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Steroids 76 (2011) 524–529 Contents lists available at ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids Rapid communication Oxysterol binding proteins (OSBPs) and their encoding genes in Arabidopsis and rice Pavan Umate Department of Biology, Washington University, Rebstock Hall, Room No. 202, Campus Box 1137, St. Louis, MO 63130, USA article info Article history: Received 10 November 2010 Received in revised form 21 December 2010 Accepted 20 January 2011 Available online 31 January 2011 Keywords: Gene family Phylogeny Protein alignment Sequence LOGO Transcriptomics abstract Cell wall deposition, biosynthesis of steroid hormones, and maintenance of membrane composition and integrity, are some of the crucial functions of sterols in plants. Followed by their synthesis in the endo- plasmic reticulum, the sterols accumulate in the plasma membrane. The concept of sterol trafficking in plant cell is not well understood. The oxysterol binding proteins are implicated in sterol transport in non-plant systems. In the study, the oxysterol binding proteins in Arabidopsis and rice are described and classified. The Arabidopsis genome encodes 12 oxysterol binding proteins-related proteins (ORPs) as compared to 6 oxysterol binding proteins (OSBPs/ORPs) in rice. The protein alignment studies reveal that amino acid sequences for oxysterol binding proteins are relatively well conserved in Arabidopsis and rice. The rice OSBPs are classified based on their phylogenetic relationship with Arabidopsis ORPs. The sequence LOGO built on LOC Os03g16690 indicated presence of fingerprint region of amino acids “EQVSHHPP” for Arabidopsis and rice OSBPs/ORPs. The organization of pleckstrin homology domain is identified in several OSBPs/ORPs in Arabidopsis and rice. The Arabidopsis oligonucleotide array data is explored to understand the expression patterns of ORPs under 17 different experimental conditions. The analysis showed the expression of ORPs in Arabidopsis is necessarily under the control of biotic stress, chemical, elicitor, hormone, light intensity, abiotic stress, and temperature conditions. The linear mean signal values for Arabidopsis ORPs revealed their relative expression patterns in different developmen- tal stages. The genes for ORP3C and ORP3B are highly expressed in all developmental stages that were analyzed. The present study thus indicates crucial functional role of the individual members of this gene family in different environmental stress conditions. © 2011 Elsevier Inc. All rights reserved. Sterols are vital cell constituents. They play role in several dynamic aspects of membrane functioning like fluidity and perme- ability, in metabolic processes related to membrane properties, and secretory trafficking events. The sterols also act as precursors for brassinosteroid (BR) hormones [1]. The impaired cell wall features, and drastic reduction in cellulose, callose, and lignin content results from defects in sterol biosynthesis [2,3]. The Arabidopsis mutants, hydra1 and fackel/hydra2, which are defective in the enzymes sterol isomerase and sterol C-14 reductase are impaired in vascu- lar patterning [4]. Apparently, sterols are linked to key aspects of development and hormone signaling in plants. The major forms of sterols like sitosterol, stigmasterol, and 24-methylcholesterol are synthesized by plants. The metabolic events linked of cellulose biosynthesis, and maintenance of mem- brane integrity are attributed to sitosterols [5,6]. The oxysterols, oxygenated derivatives of cholesterol, occur in minor amount in plants [7,8]. The oxysterols are implicated in regulating processes as diverse as Ca 2+ uptake and apoptosis, to transcriptional activ- E-mail address: pavan [email protected] ity, and cellular differentiation [9]. The mechanism for transfer of newly synthesized sterols from the endoplasmic reticulum (ER) to the plasma membrane is still unclear. In non-plant systems, sterol trafficking occurs with the aid of oxysterol binding protein (OSBP), and OSBP-related proteins (ORPs) [10–12]. Other functions are also attributed to these proteins like signaling cascades, endo- somal movement, lipid sensing, and regulation of cellular sterol distribution [13]. The OSBPs/ORPs are also involved in membrane trafficking [14], tumor metastasis [15], and cell cycle progression [16]. Most eukaryotic genomes encode for OSBPs and ORPs. The ORPs have been studied in humans [17], Drosophila [16], and fungi [14,18–21]. There are 12 OSBP/ORP genes identified in human [9,12,22,23], as well as in mouse [24], and 7 in yeast [10]. In Ara- bidopsis genome, 12 putative ORP genes have been identified [25]. Apart from oxysterols, the ORPs can also bind different lipids, including phosphoinositides, ergosterol, and cholesterol [26–30]. The pleckstrin homology (PH) domains, a conserved domain con- taining an FFAT motif (two phenylalanines embedded in an acidic tract), and ankyrin repeats are present in most ORPs [31]. The tar- geting of ORPs to cellular compartments is facilitated with the aid 0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2011.01.007

Oxysterol binding proteins (OSBPs) and their encoding genes in Arabidopsis and rice

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Page 1: Oxysterol binding proteins (OSBPs) and their encoding genes in Arabidopsis and rice

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Steroids 76 (2011) 524–529

Contents lists available at ScienceDirect

Steroids

journa l homepage: www.e lsev ier .com/ locate /s tero ids

apid communication

xysterol binding proteins (OSBPs) and their encoding genes inrabidopsis and rice

avan Umateepartment of Biology, Washington University, Rebstock Hall, Room No. 202, Campus Box 1137, St. Louis, MO 63130, USA

r t i c l e i n f o

rticle history:eceived 10 November 2010eceived in revised form1 December 2010ccepted 20 January 2011vailable online 31 January 2011

eywords:ene familyhylogenyrotein alignmentequence LOGOranscriptomics

a b s t r a c t

Cell wall deposition, biosynthesis of steroid hormones, and maintenance of membrane composition andintegrity, are some of the crucial functions of sterols in plants. Followed by their synthesis in the endo-plasmic reticulum, the sterols accumulate in the plasma membrane. The concept of sterol traffickingin plant cell is not well understood. The oxysterol binding proteins are implicated in sterol transportin non-plant systems. In the study, the oxysterol binding proteins in Arabidopsis and rice are describedand classified. The Arabidopsis genome encodes 12 oxysterol binding proteins-related proteins (ORPs)as compared to 6 oxysterol binding proteins (OSBPs/ORPs) in rice. The protein alignment studies revealthat amino acid sequences for oxysterol binding proteins are relatively well conserved in Arabidopsisand rice. The rice OSBPs are classified based on their phylogenetic relationship with Arabidopsis ORPs.The sequence LOGO built on LOC Os03g16690 indicated presence of fingerprint region of amino acids“EQVSHHPP” for Arabidopsis and rice OSBPs/ORPs. The organization of pleckstrin homology domain isidentified in several OSBPs/ORPs in Arabidopsis and rice. The Arabidopsis oligonucleotide array data is

explored to understand the expression patterns of ORPs under 17 different experimental conditions. Theanalysis showed the expression of ORPs in Arabidopsis is necessarily under the control of biotic stress,chemical, elicitor, hormone, light intensity, abiotic stress, and temperature conditions. The linear meansignal values for Arabidopsis ORPs revealed their relative expression patterns in different developmen-tal stages. The genes for ORP3C and ORP3B are highly expressed in all developmental stages that wereanalyzed. The present study thus indicates crucial functional role of the individual members of this gene

nmen

family in different enviro

Sterols are vital cell constituents. They play role in severalynamic aspects of membrane functioning like fluidity and perme-bility, in metabolic processes related to membrane properties, andecretory trafficking events. The sterols also act as precursors forrassinosteroid (BR) hormones [1]. The impaired cell wall features,nd drastic reduction in cellulose, callose, and lignin content resultsrom defects in sterol biosynthesis [2,3]. The Arabidopsis mutants,ydra1 and fackel/hydra2, which are defective in the enzymesterol isomerase and sterol C-14 reductase are impaired in vascu-ar patterning [4]. Apparently, sterols are linked to key aspects ofevelopment and hormone signaling in plants.

The major forms of sterols like sitosterol, stigmasterol, and4-methylcholesterol are synthesized by plants. The metabolicvents linked of cellulose biosynthesis, and maintenance of mem-

rane integrity are attributed to sitosterols [5,6]. The oxysterols,xygenated derivatives of cholesterol, occur in minor amount inlants [7,8]. The oxysterols are implicated in regulating processess diverse as Ca2+ uptake and apoptosis, to transcriptional activ-

E-mail address: pavan [email protected]

039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.steroids.2011.01.007

tal stress conditions.© 2011 Elsevier Inc. All rights reserved.

ity, and cellular differentiation [9]. The mechanism for transfer ofnewly synthesized sterols from the endoplasmic reticulum (ER)to the plasma membrane is still unclear. In non-plant systems,sterol trafficking occurs with the aid of oxysterol binding protein(OSBP), and OSBP-related proteins (ORPs) [10–12]. Other functionsare also attributed to these proteins like signaling cascades, endo-somal movement, lipid sensing, and regulation of cellular steroldistribution [13]. The OSBPs/ORPs are also involved in membranetrafficking [14], tumor metastasis [15], and cell cycle progression[16].

Most eukaryotic genomes encode for OSBPs and ORPs. TheORPs have been studied in humans [17], Drosophila [16], and fungi[14,18–21]. There are 12 OSBP/ORP genes identified in human[9,12,22,23], as well as in mouse [24], and 7 in yeast [10]. In Ara-bidopsis genome, 12 putative ORP genes have been identified [25].Apart from oxysterols, the ORPs can also bind different lipids,including phosphoinositides, ergosterol, and cholesterol [26–30].

The pleckstrin homology (PH) domains, a conserved domain con-taining an FFAT motif (two phenylalanines embedded in an acidictract), and ankyrin repeats are present in most ORPs [31]. The tar-geting of ORPs to cellular compartments is facilitated with the aid
Page 2: Oxysterol binding proteins (OSBPs) and their encoding genes in Arabidopsis and rice

P. Umate / Steroids 76 (2011) 524–529 525

Table 1Genomic loci encoding oxysterol binding protein-related proteins (ORPs) in Arabidopsis thaliana L.

Locus Annotation Aa Ba Ca

AT1G13170 OSBP (Oxysterol binding protein)-related protein 1D (ORP1D) 816 92331.3 6.4705AT2G31020 OSBP (Oxysterol binding protein)-related protein 1A (ORP1A) 760 87318.6 6.7662AT2G31030 OSBP (Oxysterol binding protein)-related protein 1B (ORP1B) 489 56555.7 6.778AT3G09300 OSBP (Oxysterol binding protein)-related protein 3B (ORP3B) 458 51956 4.8207AT4G08180 OSBP (Oxysterol binding protein)-related protein 1C (ORP1C) 814 92763.4 6.4778AT4G12460 OSBP (Oxysterol binding protein)-related protein 2B (ORP2B) 693 79564.9 7.709AT4G22540 OSBP (Oxysterol binding protein)-related protein 2A (ORP2A) 721 82041.6 7.046AT4G25850 OSBP (Oxysterol binding protein)-related protein 4B (ORP4B) 383 43673.7 7.0367AT4G25860 OSBP (Oxysterol binding protein)-related protein 4A (ORP4A) 386 44071.4 8.229AT5G02100 OSBP (Oxysterol binding protein)-related protein 3A (ORP3A) 453 51544.6 4.8769AT5G57240 OSBP (Oxysterol binding protein)-related protein 4C (ORP4C) 379 42968.2 9.5279AT5G59420 OSBP (Oxysterol binding protein)-related protein 3C (ORP3C) 457 51965.9 4.9524

a A, amino acids; B, molecular weight; C, isoelectric point.

F oteinsO ight. Aa s/ORPs

otat

aAftatoo(gOla8agra

TG

ig. 1. Protein alignment of oxysterol binding proteins (OSBPs) and OSBP-related prSBPs/ORPs are identified on left and numbers of amino acid residues are given on rre introduced to improve the alignment. A stretch containing the conserved OSBP

f these conserved domains [11,31,32]. A highly conserved oxys-erol binding domain is present in OSBPs in animals, fungi, Petunia,nd Arabidopsis [25]. The sterol interaction which is typical for thisype of proteins is facilitated by this [25].

In this study, the OSBPs and ORPs from Arabidopsis thaliana L.nd Oryza sativa L. (rice) are been identified (Tables 1 and 2). Therabidopsis genome encodes 12 ORPs that can be classified into 4

unctional categories, ORP1A to ORP1D, ORP2A to ORP2B, ORP3Ao ORP3C, and ORP4A to ORP4C (Table 1). The Arabidopsis ORPsre in the range from 379 to 816 amino acids (Table 1). The pro-ein alignment of Arabidopsis ORPs demonstrate the occurrencef a conserved stretch of amino acids E(Q/K)VSHHPxMS previ-usly identified as fingerprint for oxysterol binding protein [33]Fig. 1). Minor differences exist in the sequence within this fin-erprint region of Arabidopsis ORPs (Fig. 1). In rice, there are fiveSBPs localized on 3, 8, 10, and 12 chromosomes, and one ORP6 is

ocalized on chromosome eight (Table 2). Of these, the two OSBPsre localized on chromosome 3, and one each on chromosomes, 10 and 12 (Table 1). The protein sequences of rice OSBPs/ORP6

nd Arabidopsis ORPs are relatively well conserved (Fig. 1). The fin-erprint for oxysterol binding protein is also well conserved inice OSBPs (Fig. 1). The rice genome annotation project databasend resource is available at http://rice.plantbiology.msu.edu. The

able 2enomic loci encoding oxysterol binding proteins (OSBPs) in Oryza sativa L.

Chromosome Locus id Putative function/annotation

3 LOC Os03g16690 Oxysterol binding protein, putative3 LOC Os03g49770 Oxysterol binding protein, putative8 LOC Os08g05870 Oxysterol binding protein-related p8 LOC Os08g40590 Oxysterol binding protein, putative10 LOC Os10g40590 Oxysterol binding protein, putative12 LOC Os12g18770 Oxysterol binding protein, putative

a A, amino acids; B, molecular weight; C, isoelectric point.

(ORPs) in Arabidopsis and rice. The entire protein sequences aligned using ClustalX.sterisks indicate conserved amino acid changes. Gaps in the amino acid sequencesfingerprint region is shown on top of alignment.

complete genome sequence for Arabidopsis is available at the Ara-bidopsis information resource (TAIR) (http://www.arabidopsis.org).

The Arabidopsis and rice OSBPs/ORPs are phylogeneticallyclassified based on their protein sequence similarity and corre-sponding bootstrap values (Fig. 2). The phylogenetic tree showedhigh degree of similarity between the Arabidopsis ORP3B andORP3A (100 bootstrap), ORP4B and ORP4A with ORP4C (100bootstrap), ORP1A and ORP1C with ORP1B (100 bootstrap), andORP2B and ORP2A (100 bootstrap) (Fig. 2). Interestingly, therice OSBPs always emerged as separate branches in each majorclade (Fig. 2). Based on their positions and bootstrap valueswith their Arabidopsis counterparts, the rice OSBPs are classifiedas OSBP1A (LOC Os03g49770), OSBP1B (LOC Os12g18770),OSBP2A (LOC Os08g05870), OSBP2B (LOC Os10g40590),OSBP3 (LOC Os03g16690), and OSBP4 (LOC Os08g40590)(Fig. 2).

In this study, the sequence profiles for all homol-ogous OSBPs/ORPs sequences from Pfam database(http://pfam.janelia.org/family?acc=PF01237) were visualized

based on pHMM (profile Hidden Markov Model) sequence LOGO[34,35]. The sequence LOGO was built on LOC Os03g16690. Thestudy demonstrates that the fingerprint region for OSBP withamino acids “EQVSHHPP” (from amino acids 99 to 106) is promi-

Aa Ba Ca

, expressed 464 51806.1 5.1585, expressed 805 90469.3 7.3695rotein 6, putative, expressed 531 60772.9 5.2345, expressed 392 42875.7 6.6723, expressed 743 82345.2 7.1085, expressed 757 85150.8 6.8209

Page 3: Oxysterol binding proteins (OSBPs) and their encoding genes in Arabidopsis and rice

526 P. Umate / Steroids 76 (2011) 524–529

AT3G09300.ORP3B

AT5G02100 ORP3A

100

89 0.

AT5G59420.ORP3C

LOC Os03g16690

AT4G25850.ORP4B

AT4G25860.ORP4A

89

100

100

100

100

AT5G57240.ORP4C

LOC Os08g40590

AT1G13170.ORP1D

LOC Os12g18770

100

100

AT2G31020.ORP1A

AT4G08180.ORP1C

AT2G31030.ORP1B

LOC Os03g49770

AT4G12460 ORP2B

94

100

100

100

AT4G12460.ORP2B

AT4G22540.ORP2A

LOC Os08g05870

LOC Os10g40590

100

91

100

F in seqN .

nP(L

Fr

0.05

ig. 2. Phylogenetic relationship of Arabidopsis and rice OSBPs/ORPs built on proteeighbour-Joining (NJ) method and bootstrapped for 100 replicates. Scale bar = 0.05

ently represented among all homologous OSBPs occurring in thefam database (Fig. 3). Apart, a conserved stretch “KPFNPLLGETF”from amino acids 76 to 86) is also highlighted in the sequenceOGO profile (Fig. 3).

ig. 3. HMM logo generated on rice OSBP (LOC Os03g16690) with height of letter (amiegions of amino acids “KPFNPLLGETF” and “EQVSHHPP” are shown.

uences and viewed with MEGA4 program. The unrooted tree was generated using

The Gene3D database (http://gene3d.biochem.ucl.ac.uk) multi-ple sequence scan against the HMM library = v3 3 0.lib is exploredto identify the occurrence of conserved domains on proteinsequences for Arabidopsis and rice OSBPs/ORPs. The Gene3D

no acid) at each position representing the degree of conservation. The conserved

Page 4: Oxysterol binding proteins (OSBPs) and their encoding genes in Arabidopsis and rice

P. Umate / Steroids 76 (2011) 524–529 527

F namea brackv ain. T

diOstOtLTo(

ig. 4. Gene3D scan graphical results for Arabidopsis and rice OSBPs/ORPs. (A) There shown in magenta. The number of amino acids spanning the domain is given iniolet. The numbers in the parentheses represent the amino acids spanning the dom

atabase describes protein families and domain architecturesn complete genomes. The Arabidopsis ORP1A, ORP1C, ORP1D,RP2A, and ORP2B showed the presence of conserved pleck-

trin homology (PH) domain while, the analysis showed thathis domain is absent in other members like ORP1B, ORP3A,RP3B, ORP3C, ORP4A, ORP4B, and ORP4C (Fig. 4A). In rice,

he OSBPs representing LOC Os03g49770, LOC Os10g40590, and

OC Os12g18770 showed the occurrence of PH domain (Fig. 4B).he sequence analysis demonstrated that the PH domain do notccur in LOC Os03g16690, LOC Os08g05870, and LOC Os08g40590Fig. 4B).

s for the Arabidopsis ORPs are given on left. The pleckstrin homology (PH) domainsets. (B) The locus IDs for rice OSBPs are given on left. The PH domains are shown inhe gray-shaded bars simulate protein length.

Fig. 5 shows expression profile for the Arabidopsis ORPs down-loaded from genevestigator expression data resource of TAIRdatabase (http://www.arabidopsis.org). The heat map is generatedfrom log2 signal ratios for 17 different experimental plots whichcan be divided into 7 major conditions (Fig. 5). These conditionsinclude, biotic stress (Pseudomonas syringae), chemical treatment(high CO , cycloheximide, ozone), elicitor (CaCl /MgCl ), hormones

2 2 2(ABA, GA3), light intensity (dark, high light, low light), abioticstresses (cold, drought, heat, osmotic, oxidative, and salt), andtemperature stress (16 ◦C) (Fig. 5). The transcription profiling forORP4A, ORP4B, and ORP4C showed a common trend with genes
Page 5: Oxysterol binding proteins (OSBPs) and their encoding genes in Arabidopsis and rice

528 P. Umate / Steroids 76 (2011) 524–529

F mentaI n on tT

u(not

fiyflt

Fa

ig. 5. The heat map with expression analysis for 12 ORPs under 17 different experiDs and gene nomenclature is given on right. The experimental conditions are showhe figure was generated using the program Genesis [35,45,46].

pregulated under high CO2, treatment with ozone, ABA, osmoticlate), and salt (early) (Fig. 5). The ORP4A and ORP4B showed sig-ificant upregulation in drought stress condition (Fig. 5). Severalf the ORPs are upregulated under biotic stress, high light, andemperature stress (Fig. 5).

The linear mean signal values depicting the expression levelsor Arabidopsis ORPs in different developmental stages are shown

n Fig. 6. The developmental stages like germinated seed, seedling,oung rosette, developed rosette, bolting, young flower, developedower, flowers and siliques, and mature siliques are covered duringhe analysis. The expression levels for ORP3C were higher followed

ORP3C

ORP4A

ORP3A

ORP4C

ORP2A

ORP4B

ORP3B

ORP1C

ORP2B

gene

nam

e

ORP1D

ORP1A

ORP1B

0 1000 2000

mean signal v

ig. 6. A graphical representation of expression levels drawn with linear mean signal vare given on right. The error bars shown on graph represent standard error.

l conditions in Arabidopsis generated based on hierarchical clustering. The probesetop of the expression map. The scale bar represents the log2 value from −3.0 to 3.0.

by ORP3B and ORP3A as compared to other ORPs (Fig. 6). The genes,ORP1A, ORP1B, ORP4A, and ORP4B are least expressed in all devel-opmental stages (Fig. 6).

A variety of sterols are produced in plant cells [36,37]. Sterolsplay essential structural role as suggested from studies on steroldeficient Arabidopsis mutants [37–39]. Apart from their conversionto brassinosteroids (BRs), the sterols are also known to be involved

in coordinating multiple events in plant development [38,39]. Themembers of ORP family have been documented to transport sterolsand oxysterols [13]. A bona fide sterol binding protein in Ara-bidopsis, ORP3a is an oxysterol-binding protein [33]. The ORP3a is

maturesiliques flowers and siliques developed flower young flower bolting developed rosetteyoung rosette seedling germinated seed

3000 4000 5000

alue

lues on X-axis and Arabidopsis ORPs on Y-axis. The different developmental stages

Page 6: Oxysterol binding proteins (OSBPs) and their encoding genes in Arabidopsis and rice

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[45] Sturn A, Quackenbush J, Trajanoski Z. Genesis: cluster analysis of microarray

P. Umate / Steroi

ocalized at the ER [33]. The finding also suggest that ORP3a canreferentially bind sitosterol as compared to ergosterol and stig-asterol [33]. Studies with certain yeast and mammalian ORPs

ave indicated promiscuous binding of different sterols, besidesxysterols [29,40]. It was demonstrated that 12 ORPs from Ara-idopsis were expressed of which 10 were expressed in stem, leaf,eedling, and root, and 3 were expressed in pollen [25]. The PetuniaRP was localized at the plasma membrane of pollen tubes [25]. The

nteraction of Petunia inflata ORP with cytoplasmic kinase domainf a receptor-like kinase was determined [25]. The Glycine max ORPas demonstrated to be involved in salt stress response [41]. In bar-

ey, an infection with powdery mildew results in up-regulation ofn ORP expression [42].

To conclude, this study indicates that rice genome encodes foress number of OSBPs as compared to Arabidopsis. Similarly, dif-erences in the total number of members constituting a minorene family in Arabidopsis and rice were also observed previously43,44]. The OSBP family members are not been thoroughly inves-igated in rice. The more advanced knowledge on OSBPs function

ight spread light on the significant role of oxysterols in Ca2+

ptake and apoptosis, to transcriptional activity, and cellular dif-erentiation, signaling cascades, lipid sensing, and regulation ofellular sterol distribution.

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