Cloning of an Arabidopsis Patatin-Like Gene, STURDY, by Activation T-DNA Tagging · Cloning of an...

Cloning of an Arabidopsis Patatin-Like Gene,STURDY, by Activation T-DNA Tagging

Shihshieh Huang*, R. Eric Cerny, Deepti S. Bhat, and Sherri M. Brown

Mystic Research, Monsanto Company, 62 Maritime Drive, Mystic, Connecticut 06355 (S.H.); andAgricultural Sector, Monsanto Company, 700 Chesterfield Parkway North, St. Louis, Missouri 63198(R.E.C., D.S.B., S.M.B.)

Activation T-DNA tagging can generate dominant gain-of-function mutants by overexpression of a particular endogenousgene. We identified an activation-tagged mutant, sturdy, exhibiting a stiff inflorescence stem, thicker leaves, shorter siliques,larger seeds, round-shaped flowers, and delayed growth. It is most important that unlike its wild-type counterpart, thismutant is less prone to lodging. Cloning of STURDY revealed that in sturdy, there is an open reading frame containing asingle intron encoding a patatin-like homolog. The T-DNA is inserted into the 39 region of the second exon. The mutantphenotype was shown to be the result of overexpression of STURDY by mRNA analysis and transgenic studies. Preliminaryhistological studies have revealed an increase in cell number in the inflorescence stem of mutant plants; however, additionalstudies are needed to better understand the overexpression phenotype.

Lodging occurs when crop plants fail to withstandenvironmental challenges, resulting in extensive fi-nancial losses in agriculture each year throughout theworld. Crop plants fall to the ground prematurely,making them recalcitrant to mechanical harvest.Even in areas where manual harvesting is practiced,lodged plants can absorb moisture from the groundsurface, interfering with the desiccation process andcompromising the quality of the grain. In 1992, it wasreported that lodging in winter wheat cost #£130million in loss of yield alone (Berry et al., 1998) andreduced the grain quality standard (Hagberg fallingnumber) from a U.K. 5-year average of 287 to 254(Home Grown Cereal Authority, 1993). Through con-ventional breeding methods, dwarf traits have beenidentified and introgressed into various crop species,resulting in shorter plants with stiffer stems lessprone to lodging. For example, the gibberellin-insensitive semidwarf Rht genes in wheat and the dwgenes in sorghum have been used to reduce plantheight (Quinby and Karper, 1954; Gale and Yousse-fian, 1985). However, these genetic alterations inplant stature are limited to certain crops or varietiesand are often accompanied with negative pleiotropiceffects including reduced yield and lower grain pro-tein levels. The progress in recombinant DNA tech-nologies recently has provided alternative ap-proaches for improvement of agronomic crops. Weare interested in studying plant stem structure at themolecular level and identifying target genes to ge-netically modify crops with improved lodgingresistance.

In principle, there are at least three aspects inwhich genetic engineering can be applied to improve

lodging resistance. Many genes implicated in cellwall biosynthesis, composition, and lignificationhave been cloned (for review, see Boudet, 1998; Re-iter, 1998). By altering the composition of the stemcell walls, it may be possible to increase stemstrength. A change in stem strength alternativelycould come from altering the organization of thestem structure (e.g. rearrangement of vascular bun-dles). Several homeobox genes were shown to pref-erentially express in the vascular tissues of Arabidop-sis (Baima et al., 1995; Tornero et al., 1996). AnArabidopsis mutant displaying the transformation ofcollateral vascular bundles into amphivasal vascularbundles recently was identified (Zhong et al., 1999).An increase in mass could also result in a strongerstem structure. Studies of transgenic plants demon-strated the possibility of modifying the stem mass byvarying the level of auxins or cytokinins (Medford etal., 1989; Romano et al., 1991). However, no mutantor transgenic plant showing a strengthened stemphenotype with the normal plant height has beenreported.

We initiated an activation T-DNA screen to isolatemutants displaying unusual stem phenotypes. Firstdescribed by Hayashi et al. (1992), activation T-DNAtagging produces dominant mutations caused by in-creased or ectopic expression of endogenous genes.This approach allows discovery of novel genes inwhich recessive, loss-of-function mutations have noobvious phenotype. In this study, we report the re-covery of a dominant gain-of-function mutation af-fecting stem, leaf surface, silique length, seed size,flower shape, and growth rate. The mutant allele,named sturdy, was cloned and shown to be related tothe patatin family of proteins. All phenotypes ob-served in sturdy mutants are due to overexpression ofthe gene. A preliminary microscopic examination of

* Corresponding author; e-mail [email protected]; fax 860 –572–5240.

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sturdy inflorescence stems revealing their unusualstructure is presented.

RESULTS

Identification of a Dominant, Stiff-Stem Mutant,Sturdy, in an Activation T-DNA-Tagging Population

We transformed Arabidopsis plants (Columbiaecotype) with a binary vector, pMON29963, to gen-erate an activation T-DNA-tagging population. TheT-DNA region of pMON29963 contains four enhanc-ers of the cauliflower mosaic virus (CaMV)-35S pro-moter stacked near the right border, and the selectionmarker neomycin phosphotransferase II gene (NPTII)driven by the nopaline synthase promoter close tothe left border. The transformants were first germi-nated on medium containing kanamycin and resis-tant seedlings were transferred to soil for phenotypicevaluation. Under our growth conditions, Arabidop-sis plants of the Columbia ecotype can reach an av-erage height of 40 cm (Huang et al., 1998) at the

global proliferative arrest stage (Hensel et al., 1994).The stems typically cannot physically sustain growthand the plants bend toward the soil surface or lodge.In screening 4,200 transformants, we identified a sin-gle lodge-resistant plant. It appeared to have stifferand thicker inflorescence stems and branches thanwild-type plants, so it was named sturdy. This phe-notype was observed in 83 of 114 T2 progeny, sug-gesting that sturdy is a dominant mutation caused bya T-DNA insertion at a single locus (x2 5 0.3).

As shown in Figure 1, B and C, 2-week-old sturdyplants are small and dark green in color, similar togibberellin-deficient mutants. However, sturdy didnot respond significantly different than wild-typeplants when active gibberellin analogs were applied.Moreover, unlike gibberellin-insensitive mutants,sturdy plants grow to a height comparable with wild-type plants with only a slight (15%) reduction inheight. However, it takes about 55 d for sturdy plantsto reach global proliferative arrest stage comparedwith 47 d for wild-type plants grown under the same

Figure 1. The phenotype of the sturdy mutant. A, Mature mutant plant. B and C, Two-week-old mutant and wild-type plants,respectively. D and E, Mutant and wild-type flowers, respectively. F and G, Mutant siliques and wild-type siliques,respectively. H, Topical view of a mutant flower cluster.

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conditions. The growth rate was monitored throughthe plants’ life cycle by measuring leaf expansion andplant height. The most significant difference is thatsturdy mutants’ stems are an average of 40% thickerthan those of the wild-type plants. These results aresummarized in Table I and Figure 2. It appears thatsturdy develops greater stiffness in its inflorescencestems and branches through an increase in diameterand at the expense of plant height and growth rate.The sturdy mutation affects other parts of the plant aswell. Its leaves have a rougher surface, and its flow-ers and siliques are shorter (Fig. 1, D–G). The shapeof mutant flowers develops as round instead of oval.Also, the mutant flower clusters appear to be morecompact with the stigmas protruding out of theflower buds (Fig. 1H). When the length of the siliquesand number of seeds in each silique were measured(Table II), we found that mutant siliques also pro-duced fewer seeds; however, total seed weight re-mained about the same. On average, individual mu-tant seeds weighed 30% more than wild type. Wenoticed that leaves and stems of the sturdy mutant arebrittle and prone to fracture.

Cloning of the Genomic DNA of Sturdy

To clone sturdy, we first analyzed the mutant witha genomic DNA gel blot. Three different restrictionenzymes (HindIII, BglII, and SpeI) were used to digestthe genomic DNA of wild-type and mutant plantsand the CaMV-35S enhancer was used as the probefor hybridization (Fig. 3A). DNA gel-blot analysiswith three enzyme digests displayed a single bandonly on the lanes loaded with the mutant genomicDNA. This result, along with the T2 segregation ra-tio, confirmed that a single T-DNA insertion pro-duced the dominant sturdy mutation. We then usedthe same probe to screen a library constructed fromthe genomic DNA of sturdy. Three positive cloneswere isolated independently and all of them con-tained the same 12-kb genomic DNA fragment. Fig-ure 3B shows the restriction map and the sequencedregion of the fragment.

Based on previous reports, T-DNA activation tag-ging is likely to overexpress the nearest gene fromthe CaMV-35S enhancers to induce the mutantphenotypes (Hayashi et al., 1992; Kakimoto, 1996;Kardailsky et al., 1999; Weigel et al., 2000). An ap-

proximately 5.5-kb region of the genomic DNA nearthe T-DNA was sequenced to identify ORFs. Oneputative gene 2.7 kb from the CaMV-35S enhancers(Fig. 3B) was identified with two exons separated bya single intron. This putative gene is 1,234 bp andencodes 382 amino acids (Fig. 4). The CAAT andTATA boxes as well as a poly-A signal were alsoidentified. To test if this ORF was overexpressed inthe sturdy mutant, an RNA gel blot was preparedusing equal amounts of RNA (2 mg) isolated fromleaves of wild-type and mutant plants and probedwith the ORF sequence. The RNA blot shown inFigure 3C clearly indicated that the ORF was highlyexpressed in the mutant leaves but was undetectablein the leaves of wild-type plants and in a secondactivation-tagging line grown under the same condi-tion. This suggests that the putative gene identifiedby the screen is the STURDY gene.

Overexpression of the Putative STURDY cDNA in Wild-Type Plants Conferring the Sturdy Phenotype

To demonstrate that overexpression of STURDY issolely responsible for the mutant phenotypes, weisolated the sturdy cDNA by RT-PCR and constructeda transformation vector, pMON42154, containing thecDNA driven by the enhanced CaMV-35S promoter.The sequence of the cDNA fragment amplified by theprimer designed according to the genomic DNA alsoconfirmed the location of the intron. Among 49 trans-genic plants transformed with pMON42154, 26 ofthem clearly exhibited a sturdy-like phenotype. Weselected five transgenic plants from each group forfurther analysis. As shown in Figure 5, plants num-bered 2, 3, 6, 7, and 8 had the typical sturdy floralcluster, round and compact, whereas 1, 4, 5, 9, and 10appeared to be normal. When the RNA samples pre-pared from leaves were analyzed on an RNA gel blot,only those sturdy-like transgenic plants showedstrong expression of STURDY. These results confirmthat the sturdy phenotype is caused by the overex-pression of the STURDY gene identified in theT-DNA activation-tagging screen alone.

Molecular Characterization of the STURDY Gene

When the STURDY gene was used to search theGenBank sequence database by BLAST programs

Table I. Physical characterization of the sturdy plantsTwenty sturdy and wild-type plants for the measurement; each value represents the means 6 SE.

PhenotypeFlowering

Timea Terminal HeightbTime-to-Terminal

HeightStem Diameter

d cm d mm

sturdy 30 6 0.3 35.4 6 0.63 55 6 0.2 2.10 6 0.04Wild type 25 6 0.2 41.8 6 0.64 47 6 0.3 1.50 6 0.03a Number of days for the primary bolt to reach a height of 1 cm. b Height at which main stem

produced at global proliferative arrest (Hensel et al., 1994).

Sturdy Mutant in Arabidopsis

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(Altschul et al., 1997), it matched a putative gene inan Arabidopsis chromosome 3 bacteria artificial chro-mosome clone, F16M2 (accession no. AL138648). Noother significant homology was identified at the nu-cleotide level. The predicted protein sequenceshowed similarity to a group of patatin or patatin-like proteins. STURDY was compared with threeother predicted patatin-like proteins from the Arabi-dopsis genome sequences along with patatin andpatatin homologs from potato, tobacco, and rubberby protein sequence alignment using the MegAlignprogram (DNASTAR Inc., Madison, WI). As illus-trated in Figure 6, striking resemblance exists acrossthe entire sequence between STURDY and thesepatatin proteins. Patatin, a 40-kD glycoprotein, hasbeen found in all Solanum species tested (Prat et al.,

1990) and accounts for up to 40% of the total watersoluble protein present in the tubers (Racusen andFoote, 1980). In potato, patatin is a multigene familywith 10 to 18 members per haploid genome (Migneryet al., 1988; Twell and Ooms, 1988), and its expressionoften associates with tuber formation (Perl et al.,1991). However, patatin-like proteins have beenfound in other plant organs (Paiva et al., 1983; Van-canneyt et al., 1989) and other species as well (Van-canneyt et al., 1989; Ganal et al., 1991). We examinedthe genomic composition and expression pattern ofthe STURDY gene. Even under less stringent washconditions (0.53 SSC, 50°C for the high stringentwashing), no other homologous sequence of theSTURDY gene was identified (Fig. 7A). This is con-sistent with sequence search results that failed to

Table II. Physical characterization of the silique and seed produced by sturdy plantsTwenty mature siliques were randomly picked from sturdy and wild-type plants for the measurement;

each value represents the means 6 SE.

Phenotype Silique LengthNo. of Seeds within a

SiliqueTotal Seed Wt

Total No. ofSeeds

Average Seed Wt

mm mg mg

sturdy 8.0 6 0.15 47.1 6 1.35 25.3 941 0.027Wild type 15.0 6 0.33 60.9 6 2.78 24.5 1,217 0.020

Figure 2. Growth rate comparison betweensturdy and wild-type plants. A, Chart of length(cm) of the sixth leaf by days. Under our growthconditions, the fifth and sixth leaf are the initialadult leaves in most Arabidopsis plants and theycontinue to expand throughout their life cycle.We recorded the length of the sixth leaf everyother day and used these data as one of thegrowth rate indicators. B, Chart of plant height(cm) by days. The height of the plants was re-corded every other day and was also used as agrowth rate indicator.

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identify homologous Arabidopsis sequences. Also,the STURDY gene was found to be expressed in alltissues tested with highest expression levels in roots(Fig. 7B).

Microscopic Analysis of Sturdy Mutant Stems

To investigate the stem structure of the sturdy mu-tant, we sectioned and analyzed the inflorescencestems from several individual mutant and wild-typeplants. Due to the differences in their growth rate, theplant material was collected at a similar developmen-tal stage based on height (14–15 cm) rather than daysafter planting. As shown in Figure 8, which repre-sents the stem transverse sections of typical sturdy (Aand C) and wild-type (B and D) plants, the diameteris significantly greater in sturdy plants comparedwith wild type at the same magnifications. In gen-eral, sturdy appears to increase in cell number ratherthan cell size. At this developmental stage, there areeight equally spaced vascular bundles in the wild-type plants. In sturdy plants, the number of vascularbundles remains the same, but many of them appearto be expanding and branching out at the tip toward

the pith (indicated by the arrows in Fig. 8, A and C)as if it is undergoing the formation of extra vascularbundles. When stained with toluidine blue, xylemand fiber cells become blue due to the presence oflignin in cell walls. The sturdy stem has at least onemore layer of blue-staining cells than wild type (Fig.8). Therefore, the stiffness of sturdy stems could beexplained by the presence of additional layers oflignin-containing cells.

DISCUSSION

We described the isolation and characterization ofa novel dominant Arabidopsis mutant, sturdy, gener-ated by activation T-DNA tagging. The sturdy mutantwas identified by its stiff inflorescence stem thatsupported its vertical growth and prevented lodging.The phenotype also includes thicker leaves, shortersiliques, larger seeds, round flowers, and delayedgrowth. By screening the genomic library preparedfrom sturdy genomic DNA using the CaMV-35S en-hancer sequence as the probe, we discovered a puta-tive gene of 1,234 bp linked to the CaMV-35S en-hancer tetramer. This putative gene, containing a

Figure 3. Genomic characterization of thesturdy mutant and identification of the STURDYgene. A, Genomic DNA gel-blot analysis of thesturdy mutant. A DNA gel blot containing 3 mgof wild-type and mutant genomic DNA digestedwith HindIII (H), BglII (B), and SpeI (S) hybrid-ized with a CaMV-35S enhancer probe. Thedigoxigenin-labeled DNA fragments (RocheMolecular Biochemicals, Indianapolis) wereused as markers (M). B, Restriction map of thesturdy mutant allele. The map shows HindIII (H),EcoRI (E), and SpeI (S) restriction sites of a 12-kbDNA fragment flanking the right border (RB) ofthe T-DNA isolated from the mutant genomiclibrary. Indicated by the dashed line, approxi-mately 5.5 kb of the fragment near the rightborder was sequenced, and an open readingframe (ORF) separated by one intron (blackboxes) was identified. C, RNA gel-blot analysisof the sturdy mutant. An RNA gel blot loadedwith 2 mg of total leaf RNA isolated from a wildtype (1), sturdy mutant (2), and a second activa-tion tagged line (3) were hybridized with aprobe containing the sequence of the identifiedORF. The DIG-labeled RNA fragments (RocheMolecular Biochemicals) were used as markers(M). On the bottom is an ethidium bromide stainof an agarose gel loaded with the RNA (1 mg perlane) used in the gel blot.





single intron, encodes a patatin-like protein that ishighly expressed in the sturdy mutant and thereforeis likely to be the STURDY gene. Transgenic studiesconfirmed that the sturdy phenotype resulted fromthe overexpression of the STURDY gene.

Patatin is a major soluble glycoprotein found inpotato tubers (Racusen and Foote, 1980) and is en-coded by a multigene family (Mignery et al., 1988;

Twell and Ooms, 1988). Because of the diversity andcomplexity of the gene family, most of the molecularcharacterization of patatin has focused on cloningand expression of different classes of patatin genes(Bevan, et al., 1986; Rosahl et al., 1986; Pikaard et al.,1987; Koster-Topfer et al., 1989; Rocha-Sosa, et al.,1989). The expression of patatin and patatin-likegenes subsequently were detected in other plant or-

Figure 4. The nucleotide sequence of theSTURDY gene and its flanking regions. TheSTURDY ORF is shown in uppercase letters andlowercase letters refer to the flanking and intronsequences of the STURDY gene. The CAAT andTATA sequences (positions 21–60) and the pu-tative polyadenylation signal (positions 2,185–2,190) are in boldfaced font. The single-letterabbreviations for amino acids were used and anasterisk denotes a stop codon. The sequencesused as primers for reverse transcriptase (RT)-PCR to isolate the STURDY cDNA areunderlined.

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gans (Paiva et al., 1983; Vancanneyt et al., 1989) andother species as well (Vancanneyt et al., 1989; Ganalet al., 1991). Although regarded as a storage protein,patatin has enzymatic activity. It exhibits esteraseactivity on a broad range of lipid substrates (Rosahlet al., 1987; Andrews et al., 1988; Hogen andWillmitzer, 1990). These findings suggest that patatinor patatin-like proteins may possess other biologicalfunctions. We currently are investigating the enzy-matic activities of the predicted STURDY gene prod-uct. Studying STURDY should enhance our under-standing of the function(s) of patatin. This stiffinflorescence stem is the first reported phenotypecaused by overexpression of a patatin-like protein.With the progression of the Arabidopsis genome se-quencing project, many more patatin-like proteinsare being discovered in Arabidopsis. Although theymay not always share a high degree of homology atthe nucleotide level, their protein sequences clearlyindicate that they belong to the class of patatin-like

proteins. It will be interesting to see whether overex-pression of other patatin-like proteins will result in asimilar phenotype.

The cross sections show that the inflorescence stemstructure organization of the sturdy mutant retains asimilar pattern to wild type, whereas the stem massis significantly increased. It has been reported thatthe stem cell mass could be altered by varying theendogenous levels of auxins and cytokinins in trans-genic plants (Medford et al., 1989; Romano et al.,1991). In addition, a phospholipase A2 involved insignal transduction of a disease-resistant reaction ap-peared to be a patatin (Senda et al., 1996). Therefore,it could be hypothesized that STURDY plays a role inthe signal transduction pathway of hormone re-sponses. It is interesting that mutations in a receptorprotein kinase, ERECTA, cause a similar phenotypeto sturdy (Torii et al., 1996). It is suggested thatERECTA participates in the coordination of cellgrowth pattern. We currently are investigating the

Figure 5. The floral phenotype of the transgenic plants overexpressing STURDY. A, The floral phenotype of 10 independenttransgenic lines. B, RNA gel-blot analysis. RNA (6 mg) isolated from the leaves of the 10 transgenic lines was hybridized tothe STURDY cDNA probe. On the bottom is an ethidium bromide stain of an agarose gel loaded with the RNA (1 mg perlane) used in the gel blot.





relationship between ERECTA and STURDY. Modi-fications in the biochemical composition of the sturdymutant alternatively may be responsible for thestrengthened inflorescence stem phenotype. Slightlybrittle leaves and an increase in the number of lignin-containing cells in stems of the sturdy mutant indicatebiochemical alternations in cellular components byoverexpressing of STURDY. Further detailed analysison the physiology and biochemistry of the sturdymutant is required to determine the cause of themutant phenotype.

Although most genetic screens are based on mu-tants originating from loss-of-function, the activationT-DNA-tagging screen provides an alternative ap-proach to identify mutants that are caused by gain-of-function. A related approach, with a completeCaMV-35S promoter oriented outward from a trans-posable Ds element, has also been used to isolate

gain-of-function mutations (Wilson et al., 1996;Schaffer et al., 1998; Fridborg et al., 1999). Theseapproaches enhance the possibility of discoveringnew genes in pathways that have been exhaustivelystudied by conventional genetic screens, or genesthat have either no or lethal mutant phenotypeswhen inactivated. A particular useful feature of thesemethods of gene discovery is the potential for directapplication of isolated genes. For example, in a ge-netic screen for an agronomically desirable pheno-type, conventional loss-of-function approaches arelikely to isolate genes that require inactivation fortheir application. In contrast, activation T-DNA andtransposon tagging identify genes that can be appliedby overexpression. Because gene knockout technolo-gies such as antisense, chimeric gene targeting (Hohnand Puchta, 1999) and homologous recombination(Puchta and Hohn, 1996) are still commercially prob-

Figure 6. Homology between STURDY amino acid sequence and those of patain and patatin-like proteins from Arabidopsisand other plant species. A consensus sequence between homologous proteins was identified and is shown at the top of thealignment. The amino acids in black boxes are those identical to the consensus sequence, and the amino acids in gray boxesare those similar to the consensus sequence. The positions of each sequence used in the alignment are listed on the right.The accession numbers of these sequences are STURDY, AL138648; Arabidopsis patatin-like protein 1 (AtPLP1), AC004697;Arabidopsis patatin-like protein 2 (AtPLP2), AL049655; Arabidopsis patatin-like protein 3 (AtPLP3), Z99707; tobacco,U68484; rubber, AJ223038; and potato, A24142.

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lematic, overexpression remains the most usefultechnique for genetic engineering.

The patatin gene family provided a model systemfor studying developmental and metabolic gene ex-pression in the late 1980s and early 1990s. Since then,research interests in patatin have declined due to thelack of physiological relevance. By using an activa-tion T-DNA-tagging approach, we found that apatatin-like protein, STURDY, is implicated in stemstructure. Although many questions are yet to beanswered, our findings shed new light on the biolog-ical function of patatin and, hopefully, provoke re-newed interest in research related to patatin. Mean-while, we continue to study the strengthened stemphenotype caused by overexpression of STURDYand apply the technology to crop species to enhancelodging resistance.

MATERIALS AND METHODS

Plasmid Construction

The binary vector, pMON29963, used to generate theactivation T-DNA-tagging population was constructedsimilar to methods described by Hayashi et al. (1992). Itcontains four stacked enhancers each corresponding to the290 to 2342 region of the original CaMV-35S promoter(Fang et al., 1989). This enhancer repeat was inserted intothe T-DNA region 15 bp from the right border. Near the leftborder was the neomycin phosphotransferase II gene(NPTII) driven by the nopaline synthase promoter confer-ring kanamycin resistance. All other genetic elements usedin the binary vector were identical to those described by Yeet al. (1999).

PCR primers, 59-CTTCTAGAGATGGATCTCAGCAA-GG-39 and 59-CGGGATCCTTAACGGCCGTCAGCGAG-39,

were designed according to the sequences at the beginningand end of the STURDY reading frame (Fig. 4) with theaddition of restriction sites XbaI and BamHI for convenientcloning. These primers were used in a RT-PCR reaction toamplify a 1.1-kb cDNA fragment from RNA extracted fromthe leaves of sturdy mutants. The PCR product was insertedinto the TA cloning vector (Invitrogen, San Diego) forfurther manipulation. To detect any mutations generatedby PCR, the RT-PCR amplified cDNAs were sequenced andcompared with genomic DNA containing the STURDYgene. The STURDY cDNA was then inserted into the bi-nary transformation vector for Agrobacterium tumefaciens-mediated transformation into Arabidopsis. The resultingplasmid, pMON42154, contains the STURDY cDNA undertranscriptional control of an enhanced 35S promoter. The290 to 2342 region of the original CaMV-35S promoter(Fang et al., 1989) was quadrupled to produce the en-hanced 35S promoter.

Plant Material, Growth Conditions, and Transformation

Seeds of Arabidopsis ecotype Columbia (Col-0) were ob-tained from Lehle Seeds (Tucson, AZ). Plants were grown inpotting soil (Scotts, Marysville, OH) in a growth chamber at24°C with a 16-hr photoperiod (120 mmol m22 sec21). Thebinary Ti plasmids pMON29963 and pMON42154 wereintroduced into Arabidopsis via A. tumefaciens-mediatedvacuum infiltration (Bechtold et al., 1993). To select thetransgenic plants, seeds collected from vacuum-infiltratedplants were surface sterilized and germinated on kanamy-cin (40 mg L21) containing Murashige and Skoog medium(M0404, Sigma, St. Louis) supplemented with Suc (1%[w/v]) and MES [2-(N-morpholino)ethanesulfonicacid; 0.5 g L21] at pH 5.8.

Figure 7. Genomic composition and expressionpattern of the STURDY gene. A, DNA gel-blotanalysis. A DNA gel blot containing 3 mg ofEcoRI (E), BglII (Bg), SpeI (S), BamHI (Ba), andXbaI (X) digests of wild-type genomic DNAs werehybridized to the STURDY cDNA probe. B, RNAgel-blot analysis. RNA (2 mg) isolated from theleaves of sturdy mutant and RNA (20 mg) isolatedfrom leaves (L2), stems (S), flowers (F), and roots(R) of wild type were hybridized to the STURDYcDNA probe. On the bottom is an ethidium bro-mide stain of an agarose gel loaded with the RNA(1 mg per lane) used in the gel blot.





Isolation of Genomic DNA and DNA Gel-Blot Analysis

Genomic DNA was isolated from leaves of Arabidopsisplants as previously described (Coleman and Kao, 1992).Genomic DNA (3 mg) was digested with restriction en-zymes, separated on a 0.7% (w/v) agarose gel, andtransferred to nylon membranes (Roche Molecular Bio-chemicals). Prehybridization, hybridization, washing, anddetection of the membrane were conducted using the non-radioactive DIG system (Roche Molecular Biochemicals)following the manufacturer’s protocols.

Isolation of Total RNA and RNA-Blot Analysis

Total RNA was isolated from leaves, inflorescence stems,roots, and flowers of wild-type and sturdy mutants byTRIzol Reagent from Life Technologies (Gaithersburg, MD)following the manufacturer’s protocols. Total RNA sam-

ples (2, 6, or 30 mg) were electrophoresed on 1% (w/v)agarose/formaldehyde gels and transferred to nylon mem-branes (Roche Molecular Biochemicals). The blots wereanalyzed by the same DIG system described above follow-ing the manufacturer’s protocols for RNA gel-blot analysis.

Genomic Library Construction and Screening

Approximately 30 mg of genomic DNA was isolatedfrom sturdy using the same method described in the DNAgel-blot analysis. The genomic library was constructed bypartial digestion with BglII and then using the Lambda FIXII/XhoI Partial Fill-In Vector Kit (Stratagene, La Jolla, CA)following the manufacturer’s instructions. The same non-radioactive DIG system was also used for screening thelibrary. Using the CaMV-35S enhancer as a probe, threeindependent clones were isolated from about 15,000

Figure 8. Cross sections of inflorescence stems of sturdy and wild-type plants. A and B, sturdy and wild type, respectively.C and D, Close-up sturdy and wild type. Arrows point to the branching vascular bundles. Bars 5 100 mm. co, Cortex; f,interfascicular fibers; ph, phloem; pi, pith; x, xylem.

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plaques screened. All three clones were identical in se-quence and contained a 12-kb BglII cut of the genomicfragment.

Sequence Analysis

The genomic and cDNA clones were sequenced usingin-house facilities (ABI Prism 377, Perkin Elmer, Foster,CA). The STURDY gene, including its promoter regioncontaining CAAT and TATA boxes, coding region, andpoly-A signal, was identified by GENSCAN1.0, a softwarewritten by Chris Burge (Stanford University, CA) modelingArabidopsis. The STURDY coding region and its translatedprotein sequence were used to search the GenBank data-base by applying BLASTN and BLASTP 2.0.8 (Altschul etal., 1997). Homology alignment of various patatin andpatatin-like protein sequences was done by the MegAlignsoftware (DNASTAR Inc.) using the clustal method withPAM250 residue weight table. Conserved amino acid resi-dues were defined by no larger than three distance units.

Microscopy

Tissue samples were collected from first and secondinternodes of wild-type and sturdy plants (14–15 cm) in thegrowth chamber. Tissue (approximately 5-mm slices) wasvacuum infiltrated in a fixative solution containing 4%(w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde in100 mm phosphate buffer (pH 7.0). Tissue samples werethen placed in 30% (v/v) ethanol. Further processing wasdone on a Tissue-Tek VIP automated processor (SakuraFinetek, Torrance, CA) through a series of graded ethanolwash steps: 50%, 75%, and 85% (v/v) ethanol at 37°C for anhour each; 95% and 100% (v/v) ethanol at 40°C and 45°Cfor 2 h each; and finally with paraffin at 58°C for 10 h withfour solution changes. Sections 6 mm thin were cut fromembedded tissue using a microtome (Reichert Jung 2030model, Leica Co, Deerfield, IL) and mounted on ProbeonPlus slides (Fisher Scientific, Pittsburgh), which were thendried overnight at 37°C.

For toluidine blue O stain, slides were deparaffinizedusing a series of xylene and ethanol washes (5 min each) andthen rehydrated. Slides were stained for 5 min in 1% (w/v)toluidine blue O (Fisher Scientific) in 1% (w/v) Borax (Sig-ma) and then rinsed in water for 2 min and dehydratedquickly through a series of ethanol washes (95%, 95%, 100%,and 100% [v/v] for 1 min each). These were subsequentlyplaced into two changes of xylene and mounted with Per-mount (Fisher Scientific). Slides were observed under a mi-croscope (BH-2; Olympus, Melville, NY).

ACKNOWLEDGMENTS

We thank Debbie Stone, Wendi Zumalt, and Stacy Minorfor plant transformation; Mark Woerner and Richard Orn-ber for their expertise and suggestions in microscopy; andClaire CaJacob, Charles Romano, and Tedd Elich for theirvaluable comments on the manuscript.

Received June 15, 2000; returned for revision August 16,2000; accepted September 26, 2000.

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Cloning of an Arabidopsis Patatin-Like Gene, STURDY, by Activation T-DNA Tagging · Cloning of an...

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