old leaves: 1 cm to 8 cm below tip) of 45-60 primary leaves, the leaf sections were collected on buffer A. Buffer A was exchanged for buffer B and after vacuum infiltration for 10 rain the leaf sections were incubated for 1.5 h or 2.0 h at 25 ~ C (see Results). Protoplasts were collected by sedimentation at 50"9 for 10 min and washed four times with buffer A. The resulting protoplast suspension was free of epidermal tissue or epidermal protoplasts and vascular bundles. The protoplasts were tested for viability with 0.1% neutral red (Drawert 1968) and 0.2 % Evans Blue (Gaff and Okong'o-Ogola 1971). The purity of mesophyll protoplasts was furthermore determined bytheir content of contaminating epidermal flavonoids (R3, R4), which generally was below 3 % of the parental leaf sections. The protoplasts were counted in a hemocytometer (0.2 mm depth). Isolation of vacuoles. Washed and sedimented mesophyll protoplasts (2.5 9 106) were resuspended in 1.0 ml of buffer C (0.3 M sorbitol) and kept for 120 s with soft shaking; the suspension was restabilized by addition of 1.0 ml of buffer A (0.6 M sorbitol) con-

taining 20% (w/v) Ficoll-400 (final concentration 10% FicoU, 0.45 M sorbitol) and carefully mixed. A gradient was formed by successive addition of layers, each of 2.0 ml of buffer A containing 5%, 2.5% or 0% of Ficoll-400. After centrifugation at 2000 9O for 30 min the floating vacuoles could be collected from the top of the gradient and were counted in a hemocytometer (0.2 mm depth).

Enzyme extraction. Enzyme extraction from leaf sections in buffer D was done according to Schulz and Weissenbrck (1988a). Lysis of mesophyll protoplasts and vacuoles was accomplished by freezing (liquid nitrogen) and thawing in buffer D; Dowex was added with an amount of 0.18 g, Polyclar with 0.6 g, each for 1.0 9 107 protoplasts or vacuoles, and enzyme extraction was done as for the leaves. Protein content was determined according to Bradford (1976) and chlorophyll according to Bruinsma (1961). Enzyme assays. The three UDP-glucuronate: flavone-glucuronosyltransferase activities (LGT, LMT, LDT) were assayed according to Schulz and Weissenbrck (1988b), but without 2-mercaptoethanol in the assay for the LDT; the 13-glucuronidase activity was measured according to Schulz and Weissenbrck (1987). Activities were determined by measurement of the corresponding products using HPLC (column: nucleosil 100-5-C8) with a gradient from 0-50 % acetonitrile in water (1% phosphoric acid) in 10 min, For the HPLC system see Knogge and Weissenbrck (1986), ct-Mannosidase was determined as described by Boiler and Kende (1979), glucose-6-phosphate-dehydrogenase according to Bergmeyer (1979) and NADHcytochrome-c reductase according to Hodges and Leonhard (1974). Flavonoid determination. Aliquots of tissue homogenates, protoplasts and vacuoles were taken before the addition of Dowex and Polyclar and mixed with an equal volume of methanol, centrifuged and directly injected into the HPLC-column; Ra, R2, R3 and R 4 could be separated and determined individually in a gradient of 84 % water (1% phosphoric acid), 6% methanol and 10% tetrahydrofuran to 66% water (1% phosphoric acid), 16% methanol and 18% tetrahydrofurane in 15 min.

Results Isolation and characterization o f vacuoles. T h e m e t h o d for the i s o l a t i o n o f v a c u o l e s f r o m m e s o p h y l l p r o t o p l a s t s was d e v e l o p e d a n d o p t i m i z e d using 7 - d - o l d p r i m a r y leaves o f rye (13-glucuronidase activity p e r l e a f r e a c h i n g a m a x i m u m at this stage), a n d it was f o u n d to be a p p r o p r i a t e for 4 - d - o l d leaves ( g l u c u r o n o s y l t r a n s f e r a s e s at o r n e a r their m a x i m a ) as well. O f the several k n o w n m e t h o d s for the i s o l a t i o n o f vacuoles ( W a g n e r a n d S i e g e l m a n 1975; S a u n d e r s a n d C o n n 1978; B o u d e t et al. 1981 ; H e c k et al. 1981; S c h n a b l a n d K o t t m e i e r 1984; B r a y a n d Z e e v a a r t 1985; K r e i s a n d R e i n h a r d 1985) the o s m o t i c r u p t u r e o f m e s o p h y l l p r o t o p l a s t s using a h y p o t o n i c sorbitol s o l u t i o n in M e s - b u f f e r gave the best results with r e g a r d to visual p u r i t y as o b s e r v e d in the light m i c r o scope, v a c u o l e yield a n d c o n t a m i n a t i o n o f v a c u o l e s b y p r o t o p l a s t s . V a c u o l e yield c o u l d be i m p r o v e d b y the a d d i t i o n o f 5 m M E D T A to the lysis m e d i u m , a n d i m m e d i a t e r e s t a b i l i z a t i o n b y h y p e r t o n i c buffer in the F i c o l l g r a d i e n t gave a n a v e r a g e yield o f 14% (;/-d-old leaves) a n d 9.5% (4-d-old leaves). S t a i n i n g o f p r o t o p l a s t s a n d v a c u o l e s with n e u t r a l red (final c o n c e n t r a t i o n 0.002%, w/v) used for r e c o v e r y o f v a c u o l e s d i d n o t affect the d e t e r m i n e d e n z y m e activities o r f l a v o n o i d c o n t e n L cL-Mannosidase was d e t e c t e d with 104% o f its activity in m e s o p h y l l vacuoles f r o m 7-d-old leaves ( T a b l e 1) a n d

s. Anhalt and G. Weissenb6ck: Luteolin glucuronide metabolism in rye mesophyll was therefore chosen as vacuolar marker. In 4-d-old leaves, however, only 29 % of the activity of this enzyme was in the vacuoles - results similar to those of e.g. Schnabl and Kottmeier (1984) and Le-Quoc et al. (1987) - pointing to a major presence o f this enzyme outside the vacuole (Table 2). Therefore, the 4-d-old vacuoles from rye were not atypical. Glucose-6-phosphate dehydrogenase as cytosolic marker with approx. 8.5% and N A D H cytochrome-c reductase as E R marker with 11% o f the activity detected in vacuoles (Table 1, 2) showed a rather high degree of purity o f the rye mesophyll vacuoles, comparable to former descriptions for vacuoles from other plants (Boller and Kende 1979; H o p p et al. 1985). The epidermal flavonoids R3 and R,, which had already served as purity marker for rye mesophyll protoplasts, could not be found in the mesophyll vacuoles at all.

Determination of enzyme activities of the ~-glucuronidase, flavone-olucuronosyltransferases and flavonoid content of vacuoles. In vacuoles derived from mesophyll protoplasts of 7-d-old primary leaves, only 4.7% of the enzyme activity of [3-glucuronidase could be detected (protoplast preparation by 1.5 h incubation= 100%, Table 1). The

Table 1. Enzyme activities of mesophyll protoplasts and vacuoles from rye primary leaves (7 d old)

Enzyme

luteolin glucuronides R1 and Re were present at 93.5% and 112.3%, respectively, in these vacuoles (Table 3), indicating their vacuolar location. In vacuoles derived from mesophyll protoplasts of 4-d-old primary leaves, the enzymes L G T and L M T were present at very low activities comparable to that of the cytosolic marker, which is obviously a contaminant (Table 2). However, the activity o f L D T in the vacuole was 70% o f the activity in whole protoplasts. In a search for the other 30%, a 100 000 99 pellet o f lysed vacuoles was tested for this activity, but none could be found (data not shown). Thus, there is no evidence for this enzyme being associated with the tonoplast. A loss o f enzyme activity during vacuole isolation seems to be the reason for this result. Comparable to the results obtained with 7-d-old leaves, mesophyll vacuoles from 4-d-old leaves contained 96.4% R1 and 117.7% R2 o f the content o f the parental protoplasts (Table 3), confirming the vacuolar localization o f these luteolin glucuronides.

Detection of the ~-9lucuronidase. Compared with parent leaf sections, only about 25% o f the [3-glucuronidase activity could be recovered from mesophyll protoplasts

Enzyme activity p k a t " (10 6 protoplasts) -~ pkat" (106 vacuoles) -1

l~-Glucuronidase 0.53 ct-Mannosidase 57.6 Glucose-6-phosphate 1267.8 dehydrogenase NADH-cytochrome-c 158.3 reductase

Table 2. Enzyme activities of mesophyll protoplasts and vacuoles from rye primary leaves (4 d old)

Enzyme

4.7 103.8 8.7

18.5

11.7

5.3 8.2 70.3 28.8 7.8

18.19

10.2

Flavonoid content nmol- (10 6 protoplasts) -1 nmol'

R1

81.30 58.30 6.32 19.60 87.62 77.90

R2 (RI+R2)

% in vacuoles

1.24 6.60 19.33 15.10 88.45

Flavonoid Plant age 4d 7d 4d 7d 4d 7d

% in vacuoles

0.025 59.8 110.3

Enzyme activity pkat 9 (10 6 protoplasts) -1 pkat " (10 6 vacuoles) -1

LGT 23.4 LMT 80.5 LDT 27.5 ct-Mannosidase 52.4 Glucose-6-phosphate- 1132.5 dehydrogenase NADH-cytochrome-c 178.3 reductase

TaMe 3. Luteolin-7-O-diglucuronide (R2) and luteolin-7-O-diglucuronyl-4'-Oglucuronide (R1) contents of mesophyll protoplasts and vacuoles from rye primary leaves (4-d-old, 7-d-old)

85

78.37 54.50 7.06 22.00 85.43 76.50

(10 6

vacuoles) -1% in vacuoles 96.4 93.5 117.7 112.3 97.5 98.2

86

S. Anhalt and G. Weissenb6ck: Luteolin glucuronide metabolism in rye mesophyll

Table 4. Effect of prolonged digestion of 7-d-old leaf sections of rye on 13-glucuronidase activity of the resulting mesophyll protoplasts, and the effect on this activity of storage of the washed protoplasts Storage time (4~ C) of protoplasts in washing buffer after digestion at 25~ C (min)

fI-Glucuronidase (pkat 9(106 protoplasts)-1) after digestion of leaf sections for: 1.5 h

2.0 h

0 60 120

0.430 0.415 0.393

0.052 0.051 0.048

chlorophyll, respectively (protein and chlorophyll recovery 90% each). However, only 20% each of total protein and total chlorophyll, derived from lysed protoplasts and apoplastic proteins, could be detected in the digestion medium after protoplasts isolation. In conclusion, this specific distribution of the 13glucuronidase activity strongly points to an apoplastic localization of this enzyme in the mesophyll of rye prim a r y leaves.

Discussion

of rye primary leaves (7-d-old leaves, 1.5 h incubation) and only insignificantly low amounts of activity of this enzyme were found in mesophyll vacuoles (Table 1). Prolonged incubation (2.0 h) during the isolation of mesophyll protoplasts (7-d-old leaves) reduced the activity of [3-glucuronidase of protoplasts dramatically (Table 4). However, the remaining low activity of the washed protoplasts remained nearly constant during storage of the protoplasts, indicating that the loss of ]3-glucuronidase activity during protoplast isolation is p r o b a b l y not due to protein degradation (Table 4). Therefore, after isolation of the mesophyll protoplasts (7-d-old leaf sections, 2.0 h incubation) the digestion medium was tested for 13-glucuronidase activity, and approx. 90% of the activity of the control leaf sections was found in the medium (Table 5). Together with approx. 8 % of 13-glucuronidase activity found in the washed mesophyll protoplasts, the recovery of this enzyme activity was 97% of the parent leaf sections (Table 5). A contaminating glucuronidase activity was present in the digestion enzymes; its activity was a b o u t 12% of that of the incubated leaf sections, and was subtracted for the calculation (Table 5). The sedimented protoplasts were intact, as they contained approx. 70% of total protein and total

Schulz and Weissenb6ck (1988b) proposed a channeled and cytosolic synthesis of the major flavonoid of rye mesophyll, luteolin 7-O-diglucuronyl-4'-O-glucuronide, since the three flavone-glucuronosyltransferases were highly soluble and no intermediates of the sequence could be found. The luteolin 7-O-diglucuronide, accumulating at a slow rate and in a far smaller a m o u n t than the degradation of the luteolin triglucuronide would have produced, was assumed to be the product of the 13glucuronidase-catalyzed reaction. This enzyme was expected to be vacuolar, as indicated by its acidic p H o p t i m u m of 4.3, its high solubility (Schulz and Weissenb6ck 1987) and the general hydrolytic character o f vacuoles (Matile 1975; Butcher et al. 1977; M a r t y et al. 1980), especially with regard to glycosidases (Nichimura and Beevers 1978). However, our data for the 13glucuronidase are comparable to those for the coumarinyl [3-glucosidase in the mesophyll of Melilotus alba (Oba et al. 1981), which was localized in the apoplast as well. The localization of the 13-glucuronidase in the apoplastic space maintains the view that the vacuolar luteolin diglucuronide is of anabolic origin. Additionally, the recovery of 70% of the enzyme activity of L D T in the vacuoles (4-d-old leaves) can explain the presence of both luteolin glucuronides in the vacuole and the minor accu-

Table 5. Recovery of activity of the fl-glucuronidase in the digestion medium after isolation of protoplasts (2 h incubation) from 7-d-old primary leaves of rye. The cell-wall-digesting enzyme preparation

contained a contaminating glucuronidase, which had approx. 12% of the activity of the incubated leaf sections. 13-glur= 13-glucuronidase

Parameter

Control, intact leaf section Medium after digestion Protoplasts (washed) Contamination by digestion enzymes Digestion medium minus contamination Recovery: protoplasts plus net digestion medium

pkat 13-glur 9 (leaf section)- 1

% of control

Ixg protein (leaf section)- 1

% of control

Ixg chlorophyll (leaf section)- 1

% of control

2.050

100

564.8

100

59.6

100

2.080

-

534.7

-

12.9

21.6

0.160

7.8

405.8

71.8

41.3

69.3

0.245

11.9

423.1

-

0.0

0.0

1.835

89.5

111.6

19.8

12.9

21.6

1.995

97.3

517.4

91.6

54.2

90.9

S. Anhalt and G. Weissenb6ck: Luteolin glucuronide metabolism in rye mesophyll

Hoo ~ 1

oa

o.o

Luteolin

LET

UDP-g[ur -,~

UDPd

CYTOSOL

~r,.O~

' = ~ oo.

LNT

UDP"w"1 +

el b7 ~ ~l glut~O OH

OH

Luteolin 7-O-diglur

0

glut

J

OH

Luteolin 7-0-diglur

g~-o

( R2 ) 0H

LDT

0

UOP-glur~.J uop-," l gI~*r t

OH

Oi " ~ 0 - g [j~O ur~c

LUteolin 7-O-d[g[ur -4._O_glur

g~-o

( RI ) VACUOLE

t APOPLASTIC

9 g,ur--o

Lu'~eolin7-O-diglur/,'-O-glut

g~lur-O 7

glur

~

>

Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged. We thank Professor J. Willenbrink (K61n) for critical reading of the manuscript.

References

Luleo(in7-0-gtur

UDP-glur -~

gtF

87

-.~o

0

SPACE

OH H202

~x l, Polymer

Luteo(in 7-O-diglur

Fig. 1. Working scheme for the possible subcellular localization of luteolin glucuronide metabolism in the mesophyll of rye primary leaves mulation of the diglucuronide. A vacuolar localization of a sinapoylglucose" L-malate sinapoyltransferase in protoplasts f r o m cotyledons of Raphanus sativus was described by S h a r m a and Strack (1985), indicating the ability o f plant vacuoles, in principle, to perform steps in the synthesis of natural products. F r o m our data we suggest the following pathway for luteolin glucuronides in rye primary leaves (Fig. 1): Luteolin 7-O-diglucuronide is synthesized by the two transferases L G T and L M T via luteolin 7-O-glucuronide in the cytosol, and is transported into the vacuole. There, the third glucuronate moiety is linked to the 4'-position o f luteolin 7-O-diglucuronide by L D T , and the luteolin triglucuronide is stored. During further leaf development this c o m p o u n d is transported into the apoplastic space and the 4'-O-glucuronic-acid moiety is removed by the ~-glucuronidase, this being the initial step of a further turnover. At present, an apoplastic hydrogen-peroxidedependent peroxidase using luteolin diglucuronide as substrate is being characterized. T o verify this model, we are investigating the characterization of this turnover, and the immunocytochemical localization o f the 13glucuronidase and L D T , in addition to studying flavonoid transport and possible plant defense-related functions of the luteolin glucuronides.

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S. Anhalt and G. Weissenb6ck: Luteolin glucuronide metabolism in rye mesophyll

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