Suppression of a Vegetative MADS Box Gene of Potato Activates Axillary Meristem Development1

Suppression of a Vegetative MADS Box Gene of PotatoActivates Axillary Meristem Development1

Faye M. Rosin, Jennifer K. Hart, Harry Van Onckelen, and David J. Hannapel*

Interdepartmental Program in Molecular, Cellular, and Developmental Biology, Department of Horticulture,Iowa State University, Ames, Iowa 50011–1100 (F.M.R., J.K.H., D.J.H.); and University of Antwerp,Universiteitsplein 1, B–2610, Antwerpen, Belgium (H.V.O.)

Potato MADS box 1 (POTM1) is a member of the SQUAMOSA-like family of plant MADS box genes isolated from an earlystage tuber cDNA library. The RNA of POTM1 is most abundant in vegetative meristems of potato (Solanum tuberosum),accumulating specifically in the tunica and corpus layers of the meristem, the procambium, the lamina of new leaves, andnewly formed axillary meristems. Transgenic lines with reduced levels of POTM1 mRNA exhibited decreased apicaldominance accompanied by a compact growth habit and a reduction in leaf size. Suppression lines produced truncated shootclusters from stem buds and, in a model system, exhibited enhanced axillary bud growth instead of producing a tuber. Thisenhanced axillary bud growth was not the result of increased axillary bud formation. Tuber yields were reduced and rootingof cuttings was strongly inhibited in POTM1 suppression lines. Both starch accumulation and the activation of cell divisionoccurred in specific regions of the vegetative meristems of the POTM1 transgenic lines. Cytokinin levels in axillary buds ofa transgenic suppression line increased 2- to 3-fold. These results imply that POTM1 mediates the control of axillary buddevelopment by regulating cell growth in vegetative meristems.

During vegetative development, the processes ofapical growth and lateral branching are important fordetermining patterns of growth in the shoot andinflorescence. The tremendous diversity in vegetativepattern formation between plant species arises fromthese same basic processes. Interactions between theshoot apical meristem (SAM), the axillary meristems,and signaling compounds transported from the rootsare responsible for the overall pattern of shoot andbranching architecture that is observed. Althoughthese interactions are genetically determined, theyare also mediated by internal and external cues suchas hormone levels, light, or mechanical stimuli (forreview, see Sussex and Kerk, 2001). The SAM is re-sponsible for primary shoot growth, whereas lateralbranching is initiated by the development of axillarymeristems. Produced in the axils of leaves, axillarymeristems arise postembryonically and are derivedeither directly from the meristematic cells of the SAM(potato [Solanum tuberosum]) or from cells on theadaxial surface of the subtending leaf (Arabidopsis;Sussex, 1955; Schmitz and Theres, 1999). There aretwo stages of axillary meristem development: initialformation and subsequent growth. After axillarymeristem initiation, the SAM maintains its role as theprimary site of growth by inhibiting the growth ofaxillary meristems. This phenomenon, called apical

dominance, is mediated by auxin levels and putativesecond messengers that relay the auxin signal (Chat-field et al., 2000). In potato, underground stems pro-duce a specialized vegetative shoot called the stolon.The stolon meristem grows as a horizontal stem andwill develop into a tuber under favorable conditionsor a vegetative shoot if exposed to sufficient light.

A number of plant transcription factors play animportant role in regulating the development of theSAM. MADS box genes are an example of a family ofhighly conserved transcription factors that have di-verse roles in plant development. Although not ex-amined as extensively as their role in floral organidentity (Weigel and Meyerowitz, 1994; Theissen,2001), MADS box genes are important regulators ofvegetative development. JOINTLESS, a tomato (Lyco-persicon esculentum) MADS box gene, is required forthe development of a functional abscission zone intomato flowers (Mao et al., 2000). PkMADS1 from thewoody species Paulownia kawakamii is involved incontrolling vegetative organ formation. Suppressionof PkMADS1 in antisense transgenic plants disruptedthe interaction between leaf primordia and the SAM,resulting in additional leaf formation with alteredphyllotaxy at the expense of meristem maintenance(Prakash and Kumar, 2002). Transcripts of the potatoMADS box genes STMADS11 and STMADS16 arepresent in all vegetative tissues of the potato includ-ing roots and new tubers but are not detected infloral organs (Carmona et al., 1998; Garcıa-Maroto etal., 2000). Overexpression of STMADS16 in tobacco(Nicotiana tabacum) altered the architecture of the in-florescence, resulting in increased branching and in-ternode length. Whereas flowers exhibited leaf char-

1 This is journal paper no. J-3701 of the Iowa Agriculture andHome Economics Experiment Station, Ames (project no. 3703).

* Corresponding author; e-mail [email protected]; fax 515–294 –0730.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.102.012500.

Plant Physiology, April 2003, Vol. 131, pp. 1613–1622, www.plantphysiol.org © 2003 American Society of Plant Biologists 1613

acteristics, the morphology of vegetative organs wasnot affected, indicating that STMADS16 promotesvegetative development (Garcıa-Maroto et al., 2000).

Members of the SQUAMOSA (SQUA)-like familyof MADS box genes appear to have diverse roles inboth vegetative and floral development includingcontrol of floral meristem and perianth organ iden-tity (AP1; Mandel et al., 1992; CAL; Kempin et al.,1995), fruit morphogenesis (AGL8/FUL; Gu et al.,1998), and inflorescence identity (PETUNIA FLOW-ERING GENE [PFG]; Immink et al., 1999). Loss-of-function mutants of squa are characterized by theformation of shoots instead of flowers in the axils ofbracts, indicating that SQUA is involved in the tran-sition to reproductive development, thus controllingfloral meristem identity in snapdragon (Antirrhinummajus; Huijser et al., 1992). Cosuppression of PFG, aSQUA-like MADS box gene, leads to a non-floweringphenotype in which the vegetative phase is main-tained. PFG acts earlier than SQUA, because pfg mu-tants are blocked in the vegetative to inflorescencetransition, indicating that PFG is essential for inflo-rescence identity (Immink et al., 1999).

Potato MADS box 1 (POTM1) from potato was iso-lated from an early tuber cDNA library and belongsto the SQUA-like family of MADS box genes (Kangand Hannapel, 1995). POTM1 has the highest se-quence match to SCM1 from Solanum commersonii (awild potato) and PFG from petunia (Petunia hybrida)with 97% and 91% similarity over the entire proteinlength, respectively (Hart and Hannapel, 2002). Hav-ing a widespread expression pattern, POTM1 mRNAwas detected in actively growing tissues like meri-stems, roots, new leaves, and new tubers (Kang andHannapel, 1995). Like PFG, POTM1 transcripts arelocalized in tunica and corpus cells of vegetative andinflorescence meristems, leaf primordia, and the pro-cambium (Immink et al., 1999; Hart and Hannapel,2002). Despite the high sequence match and similar-ities in expression patterns, the role of POTM1 indevelopment appears to be different from the role ofPFG and other SQUA-like family members. Using atransgenic antisense approach, our results indicatethat POTM1 regulates the balance of growth betweenaxillary and apical vegetative meristems.

RESULTS

Suppression of POTM1 in Antisense Plants

POTM1 was isolated from an early-stage tubercDNA library (Kang and Hannapel, 1995) that wasconstructed from 4-d-old axillary buds induced toform tubers. At this stage, tuberization is initiatedand cells are undergoing rapid division and enlarge-ment. Forty clones forming two groups were isolatedfrom the library by using a probe containing only theconserved MADS box sequence. Further study indi-cated that the two groups most likely represent dif-ferent alleles of POTM1, because they were 99% iden-

tical at the nucleotide level and 100% identical at thededuced amino acid level (Kang and Hannapel,1995). Southern analysis indicated that POTM1 be-longs to a small gene family and is present in a lowcopy number (Kang and Hannapel, 1996). Probeswith and without the conserved MADS box sequencerecognized only select genomic bands representativeof a small gene family. On the basis of the resultsfrom the library screen and the Southern data, it ishighly probable that our transgenic antisense strat-egy is suppressing only the accumulation of RNAfrom members of the POTM1 family of MADS boxgenes.

To elucidate the function of this regulatory protein,transgenic plants with suppressed levels of POTM1mRNA expression were generated. The POTM1cDNA sequence in the antisense orientation wasplaced under the control of the cauliflower mosaicvirus 35S promoter to drive antisense transgene ex-pression in all organs of the potato. The potato cvFL-1607 was transformed by the Agrobacterium tume-faciens (strain GV2260)-mediated leaf-disc transfor-mation method (Liu et al., 1995). Transgenic plantswere screened by PCR for the presence of the trans-gene by using primers specific for the kanamycinmarker gene (nptII). A total of 34 independent trans-genic lines that were nptII positive were assayed forantisense POTM1 transcript accumulation. Accumu-lation of the POTM1 antisense mRNA was not de-tected in two of the 34 nptII-positive transgenic lines.Of the 32 lines exhibiting POTM1 antisense expres-sion, four independent transgenic lines with thegreatest levels of antisense transcript accumulationwere chosen for further analysis. Tissue cultureplants that were transformed with the nptII genealone were used as wild-type controls (WT). Sense32P-labeled RNA probes were used to examine ex-pression levels of the POTM1 antisense transgene inshoot tips of WT and independent suppression lines2, 10, 15, and 23 (Fig. 1A). Whereas independenttransgenic lines exhibited high levels of antisensetranscript accumulation, as expected, expression ofthe transgene was not detected in RNA from WTplants. Accumulation of endogenous levels ofPOTM1 mRNA were examined with gene-specificantisense 32P-labeled RNA probes. Accumulation ofPOTM1 mRNA in all four suppression lines wasreduced substantially compared with WT levels (Fig.1B). Suppression line 23 exhibited the greatest reduc-tion in native POTM1 mRNA. Visualization ofethidium bromide-stained ribosomal RNA was usedto ascertain loading and transfer (Fig. 1C).

Phenotype of POTM1 Suppression Lines

Suppression of POTM1 resulted in plants with adisrupted pattern of growth in organs both aboveand below ground. Transgenic lines with suppressedlevels of endogenous POTM1 mRNA exhibited a re-

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1614 Plant Physiol. Vol. 131, 2003

duction in plant height and leaf size (Fig. 2, A–C).Compound leaves of the most extreme phenotypes(lines 10, 15, and 23) had fewer leaflets and were lessdissected than WT leaves (Fig. 2A). Suppression linesexhibited a 2- to 3-fold reduction in the petiole lengthcompared with WT (Fig. 2B). Although the length ofthe internodes in the apical and middle regions of theplant was reduced, the greatest reduction in inter-node length was observed in more basal internodes.Suppression line 23 exhibited a 2.6� reduction ininternode length compared with WT (data notshown). This reduction in internode length and aconcomitant loss in apical dominance resulted inplants that were dwarf and compact (Fig. 2C). Sup-pression lines exhibited shoot clusters growing fromthe axillary buds of stems (Fig. 2D). Growth of theseaxillary buds was prolific, but limited, forming adense cluster of truncated shoots and stolons as wellas small tubers. Growth from axillary buds occurredin an acropetal fashion, with the oldest axillary budsat the base of the plant exhibiting increased growthfirst (data not shown). Both initiation and growth ofroots was inhibited in suppression lines (Fig. 2E).Whereas 100% of the cuttings from WT plants hadinitiated roots after 17 d, initiation of roots was in-hibited in suppression lines, with 88%, 63%, 11%, and0% of the cuttings forming roots for suppression lines2, 10, 15, and 23, respectively. Of the roots that diddevelop in suppression lines, there was an 18% to98% reduction in the total number of roots formedand a 57% to 100% reduction in the percentage that

formed lateral roots compared with roots from WTcuttings (data not shown).

A model petiole-leaf cutting system was used toexamine the tuberization potential of suppressionlines. This system is advantageous because it providesa reliable, synchronous, and uniform model for tuberdevelopment. It has been used in numerous studiesexamining discrete stages in tuber formation (Wheeleret al., 1988; Hannapel, 1991; Vreugdenhil et al., 1999).In WT plants, axillary buds of petiole cuttings fromshort-day-induced and long-day-noninduced plantswill grow out as tubers and shoots, respectively (Fig.3A; Hannapel, 1991). POTM1 mRNA accumulates intotal RNA from axillary buds of this system (Kang andHannapel, 1996). To determine the specific location ofPOTM1 mRNA accumulation, in situ hybridization in4-d-old tuberizing axillary buds was performed. Ac-cumulation of POTM1 mRNA is visualized as an or-ange stain under dark-field microscopy. There was nostaining observed in negative control sections probedwith a POTM1 sense riboprobe (Fig. 3B). POTM1 tran-scripts were localized to both corpus and tunica cellsof the shoot apical and axillary meristems. In theprimary apical meristem, POTM1 mRNA was de-tected primarily in the central and peripheral zones. Inthe axillary meristems, but not the primary apicalmeristem, POTM1 transcripts were also present in therib zone of the meristem. Accumulation of POTM1mRNA was also abundant in the procambium ofyoung leaves and the developing stem and in theadaxial cells of young leaves (Fig. 3C).

In the present study, cuttings were taken fromplants grown under short days for at least 2 weeks,inductive conditions for tuber formation. As ex-pected, axillary buds from WT plants developed intotubers (Fig. 3D, left). In suppression lines, however,there was a proliferation of nonelongating shoots(Fig. 3, D [right] and E; Table I). Whereas only onecutting of a total of 70 produced a tuber in the foursuppression lines, all 11 of the WT axillary budsexamined produced tubers after 10 d (Table I). Undergreenhouse conditions, WT plants produced an av-erage tuber yield of 266 g plant�1, whereas yieldsfrom suppression lines exhibited a reduction rangingfrom 79% to 97% (Table I). The tuber yield of sup-pression line 15 was reduced by more than 30-fold.

Whereas growth was observed in only one meri-stem in WT axillary buds, in the suppression lines,several meristems were activated (Fig. 3, D and E). Toexamine the number and placement of additionalmeristems activated in the suppression lines, scan-ning electron microscopy was performed on axillarybuds from petiole-leaf cuttings. In WT potato, oneaxillary meristem will develop in the junction of thestem and the adaxial base of the leaf. Axillary mer-istems are initiated early in development and arederived directly from the SAM. During developmentin the petiole-leaf cutting system, the primary axil-lary meristem will develop into a single tuber under

Figure 1. Accumulation of POTM1 antisense and endogenous tran-scripts in suppression lines of POTM1. Twenty-five micrograms oftotal RNA from shoot tips of WT and independent transgenic lines 2,10, 15, and 23 were loaded per lane. 32P-labeled RNA probes withthe MADS box deleted were used in the hybridizations. A, POTM1sense RNA probes were used to examine the accumulation of anti-sense POTM1. B, POTM1 antisense RNA probes were used to detectendogenous POTM1 mRNA. C, Ethidium bromide-stained rRNAbands were used to ascertain loading of lanes.

Potato MADS Box Gene Affects Tuberization and Meristem Formation

Plant Physiol. Vol. 131, 2003 1615

short days (Fig. 3A; Hannapel, 1991). A 5-d-old axil-lary bud from line 15 is shown in Figure 4A. Thepositions of the incipient leaf (P0) to the oldest leafprimordia (P7) are marked. One meristem (black ar-row) is visible at the base of the adaxial side of the P7leaf. In addition, another meristem (white arrow) isjust emerging at the base of the P6 leaf. In an axillarybud from petiole cuttings of a WT plant, no meris-tems were observed in the base of the P6 or P7 leafeven after 9 d of growth (Fig. 4B, arrows). Figure 4Cis a montage of two micrographs of the same mag-nification showing an 8-d-old axillary bud from line15. A single meristem is observed at the bases of theP7 to P10 leaves (arrows). The development of themeristem at the base of the P10 leaf is advanced withsix leaf primordia already present (Fig. 4C, right). In

addition, in the meristem subtending the P10 leaf, anaxillary meristem is just emerging at the base of theP6 leaf primordia (arrowhead). Whereas both theplacement and number of axillary meristems is nor-mal, the timing of development is precocious in thesuppression line.

To examine the morphology of the increasedgrowth observed in axillary buds of the petiole cut-tings, light microscopy was performed. Longitudinalsections from suppression line 15 and WT werestained with toludine blue to examine the histologyof 5-d-old axillary buds. At this stage of develop-ment, WT axillary buds are just initiating radialgrowth associated with tuber formation (Vreugden-hil et al., 1999). In line 15, the cells of the meristem(Fig. 5A) are more condensed compared with WT

Figure 2. Suppression of POTM1 RNA accumu-lation alters leaf and shoot morphology. A, Thefourth true leaf of WT and suppression lines 2,10, 15, and 23 are shown. Although the mor-phology has not been altered, leaf size has beenreduced. B, The length of the petiole of thefourth leaf from the apex was measured fromthree plants each of WT and POTM1 suppres-sion lines. SE is indicated for each mean. C,Seventy-five-day-old WT (left) and suppressionline 15 (right) are compared; note that line 15 isdwarf and compact. D, There is a proliferationof meristem growth in axillary buds along stemsof line 23. E, Leaf cuttings were placed in perliteunder mist for 17 d. Root initiation and growthwere inhibited in suppression lines.

Figure 3. The effect of POTM1 suppression on axillary bud development. A, Growth of axillary buds from WT plants in amodel petiole-leaf cutting system. At d 0, axillary meristems are dormant (0d). After culturing cuttings in perlite for 8 d, tubersand shoots, respectively, are produced from axillary buds of cuttings from short-day-induced (SD) and long-day-noninduced(LD) plants. B and C, In situ hybridization of POTM1 mRNA using 4-d-old axillary buds from petiole-leaf cuttings that wereinduced to tuberize. Presence of POTM1 mRNA is indicated by an orange stain under dark-field microscopy. Bar � 100 �m.B, Control section using POTM1 sense riboprobe. C, Localization of POTM1 mRNA in 4-d-old axillary buds. AP, Primaryapical meristem; AX, axillary meristem; P, procambium; *, adaxial side of young leaf. D and E, Axillary buds of petiole-leafcuttings were grown under short days for at least 19 d. Bar � 1 mm. Cuttings from WT plants produced tubers from theaxillary bud (D, left), whereas suppression lines exhibit increased meristem activity (D, right, and E). Suppression line 15 isshown in D and E. Similar results were also observed in lines 2, 10, and 23 (Table I).

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(Fig. 5B), particularly in the rib zone (arrow) of themeristem and in young leaves. Enlargements of themeristems are shown in Figure 5, C and D. Com-pared with WT, active cell division (arrows) is ob-served in line 15. Amyloplasts (arrows) are present inthe cells of both line 15 (Fig. 5E) and WT (Fig. 5F),however, there is an increased number of amylo-plasts present in line 15. On the basis of this obser-vation, the Periodic Acid-Schiff’s (PAS) reaction wasperformed on serial sections to specifically stain fornon-soluble polysaccharides, including starch andcellulose (Fig. 5, G and H). A higher magnification ofthe boxed areas in Figure 5, G and H, is shown inFigure 5, I and J. There is an increase in the numberof starch grains present in suppression line 15 (Fig.5I, arrow), particularly in the cells of the leaf. Incontrast to the elongated cells present on the abaxialside of the WT leaf (Fig. 5J, arrowhead), the abaxialleaf cells of line 15 are more irregularly shaped andless elongated (Fig. 5I, arrowheads). This decrease incell elongation in newly formed leaves may explainthe reduction in leaf size characteristic of the suppres-sion lines. Plates in Figure 5 are representative micro-graphs of numerous sections that were examined.

An increase in the number of starch grains wasobserved in light microscopy sections of suppressionline 15. To further examine starch content, the fourthand fifth expanded leaves from whole plants of WT

and suppression lines were harvested, and starch wasquantitated. The leaves of POTM1 suppression lineshad 2- to 5-fold more starch than leaves of WT plants(Fig. 6). Line 15 had the greatest increase in starch with13.1 mg starch 50 mg�1 dry weight compared with 2.6mg starch 50 mg�1 dry weight for WT leaves.

The phenotype of the POTM1 suppression linesindicates that these plants may have altered cytoki-nin levels. To test this possibility, cytokinin analysiswas performed on axillary buds of suppression line15 and control plants by using HPLC. Both zeatinand isopentenyl types of cytokinins increased 2- to3-fold in axillary buds from plants grown under bothlong-day and short-day conditions (Table II). Theshort-day axillary buds analyzed for cytokinins(buds taken directly from stock plants grown undershort-day conditions) in Table II were from the samematerial that produced the axillary bud phenotypesshown in Figures 3, D and E, 4, and 5.

DISCUSSION

POTM1 Suppression Activates AxillaryMeristem Growth

By using a transgenic approach, we demonstratethat suppression of POTM1 mRNA accumulationproduces a phenotype exhibiting reduced apical

Figure 4. POTM1 suppression induces activation of axillary meristem growth. Axillary buds from suppression line 15 andWT plants were harvested daily from short-day-induced petiole-leaf cuttings. A, A 5-d-old axillary bud from line 15. Thepositions of the incipient leaf (P0) to the oldest leaf primordia (P7) are marked. Arrows indicate developing axillary meristemsat the bases of leaf primordia. B, A 9-d-old axillary bud from the WT plant. Arrows indicate the bases of P6 and P7 leafprimordia. Note that no axillary meristems are observed. C, An 8-d-old axillary bud from line 15. Arrows indicate developingaxillary meristems. Note the advanced development of the meristem at the base of the P10 leaf (right side) with six leafprimordia and an emerging axillary meristem (arrowhead) at the base of the P6 leaf primordia subtending the P10 meristem.

Table I. Rate of tuberization in axillary buds from cuttings of POTM1 suppression mutants grownunder a short-day photoperiod and from whole plants under a long-day photoperiod

Whole plants were induced under a short-day photoperiod for at least 2 weeks, after which axillarybud cuttings were placed in a perlite/vermiculite mixture for 10 d. The axillary bud is placed below thesurface of the perlite/vermiculite mixture while the leaf remains above the surface. After 10 d, theaxillary buds were examined for growth and tuber formation. For several 100-d-old soil-grown plantsgrown under a long-day photoperiod in the greenhouse, tubers were harvested and yield per plant(grams fresh wt) with SE was calculated.

Plant TypeNo. of

CuttingsNo. Tuberized

after 10 dPhenotype of Axillary Buds

Tuber Yield Plant�1

at 100 d

g fresh wt

Wild type 11 11 Mini-tuber formation; no shoot growth 266 � 7.4Line 2 12 0 Multiple shoots; thickened stems 57 � 34Line 10 16 1 Excessive shoot and stolon growth 43 � 16Line 15 10 0 Multiple shoots; purple pigment 8.4 � 1.6Line 23 32 0 Multiple shoots 44 � 3.4



dominance, increased lateral growth, induced forma-tion of shoot clusters on the stem, increased starchaccumulation in new leaves, and a reduction in tuberformation. These changes are accompanied by in-creases in cytokinin content and in activation of ax-illary meristem growth. There are two stages of ax-illary meristem development, the initial formation ofaxillary meristems and the subsequent growth of themeristem (Shimizu-Sato and Mori, 2001). Axillarymeristem mutants usually affect one or both of thesestages of development. The origin of axillary meri-stems differs between different plant species. In po-tato and tomato, axillary meristems are derived from

meristematic cells that detach from the shoot apicalmeristem (Sussex, 1955), whereas in Arabidopsis, ax-illary meristems are initiated in leaf axils from cellson the adaxial surface of the subtending leaf (Schmitzand Theres, 1999). Growth of axillary meristems isusually inhibited by the shoot apical meristem, in aprocess called apical dominance (Shimizu-Sato andMori, 2001). Auxin restricts, whereas cytokinin pro-motes, the growth of axillary meristems (Shimizu-Sato and Mori, 2001).

The most notable phenotype caused by the sup-pression of POTM1 is the increase in axillary meri-stem growth. This is most apparent in the compact

Figure 5. Histology of axillary bud meristems in POTM1 suppression lines. Axillary buds from suppression line 15 and WTcontrols were harvested daily from short-day-induced petiole-leaf cuttings. Sections from axillary buds were stained with 1%(w/v) toludine blue (A–F) to show general morphology or with the PAS reaction (G–J) to specifically stain non-solublepolysaccharides. Negative controls for the PAS reaction in which one reagent was missing (periodic acid) showed no staining(data not shown). A, C, E, G, and I, Meristem sections of suppression line 15. B, D, F, H, and J, Meristem sections from WTplants. Bar � 100 �m in all panels. A, One of several meristems that were present in a 5-d-old axillary bud from line 15.Arrow indicates rib zone. Note that the cells are less vacuolated and more condensed. B, The only meristem present in a5-d-old axillary bud from a WT plant. Arrow indicates rib zone. C, Enlargement of the line 15 apex. Arrows indicate cellsthat are undergoing active cell division. D, Enlargement of WT apex. E, A higher magnification of the line 15 cells in theboxed area of A. Note the increased numbers of amyloplasts present in the cells of line 15 compared with WT (arrows). F,A higher magnification of the WT cells in the boxed area of B. Arrow, Amyloplasts. G, PAS staining of one of the axillarymeristems from the axillary bud cutting of suppression line 15. H, PAS staining of the primary and only meristem from anaxillary bud cutting from WT. I, A higher magnification of line 15 leaf cells in the boxed area of G. Arrow, Examples of starchgrains; arrowhead, irregular shaped cells on abaxial side. J, A higher magnification of WT cells in the boxed area of H.Arrow, Starch grains; arrowhead, elongated cells on abaxial side. Note the difference in cell shape (arrowheads) betweenthe WT (J) and line 15 (I).

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growth habit of the plant and in the proliferation ofshoot growth on the stems of whole plants and fromthe axillary buds of petiole cuttings that were in-duced to form tubers. Scanning electron microscopy(Fig. 4) indicated that initiation of meristem forma-tion does not appear to be affected by suppression ofPOTM1 because both the number and placement ofaxillary meristems is normal. In both WT and POTM1suppression lines, one axillary meristem will developin the junction of the stem and the adaxial base of theleaf. There are several examples of other genes thatregulate lateral branching in a similar way. The MAXgenes of Arabidopsis repress shoot lateral branching(Stirnberg et al., 2002). MAX1 and MAX2 appear tospecifically control axillary growth rate after axillarymeristem initiation. The arx1 mutant of Arabidopsishas a decreased sensitivity to auxin and exhibits ahighly branched shoot phenotype (Stirnberg et al.,1999). Loss of ARX1 function does not affect theformation of axillary buds but rather increases therate of development of lateral shoots. Interestingly,both MAX2 and ARX1 may be involved in theubiquitin-mediated degradation of regulatory pro-teins. In petunia, the recessive dad1-1 allele condi-tions a highly branched phenotype accompanied by areduction in apical dominance and a decrease ininternode length (Napoli, 1996). The nonallelic ramo-sus mutations of pea (Pisum sativum) cause the releaseof vegetative axillary buds leading to extensive lat-eral growth and a reduction in apical dominance(Beveridge et al., 1996). Grafting experiments be-tween WT and dad mutants or between WT and rmsmutants indicate that axillary bud growth is affectedby an unidentified substance that is transported fromthe roots (Napoli, 1996; Beveridge et al., 2000). Theresults of these studies suggest a complex interactionamong hormones and other signaling compoundsthat regulates apical dominance and lateral branch-

ing. Similar to these mutants, POTM1 acts to inhibitaxillary bud growth after bud formation. The identityof the developing meristems is plastic becauseshoots, stolons, and tubers are capable of developingfrom the same location in the leaf axil of POTM1suppression lines (Figs. 2 and 3). In potato, all threeof these organs can develop from an axillary vegeta-tive meristem (Wheeler et al., 1988).

POTM1 Suppression Lines Are Similar to Mutants ThatOverproduce Cytokinins

The POTM1 suppression phenotype has many sim-ilarities to mutants in Arabidopsis (van der Graaff etal., 2001), tobacco (Li et al., 1992; Hewelt et al., 1994;Guivarc’h et al., 2002), tomato (Groot et al., 1995),and potato (Galis et al., 1995; Machackova et al., 1997)in which the A. tumefaciens ipt gene was introduced tooverproduce cytokinin. These cytokinin overproduc-tion phenotypes include loss of apical dominance,decreased leaf size and internode length, and poorroot growth. In potato, tuberization is also affectedby expression of the A. tumefaciens ipt gene, with highlevels of cytokinins inhibiting and moderate levelspromoting tuber formation (Galis et al., 1995; Ro-manov et al., 2000). As illustrated by Sergeeva et al.(2000), the ratio of cytokinin to auxin is important fortuberization. A slight increase in the cytokinin toauxin ratio promotes tuberization. A large change inthe ratio, however, inhibits tuberization, in favor ofthe formation of short stems instead. Local expres-sion of the ipt gene in axillary buds of transgenictobacco created a strong sink and resulted in theformation of truncated, tuberizing lateral branches(Guivarc’h et al., 2002). These swollen, lateralbranches were similar in morphology to those pro-duced on the POTM1 suppression lines (Fig. 2D).

Cytokinins contribute to axillary meristem devel-opment, particularly in the release from dormancyand subsequent growth. Both exogenous applicationand the overproduction of cytokinins in transgenicplants have been shown to release axillary meristems

Figure 6. Starch accumulation in leaves of POTM1 suppressionlines. Starch content was assayed in the fourth and fifth expandedleaves from whole plants of WT and independent suppression lines 2,10, 15, and 23. Four replicates of 50 mg each of lyophilized leaftissue were assayed and SE of the mean was calculated.

Table II. Cytokinin content in axillary buds of control plants andthe POTM1 suppression line 102-15

Zeatin types include zeatin, zeatin riboside, dihydrozeatin, anddihyrozeatin riboside. Isopentenyl types include isopentenyl andisopentenyladenine. Shoot clusters for 102-15 were similar to thoseshown in Figure 2D. The short-day (SD) axillary buds were takendirectly from stock plants grown under SD conditions (8 h of light;16 h of dark) and were from the same material that produced theaxillary bud phenotypes shown in Figures 3, D and E, 4, and 5. SE wascalculated on three replicates. WT, Wild type; LD, long day.

Zeatin Types Isopentenyl Types

pmol g�1 fresh wt

WT SD axillary buds 11.5 � 1.5 11.5 � 1.5WT LD axillary buds 27.5 � 10 15.5 � 7.5102-15 SD axillary buds 30.0 � 11 20.0 � 5.5102-15 LD shoot clusters 81.5 � 33 44.0 � 17



from dormancy (for review, see Cline, 1991; McKen-zie et al., 1998; van der Graaff et al., 2001). Chatfieldet al. (2000) demonstrated that basipetal auxin trans-port was a potent inhibitor of axillary meristemgrowth. Basal application of cytokinins, however,was able to overcome the effect of apically appliedauxins, thereby promoting the growth of axillarymeristems. The lateral suppressor mutant from tomatoprevents the initiation of axillary meristem formationresulting in empty leaf axils (Schumacher et al.,1999). Expressing the A. tumefaciens ipt gene in lateralsuppressor mutants increased cytokinin levels andproduced typical cytokinin overproduction pheno-types, but did not rescue the empty leaf axil pheno-type (Groot et al., 1995). In tomato, at least, cytokininlevels affect the growth of axillary meristems but nottheir initiation. In a similar fashion, the suppressionof POTM1 mRNA accumulation in potato affects thegrowth of axillary meristems but not their initiationor location.

Creation of Multiple Sinks in POTM1Suppression Lines

In addition to releasing axillary meristems fromdormancy, cytokinins have a profound effect on themobilization of nutrients. Li et al. (1992) showed thatcytokinin activity can create a nutrient sink and thatboth 14C-labeled Suc and amino acids are mobilizedto localized sites of high cytokinin accumulation.Amyloplast development and the increased tran-scription of starch biosynthesis enzymes are specifi-cally induced by cytokinins in cultured tobacco cells(Miyazawa et al., 1999). Local synthesis of cytokininsin axillary buds of transgenic tobacco resulted in anincrease in starch accumulation in the lateral shootsthat formed (Guivarc’h et al., 2002). POTM1 suppres-sion resulted in an increase in starch accumulationand active cell division in specific cells of meristemsand leaves (Figs. 5 and 6). These events may becontrolled by the localized activity of POTM1 to limitcytokinin accumulation to specific regions of themeristem.

In suppression lines, development of normallystrong vegetative sinks like the SAM and under-ground tubers are superceded by the induced sec-ondary growth of axillary meristems. Whereas theaxillary bud from WT cuttings of a model tuber sys-tem produces a single tuber, axillary buds fromPOTM1 suppression lines produce a proliferation ofshoots. These results support the premise that therelationship between the growth of lateral organsand the apical meristem are antagonistic. In Arabi-dopsis, recessive mutants of the revoluta gene (Tal-bert et al., 1995) are characterized by longer, thickerleaves and decreased meristem formation. Growth oflateral organs like leaves is favored over growth ofmeristems, often leading to an arrest in apical mer-istem development. It is postulated that the function

of REVOLUTA is to promote apical meristem growthand to limit cell division and elongation in leaves andstems (Talbert et al., 1995). In contrast, POTM1 sup-pression lines exhibited smaller leaves and more ac-tive lateral meristem growth. POTM1 suppressionproduced an increase in cell division in meristemsand created multiple new sinks at the expense ofleaf expansion and tuber formation. An extremeexample of this was demonstrated by the increase inshoot/stolon/tuber growth arising from axillarystem buds in select suppression lines accompaniedby a concomitant reduction in underground tuberyields.

The phenotypes of the suppression lines, particu-larly activated axillary meristem growth, increasedcell division, decreased tuber yields, and selectivestarch accumulation, suggest that POTM1 may beinvolved in regulating the balance of growth in veg-etative meristems, favoring the development of adominant sink organ. The increased levels of cytoki-nins in axillary buds of suppression line 15 supportthis premise. For the aboveground portion of theplant, this sink organ is the SAM. In stolon/tuberdevelopment, the sinks are a few enlarging tubersthat form underground. In the model petiole-leafcutting tuberization system, the single tuber pro-duced is a very strong sink. Suppression of POTM1may alter the ratio of auxins and cytokinins (by in-creasing the level of cytokinins, for example) to acti-vate growth of lateral meristems at the expense of theapical or dominant shoot organ (SAM or tuber). It isconceivable that reduction in POTM1 mRNA expres-sion enhances cytokinin activity in specific cells ofthe vegetative meristem leading to increased cell di-vision in meristems and a disruption in WT source/sink interactions.

MATERIALS AND METHODS

Transformation of POTM1 Antisense Plants

The full-length POTM1 cDNA (Kang and Hannapel, 1995) in the antisenseorientation was cloned into the binary vector, pCB201 (Xiang et al., 1999),under the control of the cauliflower mosaic virus 35S promoter. This plasmidwas transformed into Agrobacterium tumefaciens strain GV2260. The leaf-disctransformation method (Liu et al., 1995) was used to transform the constructinto potato (Solanum tuberosum cv FL-1607). The presence of the transgene wasconfirmed by PCR primers specific for the kanamycin-marker gene (nptII;data not shown). A total of 34 independent transgenic lines that were nptIIpositive were screened for the accumulation of POTM1 antisense mRNA.Four independent transgenic lines that exhibited high levels of POTM1 anti-sense mRNA accumulation were selected for further analysis. Tissue cultureplants transformed with the nptII marker gene alone were used as controls(WT). For subsequent experiments, all lines were propagated vegetativelyfrom bud cuttings in vitro and transferred to soil after rooting.

RNA-Blot Hybridization

Total RNA was extracted from shoot tips of independent transgenic linesand controls with TriPure Isolation Reagent (Roche Diagnostics, Indianap-olis). Using 10 mm methyl mercury (II) hydroxide as a denaturant, 25 �g oftotal RNA was subjected to gel electrophoresis and blot transfer. POTM1with the conserved MADS box deleted was cloned into the pCRII vector(Invitrogen, Carlsbad, CA) flanked by the SP6 and T7 promoters. The vector

Rosin et al.


was linearized with XhoI or BamHI and transcribed with the SP6 or T7 RNApolymerase for sense and antisense probes, respectively. [�-32P]CTP wasincorporated into RNA probes according to the manufacture’s instructions(Promega, Madison, WI). The membrane was hybridized in ULTRAhybhybridization buffer (Ambion, Austin, TX) at 68°C for 16 h. Washing was asfollows: 2� SSC and 0.1% (w/v) SDS at room temperature for 5 min; 1� SSCand 0.1% (w/v) SDS at 68°C for 20 min; and 0.1� SSC and 0.1% (w/v) SDSat 68°C for 20 min. The film was exposed for 4 d at �80°C.

Tuber Yields from Petiole-Leaf Cuttings andWhole Plants

For petiole-leaf cuttings, whole plants were induced under a short-dayphotoperiod (8 h of light/16 h of dark) for at least 2 weeks. Cuttings werecultured in a perlite-vermiculite mix with the axillary bud placed below thesurface under a short-day photoperiod in a growth chamber (Hannapel,1991). After 10 d, the axillary buds were examined for morphology, growth,and tuber formation. Growth was allowed to continue for at least 19 d beforephotographs in Figure 3 were taken on a SZH10 Research Stereo Microscope(Olympus, Melville, NY). For whole plants, tuber yields were measuredfrom 100-d-old plants that were grown in the greenhouse under a long-dayphotoperiod (16 h of light/8 h of dark).

In Situ Hybridization

WT plants were grown under short-day conditions (8 h of light/16 h ofdark) for at least 2 weeks. In situ hybridizations with gene-specific probesfor POTM1 were performed on 4-d-old axillary buds from petiole-leafcuttings cultured as described above. In situ hybridizations were performedas described previously (Hart and Hannapel, 2002).

Light and Scanning Electron Microscopy

WT and independent suppression line 15 plants were grown undershort-day conditions (8 h of light/16 h of dark) for at least 2 weeks.Petiole-leaf cuttings were cultured as described above, and meristems wereharvested daily and fixed in 2% (w/v) paraformaldehyde and 2% (w/v)glutaraldehyde in either 0.1 m cocadylate buffer, pH 7.2, or 0.1 m sodiumphosphate buffer, pH 7.0, at 4°C for 2 to 11 d. After fixation, the tissue wasdehydrated in a graded ethanol series. Tissue for light microscopy wasembedded in LR White (Electron Microscopy Sciences, Ft. Washington, PA),and 1.4-�m sections were cut on an ultramicrotome (Reichert/Leica, Deer-field, IL). For general morphology, sections were stained with 1% (w/v)toludine blue. The PAS reaction was performed on serial sections to stain fornon-water-soluble polysaccharides. Negative controls for the PAS reaction,in which one reagent was missing (periodic acid), did not show any staining(data not shown). Bright-field microscopy was used to photodocumentsections. Tissue for scanning electron microscopy was critical point driedwith six flushes of CO2, mounted on aluminum stubs with silver paint, andcoated with gold/palladium. Specimens were examined on a scanningelectron microscope (JSM-5800LV, JEOL, Tokyo) at the Bessey MicroscopyFacility, Iowa State University.

Starch Assay

The fourth and fifth expanded leaves from whole plants of WT andindependent suppression lines were harvested in liquid N2 and lyophilized.Four replicates of 50 mg each were assayed and se of the mean wascalculated. Starch content was measured according to the manufacture’sinstructions (STA-20, Sigma-Aldrich, St. Louis).

Extraction, Purification, and Analysis of Cytokinins

Three replicate 200 mg (fresh weight) samples of axillary buds and shoottips were collected, frozen in liquid nitrogen, lypholized, and analyzed.Samples were extracted overnight in Bieleski’s solvent. Before centrifuga-tion (24,000g, 15 min, 4°C) deuterated standards for cytokinins (Apex Inter-national, Honiton, UK) were added. The extract was purified using a com-bination of solid phase and immunoaffinity purification as described by

Redig et al. (1996). Quantitative analysis of cytokinins was performed bymeans of capillary column switching on a fully automated Famos worksta-tion (LC Packings, Amsterdam) coupled to a liquid chromatography set-upconsisting of a 325S HPLC pump (Kontron Instruments, Milan), an in-lineKontron 322 UV detector, and a triple quadrupole mass spectrometer (Quat-tro II, Micromass UK Ltd, Cheshire, UK). Twenty-five-microliter samplealiquots were introduced into one dimension of the system using 10 mmCH3COONH4 as the mobile phase at a 40 �L min�1 flow rate. During 7 min,analytes were captured on a micrometer precolumn (C18, 5 �m, 500 �m i.d.� 5 mm; LC Packings) followed by a mobile phase switch for 5 min using10 mm CH3COONH4, water:methanol (30:70, v/v) at a 7 �L min�1 flow rateas mobile phase to introduce the analytes onto the analytical column (Ad-sorbosphere C18, 5 �m, 300 �m i.d. � 150 mm, Alltech, Laarne, Belgium).Cytokinin chromatograms were recorded in multiple reaction-monitoringmode using diagnostic transition ions as described by Prinsen et al. (1995).Absolute detection limits ranged from 2 fmol for isopentenyl to 15 fmol forzeatin. Results were calculated according to the principle of isotope dilutionand expressed in picomoles per gram fresh weight.

ACKNOWLEDGMENTS

We thank Mary Tymeson, Dr. Carol Foster, Beth Fatland, and Hao Chenat Iowa State University for their invaluable support and helpful discus-sions; and Dr. Harry T. Horner, Tracey Pepper, and John Mattila at theBessey Microscopy Facility, Iowa State University, for continuing supportand expertise in microscopy techniques.

Received August 2, 2002; returned for revision October 17, 2002; acceptedNovember 25, 2002.

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Suppression of a Vegetative MADS Box Gene of Potato Activates Axillary Meristem Development1

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