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Distinct Roles of the First Introns on the Expression of Arabidopsis Profilin Gene Family Members

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Distinct Roles of the First Introns on the Expression of Arabidopsis Profilin Gene Family Members
  Distinct Roles of the First Introns on the Expression ofArabidopsis Profilin Gene Family Members 1 Young-Min Jeong, Jeong-Hwan Mun 2  , Ilha Lee, Je Chang Woo, Choo Bong Hong, and Sang-Gu Kim* Department of Biological Sciences, Seoul National University, Seoul 151–742, Republic of Korea(Y.-M.J., J.-H.M., I.L., C.B.H., S.-G.K.); and Department of Biology, Mokpo National University, Jeonnam 534–729, Republic of Korea (J.C.W.) Profilin is a small actin-binding protein that regulates cellular dynamics of the actin cytoskeleton. In Arabidopsis (  Arabidopsisthaliana ), five profilins were identified. The vegetative class profilins,  PRF1 ,  PRF2 , and  PRF3 , are expressed in vegetative organs.The reproductive class profilins,  PRF4  and  PRF5 , are mainly expressed in pollen. In this study, we examined the role of the firstintron in the expression of the Arabidopsis profilin gene family using transgenic plants and a transient expression system. Intransgenic plants, we examined  PRF2  and  PRF5 , which represent vegetative and reproductive profilins. The expression of the PRF2 promoterfusedwiththe  b -glucuronidase( GUS )genewasobservedinthevascularbundles,buttransgenicplantscarryingthe PRF2 promoter-GUSwithitsfirstintronshowedconstitutiveexpressionthroughoutthevegetativetissues.However,thefirstintronof   PRF5  had little effecton the reportergene expression pattern. Transgenic plants containing  PRF5  promoter-GUS fusionwithorwithoutitsfirstintronshowedreproductivetissue-specificexpression.Tofurtherinvestigatethedifferentrolesofthefirsttwo introns on gene expression, the first introns were exchanged between  PRF2  and  PRF5 . The first intron of   PRF5  had noapparent effect on the expression pattern of the  PRF2  promoter. But, unlike the intron of   PRF5 , the first intron of   PRF2  greatlyaffectedthereproductivetissue-specificexpressionofthe PRF5 promoter,confirmingadifferentrolefortheseintrons.Theresultsof a transient expression assay indicated that the first intron of   PRF1  and  PRF2  enhances gene expression, whereas  PRF4  and PRF5  do not. These results suggest that the first introns of profilin genes are functionally distinctive and the first introns arerequired for the strong and constitutive gene expression of   PRF1  and  PRF2  in vegetative tissues. Diverse actin-binding proteins regulate cellular dy-namics of the actin cytoskeleton to perform various biological processes, such as locomotion, elongation,shape change, cytoplasmic streaming, division, anddevelopment of the cell (Bamburg et al., 1999; Kostet al., 1999; Meagher et al., 1999; Bal u ˘ ska et al., 2001).Amongtheseproteins,thesmallactin-bindingprotein,profilin (12–15 kD), plays an important role in actindynamics (Gibbon and Staiger, 2000; Kovar et al., 2000;McKinney et al., 2001). In plants, profilin was srci-nally identified in birch pollen as a potent allergen(Valenta et al., 1991), and it has been isolated andcharacterized in various plants, such as maize ( Zeamays ), tobacco ( Nicotiana tabacum ), bean ( Phaseolusvulgaris ), Arabidopsis (  Arabidopsis thaliana ), tomato( Lycopersicon esculentum ), and castor bean ( Ricinuscommunis ; Staiger et al., 1993; Mittermann et al., 1995;Vidali et al., 1995; Christensen et al., 1996; Huanget al.,1996; Yu et al., 1998; Guillen et al., 1999; Schobert et al.,2000). Plant profilins have been shown to bind G-actininvitro,andtheyarebiochemicallysimilartononplantprofilins(Valentaetal.,1993;Guillen etal.,1999;Kovaret al., 2000). Arabidopsis profilin complemented botha budding yeast ( Saccharomyces cerevisiae ) profilin de-letion mutant and a fission yeast ( Schizosaccharomyces pombe ) cdc3-124/profilin mutant, indicating that plantprofilinisfunctionallyequivalentwithyeastprofilininvivo (Christensen et al., 1996). Like many other plantcytoskeletal genes,profilinsexistasamultigenefamilyand fall into either vegetative or reproductive classes,as shown in maize and Arabidopsis (Staiger et al.,1993; Christensen et al., 1996; Huang et al., 1996). InArabidopsis, three vegetative and two reproductiveprofilins have been isolated and characterized. Pro-filins expressed in vegetative tissues are encoded by PRF1 ,  PRF2 , and  PRF3 . Both  PRF4  and  PRF5  encodereproductive class profilins that are preferentially ex-pressedinpollen(Christensenetal.,1996;Huangetal.,1996). Analysis of transgenic plants harboring  PRF1 and  PRF2  promoter-  b -glucuronidase (GUS) fusionconstructs showed that these two profilins are mainlyexpressed in the vascular bundles of vegetative tissues(Christensen et al., 1996; Ramachandran et al., 2000).Recently, Kandasamy et al. (2002) investigated thespatial and developmental expression of profilins inArabidopsis using vegetative and reproductive pro- filin-specific antibodies. They examined the root tip, sepal, and pistil using a monoclonal antibody that 1 This work was supported by the Ministry of Education andHuman Resources Development (BK21 Research Fellowship toY.-M.J.). 2 Present address: Department of Plant Pathology, University of California, Davis, CA 95616.* Corresponding author; e-mail; fax 82–2–878–7256.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors ( is:Sang-Gu Kim (, publication date, and citation information can be found  Plant Physiology,  January 2006, Vol. 140, pp. 196–209,    2005 American Society of Plant Biologists  www.plant.orgon March 5, 2014 - Published by www.plantphysiol.orgDownloaded from Copyright © 2006 American Society of Plant Biologists. All rights reserved.  reacts strongly with PRF1 and PRF2, and weakly withPRF3. Based on the results of biochemical and struc-tural localization analysis, the authors concluded thatvegetative profilins are expressed in all vegetativeorgans and tissues. The discrepancy with previouswork on profilin gene expression may reflect the pres-ence of regulatory sequences in profilin genes. How-ever, such regulatory elements have not yet beenidentifiedinprofilingenes.Recently,ithasbeenshownthatsomeintrons areinvolvedintheregulationof spa-tial or temporal expression in potato ( Solanum tuber-osum )  Sus3  (Fu et al., 1995), bean  PsaD  (Bolle et al.,1996), tobacco  Ubi.U4  (Plesse et al., 2001), petunia( Petunia hybrida )  PhADF1  (Mun et al., 2002), and Arab-idopsis  PAT1  (Rose and Last, 1997),  AGAMOUS (Deyholos and Sieburth, 2000), and  FLC  (Sheldonet al., 2002). Particularly, in the case of genes that areknown to express constitutively, such as  PAT1 , PhADF1 , and  Ubi.U4 , introns were required for theirstrong and constitutive expression (Rose and Last,1997; Plesse et al., 2001; Mun et al., 2002).In this study, to identify the regulatory elementscontrolling profilin gene expression, we examined theeffectofanintronoftheprofilingenes.Althoughgenesencoding profilins are interrupted by two introns, weonly tested the first intron, because introns that regu-late gene expression are generally located near the 5 # region of the gene (Clancy and Hannah, 2002; Rose,2002), and the first intron was about 4 times longer inlengththanthesecondintron.Ourresultsshowthatthefirst intron of vegetative profilins ( PRF1  and  PRF2 )plays an important role in strong and constitutiveexpression in vegetative tissues both in the transgenicand/or transient assay and enhances expression bothatthemRNAandattheproteinlevel.Wealsotestedthefirst intron of reproductive profilins ( PRF4  and  PRF5 )to determine whether a regulatory role of introns isconserved in reproductive profilin. In contrast with PRF1  and  PRF2 , the first intron of   PRF4  and  PRF5  didnot alter expression patterns significantly. Further-more, the first intron of   PRF2  was able to alter spatialexpressionpatternsof both  PRF2  and PRF5  promoters. RESULTSThe First Intron of  PRF2  Is Required for Strong andConstitutive  PRF2  Expression in Vegetative Tissues Although introns are removed during the mRNAmaturation process, some introns are known to en-hance or regulate gene expression in various ways. Tocharacterize the role of introns in vegetative profilingene expression, we have cloned the  PRF2  gene andgenerated two  PRF2  promoter-GUS fusion constructs.The first construct, pPRF2-5 # , includes the 1.6-kb pro-moter, 5 # -untranslated region (UTR), and the startcodon fused with the  GUS  gene in the pBI101 vector.The second construct, pPRF2e-p2i, contains the pro-moter, the entire first exon including 5 # -UTR, the firstintron, and 24 bp of the second exon (Fig. 1A). Theshort second exon was included for proper intronsplicing.Threeindependenttransgenicplantscarryingsingle copies of the pPRF2-5 #  or pPRF2e-p2i constructswereexamined to analyze the quantitative effect of thefirst intron of   PRF2  on gene expression.We analyzed the expression of the  PRF2  promoter-GUS fusion construct in various developmental stagesof transgenic Arabidopsis. In 3-d-old seedlings con-taining pPRF2-5 # , GUS staining was mainly observedin the vascular bundles of cotyledons. Some faintstaining was detected near the vascular bundles, andthere was no expression in the shoot apical meristemregion (Fig. 1B). Most parts of the root showed GUSexpression, except the root tip (Fig. 1C), and relativelystrong staining was observed in the vascular bundlesof the root (data not shown in detail). This preferentialexpression in vascular bundles was also maintainedin 15-d-old plants. In this case, GUS expression wasdetected in the vascular bundles of rosette leaves andpetioles (Fig. 1, D and E). Most of the roots werestained, but no GUS expression was observed in theroot tips. The older expanded leaves showed strongerexpression than smaller ones (Fig. 1D). In flowers, thefilaments of stamens and the veins of sepals werestained. No GUS staining was observed in anthers,pollen, carpels, or petals (Fig. 1F). In young siliques,staining was detected in the receptacle (Fig. 1G). Theseexpression patterns were largely in agreement withprevious reports (Christensen et al., 1996). However,expression of the  PRF2  promoter-GUS was completelychanged by the inclusion of the first intron within theconstruct. In 3-d-old seedlings containing pPRF2e-p2i,strong GUS staining was detected throughout theplant. Most parts of the seedlings, including the roothairs,weredeeplystained.Shootapicalmeristemsandroot tips also showed GUS expression (Fig. 1, H and I).These strong and constitutive GUS expressions wereobserved also in 15-d-old plants. The deep blue stain-ing was detected in the rosette leaves, petioles, roots,root hairs, and trichomes (Fig. 1, J and K). Almostevery part of the floral organs, such as the petals,sepals, anthers, filaments, and stigmas, were stained(Fig. 1L). In young developing siliques, the stigmaregion and receptacle were stained (Fig. 1M). Al-though these results suggest a significant role of theintron in  PRF2  gene expression, they cannot rule outthe effect of coding regions on gene expression be-cause pPRF2e-p2i has a full length of the first exonencoding 41 amino acids. Therefore, to examine theeffect of the coding region, a pPRF2e construct thatharbors the promoter region and the first exon fusedwith the  GUS  gene was generated, and the expressionpatterns of this construct were analyzed in 23 lines of T1 transgenic plants. Because of copy number varia-tions of the transgene or positional effects, there weresome variations, but most of the plants showed similarGUS expression patterns with pPRF2-5 #  (data notshown), suggesting that the first exon of   PRF2  has nosignificant effect on the gene expression pattern. Effect of the First Intron on Profilin Gene ExpressionPlant Physiol. Vol. 140, 2006 197  www.plant.orgon March 5, 2014 - Published by www.plantphysiol.orgDownloaded from Copyright © 2006 American Society of Plant Biologists. All rights reserved.  Furthermore, in a transient assay, the entire openreading frame (ORF) in the pPRF2ORFGP constructshowed no positive effect on GUS expression and theexpression level of the pPRF2geGP that contains theentire  PRF2  gene except the 3 # -UTR was similar to thatof the pPRF2e-p2iGP (Fig. 7B). These results supportthe fact that the sequences of   PRF2  mRNA or aminoacids fused to the  GUS  gene do not affect enhancedgene expression. Therefore, these data demonstrate thatthefirstintronof  PRF2 isresponsibleforthestrong Figure 1.  Histochemical analysis of GUS expressionin transgenic Arabidopsis containing pPRF2-5 #  andpPRF2e-p2i constructs. A, Schematic representationsof   PRF2  promoter-GUS fusion constructs. The boxwith  PRF2  pr is the promoter region of   PRF2 ; the boxwith e is the first exon of   PRF2 ; the black box withGUS is the GUS coding region; the line with p2i isthefirstintronof  PRF2 ;ATGisthestartcodon.BtoG,From pPRF2-5 # ; H to M, from pPRF2e-p2i; B and H,3-d-old seedlings; C and I, roots of 3-d-old seedlings;D and J, 15-d-old plants; E and K, rosette leaves;F and L, flowers from 5-week-old plants; G and M,youngsiliques.GUSstainingwasperformedfor12h.Scale bars,1 mm (B, D, E, H,J, andK), 0.2 mm(C, G,I, and M), and 0.5 mm (F and L).  Jeong et al.198 Plant Physiol. Vol. 140, 2006  www.plant.orgon March 5, 2014 - Published by www.plantphysiol.orgDownloaded from Copyright © 2006 American Society of Plant Biologists. All rights reserved.  and constitutive expression of   PRF2  throughout planttissues. The First Intron of  PRF2  Increases BothmRNA and Protein Levels To investigate the basis for different expression of pPRF2-5 #  and pPRF2e-p2i constructs, levels of mRNAand protein were analyzed. The accumulation of   GUS transcriptswasanalyzedbyreversetranscription(RT)-PCRanalyses.OnemicrogramoftotalRNAfromroots,leaves, stems, and flowers was used for RT, and PCRwas performed for 25 cycles to prevent saturation of PCR products. Sequencing of RT-PCR products of pPRF2e-p2i indicated that splicing occurred precisely(data not shown). In all tissues, the transcript level of transgenic plants carrying pPRF2e-p2i was increasedwhen compared to pPRF2-5 #  (Fig. 2), suggesting thatdifferentexpressionoccurredatthetranscriptionlevel.TheincreasedmRNAlevelswereparticularlyapparentintheleafandstem.Toexaminewhethertheincreaseintranscript level is fully reflected in the protein level,protein accumulation in the leaves was analyzed bywestern-blot analysis and GUS assay (Fig. 3). Twentymicrograms of total soluble proteins from the leaves of 4-week-old plants were used for immunoblot analy-sis with the GUS antibody. Similar to the transcript ac-cumulation pattern, the levels of protein were muchhigher in pPRF2e-p2i than pPRF2-5 #  plants. Results of fluorometric GUS assay indicated that pPRF2e-p2igene expression was enhanced by about 15-fold rela-tive to that of pPRF2-5 #  (Fig. 3). Thus, the increase intranscriptaccumulationinducedbythefirstintronwasreflected in the GUS protein level. Therefore, strongexpression of pPRF2e-p2i is correlated with increasedaccumulation of the  GUS  transcript. These resultssuggestthattheroleofthefirstintronof  PRF2 iscloselyrelated to increasing steady-state mRNA levels. The First Intron of  PRF5  Has Little Effect on PRF5  Gene Expression We demonstrated the important role of the first in-tron in gene expression of   PRF2 , the vegetative profi-lin. To determine whether the role of first introns isconserved in a reproductive profilin, we examined thefirst intron of   PRF5 . About 1.5 kb of the promoterregion of   PRF5  and the first exon were translationallyfused with the  GUS  reporter gene in a pPRF5e con-struct that tests promoter activity. To analyze theintron’s role, a pPRF5e-p5i construct, which includesall parts of pPRF5e as well as the first intron of   PRF5 with 24 bp of the second exon, was generated. Theschematic structures of pPRF5e and pPRF5e-p5i aredepicted in Figure 4A. Because we mainly focused onthe effect of the intron on the  PRF5  spatial expressionpattern, we did not isolate the single-copy lines, andmore than 10 lines of T1 plants harboring pPRF5e andpPRF5e-p5i constructswere examinedfor theanalysis.GUS histochemical analysis of transgenic plants har- boring pPRF5e showed strong expression in pollen.Staining was also observed in the stigmas and anthers.Very weak staining was detected in the upper part of filaments and petals (Table I; Fig. 4D). GUS expressionwas not detected in 3-d-old seedlings and 15-d-oldplants in most lines that were examined (Fig. 4, B andC). Although it is not clear whether GUS stainingin other floral organs is an actual expression or justdiffusion from pollen, GUS expression of pPRF5e waspredominant in pollen. A pollen-specific expressionpattern was in agreement with a previous report(Christensen et al., 1996). pPRF5e-p5i, which includesits own first intron within the construct, showedalmost identical expression with pPRF5e. GUS stain-ingwasnotobservedinthevegetativetissuesofnearlyall transgenic plants (Fig. 4, E and F). Like pPRF5e,GUS staining in flowers was mainly detected in thepollen and anthers (Table I; Fig. 4G). These resultssuggest that the first intron of   PRF5  has no significanteffect on the expression pattern of the  PRF5  gene. The Role of Introns under the Control ofHeterogeneous Promoters The analysis of transgenic plants carrying  PRF2  and PRF5  promoter-GUS fusion constructs revealed thatthe first intron of   PRF2  plays an important role in geneexpression, while the first intron of   PRF5  has littleeffect. However, it is unclear whether only the intronitself is responsible for these differences. Other factors, Figure 2.  RT-PCR analysis of transcript expression of transgenic Arabidopsis containing pPRF2-5 #  and pPRF2e-p2i constructsin various tissues. One microgram of total RNA from each organ was used for RT.  TUBULIN2  ( TUB2 ) amplified with TUB-F(5 # -CTCAAGAGGTTCTCAGCAGTA-3 # ) and TUB-R (5 # -CTCAAGAGGTTCTCAGCAGTT-3 # ) primers was used as a control. Afterelectrophoresis of PCR products, it was transferred to the nylon membrane and hybridized with  32 P-labeled  GUS   or  TUB2 probes. Effect of the First Intron on Profilin Gene ExpressionPlant Physiol. Vol. 140, 2006 199  www.plant.orgon March 5, 2014 - Published by www.plantphysiol.orgDownloaded from Copyright © 2006 American Society of Plant Biologists. All rights reserved.  such as interaction between intron and promoter,could be involved in profilin gene expression. Thus,toinvestigatetherolesofintronsindetail,intronswereexchanged between  PRF2  and  PRF5 . For the construc-tion of pPRF2e-p5i, the first intron of   PRF5  was in-serted at the 3 #  end of the first exon in the pPRF2econstruct. In the same way, the first intronof   PRF2  wasintroduced into the pPRF5e construct, thus givingpPRF5e-p2i (Fig. 5A). For efficient splicing, both in-trons were accompanied with short flanking exons.The resulting constructs were transformed into Arabi-dopsis, and the expression pattern was analyzed inmore than 20 independent T1 plants. In 3-d-old seed-lings ofpPRF2-p5i, expressionwasmainly observedincotyledons and roots (Fig. 5B). This vascular bundle-specific expression was also observed in leaves, pet-ioles, and roots of 15-d-old plants (Fig. 5C). In flowers,expression was observed in the filaments and sepals.No GUS staining was detected in pollen or in anthers(Table I; Fig. 5D). The overall expression patternsof pPRF2-p5i plants were very similar to those of pPRF2-5 #  (Fig. 1, B–G). These data indicate that thefirst intron of   PRF5  has no significant effect on theexpression of the  PRF2  promoter. However, unlikethe first intron of   PRF5 , the first intron of   PRF2  hadsignificant influence on the expression of the  PRF5 promoter. GUS histochemical analysis of pPRF5e-p2ishowed strong expression throughout the plant. Deep blue staining was observed in nearly every part of theplant,includingroothairsandtipsof3-d-oldseedlings(Fig. 5E). The expression pattern was totally differentfromthatofpPRF5eorpPRF5e-p5i(TableI;Fig.4,B–G).This strong expression was also observed in 15-d-oldplants (Fig. 5F). Most of the floral organs, includingstigmas, anthers, pollen, filaments, petals, and sepals,also showed GUS expression (Table I; Fig. 5G). Thisconstitutive expression pattern was quite similar tothat of pPRF2e-p2i (Fig. 1, H–M). Thus, the propertiesof the two introns were maintained under heteroge-neous promoters, and this confirmed the functionaldistinctiveness of the two introns. Particularly, the firstintron of   PRF2  had a significant effect on the expres-sionpatternofboththe PRF2 and PRF5 promoters,andthis demonstrates that the first intron of   PRF2  is re-sponsiblefortheconstitutiveexpressionofpPRF2e-p2iand pPRF5e-p2i. The First Intron of  PRF2  Alters the SpatialExpression Pattern of  PRF   Genes The expression of pPRF2-5 #  was mainly observed inthe vascular bundles of vegetative tissues. But faintGUS staining was detected in nonvascular tissues.In the pPRF2e-p2i plants, GUS staining is slightlystronger in the vascular tissues. In addition, increasedexpression induced by the intron was observed in theRT-PCR and western-blot analysis. Therefore, it ispossible that strong expression of pPRF2e-p2i could be a result of a quantitative increase in the express-ion pattern of pPRF2-5 # . However, the result withpPRF5-p2i suggested that the intron alters the spatialexpressionpatternofthe PRF5  promoter.Todeterminethe role of the intron in detail, GUS staining wasperformed in 3-d-old seedlings at various incubationtimes(Fig. 6).In pPRF2-5 # , GUSstaining was observedto occur weakly in the vascular bundles of cotyledonsafter 1-h staining. This vascular expression patternwas maintained and staining intensity was increasedaccording to the staining time. After 12-h staining,weak staining was observed in the vascular bundlesand nearby tissues. In pPRF2e-p2i plants, no vascularpatterning was observed in the cotyledons after 1-hincubation. The overall parts of the cotyledons wereevenly stained. This pattern was maintained afterstaining for 4, 8, and 12 h. In the roots, a differentpattern was observed and maintained in both samplesfrom the beginning. If the intron just increased ex-pression, the early stage of the staining pattern of pPRF2e-p2i would be similar to that of pPRF2-5 # .Therefore, these results suggest that the intron doesnot just increase or enhance gene expression, but in-stead is also involved in regulating spatialgene expres-sion patterns. The First Intron of  PRF2  Affects Gene Expressionin a Position-Dependent Manner The resultsabove demonstratethat the first intron of  PRF2  enhances gene expression and can induce ec- topic expression under the control of a tissue-specific Figure 3.  Western-blot analysis and determination of GUS activity intransgenic Arabidopsis containing pPRF2-5 #  and pPRF2e-p2i. A, Totalsoluble protein (20  m g) from mature leaves was loaded per lane andanalyzed using monoclonal GUS antibody. B, GUS activity is ex-pressed as pmol 4-methyl-umbelliferone min 2 1 mg 2 1 protein. Valuesare means 6 SD  ( n  5 3).  Jeong et al.200 Plant Physiol. Vol. 140, 2006  www.plant.orgon March 5, 2014 - Published by www.plantphysiol.orgDownloaded from Copyright © 2006 American Society of Plant Biologists. All rights reserved.
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