Mechanisms of hormonal regulation of endospermcap-specific gene expression in tomato seeds

Cristina Martınez-Andujar1,†, Wioletta E. Pluskota2,†, George W. Bassel1,†, Masashi Asahina1,†, Piotr Pupel2,†, Theresa T.

Nguyen1, Noriko Takeda-Kamiya3, David Toubiana4, Bing Bai4, Ryszard J. Gorecki2, Aaron Fait4, Shinjiro Yamaguchi3 and

Hiroyuki Nonogaki1,*1Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA,2Department of Plant Physiology and Biotechnology, University of Warmia and Mazury, Oczapowskiego 1A, 10-718 Olsztyn,

Poland,3RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan, and4The French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Ben

Gurion University of the Negev, Midreshet Ben Gurion, 84490, Israel

Received 3 February 2012; revised 15 March 2012; accepted 22 March 2012; published online 11 June 2012.

*For correspondence (e-mail hiro.nonogaki@oregonstate.edu).†These authors contributed equally to this work.

SUMMARY

The micropylar region of endosperm in a seed, which is adjacent to the radicle tip, is called the ‘endosperm

cap’, and is specifically activated before radicle emergence. This activation of the endosperm cap is a

widespread phenomenon among species and is a prerequisite for the completion of germination. To

understand the mechanisms of endosperm cap-specific gene expression in tomato seeds, GeneChip analysis

was performed. The major groups of endosperm cap-enriched genes were pathogenesis-, cell wall-, and

hormone-associated genes. The promoter regions of endosperm cap-enriched genes contained DNA motifs

recognized by ethylene response factors (ERFs). The tomato ERF1 (TERF1) and its experimentally verified

targets were enriched in the endosperm cap, suggesting an involvement of the ethylene response cascade in

this process. The known endosperm cap enzyme endo-b-mannanase is induced by gibberellin (GA), which is

thought to be the major hormone inducing endosperm cap-specific genes. The mechanism of endo-b-

mannanase induction by GA was also investigated using isolated, embryoless seeds. Results suggested that

GA might act indirectly on the endosperm cap. We propose that endosperm cap activation is caused by the

ethylene response of this tissue, as a consequence of mechanosensing of the increase in embryonic growth

potential by GA action.

Keywords: embryo, endosperm, ethylene, germination, gibberellin, seed, Solanum lycopersicum.

INTRODUCTION

Seed germination is completed by emergence of the em-

bryo, (the radicle, in many cases) (Bewley and Black, 1994).

Radicle emergence is determined by two opposing forces:

the growth potential of the embryo and the mechanical

resistance of the endosperm (Nonogaki, 2006; Linkies et al.,

2009). The mechanical resistance of the micropylar region of

the endosperm, called the ‘endosperm cap’ (Nonogaki and

Morohashi, 1996; Muller et al., 2010; Morris et al., 2011), is

an obstacle to radicle emergence (Watkins and Cantliffe,

1983; Groot and Karssen, 1987). The weakening of the

endosperm cap is a prerequisite for completion of seed

germination in many species (Halmer et al., 1975; Sanchez

et al., 1986; Sanchez et al., 1990; Liu et al., 2005; Arana et al.,

2007; Nonogaki et al., 2007; Linkies et al., 2009).

The endosperm cap is specifically activated during imbi-

bition in terms of gene expression (Liu et al., 2005). In

tomato (Solanum lycopersicum) seeds, the rigidity of the

endosperm cap is derived from its cell wall, which is

composed of galactomannans (Groot et al., 1988). MAN2,

an endo-b-mannanase gene, is expressed exclusively in the

endosperm cap (Nonogaki et al., 2000). Localized expression

of MAN protein and activity (Nonogaki and Morohashi, 1996;

Toorop et al., 1996) and the erosion of cell walls (Nonogaki

et al., 1992, 1998) are also observed specifically in the

ª 2012 The Authors 575The Plant Journal ª 2012 Blackwell Publishing Ltd

The Plant Journal (2012) 71, 575–586 doi: 10.1111/j.1365-313X.2012.05010.x

endosperm cap, suggesting the involvement of MAN activity

in endosperm weakening. Other cell-wall genes, such as

XET4 (encoding xyloglucan endotransglycosylase) (Chen

et al., 2002) and EXPA4 or LeEXP4 (encoding expansin A4)

(Chen and Bradford, 2000), are also expressed exclusively in

the endosperm cap, which suggests concerted actions of

multiple cell wall-modifying proteins in this tissue. Genes

encoding chitinase (Chi9) and b-1,3-glucanase (GluB) are

also expressed exclusively in the endosperm cap, although

their function is not known (Wu et al., 2001).

What directly triggers endosperm cap-specific gene

expression remains elusive. GIbberellin (GA) has been

suggested to be a major hormone for induction of some

endosperm-cap genes, including MAN2 (Nonogaki et al.,

2000), XET4 (Chen et al., 2002) and EXPA4 (Chen and

Bradford, 2000). These genes are not expressed in GA-

deficient gib-1 tomato seeds but are induced by exogenous

GA. Thus, some endosperm cap genes appear to be

regulated by GA; however, it is not known whether GA

directly affects the endosperm cap and how GA stimulates

gene expression only in the endosperm cap.

It is necessary to obtain more information about the

mechanisms underlying tissue-specific gene expression in

seeds. To this end, a GeneChip analysis was performed for

the endosperm cap and other tissues of tomato seeds. More

endosperm cap-enriched genes were identified and charac-

terized. In addition to the previously proposed GA regula-

tion, a potential involvement of ethylene response was

suggested by the GeneChip data. The possible interaction of

GA and ethylene in the regulation of endosperm cap

activation is discussed.

RESULTS

Identification of endosperm cap-specific genes by GeneChip

Emergence of the first radicle was observed approximately

40 h after the start of imbibition in the tomato seeds used

for the GeneChip analysis (Figure S1). Cell wall-associated

genes are expressed during relatively late stages of ger-

mination (Nonogaki et al., 2007). Regulatory genes induc-

ing cell wall-associated genes were expected to be

expressed at earlier stages. We attempted to detect genes

representative of both groups, and therefore selected 18 h

as a time point suitable for this purpose. Imbibed seeds

were dissected into the micropylar part and the rest of the

seed (termed the ‘lateral’ part) (Figure 1a,b). The embryos

were removed from the micropylar and lateral parts, which

were named endosperm cap (EC) and lateral endosperm

(LE) (Figure 1c). Although both parts still contained the

testa, the term ‘endosperm’ was used because the testa is

non-viable tissue in the mature tomato seed and does not

affect expression analysis. The embryo was divided into

radicle (R) and cotyledon (C) halves (Figure 1b,c). RNA

extracted from these four tissues was used for GeneChip

analysis.

When we used a stringent cut-off of >fivefold higher

expression compared to expression in any other tissues in

GeneChip data analysis, 34 EC-, four LE-, one R- and five C-

enriched genes were detected (Table S1). The genes for

which gene annotation is available are summarized in

Table 1. The number of EC-, LE-, R- and C-enriched genes

increased to 150, 135, 72 and 29, respectively, when the

analysis was expanded to >twofold enrichment (Table S2).

The cell-wall gene SlMAN2 (AF184238.1) that is known to

be expressed exclusively in the EC of tomato seeds (Non-

ogaki et al., 2000) showed >18-fold enrichment in the EC

(Table 1). Other known EC-specific genes, EXPA4

(AF059488.1) (Chen and Bradford, 2000) and XET4

(AF186777.1) (Chen et al., 2002), showed 4.2- and 4.9-fold

enrichment in the EC, respectively (Table S2). These genes

served as an excellent internal control for EC enrichment and

verified the quality of the GeneChip analysis.

The genes enriched >fivefold in the EC included other cell-

wall genes such as other expansins [EXP2 (AF096776.1),

(a)

(c)

(b) Figure 1. Tomato seed tissues used for Gene-

Chip and other expression analysis.

(a) Intact tomato seed.

(b) Left: seed dissected into micropylar and

lateral parts. Embryonic tissues were removed

from each. The micropylar and lateral embryo-

less tissues (endosperm + testa) were desig-

nated endosperm cap (EC) and lateral

endosperm (LE), respectively. Right: embryo

excised from the seed and dissected into radicle

(R) and cotyledon (C) halves.

(c) Schematic representation of the four parts of

the seeds used for GeneChip analysis.

576 Cristina Martınez-Andujar et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 575–586

EXPA6 (AF059490) and EXP11 (AJ560646.1)] and a gene

encoding endo-b-1,4-glucanase (AF308936.1) (Table 1). An-

other endo-b-1,4-glucanase gene, Cel8 (BT013727.1), was

enriched in the EC >fourfold (Table S2). The genes enriched

>twofold in the EC included a gene encoding pectin methy-

lesterase (PME, U49330.1) (Table S2). PME is involved in EC

weakening in tomato seeds (Downie et al., 1998; Sitrit et al.,

1999). The glycosyltransferase gene BI421517, which was

detected among the >fivefold EC-enriched genes (Table 1),

exhibits similarity to Arabidopsis and Populus trichocarpa

genes involved in xylan synthesis (Kong et al., 2009). These

results support the idea that cell-wall modification occurs

exclusively in the EC (Nonogaki et al., 2007; Pinto et al.,

2007).

GC-MS-based metabolite profiling also indicated EC-

specific cell-wall modification (Table S3). Although the

content of most measured metabolites was generally

higher in the LE than in the EC, a number of cell wall-

related sugars showed higher accumulation in the EC

(Figure S2). Consistent with the expression of SlMAN2

and Cel8 in the EC (Tables 1 and S2), more mannose and

glucose accumulated in the EC compared to the LE

(Figure S2). Mannose can be converted by mannose

isomerase to fructose, which also accumulated in the

EC. Another pathway competing with this one is conver-

sion of mannose to mannose 6-phosphate, less of which

was found in the EC. Fructose and glucose possibly

accumulated at the expenses of the mannose 6-phos-

phate. A gene encoding myo-inositol 1-phosphate

synthase was enriched in the EC (Table 1). This enzyme

converts D-glucose 6-phosphate to myo-inositol, which

is then converted to UDP-glucuronic acid under favor-

able redox conditions (Seitz et al., 2000). The decreased

myo-inisitol and increased glucuronic acid in EC

(Figure S2) probably reflect the function of this enzyme

in the EC.

Another group of genes found among the >fivefold EC-

enriched genes were pathogenesis-related (PR) genes

(Table 1), including NP24 (or osmotin, M21346.1) (Jia and

Martin, 1999), which showed the highest expression level of

all EC-enriched genes, a chitinase gene (BG629640) (Dan-

hash et al., 1993), PR5-like (AY257487.1) (Li et al., 2011), P23

(X70787.1, SGN U581103), which is homologous to NP24

(Rodrigo et al., 1993), and a gene (AI895341) that is similar to

elicitor-inducible genes. The >twofold EC-enriched genes

included a gene encoding b-1,3-glucanase (M80608.1) (Kan

et al., 1992) and another pathogen/wound-induced gene

(pi1, BT012973.1) (Table S2). Expression of the chitinase

gene Chi9 and the b-1,3-glucanase gene GluB in the EC

during tomato seed germination was shown previously (Wu

et al., 2001). Although chitinase and b-1,3-glucanase could

contribute to cell-wall modification through their capacity to

Table 1 Genes enriched >fivefold in the endosperm cap (EC) of 18 h imbibed tomato seeds for which gene annotation information is available

Gene annotation GenBank accession number

Relative expression

EC LE R C

NP24 protein M21346 1063 180 7 0Expansin (LeEXP2) AF096776.1 882 34 95 5Sucrose synthase L19762.1 773 80 12 9Chitinase BG629640 709 48 103 4Expansin 11 (EXP11) AJ560646.1 476 15 33 1Pathogenesis-related PR5-like protein AY257487.1 457 88 24 14Xyloglucan-specific fungal endoglucanase inhibitor AY155579.1 297 9 4 1(1, 4)-b-mannan endohydrolase (MAN2) AF184238.1 279 15 0 0Endo-b-1,4-D-glucanase AF308936.1 269 5 18 0Weakly similar to peroxidase CK720576 225 12 0 0Pathogenesis-related P23 X70787.1 200 19 1 0Similar to C3HC4-type RING finger BT012911.1 188 26 21 17Expansin (EXPA6) AF059490.1 117 5 16 4GAST1 (GA-stimulated transcript 1) BG626882 108 10 1 0Similar to peroxidase AI773309 95 16 2 1Similar to esterase AW034398 90 13 2 1Aquaporin (LePIP1) AY725511.1 69 5 4 11Asparagine synthetase AW625684 66 11 9 8Similar to glycosyltransferase BI421517 53 1 7 1Ethylene response factor (TERF1) AY044236.1 47 5 1 0Similar to IAAs AW034122 46 1 8 0Similar to myo-inositol 1-phosphate synthase (INS-1P) BT013505.1 43 8 3 2Similar to elicitor-induced gene AI895341 38 2 1 0Similar to GH3, encoding indole-3-acetic acid amido synthetase BT013446.1 37 2 2 1Aldehyde oxidase (AO1) AF258808.1 33 5 0 1

Endosperm cap genes in tomato seeds 577

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 575–586

degrade substrate polysaccharides (i.e. chitin and callose,

respectively), there is no evidence to support their involve-

ment in active degradation of endosperm cell wall (Wu et al.,

2001). Identification of the PR genes NP24 (>5.9-fold), PR5-

like (>5.1-fold) and P23 (>10.5-fold) as EC-enriched genes

(Table 1) implies that mechanisms similar to a pathogen/

wounding response are present in the EC during tomato

seed germination. In addition, expression of the gene

encoding xyloglucan-specific fungal endoglucanase inhibi-

tor (Xegip, AY155579.1), which functions in a pathogen

response in tomato (Qin et al., 2003), was enriched in the EC

(Table 1). The >fivefold EC-enriched genes also included two

genes (CK720576 and AI773309) that encode proteins similar

to peroxidase, which has been suggested to be involved in

pathogen responses in tomato seeds (Morohashi, 2002).

Consistent with these observations is the significant enrich-

ment of the Gene Ontology (GO) biological processes

‘immune system process’ (P = 0.025) and ‘defense re-

sponse’ (P = 0.0063) within the >fivefold EC-enriched gene

list.

Other EC-enriched genes were hormone-associated

genes (Table 1). GAST1 (GA-stimulated transcript 1,

BG626882), whose expression is induced in tomato shoots

by GA (Shi et al., 1992), was enriched in the EC >10-fold. It

is known that EC-enriched cell-wall proteins are GA-induc-

ible (Groot and Karssen, 1987; Chen and Bradford, 2000;

Nonogaki et al., 2000), although there is little information

about GAST involvement in EC weakening. The analysis

also identified ethylene-associated genes. The gene encod-

ing TERF1 (AY044236.1) (Huang et al., 2004), an ethylene

response factor (ERF) in tomato, was enriched >ninefold in

the EC (Table 1). The >twofold EC-enriched genes included

a gene (BG628423) that is similar to the Nicotiana tabacum

gene encoding S-adenosylmethionine synthase (SAM,

AF321140.1) (Table S2), indicative of EC-specific ethylene

biosynthesis. These results are consistent with the recent

finding of a key regulatory role of ethylene in EC weaken-

ing in seeds of other species such as Lepidium sativum

and Arabidopsis (Linkies et al., 2009). A signal transduction

protein(s) responsible for the ethylene response in EC

weakening has not yet been identified. TERF1 is a good

candidate regulator of the ethylene response in the EC. A

gene (AW034122) encoding a protein similar to auxin

signal transduction proteins (IAAs) was detected among

the >fivefold EC-enriched genes (Table 1). The tomato

genes INDOLE-3-ACETIC ACID INDUCIBLE1 (IAA1,

BI209735), IAA2 (AF022013.1) and IAA8 (BT014412.1) were

also enriched in the EC (Table S2), suggesting a possible

involvement of auxin in the EC-specific events. Interest-

ingly, a gene (BT013446.1) similar to GH3, which encodes

an IAA amido synthetase, was enriched in the EC,

suggesting EC-specific auxin conjugation. A gene encoding

an aldehyde oxidase (AO1, AF258808.1), which catalyzes

the final steps of abscisic acid (ABA) biosynthesis (Min

et al., 2000), was detected among the >fivefold EC-enriched

genes, suggesting EC-specific ABA biosynthesis in tomato

seeds.

Characterization of the endosperm cap-enriched genes

To verify the results of GeneChip analysis, we characterized

expression patterns of the identified EC-enriched genes. We

performed tissue printing to examine localization of NP24

expression. A strong signal was detected exclusively in the

EC of germinating tomato seeds (Figure 2a), verifying

enrichment of NP24 in the EC and corroborating the Gene-

Chip data. NP24 was the most strongly expressed EC-en-

riched gene in our GeneChip analysis. We also characterized

the 5¢ upstream sequence ()1903 to +321) of NP24 using the

reporter gene NP24:GFP-GUS. When transgenic seeds were

examined, GUS signals were enriched in the EC (Figure 2b),

although some signals were also observed in the lateral

endosperm (Figure 2e) and embryo (Figure 2c,d).

We also tested the tissue specificity of TERF1 expression

using RT-PCR, which confirmed its enrichment in the EC

(Figure 3a). We detected little TERF1 accumulation in dry

seeds. TERF1 mRNA accumulated when seeds were imbibed

at 4�C for 3 days (Figure 3b, 0 h). When pre-chilled seeds

were transferred to 25�C for germination, there was a delay

in TERF1 accumulation (6 h), followed by a progressive

increase until radicle emergence (Figure 3b), suggesting the

involvement of this gene in germination events.

(a) (c)

(b) (d)

(e)

Figure 2. Endosperm cap (EC)-enriched expression of NP24, a pathogenesis-

related gene, in tomato seeds.

(a) Tissue printing of an 18 h imbibed tomato seed probed with an antisense

NP24 RNA probe. The purple signal represents localization of NP24 mRNA.

(b) GUS staining of the endosperm (plus testa) of a transgenic seed

expressing NP24:GFP-GUS.

(c) GUS staining of the embryo of a transgenic seed expressing NP24:GFP-

GUS.

(d) Magnified view of the part of the embryo outlined in (c). (e) Magnified view

of the part of the endosperm outlined in (b).

578 Cristina Martınez-Andujar et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 575–586

Consensus motifs in the promoter regions of endosperm

cap-enriched PR genes

The 5¢ upstream sequences of the identified EC-enriched PR

genes NP24, P23 and PR5-like were analyzed using the

MEME suite (http://meme.sdsc.edu/meme/intro.html). Two

conserved DNA motifs, CATAGT[GT][TC][CA]AAAAGCC

GCCA[CT]ACCCCTATATAAA[CG][ACT][CG]C (motif 1) and

AGCCGCCTA (motif 2), were over-represented. The posi-

tions of motifs 1 and 2 relative to the transcription initiation

sites were similar in the three genes (Figure 4a). The 38 bp

motif 1, which contained a TATA box at its 3¢ end (Figure 4b),

was located at approximately )55. The 9 bp motif 2 was

found at approximately )155. Both motifs contained a GCC

(or AGC) box (AGCCGCC), a well-known binding site for

ERFs (Figure 4b,c). One of the ERF genes, TERF1, was en-

riched >ninefold in the EC (Table 1). TERF1 is known to

physically interact with the NP24 promoter through the GCC

motif (Huang et al., 2004). Therefore, these results strongly

suggest that TERF1 induces expression of the PR genes in

the EC.

The GCC box is bound by other ERFs. The >twofold EC-

enriched genes included Pti6 (Pseudomonas syringae pv.

tomato-interacting kinase, U89257.1). Pti4, Pti5 and Pti6

share similarities to ERFs, as they also bind to AGCCGCC

and up-regulate NP24 (Zhou et al., 1997; Jia and Martin,

1999). Therefore, Pti6 might also act together with, or

independently of, TERF1 to induce the EC-enriched PR

genes.

Analysis of the promoter regions of cell-wall genes

SlMAN2 is one of the best-characterized EC-enriched genes

in tomato seeds. Our GeneChip analysis showed high

enrichment (>18-fold) of SlMAN2 in the EC. We analyzed the

5¢ upstream sequence (742 bp) of SlMAN2 using PLACE

(http://www.dna.affrc.go.jp/PLACE/) (Higo et al., 1999). The

SlMAN2 promoter did not contain a GCC box. However, we

found two repeats of AACTAAC (positions )316 and )402)

(Figure S3a,b, Table S4), a negative strand of GTTAGTT,

which is a binding site for tomato Pti4 (an ERF) (Table S1).

This finding suggested a possible involvement of the ethyl-

ene response in regulation of SlMAN2. The 5¢ upstream se-

(a)

(b)

Figure 3. Spatial and temporal expression of TERF1 in tomato seeds.

(a) RT-PCR for TERF1 mRNA accumulation in the endosperm cap (EC), lateral

endosperm (LE), radicle-half embryo (R) and cotyledon-half embryo (C). SGN

U346908, housekeeping control gene (Exposito-Rodriguez et al., 2008).

(b) RT-PCR for TERF1 mRNA accumulation in dry tomato seeds, seeds

imbibed at 4�C for 3 days (0 h), and seeds imbibed further at 25�C.

(a)

(b)

(c)

Figure 4. Consensus DNA motifs found in the 5¢upstream sequences of EC-enriched PR genes.

(a) Schematic representation of the 5¢ upstream

sequences of NP24, P23 and PR5-like, indicating

the positions of two consensus DNA motifs.

Negative numbers indicate the positions of the

motifs relative to the predicted transcription

initiation sites (0).

(b) Conserved DNA motifs (Motifs 1 and 2) found

in the 5¢ upstream sequences of NP24, P23 and

PR5-like using the MEME suite (http://meme.sds-

c.edu/meme/intro.html). The TATA box con-

tained in motif 1 and the GCC boxes contained

in motifs 1 and 2 are underlined. TERF1 binds to

the GCC box.

(c) Alignment of motif 1- and motif 2-containing

regions of NP24, P23 and PR5-like. The symbols +

and ) on the left of the sequences indicate the

direction of the motifs relative to the promoter

sequences (plus and minus strand).

Endosperm cap genes in tomato seeds 579

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 575–586

quence of SlMAN2 contained the GARE2OSREP1 motif

(TAACGTA), a GA-responsive element. Induction of SlMAN2

in the EC of seeds of gib-1, a GA-deficient tomato mutant, by

GA has been demonstrated (Nonogaki et al., 2000). This

element may or may not be important for the ability of GA to

induce SlMAN2 (see Discussion).

Concerted action of MAN and expansins is thought to be

necessary for efficient modification of the cell wall of the EC

of tomato seeds (Nonogaki et al., 2007). The 5¢ upstream

sequence of EXP11 was also analyzed. The Pti4 binding site

found in the SlMAN2 promoter (AACTAAC, Figure S3b) was

also present in the 5¢ upstream region (-989) of EXP11 as two

overlapping sites (AACTAACTAAC) (Figure S4). The 2 kb

EXP11 promoter region contained seven repeats of ERELEE4

(A[A/T]TTCAAA), an ethylene-responsive element (Itzhaki

et al., 1994), including three repeats within the proximal

region ()231 to 0 bp). Additionally, five repeats of a similar

sequence ([A/T][A/T]TTCAAA) were found in the 2 kb pro-

moter region. These results suggest that EXP11 is controlled

by ERFs through the non-GCC ERF-binding sites. It is

possible that the ethylene response plays a critical role for

cell wall-associated genes.

Regulation of MAN in the endosperm cap by GA

The results of the GeneChip and promoter analysis strongly

suggested the involvement of the ethylene response in

regulation of EC-specific genes. On the other hand, there is

evidence to suggest that GA plays an important role in the

induction of cell wall-associated genes (Groot and Karssen,

1987; Chen and Bradford, 2000; Nonogaki et al., 2000). It is

critical to obtain information about GA regulation of the EC

genes and investigate potential interactions of ethylene and

GA in terms of seed germination control. Therefore, we

analyzed the regulation of MAN by GA in detail, using em-

bryoless seeds (i.e. isolated endosperm, EC and LE).

The EC and LE were incubated under sterile conditions for

2 days and examined for MAN activity with native PAGE

followed by activity staining, and for MAN protein accumu-

lation using the anti-MAN antibody (Nonogaki et al., 1995).

MAN protein and activity were induced by GA in both tissues

in a dose-dependent manner (Figure 5a), which supports the

idea that MAN induction in the EC of intact tomato seeds is

dependent on GA. When the EC or LE were co-incubated

with embryonic axes in water (in the absence of GA), clear

induction of MAN was observed (Figure 5b), indicating that

GA can be replaced by the embryonic axis. This result

supports the hypothesis that GA is produced in the embry-

onic axis, is secreted to the endosperm, and induces gene

expression there (Groot and Karssen, 1987). However, it

should be noted that MAN induction in intact seeds is

exclusive to the EC and does not occur in the LE of intact

tomato seeds (Nonogaki and Morohashi, 1996; Nonogaki

et al., 2000). MAN is not expressed in the EC of GA-deficient

gib-1 tomato seeds, but can be induced by GA in the mutant

seeds (Groot and Karssen, 1987; Nonogaki et al., 2000),

which also supports the GA dependency of MAN expression

during germination. However, in this case also, MAN

induction is limited to the EC (Nonogaki et al., 2000)

(Figure S5). Therefore, the results for incubated EC and LE

in the presence of GA are not fully consistent with the

mechanisms of MAN induction in intact seeds. MAN protein

(a)

(b)

(c)

Figure 5. Induction of MAN protein and activity by GA in the isolated

endosperm.

(a) Dose–response GA dependency of MAN induction in the isolated

endosperm. Isolated endosperm caps (EC) or lateral endosperm (LE) were

incubated for 2 days in the presence of various concentrations of GA3, and

subjected to native PAGE followed by activity staining or SDS–PAGE followed

by immunoblotting with the anti-MAN antibody.

(b) MAN induction by the embryonic factor examined by immunoblotting. EC

or LE were incubated in water (in the absence of GA) with (+) or without ())

embryonic axes excised from the micropylar halves of the tomato seeds

(shown on the right) imbibed for 14 h.

(c) Time course (1–4 days) of induction of MAN protein (immunoblot) and

activity in the isolated LE and EC in the presence of 10)5 M GA3.

580 Cristina Martınez-Andujar et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 575–586

and activity were detected in the EC of intact seeds after

1 day of imbibition (Nonogaki and Morohashi, 1996; Non-

ogaki et al., 2000). However, induction of MAN expression in

the incubated EC and LE by GA required 2 days, and the

protein and activity levels declined thereafter (Figure 5c).

Thus, the rate of MAN induction also differed between intact

and incubated EC.

In intact tomato seeds, different forms of MANs are

produced during germination (EC-specific 39 kDa form) and

post-germination (LE-specific 38 kDa form) (Nonogaki and

Morohashi, 1996). Although the 1 kDa difference is hard to

distinguish by the measurement of the relative mobility of

polypeptides in SDS–PAGE gels, when these two forms of

enzymes are mixed and subjected to low-Bis SDS–PAGE

(Nonogaki and Morohashi, 1996), they can be distinguished

from each other as two separate bands (Nonogaki and

Morohashi, 1996). However, when we compared MAN

proteins induced in incubated EC and LC, they were indis-

tinguishable by this method (Figure 6a). This suggests that

the MAN form induced in incubated EC is different from the

native form of MAN induced in the EC of intact seeds. We

therefore compared the MAN forms from these two origins

(incubated EC versus intact EC). As shown in Figure 6(b), we

were able to distinguish the two forms: the MAN form

induced in the incubated EC was smaller (38 kDa) than the

authentic form of MAN in the EC of intact seeds (39 kDa),

with a mixture of two exhibiting doublet bands (Figure 6b).

The size of the MAN form induced in incubated EC by GA

was similar to the size (38 kDa) of the post-germinative LC-

specific MAN. These results suggest that the responses of

the isolated tissues might not reflect the biology of intact

seeds in terms of GA response.

Many GA-inducible genes and proteins are suppressed by

ABA (Gomez-Cadenas et al., 1999; Sutoh and Yamauchi,

2003). Although EC-specific MAN is GA-inducible, exoge-

nous ABA does not inhibit the accumulation of MAN mRNA

(Nonogaki et al., 2000), protein or activity (Toorop et al.,

1996) in the EC. When isolated EC and LE are incubated in

the presence of GA and ABA, MAN production was com-

pletely suppressed (Figure 6c), which is quite different from

the response of the EC of intact tomato seeds. These results

suggest that the responses of isolated and incubated EC

differ from those of the EC in intact tomato seeds. Thus,

there is no conclusive evidence that GA supply to the

endosperm cap directly causes EC gene expression in intact

seeds. It is possible that the ethylene response plays a

predominant role in the induction of EC genes to which GA

indirectly contributes.

DISCUSSION

Involvement of the ethylene response in EC-specific gene

expression

In addition to known EC-specific PR genes, such as chitinase

(Wu and Bradford, 2003) and b-1,3-glucanase (Wu et al.,

2001), more EC-enriched PR genes were found in this study.

The analysis also suggested potential mechanistic links

among the EC genes in tomato seeds. It is highly likely that

up-regulation of the identified EC-enriched PR genes NP24,

P23 and PR5-like (Table 1) is mediated through binding of

TERF1 to the two conserved DNA sequences in their pro-

moters, which contain the well-known GCC motif (Figure 4).

Possible involvement of ERFs in the induction of b-1,3-glu-

canase, whose promoter contains the GCC motif, was sug-

gested previously for seeds of Nicotiana tabacum (Leubner-

Metzger et al., 1998). Although expression of the tobacco

ERFs was not endosperm-cap specific (Leubner-Metzger

et al., 1998), ERF–PR gene regulation cascades may function

in both tomato and tobacco seeds. Petruzzelli et al. (2003)

expanded analysis of PR proteins to several species in the

Solanaceae family, and demonstrated that b-1,3-glucanases

are expressed in seeds of all examined solanaceous species

but chitinase induction differed depending on species. It

appears that the regulatory mechanisms of seed germina-

tion are conserved even between Solanaceae and Brassica-

ceae (e.g. Arabidopsis and Lepidium) (see Linkies and

Leubner-Metzger, 2012 for comprehensive review).

Our findings of a PR response in the EC suggest that

typical defense mechanisms observed in the other parts of

plants are operational exclusively in the EC of the tomato

seed during germination. What is the biological significance

of PR gene expression exclusively in the EC? Reserve

mobilization occurs exclusively in the EC of tomato seeds

before radicle emergence (Nonogaki et al., 1998), which may

create an attractive food source for microorganisms. Rup-

ture of the endosperm is an inevitable event to complete

(a) (b)

(c)

Figure 6. Responses of isolated versus intact endosperm caps (EC) with

regard to MAN regulation by GA and ABA.

(a) Comparison of MAN forms induced by 10)5 GA3 in isolated (Iso) EC and LE

by immunoblotting with the anti-MAN antibody. No obvious differences were

detected.

(b) Comparison of MAN forms induced by 10)5 GA3 in isolated (Iso) EC versus

intact (Int) EC by immunoblotting. Note that the MAN polypeptide induced in

Iso EC is slightly smaller than the MAN peptide detected in Int EC, and that

their mixture resulted in doublet bands.

(c) ABA sensitivity of GA-induced MAN in isolated (Iso) EC and LE examined

by immunoblotting. The EC and LE were incubated with 10)5 M GA3 in the

absence ()) or presence (+) of 10)4 M ABA.

Endosperm cap genes in tomato seeds 581

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 575–586

germination but is probably one of the most risky events

during embryo emergence. PR proteins, such as P23, inhibit

growth of phytopathogenic fungi (Rodrigo et al., 1993) and

may be involved in protection against them. Expression of

PR genes in the tomato EC could be a pre-programmed

event to provide protection against ‘expected’ attack by

microorganisms during endosperm rupture, although it is

possible that this is a response-type event (see below).

Analysis of the MAN2 and EXP11 promoters revealed the

presence of multiple repeats of non-GCC binding sites for

ERFs, including Pti4, in their promoters (Figures S3 and S4).

It has been experimentally demonstrated that tomato Pti4

up-regulates AtEXP6 in Arabidopsis (Chakravarthy et al.,

2003). Although we did not detect Pti4 in the EC, Pti6 was one

of the EC-enriched genes (Table S2). These results suggest

that EC-enriched cell wall-associated genes are also con-

trolled through the ethylene response.

Direct or indirect role of GA in EC-specific gene expression

Although the GeneChip analysis strongly suggested

involvement of the ethylene response in the EC, GA is con-

sidered the major hormone inducing MAN and other cell

wall-associated enzymes in the EC. It is hypothesized that

GA is synthesized in the embryo and secreted to the endo-

sperm where it induces MAN (Groot and Karssen, 1987)

(Figure 7a). This hypothesis is supported by MAN induction

in the isolated EC by exogenous GA (Figure 5a,c) and

replacement of the effect of GA by co-incubation with

embryonic axes (Figure 5b). In Arabidopsis seeds, the GA

biosynthesis genes GA3ox1 and GA3ox2 are expressed in

the embryonic axis during germination (Yamaguchi et al.,

2001), suggesting that GA is produced in the embryo and

stimulates germination. Possible interaction of the embryo

and endosperm, in terms of MAN induction, was also pro-

posed for Arabidopsis seeds, although the factor secreted

from the embryo to the endosperm in this hypothesis is not

GA but MAN proteins themselves (Iglesias-Fernandez et al.,

2011).

GA production in the embryo and its involvement in

germination probably also occur in tomato seeds. However,

in vitro culture of isolated EC indicated that the MAN form

expressed in isolated and incubated EC was different from

the native form of MAN induced in intact wild-type tomato

seeds or intact gib-1 seeds stimulated by GA (Figures 6b and

S5). In addition, MAN induction by GA in the isolated EC was

cancelled by ABA (Figure 6c), which is not the case in the EC

of intact tomato seeds (Toorop et al., 1996; Nonogaki et al.,

2000). More importantly, MAN induction in the isolated

endosperm by GA was not limited to the EC but occurred in

the LE also. Therefore, the results of experiments using

(a) (b)

Figure 7. Schematic representation of two hypotheses regarding the mechanisms of EC-specific gene activation.

(a) Hypothesis 1: direct induction of the EC genes by GA. GA produced in the embryonic axis is secreted to the endosperm cap and induces EC-specific transcription

factors (TFs) that directly or indirectly induce EC-specific genes. How GA exclusively stimulates EC without affecting the other part of the endosperm needs to be

explained. It is possible that GA receptors are present exclusively in the EC and only the EC can respond to GA diffused within a whole seed. Alternatively, non-

diffusible secondary messenger(s) may be produced in the embryo by GA and move to the EC.

(b) Hypothesis 2: indirect induction of the EC genes by mechanosensing. GA does not stimulate the EC directly, but induces EC gene expression through its effects

on cell expansion in the embryonic axis. In this hypothesis, the growth potential of the embryo, generated through GA biosynthesis, places pressure to the EC. This

triggers mechanosensing by the EC, which mimics wounding or a pathogenesis response, a major consequence of which is the ethylene response, including

activation of TERF1. While TERF1 involvement in EC gene induction has been verified, evidence for mechanosensing remains to be obtained. Note that the major

role of ethylene signal transduction in the EC in this hypothesis and the well-known EC gene induction by GA are not mutually exclusive.

582 Cristina Martınez-Andujar et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 575–586

isolated endosperm do not provide strong evidence for

direct control of EC-specific MAN by GA in intact tomato

seeds.

No direct evidence has been obtained to date for GA

secretion from the embryo to the EC in intact tomato seeds

during germination. If GA is synthesized in the embryo of

tomato seeds (which is most likely the case) and is secreted

to the endosperm to directly induce MAN in this tissue, why

does such a diffusible signal like GA not stimulate MAN

production in the rest of the endosperm? The site of GA3ox

expression in Arabidopsis seeds is at the radicle–hypocotyl

region, or elongation zone, behind the radicle tip, which is

enclosed by the EC. The portion of LE that surrounds the

elongation zone of the embryo will be exposed to GA

produced by the embryo (Figure 7a). It is possible that GA

receptors such as GID1 (Ueguchi-Tanaka et al., 2005; Iuchi

et al., 2007; Voegele et al., 2011) are synthesized and local-

ized exclusively in the EC, and therefore only the EC can

respond to GA signal diffused through the whole seed.

However, this possibility is not well supported by our

results, because isolated LE can respond to GA and produce

MAN (Figure 5a,c). It is possible that a non-diffusible

secondary messenger (e.g. a protein) that acts downstream

of GA is specifically transported to the EC (Figure 7a).

However, such a factor has not been identified, and mech-

anisms of site-specific induction or transportation of the

messenger by GA have to be explained. Taken together, the

possibility cannot be ruled out that GA is only indirectly

involved in induction of EC-specific genes before radicle

emergence.

Integration of GA and ethylene regulation of EC-specific

gene expression

There is no doubt that GA is involved, at least indirectly, in

induction of EC-specific MAN and germination per se in to-

mato seeds, as exogenous GA clearly induces EC-specific

MAN (Nonogaki et al., 2000) and radicle emergence (Groot

and Karssen, 1987) in intact gib-1 tomato seeds. How does

GA induce EC-specific gene expression? What is the inter-

section between GA biosynthesis and ethylene response?

These important biological questions can be addressed by

integrating the findings from the GeneChip analysis and

previous (Groot and Karssen, 1987) and present studies on

the mechanisms of MAN expression into a single scheme.

GA is thought to promote germination through its effects

on both the embryo and the endosperm simultaneously,

through growth potential increase and endosperm weaken-

ing, respectively (Ni and Bradford, 1993; Nonogaki et al.,

2007). However, it is possible that GA does not directly affect

endosperm weakening and its function is primarily in the

embryo. GA promotes cell expansion in the elongation zone

of the embryo, which increases embryo growth potential

(Yamaguchi et al., 2001). The radicle cannot emerge from a

seed before the endosperm is weakened (Groot and Kars-

sen, 1987). The increase in embryo growth potential could

increase pressure inside a seed, with the radicle pressing

down onto the EC (Figure 7b). This mechanical signal may

be sensed by the EC (mechanosensing) (Monshausen and

Gilroy, 2009). Although possible pre-programming of EC

gene expression was discussed above, another possibility is

that induction of the EC-specific genes is simply a wounding

response that is the consequence of sensing the mechanical

force by the radicle tip. Although the primary role of GA is

assumed to be in the embryo in this hypothesis, this idea

and the evidence provided from the previous study (GA

dependency of EC gene induction and endosperm weaken-

ing) (Groot and Karssen, 1987) are not mutually exclusive,

because the mechanical pressure by the radicle originates

from, and is dependent on, GA synthesized in the embryonic

axis. Interestingly, xyloglucan endotransglycosylase/hydro-

lase (XTH) a typical cell wall-associated gene expressed in

the EC, is one of the ‘Touch’ (TCH) genes induced by physical

contact with plants as a consequence of mechanosensing

(Braam, 2005). It is possible that the force of the radicle tip

stimulates typical wounding responses, including induction

of TERF1 and other ERFs, which then up-regulate EC-specific

genes. In this hypothesis, unlike the traditional one, the two

events – growth potential increase and endosperm weaken-

ing – are not independent of each other but are consequen-

tial, the embryo event being upstream. It is technically

difficult to measure the pressure provided specifically by the

radicle tip in intact seeds. If methods are developed to

specifically increase the embryo growth potential in gib-1

seeds without GA application (for example, by inducible

expression of cell expansion-associated genes such as

embryonic expansin genes or XTH) in an embryo-specific

manner, it will be possible to test whether an increase in

embryo growth potential induces expression of EC genes.

More research is necessary to test this emerging hypothesis.

EXPERIMENTAL PROCEDURES

Isolation of tomato seed tissues

Seeds of tomato (Solanum lycopersicum cv. Moneymaker) wereimbibed at 25�C in the dark for 18 h (without pre-chilling) and thendissected, essentially as described previously (Nonogaki et al.,1992). Briefly, more than 1000 seeds were cut transversely to pro-duce the micropylar and lateral parts. Each part was dissected lon-gitudinally to remove embryonic tissues. The embryoless parts(endosperm plus testa) from the micropylar and lateral parts weretermed the endosperm cap (EC) and lateral endosperm (LE),respectively (Figure 1). Separately, imbibed seeds were dissectedlongitudinally to excise the embryo. The excised embryos were cutinto radicle (R) or cotyledon (C)halves (Figure 1). Three independentsets of the four tissues were used for gene expression analysis.

GeneChip analysis

RNA was extracted from three pools of the four tissues using astandard phenol extraction method (Sambrook et al., 1989). Foreach tissue, three independent hybridizations were performed. RNA

Endosperm cap genes in tomato seeds 583

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 575–586

was hybridized to the GeneChip� Tomato Genome Array (Affyme-trix, http://www.affymetrix.com/), which is designed specifically tomonitor gene expression in tomato. The comprehensive arrayconsists of over 10 000 tomato probe sets to investigate over 9200transcripts. Hybridization and other procedures were performedaccording to the Affymetrix GeneChip� Expression Analysis Tech-nical Manual. Raw data obtained from the hybridization were nor-malized by Bioconductor (http://www.bioconductor.org/) usingMAS5 and a target intensity (TGT) scaling factor equal to 100. Sta-tistical analysis (one-way ANOVA) of log2-trransformed data wasdone using MeV (MultiExperiment Viewer) software (http://www.tm4.org/mev/). Adjusted Bonferroni P value correction wasapplied. Gene annotation was analyzed using the tools at http://www.plexdb.org/modules/PD_probeset/annotation.php?GeneChip=Tomato10k.

GC-MS metabolite profiling

Metabolite profiling was performed as described previously (Lisecet al., 2006). Details are given in Supporting Experimental Proce-dures.

Tissue printing analysis

Tissue printing of germinating tomato seeds (imbibed for 18 h after3 days pre-chilling) was performed as described previously (Non-ogaki et al., 2000).

Preparation of NP24:GFP-GUS

The 5¢ upstream sequence of NP24 ()2224 to )1) was cloned into theSacI and SalI sites in shuttle vector pRJG23 (Grebenok et al., 1997)that contained the uidA (GUS) gene. The promoter:GFP-GUS con-struct in pRJG23 was cut out using SacI and SpeI, and sub-clonedinto the SacI and XbaI sites of the pGPTV-KAN binary vector (Beckeret al., 1992). GFP was not monitored in this study.

GUS analysis

GUS staining of seedlings was done as previously described (Wei-gel and Glazebrook, 2002) using 100 mM sodium phosphate buffer(pH 7.0) containing 0.1% v/v Triton X-100 and 2 mM X-Gluc (RPI Co,http://www.rpico.com/).

Expression analysis

Total RNA was extracted from various tissues (EC, LE, R and C) orwhole seeds after 0, 6, 12, 18, 24, 36 and 48 h imbibition (following3 days pre-chilling) using standard phenol/SDS extraction (Sam-brook et al., 1989). Aliquots (1 lg) of DNase-treated total RNA wereused for reverse transcription with the ImProm-II� reversetranscription system (Promega, http://www.promega.com/). Thereverse transcription product was subjected to semi-quantitativePCR using TERF1 primers (forward:5’-GATGTCAAGCCCACTAGAG-3’ and revere: 5’-CCTATGATGAAGTCATTAAAAGC-3). The follow-ing conditions were used for PCR: initial denaturation at 94�C(4 min), touchdown cycles of 94�C for 15 sec, 69–63�C for 15 sec and72�C for 30 sec) (one cycle for each temperature) and 20 cycles of94�C for 15 sec, 60�C for 15 sec and 72�C for 30 sec, followed byextension at 72�C for 7 min. A control gene (SGN U346908) wasused as a control in the semi-quantitative PCR using specific prim-ers listed in Exposito-Rodriguez et al. (2008).

Promoter analysis

The 5¢ upstream sequences of the EC-enriched genes were obtainedby nucleotide search at the National Center for BiotechnologyInformation website (http://www.ncbi.nlm.nih.gov/) and BLAST

search at the Sol Genomics Network website (http://solgenom-ics.net/). A search for conserved DNA motifs in the promoter regionwas performed using the MEME suite (http://meme.sdsc.edu/meme/intro.html) and PLACE, a database of plant cis-acting regu-latory DNA elements (http://www.dna.affrc.go.jp/PLACE/) (Higo etal., 1999).

MAN induction in the isolated endosperm

Tomato seeds (cv. First Up) were surface-sterilized in 0.25% NaOClcontaining 0.05% v/v Triton X-100 for 20 min, thoroughly rinsed andimbibed in sterile water. After 2 h, the EC and LE were separatedfrom the seeds. The isolated endosperms were laid with the cutsurface up on filter paper wetted with water or test solutions, andincubated at 28�C in the dark. The test solutions were sterilized byultrafiltration. Seeds imbibed for 14 h were cut transversely intohalves, and the embryonic axes (radical + hypocotyl, Figure 5b)remaining inside the micropylar halves were pushed out by apply-ing gentle pressure with a forceps. Forty axes prepared in this waywere co-incubated with endosperm caps (n = 40) or lateral endo-sperms (n = 5) in 0.15 ml water at 28�C in the dark. For the co-incubation assay, 40 embryonic axes were excised (Figure 5b) from14 h imbibed seeds, and added to EC or LE incubated in 100 ll ofwater.

MAN activity staining

Activity staining of MAN was performed as previously described(Nonogaki et al., 1995).

MAN immunoblot

Proteins were separated by SDS–PAGE using 10% w/v acrylamidegels as previously described (Laemmli, 1970). Native PAGE wasperformed in 7.5% w/v gels as described by Davis (1964), except thatammonium peroxydisulfate was used in place of riboflavin inthe stacking gel. Immunoblotting was performed as previouslydescribed (Nonogaki et al., 1995).

ACKNOWLEDGEMENTS

We are grateful to Yukio Morohashi (Tokyo, Japan) and Yuji Kamiya(RIKEN Plant Science Center, Yokohama, Japan) for helpful guid-ance, discussion and continuous support, and to Taku Demura andSachiko Ooyama (RIKEN Plant Science Center, Yokohama, Japan)and Tammy Chan, Jennifer Coppersmith, Natalya Goloviznina andJing Sun for technical assistance. A.F. and H.N. are grateful to theJacob Blaustein Center for Scientific Cooperation for supportingH.N.’s initial visit to Israel. This work was supported by PolishMinistry of Science and Higher Education grant numberNN301234836 (to W.E.P. and R.J.G.) and US–Israel Bi-national Sci-ence Foundation grant number 2009173 (to A.F. and H.N.).

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Data S1. Details of methods used for GC-MS metabolite profiling.Figure S1. Germination time course of the tomato seeds used forGeneChip analysis.Figure S2. Differential accumulation of metabolites in the endo-sperm cap and lateral endosperm.Figure S3. DNA motifs found in the 5¢ upstream sequence ofSlMAN2.Figure S4. The 5¢ upstream sequence of EXP11.Figure S5. Endosperm cap-specific induction of MAN in intact gib-1mutant seeds by GA.

584 Cristina Martınez-Andujar et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 575–586

Table S1. Genes expressed in 18 h imbibed tomato seeds in a tissue-specific manner (>fivefold changes).Table S2. Genes expressed in 18 h imbibed tomato seeds in a tissue-specific manner (>twofold changes).Table S3. GC-MS-based metabolite profiling data for the endospermcap (EC) and lateral endosperm (LE).Table S4. Representative motifs found in the SlMAN2 promoter.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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