isolation of mads-box genes from sweet potato - plant and cell

Plant Cell Physiol. 43(3): 314–322 (2002)

JSPP © 2002

Isolation of MADS-box Genes from Sweet Potato (Ipomoea batatas (L.) Lam.) Expressed Specifically in Vegetative Tissues

Sun-Hyung Kim, Kouichi Mizuno 1 and Tatsuhito Fujimura

Institute of Agricultural and Forest Engineering, University of Tsukuba, Tsukuba, Ibaraki, 305-8572 Japan

;

New MADS-domain genes, IbMADS3 and IbMADS4,

were isolated from pigmented and tuber-forming root tissue

in sweet potato (Ipomoea batatas L.). Both genes were

expressed preferentially in vegetative tissues, especially root

tissues; white fibrous roots, pigmented roots, and develop-

ing tuberous roots. On sequence alignment, these genes fell

into the STMADS group composed of SVP, STMADS11,

STMADS16 and AGL24, which share high sequence simi-

larity, similar expression patterns and similar function.

Transcripts of these two genes in roots were found in the

vascular cambium region. This particular expression pat-

tern of these genes may lead to a higher proliferative poten-

tial of vegetative tissues, and may facilitate tuber initiation

in sweet potato. These genes may lead to important infor-

mation on the morphogenesis of vegetative structures.

Key words: IbMADS — Ipomoea batatas — Tuber formation

— Vascular cambium.

The nucleotide sequences reported in this paper have been sub-

mitted to DDBJ, EMBL, and GenBank under accession numbers

AF345246 and AF346303.

Introduction

MADS-box genes have been cloned in many plant spe-

cies and their role as homeotic genes that control floral organ

development has been well established (Coen and Meyerowitz

1991, Davies and Schwarz-Sommer 1994, Weigel and Meye-

rowitz 1994, Mena et al. 1995, Riechmann and Meyerowitz

1997). Two conserved regions characterize MADS-box pro-

teins. One is the DNA-binding domain, designated the MADS-

box, which consists of 56 amino acid residues of conserved

motifs present in transcription factors of MCM1 in yeast (Pass-

more et al. 1988), AG in Arabidopsis (Yanofsky et al. 1990),

DEF A in Antirrhinum (Schwarz-Sommer et al. 1990), and SRF

in humans (Norman et al. 1988). The other is designated the K-

box because of its structural similarity to the coiled-coil

domain of keratin (Ma et al. 1991, Theißen et al. 1996). The K-

box domain mediates protein–protein interactions of the

MADS-box proteins to form homo- or heterodimers that facili-

tate DNA-binding to other regulatory factors (Ma et al. 1991,

Pnueli et al. 1991, Theißen et al. 1995). Two additional

domains can be distinguished in MADS proteins: the I-domain,

a weakly conserved intervening region that is located between

the MADS-box and the K-box, and the C-domain, at the C ter-

minus, which is the most variable region with regard to both

sequence and length among MADS-box proteins. The MADS-

box proteins bind DNA as dimers. The distribution of molecu-

lar determinants that control dimerization specificity varies

among different MADS proteins and these determinants are sit-

uated in the MADS, K and I domains (Riechmann et al. 1996).

Plant MADS-box genes play important roles in flower

development. Two plant homeotic genes, DEFICIENS (DEF)

of Antirrhinum majus (Schwarz-Sommer et al. 1990) and AGA-

MOUS (AG) of Arabidopsis thaliana (Yanofsky et al. 1990)

were isolated first, and subsequent isolation of additional genes

whose mutants give rise to homeotic phenotypes revealed that

they are members of the MADS-box gene family (Ma 1994,

Weigel and Meyerowitz 1994, Theißen et al. 1995). Although

most of the MADS-box genes isolated in plants are expressed

exclusively in floral tissues, some of their transcripts have been

found in various vegetative tissues (Carmona et al. 1998).

Moreover, their expression patterns have been shown to corre-

late with vegetative growth, root or fruit development, or

embryogenesis (Ma et al. 1991, Pnueli et al. 1991, Flanagan

and Ma 1994, Heck et al. 1995, Mandel et al. 1994, Rounsley

et al. 1995, Shore and Sharrocks 1995). Recently, a few genes

that are expressed in vegetative tissues have also been identi-

fied in Arabidopsis [AGL24 (GenBank accession number

AAC63140) and SVP; Hartmann et al. 2000] and in potato

(STMADS11 and STMADS16; Carmona et al. 1998).

Based on a phylogenic analysis of 33 reported MADS-box

genes and the two genes reported here, the latter two genes are

closely related to those in the STMADS subfamily; SVP,

STMADS11, STMADS16, and AGL24. Members of a subfamily

usually share high sequence similarity, similar expression pat-

terns and similar function (Theißen et al. 1996). Most of these

genes are expressed during vegetative development, which is

evidence that the STMADS subfamily might be involved in the

regulatory network that directs vegetative organogenesis dur-

ing plant growth (Garcia-Maroto et al. 2000). However, while

considerable information is available on MADS-box genes that

direct flower development, little is known about those that are

specifically expressed in vegetative tissue.

In this study, we report the cloning and molecular charac-

terization of two new MADS-box genes from sweet potato,

1 Corresponding author: E-mail, [email protected]; Fax, +81-298-53-4657.

314

MADS-box genes from sweet potato 315

IbMADS3 and IbMADS4, possible roles to the development of

the tuber of sweet potato, and the first step toward the func-

tional characterization of IbMADS genes involved in vegeta-

tive development.

Results

Sequence analysis of IbMADS3 and IbMADS4

Putative STMADS homologues were cloned from sweet

potato by PCR. Two degenerated primers corresponding to

MADS-domain conserved regions were used to amplify frag-

ments from cDNAs made from total RNA isolated from pig-

mented roots of sweet potato. After 3�-RACE, 11 clones encod-

ing the part of MADS-box and K-box region were selected

from 315 clones that were isolated by sequencing. These

cDNA fragments were stretched to the 5�-ends of the cDNA

sequence by 5�-RACE. After the full-length cDNAs were

reconstructed by perfect overlapping, two MADS-box genes

were isolated from the 11 clones as MADS-box gene frag-

ments. Both of these fragments had two motifs of the MADS-

box and K-box, suggesting that sweet potato has at least two

STMADS group of MADS-box genes. These were named

IbMADS3 (accession no. AF345246) and IbMADS4 (accession

no. AF346303).

IbMADS3 and IbMADS4 encode polypeptides consisting

of 227 and 229 amino acids, respectively, and show 64% amino

acid sequence similarity to each other, in the start of the

MADS-box and in the predicted helical coiled-coil structure of

the K-box in the central region (amino acids 93 to 160), like

other MADS-box genes (Fig. 1). The MADS-box sequences of

IbMADS3 and IbMADS4 are 100% and 93% identical to that of

STMADS16 (Carmona et al. 1998), respectively. The deduced

IbMADS4 protein is 82.5% and 89.5% identical to those of

STMADS11 and SVP, respectively (Table 1).

Phylogenetic analysis of the entire 35-plant MADS-box

gene family has shown that plant MADS-box genes can be

Fig. 1 Amino acid sequence comparison among IbMADS3, IbMADS4 and related MADS-box proteins. The deduced amino acid sequence of

proteins IbMADS3 (AF345246), IbMADS4 (AF346303), STMADS16 (AAB94005), STMADS11 (AAB94006), SVP (AAG24508) and AGL 24

(AAC63140) were aligned. Sources of the genes are indicated in parentheses as follows. At, Arabidopsis thaliana; St, Solanum tuberosum; Ib,

Ipomoea batatas. Degenerated primer (M3 and M4) site is a dotted line.

MADS-box genes from sweet potato316

classified into more than 10 groups for which all of the genes

have developed from an ancestral gene by gene duplication and

sequence diversification (Theißen et al. 1996, Hasebe and Ito

1999). IbMADS3 and IbMADS4 seem to be most closely

related to STMADS11 and STMADS16 from potato and to

AGL24 (Hartmann et al. 2000) and SVP from Arabidopsis (Fig.

2). Since STMADS11 was the first gene to be identified within

this group of MADS-box genes, it has been suggested that this

new subfamily be named the STMADS subfamily (Hartmann et

al. 2000). In this subfamily, members tend to share highly simi-

lar sequences, expression patterns and functions (Theißen et al.

1996).

Genomic analysis and expression pattern of IbMADS3 and

IbMADS4

Southern blot analysis under stringent conditions against

sweet potato genomic DNA revealed one or two hybridization

bands when digested with EcoRI, HindIII, and BamHI (Fig. 3).

Both IbMADS3 and IbMADS4 are single or low copy-number

genes in sweet potato.

Northern blot analysis was performed using total RNA

extracted from different organs of mature plants (Fig. 4). In

sweet potato, these genes are preferentially expressed in vege-

tative tissues; leaf, stem, root, and developing tuber, with

slightly different expression levels during root development.

IbMADS4 was expressed in all vegetative tissue, whereas

IbMADS3 was expressed more predominately in roots and

developing tuberous roots. Neither IbMADS3 nor IbMADS4

was expressed at a detectable level in buds or flowers (Fig. 4).

Expression of IbMADSs and their possible role during tuber

development

Developing tuberous roots of sweet potato, which had not

yet reached their final size, were sectioned into four segments

(about 2.5 cm long) on 50 d after planting. Although the rate of

shoot and root growth (especially root elongation) decreased on

40 d after planting, tuber-bulking started in pigmented roots

(Fig. 5). Developing tuberous roots were divided into four parts

with different biological activities; a distal part, two middle

parts and a proximal part (Fig. 6, segments 1, 2, 3, and 4,

respectively). The root elongates in the distal part, develops

into a starch-accumulating tuber in the middle parts, and

changes into a tuberous root in the proximal part. The diameter

of the root in the distal part is less than that in the rest of the

developing tuberous root. IbMADS3 and IbMADS4 were tran-

scribed at the highest levels in the middle parts and the distal

middle part, respectively, indicating that these IbMADS are

strongly correlated with cell proliferation in developing tubers

(Fig. 6).

To elucidate more precisely the relationship between tuber

development and the increased expression of IbMADS3 and

IbMADS4, developing tuberous roots were sectioned into three

parts; part 1, vascular cambium with a few millimeters of adja-

cent protoxylem element and epidermis; part 2, parenchyma-

tous cells; and part 3, central metaxylem cells plus parenchy-

matous cells. Low transcript levels were found in part 1 for

IbMADS3 and in parts 1 and 3 for IbMADS4 (Fig. 7). The

development of sink activity in sweet potato roots is related to

the proliferation of cells in the vascular cambium, as well as

the development of anomalous secondary and tertiary meris-

tems in tubers (Wilson and Lowe 1973). Thus, cell prolifera-

tion in tuber formation may be controlled or mediated by

IbMADS proteins in the tuber.

The expression patterns of the IbMADS3 and IbMADS4

were examined in root tissue of sweet potato grown for 50 d

using tissue print mRNA hybridization. The mRNA signals of

these genes were predominantly present in the cambial region

(Fig. 8).

Table 1 Identity of amino acid sequences, IbMADS3, IbMADS4 and other STMADS subfamily

MADS-box proteins

Letters indicate sources of sequences (accession number of NCBI): IbMADS3 (AF345246) and IbMADS4

(AF346303) from sweet potato, STMADS16 (AAB94005) and STMADS11 (AAB94006) from potato, SVP

(AAG24508) and AGL 24 (AAC63140) from Arabidopsis.

Sequence comparisonIdentify (%)

MADS I K C Overall

IbMADS3 IbMADS4 91.2 61.7 62.7 45.6 64.0

STMADS11 80.7 34.2 43.2 40.0 48.2

STMADS16 100 61.2 61.5 31.3 63.8

AGL24 89.4 50.0 59.1 50.0 57.7

SVP 91.2 84.8 72.5 53.7 69.4

IbMADS4 STMADS11 82.5 22.2 52.4 40.8 53.2

STMADS16 92.9 61.8 75.0 49.3 64.7

AGL24 91.2 53.0 61.5 42.0 61.8

SVP 89.5 53.3 64.2 43.2 56.9


Discussion

We isolated new MADS-box genes, IbMADS3 and

IbMADS4, from bulking sweet potato roots that are members of

the STMADS subfamily based on their sequences and expres-

sion patterns. Transcripts of these two IbMADS cDNAs were

detected exclusively in vegetative tissues (leaf, stem, root,

developing tuberous root, and tuber) and not in floral tissues

(Fig. 4), similar to STMADS11, STMADS16 of potato and SVP

of Arabidopsis. The sequences and expression patterns of both

genes are consistent with those of other STMADS genes.

IbMADS genes also show a very high similarity, 80% overall

and 98% within the MADS-box, to both STMADS16 from

potato and SVP from Arabidopsis (Table 1, Fig. 1). These

alignment analyses and specific expression patterns indicate

that the IbMADS-box genes of sweet potato are homologous to

other genes that are specifically expressed during vegetative

organ development.

Phylogenies and ancestral character reconstructions of

developmental regulators provide a historical framework for

studies of the evolution of developmental genetic pathways and

give useful clues regarding the molecular basis of morphologi-

cal evolution, thus linking the fields of development and evolu-

tion (Purugganan 1998). In this study, phylogenic analysis

placed these IbMADS genes into the group containing

STMADS11, STMADS16, AGL24 and SVP, but separated these

genes from other MADS-box proteins (Fig. 2). Consequently,

this STMADS subfamily may contain a new MADS-box, based

on recently reported criteria (Hartmann et al. 2000). Although

ABC clades are largely flower- and fruit-specific, and none

Fig. 2 Neighbour-joining dendrogram illustrating similarities among MADS-box proteins from dicot-species. The Neighbour-joining method

was used to generate a dendrogram of MADS-box proteins. Bootstrap values expressed as percentage (over 1,000 replicates) are shown at the cor-

responding nodes. Sources of the genes are indicated in parentheses follows. At, Arabidopsis thaliana; Am, Antirrhinum majus; Bo, Brassica

oleracea; St, Solanum tuberosum; Sa, Sinapsis alba; Le, Lycoperscon esculetum; Nt, Nicotiana tabacum; Ib, Ipomoea batatas.


appears to be expressed in roots, some genes in the A clade are

also expressed in leaves and stems (Rounsley et al. 1995,

Purugganan 1998). The newly discovered clades of MADS-box

genes, whose expression is restricted to vegetative structures,

suggests that complex genetic circuits also underlie the devel-

opment of these structures (Scheres et al. 1995). IbMADS

seems to be most closely related to STMADS11 and STMADS16

from potato and to AGL24 and SVP from Arabidopsis. Sub-

family members tend to share highly similar sequences, expres-

sion patterns and related functions (Doyle 1994, Purugganan et

al. 1995, Theißen et al. 1996). STMADS11 and STMADS16

from potato were found to be expressed during vegetative

growth (Carmona et al. 1998). Although SVP and AGL24 are

expressed in inflorescence meristems, stem, leaves and roots,

SVP transcripts disappeared prior to emergence of the sepal

primordia during flower development and may positively

regulate a repressor of flowering (Alvarez-Buylla et al. 2000).

These floral repressor genes seem to play an important role in

determining the duration of the vegetative phase (Hartmann et

al. 2000). A part from the role of IbMADS3 and IbMADS4 in

flowering, these genes seem to be exerting its function in vege-

tative phase.

In sweet potato, tuber development resulted from the

emergence of anomalous primary and secondary cambia and a

vascular cambium, which enabled rapid cell proliferation for

expanded, starch-storing, parenchymatous cells (Wilson and

Lowe 1973). In the developing tuberous root, IbMADS3 and

IbMADS4 are weakly expressed at the vascular cambium, epi-

dermis and protoxylem element (Fig. 7, segment 1). This result

also shows that differentiation of the tuberous root depended on

the division and expansion of the cells in root ontogenesis. This

particular expression pattern for the STMADS subfamily that

includes IbMADS3 and IbMADS4 may lead to a higher prolif-

erative potential of vegetative tissue, which facilitates tuber ini-

Fig. 4 Expression pattern of IbMADS3 and IbMADS4 in different organs of the sweet potato plant at 50 d after planting. (A) Each organ of

sweet potato at 50 d after planting. (B) Expression pattern of IbMADS3 and IbMADS4 in different organs of the sweet potato plant at 50 d after

planting. Northern blot analysis was performed on equal amount of total RNA (30 �g) from bud (B), flower (F), leaf (L), stem (S), white fibrous

root (W), pigmented root (R), developing tuberous root (D), and tuber (T) with gene-specific probes for IbMADS3 or IbMADS4. (C) Relative

mRNA level of IbMADS3 (C-1) and IbMADS4 (C-2). Band intensity was analyzed using the NIH Image program (National Institutes of Health,

U.S.A.) on a Macintosh computer. Ethidium bromide-staining of rRNA is shown under the figure of Northern blot hybridization.

Fig. 3 Southern blot analysis of genomic DNA digested with EcoRI

(E), HindIII (H), and BamHI (B). Filters were hybridized with probes

specific to IbMADS3 or IbMADS4 obtained from spanning about 67

amino acids of the C-terminus plus the 3�-untranslated region respec-

tively. Each lane contains 15 �g of genomic DNA digested with each

restriction enzyme. Number given on the center are DNA size markers

(�/HindIII and pGEMEXI/RsaI marker).


tiation in sweet potato.

Sweet potato roots stopped elongating at 40–50 d after

planting and started to thicken (Fig. 5). The total dry weight of

the root rapidly increased, reaching about five times the initial

value (unpublished result). Higher expressions of IbMADS3

and IbMADS4 are closely correlated with developing tuberous

root tissue, but not with elongating roots 40 d after planting.

IbMADS3 and IbMADS4 were transcribed preferentially in all

root sections, with higher levels in the middle segments (Fig. 6,

segments 1, 2, and 3), which also showed a close relationship

between IbMADS and cell proliferation during tuber bulking.

The tissue print technique was recently developed as a

rapid and convenient method to visualize the localization pro-

teins and mRNAs in plant tissues (Song et al. 1993). Many

molecules have been detected using tissue printing (Ye and

Varner 1991). In in situ hybridization, a preferential accumula-

tion of STMADS11 mRNA along the ring of the vascular bun-

dles in the stem of potato was shown (Carmona et al. 1998). On

the other hand, there is little information about the distribution

of expression of MADS-box genes in developing root tissue.

We showed that the expression of IbMADS was localized in the

epidermis and vascular cambium in the developing tuberous

root of sweet potato (Fig. 8). We propose a putative role for

IbMADS3 and IbMADS4 from these results as facilitating cell

division and expansion in the course of tuber organogenesis.

Although IbMADS3 and IbMADS4 were specifically

expressed in vegetative tissues, they showed different expres-

sion patterns (Fig. 4, 6, 7) in root tissues. The amino acid iden-

tify is very low in the C region (less than 45%) between

IbMADS3 and IbMADS4 while that in the MIK region is more

than 80%. MADS-box proteins are shown to form functional

DNA binding complexes through homo- or heterodimerization

with other MADS-box proteins (Tröbner et al. 1992). It is thus

an obvious possibility that the high similarity of expression

patterns between IbMADS3 and IbMADS4 make them candi-

dates for involvement in root organogenesis cooperatively, as

in the case of the GLOBOSA/DEFICIENS during flower forma-

tion of Antirrhinum (Tröbner et al. 1992).

In conclusion, IbMADS3 and IbMADS4 may mediate the

regulation of tuber formation via the meristematic activity of

primary cambia. The different expression patterns of the

IbMADS3 and IbMADS4 genes suggest that their products play

functionally diverse roles in development, so that these genes

may also be important in the formation of vegetative structures

in sweet potato.

Fig. 5 The relationship between growth rates of sweet potato in root

length (cm) and root weight (g). The measurements were carried out in

five samples at 20, 30, 40 and 50 d, respectively. The results are the

mean � standard error (n=5).

Fig. 6 Analysis of mRNA level in sections of developing tuberous root. (A) Developing tuberous root of about 50-day-old plants, which had not

yet reached their mature size, were sectioned transversely at about 2.5 cm intervals. (B) Northern blot with total RNA (30 �g/lane) from develop-

ing tuberous root section (1–4; section 1 is connected with the stem). (C) Relative mRNA level of IbMADS3 (C-1) and IbMADS4 (C-2). Band

intensity was analyzed using the NIH Image program (National Institutes of Health, U.S.A.) on a Macintosh computer. Ethidium bromide-staining

of rRNA is shown under the figure of Northern blot hybridization.


Materials and Methods

Plant materials

Sweet potato plants (Ipomoea batatas (L.), cv. Beniazma) grown

in a greenhouse were harvested to analyze expression of IbMADS in

different organs of the adult plants. White fibrous roots, developing

flowers, buds, leaves, stems, developing tuberous roots (5–10 mm in

diameter), pigmented roots, and the mature resting tuber were har-

vested separately. White fibrous roots, pigmented roots, and develop-

ing tuberous roots were also harvested from developing plants on 50 d.

RNA/DNA extraction

CTAB method (Chang et al. 1993) was revised for extraction of

high-quality RNA from sweet potato. A tissue sample (2–4 g FW) was

ground to a fine powder in liquid nitrogen. The frozen powder was

mixed thoroughly with 20 ml of extraction buffer (2% CTAB, 1.0 M

NaCl, 0.5% SDS, 10 mM Tris-HCl pH 8.0, and 5 mM EDTA, 2% �-

mercaptoethanol). The sample was mixed with an equal volume of

chloroform, and then centrifuged at 11,000�g for 10 min. The superna-

tant was taken up and mixed with 0.25 vol. of 10 M LiCl, and then

stored at –20�C for 4 h. Total RNA was precipitated by centrifugation

at 11,000�g for 10 min, and then resuspended in 2 ml of TE. The sus-

pension was extracted once with 1 vol. of phenol/chloroform and then

with 1 vol. chloroform. The supernatant was recovered after 20 min

centrifugation at 11,000�g, mixed with 1/4 vol. of 10 M LiCl, and

stored at –20�C overnight. The RNA was collected by centrifugation at

11,000�g for 10 min, and then dissolved in 50 �l dH2O.

DNA was isolated from sweet potato leaves as according to

Gawel and Jarret (1991).

Selection of primers and condition of PCR reaction

Conserved sequences between MADS-box genes are generated

using GENETYX (Software Development Co., Japan). Two degener-

ated primers were designed according to the conserved amino acid

sequences VLCDADV [forward primer – M3: GTI (C/T) TI TG(C/T)

GA(C/T) GCI GA (A/G) GT] and QVTFSKR [forward primer – M4:

GIC A (A/G) G TIA CIT T(C/T)(A/T) (G/C) IA A(C/T)(A/T) G] in

the MADS-box in an alignment, respectively. First strand cDNA syn-

theses from pigmented roots were performed according to the manu-

facturer instructions of the 3�-RACE (Rapid Amplification of cDNA

Ends) system (TaKaRa, Japan), and amplified with templates of cDNA

using M3 or M4 primer and 3�-RACE adaptor primer (GACTC-

GAGTCGACATCGA).

PCR cycle number, and annealing temperature were adjusted for

each MADS-domain primer set respectively. PCR amplification was

done as follows; a 50 �l of reaction mixture was assembled with 10�

GeneTaq buffer, 200 �M dNTP, 2.5 unit GeneTaq™ (Wako, Japan),

20 pmol specific primer, 20 pmol 3�-RACE adapter specific primer,

and 1 �l cDNA pool on ice. After mixing the PCR reaction contents,

reaction mixtures were heated in MJ Thermocycler (MJ, U.S.A.) to

Fig. 7 Analysis of mRNA level in sections of developing tuberous root. (A) Developing tuberous root of about 50-day-old plants which had not

yet reached their mature size were sectioned into (1) vascular cambium with a few millimeters of adjacent protoxylem element and epidermis, (2)

parenchymatous cell, and (3) central metaxylem cell plus parenchymatous cell. (B) Northern blot with total RNA (30 �g/lane) from developing

tuberous root section. (C) Relative mRNA level of IbMADS3 (C-1) and IbMADS4 (C-2). Band intensity was analyzed using the NIH Image pro-

gram (National Institutes of Health, U.S.A.) on a Macintosh computer. Ethidium bromide-staining of rRNA is shown under the figure of North-

ern blot hybridization.

Fig. 8 Tissue print detection of IbMADS3 (A) and IbMADS4 (B)

expression in sweet potato root slices. Developing tuberous root of

about 50-day-old plants which had not yet reached their mature size

were cut transversely. IbMADS3 and IbMADS4 cloned in pGEM-T

Easy Vector (Promega, U.S.A.) were used to synthesize [�32P]UTP

labeled antisense RNA probes by using SP6 RNA polymerase.


95�C for 3 min. And then, 40 cycles of amplification were performed

using the following parameters: denaturation, 30 s at 94�C, annealing,

100 s at 50�C, extension, 2 min at 72�C. The PCR products were sepa-

rated on 5% acrylamide gels. Fragments from 0.6 kb to 1.5 kb in

length were isolated and then cloned into pT7-Blue vector (TaKaRa,

Japan).

Southern and Northern blot analysis

About 10 �g genomic DNA was digested with restriction

enzymes, EcoRI, HindIII, and BamHI, then separated on a 0.8% agar-

ose gel, and transferred onto Hybond-N+ membrane (Amersham Phar-

macia Biotech, U.S.A.). The membranes were hybridized with �32P-

radiolabelled IbMADS-specific probes containing EcoRI and HindIII

digested fragments spanning about 67 amino acids of the C-terminus

plus the 3� untranslated region. Hybridization was performed under

stringent conditions (65�C) in hybridization buffer (5� Denhardt’s rea-

gent, 5� SSPE, 0.5% SDS, and 100 �g ml–1 salmon sperm DNA). A

final high-stringency wash in 0.1� SSC, 0.1% SDS, was applied for

20 min at 65�C.

For RNA blot analysis, 30 �g of total RNA in a volume of 4.6 �l

was denatured by incubation with 3.4 �l glyoxal, 2 �l sodium phos-

phate buffer (0.1 mM, pH 7.0), and 10 �l DMSO at 55�C for 1 h. The

RNA solution was chilled on ice, and then separated by electrophore-

sis through a 1.2% agarose gel with 10 mM sodium phosphate buffer

(Mizuno et al. 1993).

The separated RNA was transferred onto a Hybond-N+ nylon

membrane (Amersham Pharmacia Biotech, U.S.A.) by capillary

action. Transfer was performed for at least 16 h with transfer solution

(20� SSC). After blotting, the membrane was rinsed briefly in 2� SSC

and RNA was fixed to the membrane at 80�C for 2 h, and then hybridi-

zation was carried out. The RNA blots were pre-hybridized for at least

2 h at 65�C in 5 ml of pre-warmed hybridizing solution (5� Den-

hardt’s reagent, 5� SSPE, 0.5% SDS, and 100 �g ml–1 salmon sperm

DNA). The probes were labeled with [�32P]dCTP using Bca BEST

random labeling kit (TaKaRa, Japan). Unincorporated [�32P]dCTP was

removed by passing through a Sephadex G-50 spin column. The

labeled probes were denatured at 100�C for 3 min then added to the

hybridization solution. The membrane was hybridized with each of the

probes for 16–24 h at 65�C in 5 ml hybridizing solution, and then

washed sequentially twice for 15 min with 2� SSC and 0.1% SDS,

then with 0.1� SSC, 0.1% SDS until the background was reduced. The

membranes were exposed to X-ray film (Kodak, U.S.A.) for 24–96 h

at –80�C to intensify sensitivity. Band intensity was analyzed using the

NIH Image program (National Institutes of Health, U.S.A) on a Mac-

intosh computer.

Tissue printing

Tissue printing was followed as according to Ye and Varner

(1991) with several modifications. The fresh cut surface is slightly

blotted with Kimwipes (Kimberly-Clark Co., U.S.A.) and immedi-

ately pressed against a Hybond-N+ membrane (Amersham Pharmacia

Biotech, U.S.A.) supported with five layers of filter paper for 15–30 s.

At least three prints are prepared by repeated cutting close to the first

cut from the same plant. After printing, the membrane was baked for

2 h at 80�C. The membrane was washed in 0.2� SSC, 1% SDS for 4 h

at 65�C, prehybridized for 4 h at 68�C in 2� SSC, 1% SDS, 5� Den-

hardt’s reagent, 0.1 mg ml–1 salmon sperm DNA, and 10 mM DTT.

IbMADS3 and IbMADS4 cloned in pGEM-T Easy Vector (Promega,

U.S.A.) were used to synthesize [�32P]rUTP labeled antisense RNA

probes by using SP6 RNA polymerase, respectively. Hybridization of

probes to the membrane was carried out at 68�C for 16 h. The mem-

brane was washed with 2� SSC, 0.1% SDS at 42�C three times for

20 min each; 0.2� SSC, 0.1% SDS at 65�C twice for 30 min each.

Sequence analysis

Plasmids were purified from selected clones and sequences of

both strands of inserted DNA were determined in full length with ABI

Prism™ 310 genetic analyzer (Perkin-Elmer Applied Biosystems,

U.S.A.) using M13/pUC forward and reverse sequencing primers.

Sequences were analyzed using the GENETYX (Software Develop-

ment Co., Japan) software package. To make deduced amino acid

sequence, each cDNA was translated from the first ATG codon to the

first stop codon. The Neighbour-joining method was used to generate a

dendrogram of MADS-box proteins.

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(Received September 9, 2001; Accepted January 8, 2002)

isolation of mads-box genes from sweet potato - plant and cell

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