Sphingoid base composition of monoglucosylceramide in Brassicaceae

• JOURNAL OF • PLANT PHYSIOLOGY

J. Plant Physiol. 157. 453-456 (2000) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

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Short Communication

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Sphingoid base composition of monoglucosylceramide in Brassicaceae Hiroyuki Imai*, Yasuaki Morimoto, Kentaro Tamura Department of Biology, Konan University, 8-9-1 Okamoto, Higashinadu-ku, Kobe 658-8501, Japan

Received April 5, 2000 . Accepted June 6, 2000

Summary The sphingoid base composition of monoglucosylceramide was examined in leaves and roots of six species of the family Brassicaceae. The major components of the nine sphingoid bases we detected were 4-hydroxy-8-sphingenine [i.e. t18: 1(8E) and (8Z)] and 8-sphingenine, in both leaves and roots. The composition was unique because the relative proportions of 8-E/Z isomers in 4-hydroxy-8-sphingenine and in 8-sphingenine were different. So far, only chilling-resistant plants have been found to contain more t18: 1 (8Z) components than t18: 1(8E) [Imai et al. (1997) Biosci Biotechnol Biochem 61: 351-353]. Species of the family Brassicaceae can now be added to this list. Key words: Arabidopsis thaliana - Brassicaceae - cerebroside -long chain base - sphingolipid. Abbreviations: MGC monoglucosylceramide. - t18: 0 4-hydroxysphinganine. - t18: 1(8E) or (8Z) 4-hydroxy-8-E- or 4-hydroxy-8-Z-sphingenine. - d 18: 0 sphinganine. - d 18: 1(8E) or (8Z) 8-E- or 8-Z-sphingenine. - d18: 1(4E) 4-E-sphingenine. - d18: 2(4E,8E) or (4E,8Z) 4-E 8-E- or 4-E 8-Z-

sphingadienine.

Introduction Monoglucosylceramide (MGC) is localized in plasma membranes and tonoplasts as a major lipid class (Yoshida and Uemura 1986). It has been suggested that MGC may act in a plant's physiological reaction to low temperature, a response noted in the chilling sensitivity of mung bean hypocotyls (Yoshida et al. 1988) and the cryostability of rye plasma membrane (Lynch and Steponkus 1987). In 1988, we found, using * E-mail correspondingauthor:[email protected]

differential scanning calorimetry, that MGC species having 8-Z-unsaturated sphingoid bases had a lower phase transition temperature than those having 8-E-forms (Ohnishi et al. 1988). Continuing this research, we previously speculated that the degree of 8-Z-unsaturation of the sphingoid base would be different between chilling-resistant and chilling-sensitive plants. Although we have recently discovered that the degree of 8-Z-unsaturation in leaf MGC is not correlated with the chilling sensitivity of higher plants, we found that chillingresistant plants had more t18 : 1 (8Z) components (Fig. 1 a) than t18: 1(8E) (Fig. 1 b) (Imai et al. 1997). 0176-1617/00/157/04-453 $ 15.00/0

454

Hiroyuki Imai, Yasuaki Morimoto, Kentaro Tamura

a

Methods

JHH'O,.f" HO

H~

OH

R

OH

b

H l~$-f°

H

HN

OH

I

OH

R

Figure 1. Structures of MGC containing (a) 4-hydroxy-8-Z-sphingenine [t18 . 1(8Z)] and (b) 4-hydroxy-8-E-sphingenine [t18. 1(8£)]. R represents particularly C16 to C26 2-hydroxy fatty acid residues.

Other researchers have suggested that MGC species combining 4-hydroxy-8-sphingenines [i .e. t18: 1 (8 E) and (8 Z)] and 2-hydroxy fatty acids with very long chains, such as C 22 , C 24 , and C 26 , would set the phase transition temperature in a liposome system of unsaturated phospholipids (Yoshida et al. 1988). In addition, we recently found that the relative proportions of 8-E/Z isomers of 4-hydroxy-8-sphingenines in grapevine leaves differed among the species in respect to freezing tolerance (Kawaguchi et al. 2000). Since we know that the 4-hydroxy bases are usually bonded to very long chain 2-hydroxy fatty acids, we now think that 8-Z- and 8-E-geometrical isomerism of the 4-hydroxy sphingoid bases would be involved in a mechanism of resistance to low temperature. Here, we have analyzed the sphingoid base composition of MGC of the family Brassicaceae to determine the overall profile of MGC. No paper has previously been published on the sphingoid base composition of MGC of the family Brassicaceae. The results were unlike those found in any other family examined to date.

Materials and Methods Plant materials Seeds of Arabidopsis thaliana (L.) Heynh. ecotype Columbia were germinated in a soil mixture (Soil for Saintpaulia, Tosho Co., Yaizu, Japan) and grown under a light/dark cycle of 8/16 h at 23 'C for 8 weeks in order to have enough rosette leaves to identify sphingoid bases, including minor components. Rosette leaves and roots were used in further experiments. Seeds of Brassica napus (L.) cv. Nourin16 were germinated in vermiculite, grown under a IighVdark cycle of 16/8 h at 23 'C for 4 weeks, and then harvested for leaves and roots. Seedlings of Raphanus sativus (L) were grown under a IighVdark cycle of 16/8h at 25 'C for 7 days. Leaves and roots were taken from the seedlings. Leaves and roots of B. oleracea (L.) var. capitata (L), B. campestris (L.) var. nippoleracea (Makino) and Eutrema wasabi (Maxim.) were harvested from plants growing in farm fields.

Lipids were extracted according to the method of Bligh and Dyer (1959). Experimental procedures for purifying and checking MGC were carried out according to the method of Fujino and Ohnishi (1983). To analyze the sphingoid base composition, MGC was hydrolyzed with 1 N HCI in 90 % methanol at 80 'C for 18 h. The reaction mixture was washed twice with n-hexane and adjusted to pH 9.6 with 6 N KOH. The component sphingoid bases were extracted twice with diethyl ether, evaporated under nitrogen , and converted to fatty aldehydes by a Nal04 oxidation method (Ohnishi et al. 1983). The resulting fatty aldehydes were analyzed by gas chromatography (GC-18A, Shimazu, Kyoto, Japan) equipped with a capillary column coated with 70% cyanopropyl polysilphenylene-siloxane of 0.251lm thickness (0.25 mm i.d. x30 m; TC-70, GL Science Inc, Tokyo, Japan) and a hydrogen flame-ionization detector. The column temperature was programmed from 140 ' C (5min hold) to 200'C at 3'C min-1. Injector and detector temperatures were maintained at 220 'C. Fatty aldehydes were identified by comparing their retention times with those of authentic standards which had been prepared from the sphingoid bases of bovine brain cerebroside (Sigma) and lettuce leaf MGC (Imai et al. 1997).

Results and Discussion Figure 2 shows the capillary gas chromatogram of fatty aldehydes converted from the component sphingoid bases of Arabidopsis leaf MGC. The separation was not only on the basis of chain length, but the 4- and 8-geometrical isomers were also resolved. The sphingoid base composition of the MGC of leaves and roots from six species of Brassicaceae is shown in Table 1. No significant differences were found among three members of the genus Brassica (i.e. B. oleracea and B. campestris are thought to be parental diploids to the allotetraploid species B. napus) , or the family Brassicaceae. The major components of the nine sphingoid bases we detected were 4-hydroxy-8-sphingenine and 8-sphingenine, in both leaves and roots. These results suggest that the production levels of each sphingoid base in MGC are virtually the same in leaves and roots. All the speCies had a higher level of t18: 1(8Z) than of t18: 1(8E) . So far, only chilling-resistant plants have been found to contain more t18: 1(8Z) components than t18 : 1(8E) (Imai et al. 1997). Species of the family Brassicaceae can now be added to this list. As a preliminary experiment, we analyzed the sphingoid bases of MGC from Arabidopsis rosette leaves grown under long-day conditions (16 h light). There was essentially no difference in the relative proportions of 8-E/Z isomers of 4-hydroxy-8-sphingenines between the leaves cultured under Short-day (8 h light) conditions and those grown under the long-day conditions. Members of the same plant families are phylogenetic ally similar on the levels of 8-Z-sphingoid bases in MGC (Imai et al. 1997). We have speculated that the molecular mechanism(s) involving thedesaturation and/or isomerization at the C-8 positions of sphingoid bases would coordinately operate between dihydroxy and trihydroxy bases , with the exception

Sphingoid bases in Brassicaceae

of Chenopodiaceae (spinach and beet). These results for Brassicaceae, however, show that the 8-Z isomers are higher in trihydroxy bases and the 8-E isomers are higher in dihydroxy bases: the level of d 18: 1(8E) was higher than that of

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Table 1. Sphingoid base composition (mol 'Yo) of MGC from Brassica-

ceae. Plant

d18: 1(8Z) (Table 1), indicating that the sphingoid base pro-

Sphingoid base t1S:0 t1S:1 t1S:1 d1S:0 d1S:1 d1S:1 d1S:1 d1S:2 d1S.2 (SZ) (4E) (4E, SE) (4E, SZ) (SE) (SZ) (SE)

files of members of the family Brassicaceae are similar to those of spinach and beet. These two members of the family Chenopodiaceae, however, have dihydroxy sphingoid bases

A thaliana

in MGC that consist chiefly of 4-E-8-sphingadienine, while di-

Leaf t Root 2

hydroxy sphingoid bases in Brassicaceae MGC consist principally of 8-sphingenine. In both regards, sphingoid base

B. napus

composition of MGC from members of the family Brassica-

Leaf Root 2

ceae was distinct from that of other families. Recently, sphingoid base analysis of total sphingolipids from A. thaliana leaves was conducted by a method of direct alkaline hydrolysis that omits a procedure for lipid extraction with organic solvents (Sperling et al. 1998). Interestingly, the data indicates that A. thaliana leaves have more t18: 1(8E) components than t18: 1(8Z). MGC has been thought to be the most abundant sphingolipid in higher plants when total lipids are extracted with a wide variety of organic solvents.

31 33

56 51

10 10

2 2

33 37

56 51

S S

2

24 39

69 46

17 27

75 67

37 33

44 54

2

S 9

3S 32

55 53

t 4

5 6

t 2

B. o/eracea

Leaf Root

t t

2

5 S

2

t 2

t 2

B. campestris

Leaf Root

t t

2

3 3

R. sativus

Leaf Root

1 1

2

3

2

t 2

0

E. wasabi

Leaf t Root 1

tiS' 0, 4-hydroxysphinganine; t1S:1 (SE) or (SZ), 4-hydroxy-S-E- or 4-hydroxy-S-Z-sphingenine; diS 0, sphinganine; diS: 1(SE) or (SZ), S-E- or S-Zsphingenine; diS: 1(4E), 4-E-sphingenine; diS: 2(4E, SE) or (4E, SZ), 4-E, S-E- or 4-E, S-Z-sphingadienine; t, trace (less than 0.5 %). The values represent the averages of four determinations from two different samples.

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The results in the present study and in that of Sperling et al. (1998), however, suggest that MGC is not the most abundant sphingolipid in A. thaliana leaves. Thus, it appears that other

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complex sphingolipids, such as phytoglycolipids (Hsieh et al. 1981), are more abundant than MGC in A. thaliana leaves. Acknowledgements. We thank Prof. M. Ohnishi for his helpful sug-

5 1 I

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r---~~------~----~

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4

8

7 8 9

4

\,f \1/

12

16

20

Retention time (min) Figure 2. Gas chromatogram of the fatty aldehydes converted from the corresponding component sphingoid bases of Arabidopsis leaf MGC. Peak numbers correspond to t18: 0 (1), t18: 1(8E) (2), t18: i(8Z) (3), d18:0 (4), d181(8E) (5), di8: i(8Z) (6), d181(4E) (7), d 18: 2(4E,8E) (8) and diS: 2(4E,SZ) (9).

gestions, and are grateful to Prof. A. Shibahara and Dr. K. Yamamoto for their help during the preparation of this manuscript. This work was supported in part by the Grant for Basic Science Research Project (970659) from The Sumitomo Foundation.

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Hiroyuki Imai, Yasuaki Morimoto, Kentaro Tamura

Lynch DV, Steponkus PL (1987) Plant Physiol83: 761-767 Ohnishi M, Imai H, Kojima M, Yoshida S, Murata N, Fujino Y, Ito S (1988) In : Proceedings of ISF-JOCS World Congress 1988, Vol II , The Japan Oil Chemists' Society, Tokyo, pp 930-935 Ohnishi M, Ito S, Fujino Y (1983) Biochim Biophys Acta 752: 416-422

Sperling P, Zahringer U, Heinz E (1998) J Bioi Chem 273: 2859028596 Yosh ida S, Uemura M (1986) Plant Physiol 82: 807-812 Yoshida S, Washio K, Kenrick J, Orr G (1988) Pl ant Cell Physiol 29: 1411-1416