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Signaux et Messages Cellulaires chez les Végétaux, UMR 5546 CNRS-UPS, Pôle de Biotechnologie Végétale, BP 17 Auzeville, 31326 Castanet-Tolosan, France
1Correspondence: Signaux et Messages Cellulaires chez les Végétaux, UMR 5546 CNRS-UPS, Pôle de Biotechnologie Végétale, BP 17 Auzeville, 31326 Castanet-Tolosan, France. E-mail: ranjeva{at}cict.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONGRAVISENSINGSIGNALING PROCESSES INVOLVED IN...ORGAN SPECIFICITY OF GRAVITROPIC...DNA SEQUENCE(S) SPECIFIC FOR...CONCLUSIONS AND PROSPECTSREFERENCES |
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Key Words: auxin • calcium • gravisensing • plants • response specificity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONGRAVISENSINGSIGNALING PROCESSES INVOLVED IN...ORGAN SPECIFICITY OF GRAVITROPIC...DNA SEQUENCE(S) SPECIFIC FOR...CONCLUSIONS AND PROSPECTSREFERENCES |
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. Because
plants are sessile organisms, their success in a particular environment
will depend on their ability to integrate a complex range of external
and internal information that may vary from minute to minute.
Responding to various environmental signals such as light and water
status results generally in changing the growth direction
(1)
. These directional movements, called tropisms, shape
the plants by involving complex physiological processes and, thereby,
many components of plant cells. As a result, plants will grow either
against or according to the gradient of stimuli they have to face. Most
of the parameters of the environment may vary in quality and intensity
with the notable exception of gravity, which remains basically at the
same value as on Earth (2)
. Consequently, all the adaptive
responses of plants have been developed with a constant background of
gravitational forces. The common-sense statement that "due to
gravity" one can observe upward shoot or downward root growth
reflects in fact the result of multiple interfering processes, because
it is now known that light as well as other physical stimuli or growth
substances may modulate the gravitropic orientation of plants by
creating localized asymmetry (3
, 4
). Another
cause of complexity comes from the recognized situation that the
signaling pathways that link initial stimuli to long-term biological
responses involves common components such as changes in membrane
potential, ion fluxes, reorganization of the cytoskeleton, and other
processes (5)
. These pleiotropic responses question the
action specificity of a given stimulus and understanding exactly what
is due to "pure" gravity-dependent processes or "how gravity is
sensed" in plant systems is a very challenging scientific problem
from both the theoretical and practical points of view. Recent advances
in these fields have been reviewed in a special issue of the journal
Planta (6)
with special emphasis on the
cellular and whole-plant aspects. Considerable progress has been
achieved by combining spaceflight facilities and plant cell biology
with the development of A. thaliana as a plant model
amenable to molecular genetics and benefiting from a world-wide
concerted effort of the genome systematic sequencing (7)
. These new concepts and methodologies, along with the large amount of information accumulated over a century of dealing with plant graviperception and gravitropism, will certainly help to address the following key questions. 1) How is gravity perceived and transformed into an adaptive response by plants? 2) Is it possible to segregate gravitropism from other types of tropism? 3) What are the molecular bases explaining the differential behavior of plant organs in response to gravity? In other words, are the mechanisms of gravitropism genetically different between various organs? 4) Is there (micro)gravity-driven gene expression in plants? To date, there are obviously no definitive answers to these questions; as newcomers we will give some non-objective views on a very complex field that needs the integration of different approaches.
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GRAVISENSING |
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TOPABSTRACTINTRODUCTIONGRAVISENSINGSIGNALING PROCESSES INVOLVED IN...ORGAN SPECIFICITY OF GRAVITROPIC...DNA SEQUENCE(S) SPECIFIC FOR...CONCLUSIONS AND PROSPECTSREFERENCES |
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and references therein). These
uniquely organized cells display characteristic structures with clear
asymmetric distribution of organelles such that the endoplasmic
reticulum and a particular class of plastid accumulating starch
(cytoliths or statoliths) are located next to the distal wall opposite
to the nucleus (9
, 10
). The cytoskeletal
components are most probably implicated in 1) guiding the
transport of endomembranes by the actin microfilaments and
2) anchoring them at the distal part of the cell by the
microtubule meshwork, creating therefore a density gradient in the
cytoplasm with the statoliths acting as ballast. Recent findings
(11
, 12
) have established that the cortical
microtubule may act as a strain gauge for amplification and
stabilization of environmentally induced changes in the direction of
elongation growth. Changing the direction of the gravity vector (for
example by rotating the roots to 90°) will induce a downward movement
of the statoliths driven by the reorganization of the cytoskeletal
meshwork and a correlative asymmetric distribution of auxin, a plant
growth substance, causing reorientation of growth (13)
.
The "statocyte-statolith" concept has been strengthened by the fact
that depletion of starch through experimental manipulation disturbs the
gravisensitivity of plants (14)
and recent data resulting
from spaceflight experiments lead to the conclusion that hypocotyls of
wild-type seedlings responded to a stimulus provided by unilateral
centrifugation at 1 g, whereas those of starch-deficient
strains of A. thaliana did not (15)
. Moreover,
the response to gravity of rhizoids of the alga Chara
depends on the number of statoliths (16)
and lateral
displacements of these organelles by non-invasive micromanipulation
with "optical tweezers" elicit changes in the orientation of tip
growth in the same way as if the rhizoids were gravistimulated
(17)
. However, a significant number of studies have
established that mutants altered in the starch content of their
statocytes display clear impairment in their sensitivity to gravity but
essentially from the quantitative point of view. Thus, a mutant line of
A. thaliana with only 60% of the starch of the wild-type
has almost the same degree of sensitivity as the wild-type and even
starchless mutant plants respond slightly but significantly to gravity
(15
, 18
, 19
). Such a situation
may be consistent with the existence of an alternative gravisensing
system in plants as established, for example, for light perception,
which involves multiple photoreceptors; starch may be essential in the
amplification of the initial gravisensing step but not in the
gravisensing process itself (19)
. Such an alternate system
would implicate the existence of a functional continuity between the
cell wall, the plasma membrane, and the cytoskeleton, resembling by
some manner the mechanosensing process in animal systems
(20)
. The model is based on the clustering of
mechanosensitive calcium channels around integrin-like proteins that
allow the continuity between the extracellular matrix and the interior
of the cell (reviewed in ref. 13
). Changes in tension due to the
distribution of forces exerted by one of the components of the
supramolecular organization at the attachment sites will open
mechanosensitive calcium channels, resulting in increases in cytosolic
calcium and activation of calcium-dependent biochemical and
developmental events. Consequently, the response of plants to gravity
may be a particular aspect of the more general field of
mechanoresponses (1)
. As a matter of fact,
mechanosensitive calcium channels have been clearly characterized in
plants (21)
and have been shown to be modulated by
cytoskeletal elements. At present, even if the formal identification of
integrins remains to be achieved in plants, integrin-like antigens have
been immunolocalized in A. thaliana and Chara in
sites of gravity perception/transduction (22)
. Moreover,
recent evidence has shown that plant plasma membrane is able to bind
specifically synthetic peptides bearing an R-G-D sequence
characteristic of integrin ligands (23)
. It is important
to note that challenging Chara internodes with such peptides
abolishes the gravity-dependent polarity of cytoplasmic streaming
(24)
. The peptide is effective at the top of the cell when
the density of the cytoplasm is greater than that of the external
medium. It becomes active at the bottom when the external milieu is
more dense than the cytoplasm. Such data show that the peptide is
effective only at the site of tension and not of compression.
Application of RGDS peptide consistently inhibits calcium influx, which
has been shown to proceed more rapidly at the site of tension than at
the site of compression (24)
.
The relevance of such a model needs the formal identification of all
the molecules supposed to be implicated in the process and more
importantly, the determination of their real biological role. However,
the notion that supramolecular organization of cellular components is
crucial in signal perception and transduction in plants has been
substantiated by different experimental results. Thus, in
Fucus rhizoids, receptors of dihydropyridine derivatives,
used to probe putative calcium channels, have been shown to be
concentrated at the growing part of the tip and anchored by
microfilaments (25)
. In higher plants, voltage-operated
calcium channels may be recruited by large depolarization, presumably
by forming clusters of active channels (26)
. The process
depends on the organization of cytoskeletal elements essentially in a
microtubule meshwork, the disruption of which activates channel
activities and increases their lifetime (27)
, leading to
considerable increases in cytosolic calcium (5
,
28
).
At present it is impossible to determine which of the two models
(statocyte-statolith or integrin-like perception) is really effective
in graviperception and, in fact, they may coexist in plants. The
statocyte-statolith model might be predominantly operative in
specialized cells (e.g., the columella in the roots and the endodermal
cells around the vascular bundles in the shoot) (14)
with
the other system working in cell types poor in (or devoid of)
statoliths. In all cases, the putative gravireceptor remains to be
identified at the molecular level, possibly among the myriad of plant
orphan receptors unveiled by the genome program (7)
.
Concerning the events downstream of the initial perception of gravity,
calcium and auxin are considered to be key players (13
,
14
). Even if cytosolic calcium has not been clearly shown
to respond to gravistimulation (29)
, it is known that
compounds interfering with calmodulin (the ubiquitous calcium-binding
protein) or calcium-ATPase impair plant graviresponses
(30)
. More important, high amounts of calmodulin are
associated with amyloplasts in statocytes (31
,
32
) and expression of calmodulin itself or of
calmodulin-like genes, referred to as touch genes, is induced by
mechanical stimulation and other signals (33-35)
. By
extrapolation, it may be suggested that continuous gravitational
stimulation on statocytes may explain their higher content in
calmodulin (30)
. The crucial role of calmodulin in
gravitropic response has been illustrated by data reporting that, in
contrast to the wild-type where calmodulin transcripts increase rapidly
on gravistimulation, the converse situation has been observed in
A. thaliana agr-3 mutant (36)
.
Local accumulation of calmodulin might make some critical cellular
domains more responsive to slight changes in calcium that cannot be
monitored by conventional methods and would allow specific activation
of calcium-calmodulin-dependent enzymes (protein kinases/phosphatases)
or multimolecular association. For example, a plant-specific kinesin, a
motor protein interacting with microtubules, is also a
calmodulin-binding protein (37)
at very specific places in
the cell.
Taken together these data are consistent with the utmost importance of
the intracellular organization in generating asymmetry. However, the
extracellular compartment is certainly to be considered in the overall
process as illustrated by the growth of the pollen tube, which is
clearly oriented by the calcium gradient in the culture medium
(38)
. Even if it is not possible to directly compare root
and pollen tube growth, it remains unclear how extracellular calcium
and auxin redistribute in root cap of higher plants in response to
gravistimulation.
Concerning auxin, it is known that this plant growth substance
influences many cellular processes and regulates gravity-induced root
bending by inhibiting root cell elongation (39
,
40
). The asymmetric distribution of auxin at the
elongation zone is considered to be the causative factor of the
asymmetric growth for curvature induced by gravity (41
,
42
) and many mutants affected in their sensitivity to
auxin are impaired in their response to the gravity vector
(43)
. For example, A. thaliana seedlings
mutated within the AUX 1 gene became auxin-resistant and did
not display any root gravitropic curvature (44)
. The
AUX 1 gene has been isolated from a T-DNA tagged gene in
Arabidopsis and the polypeptide product has been shown to
share sequence similarity with a family of fungal and plant permeases
and to be localized in the root apical tissues (45)
. Of
special interest is the homology to amino acid permeases that function
as proton-driven symporter (46)
, suggesting that AUX
1 may mediate the uptake of indole-3-acetic acid, the major form
of auxin in plants, due to its similarity to tryptophan. These data
suggest that AUX 1 is most probably implicated in the auxin
uptake and in its asymmetric redistribution. It is interesting to note
that the cloned AUX 1 gene restores auxin sensitivity,
including gravisensitivity to root agravitropic of transgenic aux
1 roots.
Other aspects dealing with gravisensing and graviresponse at the whole
plant or cellular levels have been extensively discussed by Volkmann et
al. (47)
.
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SIGNALING PROCESSES INVOLVED IN GRAVITROPISM AND OTHER TROPIC RESPONSES |
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TOPABSTRACTINTRODUCTIONGRAVISENSINGSIGNALING PROCESSES INVOLVED IN...ORGAN SPECIFICITY OF GRAVITROPIC...DNA SEQUENCE(S) SPECIFIC FOR...CONCLUSIONS AND PROSPECTSREFERENCES |
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and
gravitropic responses have been considered as a particular aspect of
mechanical responses in plants (1)
. More important, it has
been clearly established that physical stimuli such as light interfere
with some gravitropic responses and may play a crucial role in the
expression of gravisensitivity; it is also known that phytochrome (a
photoreceptor) mediates the light response (3
,
48
, 49
). Thus, roots of a corn cultivar
(Merit) grow nearly horizontally in darkness. Illumination of the roots
initiates gravitropic bending that may be impaired by pharmacological
compounds known to inhibit calcium-calmodulin-dependent kinase in
animal systems (50)
. Because calcium plays an important
role in the lateral distribution of the plant growth substance auxin
(51)
the working hypothesis is that the kinase would be
associated with the establishment of auxin gradients in the root, via
the activation of a proton pump that provokes the redistribution of
auxin. A cDNA encoding a corn homolog of mammalian
calcium-calmodulin-dependent protein kinase specifically expressed in
root cap, the site for both light and gravity perception, has been
obtained (52)
. The recombinant protein referred to as MCK1
has been shown to definitely bind calmodulin and to be sensitive to
calcium-calmodulin-dependent protein kinase inhibitors at
concentrations impairing light-regulated root gravitropism. However,
the gene was constitutively expressed in both the light and the dark
(53)
so that a direct effect of light on the amount of
protein may be ruled out. Regardless of the existence of another
putative member of the calcium-calmodulin-dependent protein kinase
multigene family that would be specifically induced by light, the
observed response might be explained by the differential regulation of
the enzyme activity under dark and light conditions. Thus, in
light-requiring cultivars, illumination might initiate substantial
changes in calcium distribution (54)
, resulting in the
localized activation of calcium-calmodulin-dependent protein kinase in
particular areas where calcium reaches the threshold concentrations
necessary to induce protein kinase-CaM interactions (53
,
55
). Taking advantage of the inability of roots of
A. thaliana to penetrate a 1.5% agar gelled surface, Okada
and Shimura (56)
have devised an experimental protocol
allowing the isolation of mutants affected in their response to various
physical stimuli. Thus, when tilted from the vertical position, the
roots of seedlings growing at the surface tend to penetrate into the
agar in response to gravity. Because roots are unable to do so, their
growth pattern looks like waves when the seedlings are allowed to grow
further, the agar acting as an obstacle and as a touch stimulus. The
same wavy pattern was observed when the seedlings were grown vertically
on the surface of the gelled medium and illuminated laterally to induce
phototropism. Under these conditions, the Japanese researchers
(56)
have been able to isolate three different classes of
mutants. One class was preferentially affected in root gravitropism,
another more specifically in their response to lateral illumination,
whereas the last class remained responsive to both light and gravity
but was affected in root waving (touch response). These data suggest
that different stimuli may activate common steps in the transduction
pathway but some branches may be specific to a given stimulus. |
ORGAN SPECIFICITY OF GRAVITROPIC RESPONSE |
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TOPABSTRACTINTRODUCTIONGRAVISENSINGSIGNALING PROCESSES INVOLVED IN...ORGAN SPECIFICITY OF GRAVITROPIC...DNA SEQUENCE(S) SPECIFIC FOR...CONCLUSIONS AND PROSPECTSREFERENCES |
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. Having shown that the inflorescence stem is a
suitable material for physiological and genetic studies of shoot
gravitropism in higher plants (60)
it has been possible to
perform a comparative study of the regulatory mechanisms for
gravitropic response between different aerial organs and roots
(59
, 61
, 62
). Tropic responses
of different organs of mutagenized seeds were measured by their
curvature either after gravistimulation in the dark (graviresponse) or
by lateral illumination (phototropism) and compared with the standard
response of wild-type seedlings taken as a control. A number of mutants
with abnormal shoot gravitropic response have been isolated and genetic
analysis performed by crossing the mutant lines with wild-type plants
to determine the segregation of the inflorescence stem gravitropism in
F1 and F2 progeny. In the F1
generation, all the progeny behaved like the wild-type and displayed an
upward curvature. Abnormal gravitropic response phenotypes segregated
about one-quarter in the F2 generation to indicate that the
mutation was recessive. At least six independent loci named
SGR1 to SGR6 (for shoot gravitropism) have been
shown to be involved in shoot gravitropism in Arabidopsis
and the mutations fall into three different classes according to their
responsiveness to gravity (59
, 61
). In class
1 (e.g., sgr 1-1 and sgr 4-1 mutants) both
inflorescence stems and hypocotyls were agravitropic. Class 2
(sgr 2-1 mutant) showed no gravitropic response in
inflorescence stems but hypocotyls remain partly responsive to
gravistimulation. Class 3 (sgr 3-1, sgr 5-1, and
sgr 6-1 mutants) was characterized by a normal gravitropic
response in hypocotyls but a reduced one in inflorescence stems. It is
interesting to note that sgr mutants showed normal root
gravitropism, which has been shown to be genetically separable from
shoot gravitropism (59
, 61
). Recently,
another class of mutant referred to as RHG mutants (for root
and hypocotyl gravitropism) corresponding to a single recessive
mutation has been isolated (62)
. This class of mutant is
not affected in the inflorescence stem gravitropism but is severely
impaired in root and hypocotyl response to gravistimulation.
Taken together, these data establish that the mechanisms of
gravitropism are genetically different between different plant organs.
A common feature to all the above-mentioned classes of mutants is that
they remain responsive to phototropism. Because phototropism as well as
gravitropism are considered to be due to lateral transport of auxin
(42
, 63
), it is suggested that the
distribution of auxin for phototropic response is normal in the
mutants. Therefore, separate pathways involving possibly the same
intermediary steps are likely to be switched on by light but not by
gravity. Of special interest is the existence of apparently normal
statoliths in the statocytes of roots and hypocotyls in rhg
mutants, strengthening the idea that both SGR and
RHG gene products are involved in specific steps of gravity
perception or transduction pathways in the considered organs
(62)
.
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DNA SEQUENCE(S) SPECIFIC FOR GRAVISENSING? |
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TOPABSTRACTINTRODUCTIONGRAVISENSINGSIGNALING PROCESSES INVOLVED IN...ORGAN SPECIFICITY OF GRAVITROPIC...DNA SEQUENCE(S) SPECIFIC FOR...CONCLUSIONS AND PROSPECTSREFERENCES |
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), temperature
(65)
, drought (66)
, or plant growth
substances (67)
. This is the case also concerning
mechanical stimuli such as touch (34)
and probably all
stimuli involving induction/repression of gene expression. The common
feature to the above-mentioned situations is that one may directly
compare plants treated under different conditions with their
corresponding controls. This is not the case with long-term experiments
necessary to plant development under microgravitational conditions,
which need hardware specific to space biology. In this case,
ground-based work is not enough to detect putative genes sensitive to
(micro)gravity and hence to characterize their promoters and regulatory
binding proteins. To allow further progress in this field, large
numbers of plants grown under microgravity and appropriately fixated
for further molecular analysis will be necessary and will be one of our
most difficult problems. |
CONCLUSIONS AND PROSPECTS |
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TOPABSTRACTINTRODUCTIONGRAVISENSINGSIGNALING PROCESSES INVOLVED IN...ORGAN SPECIFICITY OF GRAVITROPIC...DNA SEQUENCE(S) SPECIFIC FOR...CONCLUSIONS AND PROSPECTSREFERENCES |
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The phenomenology of different steps implicated in graviperception and
gravitransduction is being described in detail through the use of
technically demanding protocols (10)
and the combination
of general approaches, including formal and molecular genetics. As a
result, our knowledge of the intimate mechanisms involved in the
overall process has dramatically progressed but numerous questions
remain unanswered. Thus, the isolation of different genes of interest
and, more importantly, the characterization of their biological
function will help to identify different steps of the graviresponse and
their connection with signaling pathways triggered by other stimuli. In
this way the identification of AUX 1 as a gene encoding a
permease that localizes to specific cells is of utmost importance
(45)
and sheds light on the mechanisms involved in
asymmetric auxin distribution. However, the probable presence of
statoliths in statocytes of roots and hypocotyls of rhg
mutants unable to respond to gravity (62)
makes it
necessary to define more precisely the very early steps of signal
perception and transduction. These recent findings strengthen the idea
of distinct "susceptor" and "receptor" devices for the
gravitropic response (19)
. The validity of such a
framework has to be addressed under microgravity and the search for
specifically regulated genes certainly needs the setting up of a system
allowing not only the growth of plants but also their appropriate
processing during spaceflights. More important, the study, in real
time, of different key events under microgravity requires the use of
appropriate tools. The in vivo movements of regulatory
proteins, the dynamics of cellular component reorganization, and the
changes in important compounds such as calcium, the proton, and auxin
may be envisaged during spaceflight through the use of luminescent or
fluorescent probes and detection systems, the availability of which are
anticipated (30)
. By and large, plant biology in space
would allow us to address critical questions of plant development on
Earth.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONGRAVISENSINGSIGNALING PROCESSES INVOLVED IN...ORGAN SPECIFICITY OF GRAVITROPIC...DNA SEQUENCE(S) SPECIFIC FOR...CONCLUSIONS AND PROSPECTSREFERENCES |
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