Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection...

Cutin deficiency in the tomato fruit cuticle consistentlyaffects resistance to microbial infection and biomechanicalproperties, but not transpirational water loss

Tal Isaacson1, Dylan K. Kosma2, Antonio J. Matas1, Gregory J. Buda1, Yonghua He1, Bingwu Yu3, Arika Pravitasari4,

James D. Batteas4, Ruth E. Stark3, Matthew A. Jenks2 and Jocelyn K. C. Rose1,*

1Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA,2Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907 USA,3Departments of Chemistry, College of Staten Island and City College of New York, City University of New York Graduate

Center and Institute for Macromolecular Assemblies, New York, NY 10031 USA,4Department of Chemistry, Texas A&M University, College Station, TX 77842-3012, USA

Received 29 May 2009; revised 19 June 2009; accepted 26 June 2009; published online 6 August 2009.*For correspondence (fax +1 607-2555407; e-mail [email protected]).

SUMMARY

Plant cuticles are broadly composed of two major components: polymeric cutin and a mixture of waxes, which

infiltrate the cutin matrix and also accumulate on the surface, forming an epicuticular layer. Although cuticles

are thought to play a number of important physiological roles, with the most important being to restrict water

loss from aerial plant organs, the relative contributions of cutin and waxes to cuticle function are still not well

understood. Tomato (Solanum lycopersicum) fruits provide an attractive experimental system to address this

question as, unlike other model plants such as Arabidopsis, they have a relatively thick astomatous cuticle,

providing a poreless uniform material that is easy to isolate and handle. We identified three tomato mutants,

cutin deficient 1 (cd1), cd2 and cd3, the fruit cuticles of which have a dramatic (95–98%) reduction in cutin

content and substantially altered, but distinctly different, architectures. This cutin deficiency resulted in an

increase in cuticle surface stiffness, and in the proportions of both hydrophilic and multiply bonded polymeric

constituents. Furthermore, our data suggested that there is no correlation between the amount of cutin and

the permeability of the cuticle to water, but that cutin plays an important role in protecting tissues from

microbial infection. The three cd mutations were mapped to different loci, and the cloning of CD2 revealed it

to encode a homeodomain protein, which we propose acts as a key regulator of cutin biosynthesis in tomato

fruit.

Keywords: plant cuticle, cutin, tomato fruit, transpiration, plant–microbe interactions.

INTRODUCTION

The aerial organs of all plants are covered by a thin lipophilic

layer, the cuticle, which defines their boundaries and pro-

vides protection against biotic and abiotic stresses (Jeffree,

2006; Nawrath, 2006). Although the chemical composition of

plant cuticles varies between species, and even between

organs of a single plant, they are all composed of two main

components: cutin, a polymer of mainly C16 and C18 hydroxy

fatty acids or diacids, and waxes (Jeffree, 2006; Nawrath,

2006). The higher order structural organization of these

polymers also shows a similar generic pattern: a thin epi-

cuticular wax layer, beneath which is a mixture of intracu-

ticular waxes and cutin, and within which are embedded

polysaccharides (Jeffree, 2006; Nawrath, 2006). Decades of

research have provided insights into the diversity of cuticle

composition and architecture; however, most studies have

been descriptive, comparative surveys, and relatively little is

known about the biosynthesis, transport and assembly of

cuticular compounds. Similarly, there is a limited under-

standing of the relative importance and differing roles of

waxes and cutin.

The cuticle provides a critical and typically very efficient

barrier against water loss, although it is not completely

impermeable (Burghardt and Riederer, 2006). Determining

the relative contribution of waxes and cutin to water

ª 2009 Cornell University 363Journal compilation ª 2009 Blackwell Publishing Ltd

The Plant Journal (2009) 60, 363–377 doi: 10.1111/j.1365-313X.2009.03969.x

retention has been challenging, largely because of the

difficulties in separating the polymeric constituents to yield

a material that retains its native architecture. Studies using

solvent-extracted, enzymatically isolated cuticles resulted in

the conclusion that water permeability is limited primarily

by waxes (Schonherr, 1976, 1982). However, such chemical

treatments may alter the structure of the remaining matrix,

and should be interpreted cautiously. Alternatively, a num-

ber of Arabidopsis mutants have now been identified with

cuticle phenotypes and a high permeability to water. For

example, the double mutant gpat4/gpat8 has an abnormal

cutin composition, but a normal wax profile (Li et al., 2007),

whereas wax2 shows the opposite trend (Chen et al., 2003;

Goodwin and Jenks, 2005; Rowland et al., 2007), and yet

others, such as bodyguard and fatb-ko are altered in both

cutin and waxes (Bonaventure et al., 2003, 2004; Kurdyukov

et al., 2006a). The phenotypes of these mutants suggest that

both cutin and wax are important in resisting desiccation.

However, it is important to note that in many cases defects in

stomatal morphology have been noted, which could explain

the altered water status.

Similarly, the relative importance of cutin and waxes to

resistance against microbial infection is not well under-

stood. Many fungal pathogens secrete cutinases during

infection (Kolattukudy, 1985); however, none of the knock-

out lines reported to date, in various fungal species, has

provided clear evidence that cutinase plays a direct role in

cuticle penetration (e.g. Yao and Koller, 1995; van Kan et al.,

1997; Reis et al., 2005). Evaluations of Arabidopsis mutants

with cuticle phenotypes have also provided inconclusive, or

even contradictory, information (Xiao et al., 2004; Bessire

et al., 2007; Chassot et al., 2007; Li et al., 2007; Tang et al.,

2007). Here too, interpretation of the results is complicated

by the fact that the stomata of these mutants are suggested

to be defective, and thus might provide a gateway for

pathogen entry. In summary, although some researchers

have concluded that the cuticle does not present a major

barrier to pathogens, this remains an open question.

One of the major obstacles towards functional dissection

of the cuticle has been the identification of a robust research

model. Arabidopsis stems and leaves have played this role

for more than a decade, and many cuticle-associated

mutants have been identified (Nawrath, 2006; Pollard et al.,

2008; Samuels et al., 2008). However, Arabidopsis cuticles

are extremely delicate and contain stomata (Franke et al.,

2005): two major experimental limitations. Tomato (Sola-

num lycopersicum) fruits, on the other hand, offer a system

that is potentially more suitable, as the cuticle is relatively

thick and astomatous, thereby providing a uniform intact

surface (Vogg et al., 2004). Additionally, several studies in

tomato have shown the same molecular pathways are

present in vegetative and reproductive tissues, but are

regulated by different suites of genes (Fraser et al., 1999;

Ronen et al., 2000; Galpaz et al., 2006). This can allow a

phenotype to be exhibited even if the mutation abolishes an

essential function that would result in lethality if the gene

was specific to vegetative tissues.

In this paper we describe three tomato mutant lines [cutin

deficient 1 (cd1), cd2 and cd3], the fruit cuticles of which are

severely cutin deficient, providing an excellent experimental

resource to investigate the contribution of cutin to various

aspects of cuticle function and fruit physiology. We describe

the consequences of the massive reduction in cutin on

cuticle architecture and biomechanical properties, and sus-

ceptibility of the fruit to water loss and microbial infection.

We also report on the mapping of the three mutants and the

cloning of CD2.

RESULTS

Identification of three independent tomato mutant lines

with fruit cuticle-associated phenotypes

Seeds of three mutant lines (cd1, cd2, cd3) were obtained

from the ‘Genes that Make Tomatoes’ collection (Menda

et al., 2004; http://zamir.sgn.cornell.edu/mutants) that were

annotated as having cuticle-associated phenotypes. Their

fruits had a highly glossy phenotype (Figure 1) and a ‘rub-

bery’ surface texture compared with those from the M82

background cultivar, but no visible phenotype in other

organs. The three cd lines were back-crossed with M82 and

then crossed with each other, and the segregation ratios of

the glossy trait in the progeny indicated that the mutations

occur at three different single loci. Moreover, the trait was

inherited in a recessive mode in all three lines, although it is

possible that heterozygous plants have a weaker interme-

diate phenotype.

The cd mutant fruit cuticles have dramatically reduced

cutin content

The composition of isolated fruit cuticles from three key

stages of fruit development (small green, SG; mature green,

MG; and red ripe, RR) was analyzed to determine whether

M82

cd1

cd2

cd3

Figure 1. Two red ripe-stage fruit from the cutin deficient (cd) lines cd1, cd2

and cd3, and their background line M82. Scale bar: 1 cm.

364 Tal Isaacson et al.

ª 2009 Cornell UniversityJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 363–377

the glossy phenotypes could be associated with quantitative

or qualitative differences in wax and/or cutin content. The

most striking difference between M82 and the mutant lines

was in the total level of cutin per unit surface area. At the RR

stage, when the cuticle reaches full maturity, the cutin levels

of M82 fruits were, on average, 985 lg cm)2, whereas

drastically lower quantities were detected in fruits of cd1

(50 lg cm)2), cd3 (38 lg cm)2) and cd2 (15 lg cm)2), corre-

sponding to a reduction of 95, 96 and 98%, respectively

(Table 1). This severe cutin deficiency was detected

throughout development, from the early SG stage

(Table S1). In addition, whereas the levels of cutin per sur-

face area were seen to increase as the fruit developed and

ripened in M82, the levels in the cd fruits remained relatively

constant. Interestingly, the ratios between the main cutin

monomers in the cuticles of the cd lines were similar to

those of M82, with 9(10), 16-dihydroxyhexadecanoic acid

being the most abundant monomer. One exception was

hexadecanoic acid, which was detected at much higher

levels in the cd lines at all developmental stages (Tables 1

and S1).

Explanations for the dramatic reduction in cutin content of

the enzymatically isolated cuticle might include a blockage

in the transport of cutin monomers within the cell, their

export across the plasma membrane, trafficking through the

apoplast to the cell surface or subsequent polymerization,

rather than a disruption in the biosynthetic pathway. To test

this possibility, and investigate whether the mutants accu-

mulate normal levels of cutin monomers, which are then lost

during preparation of isolated cuticles, the lipid profiles of

entire dewaxed MG fruit peels of all four genotypes were

compared. The same trend was observed as for the

enzymatically isolated cuticles, in that fruit peels of all cd

lines contained very low levels of cutin monomers com-

pared with those of M82, and among the three mutants, the

cutin content was highest in cd1 and lowest in cd2 (Table 2).

We note that although the total cutin monomer values

obtained for the peel extracts using acid-catalyzed depoly-

merization (Table 2) were slightly higher than those

obtained with the base-catalyzed depolymerization of the

enzymatically isolated cuticles (Table 1), the same pattern

was apparent.

cd fruits have normal levels of wax, but each line has a

different composition

The wax composition of M82, cd1, cd2 and cd3 was mea-

sured at the RR and MG developmental stages (Tables 3, 4,

S2 and S3). M82 fruit wax at the RR stage was dominated by

alkanes, which comprised about one-third of the total

waxes, and the average total fruit wax coverage was similar

in the mutants (Table 3).

The cd1 fruits showed altered levels of the two main

classes of waxes: alkanes and triterpenoids. A decreased

alkane content was attributed to reductions in the C31, C32

and C33 alkanes, specifically, despite elevated levels of C26–

C29 alkanes (Tables 3 and 4), whereas a higher triterpenoid

content was mainly the result of an increase in all types of

amyrins (a, b and d). The cd2 fruits also showed abnormal

levels of alkanes and triterpenoids, but the trend was

opposite to that observed for cd1. In cd2 alkanes were

elevated, again primarily because of elevated levels of C31,

C32 and C33 alkanes, and amyrin levels were reduced. Only

minor differences were detected in cd3 waxes, other than the

higher levels of unidentified triterpenoids.

The cd fruits show dramatic differences in cuticle

architecture

A range of microscopic techniques were used to determine

whether the remarkably low levels of cutin result in differ-

ences in cuticle morphology and ultrastructure. Scanning

electron microscopy (SEM) and light microscopy of fruit

pericarp cross-sections from RR and MG fruit, respectively,

Table 1 Cutin acid monomer levels (lg cm)2), and percentages, of isolated cuticles from M82, cd1, cd2 and cd3 ripe tomato fruit

M82 (%) cd1 (%) cd2 (%) cd3 (%)

Hexadecanoic 0.9 � 0.3 (0.1) 0.9 � 0.5 (1.8) 1.8 � 0.3 (12.1) 7.9 � 0.8 (20.6)16-OH Hexadecanoic 35 � 1.6 (3.7) 0.8 � 0.4 (1.6) 0.4 � 0 (2.7) 0.6 � 0.2 (1.6)10,16-diOH Hexeadecanoica 760.9 � 55 (80.2) 28.7 � 8.5 (57.6) 7 � 0.3 (47.0) 16.4 � 1.6 (42.7)Hexadecane-1,16-dioic 12 � 1.6 (1.3) 0.5 � 0.1 (1.0) 0.1 � 0 (0.7) 0.8 � 0.3 (2.1)18-OH Octadecanoic 25.6 � 9.3 (2.7) 1.5 � 1 (3.0) 0.3 � 0.1 (2.0) 1.2 � 0.2 (3.1)9,18-diOH Octadecanoic 11.2 � 0.4 (1.2) 1.4 � 0.2 (2.8) 0.3 � 0.1 (2.0) 0.4 � 0 (1.0)9,10,18-triOH Octadecanoic 5.4 � 0.7 (0.6) 2 � 1.4 (4.0) 1.2 � 0.5 (8.1) 0.3 � 0.2 (0.8)9,10,18-triOH Octadecenoicb 5.8 � 1.1 (0.6) 1.7 � 0.8 (3.4) 0 � 0 (0.0) 0.5 � 0.3 (1.3)p-coumaric 7.2 � 0.6 (0.8) 1.3 � 0.3 (2.6) 0.5 � 0.1 (3.4) 1.6 � 0.2 (4.2)m-coumaric 15.6 � 1.9 (1.6) 2 � 0.5 (4.0) 0.5 � 0.2 (3.4) 2.3 � 0.3 (6.0)Not identified 105.2 � 16 (11.1) 9 � 1.5 (18.1) 2.8 � 1.5 (18.8) 6.5 � 0.4 (16.9)Total 984.8 � 48 50 � 8.4 14.9 � 2 38.4 � 3.1

Data represent the means of three replicates � SE.aIsomer composition not determined.bDouble-bond position not determined. Previously reported at D12 carbon (Baker et al., 1982).

Cutin-deficient tomato mutants 365


revealed striking differences between the M82 fruit cuticles

and those of the cd lines (Figure 2a,b, respectively). Tomato

fruits typically have a relatively thick cuticle that surrounds

the epidermal cell layer and extends down the anticlinal

walls, often permeating through the walls of additional

underlying cell layers (Bargel and Neinhuis, 2004; Matas

et al., 2004). Dramatic differences in gross cuticle architec-

ture between the mutants and M82 were evident using either

SEM (Figure 2a) or light microscopy of sections treated with

the lipid-specific stain Sudan IV (Figure 2b). The cd1 cuticle,

although still far less substantial than that of M82, none-

theless penetrated between the cells, forming clear anticlinal

pegs, and in some cases extended into the periclinal walls

underlying the epidermal cell layer, although in no cases

were the epidermal cells surrounded with cuticular material,

as was observed in M82 (Figure 2b). In contrast, the cuticle

of cd3 typically had small protrusions, rather than true

anticlinal pegs, and little staining was detected other than in

the outer epidermal wall. The cd2 mutant showed the most

dramatic phenotype, and the cuticle comprised an extremely

thin, superficial surface layer. The differences in cuticle

architectures were also evaluated using high-resolution

three-dimensional imaging and tomography (see Buda

et al., 2009).

Transmission electron microscopy (TEM) imaging again

emphasized the major reduction in cuticle thickness in the cd

mutants, but also revealed substantial differences in the

ultrastructure of the cuticle and epidermal cell wall

(Figure 3). The M82 cuticle showed a typical gradual transi-

tion from a darker, electron-dense region (the internal

cuticular layer; ICL), to the more opaque external cuticular

layer (ECL). In contrast, the cuticles of all three cd mutants

were characterized by a thin dark layer. Differences were

also seen between the mutants, as although the electron

dense region in the cd2 cuticle had a sharply defined

interface with the underlying wall, those of cd1 and cd3

Table 2 Cutin acid monomer levels (lg cm)2) from epidermal peel of M82, cd1, cd2 and cd3 mature green (MG) fruit

M82 cd1 cd2 cd3

Hexadecanoic 16.3 � 0.5 15.3 � 0.9 13.5 � 0.3 23.9 � 2.516-OH Hexadecanoic 26.8 � 1.6 0.6 � 0.1 0.8 � 0.1 0.4 � 0.010,16-diOH Hexeadecanoica 678.6 � 28.7 59.6 � 4.5 10.3 � 3.1 19.6 � 1.3Hexadecane-1,16-dioic 6.9 � 0.3 0.6 � 0.0 0.3 � 0.0 0.4 � 0.0Octadecanoic 2.9 � 0.1 3.7 � 0.6 1.2 � 0.6 4.0 � 0.5Octadecenoic (C18:1/C18:2/C18:3) 35.9 � 5.2 32.8 � 5.9 30.9 � 5.0 27.5 � 3.018-OH Octadecanoic 223.8 � 9.9 14.2 � 1.5 5.6 � 3.9 16.2 � 1.79,18-diOH Octadecanoic 5.3 � 0.2 1.3 � 0.2 0.2 � 0.1 0.2 � 0.09,10,18-triOH Octadecanoic 10.1 � 0.7 1.0 � 0.1 4.7 � 1.2 1.2 � 0.19,10,18-triOH Octadecenoicb 6.9 � 0.2 1.1 � 0.0 1.5 � 0.3 1.0 � 0.0p-coumaric 3.3 � 0.3 1.3 � 0.3 0.7 � 0.3 1.1 � 0.3m-coumaric 23.3 � 0.3 6.2 � 0.5 1.0 � 0.1 6.4 � 0.4Not Identified 56.0 � 3.5 14.6 � 1.6 6.1 � 0.4 8.9 � 0.5Total 1096.2 � 49.2 152.48 � 9.9 77.0 � 2.5 102.0 � 2.3

As detected after acid-catalyzed (MeOH-HCl) depolymerization. Data represent means of three replicates � SE.aIsomer composition not determined.bDouble-bond position not determined. Previously reported at D12 carbon (Baker et al., 1982).

Table 3 Wax class composition of red ripe M82, cd1, cd2 and cd3 fruit

Total load Acids Aldehydes Alkanols Alkenols Alkenes

M82 8.41 � 0.59 0.51 � 0.07 0.29 � 0.09 0.43 � 0.05 0.32 � 0.14 1.22 � 0.12cd1 8.77 � 0.62 0.84 � 0.11 0.19 � 0.03 0.82 � 0.15 0.17 � 0.07 0.80 � 0.09cd2 7.72 � 0.99 0.60 � 0.11 0.28 � 0.09 0.18 � 0.05 0.03 � 0.01 0.69 � 0.29cd3 8.74 � 0.46 0.51 � 0.03 0.18 � 0.03 0.52 � 0.02 0.17 � 0.05 0.64 � 0.08

Alkanes Iso-Alkanes anteiso-Alkanes Esters Triterpenoids & Sterols Unidentified

M82 2.79 � 0.10 0.38 � 0.07 0.08 � 0.01 N.D. 1.62 � 0.18 0.77 � 0.20cd1 2.50 � 0.29 0.52 � 0.14 0.08 � 0.02 0.06 � 0.01 2.17 � 0.34 0.62 � 0.08cd2 3.94 � 0.47 0.70 � 0.07 0.11 � 0.01 N.D. 0.96 � 0.30 0.23 � 0.03cd3 2.82 � 0.18 0.28 � 0.05 0.09 � 0.02 0.19 � 0.05 2.67 � 0.27 0.67 � 0.33

Values are given as lg cm)2. Data represent the means of three or four replicates � SE; N.D., below the limit of detection.



Table 4 Wax composition of red ripe M82, cd1, cd2 and cd3 fruit

C chain length/compound M82 cd1 cd2 cd3

Alkanoic acids 16 2.5 � 0.5 6.7 � 1.0 4.7 � 1.3 11.1 � 1.918 1.6 � 0.7 4.9 � 1.4 4.5 � 1.4 5.6 � 0.820 2.3 � 0.9 1.3 � 0.7 0.8 � 0.5 0.5 � 0.122 2.0 � 0.2 8.3 � 2.0 6.7 � 2.5 2.7 � 0.224 18.7 � 5.3 19.5 � 3.1 20.6 � 5.5 10.2 � 1.926 7.7 � 2.2 12.8 � 3.4 9.5 � 2.5 5.9 � 0.328 1.2 � 0.4 4.7 � 0.6 1.9 � 0.8 1.7 � 0.230 7.5 � 1.6 5.9 � 0.5 5.1 � 1.5 6.0 � 0.532 8.0 � 0.9 19.3 � 4.6 6.3 � 0.7 7.3 � 1.0

Aldehydes 24 20.4 � 6.5 5.7 � 2.8 20.2 � 6.9 10.1 � 2.126 4.6 � 1.9 1.5 � 0.2 5.5 � 1.9 2.8 � 0.632 3.5 � 0.8 12.0 � 2.9 2.2 � 0.2 4.9 � 0.1

Alkanols 20 0.6 � 0.3 0.3 � 0.1 0.3 � 0.2 0.4 � 0.122 0.5 � 0.3 2.1 � 1.2 0.4 � 0.1 0.5 � 0.224 1.6 � 0.5 4.0 � 2.2 0.6 � 0.2 1.5 � 0.225 1.2 � 0.5 0.5 � 0.1 0.2 � 0.1 0.3 � 0.126 3.6 � 1.2 3.4 � 0.8 1.1 � 0.7 1.1 � 0.427 1.5 � 0.0 4.1 � 1.0 1.2 � 0.6 2.8 � 0.428 4.2 � 1.5 11.3 � 2.0 4.2 � 0.7 7.5 � 0.930 10.7 � 2.3 19.3 � 2.7 5.4 � 1.9 14.9 � 0.832 15.0 � 2.2 31.5 � 7.7 4.9 � 1.4 16.5 � 2.134 3.6 � 0.7 5.4 � 1.9 0.0 � 0.0 6.7 � 0.8

Alkenols 22 1.9 � 1.0 1.6 � 0.9 0.5 � 0.1 1.5 � 0.424 7.1 � 3.4 6.6 � 4.2 0.4 � 0.1 4.0 � 1.526 23.2 � 9.9 9.2 � 2.9 2.1 � 1.1 11.6 � 3.4

Alkanes 23 0.3 � 0.1 0.4 � 0.1 0.3 � 0.1 0.5 � 0.125 2.6 � 0.4 2.5 � 0.4 3.7 � 1.5 1.8 � 0.426 1.7 � 0.3 2.9 � 0.3 2.5 � 0.2 4.3 � 2.227 4.4 � 0.8 10.8 � 0.8 7.7 � 3.0 4.1 � 0.628 8.3 � 2.2 13.1 � 0.6 10.9 � 2.1 11.0 � 1.429 42.8 � 14.4 73.2 � 13.3 68.2 � 19.7 69.9 � 6.430 20.2 � 1.7 21.0 � 2.6 15.2 � 7.3 27.3 � 2.031 140.4 � 14.3 90.1 � 9.1 187.5 � 25.9 118.8 � 10.632 23.3 � 4.0 10.2 � 1.5 36.5 � 3.0 17.7 � 1.633 32.5 � 3.3 21.0 � 6.8 57.5 � 5.8 23.8 � 5.034 2.9 � 0.6 4.4 � 1.5 3.7 � 0.8 3.0 � 0.3

Alkenes 33 105.4 � 9.1 66.6 � 8.2 53.1 � 21.4 51.3 � 6.634 3.7 � 0.5 4.3 � 0.3 3.2 � 2.2 4.0 � 0.635 12.5 � 3.6 9.5 � 0.9 12.3 � 5.1 8.2 � 0.7

iso-Alkanes 29 6.4 � 1.1 11.1 � 3.6 7.3 � 1.9 5.6 � 3.330 4.5 � 1.5 4.7 � 0.9 8.0 � 0.5 3.0 � 0.531 17.4 � 2.2 21.2 � 4.5 44.2 � 5.4 15.4 � 1.932 3.8 � 0.2 3.1 � 0.8 10.8 � 1.1 3.4 � 0.233 5.7 � 4.5 12.3 � 5.3 N.D. 0.8 � 0.5

anteiso-Alkanes 30 4.1 � 1.1 3.1 � 0.8 2.7 � 0.4 3.6 � 0.531 1.0 � 0.3 1.0 � 0.2 1.4 � 0.2 0.8 � 0.232 2.9 � 0.7 3.7 � 0.9 6.4 � 0.9 4.6 � 1.2

Triterpenoids & Sterols a amyrin 39.1 � 5.5 44.6 � 5.8 14.8 � 4.6 32.7 � 4.5b amyrin 27.2 � 3.4 45.5 � 11.8 15.9 � 3.4 46.5 � 9.4d amyrin 52.4 � 5.8 73.8 � 11.5 37.1 � 21.3 51.9 � 6.7multiflorenol 8.3 � 0.5 11.4 � 1.3 3.8 � 1.5 7.5 � 0.9taraxerol 8.3 � 0.5 11.4 � 1.3 3.8 � 1.5 7.5 � 0.9taraxasterol 4.8 � 0.4 5.5 � 1.4 2.0 � 0.7 4.5 � 0.6w taraxasterol 8.5 � 0.1 8.0 � 2.0 2.9 � 1.4 6.3 � 0.9Unidentified 13.6 � 2.2 17.0 � 2.1 15.7 � 3.6 109.6 � 15.2Alkyl Esters N.D. 6.1 � 0.6 N.D. 19.4 � 4.9Unidentified 76.9 � 20.0 61.7 � 7.7 23.4 � 3.2 66.6 � 33.3

Values are given as lg cm)2 · 102. Data represent the means of three or four replicates � SE; N.D., below the limit of detection.



had intermediate layers containing dark fibrillar structures

and more diffuse interfaces.

The cd mutant cuticles exhibit differences in biomechanical

properties

Enzymatically isolated tomato cuticles were imaged using

atomic force microscopy (AFM) in tapping mode (Figure 4),

to investigate the consequences of the cutin deficiency on

the nanobiomechanical properties of the cuticle surface, and

to compare those properties between the three mutants. The

topographic images showed clear differences between

the wild-type and cd samples, which are likely to result from

the compositional differences described above. On a larger

scale (Figure 4a,d,g,j), whereas cellular outlines were visible

for M82, they were less discernable in the mutants, with the

exception of cd1, consistent with their reduced formation of

anticlinal pegs (Figure 2b). The higher resolution images

(Figure 4b,e,h,k) showed amorphous lipid clusters, typical

of fruit cuticular membranes, which were similar in all

samples. Locally, the surface roughness was dominated by

these lipid clusters with a root-mean-square roughness of

approximately 40–50 nm in all four lines (data not shown).

Because of the fragile nature of the cuticles, their biome-

chanical properties were explored by AFM nanoindentation

measurement, by applying a simple Hertzian contact

mechanics model (Round et al., 2000). From this, the elastic

modulus of each of the samples was determined, and sub-

stantial differences were seen between the M82 and cd

samples, where the M82 cuticle had a far lower Young’s

modulus value than those of the mutants (Table 5). To

compare the relative contributions of the cuticular waxes

and cutin to cuticle biomechanical properties, Soxhlet

extraction was used to dewax the tomato cuticular samples,

and the mechanics were measured by AFM nanoindenta-

tion, as described above. The dewaxed surfaces exhibited a

smooth texture (Figure 4c,f,i,l), and the Young’s modulus

values of the cd cuticles were similar to those of the M82

cuticle (Table 5) and earlier studies of isolated dewaxed to-

M82

cd1

cd2

cd3

EC

EC

Col

EC

Col

EC

Col

Col

CMAP

AP

(b)

(a)

cd2 cd3

M82 cd1

CMEC

Col Col

ColCol

ECEC

EC

Figure 2. Images of breaker-stage fruit pericarp sections from M82 and cd

lines obtained using: (a) scanning electron microscopy; and (b) light

microscopy showing cuticles stained with Sudan IV. Scale bars: (a) 10 lm;

(b) 20 lm.

Abbreviations: AP, anticlinal peg; CM, cuticular membrane; Col, collenchyma

cell; EC, epidermal cell.

M82 cd1

cd2

cd3

ECL

ICL

Cyt

Cyt

Cyt

CM

CM

CM

CM

PCWPCW

PCW

PCW

Figure 3. Transmission electron microscopy images of red ripe-stage fruit

pericarp sections from M82 and cd lines. Scale bars: 2 lm.

Abbreviations: CL, cuticular membrane; Cyt, cytoplasm; ECL, external cutic-

ular layer; ICL, internal cuticular layer; PCW, polysaccharide cell wall.



mato fruit cuticles (Round et al., 2000). This suggests that

the differences in nanobiomechanical properties of the

cuticle are not caused by significant differences in the make-

up of the polymerized cutin matrix, but rather by its interplay

with the varying lipid content.

The cd mutant fruit show increased susceptibility to

microbial infection, but not necessarily increased water loss

Despite the fact that the cd mutant cuticles were shown to be

severely cutin deficient, and to have dramatically perturbed

and distinctly different architectures, the fruits appeared

remarkably normal, other than having an increased glossi-

ness (Figure 1). Under controlled glasshouse growth con-

ditions, no differences were seen in fruit ripening or other

aspects of plant physiology, such as susceptibility to wilting.

We therefore investigated whether some of the cuticle

functions associated with protection against biotic and

abiotic stresses were compromised.

An important role of the cuticle is to limit water loss from

plant tissues, and so to determine whether the cd fruit show

altered transpiration, the weights and external appearance

(d) (e) (f)

(a) (b) (c)

(g) (h) (i)

(j) (k) (l)

Figure 4. Atomic force microscopy tapping

mode topographical images of cuticles from

red ripe fruit of M82 (a–c), cd1 (d–f), cd2 (g–i)

and cd3 (j–l), with Dz values of 6089, 115, 65,

2326, 199, 95, 6836, 157, 128, 2762, 219 and

149 nm, respectively. Samples were analyzed at

magnifications of 70 x 70 lm2 (a, d, g and j), 2 x

2 lm2 (b, e, h and k) and 2 x 2 lm2, after removal

of epicuticular waxes (c, f, i and l).

Table 5 Surface elastic modulus (Young’s modulus) valuesfor isolated cuticles from M82, cd1, cd2 and cd3 fruit, taken atdifferent locations on the surface, before and after the removal ofwaxes

Cuticles with wax Dewaxed cuticles

Low (MPa) High (MPa) Low (MPa) High (MPa)

M82 40 � 9 37 � 5 53 � 8 57 � 15cd1 240 � 33 187 � 55 22 � 3 19 � 2cd2 303 � 41 234 � 47 52 � 23 47 � 14cd3 162 � 26 195 � 26 46 � 3 49 � 2

The low value corresponds to the center of the surface depressions,whereas the high value corresponds to the ridges of the cellularoutlines on the surface.



of detached ripe fruits held at room temperature (21�C) were

evaluated over a 10-week period (Figure 5). cd1 fruits

showed rapid symptoms of desiccation within a few days

of harvest, and by 3 weeks were notably shriveled, whereas

those of cd2 and cd3 appeared similar to M82 fruits

(Figure 5a). The phenotypes correlated with water loss, as

cd1 fruits showed approximately twofold greater sustained

water loss rates than those of M82. The values for cd2 and

cd3 were only marginally greater than the wild type

(Figure 5b), and it was notable that they were also different

from each other. To further test the permeability of the fruit

cuticles to aqueous solutions, drops of Toluidine Blue

solution were placed on the surface for up to 8 hours, as

was reported in a study of the other cuticle mutants (Tanaka

et al., 2004). However, no staining of the underlying tissue

was seen in fruits from M82 or any of the cd lines (data not

shown).

Another important function of the cuticle is to protect the

plant from pathogens: to test the susceptibility of the cd

fruits to opportunistic microbes, ripe fruit were stored at

room temperature in high humidity conditions, and infec-

tion rates were recorded. Fruits of the cd lines started to

show symptoms of infection after 12 days of storage, and

most were infected by 25 days (Table 6). The first signs of

infection on any M82 fruit were not apparent until 22 days,

and by 30 days only 13% of M82 fruits were infected

(Table 6), with most fruit showing no symptoms even after

10 additional days. To test pathogen–fruit interactions in a

more controlled way, and using a tomato-associated path-

ogen of commercial importance, ripe M82 and mutant fruits

were inoculated with spores of the fungus Botrytis cinerea,

the causal agent of gray mold. Droplets of inoculum were

applied ectopically to the fruits, which were then stored at

room temperature in high humidity conditions. No infection

or any other symptoms were observed with M82 fruits,

whereas some fruits of each cd line became infected,

although cd1 fruits (15% infected) were far less susceptible

than those of cd2 or cd3 (38 and 56%, respectively) (Table 6).

NMR analysis of cd fruit cuticles suggests differences in the

organization of hydrophobic and hydrophilic domains

One factor that might contribute to the differences in

dehydration rate among the tomato fruit lines is the relative

M8 2

cd 1

cd 2

cd 3

0

10

20

30

40

50

60

70

0 10 20 30 40 50 60 70

Wei

ght l

oss

(%)

M8 2

cd 1

cd 2

cd 3

Days post-harvest

(a)

(b)

Figure 5. Analysis of transpirational water loss from M82 and cd fruit.

(a) Sets of two ripe fruits of M82, cd1, cd2 and cd3 3 weeks following fruit

detachment; (b) percentage weight loss of M82 and cd fruits over a 10-week

period.

Table 6 Percentage of fruits infected by opportunistic pathogens after harvest and storage at 100% humidity at room temperature, andpercentage of fruits that were infected by Botrytis cinerea 7 days after inoculation

Line

Opportunistic microbial infection B. cinerea inoculation

No. offruit

% infectedby 20 daysfrom harvest

% infectedby 25 daysfrom harvest

% infected by30 days fromharvest

No. offruit

% of infectedfruits

M82 30 0 (0) 3 (1) 13 (4) 34 0 (0)cd1 17 41 (7) 53 (9) 76 (13) 34 15 (5)cd2 27 37 (10) 70 (19) 93 (25) 34 38 (13)cd3 13 38 (5) 62 (8) 69 (9) 34 56 (19)

The numbers of fruits are given in parentheses.



distribution and interactions between hydrophobic and

hydrophilic domains. To test this hypothesis, the abundance

of potentially important functional groups and polymeric

interactions in isolated dewaxed cuticles from RR stage M82

and cd fruits were analyzed by NMR spectroscopy.

Typical solid-state CPMAS 13C NMR spectra for M82 and

mutant (cd2) tomato cutin are shown in Figure 6, illustrating

the ability to resolve chemically distinct carbon moieties

[C=O, aromatics/alkenes, CHO, CH2O and (CH2)n], and to

discern clear differences in relative signal intensities for

long-chain aliphatic structures (A, 30 ppm) and various

oxygenated carbon moieties (O, 50–100 ppm). High-fidelity

DPMAS experiments provided integrated signal intensity

ratios between these carbon-containing functional groups

(Figure 6). Two NMR spectral comparisons were made for

enzymatically isolated cuticles. First, the ratio of oxygenated

aliphatic carbons (CHO + CH2O) to long-chain aliphatics

[(CH2)n] (O/A; Figure 6b) was measured, and was found to

be elevated by more than twofold for each mutant: an

observation that is attributable to a large percentage of both

CHO and CH2O groups, compared with the total carbon

signal (not shown). For cd2, the O/A ratio was also increased

by an anomalously small percentage of aliphatics (not

shown). Second, this trend was similar for carbonyl groups

(C=O) to aliphatic ratios, which were higher in the mutants

than in M82, but were similar between the mutants (Fig-

ure 6c). These analyses also showed an increase in multiply

bonded or cross-linked structural elements with respect to

aliphatic chains (Figure 6d).

Finally, our observations of anomalies in chemical com-

position for the cd lines were extended to examine possible

consequences for molecular organization. For instance, we

questioned whether aliphatic chains with protruding hydro-

xyl groups or midchain cross links might pack less effi-

ciently, or if well-mixed cutin and wax constituents could

form a flexible, resilient composite. The fractions of liquid-

like alkyl chains for both enzymatically isolated M82 tomato

fruit cuticles and dewaxed cutins (Figure 6e) were at least

threefold greater in the mutant cuticles compared with M82.

For the cuticles, the greater average flexibility of the cutin

and wax alkyl chains in the cd samples (from NMR)

contrasted with enhanced surface stiffness (from AFM), a

reasonable result because the latter nanoindentation mea-

surements sense the mechanical resistance of the waxy

cuticular overlayer, which is most abundant in the mutants.

The three cd mutations map to different loci, and positional

cloning of CD2 suggests it encodes a homeodomain protein

The three mutants (cd1, cd2 and cd3) were each crossed with

the wild tomato species Solanum pimpinellifolium (LA1589),

and F2 plants were genotyped using genetic markers across

the tomato genome, whereas fruits from the F2 populations

were phenotyped for water loss rates and cuticle thickness.

CD1 was mapped to the top end of chromosome 11, CD2

was mapped to chromosome 1 and CD3 was mapped to

chromosome 8 (Figure 7a). Further fine mapping of cd2

localized it within a single BAC (SL_MboI0026F20), and

13C chemical shift (ppm)

M82 cd1c d2 cd3

(CH

O +

CH

2O)/

alip

hatic

Car

bony

l/alip

hatic

Mul

tiply

bon

ded/

alip

hatic

Fra

ctio

n of

mob

ile(C

H2)

n0

0.2

0.4

0.6

0

0.1

0.2

0

0.2

0.4

0.6

0

0.1

0.2

0.3

M82

cd2Aromatics/Alkenes

CHO

CH2O

(CH2)n

COO

200 160 120 80 40 0

(a)

(b)

(c)

(d)

(e)

Figure 6. NMR analysis of isolated red ripe fruit cuticles from M82 and the cd

lines.

(a) 75-MHz CPMAS 13C NMR spectra and peak assignments for tomato cutins

from M82 and cd2 fruits; 13C NMR peak ratios of (b) oxygenated aliphatic

carbons (CHO + CH2O); (c) carbonyl groups (C=O); (d) or multiply bonded to

long-chain aliphatics [(CH2)n]; (e), variation in the fraction of liquid-like

aliphatic chains (13C NMR resonances at 20–40 ppm), for cutins (solid) and

cuticles (empty) of M82 and cd fruits.



finally localized it to a region of approximately 8 kb

(Figure 7a). This area contained only one predicted gene,

which was annotated as a homolog of the Arabidopsis gene

ANTHOCYANINLESS2 (ANL2), a homeodomain gene that

has been associated with anthocyanin distribution in epi-

dermal cells (Kubo et al., 1999) (Figure 7b). A comparison of

the sequence of the 8-kb regions from M82 and cd2 revealed

only one nucleotide difference: a G fi A substitution that is

predicted to cause an amino acid substitution of a conserved

glycine to an arginine at position 736 of the protein (Fig-

ure 7b,c). The CD2 protein contains two predicted functional

domains: a ‘homeodomain’ DNA binding domain and a

‘StAR (steroidogenic acute regulatory protein) related lipid-

transfer (START)’ domain, the function of which in plants is

not clear, although it has been suggested to be involved in

the binding of regulatory lipids/sterols (Schrick et al., 2004).

In the Arabidopsis genome, 16 genes encode proteins with a

similar structure, forming the class-IV homeodomain-

leucine zipper family, or HD-GL2 (Nakamura et al., 2006).

Interestingly, the expression of many of these genes, and the

functions of the corresponding proteins, has been associ-

ated with the development of cells in the outer layers of the

plant (Nakamura et al., 2006). Examples include ANTHO-

CYANINLESS2 (ANL2), which was connected to anthocyanin

distribution in epidermal and subepidermal cells (Kubo

et al., 1999), and GLABRA2 (GL2), which is involved in

determining the developmental fate of trichomes and non-

root hair cells (Di Cristina et al., 1996; Rerie et al., 1994).

Moreover, promoter-GUS expression analyses of genes

from this family found that although each is expressed in

different organs, most are expressed in the outermost cell

layers (Nakamura et al., 2006). It has been suggested that

members in the HD-GL2 family regulate transcription, and,

accordingly, nuclear localization has been shown for GL2

(Szymanski et al., 1998), and DNA binding sites were found

for three members of the family: GL2 (Ohashi et al., 2003),

MERISTEM LAYER1 (ATML1; Abe et al., 2001) and PROTO-

DERMAL FACTOR2 (PDF2; Abe et al., 2003). The functions of

START domains are less known, and although they have

been shown to bind cholesterol, lutein or ceramide in

mammals, the amino acid sequences of these domains are

not sufficiently conserved with those of plants to allow for

the prediction of ligand(s) (Schrick et al., 2004). The cd2

mutation is in the C terminus of the protein, not in the

homeodomain or START domain (Figure 7c), which pro-

vides no insight into the consequences of the mutation, but

we note that the mutated glycine is completely conserved in

all HD-GL2 family members from Arabidopsis and homologs

from numerous plant species.

DISCUSSION

Three tomato mutants with a glossy fruit phenotype were

identified, and allelic tests showed that each line carries a

mutation in a different genetic locus. An analysis of the

chemical composition of fruit cuticle from the three lines

revealed a dramatic reduction in cutin content (Table 1),

resulting in extremely thin cuticles with abnormal ultra-

structures (Figures 2 and 3), and a greater surface stiffness.

NMR analysis further suggested altered molecular organi-

zation and packing, as increases in oxygen-linked, poten-

tially cross-linked, and multiply bonded carbon moieties of

the mutant cuticle constituents were observed. To our

knowledge, such a large decrease in cutin content has not

been previously described in any other mutant. We suspect

that such a reduction of cutin content in vegetative tissues

would result in lethality, or a very severe phenotype, illus-

trating the potential of the tomato fruit as a model system to

study cuticle biosynthesis, assembly and function.

The influence of the cutin matrix on cuticular transpiration

Despite the striking reduction in cutin content, the water loss

rates of cd2 and cd3 fruits were not very different from M82.

It is important to note that although the cutin content of the

fruit cuticle of all mutants was dramatically reduced, the

residual cutin was still sufficient to form a visible cuticle.

For comparison, the cutin content of the cd2 fruit cuticle,

the mutant with the lowest levels, was approximately

Chromosome 1 Chromosome 11Chromosome 8

C2_At4g11560

(a)

(b)

T1162

C2_At4g01210

TG59

BA

C M

boI026F20

TG59

~8 K

b cd2 TG294

cd3

TG497

C2_At5g04420

cd1

Gly Arg

126-183 343-558 736

HD START

Figure 7. Mapping of the cd mutants. (a) cd2 was mapped to chromosome 1

between CAPS markers T1162 and C2_At4g01210, cd3 to chromosome 8

between markers C2_At4g11560 and TG294, and cd1 to chromosome 11

between TG497 and C2_At5g04420. Fine mapping of cd2 on chromosome 1

was performed with the CAPS markers indicated on the map. BAC

MboI026F20 was selected and cd2 was mapped to a DNA fragment of

approximately 8 kb; (b) Schematic representation of CD2 showing domain

organization and the location of the cd2 mutation. aa positions are indicated

below. The total predicted length of CD2 is 821 aa.



15 lg cm)2, which is <2% of M82 fruit, and yet is still much

more than the values reported for Arabidopsis stems or

leaves (0.2–3.3 lg cm)2) (Xiao et al., 2004; Franke et al.,

2005; Kurdyukov et al., 2006b; Bird et al., 2007; Panikashvili

et al., 2007). This suggests that either the cutin matrix does

not play a central role in limiting water loss, inferring that

waxes are the primary barrier, or that the residual level was

sufficient, and that tomato fruit have far more cutin than is

necessary for resisting desiccation. Indeed, of the three

mutants, cd1 fruits showed both the highest water loss rate

and level of cutin, further demonstrating the lack of corre-

lation between cutin content and water permeability. Images

of cd1 fruit cuticles obtained by light microscopy, electron

microscopy and AFM did not reveal any microfissures, as

has been described for a more rapidly desiccating Cwp1

tomato genotype (Hovav et al., 2007), or other structural

disruptions that might explain this phenotype.

Another explanation for the high water loss rates of cd1

fruits might be the abnormal wax composition. Previous

studies have not identified a clear relationship between

wax composition and water permeability (Riederer and

Schneider, 1990; Hauke and Schreiber, 1998; Kissinger

et al., 2005), but characterization of the lecer6 tomato

mutant, which is deficient in very-long-chain fatty acid b-

ketoacyl-CoA synthase (Millar et al., 1999) suggested such

a correlation (Leide et al., 2007). lecer6 fruit cuticles have

elevated levels of amyrins but lower levels of alkanes of

chain length >30, compared with the wild type, and higher

water loss rates were reported in the mutant fruit (Vogg

et al., 2004; Leide et al., 2007). Furthermore, a negative

correlation was found between water permeance and the

levels of cuticular alkanes. Interestingly, RR-stage cd1 fruit

cuticles have similarly altered levels of amyrins and

alkanes of chain length >30 (Tables 3 and 4). Leide et al.

(2007) referred to a model in which the waxes are

organized as islands of crystalline platelets in an amor-

phous material (Geyer and Schonherr, 1990; Reynhardt

and Riederer, 1994; Riederer and Schreiber, 1995; Kosma

and Jenks, 2007), and water molecules move through the

amorphous matrix but not through the crystalline regions.

Furthermore, triterpenoids are predicted to be localized in

the amorphous matrix, and alkanes are predicted to be

localized in the crystalline regions. Hence, a reduction in

alkanes, accompanied by an increase in triterpenoids,

would result in an increase in the amorphous portion of

the wax, and a decrease in the crystals, potentially leading

to increased water flux. This model is supported by our

results, as is the idea that cutin does not function as a

significant hydrophobic barrier, but rather provides a

framework into which the intracuticular wax molecules

are deposited, providing a structure that can effectively

restrict water movement. We note that, unlike cd1, the

lecer6 has a thicker cuticle than its background genotype

(Vogg et al., 2004; Leide et al., 2007), and the two muta-

tions map to different chromosomes (data not shown), so

we exclude the possibility that cd1 is altered in the LeCER6

gene.

Cutin and plant pathogen resistance

Fruits of the cd lines showed an increased susceptibility to

opportunistic saprophytes, and to B. cinerea, under con-

trolled inoculation conditions (Table 6). Susceptibility to

pathogens has been shown to be influenced by the rough-

ness of the surface of a plant organ (Zabka et al., 2008);

however, the roughness of the cd cuticles was shown by

AFM to be similar to that of M82 (Figure 4b,e,h,k and data

not shown), so it is less likely that the increased suscepti-

bility of their fruit was caused by changes in surface

roughness as a result of the different wax composition.

Rather, the data suggest that the increased susceptibility

resulted from a massive reduction in cutin content. This

phenomenon may, at least in part, explain the relatively high

thickness of the tomato cuticle, and the relatively low

occurrence of microbial infection of nutritionally rich ripe

fruits under normal conditions.

The contributions of cutin and wax content to cuticle

biomechanical properties

Biomechanical studies of isolated whole cuticles from to-

mato fruits (Lopez-Casado et al., 2007) have suggested that

cutin imparts the cuticle with its viscoelastic behavior (low

elastic modulus and high strain values). In its absence, the

cuticle is predicted to be stiffer and less elastic. The cuticles

of the cd lines were too fragile to perform macro-scale bio-

mechanical tests, but nano-scale AFM analyses showed that

the membrane is harder in the presence of waxes, although

dewaxed samples from the mutants and M82 showed sim-

ilar biomechanical behavior. Based on the compositional

differences between the mutants and M82, the observed

changes in biomechanical behavior can most likely be

attributed to the relative cutin content, as well as the wax

composition of the samples. The high values of the surface

modulus for the cd mutants correlated with their extremely

low cutin content, and with the greater proportions of

CHO + CH2O and carbonyl species, compared with aliphat-

ics (Figure 6). In this scenario, the cuticles with the greater

proportions of these species would be more crystalline, and

would thus exhibit larger moduli.

CONCLUSIONS

The data presented here help resolve some of the questions

regarding the contribution of cutin to cuticle properties and

function; however, the identification of these cutin-deficient

mutants also provides an opportunity to better understand

cutin biosynthesis. We found cd2 to be mutated in a gene

that is presumed to encode a transcription factor of the HD-

GL2 family. This, together with the observation that most

HD-GL2 genes are predominantly expressed in surface cell



layers, supports a model wherein CD2 is a key regulator of

cutin biosynthesis in tomato fruit, and this will provide a

platform for future studies. We also anticipate that the

identification of the mutated loci for cd1 and cd3 will reveal

two more genes that are essential in this poorly understood

pathway, and genetic mapping is already underway.

EXPERIMENTAL PROCEDURES

Plant materials

Seeds of the tomato (S. lycopersicum) lines described here [wild-type cultivar M82, and the derived mutated lines e4247m1 (cd1),e4393m2 (cd2) and n3056m1 (cd3)] were obtained from the Genesthat Make Tomatoes germplasm collection (Menda et al., 2004;http://zamir.sgn.cornell.edu/mutants). Plants were grown in aglasshouse in Ithaca, New York, under 16-h of light and 8-h of dark,using standard practices. Fruits were harvested at the SG develop-mental stage when the fruit length reached 2–3 cm, at the MG stagewhen fruits reached full size, but were still green, and at the RR stage4–5 days after the color break. F2 mapping populations were grownin the field (Freeville NY, summer 2007).

Genetic mapping

Three F2 mapping populations were created by crossing each cdmutant to the wild species S. pimpinellifolium (accession LA1589).Genomic DNA was extracted from a young leaf of each seedlingaccording to the method described by Fulton et al. (1995). For cd1,94 plants of the F2 population were phenotyped for fruit water loss:21 plants that exhibited high water loss rates and nine plants thatexhibited low water loss rates were genotyped for 48 markersacross the tomato genome. For cd2, fruit cuticles from 94 plantswere phenotyped and the plants were genotyped for 60 markersacross the genome. For cd3, cuticles from 47 F2 plants were phe-notyped and 13 plants were found to have the cd3 phenotype. Ofthese, 11 plants as well as an additional seven plants that had nor-mal (S. pimpinellifolium) cuticles were genotyped for 48 markersacross the genome. Genotype and phenotype data were analyzedusing QGENE software (Nelson, 1997). For the fine mapping of cd2,940 plants of the cd2 F2 population were screened for recombina-tion events between CAPS markers T1162 (position 1.059) andC2_At4g01210 (position 1.081). Fine mapping located the cd2mutation on a single BAC (SL_MboI0026F20). Using the BACsequence (GenBank Accession AC217915), new CAPS markers weredesigned and cd2 was mapped to a region of approximately 8 kb.

Water loss measurements

A total of 41–48 fruits of each line were picked at the RR stage, andwere stored at room temperature for 10 weeks. Fruit weight wasrecorded every week, and water loss was calculated as a percentageof weight loss.

Cutin monomer and wax analysis

For compositional analysis, cuticles were enzymatically isolated(Schonherr and Riederer, 1986) from tomato fruit exocarp discs byincubation at 35�C in 2% (v/v) pectinase (EC 3.2.1.15; Sigma-Aldrich,http://www.sigmaaldrich.com), 0.1% (w/v) cellulase (EC 3.2.1.4; TCIAmerica, http://www.tciamerica.com) in 20 mM citric acid, pH 3.7,containing 0.001% (w/v) phenylmercuric nitrate to prevent microbialgrowth. Isolated cuticles were washed in an acetone series andrefluxed for 24 h in a Soxhlet apparatus with chloroform:methanol(1:1) and 50 mg L)1 butylated hydroxytoluene. After washing with

methanol to remove chloroform, cuticle composition was analyzedas described in Saladie et al. (2007).

The cutin monomer content of epidermal peels, including intra-cellular lipids, was analyzed based on a depolymerization method(Franke et al., 2005), with modification. Briefly, MG fruits weredewaxed by a 1-min submersion in chloroform. Epidermal tissueswere peeled from the fruit equator and 0.6–1-cm2 disks werepunched out, blotted dry and subjected directly to transmethylationreactions, consisting of 6.5 ml of 2.7 N methanolic hydrochloride(MeOH-HCl) containing 7% (v/v) methyl acetate at 60�C. Methylheptadecanoate was used as an internal standard. After 16 h,reactions were allowed to cool to room temperature, and wereterminated by adding 6 ml of saturated, aqueous NaCl, followed bytwo extractions (10 ml) with distilled dichloromethane to removemethyl ester monomers (Bonaventure et al., 2004). The organicphase was washed three times with 0.9% aqueous NaCl (w/v), thendried with 2,2-dimethoxypropane under nitrogen gas. Derivatiza-tion and subsequent GC analysis was performed as describedabove for cutin monomer analysis. All values represent the mean offour replicates. The presence of C18:1, C18:2 and C18:3 fatty acidsindicated that intracellular lipids and potential intracellular cutinwere not substantially removed by the 1-min chloroform submer-sion used for wax extraction.

Cuticular wax analysis was performed as described in Saladieet al. (2007) with slight modification. GC was carried out withtemperature-programmed automatic injection at 80�C, then held for2 min at 80�C, raised by 40�C min)1 to 200�C, held for 2 min at200�C, raised by 3�C min)1 to 320�C and held at 320�C for 30 min.Triterpenoids were putatively identified based on spectra describedby Bauer (2002).

Pathogen treatments

Botrytis cinerea (isolate BCT-862) isolated from tomato fruit wasgrown on potato dextrose agar (PDA) plates until they reached astate of heavy sporulation. Inoculum was prepared by gently rinsingthe plates with water, and inoculum droplets containing approxi-mately 2000 spores were applied ectopically to a group of 8–10 RRfruits of the M82 and cd lines. Fruits were stored at room temper-ature in a sealed container at saturating humidity. To assessopportunistic microbial infection, 8–10 RR fruit from the M82 and cdlines were gently rinsed with water, dried and stored as describedabove.

Scanning electron microscopy (SEM)

Cubes of RR-stage fruit pericarp were frozen in liquid propane,freeze dried, mounted on aluminum stubs and sputter coated withgold : palladium 60:40 using a Denton Desk II coater (to a metal coatthickness of approximately 10 nm). Microscopic observations at theedges of the tissue break-line were made with a Leica 440 scanningelectron microscope using an accelerating voltage of 5 kV (Leica,http://www.leica.com).

Transmission electron microscopy (TEM)

Pieces of pericarp (cubes of approximately 5-mm per side) wereexcised from the equator of three replicate fruits of each genotypeat the SG, MG and RR stages. Samples were prepared for TEMusing microwave technology (oven model 3450; Ted Pella, http://www.tedpella.com) under vacuum, to improve cuticle fixation.Samples were fixed in primary fixative containing 2.5% (v/v) glutar-aldehyde and 2% (v/v) formaldehyde in 0.05 M phosphate buffer,pH 6.8 (PB) (Karnovsky, 1965), and were then subjected to two 40-secmicrowave treatments using the low power setting, allowing 3 minbetween treatments. Samples were washed with PB and post-fixed in



1% (v/v) osmium tetroxide and 1.5% potassium ferricyanide in water,followed by one 40-sec microwave treatment with vacuum, asdescribed above, for osmium fixation. Samples were washed withwater (2 · 40-sec microwave treatments) and then dehydratedthrough a gradient ethanol series (40 sec for each change). Sampleswere infiltrated with propylene oxide-Spurr resin mixtures andpolymerized in 100% resin for 48 h at 60�C. Ultrathin sections (80–100 nm) were stained with 2% uranyl acetate and Reynold’s leadcitrate, and were viewed with a Philips CM-10 Biotwin transmissionelectron microscope (FEI Co., http://www.fei.com). Images wereanalyzed using IMAGEJ software (Abramoff et al., 2004).

Light microscopy

Light microscopy and staining were conducted as described in Budaet al. (2009), accompanying paper).

Enzymatic cuticle isolation

To obtain isolated cuticles for AFM and NMR analyses, fruit pericarpsections were incubated in a mixture of 40 U ml)1 cellulase(EC 3.2.1.4; TCI America), 10 U ml)1 pectinase (EC 3.2.1.15; TCIAmerica) in sodium citrate buffer (50 mM, pH 4.0) and 1 mM NaN3

for 7–10 days at 32�C. Cuticles were then washed in water incubatedagain in fresh enzymatic buffer until clean, and were then washed inwater again and dried at room temperature.

Atomic force microscopy (AFM)

Surface topography and nanoindentation properties of isolatedcuticular samples were determined using a commercial AFM(Alpha300 S; WITec, http://www.witec.de) that includes a confocal(Raman and fluorescence) microscope. The enzymatically isolatedcuticle samples were attached to glass slides with double-sided tapeand imaged under ambient conditions (20 � 1�C) at approximately45% relative humidity. Images were collected in both tapping modeand contact mode. Tapping mode images were collected usingultrasharp silicon AFM tips (VISTA probes; NanoScience Instru-ments, http://www.nanoscience.com), with nominal tip radii of<10 nm and a lever resonance frequency of approximately 300 kHz(force constant of approximately 40 N m)1). To determine the rep-resentative roughness of the surface, 0.5 · 0.5 lm2 AFM topo-graphic images were taken at random locations on the cuticlesurface, and the root-mean-square roughness of the surfaces werethen determined.

For contact mode imaging and nanoindentation experiments, Sitips were used (Mikromasch, http://www.spmtips.com). As thespring constants and tip dimension can vary between probes, thetip size and lever spring constant were determined experimentallyfor each cantilever tip used. The normal force constants of the leverswere determined based on the measurements of the cantileverspring constant and a standardized lever of a known spring constant(Tortonese and Kirk, 1997), and were found to exhibit a typical forceconstant of 0.35 � 0.06 N m)1. The tip shape and radius of curva-ture were approximated by the imaging of an Sr3TiO3 (305) singlecrystal (Sheiko et al., 1993), and were found to exhibit a typicalradius of curvature of 68 � 9 nm. AFM images were collected withscan sizes ranging between 2 · 2 lm2 and 70 · 70 lm2, at a typicalscan rate of 0.2 Hz.

Measurements of the surface mechanical properties wereobtained by nanoindentation measurements. Cuticle samples werefirst imaged in contact mode, and then their nanomechanicalproperties were evaluated by indentation, with reference to anideally hard surface: in this case a cleaned Si(100) wafer. Todetermine the extent of tip indentation into the cuticular sample, theslope of the force–distance spectrum during retraction was taken

while the tip was in contact, and the indentation was determined ata fixed load of 0.5 nN for each sample. Based on these indentationexperiments, the Young’s modulus of the cuticular surface could bedetermined assuming a Hertzian contact (Round et al., 2000).Measurements were made in at least 10 different locations both inthe surface depression on the cuticle and on the ridges thatcorresponded to the boundaries of the underlying cells. Soxhletextraction (Pacchiano et al., 1993) was used to dewax the tomatocuticular samples, which involved successive refluxing (approxi-mately 6 h for each solvent) with hot methanol, dichloromethaneand tetrahydrofuran (THF).

NMR analysis

Intact or dewaxed enzymatically isolated cuticles (7–10 mg ofpowdered samples) were analyzed using NMR spectroscopy. Toobtain quantitative estimates of the various carbon types, inte-grated areas were measured from direct polarization-magic-anglespinning (DPMAS) 13C NMR (Duer, 2004) using a Varian 600-MHzDirectDrive NMR System (http://www.varianinc.com), with a recycletime of 100 sec between successive acquisitions. Similar composi-tional trends for replicate tomato samples and error limits of 3–18%in repeated experiments with M82 and cd lines were establishedusing cross polarization (CPMAS) 13C NMR spectra obtained at both300 MHz (on a Varian Unityplus spectrometer) and 600 MHz (NMRSystem). These latter experiments used a 2-ms mixing time and a2-sec recycle time in order to ensure consistent cross polarizationof the carbons and spin relaxation of the 1H nuclei, betweenacquisitions, respectively. DPMAS and CPMAS NMR spectra wereacquired with a comparable signal-to-noise ratio of >50:1 for the(CH2)n groups. The two-pulse phase-modulation (TPPM) decouplingmethod and a 1H field of 50 kHz were used, unless otherwise stated.The typical spinning speeds were 8 kHz.

The proportions of liquid-like and solid-like aliphatic chaincarbons were estimated by comparing integrated areas of thespectral region between 20 and 40 ppm for DPMAS spectraacquired with low-power decoupling (1H field strength correspond-ing to 5 kHz, mobile carbons only) and high-power decoupling (1Hfield strength corresponding to 50 kHz, all carbons) during NMRsignal acquisition. To determine 1H rotating-frame relaxation times,T1q(H), the decay of the carbon signal intensity, was monitored withincreasing 13C–1H contact time (s) in a CPMAS experiment (Schaeferand Stejskal, 1979).

ACKNOWLEDGEMENTS

The authors would like to thank Dr J. Lorbeer for providing theB. cinerea culture, Dr D. Zamir for tomato germplasm, and Dr S.Tanksley and Emma Flemmig for assistance with the CD2 mapping.The project was supported by the National Research Initiative ofthe USDA Cooperative State Research, Education and ExtensionService, grant number #2006-35304-17323; by the CUAES HatchProject, NYC-184485; by the United States–Israel Binational ScienceFoundation (award #2005168); and by NSF grants #MCB-0134705,MCB-0843627 and DBI-0606595. TI was supported by a postdoctoralaward no. FI-375-2005 from the United States–Israel BinationalAgricultural Research and Development Fund.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Table S1–S3. Cutin and wax composition of M82, cd1, cd2 and cd3fruit cuticle at the small green (SG) and mature green (MG)developmental stages.



Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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Accession numbers: sequence data from this article for the cDNA of CD2 can be found in the GenBank/EMBL data libraries under

accession number GQ222185.



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