Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 https://doi.org/10.1186/s12870-021-02898-7 RESEARCH ARTICLE Open Access Structural and functional analysis of tomato sterol C22 desaturase Laura Gutiérrez-García1, Montserrat Arró1,2, Teresa Altabella1,3, Albert Ferrer1,2 and Albert Boronat1,4* Abstract Background: Sterols are structural and functional components of eukaryotic cell membranes. Plants produce a complex mixture of sterols, among which β-sitosterol, stigmasterol, campesterol, and cholesterol in some Solanaceae, are the most abundant species. Many reports have shown that the stigmasterol to β-sitosterol ratio changes during plant development and in response to stresses, suggesting that it may play a role in the regulation of these processes. In tomato (Solanum lycopersicum), changes in the stigmasterol to β-sitosterol ratio correlate with the induction of the only gene encoding sterol C22-desaturase (C22DES), the enzyme specifically involved in the conversion of β-sitosterol to stigmasterol. However, despite the biological interest of this enzyme, there is still a lack of knowledge about several relevant aspects related to its structure and function. Results: In this study we report the subcellular localization of tomato C22DES in the endoplasmic reticulum (ER) based on confocal fluorescence microscopy and cell fractionation analyses. Modeling studies have also revealed that C22DES consists of two well-differentiated domains: a single N-terminal transmembrane-helix domain (TMH) anchored in the ER-membrane and a globular (or catalytic) domain that is oriented towards the cytosol. Although TMH is sufficient for the targeting and retention of the enzyme in the ER, the globular domain may also interact and be retained in the ER in the absence of the N-terminal transmembrane domain. The observation that a truncated version of C22DES lacking the TMH is enzymatically inactive revealed that the N-terminal membrane domain is essential for enzyme activity. The in silico analysis of the TMH region of plant C22DES revealed several structural features that could be involved in substrate recognition and binding. Conclusions: Overall, this study contributes to expand the current knowledge on the structure and function of plant C22DES and to unveil novel aspects related to plant sterol metabolism. Keywords: Tomato, Sterol metabolism, Stigmasterol, β-Sitosterol, Cytochrome P450, Sterol C22-desaturase, endoplasmic reticulum Background system with a hydroxyl group at position C3, methyl Sterols are isoprenoid-derived lipids that play an essential groups at positions C10 and C13 and a side chain of vari- role in the regulation of membrane fluidity, permeability able length attached to C17 [3]. Contrary to other and function [1, 2]. Sterols share a common structure eukaryotic organisms, plants are characterized by produ- based on the cyclopentane perhydro phenanthrene ring cing a high diversity of sterols that mostly differ in the nature of the side chain attached to C17, being β- sitosterol, stigmasterol and campesterol the most abun- * Correspondence: aboronat@ub.edu 1 Center for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra, dant ones, and in some Solanaceae also cholesterol. In Barcelona, Spain plants, sterols are present in free form and also in conju- 4 Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, gated forms as steryl esters, steryl glycosides and acyl University of Barcelona, 08028 Barcelona, Spain Full list of author information is available at the end of the article steryl glycosides. While free sterols and steryl glycosides © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 Page 2 of 14 are mainly found in the plasma membrane (PM), steryl form). Although it is widely accepted that free sterol esters accumulate in cytoplasmic lipid droplets [4]. biosynthesis occurs in the ER [23–25], the participa- Sterols are known to be crucial for the function of the tion of the PM in the final steps of the sterol path- PM as they modulate its physicochemical properties, as way has not been excluded [3]. All plant CYP well as the formation of microdomains (also known as proteins described so far are membrane-bound and lipid rafts) that are involved in many relevant cellular mainly localized in the ER. However, some particular processes such as cell to cell interactions, signal trans- CYP proteins have been reported in other subcellular duction, membrane transport, protein trafficking and localizations such as mitochondria, plastids and the stress responses [5, 6]. In plants, the right function of PM [26, 27]. Therefore, the elucidation of the sub- the PM depends on the balanced levels of campesterol, cellular localization of C22DES represents a relevant β-sitosterol and stigmasterol [7]. In particular, changes issue in plant sterol metabolism. Other important as- in the β-sitosterol/stigmasterol ratio have been proposed pects related to the characterization of C22DES are to influence different developmental processes and stress the identification of structural and functional motifs responses [7]. β-Sitosterol and stigmasterol only differ in involved in the intracellular targeting of the enzyme the double bond present at position C22 in stigmasterol as well as in its membrane topology and catalytic side chain (Additional file 1: Fig. S1). However, despite function. Some of these issues have been addressed their high structural similarity these sterols have a differ- in the present work using tomato C22DES, which ential effect on the physicochemical properties of the was chosen for this study not only because this plant PM [8]. Stigmasterol-enriched membranes are less per- is one of the most important crops worldwide but meable and, therefore, show a decreased leakage [9, 10]. also because, in contrast to other plant species, to- Therefore, the level of β-stigmasterol in the PM is mato contains a single gene coding for this enzyme expected to be tightly regulated during plant develop- [11, 17]. ment and stress responses [11–13]. Stigmasterol is the end product of the 24-ethyl branch of the sterol biosynthetic pathway and is synthesized from Results β-sitosterol by the action of the enzyme sterol C22- Tomato C22DES localizes in the ER desaturase (C22DES) (Additional file 1:Fig. S1) [11, 14]. To define the subcellular location of C22DES, a chimeric C22DES, also known as CYP710, belongs to the cyto- protein containing the entire tomato C22DES coding se- chrome P450 (CYP) protein family, which includes en- quence fused at the N-terminal end of the green fluores- zymes involved in numerous biosynthetic and xenobiotic cent protein (GFP) (C22DES-GFP) was transiently pathways in all living organisms. CYP proteins share a expressed in Nicotiana benthamiana leaves. As revealed common catalytic center including a heme-iron binding by confocal fluorescence microscopy, C22DES-GFP domain. NADPH acts as the electron donor in the reac- exhibited a typical ER-like pattern. This fluorescence tion catalyzed by C22DES through the action of cyto- pattern was essentially the same observed in cells co- chrome P450 reductase, a membrane-bound protein expressing T3RE protein fused to the red fluorescent localized in the ER membrane [14–16]. C22DES is phylo- protein (RFP) (T3RE-RFP), which was used as a specific genetically related to CYP51, a CYP protein having sterol marker for ER-localization [28] (Fig. 1). Actually, mer- 14-demethylase activity. CYP51 is common to the plant, ging the fluorescence of both channels revealed a clear yeast and animal sterol biosynthetic pathways [17, 18], overlap of the two images. and there is evolutionary evidence suggesting that these To make sure that the C-terminal GFP tag was not enzymes already existed in the most ancient eukaryotes affecting neither the correct targeting of C22DES nor [19]. Since C22DES acts downstream of CYP51 in the its catalytic activity, the stigmasterol levels of the sterol biosynthesis pathway it has been proposed that it agroinfiltrated N. benthamiana leaves expressing ei- evolved from a CYP51 gene duplication [19, 20]. ther the native C22DES or the chimeric C22DES-GFP Plant C22DES was cloned and characterized at the variant were determined and compared to those of biochemical level more than one decade ago [14, 21, 22]. leaves agroinfiltrated with the empty expression vector However, there are several functional and structural that were used as a control. As shown in Fig. 2, the aspects related to this enzyme that still remain un- total stigmasterol level increased by about 75% in a known. One of them concerns the elucidation of its similar way in both cases. These results revealed that subcellular localization, an aspect that is particularly the chimeric enzyme was properly targeted to the remarkable considering that C22DES may act on β- subcellular compartment(s) were its substrate (β-sitos- sitosterol synthesized in the ER during de novo terol) is found and also provided an in vivo assay to sterol biosynthesis and/or also on β-sitosterol evaluate the enzyme activity of a set of C22DES-GFP present in the PM (either in free or conjugated derivatives described below. Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 Page 3 of 14 Fig. 1 Subcellular localization of C22DES. Confocal optical sections showing the GFP and RFP fluorescence pattern of N. benthamiana cells transiently co-expressing the C22DES-GFP fusion protein (left) and the ER marker T3RE (middle). The merge of both images is shown on the right 3D-modeling of tomato C22DES composed by an N-terminal amphipathic α-helix The tertiary structure of tomato C22DES was modeled followed by a transmembrane α-helix [29], in C22DES with 100% confidence using the single highest scoring this region is shorter and contains a single putative template of the Saccharomyces cerevisiae lanosterol 14α- transmembrane α-helix (residues 1–28), that was demethylase (CYP51) crystal structure [29], which has referred to as TMH (Fig. 3a). Moreover, an interaction previously been reported to be an ER-membrane- model of C22DES with cell membranes, generated using associated enzyme [30]. The overall tertiary structure the PPM web server of the Orientations of Proteins in predicted for C22DES was very similar to that of yeast Membranes (OPM) database [31], predicted two other CYP51 although a remarkable difference was observed membrane contact regions in the protein sequence, in the N-terminal region. While in CYP51 it is namely MCR1 (residues 38–58) and MCR2 (residues Fig. 2 In vivo enzymatic activity of C22DES and C22DES-GFP. Stigmasterol levels in total sterol fractions of N. benthamiana leaves transiently expressing C22DES and C22DES-GFP. Values are mean values ± SD of three technical replicates (n = 3). Lowercase letters indicate significant differences among mean values relative to those in leaf samples expressing the empty vector (one-way ANOVA with Dunnett’s multiple comparisons test). DW: Dry weight Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 Page 4 of 14 Fig. 3 Predicted tomato C22DES tertiary structure. a Overall predicted fold of tomato C22DES. b Predicted orientation of C22DES in a dioleoylphosphatidylcholine (DOPC) membrane. The predicted N-terminal transmembrane helix (TMH) and the membrane contact regions MCR1 and MCR2 are indicated. c and d Sequence logo of the consensus MCR1 and MCR2 sequences obtained from the alignment of the plant C22DES proteins indicated in Additional file 2: Table S1 227–233) (Fig. 3a and b). MCR1 is a proline-rich motif fluorescence compatible with a cytosolic localization located in close proximity to TMH and highly conserved with fluorescence also present the nucleus (Fig. 4b). among C22DES from different plant species (Fig. 3c) However, MCR1-GFP also showed some overlap with whereas MCR2 is less conserved (Fig. 3d) and localizes that of T3RE (Fig. 5c). These results are in agreement in the globular domain next to a long amphipathic α- with the fact that MCR1 may confer the ability of helix predicted in all plant C22DES (Fig. 3a and b). MCR1-GFP to interact with the ER, reinforcing in this These results are consistent with our previous finding way the functional role of this motif in the interaction of showing that functional tomato C22DES localizes into C22DES with the ER. The localization of green fluores- the ER membrane of N. benthamiana cells. cence in the nucleus may be explained considering that at least part of the expressed MCR1-GFP was present in TMH is sufficient to target and retain tomato C22DES in soluble form in the cytosol. the ER membrane To study the role of the ER interacting sequences pre- The globular domain of tomato C22DES interacts with the dicted in the N-terminal region of tomato C22DES ER in the absence of TMH (TMH and MCR1), the amino acid sequences containing It has been reported that the globular domain of several residues 1 to 75 (including both TMH and MCR1), resi- CYP proteins interacts with the ER membrane in the ab- dues 1 to 37 (including only TMH), and residues 28 to sence of the transmembrane domain [32–35]. To deter- 66 (including only MCR1) were fused at the N-terminal mine if this is the case in tomato C22DES, a N-terminal end of GFP (Fig. 4a). The MCR1 motif was included in truncated form of the enzyme lacking the TMH region these studies because of its close proximity to TMH and (residues 2 to 27) was fused to the N-terminal end of its predicted interaction with the ER membrane (Fig. RFP and the resulting protein (C22DESΔ2–27-RFP) 4b). The subcellular localization of the resulting transiently expressed along with TMH-GFP in N. chimeric proteins (TMH + MCR1-GFP, TMH-GFP, and benthamiana leaves. The fluorescence distribution of MCR1-GFP, respectively) was analyzed by confocal mi- C22DESΔ2–27-RFP showed a typical reticular pattern croscopy after transient expression in N. benthamiana and co-localization with TMH-GFP (Fig. 5a). These leaves. The fluorescence distribution of TMH + MCR1- results indicated that the globular domain of tomato GFP and TMH-GFP resulted in a typical ER localization C22DES was interacting and retained in the ER in the pattern (Fig. 4b). In contrast, MCR1-GFP showed a absence of TMH. Fluorescence Recovery After Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 Page 5 of 14 Fig. 4 (See legend on next page.) Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 Page 6 of 14 (See figure on previous page.) Fig. 4 Role of TMH1 in the targeting and retention of C22DES in the ER membrane. a Schematic representation of the GFP fusion constructs generated to study the role of TMH and MCR1 in the targeting and retention of C22DES in the ER. Grey boxes indicate the transmembrane helix (TMH), orange boxes correspond to the MCR1 motif and green boxes correspond to the GFP protein. The amino acid sequence of TMH and MCR1 are shown below the corresponding regions. b Confocal optical sections showing the GFP fluorescence pattern of N. benthamiana cells transiently expressing TMH + MCR1-GFP, TMH-GFP, and MCR1-GFP. The arrow indicates the cell nucleus (n). c Close-up view of the fluorescence pattern of TMH-GFP and MCR1-GFP (left), T3RE (middle) and the corresponding merged images (right) Photobleaching (FRAP) analysis [36, 37] was performed analysis using anti-GFP antibodies of the cytosolic and to reinforce these results using the brassinosteroid membrane fractions obtained from N. benthamiana receptor BRL3 fused to GFP (BRL3-GFP) [38] and GFP leaves transiently expressing TMH-GFP, C22DES-GFP as membrane-bound and cytosolic control proteins, and C22DESΔ2–27-GFP demonstrated that they were respectively. As shown in Fig. 5b, C22DESΔ2–27-RFP present in the membrane fraction (Fig. 5c and d). In the showed a recovery rate similar to that of BRL3-GFP, immunoblot analysis C22DESΔ2–27-GFP was used thus confirming its behavior as an integral membrane instead of C22DESΔ2–27-RFP because anti-GFP anti- protein. In agreement with this result, immunoblot bodies do not recognize the RFP. Fig. 5 Targeting and retention of the C22DES globular domain in the ER. a Confocal optical sections showing the fluorescence of C22DESΔ2–27- RFP (left) and TMH-GFP (middle) transiently expressed in N. benthamiana leaves. Merged images are shown on the right. b FRAP curves representing the fluorescence recovery rates of C22DESΔ2–27-RFP, BRL3-GFP, and GFP. Fluorescence recovery curves represent the best fits from normalized datasets of at least 6 independently bleached points spots. (C) Cropped images of immunoblot analysis of soluble (S) and membrane (M) cell fractions from N. benthamiana leaves transiently expressing BRL3-GFP (≈153 kDa) and GFP (≈26.8 kDa) as membrane bound and soluble control proteins, respectively. Full-length blots are presented in Additional file 6: Fig. S4. c Cropped images from immunoblot analysis of soluble (S) and membrane (M) cell fractions from N. benthamiana leaves transiently expressing C22DESΔ2–27-GFP (≈84.2 kDa), TMH-GFP (≈34 kDa), and C22DES-GFP (≈87.2 kDa). Full-length blots are shown in Additional file 7: Fig. S5 Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 Page 7 of 14 TMH is required for tomato C22DES activity agroinfiltrated with the empty expression vector (Fig. 6b), The results reported above suggested that TMH could thus indicating that TMH is necessary for enzyme activity. have other functions in addition to anchor the enzyme to the ER. To explore this possibility, C22DESΔ2–27- The N-terminal region of C22DES from different plant GFP was transiently expressed in N. benthamiana leaves species share features that may be relevant for enzyme to evaluate its enzyme activity. C22DES-GFP was activity expressed in parallel as a positive control. The expres- The sequence alignment of C22DES from different plant sion of both proteins was determined by immunoblot species showed that both the length and the sequence of analysis using anti-GFP antibodies (Fig. 6a). As expected, the TMH was poorly conserved (Fig. 7). However, a the samples expressing C22DES-GFP showed an careful inspection of these sequences revealed the pres- increase in the total stigmasterol content (Fig. 6b). ence of several common features shared by all plant However, in the case of C22DESΔ2–27-GFP, the stig- C22DES proteins. One of them was the high number of masterol content was similar to that found in the leaves threonine and serine residues in the N-terminal half of Fig. 6 Test of C22DESΔ2–27 enzymatic activity in vivo (a) Cropped image from immunoblot analysis of C22DES-GFP (≈87.24 kDa) and C22DESΔ2–27-GFP (≈84.22 kDa) of agroinfiltrated N. benthamiana leaves. Full-length blot is presented in Additional file 8: Fig. S6. b Stigmasterol levels in total sterol fractions of N. benthamiana leaves expressing C22DES-GFP and C22DESΔ2–27-GFP. Values are mean values ± SD of three technical replicates (n = 3). Lowercase letters indicate significant differences among mean values relative to those in leaf samples expressing the empty vector (one-way ANOVA with Dunnett’s multiple comparisons test) Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 Page 8 of 14 Fig. 7 Multiple sequence alignment of the N-terminal region of plant C22DES. The sequence alignment of the N-terminal region of C22DES from the 27 plant species listed in Additional file 2: Table S1 is shown. Amino acid residues are numbered on the right. Asterisks denote residues conserved in all sequences. Colons indicate conservation between amino acid groups of strongly similar properties whereas periods indicate conservation between amino acid groups of weakly similar properties. Hyphens indicate gaps introduced to optimize the alignment. The tomato TMH sequence is highlighted in blue; prolines (P) are shown in red, and serine (S) and threonine (T) residues are shown in magenta. The CRAC1 motif (including the conserved Q27 and Y30 residues) and the MCR1 sequence are also shown. The branched-chain amino-acids [leucine (L), valine (V) and isoleucine (I)] in CRAC1 are shown in green, tyrosine (Y) in cyan and the dibasic residues [arginine (R) and lysine (K)] in yellow. Soly, Solanum lycopersicum; Klni, Klebsormidium nitens; Semo, Selaginella moellendorffii; Pisy, Pinus sylvestris; Sppo, Spirodela polyrhiza; Orsa, Oryza sativa; Deca, Dendrobium catenatum; Phda, Phoenix dactylifera; Anco, Ananas comosus; Amtr, Amborella trichopoda; Bevu, Beta vulgaris; Kafe, Kalanchoe fedtschenkoi; Potr, Populus trichocarpa; Cicl, Citrus clementina; Gora, Gossypium raimondii; Arth, Arabidopsis thaliana; Frve, Fragaria vesca; Ergu, Erythranthe guttata; Cusa, Cucumis sativus; Glma, Glycine max; Paso, Papaver somniferum; Jure, Juglans regia; Nenu, Nelumbo nucifera; Daca, Daucus carota; Cyca, Cynara cardunculus; Vivi, Vitis vinifera; Eugr, Eucalyptus grandis THM (Fig. 7). This may be relevant since some studies referred to as CRAC1) also includes the conserved glu- have described the role of hydroxylated residues in pro- tamine residue indicated above. tein transmembrane domains to provide substrate speci- ficity or correct associations with other membrane The globular domain of plant C22DES contains components through interactions with the hydroxyl cholesterol recognition/interaction amino acid consensus group of their polar side chains [39, 40]. Another motifs common feature was the presence of one or more pro- The multiple alignment of plant C22DES (Additional file 2: line residues in the N-terminal half of TMH (Fig. 7). Table S1) revealed the presence of other conserved choles- Since proline residues induce a turn of about 30 degrees terol recognition/interaction amino acid consensus motifs in α-helices, the TMH sequence of most plant C22DES in the globular domain. In addition to the already may have one or more turns in their N-terminal half. described CRAC motifs, the globular domain also contains The alignment shown in Fig. 7 also showed the con- the so called CARC motifs, which correspond to a specu- servation of a glutamine and a tyrosine residue (corre- lar sequence of the CRAC motifs and are defined by the sponding to Q27 and Y30 in the tomato sequence) and consensus K/R-X1–5-Y/F-X1–5-L/V). CARC motifs may the presence of a short stretch of positively charged resi- also bind cholesterol although in opposite orientation dues between TMH and MCR1. Interestingly, the con- [42]. Two CRAC motifs (CRAC2 and CRAC4), three served tyrosine residue and the positively charged CARC motifs (CARC1, CARC2 and CARC3) and one in residues were identified as elements of a cholesterol rec- which a CRAC motif and a CARC motif overlap (CRAC3/ ognition/interaction amino acid (CRAC) motif defined CARC4) were found to be conserved in the globular by the consensus –L/V-X1–5-Y-X1–5-R/K [41] (Fig. 7). domain of plant C22DES (Additional file 3: Fig. S2). The CRAC motifs are usually found in transmembrane heli- identification of such a large number of putative ces of membrane proteins. This putative CRAC motif cholesterol-interaction motifs was surprising and led to identified in the N-terminal region of C22DES (hereafter hypothesize that, at least in some cases, it could merely Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 Page 9 of 14 reflect the laxity of these consensus motifs. Since the N-terminal transmembrane domain of CYPs remains a functional CRAC and CARC motifs reported in the controversial issue. Thus, while some recombinant CYPs globular domain of membrane proteins are found in lacking the N-terminal anchor region have been α-helices [43, 44] it is likely that only those motifs lo- reported to be active in vitro, no activity could be cated in predicted α-helices (CARC2, CARC3, CRAC2 detected in in vivo assays [34, 51, 52]. The differential and CRAC3/CARC4) (Additional file 4: Fig. S3) might behavior of the N-terminal region of CYPs may be have a functional role in C22DES. Among them, related, at least in part, with the nature and subcellular CARC2, CARC3, and CRAC3/CARC4 could be of special availability of their substrates. This would explain why interest as they are present in amphipathic α-helices and some CYPs show activity under in vitro test conditions located near the catalytic site in the upper part of the in which the substrate is fully available, but not under globular domain (Additional file 4: Fig. S3). in vivo assay conditions where the availability of sub- strate may be a limiting factor. Furthermore, it has been Discussion proposed that the N-terminal membrane domain of The results obtained in this work demonstrate that to- some CYPs participates in the correct positioning of the mato C22DES is an ER-resident protein. The 3D model- globular domain with respect to the membrane during ing of tomato C22DES using the crystal structure of the catalysis. Thus, it has been reported that the transient phylogenetically related lanosterol 14α-demethylase tilting of the globular domain is an essential requirement (CYP51), an enzyme also involved in sterol metabolism to allow the interaction of CYPs with their substrates thought to be one of the most ancient and conserved when located within the hydrophobic core of the ER P450s across the kingdoms, predicted the presence of an membrane [53, 54]. In the particular case of C22DES, it N-terminal hydrophobic transmembrane domain and can be speculated that it could not interact with its sub- two short sequences (MCR1 and MCR2) located in the strate (β-sitosterol) unless TMH provides the right globular domain which also interact with the ER mem- anchoring of the globular domain to allow its tilting brane (Fig. 3). Subcellular localization studies using within the ER membrane during catalysis. C22DES derivatives fused to GFP indicated that TMH is Despite the essential role of TMH in tomato C22DES sufficient for the targeting and retention of C22DES in activity, it was surprising to find that both the length the ER (Fig. 4 and 5d). These studies also revealed that and the amino acid sequence of the N-terminal region the globular domain can interact and be retained in the of plant C22DES was poorly conserved (Fig. 7). How- ER membrane in the absence of TMH (Fig. 5). The ever, a detailed inspection of these sequences revealed sequence conservation of MCR1 and MCR2 among two conserved features that could be relevant for plant C22DES (Figs. 3c and d) supports a role for these enzyme activity: i) the presence of a cholesterol recogni- sequences in the interaction with the ER membrane. tion/interaction amino-acid consensus (CRAC) motif, The mechanisms underlying the interaction and reten- and ii) an enrichment of serine and threonine residues tion of the globular domain of C22DES in the ER remain in the N-terminal half of TMH. Cholesterol-binding to be characterized. However, it is likely that they could domains have been the focus of many studies involving involve the hydrophobic interaction of MCR1 and computational methods to explore the transmembrane MCR2 with the ER membrane and/or its interaction regions of animal proteins for which there is good evi- with other ER resident proteins such as NADPH-cyto- dence of their interaction with cholesterol [41, 42]. The chrome P450 reductase, which is required for the func- first motif to be identified was termed cholesterol recog- tion of CYPs [27, 45]. The interaction between some nition/interaction amino-acid consensus (CRAC) and CYP proteins to form heterodimers, as well as between fulfills the consensus (L/V)-X1–5-(Y)-X1–5-(R/K), (where CYPs and other proteins such as cytochrome b5 and X is any amino acid) [41, 42, 55]. Another cholesterol- UDP-glucuronosyltransferase (UGT)1A has also been binding motif named CARC corresponds to the mirror reported [46–50]. version of the CRAC motif with the consensus sequence The observation that the globular domain of tomato (K/R)-X1–5-(Y/F)-X1–5-(L/V) [42, 55]. Despite the CRAC C22DES was enzymatically inactive despite behaving as motif was initially identified and characterized in animal an integral membrane protein (Fig. 6) revealed that proteins [55–58], its cholesterol-binding function has TMH was also required for enzyme activity. This result also been demonstrated in plants [59]. Interestingly, a was somehow unexpected considering the recent work CRAC motif able to interact with β-sitosterol has of Gnanasekaran et al. (2015) showing that the N- recently been described in the type 1 cholecystokinin terminal region of CYP720B4, a plant cytochrome P450 receptor [60]. Thus, it is likely the CRAC1 motif present involved in isopimeric acid biosynthesis, was not essen- in the N-terminal region of plant C22DES (CRAC1) may tial for the activity of this enzyme when expressed in N. contribute to the interaction of the enzyme with the β- benthamiana leaves. However, the functional role of the sitosterol present in the ER membrane. Another feature Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 Page 10 of 14 reported in cholesterol-binding regions is the presence very challenging topic in the field of plant biology and of serine and threonine residues. Furthermore, the struc- biotechnology [11, 77]. In this sense, the progress in this tural analysis of several cholesterol-binding proteins has field should certainly benefit from the new advances in led to propose that the hydrophilic side-chain of these the characterization of C22DES at the genetic, cellular amino acids may interact with the C3-hydroxyl group of and biochemical levels. Although the structural and the cholesterol molecule [61]. Thus, it is likely that the functional studies carried out in the present work repre- serine and threonine residues present in the N-terminal sent a significant contribution towards this end, we be- half of TMH could enhance the interaction of C22DES lieve that future research in this area should pay special with β-sitosterol in the ER, facilitating in this way its attention to better understand the cellular and molecular interaction with the CRAC1 motif. mechanisms involved in the regulation of C22DES activ- The localization of C22DES in the ER was not unex- ity considering the different pools of β-sitosterol present pected considering that other enzymes involved in sterol in the plant cell endomembrane system. biosynthesis, such as SMT1, CPI1, HYD1, and DWF/ DIM [5, 62–64] as well as cytochrome P450 reductase, Conclusion the physiological redox partner of cytochrome P450s, Tomato C22DES is an integral ER-membrane protein also localize in this cellular compartment [65]. However, having a single transmembrane α-helix at the N- the localization of C22DES in the ER raises the question terminal end (TMH). Two short sequences able to inter- about how this enzyme can act on the major cellular act with the ER membrane (MCR1 and MCR2) have also pool of β-sitosterol present in the PM. It is likely that been predicted in the globular domain. TMH is suffi- this process may involve the capacity of the ER to phys- cient for the targeting and retention of the enzyme in ically interact with the PM at structures known as ER- the ER-membrane. However, the globular domain can PM contact sites in which both membranes are in close also interact and be retained in the ER-membrane in the contact [66–68]. The possibility that enzymes located in absence of TMH, which is required for C22DES activity one membrane may act on substrates present in a differ- in vivo. The TMH region contains a highly conserved ent cell membrane, the so-called in trans activity, is not cholesterol recognition/interaction amino-acid consen- unprecedented [69–72]. Interestingly, specific ER-PM sus (CRAC) motif and is enriched in threonine and contact sites involved in the regulation of lipid- serine residues. These features may be relevant for the homeostasis, including phospholipids and sterols, have recognition and uptake of the β-sitosterol present in the recently been reported [73]. Furthermore, enzymes in- ER membrane to the catalytic site of the enzyme. Over- volved in lipid biosynthesis have been localized at ER- all, the results presented here suggest the existence of a PM contact sites and shown to contribute to their for- complex pattern of interactions of C22DES with the ER- mation [72, 74]. However, and to the best of our know- membrane which are essential for proper enzyme func- ledge, the involvement of ER-PM contact sites in sterol tion. The molecular mechanisms underlying the inter- or lipid homeostasis has not yet been reported in plants, action of C22DES with the major cellular pool of β- although it has been suggested that the PM-localized to- sitosterol present in the PM remain unknown and de- mato Acyl-CoA:sterol acyltransferase (SlASAT1) may serve further studies. act in trans on its substrate cycloartenol in the ER lipid bilayer at ER–PM contact sites to produce cycloartenyl Methods esters [75]. The existence of several conserved CRAC/ Plant material CARC motifs in the globular domain of plant C22DES Nicotiana benthamiana plants (obtained from the Plant suggests that they could have a role in the interaction of Growth Service of the Center for Research in Agricul- the enzyme with the β-sitosterol present in the PM. In tural Genomics, Barcelona) were grown under standard this respect, the localization of CARC2, CARC3 and greenhouse conditions (14 h light at 26 ± 1 °C and 10 h CRAC3/CARC4 in amphypathic α-helices may be rele- dark at 21 ± 1 °C) in individual pots of 12 cm of vant considering that this type of structures has been de- diameter. scribed to serve not only as membrane interaction domains but also as lipid binding sites [76]. The study of Cloning and plasmid constructions the contribution of these CARC and CRAC motifs in the All the protein-coding sequences lacking the stop codon activity of C22DES on the β-sitosterol present in the PM used for in-frame fusions of tomato C22DES with the represents an interesting issue in further studies dealing GFP and RFP were amplified by PCR using 35S:C22DES with the functional characterization of this enzyme and plasmid as a template, which was previously obtained in the regulation of plant sterol metabolism. the laboratory and contained the open reading frame Cellular and metabolic engineering approaches aimed coding for the C22DES (GenBank: NM_001247585). All at modifying stigmasterol levels in plants represent a the PCR reactions were performed using high fidelity Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 Page 11 of 14 AccuPrime™ Taq DNA polymerase (Invitrogen) and spe- confocal laser-scanning microscope using the 60x water cific primer pairs (Additional file 5: Table S2). Amplifi- immersion NA: 1.20 objective. The emission windows cation products were purified and cloned into for fluorescence visualization and the conditions used pDONR207 donor vector using Gateway® technology for image acquisition have been described in Ramírez- (Invitrogen) and the resulting pENTRY plasmids trans- Estrada et al. [83]. Fluorescence recovery after photo- formed into chemically competent Top10 E. coli cells, bleaching (FRAP) analysis were also performed as which were used for all cloning steps. The cDNA se- described in Ramírez-Estrada et al. [83] quences in the resulting pENTRY plasmids were se- quenced to confirm the absence of mutations derived Sterol analysis from the amplification process. The verified sequences N. benthamiana leaves from three independently agroin- were sub-cloned into the binary vectors pEarleyGate103 filtrated plants were frozen in liquid nitrogen, ground [78] and pGWB454 [79] using Gateway® technology to with a mortar and pestle and lyophilized. Samples (30 respectively generate GFP and RFP fusions at the C- mg) were mixed with internal standards [2.5 μg of cho- terminus under the control of the CaMV35S promoter. lestanol, 5 μg of palmitoyl-cholestanol, 5 μg of The obtained constructs were confirmed by restriction cholestanyl-β-D-glucoside and 5 μg of palmitoyl-β-D- mapping and DNA sequence analysis. glucosyl-cholestanol in chloroform-methanol (2:1)] and extracted with chloroform-methanol (2:1) as indicated in Agroinfiltration of N. benthamiana leaves Ramírez-Estrada et al. [83]. Free sterol, steryl ester, steryl Subcellular localization assays were performed by ex- glucoside and acyl steryl glcusoside fractions were sepa- pression of C22DES fusions with GFP or RFP proteins rated by TLC and their sterol composition determined in leaves of 3–5-week-old N. benthamiana plants grown by GC-MS as described in Lara et al. (2018) [75]. Total under standard greenhouse conditions (14 h light at stigmaterol levels were calculated from those present in 26 ± 1 °C and 10 h dark at 21 ± 1 °C) in individual pots of the four sterol fractions extracted from each sample. 12 cm diameter. Leaves were infiltrated with suspensions of the different GV3101 A. tumefaciens strains har- Immunoblot analysis boring the corresponding recombinant expression Soluble (S) and membrane (M) protein fractions from N. plasmids [80, 81], which were prepared as follows. A benthamiana agroinfiltrated leaves were obtained from single positive colony per construct was inoculated approximately 10 g of tissue samples as previously into 3 mL of YEB medium supplemented with the reported [83]. Protein concentration was determined as right antibiotics (rifampicin 50 μg/ml, gentamycin described in Bradford et al., 1976 [84]. Equivalent pro- 25 μg/ml and the plasmid selective antibiotic) and in- tein amounts of the T fractions (samples before ultra- cubated overnight at 28 °C and at 250 rpm in a con- centrifugation) (around 20 μg), M fractions (around tinuous rotary shaker. A 1:100 dilution of the 5 μg) and S fractions (around 20 μg) from each leaf sam- overnight culture was inoculated into 25 mL of YEB ple were fractionated by 10% polyacrylamide-SDS gel medium containing the same antibiotics and incu- electrophoresis TGX™ FastCast™ Gel (Bio-Rad). After bated under the same conditions. The culture was SDS-PAGE, the proteins were transferred to a 0.45 μm centrifuged at 5.000 rpm for 15 min at 4 °C and the nitrocellulose membrane (Amersham, GE Healthcare) bacterial pellets resuspended in infiltration buffer (10 using the Trans-Blot® Turbo™ Transfer system (Bio-Rad). mM MES, pH 5.6, 10 mM MgSO4 and 150 μM aceto- Immunoblots using a rabbit anti-GFP antibody (Invitro- syringone) to reach a final OD600 of 1. Cultures of gen) were performed as described in Ramírez-Estrada the transformed A. tumefaciens strains were separately et al. [83]. mixed with a culture of A. tumefaciens strain express- ing HC-Pro [82] in a 1:1 ratio and infiltrated in the In silico analysis of protein structure abaxial part of N. benthamiana leaves using a syringe. The 3D structure of the C22DES (NP_001234514.1) was For co-expression analysis, the strains harboring the modeled using Phyre2 fold recognition server [85] different expression plasmids were mixed in equal (http://www.sbg.bio.ic.ac.uk/phyre2). The tertiary struc- proportions and also with HC-Pro (the mix never ture was predicted using the Lanosterol 14α- reaching an OD600 higher than 1). Agroinfiltrated Demethylase (Erg11p) of Saccharomyces cerevisiae [Pro- plants were let to grow for 3–4 days under the green- tein Data Bank (PDB) ID: c4lxjA] as a template with house conditions indicated above. 100% confidence. For membrane-protein interactions, the predicted 3D models were orientated using PPM Confocal microscopy web server from the Orientations of Proteins in Mem- Agroinfiltrated leaves were cut into small pieces and the branes (OPM) database [31] (https://opm.phar.umich. abaxial epidermis analyzed with an Olympus FV 1000 edu/ppm_server). Gutiérrez-García et al. BMC Plant Biology (2021) 21:141 Page 12 of 14 Protein sequence analysis Acknowledgements S. lycopersicum C22DES protein sequence was retrieved LGG received a doctoral fellowship (Ref. BES-2014-070425) from the Minis- terio de Economia y Competitividad (Spain). We also thank the staff of the from the SolGenomics Net work we bs ite (h tt p :// Microscopy and Imaging, Genomics and Plant Growth facilities of the Centre solgenomics.net/) and used as query to search for other for Research in Agricultural Genomics (CRAG). plant species homologs using the BLAST tool on the Phytozome (https://phytozome.jgi.doe.gov), GenomeNet Authors’ contributions AB, TA and AF conceived and designed the research; LGG conducted the (https://www.genome.jp/), NCBI (https://www.ncbi.nlm. experimental work with the contribution of MA in the sterol analysis. LGG nih.gov/), PLAZA (https://bioinformatics.psb.ugent.be/ and AB wrote the manuscript. All authors have read and approved the plaza/) and the EnsemblPlants (http://plants.ensembl. manuscript. org) websites. The accession numbers of the used homo- Funding logs are listed in Additional file 2: Table S1. Protein This work was funded by grants AGL2017–88842-R from FEDER/Ministerio de alignments were performed using ClustalX v 2.0 [86] Ciencia, Innovación y Universidades-Agencia Estatal de Investigación (Spain), with default settings and the GeneDoc software was used 2017SGR710 from the Generalitat de Catalunya, and by the CERCA Programme of the Generalitat de Catalunya. We also acknowledge financial for alignment visualizations and manual edition. The cri- support from the Spanish Ministerio de Economía y Competitividad through teria for sequence inclusion on the final alignment was the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016–2019 choosing one C22DES from each plant family, preferably (SEV-2015- 0533). The funders have not been involved in the design of the study, in the collection, analysis, and interpretation of data and in writing the those in which C22DES was a single-copy gene. For the manuscript. plant species with more than one C22DES paralog, the selection of just one of them was based on the highest Availability of data and materials similarity of each paralog with those C22DES from the All data generated or analysed during this study are included in this published article [and its supplementary information files]. The datasets used single-copy species. and/or analysed during the current study are available from the For sequence logo generation, WebLogo web server corresponding author on reasonable request. was used [87] (https://weblogo.berkeley.edu/). Declarations Abbreviations Ethics approval and consent to participate BLAST: Basic local alignment search tool; C22DES: Sterol C22-desaturase; Not aplicable. CARC: CRAC specular sequence; CRAC: Cholesterol recognition/interaction amino acid consensus; CYP: Cytochrome P450; DW: Dry weight; Consent for publication ER: Endoplasmic reticulum; FRAP: Fluorescence recovery after Not aplicable. photobleaching; GC-MS: Gas chromatography-mass spectrometry; GFP: Green fluorescent protein; HCPRO: HC-Pro silencing supressor; MCR: Membrane contact region; OD600: Optic density at 600 nm; Competing interests OPM: Orientation of proteins in membranes; PCR: Polymerase chain reaction; The authors declare that they have no competing interests. PM: Plasma membrane; RFP: Red fluorescent protein; SD: Standard deviation; SDS: Sodium Dodecyl Sulfate; SDS-PAGE: SDS-Polyacrylamide Gel Author details 1 Electrophoresis; TLC: Thin layer chromatography; TMH: Transmembrane helix Center for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra, motif Barcelona, Spain. 2Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain. 3Department of Biology, Healthcare and the Environment, Faculty of Supplementary Information Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, The online version contains supplementary material available at https://doi. Spain. 4Department of Biochemistry and Molecular Biomedicine, Faculty of org/10.1186/s12870-021-02898-7. Biology, University of Barcelona, 08028 Barcelona, Spain. Additional file 1: Supplementary Figure S1, Reaction catalyzed by Received: 21 October 2020 Accepted: 21 February 2021 C22DES. 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