Arabidopsis membrane steroid binding protein 1 is involved in inhibition of cell elongation

Abstract

A putative Membrane Steroid Binding Protein (designated MSBP1) was identified and functionally characterized as a negative regulator of cell elongation in Arabidopsis thaliana. The MSBP1 gene encodes a 220-amino acid protein that can bind to progesterone, 5-dihydrotestosterone, 24-epi-brassinolide (24-eBL), and stigmasterol with different affinities in vitro. Transgenic plants overexpressing MSBP1 showed short hypocotyl phenotype and increased steroid binding capacity in membrane fractions, whereas antisense MSBP1 transgenic plants showed long hypocotyl phenotypes and reduced steroid binding capacity, indicating that MSBP1 negatively regulates hypocotyl elongation. The reduced cell elongation of MSBP1-overexpressing plants was correlated with altered expression of genes involved in cell elongation, such as expansins and extensins, indicating that enhanced MSBP1 affected a regulatory pathway for cell elongation. Suppression or overexpression of MSBP1 resulted in enhanced or reduced sensitivities, respectively, to exogenous progesterone and 24-eBL, suggesting a negative role of MSBP1 in steroid signaling. Expression of MSBP1 in hypocotyls is suppressed by darkness and activated by light, suggesting that MSBP1, as a negative regulator of cell elongation, plays a role in plant photomorphogenesis. This study demonstrates the functional roles of a steroid binding protein in growth regulation in higher plants.

Figure 1.

Structure of MSBP1. (A) Exon/intron structure of the MSBP1 gene. Two exons (381 and 525 bp; boxed) were identified via comparison of the MSBP1 cDNA and genomic sequences. Numbers indicate sizes (in nucleotides). (B) Schematic representation of the SBPs, indicating conserved transmembrane and steroid binding regions. Accession numbers are Q95250 (PGC1_PIG) and O15173 (PGC2_HUMAN). Numbers indicate positions of amino acid residues. (C) Multiple amino acid sequence alignment of MSBP1 and selected PGC1_PIG and PGC2_HUMAN. Alignments are colored using the ClustalX scheme in Jalview. Orange, Gly (G); light green, Pro (P); blue, small and hydrophobic amino acids (A, V, L, I, M, F, and W); dark green, hydroxyl and amine amino acids (S, T, N, and Q); magenta, negatively charged amino acids (D and E); red, positively charged amino acids (R and K); dark blue, His (H) and Tyr (Y). The α-helix and β-sheet are indicated as magenta rectangles and yellow arrows, respectively. Note that the secondary structure of the steroid binding region is highly conserved. (D) Predicted stereostructure of MSBP1 (right panel) and PGC1_PIG (left panel). The steroid binding domain consists of a mixed α+β structure with two pairs of α-helices forming a binding pocket to one side of β-sheets. The prediction was performed at http://swissmodel.expasy.org and viewed with the program of Deep View Swiss-Pdb Viewer written by Nicolas Guex, Alexandre Diemand, Manuel C. Peitsch, and Torsten Schwede. Arrow indicates the putative binding pocket.

Figure 2.

Steoid Binding Assays for MSBP1. (A) Expression and purification of recombinant MSBP1. M. mass, molecular mass marker; lanes 1, 2, and 3, uninduced or induced crude extract and purified recombinant control proteins (AtIPK2α); lanes 5, 6, and 7, uninduced or induced crude extract and purified MSBP1. Induction was performed with the supplementation of IPTG (1 mM in final concentration) for 2.5 h. Arrow shows the position of recombinant MSBP1. (B) Recombinant MSBP1 binds progesterone in vitro. The K_d value and B_max were calculated from data of three independent experiments. AtIPK2α, which was used as control protein, showed no binding to labeled progesterone. (C) Binding of MSBP1 to [³H]-progesterone was competitively inhibited by unlabeled progesterone or steroid molecules 5α-DHT, 24-eBL, and stigmasterol but not by ABA. IC₅₀ showed the different binding affinity of MSBP1 to various steroids.

Figure 3.

Expression Pattern Analysis of MSBP1 in Various Tissues and Differential Expression of which under Light and Darkness. (A) Semiquantitative RT-PCR analysis revealed constitutive expression of MSBP1 in various tissues, including cotyledon, stem, root, leaf, and flower. Actin2 was used as a positive internal control. (B) Promoter-reporter gene fusion studies indicated that MSBP1 was expressed in Arabidopsis seedlings and root tip (a), leaf (b), stem (c), petal (d), base of flower (e), and stigma (f). (C) Semiquantitative RT-PCR analysis revealed that expression of MSBP1 was not altered after treatment of seedlings with osmotic stresses and plant hormones, including auxin, BR, gibberellin, ABA, and cytokinin, but was severely suppressed in darkness (top panel). Detailed studies of MSBP1 expression indicated normal transcript levels under light and very low levels in darkness and induction when seedlings were transferred to light. By contrast, MSBP1 expression was suppressed when seedlings were transferred from light to darkness (bottom panel). M, DNA marker; C, untreated control; L, light; D, darkness; L-D, the seedlings were transferred from light to darkness for 24, 48, and 72 h; D-L, the seedlings were transferred from darkness to light for 24, 48, and 72 h. The Actin2 gene was used as a positive internal control. (D) Differential expression of MSBP1-GUS in hypocotyls under dark and light conditions. After 2 d of germination, MSBP1-GUS was strongly expressed in light and obviously suppressed by dark. Bar = 1 mm. (E) MSBP1 expression was not detected in hypocotyls after germination for 5, 6, and 7 d under darkness but was induced by transferring 4-d-old dark-grown seedlings into the light condition for 1 (Day 5, Dark→light), 2 (Day 6, Dark→light), and 3 d (Day 7, Dark→light). By contrast, expression of MSBP1 (L) was suppressed when 4-d-old light-grown seedlings were transferred to darkness for 1 (Day 5, Light→dark), 2 (Day 6, Light→dark), and 3 d (Day 7, Light→dark). Bar = 1 mm. (F) Expression of MSBP1-GUS under various light conditions (continuous blue light, red light, and far-red light). Seedlings were grown in each light condition for 6 d.

Figure 4.

Enhanced Expression of MSBP1 Results in Shortened Hypocotyls Because of Reduced Cell Elongation, whereas Its Suppressed Expression Results in Elongated Hypocotyls with Increased Cell Length. (A) Overexpression of MSBP1 in transgenic plants harboring CaMV35S:O-MSBP1. Expression of MSBP1 RNA was analyzed by RT-PCR in wild-type plants (labeled as C in [A] to [H]) and independent transgenic lines (O-L1, O-L2, O-L3, and O-L4). The Actin2 gene was used as an internal control. (B) Deficiency of MSBP1 in transgenic plants harboring CaMV35S:A-MSBP1. Expression of MSBP1 RNA was analyzed by RT-PCR in wild-type (C) and independent transgenic lines (A-L1, A-L2, A-L3, and A-L4). External primers at the 5′ and 3′ untranslated regions were used for detecting sense MSBP1 transcript, and internal primers were used to detect both sense and antisense transcripts. The Actin2 gene was used as an internal control. (C) The membrane fractions from MSBP1-overexpressing plants presented a higher binding capacity to progesterone than that from wild-type plants (B_max, 63.0 ± 5.4 compared with 35.8 ± 3.8 fmol/μg·membrane protein), with the similar K_d constant (K_d = 36.7 ± 2.5 nM compared with 31.5 ± 4.6 nM), whereas the binding capacity of the membrane fractions from MSBP1-deficient plants indicated the presence of other unknown binding proteins (B_max, 23.9 ± 2.5 fmol/μg·membrane protein). For information of transgenic plants, please refer to Figures 4A and 4B. (D) Growth of control and transgenic plants in darkness for 4 (left) and 6 (right) d. Heteroscedastic t test analysis showed the changes of P-value < 0.01 (**). Error bar represents se. (E) Growth and relative growth rates of control and transgenic plants in darkness for 4 d (open bars) then transferred to light for 4 d (closed bars, left panels) and under light for 6 d (right). Heteroscedastic t test analysis showed the changes of P-value < 0.05 (*) and P-value < 0.01(**). Error bar represents se. (F) Calculations of cell length (left) and longitudinal section (right) indicate decreased cell elongation in transgenic plants overexpressing MSBP1. Six-day-old seedlings under dark conditions were used. Heteroscedastic t test analysis showed the changes of P-value < 0.01 (**). Bar = 100 μm. Error bar represents se. (G) Calculations of cell length (left) and longitudinal section (right) indicate enhanced cell elongation in MSBP1-deficient transgenic plants. Seedlings were grown for 6 d under light conditions. Heteroscedastic t test analysis showed the changes of P-value < 0.05 (*). Bar = 100 μm. Error bar represents se. (H) RT-PCR analyses of the expression of cell division- and cell elongation–related genes in MSBP1-sense (O-MSBP1) or -antisense (A-MSBP1) transgenic plants. O-L1, O-L2, A-L1, and A-L3, independent transgenic lines.

Figure 5.

Transgenic Plants Overexpressing or Deficient for MSBP1 Showed Altered Sensitivities to Exogenous Progesterone and 24-eBL. Seven-day-old seedlings grown under light or darkness conditions were measured. Error bar represents se. (A) MSBP1-overexpressing (O-MSBP1) plants showed reduced sensitivities to 24-eBL under light conditions, and MSBP1-deficient plants (A-MSBP1) showed increased sensitivity. Seedlings were grown on media containing various concentration of 24-eBL for 7 d. The relative promotion ratios (%) are shown at bottom (B) Effect of 24-eBL on hypocotyl lengths of wild-type (C), A-MSBP1, and O-MSBP1 plants in the dark. Seedlings were grown on media containing various concentration of 24-eBL for 7 d. The relative inhibition ratios (%) are shown at bottom. (C) Effect of 24-eBL on hypocotyl lengths of wild-type (C), A-MSBP1, and O-MSBP1 plants grown under light conditions. Seedlings were grown on media containing various concentration of progesterone for 7 d. The relative promotion ratios (%) are shown at bottom. (D) Increasing progesterone inhibited the hypocotyls elongation of the wild-type (C) and MSBP1-deficient plants but not the O-MSBP1 plants under darkness. Seedlings were grown in the dark on media containing various concentration of progesterone for 7 d. The relative inhibition ratios (%) are shown at bottom.

Figure 6.

MSBP1 Localizes to the Plasma Membrane. Green fluorescence in transgenic plants harboring mock vector (CaMV35S:mGFP5) shows that mGFP5 is ubiquitously distributed in the cell cytoplasm ([A], hypocotyl cells; [C], root-tip cells), whereas that in transgenic plants expressing CaMV35S:MSBP1:mGFP5 is localized to the plasma membrane ([B], hypocotyl cells; [D], root-tip cells). Bars = 20 μm.

Figure 7.

A Possibly Conserved Function of MSBP1 in Higher Plants. (A) A functional model for MSBP1. Transcription of MSBP1 is activated by light signaling. The MSBP1 protein is localized to the plasma membrane. MSBP1 may perceive a growth-inhibiting steroid signal. Alternatively, MSBP1 may sequester or transport a growth-promoting steroid away from its site of action (such as inside versus outside of the cell). (B) Presence of multiple SBPs in higher plants. Alignment of MSBP1 with other predicted SBPs found in the genomes of Arabidopsis and O. sativa. Accession numbers are as follows: At2g24940, At3g38890, At4g14965, NM_197219, and NM_197217. The transmembrane and steroid binding regions are indicated.