Light-dependent death of maize lls1 cells is mediated by mature chloroplasts

Article (PDF Available)inPlant physiology 130(4):1894-907 · January 2003with44 Reads
DOI: 10.1104/pp.008441 · Source: PubMed
We reported previously the isolation of a novel cell death-suppressing gene from maize (Zea mays) encoded by the Lls1 (Lethal leaf spot-1) gene. Although the exact metabolic function of LLS1 remains elusive, here we provide insight into mechanisms that underlie the initiation and propagation of cell death associated with lls1 lesions. Our data indicate that lls1 lesions are triggered in response to a cell-damaging event caused by any biotic or abiotic agent or intrinsic metabolic imbalance--as long as the leaf tissue is developmentally competent to develop lls1 lesions. Continued expansion of these lesions, however, depends on the availability of light, with fluence rate being more important than spectral quality. Double-mutant analysis of lls1 with two maize mutants oil-yellow and iojap, both compromised photosynthetically and unable to accumulate normal levels of chlorophyll, indicated that it was the light harvested by the plant that energized lls1 lesion development. Chloroplasts appear to be the key mediators of lls1 cell death; their swelling and distortion occurs before any other changes normally associated with dying cells. In agreement with these results are indications that LLS1 is a chloroplast-localized protein whose transcript was detected only in green tissues. The propagative nature of light-dependent lls1 lesions predicts that cell death associated with these lesions is caused by a mobile agent such as reactive oxidative species. LLS1 may act to prevent reactive oxidative species formation or serve to remove a cell death mediator so as to maintain chloroplast integrity and cell survival.
Light-Dependent Death of Maize lls1 Cells Is Mediated by
Mature Chloroplasts
John Gray
*, Diane Janick-Buckner
, Brent Buckner, Pam S. Close, and Gurmukh S. Johal
Department of Biological Sciences, The University of Toledo, Toledo, Ohio 43606 (J.G.); Division of Science,
Truman State University, Kirksville, Missouri 63501 (D.J.-B., B.B.); Hickman High School, Columbia,
Missouri 65202 (P.S.C.); and Department of Botany and Plant Pathology, Purdue University, West Lafayette,
Indiana 47907 (G.S.J.)
We reported previously the isolation of a novel cell death-suppressing gene from maize (Zea mays) encoded by the Lls1
(Lethal leaf spot-1) gene. Although the exact metabolic function of LLS1 remains elusive, here we provide insight into
mechanisms that underlie the initiation and propagation of cell death associated with lls1 lesions. Our data indicate that lls1
lesions are triggered in response to a cell-damaging event caused by any biotic or abiotic agent or intrinsic metabolic
imbalance—as long as the leaf tissue is developmentally competent to develop lls1 lesions. Continued expansion of these
lesions, however, depends on the availability of light, with fluence rate being more important than spectral quality.
Double-mutant analysis of lls1 with two maize mutants oil-yellow and iojap, both compromised photosynthetically and
unable to accumulate normal levels of chlorophyll, indicated that it was the light harvested by the plant that energized lls1
lesion development. Chloroplasts appear to be the key mediators of lls1 cell death; their swelling and distortion occurs before
any other changes normally associated with dying cells. In agreement with these results are indications that LLS1 is a
chloroplast-localized protein whose transcript was detected only in green tissues. The propagative nature of light-dependent
lls1 lesions predicts that cell death associated with these lesions is caused by a mobile agent such as reactive oxidative
species. LLS1 may act to prevent reactive oxidative species formation or serve to remove a cell death mediator so as to
maintain chloroplast integrity and cell survival.
lls1 (lethal leaf spot-1) is a maize (Zea mays) muta-
tion, characterized by the formation of necrotic spots
that expand continuously to kill the entire leaf and
eventually the whole plant. The developmentally
programmed phenotype of lls1 manifests in a cell
autonomous fashion as evidenced by the discrete
border between mutant and revertant tissue in sec-
tored plants (Gray et al., 1997) and is suggestive of
the involvement of an endogenous program in lls1
cell death. Because this mutation is inherited in a
strictly recessive fashion, it is likely that the wild-
type Lls1 gene functions to positively maintain cell
homeostasis (Ullstrup and Troyer, 1967; Johal et al.,
1994). The Lls1 gene has been cloned. Although it
appears to encode a novel protein specific to plants,
it does have two motifs, a Rieske-type Fe-sulfur cen-
ter and a mononuclear non-heme Fe-binding site,
that are found in the aromatic ring-hydroxylating
dioxygenases of bacteria. Because of the fact that the
biochemical function of these enzymes is to degrade
aromatic hydrocarbons, we hypothesized previously
that LLS1 may also work by breaking down a phe-
nolic mediator of cell death in plants (Gray et al.,
1997). This proposal remains contentious, however,
because the nature of the substrate, if any, for LLS1
remains unknown and we have now found these
motifs in a small family of plant enzymes, two of
which are known to function in chlorophyll b and Gly
betaine biosynthesis (below).
The lls1 mutation belongs to a class of defects in
plants called disease lesions mimics in which lesions
resembling infection sites are formed in the absence
of a pathogen (Greenberg, 1997; Morel and Dangl,
1997; Gray and Johal, 1998; Buckner et al., 2000).
Several of these genes have now been cloned in an-
ticipation of identifying molecular components that
play a direct role in controlling cell death and the
hypersensitive response (HR) in plants (Morris et al.,
1998; Kliebenstein et al., 1999; Yin et al., 2000). These
studies reveal that lesion mimic genes encode a va-
riety of functions including membrane receptors (Mlo
and Rp1), a putative transcription factor regulating
superoxide dismutase (Lsd1), and salicylate and
sphingolipid signaling (Acd6 and Acd11; Hu et al.,
1996; Buschges et al., 1997; Collins et al., 1999; Devoto
et al., 1999; Kliebenstein et al., 1999; Rate et al., 1999;
Sun et al., 2001; Broderson et al., 2002). The genes
Major funding for this work was provided by the National
Science Foundation (grant no. MCB–9729608 to G.S.J.), and minor
funding provided by the U.S. Department of Agriculture (grant no.
200001465 to J.G.) and by The University of Toledo (laboratory
startup funds to J.G.). This is journal paper no. 16,872 of the
Purdue University Agricultural Research Programs.
These authors contributed equally to the paper.
* Corresponding author; e-mail; fax
Article, publication date, and citation information can be found
1894 Plant Physiology, December 2002, Vol. 130, pp. 1894–1907, © 2002 American Society of Plant Biologists
underlying other lesion mimic phenotypes appear to
play a more direct role in maintaining cellular ho-
meostasis. Blockage of metabolic processes such as
the synthesis or degradation of chlorophyll (by Les22-
encoding uroporphyrinogen decarboxylase and acd2-
encoding red chlorophyll catabolite reductase, re-
spectively), results in the accumulation of porphyrin
intermediates that become toxic free radicals when
cells are exposed to excess light (Hu et al., 1998;
Molina et al., 1999; Ishikawa et al., 2001; Mach et
al., 2001). A deficiency in fatty acid biosynthesis (by
the mod1 gene encoding an enoyl-acyl carrier pro-
tein reductase) causes pleiotropic effects on plant
growth and results in premature cell death (Mou
et al., 2000).
Although there is a ubiquitous association of these
mutants with tissue death, careful examination is
required to determine which ones may represent de-
fects in genes and mechanisms that control pro-
grammed cell death (PCD; Greenberg, 1997; Morel
and Dangl, 1997; Gray and Johal, 1998; Buckner et al.,
2000, Mou et al., 2000). Our knowledge of how plants
might accomplish PCD remains rudimentary. In an-
imal studies, the cellular features of PCD events such
as cell shrinkage, membrane blebbing, and phagocy-
tosis by neighboring cells are widely recognizable
(Haecker, 2000). The nDNA of dying cells is de-
graded into oligonucleosomal ladders. A class of Cys
proteases termed caspases enables such dismantling
of the cell. Produced as zymogens, caspases are kept
in check directly or indirectly by suppressors of cell
death. Some of these suppressors of cell death do so
by maintaining the integrity of mitochondria (Hen-
gartner, 2000). When animal cells are accidentally
injured they exhibit a swelling of the cell and or-
ganelles known as oncosis evidenced later by chro-
matin clumping and phagocytosis (Majno and Joris,
1995). The extent to which similar molecular compo-
nents participate in the apoptotic and oncotic cell
death pathways is not yet fully known.
Although studies of lesion mimics have identified
genes and mechanisms that are largely unique to
plants, only in a few cases have the details of how cells
affected in each of these mutants undergo cell death.
Do they follow the paradigm of apoptotic cell death,
which is shown to be conserved biologically across the
entire animal kingdom, or do they follow a sequence
of events that is unique to plants? The present inves-
tigation was undertaken to explore the nature of cell
death mechanisms that underlie the initiation and
propagation of lls1 lesions. The results obtained
clearly support a central role for light absorbed by
chloroplasts in lls1 cell death. Although the results do
not indicate that lls1 cells undergo a PCD event, they
do prompt the idea that the regulation of chloroplast
integrity should be considered as a possible means
by which some cell death events may be controlled in
lls1 Lesions Exhibit Some Features of Cell Death
Induced by Infectious Agents
Plants often respond to pathogens by unleashing a
cell death program at or around the site of infection
(Hammond-Kosack and Jones, 1996). This cell death
reaction, often associated with HR, results in cellular
collapse and the formation of necrotic lesions. Ne-
crotic regions where irreversible membrane damage
or cell death has occurred are apparent when lls1
lesions are stained with trypan blue (Fig. 1, A and B;
Dietrich et al., 1994). Two cytological markers that
are almost always found associated with the HR are
the deposition of callose and the accumulation of
autofluorescent lesions in and around infection sites
(Aist et al., 1988; Koga et al., 1988). To assess whether
lls1 lesions exhibit features characteristic of HR le-
sions, we monitored the induction of these responses
during the development of lls1 lesions. Callose dep-
osition was observed in most cells within lls1 lesions.
The BS cells were the first to form callose, deposited
as plugs in the plasmodesmatal pit fields (Fig. 1, C
and D). This callose response was observed several
vascular bundles distant from the actual lesion site,
indicating that a stress signal emanating from the
dying cells triggered the response. Autofluorescence,
which is thought to reflect accumulation of stress-
related phenolic compounds, was also observed in
lls1 lesions (Fig. 1, E and F). It appeared to be re-
stricted to dying tissue. These results show that lls1
lesions exhibit some features of cell death that are
associated with the HR. We also attempted to discern
if DNA fragmentation, which is a marker of some
plant PCD events, occurs in lls1 dying cells. We did
not detect an oligonucleosomal ladder in lls1 lesioned
leaves but this may have been because of the asyn-
chronicity of rapidly dying cells in these lesions (data
not shown).
Cellular Damage Is Required for lls1 Lesion Initiation
Lesion mimics have been categorized into two
classes: those in which lesions stay small and discrete
(initiative class), and those in which lesions, once
formed, continue to expand (propagative class). The
initiative class mutants may have defects that inap-
propriately trigger a cell death program, whereas
mutants of the propagative class may have defects in
negative regulators of cell death (Walbot et al., 1983;
Greenberg and Ausubel, 1993; Dietrich et al., 1994;
Johal et al., 1994). We described previously how the
lls1 phenotype follows a developmental gradient
with lesions forming first near the tip of the oldest
leaf and then gradually moving downward toward
its base (Johal et al., 1994; Gray et al., 1997). This
pattern is repeated progressively on every leaf up the
plant (Fig. 1G). However, within a region of a leaf
that has attained developmental competence, lls1 le-
Mediation of lls1 Cell Death by Mature Chloroplasts
Plant Physiol. Vol. 130, 2002 1895
Figure 1. Histological features and inducible expression of lls1 lesion phenotype during development. A, White light
trans-illumination of an lls1 lesion (5). B, Trypan blue staining in area of necrosis of same lesion, as in A. C, Callose
plugging of plasmadesmatal fields in BS cells of an lls1 plant. Picture shows aniline blue-stained unsectioned leaf tissue
observed under UV light. Black arrow indicates an individual BS cell outside the periphery of the observable dying cells
(which is to the left of the field) that has begun to deposit callose in the plasmadesmatal fields. The white arrow indicates
the location of a neighboring xylem cell in the vascular bundle. D, Cartoon indicating bundle sheath (BS) and xylem cell
boundaries in A. The plasmadesmatal pit fields, containing up to 100 plasmadesmata/pit fields, are highlighted for one cell
in blue. E, White light trans-illumination of an lls1 lesion (40). F, Blue-light autofluorescence of same lesion as in E. G,
Developmental expression of lls1 lesions. Field-grown lls1-ref/lls1-ref plant at the nine-leaf stage. H, Close-up view of typical
spreading lesions on an expanded leaf of an lls1-ref/lls1-ref plant grown under field conditions. Occasionally, the concentric
rings exhibit a dark coloration as shown here. I, Close-up view of field-grown lls1 lesions showing the concentric ring
appearance of lesions and elongate shape of lesions. J, Time lapse series showing expansion of an individual lls1 lesion over
a period of 96 h. The ruler markings in each picture are 1 mm apart. K, Lesions induced in an lls1/lls1 plant 7 d after infection
with a nonpathogenic strain of C. carbonum. L, Lesions induced in a region of an lls1/lls1 leaf by pinprick wounding 4 d
earlier. This region of the leaf was competent for lesion formation but normal spontaneous lesions had not yet progressed
to this region of the leaf. M, Spontaneous lesion development on an lls1/lls1 plant from a population segregating for Les101
and lls1. Lesions exhibit a low density. N, Spontaneous lesion development on an Les101 plant from a population
segregating for Les101 and lls1. Lesions exhibit a high density. O, Spontaneous lesion development on an Les101/ lls1/lls1
plant from a population segregating for Les101 and lls1. Lesions are initiated as les101 lesions and progress to an lls1
phenotype with a density that of Les101 lesions.
Gray et al.
1896 Plant Physiol. Vol. 130, 2002
sions appear to form in a random pattern (Fig. 1H).
Once initiated, lls1 lesions then continue to expand
and often appear to be slowed by leaf veins leading
to a longitudinal appearance (Fig. 1, I and J). This
expansion was measured over time (Fig. 1J) and it is
estimated that longitudinal expansion (7.7 mm d
occurs approximately 5.6 times faster than latitudinal
expansion (1.8 mm d
). The continuous lesion ex
pansion implies that lls1 mutation is defective in a
mechanism involved in the containment of cell death.
The impedance of the spread of cell death by vascular
bundles suggests dilution of a diffusible cell death-
promoting factor or alternatively the reduced pro-
duction of the factor by non-photosynthetic vascular
cells. These observations prompted the closer exam-
ination of the immediate events that underlie lls1
lesion formation.
To address this, we treated mutant plants in a
variety of ways. First, we asked how lls1 plants react
to pathogens that trigger HR. This was accomplished
by inoculating lls1 seedlings with an avirulent strain
of Cochliobolus carbonum that induces only necrotic
flecks at the site of penetration (Johal and Briggs,
1992). Initially, the tissue reacted by producing
flecks, which appeared at the same rate and intensity
as they did on wild-type siblings. However, as the
competence to form lls1 lesions developed, most sites
expanded to form lls1 lesions on mutant leaves (Fig.
1K). Inoculations with other maize pathogens, in-
cluding C. heterostrophus and Puccinia sorghi, gave
similar results (data not shown), which indicated that
some general stress associated with pathogen inva-
sion probably provided a trigger for lls1 lesion
To test whether this stress was caused by physical
injury associated with fungal penetration, lls1 leaves
were wounded by pinpricks. Like infection sites, all
wound sites transformed into lls1 lesions when the
tissue acquired the potential to express the mutant
phenotype (Fig. 1L), suggesting that a damaged or
dying cell can serve as the trigger for lls1 lesion
initiation. This conclusion is supported by the addi-
tive phenotype of double mutants in which lls1 was
coupled either with Les-101 or Les-22. The latter are
two dominant Les mutations of the initiation class,
whose production of lesions preceded those caused
by lls1 (Fig. 1, MO; Johal et al., 1994). In the case of
Les101, lesions also form at a much higher density
(136.3 21.2 lesions/unit area
) than spontaneous
lls1 lesions on similarly aged leaves (2.5 1.4 le-
sions/unit area
). We inspected Les101/lls1 double
mutants over successive days and monitored the pro-
gression of individual lesions in a time lapse fashion.
The phenotype was initially of the respective Les
type, however, as the leaf acquired developmental
competence to express lls1 many Les lesions trans-
formed into lls1-like lesions (Fig. 1O; an increased lls1
lesion density of 19.5 5.9 lesions/unit area
) that
rapidly expanded to overlap with other Les lesions
and consume the whole leaf. Taken together, these
results clearly indicate that biotic or abiotic cellular
damage, irrespective of the nature of the agent that
caused it, serves as a signal to initiate an lls1 lesion.
Even so-called spontaneous lesions that form on
lls1 plants in the apparent absence of any wounding
(e.g. in a growth chamber) may reflect an unidenti-
fied endogenous stress such as the accumulation of a
phototoxic metabolite (see below).
Light Harvested by Photosynthetic Tissue Is
Required for lls1 Lesion Formation
The lls1 lesions typically exhibit alternating circles
of light and dark necrotic tissue, suggesting that
expansion of lls1 lesions is subject to influence by
environmental factors (Fig. 2A). Because concentric
rings largely disappear when lls1 mutants are grown
under continuous illumination (Fig. 2B), light may
play a role in the expression of lls1 lesions. To inves-
tigate this possibility, maize leaves were covered
with aluminum foil at different stages of lesion de-
velopment. Macroscopic lesions did not form on cov-
ered parts of the leaf, and the ones that had already
initiated stopped growing once the leaf was covered
(Fig. 2C). We have not determined absolutely that
microscopic cell death is inhibited in the dark but the
growth of lesions induced by mechanical wounding
was similarly impeded (data not shown), indicating
that light plays a deciding role at least in lls1 lesion
expansion, if not also lesion induction.
To investigate whether it was the quantity or qual-
ity of light that allowed lls1 lesion expansion, mutant
leaves were covered with plastic filters that allowed
transmission of different wavelengths of light. Except
for the far-red filter, which transmitted less than 1%
of incident sunlight (approximately 7
mol m
none of the filters could prevent lesion progression
under full exterior sunlight (1,6001,700
mol m
). Under greenhouse conditions where overall in
cident light was approximately 25% of full sunlight,
filters that transmitted approximately 40
mol m
or less provided a protective function (Fig. 2D).
These results are consistent with the conclusion that
lls1 lesions are promoted by a higher fluence rate of
incident light, but they do not reveal if it was specif-
ically the light harvested for photosynthesis that was
responsible for lls1 lesion growth.
We used a genetic approach to further explore this
question. We generated double mutants of lls1 with
ij1 (iojap-1), NCS2 (non-chromosomal stripes), and Oy1–
700 (Oil yellow-1), which are all compromised in their
ability to capture light or photosynthesize effectively.
ij1 is a recessive nuclear mutation that produces al-
bino (homoplastidic) and pale green (heteroplastidic)
stripes on an otherwise normal green leaf (Han et al.,
1992). Chloroplasts affected in ij1 do not contain
ribosomes, largely lack thylakoids, and exhibit unde-
tectable levels of ribulose-biphosphate carboxylase
Mediation of lls1 Cell Death by Mature Chloroplasts
Plant Physiol. Vol. 130, 2002 1897
and ATPase activity (Thompson et al., 1983). Pale-
green or albino stripes of NCS2, a maternally inher-
ited defect, result from a deficiency of PSI (Marien-
feld and Newton, 1994). In both of these variegated
backgrounds, lls1 lesions formed only in normal
green tissues and failed to initiate or develop in
either the pale green or albino sectors (Fig. 2, EG
and IK). Moreover, lls1 lesions that formed in green
tissues quickly stopped expanding when they
reached albino or pale-green sectors (Fig. 2, E, F, I,
Figure 2. Requirement of light for lesion development and suppression of the lls1 phenotype in photosynthetically
compromised mutants. A, Multiple initiation points for cell death during diurnal cycle. Image shows close-up view of a dead
lls1/lls1 lesioned leaf that was grown with an approximately 16-h-light/8-h-dark cycle. The center of each set of concentric
rings represents the initiation points of cell death in this leaf region. B, “Wave” of cell death during continuous illumination.
Image shows close-up view of a dying lls1/lls1 lesioned leaf that was grown under continuous illumination in a growth
chamber. Cell death is seen to process as a wave from the tip of the leaf toward the base as opposed to a confluent “leaf
spot” pattern. C, Requirement of light for lesion formation in an lls1/lls1 plant. The leaf shown was protected by wrapping
aluminum foil around the region indicated by the arrow. Lesions developed in the region exposed to light but not in the
protected region. A similar protective effect was observed for lesions induced by pinprick wounding (not shown). D, Light
intensity versus wavelength experimental arrangement. Leaf regions not yet exhibiting lesions were protected by foil or a
transparent plexiglass filter (red for the leaf shown here) around the region indicated by the arrow. After lesion formation on
the lower side of the covered region, the filter was removed and the underlying tissue examined for lesions. Here, a red
plexiglass filter 3 mm in thickness prevented lesion formation. E, Suppression of lls1 lesion formation in pale-green or albino
sectors of an iojap (ij) mutant. Lesions developed in an lls1/lls1 ij/ij plant but only in dark-green tissue. Lesions formed on
either side of pale green or albino sectors but never within the albino sectors. F, Suppression of lls1 lesion formation in
pale-green or albino sectors of an iojap (ij) mutant. Lesions developing in a narrow green sector propagate lengthways but
not into the neighboring albino sectors. G, Suppression of lls1 lesion formation in pale-green or albino sectors of an iojap
(ij) mutant. In this instance, an lls1 lesion appears to “traverse” a narrow pale green sector (arrow). H, Les4 lesions will form
in the albino sectors of an iojap (ij) mutant. The lesions of the dominant lesion mimic les4 formed in both pale-green and
albino sectors (shown) of a Les4/ ij/ij double mutant plant. I and J, Albino sector of an ij/lls1 leaf traversed by an lls1 lesion
is still alive. The dead tissue of a leaf section viewed by white light in I is revealed by trypan blue staining in J. K, Suppression
of lls1 lesion formation in pale-green sectors of an ncs7 mutant. Lesions developed in an lls1/lls1 NCS2 plant but only in
dark-green tissue. L, Suppression of lls1 lesion formation in an oy1-700 mutant. Similarly positioned leaves from field-grown
plants of the same age are compared from a population segregating for lls1 and oy1-700. The plant on the right (lls1/lls1
Oy1-700) exhibits typical lls1 lesion development, whereas the plant on the left (lls1/lls1 oy1-700/) exhibited smaller
lesions that did eventually coalesce but at a greatly reduced rate. The reduction of lesion formation in lls1/lls1 oy1-700/
plants often caused the suppression of lls1/lls1 lethality (permitting seed set).
Gray et al.
1898 Plant Physiol. Vol. 130, 2002
and J). An exception to this trend, however, was
noted when such chlorophyll-deficient sectors were
narrow. In these situations, although the lls1 lesion
still failed to annihilate the albino tissue, it was able
to traverse such narrow stripes and resume growth in
green tissue on the other side (Fig. 2G). The lack of
trypan blue staining in the traversed albino tissue
indicates that the cells therein are still alive (Fig. 2, I
and J). Apparently, the signal(s) mediating the prop-
agation of lls1 cell death is somewhat diffusible, but
the fact that lls1 lesions fail to develop or expand into
nongreen tissue indicates that some activity related
to light harvesting or photosynthesis is important in
expression of lls1 lesions. That this is an lls1-specific
phenomenon is indicated by the observation that Les4
lesions are not suppressed in albino areas (Fig. 2H).
The interpretation that some factor or activity as-
sociated with light harvesting by photosynthetic pig-
ments is required for lls1 lesion development is fur-
ther supported by the behavior of lls1 in the Oy1-700
background. Oy1-700 is a semidominant mutation
with a defect in Mg-chelatase activity (Polacco and
Walden, 1987; Neuffer et al., 1997). As a result, the
mutant has reduced chlorophyll levels, only 30% to
40% compared with normal plants. In fact, chloro-
phyll b is largely absent in this mutant. On leaves of
lls1 mutants that are heterozygous for Oy1-700, le-
sions initiated at a similar frequency (1.8 1.5 le-
sions/unit area
) to those in wild-type tissue (1.5
1.6 lesions/unit area
). However these lesions ex
panded at a greatly reduced rate than in normal lls1
mutants (Fig. 2L). Even though lesions may eventu-
ally coalesce in older leaves of lls1/Oy1-700 double
mutants, plants often survive to maturity under field
conditions, allowing maintenance of lls1 mutants in
homozygous conditions.
Death of lls1 Cells Is Mediated by Chloroplasts
To characterize cellular events that might be caus-
ally involved in the death of lls1 cells, cells in and
around freshly induced lesions were examined by
electron microscopy. To make sure that cells of sim-
ilar age and developmental status were compared,
lesions were induced intentionally by puncturing
leaves that had just acquired developmental compe-
tence to form lls1 lesions. Mutant cells from wound
sites were compared with uninjured cells from the
same leaf, as well as with cells sampled from equiv-
alent punctured and uninjured sites of wild-type
leaves. Although tissue was examined at 21 and 42 h
post-wounding, only images collected at 21 h are
shown. This is because similar ultrastructural
changes were found at both time points, even though
the lesions had progressed farther from the site of
wounding in the 42-h post-wounding tissue.
Both mesophyll and BS cells taken from intact sites
of lls1 mutant leaves were almost indistinguishable
from equivalent cells of wild-type leaves. These leaf
cells exhibited the typical dimorphic anatomy char-
acteristic of C4 metabolism, meaning that the starch-
less, oval chloroplasts of mesophyll cells were highly
granal, whereas the starch-filled, cigar-shaped chlo-
roplasts of BS cells were devoid of grana (Figs. 3C
and 4C). One difference that existed between the
mutant and wild-type cells was the number and size
of starch grains in chloroplasts of BS cells, both of
which were significantly abated in mutant cells
(6.2 3.7 granules choroplast
) compared with the
wild type (10.3 3.3 granules choroplast
; P
0.0023, n 11 and 26 chloroplasts, respectively).
Very striking changes, however, were observed in
cells when the mutant leaf was wounded to incite the
lls1 lesion. The most prominent of these changes was
an alteration in the structure of chloroplasts of me-
sophyll and BS cells alike, although the type of
change differed in the two cell types. Chloroplasts in
mesophyll cells adjacent to the wound-induced lls1
lesion were highly swollen, whereas their thylakoid
membranes possessed a relatively normal organiza-
tion (Fig. 4D). In contrast, chloroplasts of BS cells
adjacent to the wound were grossly distorted and
their thylakoid membranes appeared to have folded
over upon themselves (Fig. 3D). Chloroplasts of BS
and mesophyll cells adjacent to the wound in wild-
type leaves displayed a relatively normal structure
(Figs. 3B and 4B).
A number of other fine structure changes were
witnessed in mutant cells from lls1 lesions, including
an increase in and marginalization of heterochroma-
tin in BS nuclei, a frequently observed hallmark of
apoptosis in animals (Figs. 3, D and F). Some increase
in heterochromatin was also observed in mutant me-
sophyll cell nuclei, but the increase was not as great
as that observed in BS nuclei (data not shown). The
most common morphological change noted in plant
PCD studies, namely the presence of small cytoplas-
mic vacuoles in dying cells, followed by cytoplasmic
condensation (Bestwick et al., 1997; Kosslak et al.,
1997; Mittler et al., 1997; Partington et al., 1999; Wang
et al., 1999) was observed in mesophyll cells. In ad-
dition, the central vacuole seemed to have disap-
peared in all cells adjacent to pinprick wounds, sug-
gesting that tonoplast collapse (Jones, 2001) may also
be a feature of cell death in lls1 plants.
Extensive examination of sections taken from lls1
plants indicated that although cellular alterations
discussed in the preceding paragraph do eventually
emerge in all cells within lls1 lesions, they occur
relatively late in the sequence of events leading to cell
death. In contrast, change in chloroplast structure
was not only the most conspicuous, but also the first
to manifest in lls1 cells. As demonstrated in Figure 4,
A and B, for mesophyll cells, swelling of the chloro-
plast envelope initiates well ahead of the lesion front.
A similar gradient of events was witnessed with
chloroplasts of mutant BS cells (data not shown).
Intriguingly, mitochondria, the organelle that seems
Mediation of lls1 Cell Death by Mature Chloroplasts
Plant Physiol. Vol. 130, 2002 1899
to integrate most apoptotic cell deaths in animals,
maintain a normal structure even in cells displaying
dramatically altered chloroplast structure (Figs. 3D
and 4D; data not shown). Likewise, integrity of Golgi
and endoplasmic reticulum remains unaffected in
dying lls1 cells. A number of other hallmarks of
apoptosis that are not witnessed in dying lls1 cells
include blebbing of membranes and fragmentation of
cells into vesicles (apoptotic bodies). These observa-
tions suggest that lls1 cells collapse because of a cell
death process other than apoptosis.
The LLS1 Gene Is Expressed in Photosynthetic Tissues
The suppression of the lls1 phenotype in non-
photosynthetic tissues suggests that the product of
the Lls1 gene may only be required in photosynthetic
tissues. We examined various maize tissues for the
presence of the Lls1 transcript. First, we determined
by Southern blot that the Lls1 gene is a single-copy
gene in maize as evidenced by the detection of a
DNA fingerprint that is explicable by the sequence of
the cloned B73 Lls1 gene alone (Fig. 5, A and B).
Southern blots washed at low stringencies did not
reveal any other significant cross-hybridizing bands,
so we could use the Lls1 cDNA as a unique probe for
northern analysis. The Lls1 message was almost un-
detectable in leaves when total RNA was used for
northern-blot analysis (data not shown). However,
using enriched poly(A
) mRNA the Lls1 transcript
was readily detectable in young and old leaves of a
mature B73 plant (Fig. 5D) and a low level of tran-
script was also detectable in the leaf sheath of plants.
However, we did not detect the Lls1 message in any
non-photosynthetic tissues including young tassels,
silks, ear tissue, and roots. Thus, we conclude that
Figure 3. Transmission electron microscopy of BS cell chloroplasts and nuclei in injured and uninjured (21 h post-pinprick
wounding) wild-type and lls1 leaves. A, BS cell in uninjured wild-type leaf tissue. B, BS cell adjoining dead cells in injured
wild-type leaf tissue. Asterisk indicates location of dead cell. C, BS cell in uninjured lls1 tissue. D, BS cell adjoining dead
cell in injured lls1 leaf tissue. An increased amount of heterochromatin (arrowhead) is present in the nucleus. Note also the
apparent folding of the thylakoid membranes (arrow). E, Nucleus of a BS cell adjoining a dead cell in injured wild-type leaf
tissue. Asterisk indicates location of dead cell. Note the small amount of heterochromatin. F, Nucleus of a BS cell adjoining
a dead cell in injured lls1 leaf tissue. A large amount of heterochromatin is present in this nucleus (arrowheads). Bars 1
m. n, Nucleus; Nu, nucleolus; C, chloroplast; S, starch granule; M, mitochondrion; V, vacuole; R, rough endoplasmic
reticulum; IS, intercellular space.
Gray et al.
1900 Plant Physiol. Vol. 130, 2002
expression of the Lls1 gene occurs mainly in the
photosynthetic tissues that were demonstrated to be
prone to lesion formation in mutant plants.
LLS1 Is Most Closely Related to Other Chloroplast and
Cyanobacterial Proteins Containing Non-Heme
Iron-Binding Motifs
Examination of the LLS1 amino acid sequence and
that of its Arabidopsis ortholog ACD1 (J. Gray, un-
published data) reveals that even though these pro-
teins have high 72% overall amino acid identity, they
exhibit weak homology at the amino terminus (Fig.
6A). However, using the algorithm ChloroP algo-
rithm (Emanuelsson et al., 1999), both of these pro-
teins are predicted to contain a conserved chloroplast
transit-peptide cleavage site (Fig. 6A), suggesting
that these proteins are targeted to the chloroplast.
The LLS1/ACD1 proteins contain two conserved
functional motifs, a Rieske Fe-sulfur coordinating
center and a non-heme mononuclear Fe-binding site.
Previously, we identified these motifs only in bacte-
rial aromatic ring-hydroxylating dioxygenases,
which catalyze the opening of a phenolic ring (Gray
et al., 1997). A survey of the complete Arabidopsis
genome indicates that there are a total of five Arabi-
dopsis proteins that contain the same two motifs and
four of these plant genes have now been cloned and
studied. In addition to ACD1, these genes include
Cmo (choline monooxygenase), involved in the pro-
duction of the osmoprotectant betaine (Rathina-
sabapathi et al., 1997); Cao (chlorophyll a oxygenase),
Figure 4. Transmission electron microscopy of mesophyll cell chloroplasts and nuclei in injured and uninjured (21 h
post-pinprick wounding) wild-type and lls1 leaves. A, Mesophyll cell chloroplasts in uninjured wild-type leaf tissue. B,
Mesophyll cell chloroplasts adjoining dead cells in injured wild-type leaf tissue. C, Mesophyll cell chloroplasts in uninjured
lls1 leaf tissue. Chloroplasts exhibit granal stacking (not observed in Fig. 3C). D, Mesophyll cell chloroplasts adjoining dead
cells in injured lls1 leaf tissue. Chloroplasts are dramatically swollen as seen by the location of the chloroplast envelope
(arrowheads). The cytoplasm is vacuolated (asterisks), although endoplasmic reticulum, mitochondria, and Golgi (arrows) appear
normal in structure. E, Portions of three mesophyll cells in injured lls1 leaf. The chloroplasts in the cell closest to the injury
(asterisk) are greatly swollen. F, Fine detail of one of the chloroplasts in the adjoining cell. No other ultrastructural alterations are
apparent in this cell except for the swelling visible on one of the cells chloroplasts (arrowhead). Bars 1
m. C, Chloroplast; S,
starch granule; M, mitochondrion; V, vacuole; R, rough endoplasmic reticulum; G, Golgi apparatus; IS, intercellular space.
Mediation of lls1 Cell Death by Mature Chloroplasts
Plant Physiol. Vol. 130, 2002 1901
involved in the conversion of chlorophyll a to chlo-
rophyll b (Tanaka et al., 1998); and Tic55, which codes
for a 55-kD product suspected to be involved in the
translocation of proteins to the inner chloroplast
membrane (Caliebe et al., 1997). The last is an lls1-like
gene on chromosome 4 (see below). Because CMO
and CAO are definitely not involved in phenolic
metabolism, questions can be raised as to whether
the function of LLS1 would be to degrade a phenolic
substrate. A phylogenetic comparison between these
proteins indicates that the LLS1 protein is not a clear
homolog of any of these plant proteins (Fig. 6B). In
Arabidopsis, there are genes more homologous to
CMO and CAO and these are distinct from the Lls1
ortholog Acd1 (Fig. 6B; J. Gray and S. Reinbothe,
unpublished data). In rice (Oryza sativa) and Arabi-
dopsis, we have identified another gene (lls1-like)
that is more closely related to Lls1 than Cao, Cmo,or
Tic55. The LLS1-LLS1-like clade also contains several
open reading frames from the cyanobacteria Synecho-
cystis sp. PCC 6803 and Anabaena sp. PCC 7120,
which have as yet unidentified functions. Only two
bacterial phenolic dioxygenases are included in this
analysis and these clustered in the CMO clade. Other
bacterial dioxygenases proved to be more distantly
related than any of the proteins included here and
were omitted from this cladogram. Our earlier study
indicated that the relationship between LLS1 and
these bacterial phenolic dioxygenases is strictly lim-
ited to the regions containing the Fe-binding motifs
(Gray et al., 1997).
The LLS1-LLS1-like clade appears to be phyloge-
netically equidistant from the CMO, CAO, and TIC55
clades, suggesting that in plants these two Fe-binding
motifs have been recruited toward diverse metabolic
functions. Another important feature of this analysis
is that of all of the plant proteins known to possess
these particular motifs (CAO, CMO, and TIC55) are
known to be localized to the chloroplast or they are
closely related to genes found in cyanobacteria
which in turn are related to the ancestral chloroplast
endosymbiont. Together, these observations lend
support to the proposition that LLS1 evolved to pro-
vide its positive homeostatic function within the
Previous studies on lls1 demonstrated that the de-
velopmentally specified phenotype of this mutation
results from a cell death program that manifests in-
tracellularly in a cell-autonomous manner (Johal et
al., 1995; Gray et al., 1997; Simmons et al., 1998). The
work presented here provides insights into factors
and mechanisms that mediate the initiation and
propagation of lls1 lesions and the nature of cellular
events that result in the demise of an lls1 cell.
Loss of Chloroplast Structural Integrity Is a Key
Event in lls1 Cell Death
A key finding of this study is that the loss of
structural integrity of chloroplasts is the most con-
spicuous feature of dying lls1 cells, and the swelling
of this organelle appears to be the first discernible
structural event that takes place in mesophyll cells
Figure 5. The Lls1 gene is a single-copy gene and the Lls1 transcript
is expressed in photosynthetic tissues. A, Southern-blot analysis.
Maize B73 DNA was digested with the indicated enzymes, blotted,
and probed with an Lls1 cDNA probe. Most lanes exhibit a single
band and those with multiple bands are explicable by multiple
restriction sites in the B73 structural gene. Size standards are indi-
cated in kb. B, Gene structure of the maize Lls1 gene. The intron/
exon structure of the Lls1 gene was determined by comparing the
sequence of the B73 genomic sequence with an Lls1 cDNA se-
quence. Blocks indicate exons. Restriction sites: RI, EcoRI; HIII,
HindIII; PI, PstI; and SI, SalI. C, Poly(A
)-enriched RNA from the
various maize tissues was subjected to northern analysis using maize
Lls1 cDNA as probe. One microgram of poly(A
) RNA was loaded
per lane except for root tissue, which was deliberately overloaded.
Picture of ethidium bromide-stained gel shows near equal loading of
samples from indicated tissues of a mature 13-leaf B73 plant. D,
Northern blot showing that a unique lls1 transcript is detectable in
fully photosynthetic green leaves and at a lower level in leaf sheath
but not in other tissues. E, To normalize RNA loading, the blot was
stripped and rehybridized with a maize actin probe. This experiment
was repeated twice with similar results.
Gray et al.
1902 Plant Physiol. Vol. 130, 2002
before they die. Chloroplast swelling reflects the loss
of differential permeability of its envelope mem-
branes and may result from changes occurring within
the chloroplast such as photooxidation, a change in
pH, or a loss of energy production (Wise and Cook,
1998; Mostowska, 1999). An intriguing difference
was noticed in the way lls1 affected chloroplasts of
the two cell types of the maize leaf. Although grossly
disorganized, there was less evidence of chloroplast
swelling in the BS cells of lls1 leaves. This may be
because of the differential sensitivities of the chloro-
plast types to ROS or a differential ability of the BS
chloroplasts to produce ROS because they lack the
oxygen-generating PSII found in mesophyll chloro-
plasts (Pfuendel and Meister, 1996; Kingston-Smith
and Foyer, 2000). Lower ROS production could result
in less damage to BS cells and this could explain why
the autocatalytic spread of lls1 lesions is retarded
near vascular elements.
In animals, mitochondrial membrane changes re-
sulting in mitochondrial swelling have been noted in
apoptotic cells that aid the generation of ROS and in
the release of a number of apoptogenic factors (such
as cytochrome c and apoptosis-inducing factor) from
the intermembrane space of mitochondria to the cy-
tosol (Hengartner, 2000; Simon et al., 2000; Von Ah-
sen et al., 2000). It is tempting to think that similar
cell death promoting factors may be released in a
parallel fashion from chloroplasts although so far
none have been identified. Distortion of the chloro-
plast has been reported in other plant cell deaths
(Mou et al., 2000), but the timing of such change has
not been established and it may be a late event. In
this study, however, we document that this change is
an early and apparently causal event in the cell death
LLS1 Suppresses Spread of Cell Death Initiated by
Multiple Biotic and Abiotic Factors
We found that lls1 lesions can be triggered by
cellular damage inflicted by a number of agents such
as pathogen ingress, physical wounding, and meta-
bolic disorder caused by another genetic lesion. The
Figure 6. Predictive targeting of LLS1 to chloro-
plast and phylogenetic comparison of LLS1 with
non-heme Fe-binding proteins from bacteria
and plants. A, Alignment of amino termini of
LLS1 and ACD1 proteins shows low conserva-
tion of sequence in this region. Arrow indicates
the conserved cleavage site for a chloroplast
transit peptide as predicted using the ChloroP
algorithm. B, Cladogram of consensus tree ob-
tained from maximal parsimony bootstrap anal-
ysis using the indicated proteins as operational
taxonomic units. The consensus tree recon-
structs the evolutionary relationship between
LLS1 and other non-heme Fe-binding proteins.
The proteins are labeled by species name or
bacterial strain number in which they are found
(for accession nos. and biocomputational meth-
ods, see Materials and Methods). Clades of
related proteins are color shaded as follows: red,
LLS1 and LLS1-like homologs in various plants
and cyanobacteria; black, bacterial ring hy-
droxylating enzymes; purple, plant choline
monooxygenase (CMO) enzymes; green, plant
and cyanobacterial chlorophyll a oxygenase
CAO enzymes; blue, pea (Pisum sativum) TIC55
Rieske Fe-sulfur protein putatively associated
with transport through inner chloroplast mem-
brane and related plant proteins.
Mediation of lls1 Cell Death by Mature Chloroplasts
Plant Physiol. Vol. 130, 2002 1903
convergence of multiple triggers, inducing lls1 cell
death, might be explained if lls1 cells are predisposed
to damage because a cell compartment such as the
chloroplast is already compromised and sensitized
toward a common cell death mediator. One class of
candidates that fits well in this role are ROS, which
are known to act as partly diffusible stress signals
during diverse provocations in plants, including HR,
lesion mimicry, and wounding (Hippeli et al., 1999;
Jabs, 1999; Kliebenstein et al., 1999; Mittler et al.,
1999; Heath, 2000). For example, loss-of-function mu-
tations of the Arabidopsis Lsd1 fail to up-regulate
superoxide dismutases in response to salicylic acid
signaling. Failure to detoxify accumulating superox-
ide gives rise to propagative lesions similar to the
maize lls1 mutation (Kliebenstein et al., 1999). In
contrast to lls1, however, lsd1 lesions cannot be in-
duced by mechanical wounding, indicating different
modes of location or operation for the gene products.
It may be that LLS1, like LSD1, is also suppressing
the production or action of ROS but it may operate
more directly and in a specific cell compartment such
as the chloroplast.
Propagation of Cell Death in lls1 Lesions Is
Light Dependent
Although a cellular injury is required for initiation
of an lls1 lesion, its enlargement exclusively depends
on the availability of light. The data with light filters
and albino-sectored plants indicate that only light
above a certain fluence rate that is captured by green
photosynthetic tissue is driving the expression of lls1
lesions. Light is known to exacerbate cell death in
plants and this has been documented in the case of
HR, in many lesion mimic mutations (below), in bar-
ley (Hordeum vulgare) aleurone cells, and in
fumonisin-induced cell death in Arabidopsis (Jabs et
al., 1996; Hu et al., 1998; Mock et al., 1998; Shirasu
and Schulze-Lefert, 2000; Stone et al., 2000; Mach et
al., 2001). One way that cell death is promoted is
through the production of free radicals by light (Hip-
peli et al., 1999; Jabs, 1999). For instance, in the case
of Les22 plants, the production of excess porphyrin
free radicals is the precipitating cause of cell death
(Hu et al., 1998; Mock et al., 1998). Similarly, in acd2
plants, it is the photo-activation of the red chloro-
phyll catabolite that triggers free radical production
and subsequent cell death (Mach et al., 2001). The fact
that lls1 lesions do not develop in cells lacking chlo-
rophyll is consistent with the idea that death of lls1
cells occurs in a similar fashion. The inability to
remove photo-activatable chlorophyll intermediates
could explain the developmentally regulated and
wound-induced formation of lesions only in green
tissue. Support for this interpretation is also reflected
in the reduction of the lls1 phenotype in chlorophyll-
deficient lls1/Oy and lls1/ij double mutants. Inability
to remove photo-activated pigments could also result
in the autocatalytic production of ROS and such an
event is compatible with the observation that the
signal causing propagation of lls1 lesions is some-
what diffusible (Johal et al., 1995; Dangl et al., 1996).
LLS1 Is Most Closely Related to Chloroplast and
Cyanobacterial Proteins
Because chloroplast structural alterations appear to
be an early feature of lls1 cell death, it is likely that
chloroplasts are the primary site of action of LLS1.
The fact that we could detect the Lls1 transcript only
in photosynthetic tissue supports this proposition.
We also found that the LLS1 protein and the dicot
ortholog from Arabidopsis (ACD1) are both pre-
dicted to contain a conserved chloroplast transit pep-
tide cleavage site. The LLS1 gene itself is highly
conserved in all plant species examined thus far and
the LLS1 protein is closely related to four other plant
proteins that are known to function in the chloro-
plast. None of these are known to be involved in
phenolic metabolism, which weakens our original
hypothesis that LLS1 might remove a phototoxic
phenolic compound (Gray et al., 1997). An alternative
possibility is that LLS1 catalyzes the removal of an-
other photosensitive metabolite (chlorophyll?) or
regulates such a process by virtue of the redox-
sensing Fe-binding motifs that it contains. Our phy-
logenetic comparison suggests that the LLS1-related
plant proteins have been recruited toward diverse
metabolic functions but these functions appear re-
stricted to the chloroplast compartment and may
have evolved from ancestral endosymbiont genes.
In conclusion, we present evidence to show that
LLS1 functions in plants to protect the integrity of the
chloroplast compartment after biotic or abiotic stress.
In the absence of this protective function, light en-
ergy is used directly or indirectly to produce a cell
death mediator (possibly ROS or a phototoxic chlo-
rophyll intermediate) that damages the chloroplast.
Chloroplast destabilization then plays a central role
in precipitating propagative cell death because leak-
age of these cell death mediators triggers death in
neighboring photosynthetic cells. The protective
function of LLS1 may have evolved in ancestral cya-
nobacteria and is conserved now in all photosyn-
thetic organisms. We believe that whether or not
chloroplast dysfunction is regulated during PCD
events, it will be a useful model for reexamining
other forms of rapid cell death in plants, particularly
those that occur in photosynthetic tissue.
Plant Material
The reference allele for lls1 was obtained from the Maize Genetics Co-
operation Stock Center (University of Illinois, Urbana-Champaign). The
Gray et al.
1904 Plant Physiol. Vol. 130, 2002
NCS2 mutation was provided by Kathy J. Newton (University of Missouri,
Columbia). The oy-700 mutation was provided by Gerald Neuffer (Univer-
sity of Missouri, Columbia). The ij mutation was provided by Edward Coe
Jr. (U.S. Department of Agriculture, University of Missouri, Columbia).
Histochemistry and Autofluorescence Microscopy
Leaves for callose studies were prepared for examination according to the
method of Eschrich and Currier (1964). Leaf pieces were mounted in Moviol
and examined using UV epifluorescence (excitation filter, 365 nm; dichroic
mirror, 395 nm; and barrier filter, 420 nm). Captured images were color
enhanced using Adobe Photoshop (Adobe Systems, San Jose, CA). Whole
leaf pieces were examined directly for blue light autofluorescence using a
standard fluorescein isothiocyanate filter set (excitation filter, 450490 nm;
dichroic mirror, 505 nm; and barrier filter, 520 nm). Trypan blue staining
was performed on fresh leaf sections as described by Yin et al. (2000), except
that we stained and then destained for 12 h.
Electron Microscopy
The seventh leaves of 4-week-old wild-type (Lls1/Lls1 or Lls1/lls1) and
homozygous lls1 plants were wounded via pinpricking on one side of the
mid-rib. At 21 and 42 h, leaf tissue was excised at the wound and, for
uninjured tissue, on the opposite side of the mid-rib. Two wild-type and two
lls1 plants were examined at both time points. Tissue was excised under and
fixed in 2.5% (v/v) glutaraldehyde in 100 mm sodium cacodylate buffer (pH
6.9)for2.5hat4°C. Tissue samples were postfixed for2hin1%(w/v) OsO
and 100 mm sodium cacodylate buffer (pH 6.9). The tissues were then
stained en bloc in 2% (w/v) aqueous uranyl acetate for 1 h, washed in
deionized, distilled water, and dehydrated through an ethanol series. After
infiltration through a graded propylene oxide/Spurrs epoxy resin series,
the tissues were embedded in 100% (w/v) Spurrs epoxy resin and poly-
merized at 60°C for 24 h. Ultrathin sections were prepared using a diamond
knife on an 8800 Ultratome III (LKB Instruments, Inc., Gaithersburg, MD),
and stained with uranyl acetate and lead citrate. The stained sections were
examined on a JEM-1200EX transmission electron microscope (JEOL, Ltd.,
Akishima, Japan). Images were recorded on 4489 film (Eastman-Kodak,
Rochester, NY). The statistical significance of variations in the number of
starch granules per chloroplast was performed by applying a Student t test
analysis (two tailed, unpaired) on 11 mutant and 26 normal chloroplasts
images, respectively.
Southern- and Northern-Blot Analyses
DNA was isolated from B73 maize (Zea mays) leaf tissue using a
cetyltrimethylammoniumbromide-based method (Hulbert and Bennetzen,
1991). Southern-blot analysis was performed essentially as described by
Gardiner et al. (1993) and the blot hybridized using a partial Lls1 cDNA
clone (pJG200) as a probe. Tissue for RNA isolation was frozen in liquid
nitrogen, ground to a fine powder, and added to premeasured denaturation
and extraction solution (2.0 m guanidine thiocyanate, 0.6 m ammonium
thiocyanate, 0.2 m sodium acetate [from 2 m stock, pH 4.0], 8% [w/v]
glycerol, and 50% [w/v] phenol [water saturated, pH 4.3 0.3]). Samples
were vortexed and organic phase separation was effected by the addition of
0.2 volumes of chloroform per volume of extraction solution employed.
RNA was isopropanol precipitated from the aqueous phase, washed with
70% (w/v) ethanol, and resuspended in RNAase-free water. Poly(A
enriched RNA was isolated from total RNA samples using oligo(dT)-
cellulose [MicroPoly(A) Pure Kit, Ambion, Austin, TX]. RNA samples were
subjected to northern-blot analysis using a 50% (w/v) formamide hybrid-
ization solution (Ausubel et al., 1994). The same cDNA probe as above was
used to detect the maize lls1 transcript.
Analysis of Light Requirement for lls1
Lesion Development
To determine the spectral range of light required for lesion formation,
sections of leaves were clamped between 0.125-inch plexiglas GM filters
held in place by a metal stand with a side arm clamp. The following
transparent filters were used: plexiglas GM 2,423 (red), 2,711 (far red), 2,424
(blue), 2,092 (green), 2,208 (yellow), and 2,422 (amber) or clear (Cope Plastics
Inc., St. Louis). Transmission spectra of filters were determined by examin-
ing small sections of filters in a spectrophotometer. Leaf sections of green-
house or field-grown plants were covered in aluminum foil to completely
reflect incident light. After complete lesioning of a leaf, filters were removed
to observe if lesioning had occurred in the covered region. For the estima-
tions of lesion densities in lls1, Les, and Oy/ leaves, the leaves were
photographed side by side and lesion density expressed per unit area
these photographs. The numbers of lesions were counted in equivalent
regions along the length of lesioned leaves or similar developmental age and
Biocomputational Methods and Data Sources
The amino acid sequences of 28 listed enzymes (below) were predicted
from available GenBank files. The neighborhood search algorithm BLAST
(Altschul et al., 1990) was employed for database searches using the World
Wide Web BLAST servers of the National Center for Biotechnology Infor-
mation and The Arabidopsis Information Resource. In addition, rice (Oryza
sativa) genomic sequences were retrieved from the Rice Genome Database
( and from the Syngenta Torrey Mesa Research
Institute rice genome project ( The ChloroP
(Emanuelsson et al., 1999) algorithm was employed to predict the cellular
localization of proteins. The entire proteins were aligned using with the
ClustalV method with PAM250 residue weights within the MegAlign pro-
gram (DNAStar, Madison, WI). Unrooted cladograms were generated by
using the PAUP 4.0b10 program (Swofford, 2001). The bootstrap method
was performed for 100 replicates with a maximum parsimony criterion. All
characters were weighted equally. Starting trees were obtained by random
stepwise addition and the tree-bisection-reconnection algorithm was used
for branch swapping.
Accession Numbers
Accession numbers used for the CMO (choline monooxygenase) clade are as
follows: Arabidopsis CMO precursor, T08550; spinach (Spinacia oleracea)
CMO, T09214; beet (Beta vulgaris) CMO precursor, T14542; and rice CMO,
AAAA00000000 (contig no. 180). Accession numbers used for the CAO (chlo-
rophyll a oxygenase, chlorophyll b synthase) clade are as follows: Arabidopsis
CAO (Arabidopsis), AAD54323; rice CAO, AAAA00000000 (contig no. 19995;
Yu et al., 2002), AC087599, D48708, BAA82479, and AB021310; Dunaliella salina
CAO, BAA82481; Chlamydomonas reinhardii CAO, BAA33964; liverwort (March-
antia polymorpha) CAO, BAA82480; Prochloron didemni CAO, BAA82483; and
Prochlorothrix hollandica CAO, BAA82482. Accession numbers used for the LLs1
and LLs1-like clade are as follows: maize LLS1 lethal leaf-spot 1, U77346 (genomic
clone pJG201) and AAC49676 (partial cDNA clone pJG200; Gray et al., 1997);
Arabidopsis ACD1 accelerated cell death 1 (Lls1 ortholog), AL391254, protein
identification CAC03538.1; Rice LLS1 homolog, AAAA00000000 (contig no.
6404 and Syngenta contig no. CLB11460.2,; Goff et
al., 2002); Medicago truncatula LLS1 homolog, contig of overlapping expressed
sequence tags AW257191, BE248884, BE249137, BF005787, BF633506, BF634038,
BF634446, BF636009, and BF642558; Arabidopsis LLS1-like gene L73G19.30,
AL050400; Anabaena sp. alr4354 LLs1-like homolog (Nostoc sp. PCC 7120), NP
488394; Anabaena sp. alr7348 LLs1-like homolog (Nostoc sp. PCC 7120),
NP 490454; Anabaena sp. alr2097 LLs1-like homolog (Nostoc sp. PCC 7120), NP
486137; and Anabaena sp. alr5007 LLs1-like homolog (Nostoc sp. PCC 7120),
NP 489047. Accession numbers used for the Tic55 clade are as follows: pea
(Pisum sativum) TIC55 (55-kD Rieske [2Fe-2S] Fe-sulfur protein putatively
associated with transport through inner chloroplast membrane), AC006585;
Arabidopsis TIC55 homolog, AC006585 and contig of overlapping expressed
sequence tags AI995341, AV439633, AV442063, BE038210, BE528579, and
protein identification AAD23030.1; and rice TIC55 homolog, AAAA00000000
(contig no. 29820). Accession numbers used for other sequences are as follows:
G7 NahAc naphthalene 1,2-dioxygenase Fe-sulfur oxygenase component
large chain, Pseudomonas putida (strain G7), JN0644; 9816-4 NahAc naphtha-
lene dioxygenase ISP alpha subunit (Pseudomonas sp.), AAA92141; Synecho-
cystis sp. PCC6803 slr1747 hypothetical protein, BAA17786.1; and Synechocys-
tis sp. PCC6803 sll1869 hypothetical protein putative 3-chlorobenzoate-3,4-
dioxygenase, BAA18227.
Mediation of lls1 Cell Death by Mature Chloroplasts
Plant Physiol. Vol. 130, 2002 1905
Distribution of Materials
Upon request, all novel materials described in this publication will be
made available in a timely manner for noncommercial research purposes,
subject to the requisite permission from any third party owners of all or
parts of the material. Obtaining any permissions will be the responsibility of
the requestor.
All electron microscopy work was carried out at the Electron Microscopy
Core Facility (University of Missouri, Columbia). The authors wish to
acknowledge Cheryl Jensen (Electron Microscopy Core Facility) for thin
sectioning all samples used in the study. The electron microscopy work
described in this manuscript was carried out by D.J.-B. and B.B. while they
were on sabbatical leave in the laboratory of G.S.J. in the Department of
Agronomy at the University of Missouri (Columbia). The authors thank
Stephen Goldman (University of Toledo, Plant Science Growth Center) for
the use of facilities used for some of the analysis in this research. We thank
Yang Manli (University of Toledo) for technical assistance in microscopic
Received May 13, 2002; returned for revision June 25, 2002; accepted August
30, 2002.
Aist JR, Gold RE, Bayles CJ, Morrison GH, Chandra S, Israel HW (1988)
Evidence that molecular components of papillae may be involved in ml-o
resistance to barley powdery mildew. Physiol Mol Plant Pathol 33: 1732
Altschul SF, Gish W, Miller W, Myers E, Lipman D (1990) Basic local
alignment search tool. J Mol Biol 215: 403410
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA,
Struhl K (1994) Current Protocols in Molecular Biology. John Wiley &
Sons, Inc., New York
Bestwick CS, Brown IR, Bennett MHR, Mansfield JW (1997) Localization
of hydrogen peroxide accumulation during the hypersensitive reaction of
lettuce cells to Pseudomonas syringae pv phaseolicola. Plant Cell 9: 209221
Brodersen P, Petersen M, Pike HM, Olszak B, Skov S, Odum N, Jorgensen
LB, Brown RE, Mundy J (2002) Knockout of Arabidopsis ACCELERATED-
CELL-DEATH11 encoding a sphingosine transfer protein causes activation
of programmed cell death and defense. Genes Dev 16: 490502
Buckner B, Johal GS, Janick-Buckner D (2000) Cell death in maize. Physiol
Plant 108: 231239
Bu¨ schges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A,
Van Daelen R, Van Der Lee T, Diergarde P, Groenendijk J et al. (1997)
The barley Mlo gene: a novel control element of plant pathogen resis-
tance. Cell 88: 695705
Caliebe A, Grimm R, Kaiser G, Lubeck J, Soll J, Heins L (1997) The
chloroplastic protein import machinery contains a Rieske-type iron-
sulfur cluster and a mononuclear iron-binding protein. EMBO J 16:
Collins N, Drake J, Ayliffe M, Sun Q, Ellis J, Hulbert S, Pryor T (1999)
Molecular characterization of the maize Rp1-D rust resistance haplotype
and its mutants. Plant Cell 11: 13651376
Dangl JL, Dietrich RA, Richberg MH (1996) Death dont have no mercy:
cell death programs in plant-microbe interactions. Plant Cell 8: 17931807
Devoto A, Piffanelli P, Nilsson I, Wallin E, Panstruga R, von Heijne G,
Schulze-Lefert P (1999) Topology, subcellular localization, and sequence
diversity of the Mlo family in plants. J Biol Chem 274: 3499335004
Dietrich RA, Delaney TP, Uknes SJ, Ward ER, Ryals JA, Dangl JL (1994)
Arabidopsis mutants simulating disease resistance response. Cell 77:
Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural
network-based method for predicting chloroplast transit peptides and
their cleavage sites. Prot Sci 8: 978984
Eschrich W, Currier HB (1964) Identification of callose by its diachrome and
flourochrome reactions. Stain Technol 39: 303307
Gardiner JM, Coe EH, Melia-Hancock S, Hoisington DA, Chao S (1993).
Development of a core RFLP map in maize using an immortalized F2
population. Genetics 134: 917930
Goff SA, Ricke D, Lan T-H, Presting G, Wang R, Dunn M, Glazebrook J,
Sessions A, Oeller P, Varma H et al. (2002) A draft sequence of the rice
genome (Oryza sativa L. ssp. japonica). Science 296: 92100
Gray J, Close PS, Briggs SP, Johal GS (1997) A novel suppressor of cell
death in plants encoded by the Lls1 gene of maize. Cell 89: 2531
Gray J, Johal GS (1998) Programmed cell death in plants. In M Anderson,
J Roberts, eds, Arabidopsis. Sheffield Academic Press, Sheffield, UK, pp
Greenberg JT (1997) Programmed cell death in plant-pathogen interactions.
Annu Rev Plant Physiol Plant Mol Biol 48: 525545
Greenberg JT, Ausubel FM (1993) Arabidopsis mutants compromised for
the control of cellular damage during pathogenesis and aging. Plant J 4:
Haecker G (2000) The morphology of apoptosis. Cell Tissue Res 301: 517
Hammond-Kosack KE, Jones JDG (1996) Resistance gene-dependent plant
defense responses. Plant Cell 8: 17731791
Han CD, Coe EH, Martienssen RA (1992) Molecular cloning and charac-
terization of iojap (ij), a pattern striping gene of maize. EMBO J 11:
Heath MC (2000) Hypersensitive response-related death. Plant Mol Biol 44:
Hengartner MO (2000) The biochemistry of apoptosis. Nature 407: 770776
Hippeli S, Heiser I, Elstner EF (1999) Activated oxygen and free oxygen
radicals in pathology: new insights and analogies between animals and
plants. Plant Physiol Biochem 37: 167178
Hu G, Richter TE, Hulbert SH, Pryor T (1996) Disease lesion mimicry
caused by mutations in the rust resistance gene rp1. Plant Cell 8:
Hu G, Yalpani N, Briggs SP, Johal GS (1998) A porphyrin pathway im-
pairment is responsible for the phenotype of a dominant disease lesion
mimic mutant of maize. Plant Cell 10: 10951105
Hulbert SH, Bennetzen JL (1991) Recombination at the Rp1 locus of maize.
Mol Gen Genet 226: 377382
Ishikawa A, Okamoto H, Iwasaki Y, Asahi T (2001) A deficiency of cop-
roporphyrinogen III oxidase causes lesion formation in Arabidopsis.
Plant J 27: 8999
Jabs T (1999) Reactive oxygen intermediates as mediators of programmed
cell death in plants and animals. Biochem Pharmacol 57: 231245
Jabs T, Dietrich RA, Dangl JL (1996) Initiation of runaway cell death in an
Arabidopsis mutant by extracellular superoxide. Science 273: 18531856
Johal GS, Briggs SP (1992) Reductase activity encoded by the HM1 disease
resistance gene in maize. Science 258: 985987
Johal GS, Hulbert S, Briggs SP (1995) Disease lesion mimics of maize: a
model for cell death in plants. Bioessays 17: 685692
Johal GS, Lee EA, Close PS, Coe EH, Neuffer MG, Briggs SP (1994) A tale
of two mimics: transposon mutagenesis and characterization of two
disease lesion mimic mutations of maize. Maydica 39: 6976
Jones AM (2001) Programmed cell death in development and defense. Plant
Physiol 125: 9497
Kingston-Smith AH, Foyer CH (2000) Bundle sheath proteins are more
sensitive to oxidative damage than those of the mesophyll in maize
leaves exposed to paraquat or low temperatures. J Exp Bot 51: 123130
Kliebenstein DJ, Dietrich RA, Martin AC, Last RL, Dangl JL (1999) LSD1
regulates salicylic acid induction of copper zinc superoxide dismutase in
Arabidopsis thaliana. Mol Plant-Microbe Interact 12: 10221026
Koga H, Zeyen RJ, Bushnell WR, Ahlstrand GG (1988) Hypersensitive cell
death, autofluorescence, and insoluble silicon accumulation in barley leaf
epidermal cells under attack by Ersiphe graminis f. sp. hordei. Physiol Mol
Plant Pathol 32: 395409
Kosslak RM, Chamberlin MA, Palmer RG, Bowen BA (1997) Programmed
cell death in the root cortex of soybean root necrosis mutants. Plant J 11:
Mach JM, Castillo AR, Hoogstraten R, Greenberg JT (2001) The
Arabidopsis-accelerated cell death gene ACD2 encodes red chlorophyll
catabolite reductase and suppresses the spread of disease symptoms.
Proc Natl Acad Sci USA 98: 771776
Majno G, Joris I (1995) Apoptosis, oncosis, and necrosis. An overview of
cell death. Am J Pathol 146: 315
Marienfeld JR, Newton KJ (1994) The maize NCS2 abnormal growth mu-
tant has a chimeric nad4-nad7 mitochondrial gene and is associated with
reduced complex I function. Genetics 138: 855863
Mittler R, Herr EH, Orvar BL, van Camp W, Willekens H, Inze´ D, Ellis BE
(1999) Transgenic tobacco plants with reduced capability to detoxify
Gray et al.
1906 Plant Physiol. Vol. 130, 2002
reactive oxygen intermediates are hyperresponsive to pathogen infection.
Proc Natl Acad Sci USA 96: 1416514170
Mittler R, Simon L, Lam E (1997) Pathogen-induced programmed cell death
in tobacco. J Cell Sci 110: 13331344
Mock HP, Keetman U, Kruse E, Rank B, Grimm B (1998) Defense responses
to tetrapyrrole-induced oxidative stress in transgenic plants with re-
duced uroporphyrinogen decarboxylase or coproporphyrinogen oxidase
activity. Plant Physiol 116: 107116
Molina A, Volrath S, Guyer D, Maleck K, Ryals J, Ward E (1999) Inhibition
of protoporphyrinogen oxidase expression in Arabidopsis causes a
lesion-mimic phenotype that induces systemic acquired resistance. Plant
J 17: 667678
Morel J-B, Dangl JF (1997) The hypersensitive response and the induction
of cell death in plants. Cell Death Diff 4: 671683
Morris SW, Vernooij B, Titatarn S, Starrett M, Thomas S, Wiltse CC,
Frederiksen RA, Bhandhufalck A, Hulbert S, Uknes S (1998) Induced
resistance responses in maize. Mol Plant-Microbe Interact 11: 643658
Mostowska A (1999) Response of chloroplast structure to photodynamic
herbicides and high oxygen. Z Naturforsch C J Biosci 54: 621628
Mou Z, He Y, Dai Y, Liu X, Li J (2000) Deficiency in fatty acid synthase leads
to premature cell death and dramatic alterations in plant morphology.
Plant Cell 12: 405417
Neuffer MG, Coe EH, Wessler SR (1997) Gene descriptions. In Mutants of
Maize. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp
Partington JC, Smith C, Bolwell GP (1999) Changes in the location of
polyphenol oxidase in potato (Solanum tuberosum L.) tuber during cell
death in response to impact injury: comparison with wound tissue.
Planta 207: 449460
Pfuendel E, Meister A (1996) Flow cytometry of mesophyll and bundle
sheath chloroplast thylakoids of maize (Zea mays L.). Cytometry 23:
Polacco ML, Walden DB (1987) Genetic, developmental, and environmental
influences on Oy-700 expression in maize. J Hered 78: 8186
Rate DN, Cuenca JV, Bowman GR, Guttman DS, and Greenberg JT (1999)
The gain-of-function Arabidopsis acd6 mutant reveals novel regulation
and function of the salicylic acid signaling pathway in controlling cell
death, defenses, and cell growth. Plant Cell 11: 16951708
Rathinasabapathi B, Burnet M, Russell BL, Gage DA, Liao P-C, Nye GJ,
Scott P, Golbeck JH, Hanson AD (1997) Choline monooxygenase, an
unusual iron-sulfur enzyme catalyzing the first step of glycine betaine
synthesis in plants: prosthetic group characterization and cDNA cloning.
Proc Natl Acad Sci USA 94: 34543458
Shirasu K, Schulze-Lefert P (2000) Regulators of cell death in disease
resistance. Plant Mol Biol 44: 371385
Simmons C, Hantke S, Grant S, Johal GS, Briggs S (1998) The maize lethal
leaf spot1 mutant has elevated resistance to fungal infection at the leaf
epidermis. Mol Plant-Microbe Interact 472: 11101118
Simon H-U, Haj-Yehia A, Levi-Schaffer F (2000) Role of reactive oxygen
species (ROS) in apoptosis induction. Apoptosis 5: 415418
Stone JM, Heard JE, Asai T, Ausubel FM (2000) Simulation of fungal-
mediated cell death by fumonisin B1 and selection of fumonisin B1-
resistant (fbr) Arabidopsis mutants. Plant Cell 12: 18111822
Sun Q, Collins NC, Ayliffe M, Smith SM, Drake J, Pryor T, Hulbert SH
(2001) Recombination between paralogues at the rp1 rust resistance locus
in maize. Genetics 158: 423438
Swofford DL (2001) PAUP*: Phylogenetic Analysis Using Parsimony (*and
Other Methods. Sinauer Associates, Sunderland, MA
Tanaka A, Ito H, Tanaka R, Tanaka N, Yoshida K, Okada K (1998)
Chlorophyll a oxygenase is involved in chlorophyll b formation from
chlorophyll a. Proc Natl Acad Sci USA 95: 1271912723
Thompson D, Walbot V, Coe EHJ (1983) Plastid development in iojap- and
chloroplast mutator-affected maize plants. Am J Bot 70: 940950
Ullstrup AJ, Troyer AF (1967) A lethal leaf spot of maize. Phytopathology
57: 12821283
Von Ahsen O, Waterhouse NJ, Kuwana T, Newmeyer DD, Green DR (2000)
The harmless release of cytochrome c. Cell Death Diff 7: 11921199
Walbot V, Hoisington DA, Neuffer MG (1983) Disease lesion mimic mu-
tations. In T Kosuge, CP Meredith, A Hollaender, eds, Genetic Engineer-
ing of Plants. Plenum Publishing Corp., New York, pp 431442
Wang M, Hoekstra S, van Bergen S, Lamers GEM, Oppedijk BJ, van der
Heijden MW, de Priester W, Schilperoort RA (1999) Apoptosis in de-
veloping anthers and the role of ABA in this process during androgenesis
in Hordeum vulgare L. Plant Mol Biol 39: 489501
Wise RR, Cook WB (1998) Development of ultrastructural damage to chlo-
roplasts in a plastoquinone-deficient mutant of maize. Environ Exp Bot
40: 221228
Yin Z, Chen J, Zeng L, Goh M, Leung H, Khush GS, Wang G-L (2000)
Characterizing rice lesion mimic mutants and identifying a mutant with
broad-spectrum resistance to rice blast and bacterial blight. Mol Plant-
Microbe Interact 13: 869876
Yu J, Hu S, Wang J, Wong GK-S, Li S, Liu B, Deng Y, Dai L, Zhou Y, Zhang
Xetal.(2002) A draft sequence of the rice genome (Oryza sativa L. ssp.
indica). Science 296: 7992
Mediation of lls1 Cell Death by Mature Chloroplasts
Plant Physiol. Vol. 130, 2002 1907
    • "In the NAM population analysis, of the 18 associated SNPs, 6, 3 and 9 associated loci 160 were localized within coding sequence, less than 5 kbp from the coding sequence, and between response, cell wall modification and vesicle trafficking/remodeling (Table 1). Gray et al., 2002; Penning et al., 170 2004; Negeri et al., 2013). To establish if variation in light intensity had an effect on the severity were consistently detected in the non-flecking lines, with significantly elevated levels detected in 184 the lines with low flecking. "
    [Show abstract] [Hide abstract] ABSTRACT: Physiological leaf spotting, or flecking, is a mild lesion phenotype observed on the leaves of several commonly used maize inbred lines and has been anecdotally linked to enhanced broad-spectrum disease resistance. Flecking was assessed in the maize nested association mapping (NAM) population, comprising 4998 recombinant inbred lines from 25 bi-parental families, and in an association population comprised of 279 diverse maize inbreds. Joint family linkage analysis was conducted with 7386 markers in the NAM population. Genome-wide association tests were performed with 26.5 million SNPs in NAM population and with 246,497 SNPs in the association population, resulting in the identification of, respectively, 18 and three loci associated with variaton in flecking,. Many of the candidate genes co-localizing with associated SNPs are similar to genes that function in plant defense response via cell wall modification, salicylic acid and jasmonic acid-dependent pathways, redox homeostasis, stress response and vesicle trafficking/remodeling. Significant positive correlations were found between increased flecking, stronger defense response and increased disease resistance and increased pest resistance. A non-linear realtionship with total kernel weight was also observed whereby lines with relatively high levels levels of flecking had, on average, lower total kernel weight. We present evidence suggesting that mild flecking could be used as a selection criterion for breeding programs trying to incorporate broad-spectrum disease resistance.
    Article · Sep 2016
    • "The Rieske-type 307 iron-sulfur cluster and the mononuclear iron site present in this protein were considered as parts of a 308 hypothetical electron transport chain at the inner envelope with potential final transfer of electrons to 309 oxygen, thus integrating protein import to the redox regulatory network of chloroplasts (Bölter et al., 310 2015). However, potential substrates for TIC55 that, as a genuine Rieske-type oxygenase (Ferraro et al., 311 2005)The Arabidopsis genome encodes five Rieske-type oxygenases (Gray et al., 2002). With the identification 316 of TIC55 as a phyllobilin hydroxylase reported here, four out of these five have been attributed to 317 chlorophyll metabolism. "
    [Show abstract] [Hide abstract] ABSTRACT: Chlorophyll degradation is the most obvious hallmark of leaf senescence. Phyllobilins, linear tetrapyrroles that are derived from opening of the chlorin macrocycle by the Rieske-type oxygenase PHEOPHORBIDE a OXYGENASE (PAO), are the end products of chlorophyll degradation. Phyllobilins carry defined modifications at several peripheral positions within the tetrapyrrole backbone. While most of these modifications are species-specific, hydroxylation at the C32 position is commonly found in all species analyzed to date. We demonstrate that this hydroxylation occurs in senescent chloroplasts of Arabidopsis thaliana. Using bell pepper (Capsicum annuum) chromoplasts, we establish that phyllobilin hydroxylation is catalyzed by a membrane-bound, molecular oxygen- and ferredoxin-dependent activity. As these features resemble the requirements of PAO, we considered membrane-bound Rieske-type oxygenases as potential candidates. Analysis of mutants of the two Arabidopsis Rieske-type oxygenases (besides PAO), uncovered that phyllobilin hydroxylation depends on TRANSLOCON AT THE INNER CHLOROPLAST ENVELOPE 55 (TIC55). Our work demonstrates a catalytic activity for TIC55, which in the past has been considered as a redox sensor of protein import into plastids. Given the wide evolutionary distribution of both PAO and TIC55, we consider that chlorophyll degradation likely co-evolved with land plants.
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    • "Light has been reported as an important stimulus in some LMs [13,[56][57][58][59] . Therefore a shading assay was performed in the greenhouse of IDGB, CAS to test whether lesion formation in the lm3 mutant was dependent on light. "
    [Show abstract] [Hide abstract] ABSTRACT: Lesion mimics (LMs) that exhibit spontaneous disease-like lesions in the absence of pathogen attack might confer enhanced plant disease resistance to a wide range of pathogens. The LM mutant, lm3 was derived from a single naturally mutated individual in the F1 population of a 3-1/Jing411 cross, backcrossed six times with 3-1 as the recurrent parent and subsequently self-pollinated twice. The leaves of young seedlings of the lm3 mutant exhibited small, discrete white lesions under natural field conditions. The lesions first appeared at the leaf tips and subsequently expanded throughout the entire leaf blade to the leaf sheath. The lesions were initiated through light intensity and day length. Histochemical staining revealed that lesion formation might reflect programmed cell death (PCD) and abnormal accumulation of reactive oxygen species (ROS). The chlorophyll content in the mutant was significantly lower than that in wildtype, and the ratio of chlorophyll a/b was increased significantly in the mutant compared with wildtype, indicating that lm3 showed impairment of the biosynthesis or degradation of chlorophyll, and that Chlorophyll b was prone to damage during lesion formation. The lm3 mutant exhibited enhanced resistance to wheat powdery mildew fungus (Blumeria graminis f. sp. tritici; Bgt) infection, which was consistent with the increased expression of seven pathogenesis-related (PR) and two wheat chemically induced (WCI) genes involved in the defense-related reaction. Genetic analysis showed that the mutation was controlled through a single partially dominant gene, which was closely linked to Xbarc203 on chromosome 3BL; this gene was delimited to a 40 Mb region between SSR3B450.37 and SSR3B492.6 using a large derived segregating population and the available Chinese Spring chromosome 3B genome sequence. Taken together, our results provide information regarding the identification of a novel wheat LM gene, which will facilitate the additional fine-mapping and cloning of the gene to understand the mechanism underlying LM initiation and disease resistance in common wheat.
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