Identification of marneral synthase, which is critical for growth and development in Arabidopsis

Authors


For correspondence (e-mail mcsuh@chonnam.ac.kr).

Summary

Plants produce structurally diverse triterpenoids, which are important for their life and survival. Most triterpenoids and sterols share a common biosynthetic intermediate, 2,3-oxidosqualene (OS), which is cyclized by 2,3-oxidosqualene cyclase (OSC). To investigate the role of an OSC, marneral synthase 1 (MRN1), in planta, we characterized a Arabidopsis mrn1 knock-out mutant displaying round-shaped leaves, late flowering, and delayed embryogenesis. Reduced growth of mrn1 was caused by inhibition of cell expansion and elongation. Marnerol, a reduced form of marneral, was detected in Arabidopsis overexpressing MRN1, but not in the wild type or mrn1. Alterations in the levels of sterols and triterpenols and defects in membrane integrity and permeability were observed in the mrn1. In addition, GUS expression, under the control of the MRN1 gene promoter, was specifically detected in shoot and root apical meristems, which are responsible for primary growth, and the mRNA expression of Arabidopsis clade II OSCs was preferentially observed in roots and siliques containing developing seeds. The eGFP:MRN1 was localized to the endoplasmic reticulum in tobacco protoplasts. Taken together, this report provides evidence that the unusual triterpenoid pathway via marneral synthase is important for the growth and development of Arabidopsis.

Introduction

Plants produce more than 20 000 triterpenoids with chemically diverse structures. During plant growth and development, triterpenoids are not only essential precursors for cell membranes and steroid hormones (Nes and Heftmann, 1981; Benveniste, 2004; Suzuki et al., 2006) but also play roles in defense against pathogen attack (Phillips et al., 2006) and in protection against environmental stresses (Wang et al., 2010). In addition, plant triterpenoids are widely used as medicines due to their pharmacological properties, such as anti-cancer, anti-inflammatory, and anti-oxidant activities, and anti-plasmodial activity against human malarial parasites (Waller and Yamasaki, 1996; Benoit-Vical et al., 2003; Oliveira et al., 2005; Lima-Junior et al., 2006; Holanda Pinto et al., 2008; Shai et al., 2008; Augustin et al., 2011; Osbourn et al., 2011).

Triterpenoids are known to be synthesized via the condensation of five-carbon isoprenoid precursors derived from the mevalonate (MVA) pathway in plants (McGarvey and Croteau, 1995). Farnesyl diphosphate synthase catalyzes the production of farnesyl diphosphate (FPP) from isopentenyl diphosphate and dimethylallyl diphosphate. Squalene synthase converts FPP to squalene, which is subsequently oxidized into 2,3-oxidosqualene (OS) by squalene epoxidase. Finally, the biosynthetic intermediate OS is regio- and stereo-specifically cyclized by various OS cyclases (OSCs) (Abe et al., 1993; Abe, 2007). Thus, most triterpenoids have pentacyclic and tetracyclic carbon skeletons, but some others contain acyclic, monocyclic, bicyclic, tricyclic, and hexacyclic structures (Xu et al., 2004; Ohyama et al., 2008).

Plants encode multiple OSCs containing distinct product specificity that form diverse triterpene skeletons. To date, more than 40 OSCs, including β-amyrin synthase and lupeol synthase, have been isolated (Phillips et al., 2006; Shibuya et al., 2007, 2009). Mutagenesis studies on OSCs have demonstrated that their product specificity can be altered by only one amino acid substitution on OSCs (Kushiro et al., 2000). Arabidopsis harbors 13 OSC genes, which have been well characterized in a yeast mutant lacking lanosterol synthase activity (Morlacchi et al., 2009). Eight of these genes encode enzymes producing a predominant triterpenoid, such as cycloartenol, lanosterol, marneral, camelliol C, β-amyrin, arabidiol, thalianol, or tirucalladienol (Corey et al., 1993; Fazio et al., 2004; Suzuki et al., 2006; Xiang et al., 2006; Xiong et al., 2006; Shibuya et al., 2009). Five other OSCs have been shown to be multifunctional enzymes involved in the formation of several triterpenoids (Herrera et al., 1998; Kushiro et al., 2000; Segura et al., 2000; Husselstein-Muller et al., 2001; Ebizuka et al., 2003; Kolesnikova et al., 2007; Lodeiro et al., 2007; Shibuya et al., 2007; Morlacchi et al., 2009).

The most well-characterized OSC product is cycloartenol, which is synthesized by a cycloartenol synthase 1 (CAS1) and is a precursor for the synthesis of sterols and steroid hormones (Corey et al., 1993). In planta roles of cycloartenol were confirmed by the characterization of cas1 knock-out or knock-down mutants. A cas1 knock-out mutant is a male gametophyte-lethal mutant due to the depletion of sterols, indicating that CAS1 is essential for cell viability. Analysis of weak or conditional cas1 mutants further potentiated the function of CAS1 in plastidial thylakoid membrane biogenesis (Babiychuk et al., 2008). In contrast, no significant morphological phenotypes were observed in the Arabidopsis lanosterol synthase (las) knock-out mutant (Suzuki et al., 2006), revealing that plants preferentially utilize cycloartenol over lanosterol, which is a precursor for the biosynthesis of membrane sterols and steroid hormones in animals and fungi. Recently, the growth of the above-ground parts of plants overexpressing thalianol synthase (THAS) or marneral synthase (MRN1) was shown to be significantly inhibited (Field and Osbourn, 2008; Field et al., 2011), suggesting that an excess amount of thalianol, marnerol, or their derivatives might be detrimental to plant growth. Interestingly, plants overexpressing THAS have longer roots than the wild-type and thas knock-out lines, which suggests organ-specific effects of thalianol on plant growth (Field and Osbourn, 2008). A triterpene lupeol was reported to be also involved in moderating cell proliferation in legume (Lotus japonicus) roots through the β-catenin-mediated signaling pathway (Delis et al., 2011).

New plant triterpenoids have been identified by Arabidopsis genome mining (Fazio et al., 2004). For example, the Arabidopsis MRN1 gene encodes an enzyme that produces marneral, which was not isolated from any natural source but has been suggested as an intermediate in the biosynthetic pathway of iridals in Iridaceae (Marner, 1997; Xiong et al., 2006). The enzymatic reaction of MRN1 proceeds through the cyclization of OS to a bicyclic intermediate, which undergoes 1,2-shifts to a C5 cation, and then ring A is cleaved to generate a monocyclic aldehyde, marneral (Xiong et al., 2006). The operon-like gene cluster encoding enzymes involved in the synthesis of marneral, and its oxidized product marnerol, was identified in the Arabidopsis genome (Field et al., 2011).

In this study, an Arabidopsis mrn1 knock-out mutant showing round-shaped leaves and late flowering was isolated under long-day conditions (16/8 h, light/dark). The late-flowering phenotype was much more severe under short-day conditions (12/12 h, light/dark) than under long-day conditions. To better understand the biological roles of marneral synthase, phenotypes, gene expression, the sterol and triterpenol content, and ion and chlorophyll leakage of the loss-of-function mrn1 mutant were compared with those of the wild type. The amounts of marnerol were measured in the wild type, mrn1, a complementation line of mrn1, and transgenic Arabidopsis overexpressing MRN1. In addition, the spatial and temporal expression of the MRN1 gene and subcellular localization of the MRN1 protein were analyzed.

Results

Isolation of a late-flowering mutant with round-shaped rosette leaves

To better understand the biological roles of unidentified genes, T-DNA inserted Arabidopsis activation tagging lines were generated by transformation with the pSKI015 vector (Weigel et al., 2000) and were screened to isolate Arabidopsis mutants showing abnormal phenotypes under long-day conditions. From this screening, a late-flowering mutant with round-shaped rosette leaves was isolated (Figure 1a). The mutant was named mrn1 (marneral synthase 1) after the T-DNA inserted site was identified. The late-flowering phenotype was much severer under short days (12/12 h, light/dark) than under long days (16/8 h, light/dark) (Figure 1b). Under long days, the numbers of rosette leaves of the wild type and mrn1 were 9 and 14, respectively (Figure 1c). A RT-PCR analysis of the transcripts of the genes related to flowering time control in the wild type and mrn1 showed that the transcript levels of various flowering time genes were affected (Figure S1). Among them, the transcript levels of the FLC (Flowering Locus C) gene encoding a repressor of flowering were highly elevated, indicating that the late-flowering phenotype of mrn1 might be in part due to overexpression of FLC. The ratio of leaf blade length to width was decreased by approximately 45% in the mutant when compared with that of the wild type, causing round-shaped leaves in mrn1 (Figure 1d,e). Each organ appeared normal in the flowers of mrn1 except for the short anther filaments (Figure 1f). Markedly short mrn1 siliques were frequently observed at the initial flowering stage (Figure 1g), but the severity of this abnormal phenotype was reduced at the late stage of flowering. No or a few developing seeds were contained in very short or short mrn1 siliques, respectively, whereas developing seeds in the average sized mrn1 siliques appeared normal (Figure 1h).

Figure 1.

 Growth and morphology of Arabidopsis wild type and mrn1.
(a) Five-week-old wild type (WT) and mrn1 grown under long-day conditions. Scale bar = 1 cm.
(b) Three-month-old wild type and mrn1 grown under short-day condition. Scale bar = 1 cm.
(c) Rosette leaf numbers of 4-week-old wild type and mrn1l. Bars indicate standard deviation of the mean (= 20).
(d) Leaves of 3-week-old wild type and mrn1. Scale bar = 5 mm.
(e) The ratio of the width to the length of the primary rosette leaf from 3-week-old wild type and mrn1. Bars indicate the standard deviation of the mean. Statistically treated using Student’s t-test (*< 0.01) (= 20).
(f) Flowers of wild type and mrn1. Scale bar = 1 mm.
(g) Mature siliques of wild type and mrn1. Scale bar = 5 mm.
(h) Developing seeds of wild type and mrn1. Scale bar = 5 mm.

The mrn1 mutant displays aberrant seed morphology, a low germination rate, and root growth retardation

Since the seed morphology of mrn1 was very diverse under the anatomical microscope, wild-type and mrn1 seeds were further analyzed using scanning electron microscopy. Wild-type seeds had a normal oblong shape and the epidermal layer of the seed coat appeared hexagonal, with thickened radical cell walls and a central, volcano-shaped structure known as the columella. In contrast, the morphology of mrn1 seeds varied from the almost normal oblong shape to a variety of aberrant shapes. The moderately and severely aberrant shapes comprised 20 and 10% of the total seeds, respectively (Figure 2a). The abnormal phenotype of seeds might result from defective pollination due to short anther filaments. Although pollination was interrupted at the initial flowering stage, the mrn1 plants were eventually fertilized at the later stage.

Figure 2.

 Morphology and germination of Arabidopsis wild type and mrn1 seeds.
(a) Scanning electron microscope image of the wild type (WT) and mrn1 seeds. mrn1 seeds exhibited diverse aberrant shapes, such as normal, irregular, and completely shrunken shapes. Scale bar = 100 μm.
(b) Germination rate and root lengths of the wild type and mrn1. At 5 days after germination, mrn1 was classified into four groups; ungerminated seeds (I), non-green cotyledonary seedlings (II), green cotyledonary seedlings with moderate retardation of root growth (III), and green cotyledonary seedlings with severe retardation of root growth (IV) (= 100).

When wild-type and mrn1 seeds were germinated, approximately 10% of mrn1 seeds did not germinate under the conditions where complete germination of the wild-type seeds occurred (Figure 2b, I). Approximately 20% of mrn1 seeds contained the radicle, but did not produce normal green cotyledons (Figure 2b, II). Although approximately 30% of mrn1 seeds germinated, these seeds presented significant reduction of root growth (Figure 2b, III). Finally, the remainder of the mutant seeds (approximately 40% of mrn1 seeds) appeared almost normal, but root growth was inhibited by 5% (Figure 2b, IV).

Delayed embryo development of the mrn1 mutant

The observations on aberrant seed morphology, low germination rate, and root growth inhibition prompted us to investigate embryo development of the mrn1 mutant. Developing embryos from the wild type and mrn1 were harvested at 3–13 days after flowering (DAF) and were classified into six developmental stages: globular (i), heart (ii), torpedo (iii), late-torpedo (iv), walking-stick (v), and mature (vi) stages (Figure 3a). The percentage of developing embryos involved in each developmental stage at each DAF was measured. In this experiment, embryo-less seeds or embryos that failed to develop further at very early developmental stages were excluded. At 3 DAF, 15% of the wild-type embryos were in the heart stage, but all embryos of the mrn1 were in the globular stage. Approximately 80% of the wild-type embryos were in the torpedo stage, but more than 95% of the embryos were still in the heart stage at 5 DAF, indicating that delayed embryo development was prominent between the globular and torpedo stages. After 5 DAF, the rate of embryo development between the wild-type and the mrn1 was similar and almost all embryos from the wild-type and mrn1 were in the mature stage after 9 DAF (Figure 3b).

Figure 3.

 Embryo development of Arabidopsis wild-type and mrn1.
(a) Differential interference contrast images of developing embryos in the wild type. Embryos were harvested at different developmental stages. Each development stage of the embryos was classified into globular (I), heart (II), torpedo (III), late-torpedo (IV), walking-stick (V), and mature (VI) stages. Scale bar = 100 μm.
(b) Relative percentage of wild-type (W) and mrn1 (m) embryos in each developmental stage. Approximately 200 embryos were examined from five to ten siliques, which were tagged on the day after flowering (DAF) and the results are statistically significant (< 0.01).

mrn1 is a single recessive mutant

To determine the T-DNA insertion site in the isolated mrn1, genomic DNA was isolated from 3-week-old mrn1 leaves and thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) analysis was carried out. T-DNA was inserted between the T (2297) and T (2298) nucleotides in the seventh intron of a gene (At5g42600), which was annotated as MRN1 (Figure 4a). Because an activation tagging vector was introduced, the expression levels of the genes located within 20 kb upstream and downstream of the T-DNA insertion site were analyzed by RT-PCR using 2-week-old wild-type and mrn1 roots. The transcript levels of the genes near MRN1 in the mrn1 mutant were not altered when compared with those in the wild type, while the MRN1 transcripts were not detected in mrn1 roots (Figure 4b). To obtain a mrn1 allele, another T-DNA insertion line (SALK_152492) was analyzed. T-DNA was inserted in the third intron of the MRN1 gene, but no significant alteration in the MRN1 transcript levels was observed in this line compared with the wild-type (Figure S2).

Figure 4.

mrn1 generated by T-DNA based activation tagging and complementation of mrn1.
(a) Structure of the At5g42600 gene (MRN1) carrying the T-DNA insertion. Boxes and lines indicate exon and intron of the MRN1 gene, respectively. RB, right border region; LB, left border region. The numbers indicate the number of nucleotides from starting ATG codon of the MRN1 gene.
(b) The RT-PCR analysis of MRN1 and its neighboring genes in wild type (WT) and mrn1. Total RNA was isolated from 10-day-old wild-type and mrn1 seedlings and subjected to RT-PCR analysis. The Actin7 gene was used as a quantitative and qualitative control.
(c) Southern blot analysis of the MRN1 gene in WT and mrn1. E, EcoRI; H, Hind III; X, Xba I.
(d) mrn1 was complemented by the introduction of MRN1pro:MRN1 genomic DNA in pBIN19 vector. Total RNA was isolated from Arabidopsis WT, mrn1, and complementation lines (MRN1/mrn1; #7, #8, and #12) of mrn1 and subsequently subjected in RT-PCR analysis.
(e) Phenotypes of 5-week-old WT, mrn1, and MRN1/mrn1 (#7) Arabidopsis plants.

To identify the copy number of T-DNA insertions in the mrn1, genomic DNAs isolated from 3-week-old wild-type and mrn1 leaves were digested with EcoRI, HindIII, and XbaI restriction enzymes followed by Southern blot analysis using the biotin-labeled phosphinothricin acetyl transferase (PAT) gene as the probe. As shown in Figure 4c, one significant band was detected at 5.4 kb in the EcoRI-digestion lane, 8.1 kb in the HindIII-digestion lane, and 4.7 kb in the XbaI-digestion lane in mrn1, but not in the wild type, showing that a single copy of T-DNA was inserted in mrn1. This result was confirmed by back-crossing of mrn1 with the wild-type plant. When F2 seeds were germinated on MS media containing phosphinothricin (PPT), the ratio of PPT-resistant and PPT-sensitive seedlings of F2 progenies was approximately 3:1 and one-third of PPT-resistant seedlings showed an abnormal mutant phenotype (Table S1). These results demonstrated that mrn1 is the mutant harboring a recessive mutation in a single locus.

To confirm that the phenotypes in mrn1 were due to a recessive mutation in the MRN1 gene and to exclude the effect of the second site mutations in the mrn1, we expressed MRN1 under the control of its own promoter harboring an approximately 5.6 kb region obtained from the MFO20 bacterial artificial chromosome (BAC) library (http://www.arabidopsis.org/servlets/TairObject?type=assembly_unit&id=1376) (Bevan, 1984) in mrn1. The expression of the MRN1 gene was confirmed by RT-PCR (Figure 4d). The abnormal phenotypes of mrn1 relative to the wild type, including round-shaped leaf, late flowering, and short siliques, were completely rescued (Figure 4e).

Cell expansion or elongation is inhibited in leaves and root and shoot apical meristems of mrn1

Because mrn1 displayed round-shaped leaves, the adaxial side of the rosette leaves of 3-week-old wild type and mrn1 were visualized using a Leica DM2500 microscope (after removing the chlorophyll), which was subsequently used to measure the cell number and cell size of the leaves (Figure 5a). mrn1 leaves were approximately twice as small as the wild-type leaves. Because the number of epidermal cells were almost the same in each leaf of the wild-type and mrn1 (Figure 5b), the size of epidermal cells in mrn1 was approximately twice as small as the wild-type cells (Figure 5c). This phenotype was also observed in epidermal and mesophyll cells of the primary leaves of 7-day-old seedlings (Figure 5d). When the root meristem and elongation zones of 7-day-old seedlings were examined, the number of cells in the cortical cell files within the proximal meristems of mrn1 was increased by about 38% when compared with that of the wild type (Figure 5e,f), indicating that cell expansion of cortical cell files is severely inhibited. We further examined the shoot apical meristems (SAM), where cell division and expansion actively occur. Similarly, no difference in cell division was observed in SAMs of the wild type and mrn1, but the formation of SAM in mrn1 was delayed by approximately 3 days when compared with the wild type (Figure 5g). Furthermore, expression of cell division-related genes (Kim et al., 2006b) was not significantly altered in mrn1 when compared with the wild type (Figure S3). These results indicate that cell expansion and elongation are inhibited in leaves and root and SAMs of the mrn1.

Figure 5.

 Cell size and numbers in the epidermal and mesophyll cells in leaves, cortical cell files in roots, and shoot apical meristems of wild type (WT) and mrn1.
(a) The adaxial surface of 3-week-old WT and mrn1 leaves. Scale bars = 20 μm.
(b), (c) Cell number (b) and cell size (c) of 3-week-old WT and mrn1 were measured from three leaves. At least 30 adaxial epidermal cells from each leaf were measured, and averaged. Bars indicate standard deviation of the mean. The values were statistically treated using Student’s t-test (*< 0.01).
(d) The epidermal (arrow) and mesophyll cells of 10-day-old WT and mrn1 leaves. Scale bars = 20 μm.
(e) Confocal microscopy image of meristematic zones of 7-day-old WT and mrn1 roots. The cortical cell files within the proximal meristems are marked with a white triangle (meristem) and with an arrow (transition zone). Scale bars = 20 μm.
(f) Number of cells before the first rapidly elongated cell in the cortical cell files were counted in 9-day-old WT and mrn1 roots (= 10).
(g) Shoot apical meristems of 7-day-old (the upper panels) and 10-day-old (the lower panels) WT and mrn1. Scale bars = 20 μm.
(h) The transcript levels of genes related to cell expansion or elongation. Total RNA was isolated from 3-week-old WT and mrn1 leaves and subjected for RT-PCR analysis. The Actin7 gene was used as a quantitative and qualitative control.

To investigate whether the inhibition of cell expansion in the leaves of mrn1 could be related to the altered expression of genes involved in cell expansion, total RNAs isolated from 3-week-old wild-type and mrn1 leaves were subjected to RT-PCR to analyze the expression of expansin genes encoding cell-wall loosening proteins that promote cell enlargement. However, in mrn1, the expression levels of the EXP5, EXP6, and EXP10 genes were not altered when compared with the wild type (Figure 5h). In addition, the expression of genes involved in sterol and brassinosteroid biosynthesis was not altered in the mrn1 (Figure S4).

Marnerol was detected in transgenic Arabidopsis overexpressing MRN1

To better understand the biochemical role of MRN1-encoded proteins, the amounts of marnerol, a stable reduction product of marneral (Xiong et al., 2006), were measured by gas chromatography–mass spectrometry (GC-MS) in wild-type, mrn1, a complementation line of mrn1, and transgenic Arabidopsis overexpressing MRN1. Arabidopsis overexpressing MRN1 displayed a growth inhibition phenotype as observed by Field et al. (2011). Because marneral is known to be very unstable in vitro, marnerol was used as a standard in this measurement. A marnerol peak was detected in the MRN1 overexpression lines in GC-MS, but not in the wild type, mrn1, and the complementation line of mrn1 (Figure 6a–d). The mass spectrum of marnerol in the leaves of MRN1 overexpression line 9 matched with that of marnerol in the standard (Figure 6e,f). The amount of marnerol was measured within the range of 1–100 μg in 100 mg lyophilized seedlings, leaves, and roots (Table 1).

Figure 6.

 Gas chromatography–mass spectrometry (GC-MS) chromatograms and mass spectrum of marnerol fraction from Arabidopsis wild type (WT), mrn1, a complementation line of mrn1, and transgenic lines overexpressing MRN1.
Extracts from each sample were analyzed by GC-MS: TIC, total ion chromatograms; m/z 500, extracted ion chromatograms at a mass-charge ratio (m/z) of 500. Wild-type, mrn1 (knock-out), a complementation line of mrn1 (MRN1/mrn1), and transgenic lines overexpressing MRN1 (OX-2; OX-7; OX-9). (a) Ten-day-old seedlings. (b) Three-week-old leaves. (c) Six-week-old roots. (d) Manerol as a standard. (e) Mass spectrum of manerol in OX-9 leaf. (f) Mass spectrum of manerol in a standard.

Table 1. Quantification of endogenous manerols in Arabidopsis: wild-type, mrn1, complementation line of mrn1, and MRN1 overexpression lines
 SeedlingsLeavesRoots
  1. Ten-day-old seedlings, leaves of 3-week-old plants, and roots of 6-week-old plants were freeze-dried and used for marnerol analysis. Values (μg 100 mg−1 dry weight) are expressed as an average of three independent experiments. Parenthesis indicates standard deviation. nd = not detected. MRN1/mrn1; complementation lines of mrn1, OX-2, OX-7, and OX-9 transgenic Arabidopsis lines overexpressing MRN1.

Wild typendndnd
mrn1 ndndnd
MRN1/mrn1 ndndnd
OX-23.0 (0.09)23.3 (1.73)0.9 (0.05)
OX-77.4 (0.14)1.1 (0.03)2.4 (0.15)
OX-917.2 (0.20)108.9 (3.33)1.5 (0.03)

The levels of sterols and triterpenols are altered in the mrn1 mutant

As MRN1 shares the common biosynthetic intermediate, OS, with CAS1, we investigated whether the composition and content of sterols and triterpenols are altered in mrn1. Total sterols and triterpenols were extracted from the leaves of 3-week-old wild type and mrn1 and roots of 6-week-old wild type and mrn1, and were analyzed by GC-MS. As shown in Table S2, the total content of major sterols was approximately 2000-fold higher than that of major triterpenols in both leaves and roots. The total amount of sterols was decreased in mrn1 leaves and roots to approximately 11% and 10%, respectively, compared with the wild type. In contrast, the total amount of triterpenols in roots was increased by approximately 5% in mrn1 when compared with the wild type (Table S2). When the relative content of major sterols and triterpenols was compared between wild-type and mrn1 leaves and roots, the amounts of β-sitosterol were increased by approximately 20%, but the amounts of stigmasterol were reduced by approximately 20% in mrn1 roots compared with wild-type roots (Figure 7a). In addition, the α-amyrin content was decreased by approximately 15 and 3% in mrn1 leaves and roots, respectively, relative to wild type. Whereas the lupeol content was increased by approximately 15 and 2% in mrn1 leaves and roots, respectively, when compared with wild type (Figure 7b).

Figure 7.

 Relative content of major sterols and triterpenols in Arabidopsis.
Relative content of major sterols (a) and triterpenols (b) in Arabidopsis wild-type (WT) and mrn1 mutant leaves and roots. Leaves of 3-week-old plants and roots of 6-week-old plants were freeze-dried and used for sterol and triterpenol analyses. Values are an average of three independent experiments. Bars indicate standard error of the mean (*< 0.01).

Changes in membrane integrity and permeability were observed in mrn1 mutant

Sterols are incorporated into membranes and regulate the mobility of phospholipid fatty acid chains in plant cells (Hartmann, 1998). Triterpenols have also been proposed to act as structural components of membranes (Nes and Heftmann, 1981). We observed altered amounts of sterols and triterpenoids in mrn (Figure 7). Therefore, we investigated whether the mrn1 mutation affected membrane integrity and permeability by using ion leakage assays for the leaves and roots of 3-week-old wild type, mrn1, and the complementation line of mrn1. The relative electrolyte leakages of the mrn1 leaves and roots were approximately twofold and threefold higher than that of the wild-type leaves and roots, respectively, suggesting that the membrane integrity of the mrn1 leaves and roots is defective. The ion leakages of the complementation line were almost rescued to the levels of the wild type in leaves and roots (Figure 8a).

Figure 8.

 Membrane integrity and permeability analyses of Arabidopsis wild type (WT) and mrn1.
(a) Membrane ion leakage was determined by measuring electrolytes that had leaked using 3-week-old wild-type (WT) and mrn1 leaves and roots in 400 mm mannitol solution. Bars indicate standard error of the mean (= 10). Differences between WT and mrn1 were significant at < 0.01.
(b) Chlorophyll leaching test of WT and mrn1. Each value is an average of five replicates. Bars indicate the standard error of the mean. Differences between Arabidopsis WT and mrn1 were significant at asterisks (*< 0.01).

In addition, membrane permeability was also determined using the chlorophyll leaching assay. Leaves from 3-week-old wild-type and mrn1 were submerged in 80% ethanol for different time periods, and the concentration of chlorophylls in the solution was determined. Chlorophyll leaching occurred more rapidly in mrn1 leaves than the wild-type, but the complementation line of mrn1 showed the wild-type levels of membrane permeability (Figure 8b).

The MRN1 gene is specifically expressed in shoot and root apical meristems and induced by osmotic stress and abscisic acid

To investigate the temporal and spatial expression patterns of the MRN1 gene, transgenic Arabidopsis harboring approximately 5.4 kb of the promoter region fused to a GUS reporter gene was generated. Expression of GUS was exclusively detected in the meristematic zones of shoots and roots (Figure 9a), indicating the potential role of MRN1 in cell division and expansion.

Figure 9.

 Expression of the MRN1 gene in Arabidopsis.
(a) The GUS staining of 10-day-old (left) and 3-week-old (right) Arabidopsis plants transformed with the MRN1pro:GUS construct.
(b) The transcript levels of OSCs in various Arabidopsis organs. R, roots; YS, young seedlings; L, rosette leaves; S, stems; F, flowers; Si, siliques.

In addition, the expression pattern of six other OSCs, which are in clade II including MRN1 in the phylogenetic tree of Arabidopsis OSC family (Field and Osbourn, 2008), was analyzed to gain further insights into their functional relationship. The CAS1 gene that is not in clade II was used as a negative control. Total RNAs isolated from roots, young seedlings, leaves, stems, flowers, and siliques were subjected to RT-PCR analysis. The expression pattern of the MRN1 gene, which is specifically expressed in roots and seedlings, was similar to that of THAS/PEN4, pentacyclic triterpene synthase 3 (PEN3), and PEN1 genes. Expression of the PEN2 transcripts was observed in roots, young seedlings, and siliques, whereas PEN6 was specifically expressed in siliques (Figure 9b). In contrast to the ubiquitous expression of CAS1 in various organs, OSC genes in clade II were preferentially expressed in roots and siliques containing developing seeds, suggesting that the products synthesized by the OSCs or their metabolites might be important in non-photosynthetic tissues.

MRN1 is localized to the endoplasmic reticulum

When Arabidopsis CAS1 and lanosterol synthase (LAS) from Trypanosoma cruzi were expressed in yeast, Arabidopsis CAS1 and T. cruzi LAS were mainly observed in lipid particles, which are compartments containing high levels of triacylglycerols and steryl esters and harboring several enzymes for lipid metabolism (Milla et al., 2002). Some OSC activities were also retained in the microsomes of the yeast strains. The brassinosteroid biosynthetic enzymes DWF1 and DWF4 were reported to be located in the endoplasmic reticulum (ER) (Klahre et al., 1998; Kim et al., 2006a). However, the subcellular localization of Arabidopsis OSCs has not yet been identified in plants. To investigate the subcellular localization of MRN1, the plasmid 35Spro:eGFP:MRN1 encoding enhanced green fluorescence protein (eGFP) fused to N-terminal region of MRN1 under the control of the CaMV35S promoter was introduced into tobacco leaf protoplasts, and the subcellular localization of eGFP:MRN1 fusion proteins was analyzed by confocal laser scanning microscopy. The green fluorescent signals from the eGFP:MRN1 were merged with the red fluorescent signals from the ER marker, Brassica rapa fatty acid desaturase 2:RFP (BrFAD2:RFP, Jung et al., 2011) (Figure 10a–c), suggesting that MRN1 is localized in the ER. In addition, THAS, which belongs to Arabidopsis clade II OSCs, including MRN1, was fused to eGFP and introduced into tobacco protoplasts. The green fluorescent signals from eGFP:THAS were also observed in the ER (Figure 10d–f).

Figure 10.

 Subcellular localization of eGFP:MRN1 and eGFP:THAS. Subcellular localization of MRN1 in protoplasts from tobacco leaves. The 35Spro:eGFP:MRN1 or 35Spro:eGFP:THAS construct was transfected with 35Spro:BrFAD2:RFP and visualized by laser confocal microscopy. Scale bars = 10 μm: (a) eGFP:MRN1; (b), (e) BrFAD2:RFP; (c), (f) the merged image; (d) eGFP:THAS.

Discussion

According to previous reports (Marner and Longerich, 1992; Marner, 1997), the biosynthetic pathway of unusual triterpenoids, iridals in Iridaceae as the precursors of the irones, which are the source of the violet-like scent in orris oil, has been hypothesized. The MRN1 gene was identified to encode marneral synthase, which generates an unusual monocyclic triterpene aldehyde, marneral, by expressing Arabidopsis OSCs in the yeast mutant lacking both squalene epoxidase and lanosterol synthase (Xiong et al., 2006). Recently, MRN1 (At5g42600), MRO (At5g42590), and CYP705A12 (At5g42580) genes were found to be located in the Arabidopsis genome as an operon-like gene cluster (Field et al., 2011). In this study, through characterization of the mrn1 mutant (Figure S5) we identified the unusual triterpenoid biosynthetic pathway via marneral, which seems critical for growth and development in Arabidopsis. Our observations also suggest that this unusual triterpenoid pathway may be important for growth and development of Brassicaceae, given that Arabidopsis clade II OSCs, including MRN 1, are confined to this plant family.

The expression of GUS, under the control of the MRN1 promoter, was only detected in shoot and root apical meristematic cells where cell division and expansion actively occur. The expression patterns of the MRN1 gene are consistent with the abnormal phenotypes of the mrn1 mutant, such as inhibited growth and delayed development. Interestingly, the growth-inhibition phenotype was also observed in plants overexpressing MRN1 (Field et al., 2011). Co-overexpression of MRN1 and MRO encoding marneral oxidase resulted in the loss of the marnerol peak and the production of desaturated hydroxy-marnerol, which might be responsible for extreme dwarfism (Field et al., 2011). Taken together, these observations indicated that lack or excess of marnerols or their metabolites is detrimental to the normal growth and development of Arabidopsis.

The atexp5 knock-out mutant displayed shorter roots and hypocotyls and smaller rosette leaves, and the expression of the EXP5 gene was shown to be controlled by brassinosteroids (Park et al., 2010). Furthermore, antisense expression of the EXP10 gene also produced smaller rosette leaves with shorter petioles and leaf blades (Cho and Cosgrove, 2000). However, loss-of-function mutations in MRN1 caused inhibition of cell expansion and elongation without affecting the expression levels of EXP5 and EXP10 genes. Although the exact roles of the marnerols and their metabolites in cell expansion and elongation are still unknown, cell expansion and elongation may be fine-tuned by marnerols and their metabolites, acting as hormone-like substances, such as a terpenoid lactone, strigolactone, which has been shown to be involved in inhibition of shoot branching (Umehara et al., 2008) and in stimulating both growth of mycorrhizal fungi (Akiyama and Hayashi, 2006) and the germination of parasitic plant seeds (Cook et al., 1966). In addition, no significant alteration of EXP5 gene expression was observed in mrn1, indirectly indicating that the amount of brassinosteroids was not altered in the mutant. The hypothesis was supported by the finding that the expression of genes involved in sterol and brassinosteroid biosynthesis was not altered in mrn1.

Another explanation for the inhibition of cell expansion and elongation of mrn1 is that marnerols or their metabolites may be used as membrane constituents, based on the fact that triterpenoids, which are structurally equivalent to sterols, could function as substitutes for sterols in cyanobacteria (Rohmer et al., 1979), Tetrahymena pyriformis (Nes and Heftmann, 1981), and Iris germanica rhizomes (Bonfils et al., 1995). Moreover, marnerol, thalianol, and arabidol as well as the monocyclic iridals have the appropriate structural features required for membrane constituents, including an amphiphilic character like sterols, a mobile and flexible side chain, and possible desaturation and alkylation of the side chain (Marner, 1997; Morlacchi et al., 2009). No marnerol peak was detected in GC-MS of wild-type roots, seedlings, or leaves; however, marnerols or their metabolites could be present in the restricted regions of membranes, such as ‘lipid raft’. If marneral, marnerol, or their metabolites can be specifically or temporally used in meristematic zones that are not fully differentiated for the production of sterols, it would be an efficient usage of metabolites in plants. To address this hypothesis, whether or not the fluorescent marnerols can be incorporated in artificial or natural membranes should be further investigated.

In addition, a decline in membrane integrity was shown to lead to an increase in ion leakage and loss of selective permeability, and finally, cell death, which was accompanied by senescence in plants. Arabidopsis fackel/hydra2, hydra1, cyp51A2, and cas1 mutants that are defective in the sterol biosynthetic pathway exhibited seedling-lethal phenotypes and abnormal developmental patterns (Lindsey et al., 2003; Schaller, 2003; Kim et al., 2005; Babiychuk et al., 2008). The cyp51A2 mutant phenotype is associated with a lack of membrane integrity, due to a deficiency in sterols (Kim et al., 2005). Sitosterol and 24-methylcholesterol can regulate membrane fluidity and permeability through differential interactions with phospholipids (Hartmann, 1998). Therefore, it remains to be studied if defects in membrane integrity and permeability of the mrn1 mutant are associated with the altered levels of some sterols or other unknown factors.

Until now, no evidence for subcellular localization of Arabidopsis OSCs has been reported in plants. Only Arabidopsis CAS1 activity has been observed in lipid particles and the microsomes of yeast (Milla et al., 2002). Although none of the Arabidopsis OSCs, including MRN1 and THAS, harbor membrane-spanning residues or KDEL-like ER retention signals, eGFP:MRN1 and eGFP:THAS under the control of CaMV 35S promoter were localized to the ER network in tobacco protoplasts. Interestingly, GFP signals from the eGFP:MRN1 and eGFP:THAS constructs were merged with red fluorescent protein (RFP) signals from BrFAD2:RFP (Jung et al., 2011) in most regions of the ER network, but some regions were not overlapped, suggesting that the specialized compartments for the synthesis of marneral and thalianol and for fatty acid desaturation are present in the ER network. Even though the green spot regions from the eGFP:MRN1 construct were not merged with the lipid particles, which were stained with Nile Red dye, this result could be further confirmed by the subcellular localization of eGFP:MRN1 under the control of the native promoter in Arabidopsis.

Taken together, this study collectively supports that the triterpenoid biosynthetic pathway via marneral synthase is important for plant growth and development. This study helps us to understand the role of the marnerols and their metabolites in cell expansion and elongation of plants.

Experimental Procedures

Plant materials, growth conditions, and mutant screening

Arabidopsis thaliana (ecotype Col-0) plants were grown at 23°C in a growth room under long-day conditions (16/8 h, light/dark) or short-day conditions (12/12 h, light/dark). For abiotic stress and hormone treatment, 10-day-old seedlings were grown on half-strength solid MS medium (Murashige and Skoog, 1962) and then transferred to MS liquid medium containing 100 μm ABA, 200 mm mannitol, and 200 mm NaCl for 6 h. For drought stress, 10-day-old seedlings were transferred to filter paper and air-dried for 1 h.

To measure the growth of wild-type and mrn1 plants, Petri dishes with the plated seeds (100 seeds each) were incubated vertically. Five-day-old seedlings were used to measure root length and to analyze the germination rate. To generate activation-tagging plants, the pSKI015 vector was introduced into Arabidopsis plants using Agrobacterium tumefaciens strain GV3101 (Koncz and Schell, 1986) by the floral dip method (Clough and Bent, 1998). Transformed plants were selected on soil using Finale [BASTA, 1:1000 (v/v)]. The T-DNA insertion locus was identified using high-throughput TAIL-PCR analysis (Liu et al., 1995). Genomic DNA was extracted from Arabidopsis leaves (Murray and Thompson, 1980) and digested with EcoRI, HindIII, and XbaI restriction enzymes. The biotin-labeled phosphinothricin acetyl transferase (PAT) gene as a probe was amplified by PCR using bar-F1 and bar-R1 primers (Table S3). The signals were visualized by a chemiluminescent detection system for biotin-labeled DNA with CDP-Star® substrate (Applied Biosystems, http://www.appliedbiosystems.com/).

Genetic analysis

Crosses were performed by unopened buds and the pistils were used as recipients for pollen. The homo line of the mrn1 mutant was reciprocally crossed with wild-type (Col-0) plants. Genetic segregation in F2 seeds was analyzed for BASTA resistance.

Construction of binary vectors

To isolate the promoter of MRN1-a, approximately 1.8 kb of the MRN1 promoter of the genomic DNA (Col-0) was amplified using At5g42600P F1 and At5g42600P R1 primers and digested with the XbaI/SmaI and then inserted into the XbaI/SmaI digested pBI101 vector. Approximately 3.6 kb of the MRN1-b promoter region was obtained from Arabidopsis BAC clone MFO20 by XbaI digestion and inserted into the pBI101 vector containing 1.8 kb of the MRN1 promoter. Finally, the 5.4 kb MRN1 promoter was used in the MRN1pro:GUS construct. To generate the complementation construct, the 8.8 kb genomic DNA of MRN1 from Arabidopsis BAC clone MFO20 was digested with EcoRI and then cloned into the pBIN19 vector (Bevan, 1984). To identify the subcellular localization of MRN1, full-length cDNA of MRN1 was amplified from a cDNA pool of 2-week-old roots using At5g42600F3 and At5g42600R2 primers. Amplified MRN1 was digested with SmaI and then inserted into pBI221 containing eGFP.

Analysis by RT-PCR and quantitative RT-PCR

Total RNA was isolated from various Arabidopsis tissues and 10-day-old seedlings treated with abiotic stresses using the TRIzol reagent (Sigma-Aldrich, http://www.sigmaaldrich.com/). A total of 2 μg RNA was reverse transcribed using SuperScript® II (Invitrogen, http://www.invitrogen.com/) and subjected to PCR. The transcript levels were quantified using a Rotor-Gene 2000 real-time thermal cycling system (Corbett Research, http://www.corbettlifescience.com/) with a QuantiTect SYBR Green RT-PCR kit (Qiagen, http://www.qiagen.com/).

Light and scanning electron microscopy

To visualize developing embryos, samples were fixed in an ethanol:acetic acid (9:1) solution overnight, followed by successive washes in 90% and 70% (v/v) ethanol for 2 h as described previously (Berleth and Jürgens, 1993). For cell size measurements, leaves were incubated in methanol overnight to remove chlorophyll and subsequently placed in lactic acid. Embryos and leaves were visualized using a Leica DM2500 microscope (http://www.leica.com/). For scanning electron microscopy, the dry seeds were mounted on stubs with double-sided carbon tape and coated with gold particles using a model K550 sputter coater (Emitech, http://www.emitechinc.com/). Specimens were observed with a Hitachi S2400 scanning electron microscope at an accelerating voltage of 15 kV (http://www.hitachi.com/).

Semi-thin sections

To obtain semi-thin sections, part of the shoot apical meristems was first immersed in a fixative [3% glutaraldehyde in PBST (130 mm NaCl, 70 mm Na2HPO4, 30 mm NaH2PO4 and 0.01% Tween 20)] at 4°C overnight, rinsed with PBST buffer, post-fixed with 1% OsO4 at 4°C overnight, and washed again with PBST buffer. The samples were dehydrated through an ethanol series and embedded in Spurr’s resin. Semi-thin sections (1 μm) were cut on a model MT990 microtome (RMC, http://www.rmcproducts.com/) using a glass knife and stained with 0.1% (w/v) toluidine blue O.

Histochemical GUS analysis

Histochemical analysis of 10- or 21-day-old MRN1pro:GUS plants was carried out as described by Jefferson et al. (1987). To visualize the cross sections of the root, samples were embedded in LR White resin (London Resin Company, http://www.londonresin.com/) and then sliced into 15 μm-thick sections using a model MT990 microtome (RMC). The tissue sections were observed using a L2 light microscope (Leica).

Subcellular localization of MRN1

To express transient fluorescent fusion proteins, protoplasts from tobacco leaves and PEG–calcium transfection of plasmids were isolated as described (Yoo et al., 2007). The images were collected using a TCS SP5 laser confocal scanning microscope (Leica). For GFP and RFP, the excitation wavelengths were 488 and 561 nm, respectively, and the emitted fluorescence was collected at 494–540 and 570–620 nm, respectively.

Synthesis of (+)marnerol

Manerol was synthesized as described by Corbu et al. (2009). The common intermediate (a sesquiterpene) was prepared on a large scale using our chiral pool-based marnerol synthesis starting from plant-derived pulegone (a monoterpene). The sesquiterpenoid intermediate was converted into the marnerol precursor via a two-step transformation involving Swern oxidation followed by Wittig olefination. Segment coupling with the farnesyl chain precursor was then achieved through a B-alkyl Suzuki–Miyaura protocol.

Quantification of sterols, triterpenols, and marnerol

For quantification of sterols, freeze-dried plant tissues were extracted with chloroform/methanol (1:2, v/v). 1-Hexacosanol was added as an internal standard. After evaporation of the solvent under a N2 stream, sterols were recovered in chloroform and filtered to remove contaminated aqueous or polar materials, and then dried. Saponification was performed with 20% KOH in 80% ethanol for 1 h at 90°C and then 2 ml water was added. The sterols were extracted three times each with 3 ml of diethyl ether. The organic phase was evaporated and silylated with pyridine and N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) for 20 min at 100°C. Gas chromatography-mass spectrometry analyses were performed on a GCMS-QP2010 apparatus (Shimadzu, http://www.shimadzu.com/) fitted with a HP-5 column (Agilent J & W Scientific, http://www.agilent.com/). The analytical conditions were as follows: Electron Impact (EI) (70 eV); source temperature 280°C; injection temperature 180°C; column temperature program 180°C for 5 min, then raised to 300°C at a rate of 2°C min−1, and held at this temperature for 10 min; carrier gas He; flow rate 1.0 ml min−1. The content of each sterol compound was calculated from the peak area ratio between of the internal standard (1-hexancosanol). Quantification of triterpenoids was performed as described by Ohyama et al. (2007). The synthesized β-amyrin, α-amyrin, and lupeol were used as internal standards (Ohyama et al., 2007). The extracts were separated on silica-gel preparative TLC plates with hexane:ethyl acetate (5:1). The triterpenoids fraction was trimethylsilylated with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) at 80°C for 30 min and analyzed using GC-MS (GC: 6890A, Agilent Technologies, http://www.agilent.com/; MS: JMS-AM SUN200, JEOL, http://www.jeol.com/) with a DB-1 column (J&W Scientific). The analytical condition was as follows: EI (70 eV); source and injection temperatures 250°C; column temperature program 80°C for 1 min, then raised to 280°C at a rate of 20°C min−1, and held at this temperature for 15 min; carrier gas He; flow rate 1.0 ml min−1. Quantification of marnerol was calculated from ratio of peak area at m/z 457 using a calibration curve of authentic compound (with coefficient of determination: r2 > 0.998).

Acknowledgements

We would like to express our gratitude to Chae Eun Yim for technical help and Ji Hoon Ahn (Korea University) for providing the pSKI015 vector. This work was supported by grants from the World Class University Project (R31-2009-000-20025-0), the National Research Foundation of Korea and the Next-Generation BioGreen 21 Program (no. PJ008203), and the Rural Development Administration, Republic of Korea.

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