Dolichol biosynthesis and its effects on the unfolded protein response and abiotic stress resistance in Arabidopsis

Abstract

Dolichols are long-chain unsaturated polyisoprenoids with multiple cellular functions, such as serving as lipid carriers of sugars used for protein glycosylation, which affects protein trafficking in the endoplasmic reticulum. The biological functions of dolichols in plants are largely unknown. We isolated an Arabidopsis thaliana mutant, lew1 (for leaf wilting1), that showed a leaf-wilting phenotype under normal growth conditions. LEW1 encoded a cis-prenyltransferase, which when expressed in Escherichia coli catalyzed the formation of dolichol with a chain length around C(80) in an in vitro assay. The lew1 mutation reduced the total plant content of main dolichols by approximately 85% and caused protein glycosylation defects. The mutation also impaired plasma membrane integrity, causing electrolyte leakage, lower turgor, reduced stomatal conductance, and increased drought resistance. Interestingly, drought stress in the lew1 mutant induced higher expression of the unfolded protein response pathway genes BINDING PROTEIN and BASIC DOMAIN/LEUCINE ZIPPER60 as well as earlier expression of the stress-responsive genes RD29A and COR47. The lew1 mutant was more sensitive to dark treatment, but this dark sensitivity was suppressed by drought treatment. Our data suggest that LEW1 catalyzes dolichol biosynthesis and that dolichol is important for plant responses to endoplasmic reticulum stress, drought, and dark-induced senescence in Arabidopsis.

Figure 1.

The Phenotypes of the lew1 Mutant. (A) Wild-type and lew1 mutant plants grown in soil. Left, lew1 (arrows point to wilted parts of leaves); right, the wild type. (B) Comparison of root:shoot ratio. Data are means ± se (n = 41). (C) Lp_r of wild-type and lew1 roots. The numbers in parentheses indicate the numbers of plants analyzed. Data are means ± se. (D) and (E) Effects of aquaporin inhibitors on Lp_r of wild-type and lew1 plants: azide at 1 mM, 30 min (D) and mercury at 50 μM, 60 min (E). Data are means ± se. The numbers in parentheses indicate the numbers of plants analyzed. (F) Structure of xylem in leaves (top row) and stems (bottom row) of wild-type and lew1 plants. Arrows point to xylem. Bars = 5 μm. (G) Grafting experiments. Left, lew1 shoot with wild-type root; right, wild-type shoot with lew1 root.

Figure 2.

Analysis of Drought Resistance and Related Parameters in lew1. (A) Comparison of wild-type and lew1 plant growth under normal (left) and drought stress (right) conditions. For drought treatment, 3-week-old seedlings were subjected to water withholding for 2 weeks. (B) Stomatal conductance in the wild type and lew1. Data are means ± se. The numbers in parentheses indicate the numbers of analyzed plants. Two to four leaves were used per plant for the wild type, and one to two leaves were used per plant for lew1. (C) Water loss from detached leaves. Three independent experiments were performed, and 15 leaves per genotype were used in each experiment. Data are means ± se (Student's t test; ** statistically significant difference [P < 0.01]). (D) Stomata closure rates of the wild type and lew1. For lew1 plants, stomata from wilting parts were measured. About 40 stomata were measured in each of three experiments for both the wild type and lew1. Data are means ± se (Student's t test; ** statistically significant difference [P < 0.05]). (E) Stomata comparison between the wild type and lew1. The stomata on the wilting part of a leaf are shown. Bars = 20 μm. (F) Comparison of stomata in the wild type and lew1 under conditions that promote full stomatal opening. The stomata from the wilting part of a lew1 leaf were not open under conditions when all stomata in the wild-type leaf were open. Bars = 20 μm. (G) Comparison of stomata in the wild type and lew1. Stomata on nonwilting parts in the leaf base of lew1 are shown. Bars = 20 μm. (H) Osmotic potential in the wild type and lew1 under normal growth conditions. Values are means ± se, n = 3 (Student's t test; * statistically significant difference [P < 0.05]). (I) Comparison of leaf electrolyte leakage between lew1 and the wild type. Values are means ± se, n = 3 (Student's t test; * statistically significant difference [P < 0.05]). (J) Typical recording trace showing measurements of turgor in epidermal cells of a wild-type leaf. Following successful impalement of a cell with the pressure probe, the intracellular recording was continued until a stationary turgor was reached. The trace shows successive recordings in three individual cells. (K) Mean (±se) epidermal cell turgor in areas of leaves of wild-type and lew1 plants showing no obvious sign of wilting (wild type, n = 22 cells; lew1, n = 21 cells). There is a statistically significant difference (P = 0.0005) between the two genotypes.

Figure 3.

Position Cloning of LEW1 and Sequence Analysis of Long-Chain cis-Prenyltransferases. (A) Positional cloning of LEW1. The LEW1 locus was mapped to between two polymorphic markers on BAC clones T23J18 (recombinants, 11 of 1638) and T28K15 (recombinants, 17 of 1638). Further mapping delimited the LEW1 locus to a region within BACs F12F1 (recombinants, 3 of 1638) and T28K15. All candidate open reading frames were sequenced in this region, and only one mutation (G159A) was found in AT1G11755. (B) (a) Complementation of the lew1 mutant. Shown are the wild type (gl1), the lew1 mutant, the lew1 mutant transformed with 35S-LEW1 (cDNA), the wild type (Columbia [Col]), and a heterozygous plant of lew1 crossed with a line in which the LEW1 gene was disrupted by a T-DNA insertion. The T-DNA was inserted at position 205 counting from the first putative ATG of AT1G11755. (b) RT-PCR analysis of LEW1 transcripts in the wild type, lew1, and a heterozygous plant of lew1 crossed with the T-DNA line. rRNA is shown as a control for loading. (C) Alignment of long-chain cis-prenyltransferases from different organisms: rice, Os02g0197700 and Os03g0197000; human, NP_612468; mouse, BAE39779; Xenopus, NP_001016090; fruit fly, AAF51407; E. coli, P60472; Micrococcus luteus, BAA31993; Saccharomyces cerevisiae, BAA36577 (RER2); Arabidopsis, AT2G23410 and AT1G11755 (LEW1). (D) Phylogenetic tree of LEW1 and related proteins from different organisms: Arabidopsis, At2g23400, At5g58780, At5g58782, At5g58784, At5g58770, At5g60510, At5g60500, and At2g17570; rice, Os01g0857200, Os06g0167400, and Os07g0607700; monkey, XP_001110733; dog, XP_541219; Aedes aegypti, EAT38464; rat, XP_001058252; S. cerevisiae, NP_013819/SRT1 and NP_010088; the others are the same as in (C). The phylogenetic tree was constructed on the alignment using MEGA (version 4.0) (see Supplemental Data Set 1 online). Bootstrap values were calculated from 1000 trials and are shown at each node. The extent of divergence according to the scale (relative units) is shown at bottom.

Figure 4.

Biochemical Characterization of LEW1 and Complementation of the Yeast rer2 Mutant. (A) Ectopic expression of LEW1 in E. coli. Proteins extracted from E. coli cells were analyzed on 12% SDS-PAGE gels and stained with Coomassie blue. The arrow points to the expressed LEW1 protein in E. coli. M, marker; Con, empty vector control; LEW1, isopropylthio-β-galactoside–induced LEW1. (B) TLC analysis of LEW1 reaction products. [¹⁴C]IPP and [¹⁴C]FPP were incubated with total proteins isolated from E. coli strains transformed with either the empty vector (Con) or LEW1. At the completion of the reaction, lipid products were extracted and subjected to TLC analysis. Solanesols C₄₅ and C₉₀ were used as standards. A C₅₅ band (representing a product that can be synthesized by the native cis-prenyltransferase in E. coli) was equally detected in E. coli transformed with either empty vector or LEW1, indicating that the reactions were successful. Ori, original spot. (C) LEW1 partially complemented the temperature-sensitive phenotype of the yeast rer2 mutant. rer2 cells transformed with an empty vector, LEW1, or DPS (AT2G23410) were grown overnight. Serial decimal dilutions were spotted onto plates of synthetic dropout medium with all amino acids (URA+) or excluding URA (URA−). URA is a selection marker of the transformed clone. Plates were incubated at the indicated temperatures and photographed after 4 d.

Figure 5.

Analysis of the Relative Dolichol Contents in Wild-Type, lew1, and LEW1 Overexpression Plants by LC-MS. The peak area in the wild type was taken as 100%, and the relative contents in other plants were compared with that in the wild type. (A) The dolichol standards (left, Dol-C₇₅; right, Dol-C₈₀). Arrows indicate the peaks. (B) The wild type. (C) lew1 mutant. (D) Overexpression line 14 (OE14). (E) Overexpression line 34 (OE34). (F) RNA gel blot analysis of LEW1 expression in OE14 and OE34. Due to its low expression, LEW1 transcript could not be detected in the wild type and the lew1 mutant by RNA gel blot under the conditions used. OE15 and OE21, two other transgenic lines not used in this study, are also included. The bottom panel shows an RNA gel as a loading control.

Figure 6.

lew1 Impairs Protein N-Glycosylation, and lew1 Plants Are Hypersensitive to Tunicamycin. (A) Comparison of protein glycosylation patterns between the wild type and lew1 by PAS staining. Thirty micrograms of total protein were resolved by SDS-PAGE and detected by PAS staining. The arrow points to the bands missing from lew1. (B) Concanavalin A–Sepharose binding assay for N-glycosylated proteins. Total proteins extracted from the wild type and lew1 were incubated with concanavalin A–Sepharose, and the bound proteins were eluted, treated with (+) or without (−) endoglycosidase H (Endo H), resolved by SDS-PAGE, and stained with Coomassie blue. The arrows at left point to the two protein bands found in the wild type but not in lew1. These two bands were recovered and analyzed by MALDI-TOF MS. The top one corresponds to SKU5, and the bottom one corresponds to TTG1. Endoglycosidase H treatment resolved the proteins found in both the wild type and lew1. M, marker. (C) lew1 seedlings are sensitive to tunicamycin (TM). Seedlings were germinated on MS medium or MS medium containing 0.1 μg/mL tunicamycin and grown for 10 d in a growth chamber. WT, wild type (gl1); lew1, lew1 mutant; 35S-LEW1, a wild-type line overexpressing LEW1 under the control of the 35S promoter (line 14); lew+35S-LEW1, a lew1 mutant line overexpressing LEW1 under the control of the 35S promoter (line 5).

Figure 7.

lew1 Plants Are Hypersensitive to Dark. (A) Wild-type and lew1 seedlings (3 weeks old) were grown in light (top row) or dark (bottom row) at 22°C for 10 d. Also shown is a lew1 transgenic line overexpressing LEW1 (line 5) and a wild-type transgenic line overexpressing LEW1 (line 14). (B) Electrolyte leakage assay of wild-type and lew1 plants in the dark. Two-week-old seedlings were grown in soil in the dark at 22°C for the indicated times, and the leaves were taken for ion leakage assays. Results were from three independent experiments. Data are means ± se (* P < 0.05, ** P < 0.01). (C) Tunicamycin treatment accelerated dark-induced senescence in lew1. Three-week-old seedlings grown in soil were sprayed with water (Dark) or with 0.1 μg/mL tunicamycin (Dark+TM) and then incubated in the dark for 10 d before photographs were taken. (D) Expression of dark-inducible genes in lew1 and the wild type. Two-week-old seedlings were grown in the dark for 24 or 48 h, and total RNA was analyzed by RNA gel blot. Also shown are two wild-type transgenic lines (OE14 and OE34) overexpressing LEW1 (35S-LEW1). The two bottom panels show rRNA and tubulin as loading controls.

Figure 8.

Drought or Osmotic Stress Improves the Survival of lew1 Plants in the Dark or under Weak Light (A) Ten-day-old seedlings in soil were subjected to water withholding for 7 d to give a modest drought treatment (Drought) in the light and then incubated in the dark for 15 or 20 d, or in the light for 15 d, without watering. Wild-type but not lew1 plants died from drought treatment for 15 d in the light. All lew1 plants died from drought at 20 d in the light. Plants without drought treatment (Watered) were kept in the light for 15 d or in the dark for 15 or 20 d. (B) After 20 d, the drought-treated plants in the dark were watered and moved, together with plants without drought treatment in the dark, to light for an additional 3 d. No lew1 plants without drought treatment in the dark survived, but all lew1 plants with drought treatment in the dark survived. No wild-type plants died under the conditions used. (C) Osmotic stress imposed by mannitol, NaCl, or glycerol increased the survival of lew1 seedlings in MS medium without sucrose under weak light. Four-day-old seedlings grown on MS medium with 3% sucrose were moved to MS medium without sucrose (−), with sucrose (3%), or without sucrose but with 100 mM mannitol, 180 mM NaCl, or 1.5% glycerol and cultured for 7 d. All lew1 seedlings on MS medium without sucrose died, while all wild-type seedlings survived. In other treatment conditions, all lew1 and wild-type seedlings survived. (D) lew1 seedlings are more tolerant to high-salt stress than wild-type seedlings. Four-day-old seedlings were moved to MS medium containing 3% sucrose with different concentrations of NaCl (0, 75, 120, or 180 mM) and cultured for 10 d. There was no clear growth difference at 0, 75, and 120 mM NaCl between the wild type and lew1. However, at 180 mM NaCl, lew1 seedlings grew better than wild-type seedlings.

Figure 9.

RNA Gel Blot Analysis of Stress-Inducible Genes under Different Stress Conditions. (A) The transcripts of BiP and bZIP60 were induced to higher levels in lew1 than in the wild type by drought stress. Four-week-old seedlings were removed from soil and placed on a laboratory bench for 1 h (drought 1 [Dro1]). Then, seedlings were covered with a transparent film (to slow water loss) for 1 or 4 h (drought 2 h and 5 h [Dro2 and Dro5, respectively]). Total RNA from each treatment was used for RNA gel blot analysis. RNA from shoots without treatment was used as a control at 0 h (Con). rRNAs stained with ethidium bromide were used as loading controls. (B) More BiP transcripts accumulated in lew1 than in the wild type under mannitol treatment. Four-week-old seedlings from soil were dipped into solution containing 300 mM mannitol for 1, 2, or 6 h (Man1, Man2, and Man6, respectively), and total RNA was analyzed by RNA gel blot. rRNAs stained with ethidium bromide served as loading controls. RNA from shoots without treatment was used as a control at 0 h (0). (C) The transcripts of RD29A and COR47 were induced to higher levels in lew1 than in the wild type by drought stress. Drought treatment was done as in (A). rRNA was used as a loading control. (D) Four-week-old seedlings were treated with 150 mM NaCl for 0.5 h (NaCl0.5) or 1 h (NaCl1) or with 300 mM mannitol for 1 h (Man1) or 2 h (Man2). rRNAs stained with ethidium bromide served as loading controls. RNA from shoots without treatment was used as a control at 0 h (Con). (E) Two-week-old seedlings grown on MS medium were treated with 0.1 μg/mL tunicamycin for different times, and total RNA was analyzed by RNA gel blot. rRNAs stained with ethidium bromide served as loading controls.