Chemical phenotypes of the hmg1 and hmg2 mutants of Arabidopsis demonstrate the in-planta role of HMG-CoA reductase in triterpene biosynthesis

Article abstract of DOI:10.1248/cpb.55.1518

Plants produce a wide variety of cyclic triterpenes, such as sterols and triterpenoids, which are the major products of the mevalonate (MVA) pathway. It is important to understand the physiological functions of HMG-CoA reductase (HMGR) because HMGR is the rate-limiting enzyme in the MVA pathway. We have previously isolated Arabidopsis mutants in HMG1 and HMG2. Although the biochemical function of HMGR2 has been thought to be almost equal to that of HMGR1, based on similarities in their sequences, the phenotypes of mutants in these genes are quite different. Whereas hmg2 shows no abnormal phenotype under normal growth conditions, hmg1 shows pleiotropic phenotypes, including dwarfing, early senescence, and male sterility. We previously postulated that the 50% decrease in the sterol content of hmg1, as compared to that in the wild type, was a cause of these phenotypes,¹⁾ but comprehensive triterpene profiles of these mutants had not yet been determined. Here, we present the triterpene profiles of hmg1 and hmg2. In contrast to hmg1, hmg2 showed a sterol content 15% lower than that of the wild type. A precise triterpenoid quantification using synthesized deuterated compounds of β-amyrin (1), α-amyrin (2), and lupeol (3) showed that the levels of triterpenoids in hmg1 and hmg2 were 65% and 25% lower than in the wild type (WT), respectively. These results demonstrate that HMGR2 as well as HMGR1 is responsible for the biosynthesis of triterpenes in spite of the lack of visible phenotypes in hmg2.

Full text of DOI:10.1248/cpb.55.1518

1518
Notes
Chem. Pharm. Bull. 55(10) 1518—1521 (2007)
Vol. 55, No. 10
Chemical Phenotypes of the hmg1 and hmg2 Mutants of Arabidopsis
Demonstrate the In-planta Role of HMG-CoA Reductase in
Triterpene Biosynthesis
a
a
b
a
,a,c
*
Kiyoshi OHYAMA, Masashi SUZUKI, Kazuo MASUDA, Shigeo YOSHIDA, and Toshiya MURANAKA
a RIKEN Plant Science Center; Tsurumi-ku, Yokohama, Kanagawa 230–0045, Japan: b Showa Pharmaceutical University;
c
Machida, Tokyo 194–8543, Japan: and Kihara Institute for Biological Research, Yokohama City University; Yokohama,
Kanagawa 244–0813, Japan. Received April 13, 2007; accepted July 9, 2007; published online July 18, 2007
Plants produce a wide variety of cyclic triterpenes, such as sterols and triterpenoids, which are the major
products of the mevalonate (MVA) pathway. It is important to understand the physiological functions of HMG-
CoA reductase (HMGR) because HMGR is the rate-limiting enzyme in the MVA pathway. We have previously
isolated Arabidopsis mutants in HMG1 and HMG2. Although the biochemical function of HMGR2 has been
thought to be almost equal to that of HMGR1, based on similarities in their sequences, the phenotypes of mu-
tants in these genes are quite different. Whereas hmg2 shows no abnormal phenotype under normal growth con-
ditions, hmg1 shows pleiotropic phenotypes, including dwarfing, early senescence, and male sterility. We previ-
ously postulated that the 50% decrease in the sterol content of hmg1, as compared to that in the wild type, was a
cause of these phenotypes,1) but comprehensive triterpene profiles of these mutants had not yet been determined.
Here, we present the triterpene profiles of hmg1 and hmg2. In contrast to hmg1, hmg2 showed a sterol content
15% lower than that of the wild type. A precise triterpenoid quantification using synthesized deuterated com-
pounds of b-amyrin (1), a-amyrin (2), and lupeol (3) showed that the levels of triterpenoids in hmg1 and hmg2
were 65% and 25% lower than in the wild type (WT), respectively. These results demonstrate that HMGR2 as
well as HMGR1 is responsible for the biosynthesis of triterpenes in spite of the lack of visible phenotypes in
hmg2.
Key words HMG-CoA reductase; sterol; triterpenoid; synthesis; Arabidopsis; amyrin
The plant natural products that are derived from squalene logical activities. In particular, their glycosides are often ac-
can be divided into two groups: steroids derived from cy- tive constituents of important traditional medicines.9) Many
cloartenol (4), such as phytosterols and brassinosteroids, and studies on the biosynthesis of these compounds have focus-
triterpenoids, such as b-amyrin (1) and lupeol (3) (Fig. 1). ed on the biosynthesis of brassinosteroids from campes-
Phytosterols are highly diverse and are important for plant terol.10,11) In addition, genes encoding glycosyltransferases,
growth and development as structural components of the which are involved in steroidal saponin biosynthesis, have
plasma membrane and biosynthetic intermediates of brassi- been identified.12) However, it is not yet known how the ma-
nosteroids. Recently, lanosterol synthases were identified in jority of these compounds are regulated in response to devel-
several plants.2—4) Since this new enzyme of sterol backbone opment or environmental conditions.
synthesis has been identified, the known diversity of phytos-
Plants biosynthesize isopentenyl diphosphate (IPP) and its
terols has increased even more. Triterpenoids are plant-char- isomer dimethylallyl diphosphate (DMAPP), common pre-
acteristic isoprenoids; they also form a large, structurally di- cursors of isoprenoids, via two pathways: the cytosolic
verse group of natural products5—7) with more than 100 dif- mevalonate (MVA) pathway and the plastidic 2-C-methyl-D-
ferent carbon skeletons.8) Some have a variety of pharmaco- erythritol-4-phosphate (MEP) pathway. The metabolic flow
Fig. 1. Biosynthetic Pathways of Steroids and Triterpenoids in Plants
∗ To whom correspondence should be addressed. e-mail: muranaka@yokohama-cu.ac.jp
© 2007 Pharmaceutical Society of Japan

October 2007
1519
between these pathways has been recently reported.13—15)
Steroids and triterpenoids, which are the main MVA-derived
isoprenoidal endproducts, are biosynthesized from acetyl-
CoA at several steps. A few functional analyses of the en-
1.030 (3H, s, H3-26), 1.680 (3H, s, H3-30), 1.913 (1H, m, H-21), 2.372 (1H,
ddd, Jꢀ11.0, 11.0, 6.0 Hz, H-19), 3.186 (1H, dd, Jꢀ11.5, 5.0 Hz, H-3), 4.57
(1H, bs, H-29), 4.69 (1H, bs, H-29); 13C-NMR (CDCl3, 125 MHz) d: 14.54
(C-27), 15.36 (C-24), 15.97 (C-26), 16.11 (C-25), 18.32 (C-26), 19.30 (C-
30), 20.93 (C-11), 25.15 (C-12), 27.41 (C-2), 27.45 (C-15), 27.98 (C-23),
zymes responsible for these steps in planta have been re- 29.85 (C-21), 34.28 (C-7), 35.50 (C-16), 37.17 (C-10), 38.06 (C-13), 38.71
ported.16) As HMG-CoA reductase (HMGR) is the rate-limit-
(C-1), 38.85 (C-4), 39.91 (C-22), 40.83 (C-8), 42.76 (C-14), 42.82 (C-17),
47.99 (C-19), 48.28 (C-18), 50.44 (C-9), 55.30 (C-5), 78.98 (C-3), 109.30
(C-20), 150.96 (C-29). EI-MS 70 eV, m/z (rel. int.): 501 (7.0), 486 (1.9), 411
(1.4), 396 (2.3), 372 (4.9), 328 (1.0), 279 (4.7), 260 (3.0), 248 (2.1), 234
ing enzyme1,17,18) of the MVA pathway, many plant HMGRs
have been characterized. Two genes encoding HMGRs,
HMG119,20) and HMG2,20) exist in the Arabidopsis thaliana
(9.1), 221 (27), 206, (36), 189 (100).
Tissue Extraction and GC-MS Quantification of Triterpenoids
Freeze-dried plant tissues were extracted three times with CHCl3–MeOH
(1 : 1). [3,28,28,28-2H4]b-Amyrin (8), [28,28,28-2H3]a-amyrin (9), and
[28,28,28-2H3]lupeol (10) (4 mg gꢁ1 dry wt. in each triterpenoid) were used
as internal standards and added directly to the sample homogenate. The ex-
tracts were dried and chromatographed on a silica-gel cartridge column
(Sep-Pak® Vac 500 mg, Waters) with hexane–EtOAc (2 : 1) and CHCl3–
MeOH (1 : 1). The hexane–EtOAc eluent was dried and saponified with 1 ml
each of MeOH and 20% KOH aq. for 1 h at 80 °C. The CHCl3–MeOH eluent
was dried in a rotary evaporator. The residue and the extraction debris were
combined and hydrolyzed with 1 ml each of MeOH and 4 N HCl for 1 h at
80 °C. These reaction mixtures were then extracted three times with 2 ml of
hexane, and the combined hexane layer was evaporated to dryness. The
residue was separated on silica-gel preparative TLC plates developed twice
with hexane–EtOAc (5 : 1) to give the triterpenoid fraction. The triterpenoid
fraction was trimethylsilylated with N-methyl-N-trimethylsilyltrifluoroac-
etamide at 80 °C for 30 min and analyzed using GC-MS. GC-MS analysis
genome. The structure and expression of the Arabidopsis
HMG1 and HMG2 genes have been analyzed. To manipulate
the synthesis of phytosterols and triterpenoids, and to under-
stand the physiological roles of these compounds, we have
isolated and characterized Arabidopsis T-DNA insertion mu-
tants in HMG1 and HMG2. The hmg1 mutant shows dwarf-
ing, male sterility, and early senescence. The sterol levels in
hmg1 are approximately 50% lower than in the WT.1) In con-
trast, hmg2 shows no unusual visible phenotypes under nor-
mal growth conditions. To understand why hmg2 appears
normal, it is important that the triterpene contents in these
mutants be investigated. In this study, we determined the
metabolic profiles of sterols and triterpenoids in WT lines
and the mutants. To investigate these chemical profiles using
the same plant samples, the sterol profile of hmg1 was reana- was carried out on a mass spectrometer (JMS-AM SUN200, JEOL) con-
nected to a gas chromatograph (6890A, Agilent Technologies) with an Rtx-
5MS capillary column (15 mꢂ0.25 mm, 0.25-mM film thickness, Restek).
The analytical conditions are as follows: EI (70 eV), source temperature
lyzed. The triterpenoid levels in the WT (Wassilewskija
(WS), Columbia (Col)) and the mutants (hmg1, hmg2) were
determined by GC-MS analysis using synthetic labeled triter-
penoids as internal standards.
250 °C, injection temperature 250 °C, column temperature program: 80 °C
for 1 min, then raised to 280 °C at a rate of 10 °C/min, and held at this tem-
perature for 15 min; interface temperature 280 °C, carrier gas He, flow rate
1 ml/min, splitless injection. The levels of endogenous 1 and 2 were calcu-
lated from the peak area ratios of m/z 221 for the internal standards and m/z
218 for the endogenous compounds. The level of endogenous 3 was calcu-
lated from the peak area ratios of molecular ions for 10 and endogenous 3.
Experimental
General 1H- and 13C-NMR spectra were recorded on a JEOL Alpha-500
spectrometer in CDCl3. Analytical TLC was carried out on Merck silica-gel
60F-254 plates (0.25 mm precoated). Oleanolic acid (5), ursolic acid (6), be-
tulinic acid (7), and lupeol (3) were purchased from Sigma. b-Amyrin (1)
and a-amyrin (2) were purchased from Apin Chemicals Limited. LiAlD4
(98% D) and NaBD4 (98% D) were purchased from Acros Organics and
Aldrich, respectively. The structures of all of the endogenous triterpenoids in
Arabidopsis were confirmed by comparing their retention times in GC and
MS spectra with those of authentic samples.
Results and Discussion
Quantification of Sterols in hmg1 and hmg2 The
sterols in two-week-old seedlings of WT lines and the mu-
tants were quantified as described.1) GC chromatogram of
sterol fraction in WT (Col) was shown in Fig. 2 representa-
tively. The sterol content of the hmg2 mutant was approxi-
mately 15% lower than that of the WT (Table 1). This result
indicates that a 15% decrease in sterols does not lead to visi-
Plant Materials Seeds of Arabidopsis thaliana (L.) HEYNH. ecotypes
WS and Col, and the mutants hmg1 (WS background) and hmg2 (Col back-
ground), were germinated on agar plates containing 1X MS medium (Invit-
rogen) and 3% sucrose.
[3,28,28,28-2H4]b-Amyrin (8): 1H-NMR (CDCl3, 500 MHz) d: 0.791 (3H,
s, H3-24), 0.871 (6H, s, H3-29, -30), 0.938 (3H, s, H3-25), 0.967 (3H, s, H3- ble phenotypes. The sterol levels in hmg2 were much greater
26), 0.996 (3H, s, H3-23), 1.134 (3H, s, H3-27), 5.183 (1H, dd, Jꢀ3.5,
than those in hmg1. This result corresponds to the finding
3.5 Hz, H-12). 13C-NMR (CDCl3, 125 MHz) d: 15.48 (C-25), 15.56 (C-24),
that the expression of HMG1 is much higher than that of
16.80 (C-26), 18.38 (C-6), 23.53 (C-11), 23.70 (C-30), 25.99 (C-27), 26.16
HMG2.21) As shown in Table 1, neither the hmg2 nor the
(C-15), 26.89 (C-16), 27.14 (C-2), 28.06 (C-23), 31.09 (C-20), 32.26 (C-17),
hmg1 mutation affects the sterol composition. The HMGR
activity has no influence on the composition of sterols but af-
fects, instead, the total sterol content.
32.66 (C-7), 33.35 (C-29), 34.72 (C-21), 36.95 (C-10), 37.05 (C-22), 38.60
(C-1), 38.69 (C-4), 39.80 (C-8), 41.73 (C-14), 46.81 (C-19), 47.17 (C-18),
47.64 (C-9), 55.19 (C-5), 78.50 (t, Jꢀ21.63 Hz, C-3), 121.71 (C-12),
145.21(C-13); EI-MS 70 eV (TMS ether), m/z (rel. int.): 502 (0.7), 487 (0.3),
397 (0.3), 280 (1.1), 260 (0.4), 246 (0.8), 221 (100), 206 (34), 190 (27).
1
[28,28,28-2H3]a-Amyrin (9): H-NMR (CDCl3, 500 MHz) d: 0.793 (3H,
d, Jꢀ5.5 Hz, H3-29), 0.795 (3H, s, H3-24), 0.914 (3H, bs, H3-30), 0.956 (3H,
s, H3-25), 0.999 (3H, s, H3-23), 1.009 (3H, s, H3-26), 1.071 (3H, s, H3-27),
3.224 (1H, dd, Jꢀ11.0, 5.0 Hz), 5.17 (1H, dd, Jꢀ3.5, 3.5 Hz); 13C-NMR
(CDCl3, 125 MHz) d: 15.62 (C-24), 15.68 (C-25), 16.85 (C-26), 17.46 (C-
29), 18.36 (C-6), 21.40 (C-30), 23.27 (C-27), 23.37 (C-11), 26.63 (C-15),
27.28 (C-2), 28.06 (C-16), 28.13 (C-23), 31.25 (C-21), 32.94 (C-7), 33.53
(C-17), 36.91 (C-10), 38.78 (C-1), 38.80 (C-4), 39.62 (C-20), 39.65 (C-21),
40.02 (C-8), 41.44 (C-22), 42.09 (C-14), 47.73 (C-9), 55.19 (C-5), 59.03 (C-
18), 79.04 (C-3), 124.40 (C-12), 139.61 (C-13). EI-MS 70 eV, m/z (rel. int.):
501 (1.1), 486 (0.1), 396 (0.4), 279 (1.5), 260 (0.9), 246 (0.3), 234 (0.9), 221
(100), 206 (21), 190 (20).
Fig. 2. GC Chromatogram of Sterol Fraction in the WT (Col) of Ara-
bidopsis
[2H7]cholesterol was used as an internal standard.
1
[28,28,28-2H3]Lupeol (10): H-NMR (CDCl3, 500 MHz) d: 0.760 (3H, s,
H3-24), 0.829 (3H, s, H3-25), 0.944 (3H, s, H3-27), 0.967 (3H, s, H3-23),

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Vol. 55, No. 10
Synthesis of Deuterated Triterpenoid Internal Stan- (7), respectively, using a similar synthesis strategy. Unam-
1
dards The triterpenoid contents in hmg1 and hmg2 were biguous assignments of the H and 13C chemical shifts of 1,
determined. For standards, we synthesized deuterated triter- 2, and 3 were confirmed by 2D NMR analysis. A comparison
penoids, as stable isotope-labeled triterpenoids suitable for with the NMR and MS spectra of the parent triterpenoids
use as internal standards in mass spectrometry analysis were (1—3) confirmed the chemical structures of each synthetic
not available commercially. As b-amyrin (1) and a-amyrin compound (8—10).
(2) had been identified in Arabidopsis leaf and stem epicutic-
Quantification of Triterpenoids in hmg1 and hmg2
ular waxes,22) and 1, 2, and lupeol (3) had been identified in The triterpenoid levels in two-week-old seedlings of WT
Arabidopsis callus and rosette leaves,23) we synthesized lines (ecotype WS, Col) and the mutants were determined
deuterated triterpenoids of these compounds. The syntheses using the synthetic compounds as internal standards. The
of [2,2,3-2H3]b-amyrin24) and [28,28-2H2]b-amyrin25) have seedlings were extracted with a CHCl3–MeOH solution, and
been reported. When using isotope-labeled triterpenoids for the extracts were subjected to chromatography. Esterified and
quantitative GC-MS analysis, the position of the isotope glycosylated fractions were hydrolyzed with KOH and HCl,
label is important. Triterpenoids with a D12 double bond, respectively. All free triterpenoids were analyzed using GC-
such as 1 and 2, undergo a retro-Diels-Alder cleavage to MS after conversion to a trimethylsilyl derivative. GC chro-
form two fragment ions containing the ABC* and C*DE matogram of triterpenoid fraction in WT (Col) was shown in
rings (C* indicates the presence of a portion of ring C only) Fig. 4 representatively. The levels of endogenous 1 and 2
using electron impact ionization.26) As the most abundant were calculated from the peak area ratios of m/z 221 for the
fragment ion in the mass spectra of 1 and 2 appears at m/z internal standards and m/z 218 for the endogenous com-
218, due to the C*DE rings, the isotope label must be on pounds. The level of endogenous 3 was calculated from the
rings D or E. Therefore, we referred to and modified the peak area ratios of molecular ions for trideuterated 3 and en-
[28,28-2H2]b-amyrin synthesis method, and began our syn- dogenous 3. As shown in Table 1, b-amyrin (1), a-amyrin
thesis from oleanolic acid (5). The synthesis of [3,28,28,28- (2), and lupeol (3) were found in WT seedlings. This is con-
2H4]b-amyrin (8) is outlined in Fig. 3. We also synthesized sistent with the report of Husselstein-Muller et al.23) Al-
deuterated 2 and 3 from ursolic acid (6) and betulinic acid though these three triterpenoids were also found in the mu-
tants, the levels of these triterpenoids in the hmg1 and hmg2
Table 1. Quantification of Endogenous Sterols and Triterpenoids in two
WT Accessions and the hmg1 and hmg2 Mutants
mutants were approximately 65% and 25% lower than in the
WT, respectively. These results suggest that both HMGR1
and HMGR2 participate in triterpenoid biosynthesis. These
results were first observed experimentally. The decrease in
the level of 3 in hmg1 was greater than that of 1 and 2.
Eleven oxidosqualene cyclases are involved in triterpenoid
biosynthesis in Arabidopsis. To elucidate the cause of the
varying decreases in the levels of individual triterpenoids, the
enzymes should be analyzed comprehensively. It is interest-
ing to note that the decrease in the sterol levels was lower
than that of the triterpenoid levels. Although triterpenoids are
secondary metabolites, sterols are indispensable compounds
because they are constituents of the cell membrane and
biosynthetic intermediates of phytohormones, and are in-
volved in cell maintenance. It is possible that the restricted
amounts of squalene are cyclized into sterols in preference to
triterpenoids in the hmg1 and hmg2 mutants.
Sterols
(mg/100 mg
WT (WS)
hmg1
WT (Col)
hmg2
dry weight)
Cholesterol
Campesterol
Stigmasterol
Sitosterol
4.65 (0.44)
44.2 (2.99)
19.5 (2.48)
155 (5.46)
4.78 (0.20)
0.68 (0.18)
2.15 (0.67)
231 (11.1)
3.46 (0.53)
27.6 (1.39)
5.23 (0.25)
72.1 (6.13)
1.33 (0.16)
0.39 (0.08)
1.15 (0.13)
111 (7.84)
2.21 (0.02)
48.2 (2.75)
20.8 (1.08)
239 (16.0)
4.15 (0.33)
0.73 (0.04)
3.55 (0.20)
319 (19.7)
1.99 (0.16)
41.5 (0.76)
21.2 (0.97)
194 (8.13)
2.94 (0.11)
0.31 (0.04)
2.66 (0.03)
264 (10.4)
Brassicasterol
Campestanol
Sitostanol
Total
Triterpenoids
(ng/100 mg
dry weight)
WT (WS)
hmg1
WT (Col)
hmg2
HMGR2 Functions Similarly to HMGR Since the
phenotype of the hmg2 mutant is like that of the WT, it was
possible that the sterol and triterpenoid contents of hmg2
were also similar to those of the WT. However, the levels of
sterols and triterpenoids in hmg2 were approximately 15%
and 25% lower than in the WT, respectively. The significant
1
2
3
215 (2.74)
77.5 (0.39)
172 (13.8)
465 (16.9)
86.1 (9.02)
36.4 (2.80)
34.1 (2.01)
157 (12.2)
178 (6.00)
62.2 (0.86)
138 (9.12)
378 (9.57)
128 (2.65)
46.2 (2.74)
100 (6.64)
275 (6.05)
Total
Values are averages from three samples. Values in parentheses are standard devia-
tions.
Fig. 3. Total Synthesis of [3,28,28,28-2H4]b-Amyrin (8)

October 2007
1521
Acknowledgements We thank Ms. Yukiko Kamide and Ms. Hiromi
Hashinokuchi (Plant Science Center, RIKEN) for preparing plant materials.
This work was supported in part by a Grant-in-Aid (number 17510188) for
Scientific Research (C) to T. M.
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planta functions of these genes. The other is analysis of mu-
tants that show resistance to inhibitors of triterpene biosyn-
2252—2256 (1973).
thesis. We recently reported the mutant loi1, which is resist-
ant to lovastatin. The amount of sterols in loi1 is less affected
by lovastatin than that in the WT.27) The analysis of such
chemical phenotypes is expected to be increasingly impor-
tant in the future.
26) Burnouf-Radosevich M., Delfel N. E., England R., Phytochemistry,
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27) Kobayashi K., Suzuki M., Tang J., Nagata N., Ohyama K., Seki H.,
Kiuchi R., Kaneko Y., Nakazawa M., Matsui M., Matsumoto S.,
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