Subcellular Localizations of AS1 and AS2 Suggest Their Common and Distinct Roles in Plant Development

Journal of Integrative Plant Biology 2008, 50 (7): 897–905

Subcellular Localizations of AS1 and AS2 Suggest TheirCommon and Distinct Roles in Plant Development

Yan Zhu1, Ziyu Li1, Ben Xu2, Hongda Li1, Lingjian Wang2, Aiwu Dong1 and Hai Huang2∗

(1State Key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China;2National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences,

the Chinese Academy of Sciences, Shanghai 200032, China)

Abstract

During leaf organogenesis, a critical step for normal leaf primordium initiation is the repression of the class 1 KNOTTED1-like homeobox (KNOX ) genes. After leaf primordia are formed, they must establish polarity for normal leaf morphogenesis.Recent studies have led to the identification of a number of genes that participate in the class 1 KNOX gene repressionand/or the leaf polarity establishment. ASTMMETRIC LEAVES1 and 2 (AS1 and AS2) are two of these genes, which arecritical for both of these two processes. As a first step towards understanding the molecular genetic basis of the AS1-AS2action, we determined the subcellular localizations of the two proteins in both tobacco BY2 cells and Arabidopsis plants,by fusing them to yellow/cyan fluorescent protein (YFP/CFP). Our data showed that AS1 and AS2 alone were predominantlylocalized in the nucleolus and the nucleoplasm, respectively. The presence of both AS1 and AS2 proteins in the sameinterphase cell demonstrated their co-localization in both nucleolus and nucleoplasm. In addition, AS1 alone was able toassociate with the condensed chromosome in the metaphase cell. Our data suggest that AS1, AS2 and the AS1-AS2 proteincomplex may have distinct functions, which are all required for normal plant development.

Key words: Arabidopsis; asymmetric leaves1/2; leaf development; subcellular localization.

Zhu Y, Li Z, Xu B, Li H, Wang L, Dong A, Huang H (2008). Subcellular localizations of AS1 and AS2 suggest their common and distinct roles inplant development. J. Integr. Plant Biol. 50(7), 897–905.

Available online at www.jipb.net

Leaf organogenesis occurs within the peripheral zone of theshoot apical meristem (SAM). Two critical biological processesare essential for leaf primordium initiation and subsequentdevelopment: the specification of founder cells within the SAMperipheral zone and the establishment of leaf polarity (Byrneet al. 2000; Eshed et al. 2001; Bowman et al. 2002). Thefounder cells from which leaf primordia arise are specified bydownregulation of a set of highly conserved class 1 knotted1-

Received 7 Apr. 2008 Accepted 11 Apr. 2008

Supported by the Scientific and Technological Council Foundation of Shang-

hai (04JC14017), the National Talent Training Fund in Basic Research of

China (J0630643) to A. Dong, the National Natural Science Foundation of

China (30630041) and the Chinese Academy of Sciences (KSCX2-YW-N-

016) to H. Huang.∗Author for correspondence.

Tel: +86 21 5492 4088;

Fax: +86 21 5492 4015;

E-mail: <[email protected]>.

C© 2008 Institute of Botany, the Chinese Academy of Sciences

doi: 10.1111/j.1744-7909.2008.00693.x

like homeobox (KNOX ) genes that are originally expressed inthe SAM (Waites et al. 1998). After leaf primordia initiate, theyneed to be patterned along adaxial-abaxial, proximodistal andmediolateral axes (Byrne et al. 2000; Hudson 2000).

Recent studies on leaf development have led to significantadvances in understanding the regulation of these two leafdevelopmental processes. In Arabidopsis, class 1 KNOX genesconsist of four members: STM, BP, KNAT2 and KNAT6. Ithas become clear that downregulation of class 1 KNOX genesin the leaf primordium initiation stage and in subsequent leafdevelopment requires functions from a number of regulatorygenes (Hake et al. 2004; Scofield and Murray 2006) and planthormone actions (see Li et al. 2007 for review). In addition, acomplex regulatory network has also been identified and exten-sively characterized for leaf adaxial-abaxial polarity formation inArabidopsis (Kidner and Timmermans 2007; Xu et al. 2007).

The Arabidopsis genes, AS1 and AS2, are required for bothclass 1 KNOX gene repression and leaf polarity establishment.AS1 encodes a protein that contains an R2-R3 MYB-domain(Byrne et al. 2000) while AS2 encodes a plant-specific leucine-zipper protein (Iwakawa et al. 2002) that associates with AS1(Xu et al. 2003). Given that AS1 and AS2 might regulate the

898 Journal of Integrative Plant Biology Vol. 50 No. 7 2008

same genetic pathways during leaf morphogenesis, mutations ineither AS1 or AS2 gene could result in similar plant phenotypes.For example, both as1 and as2 mutant plants in the Landsbergerecta accession produce a proportion of leaves with petiolesgrowing underneath blades, forming a lotus leaf structure (Sunet al. 2002; Xu et al. 2002). In extreme cases, both mutantseven form some radially symmetric leaves (Xu et al. 2003;Qi et al. 2004). Although most leaves in these two mutantsare expanded, some of them are lobed at the leaf margin(Byrne et al. 2000; Semiarti et al. 2001; Sun et al. 2002),resembling those in 35S::BP transgenic plants (Chuck et al.1996).

Molecular genetic analyses have revealed that AS1 andAS2 genes repress BP, KNAT2, KNAT6, MIR165/166, FIL andKAN1, and upregulate REV , PHB and PHV genes (Byrne et al.2000; Semiarti et al. 2001; Xu et al. 2003; Li et al. 2005). A recentstudy also showed that AS1 protein was able to interact withseveral other proteins, including HIRA, a homolog of the histonechaperone, indicating that AS1-AS2 may function in chromatinremodeling for heterochromatic and euchromatic gene silencing(Phelps-Durr et al. 2005). While AS1 and AS2 functions areconsidered to be critical in leaf development, little is known aboutthe molecular mechanisms by which AS1-AS2 act to regulateother components. As a first step towards elucidating themolecular basis of the AS1-AS2 action, we analyzed subcellularlocalization for AS1 and AS2 proteins. Previous studies haveshown that AS2 and rough sheaths2 (RS2), a maize homologof AS1, are both located in the nucleus (Iwakawa et al. 2002;Theodoris et al. 2003), providing important information abouttheir functions. Recent work has also demonstrated that AS1and AS2 are co-localized in the nuclear bodies (Ueno et al.2007). Here we show more detailed analyses of AS1 and AS2subcellular localizations. We report that AS1 and AS2 aloneare preferentially localized in the nucleolus and nucleoplasm,respectively, and that AS1 and AS2 together in the sameinterphase cell are present in both nucleolus and nucleoplasm.We propose that AS1, AS2 and the AS1-AS2 protein complexmay play distinct roles that are all required for normal plantdevelopment.

Results

AS1-YFP and AS2-CFP fusion proteins are biologicallyactive

In order to investigate subcellular localizations of the AS1 andAS2 proteins, we generated fusion constructs in which the AS1and AS2 coding sequences were fused to those of YFP andCFP genes, respectively, under the control of an estradiol-inducible promoter (ES) (Zuo et al. 2000). To determine whetherthe AS1-YFP and AS2-CFP fusion proteins were biologicallyactive, we first transformed these two constructs into as1 and

as2 mutant or wild-type plants, and analyzed phenotypes ofthe transgenic plants. We previously reported that AS1 ex-pression driven by the 35S promoter rescued as1-101 mutantphenotypes, whereas the same construct transformed into wild-type Landsberg erecta (Ler) plants resulted in plants with onlyminor phenotypic changes (Sun et al. 2002). Previous dataalso demonstrated that the 35S::AS2 transgenic plants had veryseverely abnormal phenotypes, with all rosette leaves up-curled(Iwakawa et al. 2002; Xu et al. 2003).

Similar to the 35S::AS1/as1-101 plants, the ES::AS1-YFP/as1-101 plants nearly complemented the as1 phenotypesupon estradiol induction. Compared with the wild-type plants(Figure 1A), as1-101 produced broader rosette leaves, espe-cially at the bottom portion of the blade (Figure 1B, arrow). Inaddition, as1-101 leaves were not flat (Figure 1B, arrowheads),with leaf margins being curled downwards. By contrast, thesetypical as1 leaf phenotypes were not observed in the ES::AS1-YFP/as1-101 transgenic plants (Figure 1D). We also analyzedES::AS1-YFP/Ler transgenic plants, and found that, after induc-tion, these plants were also similar to those of the 35S::AS1/Lertransgenic plants (Sun et al. 2002), with a slightly dwarfishplant stature (Figure 1C). As compared with the as2-101 mutantplants (Figure 1E), the estradiol-induced ES::AS2-CFP/as2-101transgenic plants showed severely abnormal phenotypes, andall rosette leaves were curled upwards (Figure 1F, inset). Byanalyzing ES::AS1-YFP and ES::AS2-CFP transgenic plants,our results indicate that these two constructs can both producefunctional AS1 and AS2 proteins.

Subcellular localizations of AS1 and AS2 in tobaccoBY2 cells

To determine subcellular localizations of AS1 and AS2 pro-teins, we first introduced two estradiol-inducible constructs,ES::AS1-YFP and ES::AS2-CFP, into tobacco BY2 cells, re-spectively. Cell lines that showed stable AS1-YFP or AS2-CFP expression after estradiol induction were selected forfurther analyses. Our previous data on yeast two-hybrid andin vitro biochemical assays demonstrated that AS1 and AS2could form a protein complex (Xu et al. 2003), suggesting thatthese two proteins must share the same subcellular localization.Contrary to our expectations, AS1 and AS2 alone showeddifferent localization patterns in the interphase BY2 cells. AS1-YFP was non-uniformly localized in the nucleolus with a fewspeckles (Figure 2A), whereas AS2-CFP was detected pre-dominantly in the nucleoplasm and weakly in the cytoplasm(Figure 2B). To test the possibility that when the AS1 andAS2 proteins were together in the same interphase cell theymight change their localization patterns, we co-transformedES::AS1-YFP and ES::AS2-CFP into the BY2 cell and analyzedcell lines that stably produced both AS1-YFP and AS2-CFPproteins. Our data showed that, in these lines, AS1-YFP and

Asymmetric Leaves1/2 Subcellular Localizations 899

Figure 1. Phenotypes of transgenic plants carrying ES::AS1-YFP and

ES::AS2-CFP indicate that the AS1 and AS2 fusion proteins are biolog-

ically active.

Plants of 2 week-old wild-type Ler (A), as1-101 (B), T2 ES::AS1-YFP/Ler

(C), T2 ES::AS1-YFP/as1-101 (D), as2-101 (E) and T2 ES::AS2-

CFP/as2-101 (F) were grown on MS medium containing 4 μM estradiol.

A total of 12 ES::AS1-YFP/as1-101 and seven ES::AS2-CFP/as2-101

T2 transgenic lines were analyzed, and the overall phenotypes in the

corresponding transgenic plants were similar.

Bars indicate 1 mm in (A–F).

AS2-CFP proteins indeed co-localized in both nucleolus andnucleoplasm (Figure 2C). These results indicate that AS1 andAS2 are individually able to alter the distribution of the other incells.

To verify that the AS1-YFP and AS2-CFP co-localization wasnot caused by an interaction between YFP and CFP, we ana-lyzed cell lines that contained the co-expressed CFP-NtNAP1;3and NtCYCB1;1-YFP fusion proteins (Figure 2D). Previousdata showed that, during the interphase of cell cycle, CFP-NtNAP1;3 was localized in the cytoplasm while NtCYCB1;1-YFPappeared in both nucleus and cytoplasm (Criqui et al. 2001;Dong et al. 2003). Our results revealed that, in the interphaseBY2 cell expressing both CFP-NtNAP1;3 and NtCYCB1;1-YFP,YFP signals could be detected in the nucleus and cytoplasm,

whereas CFP signals were visualized only in the cytoplasm(Figure 2D). This pattern is similar to that found with the twofusion proteins alone in the tobacco BY2 cells (Criqui et al. 2001;Dong et al. 2003). Previous data also showed that, before cellsexited mitosis, NtCYCB1;1-YFP was degraded and the YFPsignals were diminished (Criqui et al. 2001). Similarly, in thetelophase/G1 stages of the BY2 cells which carried the CFP-NtNAP1;3 and NtCYCB1;1-YFP fusion constructs (Figure 2D),only CFP-NtNAP1;3 signals were recorded.

Furthermore, we showed co-localization of the NtCYCB1;1-YFP and CFP-NtNAP1;3 fusion proteins in the mitotic BY2 cell. Ithas previously been shown that, in the metaphase of cell cycle,the NtCYCB1;1 protein was associated with the condensedchromosome, but the NtNAP1;3 protein was distributed around,but not on the chromosome (Criqui et al. 2001; Dong et al.2003). Our results showed that the protein pattern producedby co-expression of CFP-NtNAP1;3 and NtCYCB1;1-YFP inthe same metaphase cell was very similar to that arising fromindependent expression of each protein (Figure 2E), suggestingthat the localization of the two proteins was independent. Allthese results indicate that the subcellular co-localization of theAS1-YFP and AS2-CFP fusion proteins depends on the AS1-AS2 interaction, but not the YFP and CFP interaction.

AS1 associates with condensed chromosomein the metaphase cell

In addition to the subcellular localizations of AS1 and AS2 inthe interphase, their distribution patterns in other phases ofthe cell cycle may be also informative for fully understandingtheir molecular functions. To this end, we analyzed the AS1localization pattern in the metaphase, using a previously re-ported metaphase marker YFP-NtSET1 (Yu et al. 2004). Todistinguish AS1 fusion protein from YFP-NtSET1 in the sameBY2 cell, we generated an additional fusion construct, ES::AS1-CFP. Similar to ES::AS1-YFP, the ES::AS1-CFP construct wasalso able to complement as1 phenotypes (data not shown),and was localized in the nucleolus of the interphase BY2 cell(Figure 3A, upper panels). As reported previously, YFP-NtSET1showed a non-uniform distribution in the nucleoplasm of theinterphase cells (Yu et al. 2004) (Figure 3A, lower panels).However, in the metaphase, YFP-NtSET1 became associateduniformly with the condensed chromosome (Yu et al. 2004)(Figure 3B, lower panels). Interestingly, the AS1-CFP fusionprotein appeared also on the condensed chromosome re-gion, but was dispersed into several concentrated speckles(Figure 3B, upper panels). We have observed approximately100 independent metaphase cells, and found that this AS1pattern was very consistent.

Because the cultured BY2 cells usually only contain asmall proportion of mitotic cells and AS1-CFP/YFP signals inthe metaphase are very weak, it is difficult to find signals in


Figure 2. Localizations of AS1-YFP and AS2-CFP fusion proteins.

AS1-YFP was localized in the nucleolus but the distribution was not uniform (A), whereas AS2-CFP was located uniformly in the nucleoplasm

and weakly in the cytoplasm (B). AS1-YFP and AS2-CFP were localized in both nucleoplasm and nucleolus when AS1-YFP and AS2-CFP were

co-expressed (C). Note that speckles appeared in nucleolar domains. In the interphase cells, NtCYCB1;1-YFP was found in both cytoplasm and

nucleus while CFP-NtNAP1;3 was localized only in the cytoplasm (D). Note that NtCYCB1-YFP and CFP-NtNAP1;3 co-expressed in one cell did not

cause a cross-detection of CFP and YFP signals; and NtCYCB1;1-YFP signals could not be detected in the telophase/G1 phases. In, interphase; Te,

telophase/G1 phase. In metaphase, NtCYCB1;1-YFP associated with condensed chromosome, while CFP-NTNAP1;3 was around the chromosome

(E), indicating that CFP and YFP signals in this cell stage were not cross-detected. More than three independent cell lines harboring each of the listed

constructs were examined, and the consistent subcellular localization patterns of each fusion protein are shown. YFP fluorescence (yellow), CFP

fluorescence (blue) and bright-field differential interference contrast (DIC) are shown. The last images (A–E) are the merged ones.

Bars indicate 10 μm in (A–E).

metaphase cells that express AS1 fusion protein alone. Toconfirm that AS1 is able to bind chromosomes during mito-sis, we co-transformed ES::AS1-CFP with an additional twofusion protein constructs, ES::YFP-NtNAP1;3 or ES::AtMYC2.Both YFP-NtNAP1;3 and YFP-AtMYC2 proteins differed in their

distribution patterns in cells between interphase andmetaphase. In interphase, YFP-NtNAP1;3 was located in thecytoplasm (Figure 3C), and YFP-AtMYC2 was distributed inthe nucleoplasm and central nucleolus (Figure 3D). In themetaphase, however, relatively strong signals from both fusion


Figure 3. AS1-associated chromosome during mitosis.

In the interphase, AS1-CFP and YFP-NtSET1 did not show a co-localized pattern, with AS1-CFP in the nucleolus and YFP-NtSET1 non-uniformly

located in the nucleoplasm (A). In the metaphase, YFP-NtSET1 signals were fully associated with the condensed chromosome, while AS1-CFP signals

were also associated with chromosome, but forming small speckles (B). In the metaphase cell, YFP-NtNAP1;3 signals surrounded the chromosome,

within which AS1-CFP signals were concentrated in speckles (arrowhead) (C). Note that of the two neighboring cells in (C), one of which is in the

interphase (In) and the other is in the metaphase (Me). Similarly, the YFP-AtMYC2 signals could mark the chromosome region of a metaphase cell

(Me), and within this region speckles of the AS1-CFP signals were detected (D). More than three independent cell lines harboring each of the listed

constructs were examined, and the consistent subcellular localization patterns are shown. YFP fluorescence (yellow), CFP fluorescence (blue) and

bright-field differential interference contrast (DIC) are shown. The last images (A–D) are the merged ones.

Bars indicate 10 μm in (A–D).


Figure 4. Localization of AS1 and AS2 fusion proteins in Arabidopsis.

Root or leaf tissues from 10-d-old transgenic plants were analyzed after estradiol induction for 6–12 h. AS1-YFP localized in the nucleolus in both root

(A) and leaf (B) cells. However, different patterns were found for AS2-YFP distribution (C,D). In roots, AS2-YFP was in the nucleoplasm (C). In

leaves, AS2-YFP signals were detected either in nucleoplasm (arrowhead) (D) and uniformly or non-uniformly located in nuclei (D,E). In any cases,

AS2-YFP signals were found in cytoplasm (C–E). Three independent transgenic plants harboring each of the listed constructs were examined, and

the consistent subcellular localization patterns are shown. YFP fluorescence (green) and bright-field differential interference contrast (DIC) are shown.

The last images (A–E) are the merged ones.

Bars indicate 20 μm in (A–E).

proteins surrounded the chromosome (Figure 3C,D), so thatthe metaphase cells could be relatively easily identified. Usingthis method, we analyzed several metaphase cells, and foundAS1-CFP signals were all associated with the chromosome inthe concentrated speckles (Figure 3C,D), further confirming thisAS1 localization pattern in the metaphase. We also analyzed theAS2-CFP localization in the metaphase cells, but the distributionpattern similar to that of AS1-CFP was not observed (data notshown). These results suggest that AS1 may have specificfunctions in different cell stages.

Subcellular localizations of AS1 and AS2 in Arabidopsis

To examine subcellular localizations of AS1 and AS2 in Ara-bidopsis plants and to compare signal intensity for the AS1

and AS2 fusion proteins, we generated an additional ES::AS2-YFP fusion construct and introduced both ES::AS1-YFP andES::AS2-YFP constructs into wild-type Arabidopsis Ler . Intobacco BY2 cells, the localization pattern between AS2-CFPand AS2-YFP was the same and ES::AS2-YFP transgenicplants showed severely abnormal phenotypes similar to thosetransformed with ES::AS2-CFP after estradiol induction (datanot shown). In the root of the transgenic Arabidopsis plants,localization patterns of AS1-YFP and AS2-YFP resembled thosein the interphase BY2 cells, with AS1 in the nucleolus andAS2 predominantly in the nucleoplasm (Figure 4A,C). However,while the AS1-YFP pattern in leaves was also similar to that ofAS1-YFP alone in the BY2 cell (Figure 4B), AS2-YFP localiza-tions showed several patterns, some of which were differentfrom those in the BY2 cells (Figure 4D,E).


Among different leaf tissues examined, while some cellsexhibited the AS2 distribution pattern similar to that in theBY2 cell (Figure 4D, arrowhead), most cells showed a uniformdistribution of AS2-YFP in the nucleus (Figure 4D). We foundthat this type of AS2 signal was due to a failure of focusing,as nuclei in leaf cells were not always at the same horizontallevel. In addition, a proportion of Arabidopsis leaf cells producedAS2-YFP signals throughout the nuclei with speckles in somenuclear regions (Figure 4E, arrowheads). This pattern is similarto that in BY2 cells with co-localization of AS1-YFP and AS2-CFP (Figure 2C), suggesting that those Arabidopsis cells maycontain a higher level of endogenous AS1 proteins. Theseresults indicate that the localization patterns of the AS1 andAS2 fusion proteins in Arabidopsis and in tobacco BY2 cells aregenerally very similar.

Discussion

Previous studies have demonstrated that AS1 and AS2 areimportant genes in controlling leaf development, not only forKNOX gene suppression but also for leaf polarity establishment.During leaf development, AS1 is expressed throughout leafprimordia (Byrne et al. 2000). Although the AS2 expressionpattern in leaf primordia or developed leaves has not beenreported, this gene was expressed in the adaxial domain ofcotyledons in embryos (Iwakawa et al. 2002). It is thereforegenerally thought that AS2 is a leaf adaxial-specific gene, butis expressed at a very low level in the adaxial leaf domainso that common in situ hybridization methods fail to detect itstranscripts. AS1 and AS2 can form a protein complex, evidencedby yeast two-hybrid experiments and in vitro biochemical assays(Xu et al. 2003). Based on the fact that transcript distribution ofAS1 and AS2 do not completely overlap, it is possible that,in addition to the AS1-AS2 complex, AS1 and AS2 may haveadditional distinct functions in plant development.

Previous studies on RS2 and AS2 localizations demonstratedthat both proteins were located in the nucleus, providing usefulinformation for interpreting their respective functions (Iwakawaet al. 2002; Theodoris et al. 2003). However, experiments withexpression of monocot RS2 gene in Arabidopsis or analysis oftransient expression of AS2 in onion epidermal cells, may notyield the critical information needed for better understandingAS1 and AS2 functions. Recent studies have shown an inplanta AS1 and AS2 localization, with both proteins being foundin nuclear bodies (Ueno et al. 2007); however, more detailedsubcellular AS1-AS2 localization is still required. Our datashowed that AS1 and AS2 have their preferential subcellular lo-cations. In the interphase BY2 cells, AS1 alone was exclusivelylocated in the nucleolus, within which some AS1 signals wereconcentrated to form speckles. These speckles may be equiva-lent to the nuclear bodies observed previously (Theodoris et al.2003; Ueno et al. 2007). AS2 was distributed uniformly in both

nucleoplasm and cytoplasm, when it was alone. AS1 and AS2were co-localized in both nucleolus and nucleoplasm with somespeckles, when they were present in the same interphase cell.The AS1-AS2 protein complex may play very important rolesin KNOX gene repression and polarity formation, because lossof function in each of these two genes results in similar leafphenotypic changes. However, in Arabidopsis, AS1 and AS2are not always present in the same cell and therefore, whenpresent alone, they may have some regulatory functions distinctfrom those of the AS1-AS2 complex.

If the presence of AS1 and AS2 in the same cell can resultin the two proteins being co-localized in both nucleoplasm andnucleolus, it is reasonable to expect that transgenic plants carry-ing either AS1-YFP or AS2-YFP should show the co-localizationpattern because of the endogenous AS1 and AS2 proteins.However, our results revealed that only cells in some leavesof the ES::AS2-YFP transgenic plants had this co-localizationpattern (Figure 4E). By analyzing the published data for AS1and AS2 expression, we found that the AS1 and AS2 subcellularlocalization patterns that we observed in the transgenic plantswere very consistent with a previous systematic characterizationof AS1 and AS2 transcript distribution (Lin et al. 2003). TheAS2 transcription level was very low in roots and leaf blades,and thus the ES::AS1-YFP plants only showed the AS1-YFPlocalization pattern similar to that with AS1-YFP alone in BY2cells. Similarly, the AS1 transcript level was very low in the root(Lin et al. 2003), and therefore the AS2-YFP localization patternin roots of the ES::AS2-YFP transgenic plants resembled thatof AS2-CFP alone in BY2 cells. By contrast, the AS1 transcriptlevel was relatively high in leaf blades of wild-type plants (Linet al. 2003), and some leaf cells of the ES::AS2-YFP transgenicplants showed an AS2 pattern similar to that in BY2 cells, inwhich both AS1-YFP and AS2-CFP were co-localized.

Because most gene expression and regulation occurs in thenucleoplasm of the interphase cells, our results provide a strongindication that the regulatory function of the AS1-AS2 complex islargely dependent on the presence of AS2. This hypothesis wassupported by previous phenotypic analyses of 35S::AS1 and35S::AS2 transgenic plants. Plants with overexpression of AS1did not show obvious defects in leaf polarity, nor abnormalities inKNOX repression (Sun et al. 2002). In contrast, overexpressionof AS2 resulted in plants with up-curled leaves and down-pointing siliques, reflecting a severe aberrant adaxial-abaxialpolarity in leaves and downregulation of BP, a gene in the class1 KNOX family (Iwakawa et al. 2002; Lin et al. 2003; Xu et al.2003).

Our results also showed that AS1 is able to bind chromo-somes in the metaphase of cell cycle, with the help of an-other three marker proteins, YFP-NtSET1, YFP-NtNAP1;3 andYFP-AtMYC2. Among these three fusion proteins, YFP-NtSET1was known to be associated with chromosomes in themetaphase (Yu et al. 2004). The other two fusion proteinswere found to surround the chromosome in the metaphase


cells, showing relatively strong fluorescent signals. In the in-terphase cells, AS1 and these three fusion proteins had theirown distribution pattern. In the mitotic cells, signals from theAS1 fusion proteins were very weak, and the identification ofthe mitotic cells among a vast majority of interphase cells wasvery difficult. Based on the specific distribution patterns of thesethree marker proteins, the metaphase cells could be relativelyeasily identified. In all metaphase cells analyzed, speckled AS1signals could be noted within the region where the condensedchromosomes were present. That AS1 but not AS2 could bindchromosomes further supports our hypothesis that AS1 alonemay have some additional functions different to those of AS2and the AS1-AS2 protein complex.

The significance of the nucleolus-located interphase AS1 pro-tein is not yet clear. The nucleolus is the place where ribosomebiogenesis is processed, from the synthesis of ribosomal RNAsto their assembly into ribosomal subunits. However, emergingevidence also shows that, in addition to this key function of thenucleolus, the nucleoli play unconventional and nonstandardroles, particularly in biogenesis of other RNA-containing cellularmachinery, stress sensing and control of cell activity (Raskaet al. 2006). Further exploration of the role of AS1 in thenucleolus is now required.

Materials and Methods

Plant growth and cell culture

Transgenic Arabidopsis plants were grown on solid Murashigeand Skoog medium M0255 (Dushefa, the Netherlands), supple-mented with 0.9% sucrose at 21 ◦C with a light : dark cycle of16:8 h. BY2 cell lines were maintained by weekly subculture aspreviously described (Nagata et al. 1992).

Constructs and plant transformation

Polymerase chain reaction (PCR)-amplified fragments of AS1and AS2 coding regions were cloned into vectors pEYFP-EYFP and pECFP-ECFP (Yu et al. 2004) to yield AS1-YFPand AS2-CFP fusions, respectively, and insertion fragmentsin the resulted constructs were sequencing verified. The AS1-YFP and AS2-CFP fragments were then subcloned into vec-tors pER10 (with kanamycin resistance selection) and pER8(with hygromycin resistance selection) for plant transforma-tion, resulting in ES::AS1-YFP and ES::AS2-CFP, respec-tively. The same methods were used to obtain ES::AS1-CFP, ES::AS2-YFP and ES::YFP-AtMYC2. PCR primers usedin amplifying cDNA sequences for construction were as fol-lowing: 5′-atgtcgacATGAAAGAGAGACAACGTTGG and 3′-atccatgggGGGGCGGTCTAATCTGCAAC (for AS1-YFP andAS1-CFP); 5′-atgtcgacATGGCATCTTCTTCAACAAACTC and

3′-atccatgggAGACGGATCAACAGTACGGC (for AS2-CFP andAS2-YFP); and 5′-atcccgggTAATGACTGATTACCGGCTAC-AAC and 3′-atactagtTTAACCGATTTTTGAAATCAAACTTG(for YFP-AtMYC2). In each of the above sequences, the lower-case letters represent additional nucleotides to introduce restric-tion sites. Constructs ES::YFP-NtNAP1;3, ES::CFP-NtNAP1;3,ES::YFP-NtSET1 and ES::NtCYCB1;1-YFP were obtained asdescribed previously (Shen 2001; Dong et al. 2005). Transgenictobacco BY2 cells and Arabidopsis plants carrying differentconstructs for fusion proteins were obtained by Agrobacterium-mediated transformation, according to a previously describedmethod (Shen 2001). For each construct, at least 24 indepen-dent transgenic tobacco BY2 cell lines were selected to testthe YFP fluorescence, among which three lines were usedfor the localization analysis. Induction of protein expressionfor transgenic Arabidopsis plants and tobacco BY2 cells wasperformed with 4 μmol/L estradiol according to a previouslydescribed method (Zuo et al. 2000).

Microscopy

The leaves and roots from transgenic Arabidopsis or tobaccoBY2 cells were analyzed and photographed using a Zeiss LSM(510) confocal microscope (Carl Zeiss, Jena, Germany).

Acknowledgements

We thank Dr N.H. Chua for providing vectors pER8 and pER10.

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(Handling editor: Chun-Ming Liu)

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Subcellular Localizations of AS1 and AS2 Suggest Their Common and Distinct Roles in Plant Development