Jekyll Encodes a Novel Protein Involved in the Sexual
Reproduction of Barley
Volodymyr Radchuk,aLjudmilla Borisjuk,a,1Ruslana Radchuk,aHans-Henning Steinbiss,bHardy Rolletschek,a
Sylvia Broeders,a,2and Ulrich Wobusa
aLeibniz-Institut fu ¨r Pflanzengenetik und Kulturpflanzenforschung, D-06466 Gatersleben, Germany
bMax-Planck-Institut fu ¨r Zu ¨chtungsforschung, D-50829 Ko ¨ln, Germany
Cereal seed development depends on the intimate interaction of filial and maternal tissues, ensuring nourishment of the new
generation. The gene jekyll, which was identified in barley (Hordeum vulgare), is preferentially expressed in the nurse tissues.
JEKYLL shares partial similarity with the scorpion Cn4 toxin and is toxic when ectopically expressed in Escherichia coli and
tobacco (Nicotiana tabacum). In barley, jekyll is upregulated in cells destined for autolysis. The gene generates a gradient of
expression in the nucellar projection, which mediates the maternal–filial interaction during seed filling. Downregulation of
differentiation of the nucellar projection and drives the programmed cell death necessary for its proper function. We further
biosynthesis in endosperm and embryo.
In contrast with the situation in animals, the tight interaction
between the mother plant and its developing offspring during
embryogenesis does not involve a vascular connection. Rather,
intercellular exchange is effected via the apoplasm (Wang et al.,
1994; Patrick and Offler, 2001). Thus, specific maternal plant
tissues (so-called nurse tissues) act as a bridge between the two
generations and provide the environment for the developing
new organism is generated by programmed cell death (PCD) of
nurse tissues, and the contents of dying cells are remobilized to
and dicots (Smart, 1994; Wu and Cheung, 2000; Greenwood
et al., 2005). The need for this source of nourishment is partic-
ularly critical during periods when the external environment de-
stabilizes steady state source/sink relationships. In extreme
situations, these can directly affect seed set, with the immediate
postfertilization period being particularly sensitive to metabolic
this time depresses endosperm cell division and endoreduplica-
tion and inhibits other cellular events that precede the synthesis
of storage products (Ober et al., 1991; Cheikh and Jones, 1994;
Artlip et al., 1995). While the mechanics of these adjustments
vary, the maternal nurse function is universally important.
tissues is poorly understood (Lopes and Larkins, 1993; Lam,
2004). Its key role in plant sexual reproduction, however, has
been confirmed repeatedly in studies of several female-sterile
sterile (Chaudhury, 1993) mutants. The interdependence be-
depends on both the identity of the gametophyte and the de-
velopmental stage of the filial organism (Norstog, 1974; Thorne,
1985; Dominguez et al., 2001). The genes involved in these
1999; Wang et al., 2003). Poaceae species provide the majority
of carbohydrates for the human diet, but only a small number of
genes active in the nurse tissues of cereal seeds have been
identified to date (Doan et al., 1996; Chen and Foolad, 1997;
Sturaro et al., 1998; Drea et al., 2005; Greenwood et al., 2005).
their potential strategic significance for crop improvement, al-
though likely, has yet to be experimentally proven.
to identify genes active during barley (Hordeum vulgare) seed
development and have isolated a cDNA sequence that is highly
expressed during early development (Sreenivasulu et al., 2002).
The gene is abundant in the nurse tissues of both the male and
1To whom correspondence should be addressed. E-mail borysyuk
@ipk-gatersleben.de; fax 49-39482-5500.
2Current address: European Commission, Joint Research Centre,
Institute for Reference Materials and Measurements, RM Unit, 2440
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instruction for Authors (www.plantcell.org) is: Ljudmilla Borisjuk
WOnline version contains Web-only data.
OAOpen Access articles can be viewed online without a subscription.
Article, publication date, and citation information can be found at
The Plant Cell, Vol. 18, 1652–1666, July 2006, www.plantcell.org ª 2006 American Society of Plant Biologists
female sporophyte, reaching an expression maximum during the
differentiation of the nucellar projection. This tissue provides the
main conduit of solutes to zygotic tissues and is therefore critical
for the process of seed filling in barley and wheat (Triticum
aestivum) (Duffus and Cochrane, 1993; Wang et al., 1994). The
conflicting role of this gene in the life and death of reproductive
nurse tissues inspired the name Jekyll, in reference to the
schizophrenic character of the Robert Louis Stevenson novel.
Jekyll Is a Single-Copy Gene in Barley, Encoding a Novel
Class of Small Proteins
length mRNA, a phage cDNA library generated from developing
barley caryopses (Weschke et al., 2000) was used to isolate nine
clones, each with the same sequence and length as HY09L21.
From a homology search of a large barley EST set (Kunne et al.,
2005), 78 entries, all identical to the previously cloned se-
quences, were identified. By genome walking, a fragment 1335
bp upstream of the translation start codon with a TATA-box
(GCTATAA) 39 to 46 bp upstream of the putative transcription
startwasisolated.JekyllcDNA encodesapredicted cationic and
amphipathic 140-residue protein (Figure 1A) with a secondary
structure composed of two a-chains and one b-sheet followed
by three a-chains (aabaaa-fold). Neither the cDNA nor the
protein sequence share any significant similarity to any known
sequences deposited in the public domain. The protein has the
following features:a25–aminoacidputative signalpeptideatthe
N terminus, three almost perfect direct repeats in the C-terminal
region,and aCys-rich domain(including8ofthe10Cysresidues
present in the complete sequence) in the central part of the
protein. The predicted translation product is also rich in Ala (18
region (Figure 1B); this encodes toxin 4 from the scorpion
Centruroides noxius (Vazquez et al., 1993).
DNA gel blot analysis showed that jekyll is a single-copy gene
in barley (data not shown). Homologous sequences are present
in the close relatives wheat and rye but not in other plants (see
Supplemental Figures 1A and 1B online). Four wheat ESTs with
strong similarity to jekyll were identified in the public database,
and these were used to construct two contigs, the derived
and 52.3 and 55.1% similarity to barley JEKYLL. The domain
structure of the predicted wheat proteins is similar to that of
barley; in particular, both possess a nearly identical 25-residue
signal peptide sequence at the N terminus and a highly con-
served C terminus. The central portions of the proteins are var-
iable, but the positions of the Cys residues are well conserved
(see Supplemental Figure 1C online).
Jekyll mRNA Is Abundant in Short-Lived Nurse Tissues
in the Reproductive Organs of Barley
RNA gel blot hybridization experiments showed that jekyll in
barley is represented by a single ;900-bp product, consistent
with the length of the cloned cDNA. The sequence was ex-
pressed in developing flowers (very weak in anthers and high in
gynoecium) and throughout all stages of caryopsis development
(peaking at 4 d after flowering [DAF]; Figure 2A) but not in any
other plant organs (Figure 2B) or in germinating seeds (data not
the inner cell layers of the nucellus (Figure 3A). At later stages of
seed development, jekyll expression spread outwards and was
followed by the autolysis of highly vacuolated cells. The disinte-
gration of lysed cells provides space for the enlarging endo-
sperm cavity (Norstog, 1974), and the process continued in the
dorsal and lateral sides of the seed until 4 DAF (Figures 3B and
3C). In the crease region, the nucellus remained intact, where it
forms the nucellar projection (Figure 3D). The peak of jekyll
expression (4 to 6 DAF) coincided temporally and spatially both
Figure 1. Structure of jekyll cDNA and Its Deduced Protein.
(A) Nucleotide sequence of jekyll cDNA and its deduced amino acid
sequence. The putative signal peptide is shown in bold. Cys residues in
the Cys-rich domain are shaded in gray. Continuous arrows mark nearly
perfect repeats, and dashed arrows label less-conserved repeats. The
stop codon is indicated by an asterisk.
(B) Amino acid alignment of similar regions of JEKYLL (Jek) and Toxin 4
(Tx4) from the scorpion C. noxius. The consensus sequence (Cons.) is
shown below in bold. Matched Cys residues are shaded in gray.
Jekyll in Barley 1653
with the differentiation of parenchyma tissues within the nucellar
projection and with the vacuolization of the nucellar epidermis
(Linnestad et al., 1998). Expression levels remained low in the
undifferentiated cells adjacent to the vascular tissues and in
the epidermis within the ventral region of the caryopsis (Fig-
ures 3E and 3F). Over time, jekyll expression decreased, but
the spatial pattern of expression was preserved (data not
Accumulation of JEKYLL Is Coupled with the
Differentiation of Nucellar Tissue
Immunodetectionshowedthat JEKYLL was present in nucellar
tissues before anthesis. In particular, it was concentrated in
the fully expanded cells in the central part of the ovule (Figure
4A). At 2 to 4 DAF, its level increased dramatically in the cells of
the nucellar projection (Figures 4B and 4C), and beyond this
stage, its accumulation became progressively restricted to the
nucellar projection tissue (Figure 4D). The appearance of
JEKYLL in the nucellar epidermis coincided with the vacuoli-
zation of the epidermal cells in the crease. The protein was
barely detectable either in the distal part or in the small mitotic
basal cells of the nucellar projection but was present in
expanding cells, reaching a maximal concentration in highly
vacuolated cells with the morphology of transfer cells (Figures
4D and 4E). Small concentrations were present in the partially
autolyzed tissues at the margins of the nucellar projection, but
the protein was absent in the attached endosperm (Figures 4E
and 4F). Thus, levels of JEKYLL coincided both temporally and
spatially with transcript levels. The protein accumulated in
concert with the differentiation of nucellar tissues, with max-
imal accumulation in expanding cells being rapidly followed by
Jekyll Expression Inhibits the Growth of Escherichia coli
and Promotes Cell Autolysis in Transgenic Tobacco
In JEKYLL overexpressing E. coli, cell growth stopped following
isopropyl-1-thio-b-D-galactopyranoside (IPTG) induction of pro-
tein synthesis. To define the peptide domain(s) responsible for
Figure 2. jekyll Transcripts Are Present in Flowers and throughout
(A) Total RNA (10 mg per line) from caryopses at various developmental
stages was hybridized with the jekyll probe (above) and with 25S rDNA
(below) as a quantitative control.
(B) Total RNA (10 mg per line) from various barley tissues was hybridized
with jekyll (above) and 25S rDNA probe (below).
Figure 3. Localization of jekyll Expression in Developing Caryopses.
(A) and (B) jekyll expression patterns in the nucellar tissues shortly after
pollination (A) and 2 DAF (B). Hybridization sites are visualized as a white
(C) Expansion of expression toward the integuments (arrows).
(D) The nucellar projection at 4 DAF.
(E) and (F) A gradient of jekyll expression within the nucellar projection 6
and 8 DAF; pjekyll:GFP (see Methods).
e, endosperm; ec, endospermal cavity; es, embryo sac; ii, integuments; n,
several layers of nucellar cells; ne, nucellar epidermis; np, nucellar projec-
tion; p, pericarp; tc, transfer cells, vs, vascular tissues. Bars ¼ 120 mm.
1654 The Plant Cell
this growth inhibition,constructs consisting of the Cys-richregion
eitheraloneor in combination with other structural domains were
prepared (see Supplemental Figure 2 online, inset) and trans-
formed into DL21 E. coli cells. A drastic inhibition of growth was
associated with the expression, even on its own, of the Cys-rich
domain (see Supplemental Figure 2 online). Since other domains
were ineffective, the Cys-rich domain must itself be responsible
for cell death.
When JEKYLL cDNA was expressed ectopically in tobacco
(Nicotiana tabacum) plants, driven by an ethanol-inducible pro-
moter, plants wilted within 24 h of induction and later died,
whereas control plants (the wild type and plants expressing
b-glucuronidase (GUS) under the same promoter) continued to
grow normally (see Supplemental Figure 3 online). The activation
of GUS expression by ethanol occurred first in the epidermal cell
layer of the root elongation region and in the root hairs. Later, the
expression spread to the cortex and finally moved toward the
uptake and transport, which is performed by epidermal cells and
root hairs specialized for this function (Gilroy and Jones, 2000).
Likewise, JEKYLL was expressed first in the epidermal cells and
in the root cortex of plants (Figure 5C, inset). Cells accumulating
the protein underwent rapid expansion, following which they
self-destructed (Figure 5C). The loss of root hairs was the first
5G). Following the disruption of root function, the stem and
leaves started to wilt. After ;48 h, roots were completely mac-
independently transformed transgenic tobacco lines (12 to 20
plants per line).
Root hair and whole plant growth were restored when jekyll
expression was turned off by removing plants to an inducer-free
medium. Thus, the primary effect of jekyll expression appears to
be to damage the epidermis and root hairs, and the subsequent
loss of turgor and eventual plant death are the result of failure in
root function. The expression of jekyll evidently prevents epider-
mal cell development (root hair building and/or growth) and
dramatically undermines cell integrity.
RNA Interference–Mediated Downregulation of Jekyll
Impairs Flower and Seed Development
A group of 46 independent barley transformants were generated
using Agrobacterium tumefaciens harboring pVECNpass con-
struct to achieve RNA interference (RNAi)–mediated jekyll re-
pression. No phenotypic differences between transgenic and
wild-type plants were observed until flowering. Later on, the
extent of phenotypic alteration was dependent on the degree
of jekyll downregulation. Four T2 lines exhibiting a weak (N18),
moderate (N61 and N81), and strong (N91) phenotype were
chosen for detailed investigation. Although the number of flow-
ers, as well as their morphology, remained unchanged (Figure
d longer than the wild-type ones, and both gynoecia and anthers
exhibited size irregularities. This phenotype was strongly ex-
assess possible effects on carbohydrate uptake of the flowers,
14C-sucrose was fed to the stem of both transgenic and
Figure 4. Immunodetection of JEKYLL in Longitudinal Sections of the
Gynoecium and the Transverse Sections of the Developing Caryopsis.
The target is present in the gynoecium (A) and developing caryopsis ([B]
to [F]), as visualized by blue staining (left panel) of the tissue section (right
panels; phase contrast microscopy). Abbreviations are as in Figure 3.
Bars ¼ 300 mm.
Jekyll in Barley1655
wild-type plants. Wild-type and transgenic flowers differed in
isotope incorporation after 48 h by >10-fold (Figure 6B), indicat-
ing a significant decrease in the sink strength of jekyll down-
regulated flowers. The expression of jekyll, as measured by RNA
gel blot hybridization, varied between transgenic lines from
almost 100 to 20% of the wild-type mRNA level (Figure 6C).
The extent of mRNA repression was consistent with the severity
of phenotype (Table 1, Figures 6D and 6E). Developing caryop-
ses were characterized by a reduction in their dorso-ventral
diameter (Figure 6E). However, despite showing retarded growth,
transgenic endosperms were histologically normal (Duffus and
all transgenics (Table 1); in addition, pericarp structure was
subtly changed. In extreme cases, additional ear cavities were
observed, reflecting an imbalance in the growth of the pericarp
and endosperm (Figure 6E, arrow in N91). Strongly affected
caryopses died during the prestorage or early storage phases.
Lastly, the number of seeds reaching maturity was lower in all
transgenics compared with the wild type, varying from;90% of
it appears that jekyll downregulation leads to impaired growth of
the reproductive organs and to a decrease in final seed yield.
The Differentiation Gradient Is Altered in the Nucellar
Projection of Transgenic Barley Seeds
We performed comparative analysis of tissue structures in wild-
type andtransgenic plant lines with respect tothe different levels
of jekyll repression (Figure 7). Four tissue types along the radial
and Cochrane, 1993) and coded in colors (Figure 7A): (1) the
basal region facing the main vascular bundle; (2) the middle zone
was little change in zonation pattern (cf. Figures 7A and 7B).
However, the basal region adjacent to the vascular tissue, which
transport cell regions was decreased and the amount of cell
debris reduced. These tendencies were exaggerated in line N61,
in which the basal region featured an additional three cell layers.
Cell elongation was dramatically reduced in the central part of
the nucellar projection, and the cell debris region was barely
recognizable (Figure 7C). Structural changes were expressed
even more noticeably in line N91 (Figure 7D), where the nucellar
projection was formed mostly by small cells (at least 10 addi-
tional cell layers) and irregularly expanded cells, which did not
show transfer cell morphology. The region of cell debris was
absent, and the nucellar projection had lost its regular tissue
structure, instead being transformed into a massive callus-like
organ. In this extreme case, the endosperm barely reached the
cellularization stage, and further development of the caryopsis
failed. Thus, the downregulation of jekyll has its primary impact
on cell fate in the nucellar projection by affecting cell differenti-
ation/expansion and preventing autolysis.
Proliferation of Endosperm Nuclei Is Delayed in Transgenic
Caryopses with an Aberrant Nucellar Projection
The decrease in size and fresh weight of transgenic caryopses
(Table 1) suggested perturbations in endosperm cell number
and/or in the endoreduplication levels (Brunori et al., 1993). Both
comparative flow cytometry and a conventional histological study
of wild-type and transgenic caryopses showed that, at 4 DAF,
cell number per caryopsis did not differ significantly from one
another. This suggested that variation in cell expansion was re-
sponsible for the variable caryopsis size of transgenics at the
type caryopses was doubled; bycontrast, neither transgenic line
Figure 5. Alcohol-Induced jekyll Expression in Transformed Tobacco
(A) and (B) Spatial patterns of promoter activity after induction with
ethanol in roots of tobacco transformed with pAlc-GUS grown under
hydroponics. GUS staining in the epidermis ([A]; arrows) and root hairs
([B]; arrows) as primary sites of promoter activity. Bars ¼ 40 mm.
(C) Disintegration of the outer cell layers in roots of transgenics after the
ethanol induction coincides spatially with JEKYLL immunodetected with
specific antibodies (shown in inset). Extensive cell damage within two or
three outer cell layers of the root are indicated in red. Bar ¼ 150 mm.
(D) to (G) Root hairs of wild-type plants ([D] and [F]) and severe reduction
of root hair growth in transgenics expressing JEKYLL ([E] and [G]).
Bars ¼ 1 mm.
1656 The Plant Cell
Figure 6. Flower and Seed Phenotypes Associated with Repression of Jekyll.
Jekyll in Barley 1657
achieved this level of cell multiplication (Figures 8A and 8B).
Thus, cell proliferation in transgenic caryopses was impaired
starting after the endosperm cellularization stage. The maternal
tissue is targeted by jekyll downregulation, and the filial tissues
are thereby indirectly affected. Flow cytometry (Arumuganathan
and Earle, 1991) was exploited to distinguish nuclei from the
DAF, the ratio cell numbers in the pericarp and endosperm were
identical in wild-type and transgenic plants. The predominant
nuclei were 2C and 4C, since maternal tissue occupies the
number of endosperm nuclei (3C to 12C) exceeded the number
population (Figure 8C, right panel). By contrast, in all the trans-
genics, the number of endosperm nuclei was much lower (Figure
8D). In the small caryopses of one transgenic line, the number
wasreduced byupto99%,butinlarger ones,theeffect wasless
extreme (27 to 41% reduction; Figure 8D, bottom panels). Thus,
but not pericarp (maternal) cells.
the proportion 4:2:1 (Figure 8C). Cells with 3C and 6C nuclei are
likely mitotic, while 12C nuclei are characteristic of cells entering
endopolyploidization (Engelen-Eigles et al., 2001). In transgenic
plants, the proportion of 12C cells was substantially reduced
compared with the wild type (Figure 8D).
jor effect of jekyll downregulation, while proper JEKYLL function
in the nucellar projection ensures the developmentally predeter-
mined proliferation of endosperm.
Storage Patterns in Transgenic Caryopses Reveal Defects
in Flux Exchange between Pericarp and Endosperm
The cellular parameters of the endosperm and its carbohydrate
uptake ability determine the starch storage capacity of the cary-
opsis (Zinselmeier et al., 1995; Smidansky et al., 2002). Starch
a combination of biochemical assay (Figure 9A), expression of
the endosperm-specific small subunit of AGPase, a key enzyme
of starch synthesis (Figure 9B), and histochemical tissue staining
(Figures 9C to 9H). In the wild type, transient accumulation of
starch began in the maternal tissues of the young caryopsis
(nucellar tissues and pericarp) but was utilized during further
development (Figures 9D and 9E). The main starch storage is
typically initiated in the endosperm (Simmonds and O’Brien,
1981; Wobus et al., 2004). The opposite trend was apparent in
jekyll downregulated plants, where an increased density of
starch grains was observed within the pericarp and integuments
and some starch remained in the nucellus until 12 DAF. In line
N91, starch content was actually increased, accompanied by a
callus-like growth of the nucellar projection (Figures 9F to 9H).
The prolonged deposition of starch in the nucellus of transgenic
plants was associated with a delay in carbohydrate accumula-
of starch in the endosperm was negligible compared with that
achieved in wild-type plants.
The storage of starch in the endosperm is associated with the
2003). In all transgenics, the expression of the endosperm-
specific small subunit of AGPase was delayed, and its level was
phase, starch accumulation rate in these lines was twofold to
threefold lower than in wild-type plants. By 12 DAF, the starch
content in the transgenics was only half that in the wild type
(Figure 9A); as a result, the starch content of transgenic seeds
was reduced at maturity (Table 1), despite the longer period (3 to
5 weeks) required for maturation.
Starch accumulation in the endosperm relies on assimilate
delivery fromthepericarp via thenucellar projection (Wang etal.,
1994; Tomlinson and Denyer, 2003; Wobus et al., 2004). There-
resources for endosperm growth and storage product accumu-
lation, especially during the immediate postfertilization period
(Schussler and Westgate, 1995). In this study, the main storage
process in transgenic endosperms was slowed, but at the same
time, there was evidence for prolonged transient storage in the
transgenic pericarp. We infer that this reflects damage to the
bridge between the sites of metabolite provision (pericarp) and
projection, which is the route for metabolite delivery and the site
of jekyll expression, is compromised in jekyll downregulated
Jekyll Encodes a Toxic Protein Essential
for Sexual Reproduction
We have described the isolation and functional characterization
of jekyll, which is highly expressed in the developing barley
Because of low molecular weight, JEKYLL belongs to a group of
small proteins, widely represented in both plants and animals,
Figure 6. (continued).
(A) Reduction of gynoecium and anther size in transgenics.
(B) Uptake of sucrose (in dots per minute [dpm]) by developing flowers in the wild type and line N91 (averaged from 15 independent measurements and
represented in a logarithmic scale; means 6 SD are shown).
(C) Total RNA from the wild type and transgenics at 4, 8, and 12 DAF hybridized with jekyll probe (above) and 25S rDNA (below).
(D) Changes in size and form of 4-DAF caryopses of the wild type and transgenics.
(E) Decrease in size and change in morphology of 12-DAF wild-type and transgenic caryopses. Orientations of anatomical cross sections are marked by
dashed lines (a). Changes in seed anatomy are indicated by red arrows. e, endosperm; p, pericarp. Bars ¼ 1 mm.
1658The Plant Cell
involved in the regulation of various cellular functions (Waters
et al., 1996). Like JEKYLL, many of these proteins are develop-
mentally regulated (Coca et al., 1994; DeRocher and Vierling,
1994; Waters et al., 1996; Wehmeyer et al., 1996). A well-studied
example is the defensins, which have a distinct function in either
seed maturation or the defense response (Thomma et al., 2002).
They are small cationic, amphypathic peptides with eight Cys
residues and are structurally similar to the first antifungal peptide
isolated from barley (Mendez et al., 1990; Thomma et al., 2002).
However, JEKYLL shows no similarity to the defensins (or any
other known plant proteins) at the level of either amino acid
composition or secondary structure. Curiously, it shares some
features with a scorpion toxin, the most conspicuous of which is
position of the six Cys residues in the domain that has a toxic
effect both on E. coli and tobacco. Whether the basis of this
toxicity of JEKYLL is similar to that of the scorpion toxin (thought
channel function; Rodrı ´guez de la Vega and Possani, 2005) is
The expression of jekyll is associated with cell death in barley.
The gene is upregulated only in deteriorating maternal tissues,
which border and nurse the new generation in the male and
female sporophytes. This tissue deathensures the survivalof the
newly growing organism and is precisely regulated. As occurs
also when jekyll is suppressed, any interference in development
(Mariani et al., 1990) or metabolism (Goetz et al., 1999) of the
nurse tissue leads to growth arrest and/or male sterility. In the
female sporophyte, jekyll is upregulated precisely in those nu-
cellar cell layers that are attached to the gametophyte and
programmed for subsequent autolysis. Nucellar PCD is critical
(Wu and Cheung, 2000).
The impact of JEKYLL on filial tissue development has been
ers of transgenic barley plants received less carbohydrate from
the maternal tissue, needed longer to develop, and were smaller
in size. Transgenic lines with a strong phenotype produced few
mature seeds. These observations underline the crucial role of
JEKYLL in flower development and, consequently, seed set.
Since the first description of nurse tissues of barley (Norstog,
1974), only three genes expressed in the nucellus have been
identified: NUC1, with no information on its function (Doan et al.,
1996); nucellin, a homolog of the dicot vacuolar-processing pro-
tease (Chen and Foolad, 1997; Linnestad et al., 1998); and
Hvex1, a putative extensin (Sturaro et al., 1998). Whether these
genes are expressed in the male sporophyte is as yet unknown,
as is their contribution to nurse function and/or sexual repro-
duction. In this work, the expression of jekyll in nurse tissues and
its functional involvement in the sexual reproduction of barley
has been experimentally proven.
JEKYLL Governs Cell Differentiation in the Nucellus and Is
Required for the Establishment of the Nucellar Projection
What is the role of JEKYLL in the establishment of nurse tissues?
jekyll expression was followed in the nucellus of wild-type cary-
opses (developmentally controlled expression), in roots of trans-
genictobacco plants(induced expression),andinthenucellus of
transgenic caryopses (RNAi-mediated downregulated expres-
sion). In the nucellus, the accumulation of JEKYLL was associ-
ated with structural changes in the nucellar projection: first in
finally reaching a maximum in elongated cells shortly before their
autolysis. The autolysis of cells was also observed in tobacco
epidermal cells where jekyll expression was induced: these cells
stopped dividing, underwent uncontrolled cell expansion, and
finally died, instead of the normal process of acquiring the de-
2000). Thus, jekyll switched epidermal cell fate to autolysis.
The opposite effect occurred in transgenic barley seeds with
reduced jekyll expression, where the larger number of small cells
present in the nucellar projection suggested a prolongation of
showing a slightly reduced level of jekyll expression (e.g., line
N18), normal levels of mitotic activity and cell expansion are
present in the nucellar projection, but autolysis is hampered.
Where jekyll is more repressed (line N61) there was a clear de-
crease in cell expansion and a slower autolysis, while mitotic
was severely downregulated (line N91), mitotic cells could not
be switched into differentiation and expansion. Instead, they
generated a callus-like growth and neither differentiated nor
We suggest that jekyll expression is sufficient to terminate cell
proliferation, but higher amounts of the protein are necessary to
release/facilitate expansion and autolysis. Plants do not appear
Table 1. Phenotypic Characteristics of Selected Lines of T2 Transgenic Barley Plants Transformed with the Construct pVECNpass
Level of jekyll
Number of Spikes
Number of Corns
56.4 6 2.3a
52.0 6 2.0a
49.3 6 3.3b
52.4 6 3.2a
51.4 6 7.7a
18.0 6 2.4a
17.7 6 2.4a
12.8 6 4.8a
15.0 6 4.0a
15.2 6 5.0a
21.6 6 3.8a
20.2 6 4.9a
13.4 6 2.8b
6.8 6 4.6c
5.2 6 3.3c
4.42 6 0.67a
3.04 6 0.05b
2.10 6 0.36c
2.14 6 0.23c
2.02 6 0.38c
455 6 46a
365 6 26b
340 6 18b
361 6 63b
The values are given as means 6 SD. Values followed by the same letter within a column are not significantly different at P < 0.05. ND, not detected.
aLevels of jekyll mRNA expression were estimated by RNA gel blot analysis at 4 DAF. Hybridization signals were quantified as described (Weschke
et al., 2000) and given in relative units as a percentage of expression to the wild type estimated as 100%.
Jekyll in Barley1659
able to compensate for jekyll downregulation, which suggests an
important function for JEKYLL. Overall, JEKYLL seems to be
necessary for the establishment of the nucellus structure in the
barley caryopsis, presumably via its involvement in the termina-
tion of mitotic activity and the facilitation of cell expansion,
followed by cell autolysis.
JEKYLL Controls Seed Filling by Promoting the
Nourishment of the Endosperm
In wild-type plants, the high metabolic activity of short-lived
nurse tissues first attracts a flow of metabolite (Andersen et al.,
2002; Sun et al., 2004) and later ensures that sufficient nutrient is
available for the gametophytic and filial tissues by releasing cell
contents through autolysis (Wu and Cheung, 2000; Wang et al.,
2003). The architecture of the nucellar projection reflects these
abilities in that proliferating cells are positioned in the upper part
part (solute translocation), and autolysing cells in the lower part,
adjacent to the endosperm cavity (solute release). Both this dif-
ferentiation gradient (Wang et al., 1994) and the gradient of jekyll
expression are arranged along the metabolite transport route
(Duffus and Cochrane, 1993) and act to guarantee metabolite
flow and release toward proliferating filial tissues.
The critical role of jekyll in the functional establishment of the
nucellar projection and its significant impact on barley seed de-
velopment and assimilate storage have been elucidated by the
a decelerated/failed autolysis of nurse tissues leads to reduced
amounts of cell debris being released into the apoplast, thus
diminishing nutrient availability for reutilization by filial tissues.
The same is true for secretion products delivered by the Golgi
apparatus and the endoplasmic reticulum from degenerating
cells, which retain their integrity even after the autolytic digestion
diffusion within the nucellar projection are compromised be-
cause the cell elongation and differentiation steps essential for
adapting transfer cell function (Patrick and Offler, 2001) were
defective. (3) Cell proliferation and starch accumulation in the
nucellus confirm its own biosynthetic activity dominating over
degradation processes, which commonly contribute to nourish-
ment (Norstog, 1974). In terms of the maternal/filial interaction,
these changes suggest a functional shift from provision of nutri-
of the new generation, especially during sink/source adjustment
Figure 7. Changes in the Cellular Structure of the Nucellar Projection Is
Associated with Repression of jekyll.
(A) Cross section of the nucellar projection of a 12-DAF wild-type
caryopsis. The basal region adheres to the vascular tissue and is visible
as one layer of small dense cytoplasmic cells (type I) traced in blue. One
to two layers of elongated cells (type II) are in yellow. Transfer cells (type
III), distinguished by maximal expansion along the radial axis, are in
turquoise. Autolysing cells (type IV) and the cell debris region occupy the
endosperm cavity and are shown in violet/red.
(B) to (D) The nucellar projection of transgenic lines N18, N61, and N91 at
12 DAF. The relative distribution of the corresponding regions of trans-
genic caryopses is represented by color bars (below). Bar ¼ 200 mm.
e, endosperm; ec, endospermal cavity; ne, nucellar epidermis; p, peri-
carp, vs, vascular tissues.
1660 The Plant Cell
episodes. Thus, when JEKYLL control over cell fate and autol-
ysis/death in nucellar tissues is lost, structural changes in the
nucellar projection are induced that interfere with its function as
the main assimilate transport route.
The delivery of nutrients is known to have a major impact on
the proliferation and onset of endopolyploidization in the endo-
over, many nutritional compounds perform regulatory or signal
et al., 2002; Koch, 2004), and hormone transport commonly
occurs via the same transport route (Kamboj et al., 1998; Yang
et al., 2002). In this context, an operational nucellar projection is
required for endosperm development. In our experiments, all
caryopses with defective jekyll function developed a lower sink
Figure 8. Ploidy Level and Total Cell Numbers in Developing Caryopses of Wild-Type and Transgenic Plants.
(A) The various ploidy levels present in the developing caryopsis.
(B) Total cell number per caryopsis in the wild type and transgenics 4 and 12 DAF. Values given as means 6 SD (n ¼ 5).
(C) Flow cytometric analysis of nuclei from wild-type caryopses 4 (left) and 12 DAF (right).
(D) Flow cytometric analysis of nuclei from transgenic caryopses 12 DAF.
Jekyll in Barley1661
and a reduced storage capacity during the major assimilate stor-
age period. They contained less starch at maturity, were of small
size, and the plants yielded less harvestable seeds than the wild
type. Hence, we propose that jekyll represents a further mech-
anism for maternal control (Jarvi and Eslick, 1975; Lynch and
Walsh, 1998; Alleman and Doctor, 2000) during seed develop-
Jekyll-like genes are restricted to species, such as barley,
wheat, and rye (Secale cereale), which form a massive nucellar
projection during the onset of seed filling. Similar sequences
were not found in plants that, although possessing a nucellus,
do not build a nucellar projection. Endogenous upregulation of
jekyll is developmentally controlled and coincides with the
massive proliferation of endosperm nuclei. During the later
developmental stages, when the endosperm becomes the ma-
et al., 2002; Borisjuk et al., 2004), the need for its nourishment
is superfluous, and jekyll expression is reduced. The size of
the mature seed is however predetermined at an early devel-
opmental stage (Brunori et al., 1993; Engelen-Eigles et al., 2001).
In conclusion, we suggest that jekyll is a part of a regulatory
system governing cell fate in nurse tissues and especially in
the nucellar projection of developing caryopsis. Its involvement
in, and its crucial impact on, the maternal/filial interaction has
been established during sexual reproduction and early seed
filling in barley.
Figure 9. Effect of Jekyll Repression on Starch Content and Starch Distribution during Seed Development.
(A) Starch content in developing caryopses 0, 4, and 12 DAF. Error bars indicate SD. FW, fresh weight.
(B) Hybridization of total RNA from wild-type and transgenic caryopses 4, 8, and 12 DAF with the endosperm-specific small subunit of ADP-glucose
(C) to (E) Iodine staining of cross sections of wild-type seeds. Transient starch accumulation is visible in the nucellus 2 DAF (C) and pericarp 4 DAF (D),
followed by starch accumulation in endosperm at 8 DAF (E). Note the absence of starch in the nucellar projection.
(F) to (H) Similar staining of caryopses of transgenic N91 shows starch deposition within the nucellar projection at 4 DAF (F). The area of starch
deposition increases with time, caryopsis at 6 DAF (G). At 12 DAF, starch is deposited primarily in the pericarp of the transgenic caryopsis (H) but not in
the endosperm. Abbreviations are as in Figure 3. Bars ¼ 300 mm.
1662The Plant Cell
Wild-type and transgenic barley (Hordeum vulgare) and tobacco (Nico-
tiana tabacum) plantswere grown understandardgreenhouseconditions
at 188C (for barley) or 248C (for tobacco) with 16 h of light and a relative air
humidity of 60%. Determination of developmental stages for developing
barley seeds and tissue isolations were performed as described
(Weschke et al., 2000).
Cloning of Jekyll and Sequence Analysis
used to screen a seed-specific phage cDNA library (Weschke et al.,
2000). Positive cloneswere selected, sequenced, and compared withthe
entire clone. Additionally, search of the EST collection from Leibniz-
Institut fu ¨r Pflanzengenetik und Kulturpflanzenforschung (http://gprc.ipk-
gatersleben.de) resulted in a number of additional ESTs, identical to the
previous clones. Promoter region was isolated using a GenomeWalker kit
(BD Biosciences). EST clones from wheat (Triticum aestivum) were found
and BJ247336) (http://www.shigen.nig.ac.jp/wheat/komugi/top/top.jsp).
DNA and RNA Procedures
Standard DNA and RNA cloning procedures were performed as de-
scribed by Sambrook et al. (1989). DNA gel blot analysis from plant total
leaf DNA (10mg per probe) digested with appropriate restriction enzymes
was performed as previously described (Radchuk et al., 2005). The jekyll
fragment derived in PCR with the oligos 59-CGTGGATCCGATCTCCAC-
was usedasa probe.Total RNAwas extracted fromtissuesamplesusing
as described (Radchuk et al., 2005).
The RNAi construction pVECNpass for barley stable transformation was
generated and consisted of the jekyll promoter together with the
59-upstream region (1332 nucleotides), sense fragment of jekyll (259 nu-
(199 nucleotides), and antisense fragment of jekyll (259 nucleotides). The
pUC19 vector using ofthe specific restriction sites.The used primers and
restriction sites were as follows: for jekyll promoter and 59-upstream
region, 59-TACTCGAGGGCACGCGTGGTCGACG-39 (XhoI underlined,
as are further restriction sites ) and 59-GAGCCACTAGTGCGCGATCGA-
GCTTGC-39 (SpeI); for jekyll sense fragment, 59-GCACTAGTGGCTCGC-
GGTGGGAAGG-39 (SpeI) and 59-TGCAGCAACAGATCTAGTGTCCTC-
GTC-39 (BglII); for jekyll antisense fragment 59-GCTCTAGAGGCTCGCG-
GTGGGAAGG-39 (XbaI) and 59-TGCAGCAACGGATTCAGTGTCCTC-
GTC-39 (BamHI). The whole cassette was cut out from pUC19 with PstI
enzyme and cloned into the appropriate restriction site of intermediate
vector pBluescript SK, and then, by cutting out with ApaI-NotI restriction
enzymes, the cassette was introduced into corresponding sites of the
binary vector pWVec8 (Wang et al., 2001).
Attempts to create a vector overexpressing jekyll gene driven by a
constitutive 35S promoter were unsuccessful because transformed
Escherichia coli cells did not grow after transformation with the resulted
construct. Therefore, for stable overexpression of JEKYLL in plants, a
construct was generated consisting of jekyll gene in an alcohol-inducible
expressionsystem(Caddick et al.,1998). For this, the codingpart of jekyll
was PCR amplified with the primers 59-GCAAGGATCCATGGCGGC-
TCGCGGTGGGAA-39 (BamHI) and 59-GAATGGATCCTCAGCGACATT-
GAACTCGCCGTG-39 (BamHI) and cloned into the BamHI restriction site
of the vector pACN (Caddick et al., 1998) between the modified AlcA
promoter and nos terminator. The sense orientation was checked by
restriction analysis and sequencing. The whole cassette was then cut out
by HindIII endonuclease and integrated into the appropriate site of binary
alcohol inducible system binSRNA to generate the pAlc-jekyll construct
used for tobacco plant transformation. The construct pAlc-GUS was
kindly provided by U. Sonnewald.
For overexpression of jekyll in E. coli, the coding region of jekyll without
signal peptide was PCR amplified using theprimerpair59-CGTGGATCC-
GATCTCCACAAGTGCTTCTG-39 (BamHI is underlined) and 59-GAAG-
into the bacterial expression vector pET23a between BamHI-XhoI restric-
tion sites, resulting in the vector pJ2.
For functional characterization of different JEKYLL protein domains,
full-length and different truncated sequence fragments were cloned in
frame into the pET23a vector between BamHI and XhoI insertion sites.
The fragments were amplified by PCR using the following primers:
for construct pJ1, 59- GCAAGGATCCATGGCGGCTCGCGGTGGGAA-39
(BamHI) and 59-GAAGCTCGAGGCGACATTGAACTCGCCGTG-39 (XhoI);
for pJ3, 59-GCAAGGATCCATGGCGGCTCGCGGTGGGAA-39 (BamHI)
and 59-CGTGCTCGAGTCACTCTGCAGCAAC-39 (XhoI); for pJ4, 59-GAT-
GGATCCAAGTGCTTCTGCGGGT-39 (BamHI) and 59-GAGTACTCTCGA-
GCCGGCACCCAAAG-39 (XhoI); for pJ5, 59-CCTGGATCCATGTGTT-
TCTTTGGGTGC (BamHI) and 59-ACTTGCCGTGCTCGAGACCCTC-
TGC-39 (XhoI); and for pJ6, 59-GATGGGATCCGCCGATCATGTCAA-39
(BamHI) and 59-CAGGCTCGAGCATGCTGCCCTCAG-39 (XhoI).
To create the pjekyll:green fluorescent protein (GFP) construct con-
sisting of GFP driven by the jekyll promoter, the sequence of the jekyll
promoter was inserted using blunt ends in front of the gfp sequence. The
whole cassette was then cut out from the plasmid with the SfiI restriction
enzyme and cloned into the same restriction site of the p6U vector (DNA
Cloning Service), resulting in the pjekyll:GFP construct.
Plant Transformation Procedures
Golden Promise essentially as described (Wang et al., 2001). Genetic
described by Kumlehn et al. (2006).
Tobacco plants (ecotype Havana) were transformed with the construct
pAlc-jekyll by A. tumefaciens strain EHA105 as previously described
(Ba ¨umleinet al.,1991). Transgenictobaccoplants carrying theGUSgene
under alcohol inducible promoter was kindly supplied by U. Sonnewald.
T2 transgenic tobacco plants with one copy of the transgene were used
for analyses. To establish hydroponic plant cultures, the seedlings were
planted in sterile sand and adopted to the greenhouse conditions. The
plants were removed, cut out from the roots, and placed in darkened
glass vessels containing liquid sterile Murashige and Skoog medium
(Murashige and Skoog, 1962) without sugars and phytohormones. Plants
built new roots in 7 to 10 d and were used for induction of transgene
expression. To activate the alcohol-inducible promoter, tobacco plants
were placed in new Murashige and Skoog medium supplemented with
2% ethanol and left for up to 7 d.
Overexpression of JEKYLL in E. coli, Microsequencing,
and Antibody Production
The plasmid pJ2 was introduced in E. coli strain BL21 (DE3) pLysS
(Novagen). Expression of the recombinant protein was induced by in-
cubation of transformed E. coli cells for 3 h at 378C in Luria-Bertani
medium supplemented with 0.5 mM IPTG and purified through one-step
Jekyll in Barley1663
affinity chromatography on nickel-nitrilotriacetic acid agarose resins
using attached His tag. Proper production of JEKYLL in E. coli cells
was checked by partial protein sequencing of the first 10 amino acids of
the isolated protein. The overexpressed protein was dissolved in 8 M
urea, purified using Ni-NTA resins (Qiagen), eluted at pH 5.0 according to
manufacturer’s instructions, and dialyzed three times against PBS, pH
8.0. Two hundred micrograms of the purified protein was used to im-
munize rabbits intradermally and boosted after 2 weeks with the same
antigen. Polyclonal anti-Jekyll antibodies were produced according to
Harlow and Lane (1988) and tested by protein gel blot hybridization in a
dilution of 1:1000. Specificity of binding was ascertained by competition
with a 100-fold molar excess of respective antigens.
Expression of Different Protein Domains in E. coli Cells
To study toxic capacities of different domains of JEKYLL, the constructs
pJ1 to pJ6 were transformed into the E. coli strain BL21 and grown at
378C in Luria-Bertani medium supplemented with 100 mg/mL ampicillin
and 100 mg/mL IPTG for 8 h. The rate of bacterial growth was estimated
every hour by measurement of optical density at A600.
Caryopses were fixed in 2.5% glutaraldehyde and 50 mM sodium
cacodylate, pH 7.0, or in 4% (w/v) paraformaldehyde and 50 mM po-
tassium phosphate buffer, pH 7.0, under slight vacuum for 4 h at room
temperature, rinsed in cacodylate buffer, dehydrated, and embedded in
microtome, transferred on poly-L-lysine–treated slides (Sigma Diagnos-
tics), and dried overnight at 458C. As a general stain, toluidine blue was
used according to Gerlach (1977) and Carson (1990). In situ hybridization
was performed according to Borisjuk et al. (1995). The same cDNA
fragmentasfor blothybridizationswasusedasaprobeafterlabeling with
[33P]dCTP. The immunostaining and structural investigations were per-
formed using microsections of seeds and roots embedded in butyl-
methyl methacrylate (Baskin et al., 1992). Briefly, barley caryopses were
sliced into small pieces and fixed 1.5 to 3 h in 4% paraformaldehyde in
PBS with 10 mm DTT, washed in PBS with DTT for 2 h, and dehydrated in
ethanol series. The embedding in butyl-methyl methacrylate was fol-
lowed by polymerization at 208C for 48 h under UV light. Sections of 3- to
5-mm thickness were cut on a microtome (RM 2165; Leica). After de-
embedding with acetone and rehydration, the sections were stained with
toluidine blue and/or used for immunodetection. The immunolocalization
antibody using a corresponding VASTASTAIN ABC-AP kit (Alkaline
Phosphatase Substrate Kit III). The histochemical detection of GUS
microscopically by an Axioscope (Carl Zeiss).
Flow Cytometry Analysis
Suspensions of nuclei were obtained by chopping plant tissues with a
Triton X-100, and 107 g MgCl23 6H2O, pH 7.0). The 49,6-diamidino-2-
phenylindole (Sigma-Aldrich) at a final concentration of 50 mg mL?1was
added to the nuclei suspension 30 min before analysis. Nuclear DNA
content was measured using a flow cytometer. The Foulgen staining was
performed for localization of nuclei in situ within the tissue sections of the
For measurement of14C-sucrose uptake, stems of barley plants were cut
;25 cm below spikes and put into 100 mL of solution containing 10 mM
pH 7.0, and 500 mL [U-14C] sucrose (7.4 MBq mL?1; Amersham-Buchler).
After 24 h of incubation in the light (;400 mmol m?2s?1), the flowers of
in liquid nitrogen. Subsequently, the plant material was homogenized in
2 mL methanol (60% [v/v]). Radioactivity was determined by a liquid
scintillation counter (Rotiszint). Counts were corrected for background
and quenching by external standards. Starch determination was per-
formed essentially as described (Rolletschek et al., 2005).
Searches of the National Center for Biotechnology Information databases
were performed with BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Align-
Sequence data were analyzed using Lasergene software (DNAstar).
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession number AM261729.
The following materials are available in the online version of this article.
Supplemental Figure 1. Genomic Organization of Jekyll in Barley and
Supplemental Figure 2. The Influence of JEKYLL Domains on
Growth of E. coli Cells.
Supplemental Figure 3. Influence of Jekyll on Phenotype of Trans-
genic Tobacco Plants Seven Days after Induction of the Gene Ex-
This work was supported in part by the projects GABI-SEED (FKZ
0312282) and GABI-SEED 2 (FKZ 0313115) of the German Ministry for
antibody production, W. Weschke for advice during molecular biological
work, M. Hajirezaei for technical support during the14C-isotope studies,
and T. Sharbel and H. Block for advice and help with flow cytometry. We
appreciate the preliminary work of S. Gubatz on immunodetection and
thank U. Siebert, A. Stegmann, G. Einert, E. Fessel, and K. Blaschek
for excellent technical assistance. Special thanks to S. Schulze and
K. Davidsen (Max-Planck-Institut) for stable barley transformation and to
U. Tiemann and K. Lipfert for excellent artwork.
Received January 20, 2006; revised April 10, 2006; accepted May 10,
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1666The Plant Cell