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Members of this clade are also found in
gymnosperms but not ferns, suggesting a role
in seed plant evolution10,11. One role of the
foundation member, AG, is to specify the identity of carpels, an evolutionary novelty specific to the angiosperms10,11. Gene duplications
creating the SHP genes could then have led to
the evolution of dehiscence in the specialized
fruits derived from the carpels and characteristic of Arabidopsis and other members of the
Brassicaceae.
The three closely related SEPALLATA
genes fall within their own clade4,10,11.
Originally named after the founding member
AGL2, it now seems appropriate to rename
this the SEPALLATA clade (Fig. 1). Again,
this includes at least one gymnosperm MADS
sequence, implying that one or more members
played an ancient role in sporocarp development10,11. The fact that SEP genes are
involved in specifying development of three
floral whorls (one perianth and both reproductive whorls) in the angiosperm flower is
consistent with such an ancient role.
Practical implications
Pod shatter before harvest is an important
agronomic problem with cruciferous crops,
especially canola (Brassica napus)13. One
consequence of uncovering the role of
SHATTERPROOF genes2, and of the FRUITFULL gene that regulates their expression6, is
that tools now exist to modify the phenotype
of the seed pod. It is possible that judicious
modulation of the time and place of expression of these genes, and/or their targets, will
allow seeds of canola to remain firmly encased
until harvesting is ready to be carried out2,6.
Acknowledgements
I thank Marty Yanofsky, Sarah Liljegren and
Soraya Pelaz for helpful comments, and for
providing photographs of mutant plants.
David Smyth
Dept of Biological Sciences, Monash University,
Melbourne, Vic 3800, Australia
(tel 161 3 9905 3861; fax 161 3 9905 5613;
e-mail [email protected])
References
01 Pelaz, S. et al. (2000) B and C floral organ
identity functions require SEPALLATA MADSbox genes. Nature 405, 200–203
02 Liljegren, S.J. et al. (2000) SHATTERPROOF
MADS-box genes control seed dispersal in
Arabidopsis. Nature 404, 766–770
03 Riechmann, J.L. and Meyerowitz, E.M.
(1997) MADS domain proteins in plant
development. Biol. Chem. 378, 1079–1101
04 Alvarez-Bullya, E.R. et al. (2000) An
ancestral MADS-box gene duplication occurred
before the divergence of plants and animals.
Proc. Natl. Acad. Sci. U. S. A. 97, 5328–5333
05 Ma, H. et al. (1991) AGL1–AGL6, an
Arabidopsis gene family with similarity to floral
homeotic and transcription factor genes. Genes
Dev. 5, 484–495
06 Gu, Q. et al. (1998) The FRUITFULL MADSbox gene mediates cell differentiation during
Arabidopsis fruit development. Development 125,
1509–1517
07 Pneuli, L. et al. (1994) The TM5 MADS box
gene mediates organ differentiation in the three
inner whorls of tomato flowers. Plant Cell 6,
175–186
08 Angenent, G.C. et al. (1994) Co-suppression of
the petunia homeotic gene fbp2 affects the
identity of the generative meristem. Plant J. 5,
33–44
09 Ferrándiz, C. et al. FRUITFULL negatively
regulates the SHATTERPROOF genes during
Arabidopsis fruit development. Science (in press)
10 Hasebe, M. (1999) Evolution of reproductive
organs in land plants. J. Plant Res. 112,
463–474
11 Theissen, G. et al. (2000) A short history of
MADS-box genes in plants. Plant Mol. Biol. 42,
115–149
12 Ferrándiz, C. et al. (2000) Redundant
regulation of meristem identity and plant
architecture by FRUITFULL, APETALA1 and
CAULIFLOWER. Development 127, 725–734
13 Spence, J. et al. (1996) ‘Pod shatter’ in
Arabidopsis thaliana, Brassica napus and
B. juncea. J. Microsc. 181, 195–203
The dawn of plant salt tolerance genetics
Recent reports from Jian Kang Zhu et al. have
provided clear evidence for a signal transduction pathway that mediates salt tolerance of
plants by controlling ion homeostasis. Earlier
research had identified several salt overly sensitive (sos) mutants of Arabidopsis thaliana
ecotype Columbia, based on Na1/Li1 hypersensitivity1. Genetic complementation studies
allowed the mutants to be placed into five
allelic groups, sos 1–sos 5 (Refs 1,2). Phenotypic additivity analyses indicate that SOS1,
SOS2 and SOS3 function in a common pathway, with SOS1 being epistatic to the other
loci. Recently, mapped-based cloning and gene
complementation has identified the genes at
these three loci. SOS3 is a Ca21-binding protein that contains EF-hand structures and a
myristoylation site in the N terminus3,4. It has
greatest sequence homology with yeast calcineurin subunit B and with animal neuronal
Ca21 sensors. SOS2 encodes a serine/threonine
kinase with a catalytic domain similar to the
yeast sucrose nonfermenting (SNF1) kinase
and the mammalian AMP-activated protein
kinase (AMPK), but is regulated differently
through a distinctive C-terminal regulatory
domain5. SOS1 is a putative plasma membrane
Na1/H1 antiporter resembling the mammalian
NHE and bacterial NhaP exchangers6.
SOS signal pathway
Molecular interaction, activation and site-specific mutant structure–function analyses indicate that SOS3 is required for the activation of
the SOS2 kinase7. It is envisaged that SOS3 is
the Ca21 sensor in a complex that is necessary
for kinase activation. Myristoylation of SOS3
is required also for function in planta4.
Mutations in SOS2 or SOS3 downregulate salt
induction of SOS1 mRNA, indicating modulation of SOS1 transcription through some, as
yet unknown, transcription factor6 (Fig. 1).
However, mammalian NHE antiporters are
regulated by phosphorylation, therefore SOS2
might regulate SOS1 additionally, independent of the transcription factor.
Control of downstream effectors
Participation of the Ca21-dependent SOS3 as
the most upstream component of the known
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
SOS pathway indicates that salt-stressresponse in plants, however perceived,
involves a short regulatory cascade that
includes elevated cytosolic Ca21 levels as an
early event of the process2,8. As in yeast, the
salt sensor and the initial signaling components controlling ion homeostasis have yet to
be identified9. Presumably, Ca21 flux in
microdomains near membranes initiate a signal, which implicates involvement of specific
channels and tethered molecules that regulate
channel function and transduce the signal
intracellularly10,11. It is conceivable that SOS3
is one of the tethered molecules, and it might
be useful to study how a local salt-induced
Ca21 oscillation can activate a stress signal
transduction pathway.
A dual level of ion homeostasis regulation
involving both transcriptional and post-translational mechanisms occurs in Saccharomyces
cerevisiae12,13. This dual level of control over
ion fluxes might be needed to allow survival
upon both rapid stress imposition and continued growth during sustained elevated
levels of external Na1. In the yeast salt-stress
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Xylem
NHE
Na
Root
SOS1
H+
Na+
?
TF
H+
Vacuole
Na+
Na+
?
Na+
SOS1
SOS2
?
Na+
Na+
Na+
NHE
NHE
H+
H+
Na+
Na+
Na+
Na+
+
Na+
Na+
Na
Na+
Na+
Transcriptional
activation of SOS1
(and other effectors)
+
Leaf
Ca2+
SOS3
H+
Vacuole
Vacuole
Casparian strip
Trends in Plant Science
Fig. 1. Model depicting the role of the salt overly sensitive (SOS) pathway in mediating
salinity tolerance by controlling Na1 flux through SOS1. Abbreviations: TF, transcription
factor; NHE, tonoplast Na1/H1 antiporter. Arrows with black arrowheads represent Na1
movement through unidentified flux system(s). Arrows with white arrow heads represent an
antiporter system. Adapted from Ref. 8.
signal pathway, the PP2B phosphatase calcineurin is a pivotal intermediate that controls
transcription and activation of the ENA1 Ptype ATPase (Ref. 12; J.M. Pardo, unpublished), which is primarily responsible for
Na1/Li1 efflux across the plasma membrane.
It appears that the SOS3 protein might play a
functionally similar role by both activating the
transcription and in situ activity of the plasma
membrane antiporter SOS1. An interesting
divergence from the yeast model appears to be
the activation through phosphorylation
by SOS2 in plants and dephosphorylation by
calcineurin in yeast.
SOS pathway is probably
multifunctional
The calcineurin pathway in yeast is essential
for regulation of key life cycle processes other
than salinity tolerance, suggesting that salt
stress response and adaptation are integrated
into cellular homeostasis14,15. To date, the only
salt tolerance effector controlled by the SOS
pathway to have been identified is SOS1.
However, the existence of numerous family
members of both SOS2 and SOS3 in the
Arabidopsis genome3,5–7,16 strongly indicates a
focal involvement of SOS-family genes in
other aspects of plant growth and development
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August 2000, Vol. 5, No. 8
that are modulated by salt or even by other
environmental cues. Different environmental
perturbations could generate signals that are
transduced through regulatory circuits that are
composed of various members of the SOS
gene families. Output from these pathways
might also have rather specialized functions in
stress adaptation processes, for example,
determinants that modulate ion fluxes in
the case of SOS2 and SOS3. In addition,
more than one signal pathway might control
the same output components. For example,
calcineurin, HOG (high osmotic glycerol)
signal pathways and, apparently, Ca21 signaling independent of calcineurin all regulate the
activation of ENA1 (Refs 13,17). This redundancy probably reflects the multiple etiologies
and pathologies associated with environmental perturbations, and the required integration
that is intrinsic to cellular function.
Identification of SOS determinants that
are most essential for plant salt tolerance
The existence of multigene families of SOS
components reminds us that screening directly
for phenotype change might be the approach
that most rapidly identifies the family members that are most important to a particular
phenotype. In fact, multiple alleles of sos1 and
sos2 were identified in the hypersensitivity
screen but not mutations to other members of
the SOS1 or SOS2 families1. Furthermore,
genetic, molecular interaction and activation
data indicate that only specific isologs of the
SOS families are pivotal determinants of salt
stress adaptation. That is, all family members
are probably not crucial to salt tolerance, at
least during the developmental stage in which
the hypersensitive mutants were identified.
Functional interaction of specific family
members must be confined to unique cell
types expressing those members. The location
of these cells in the organism de facto identifies sites of essential physiology necessary for
whole plant adaptation and might provide
important clues to the essential physiology of
tolerance. A tentative model of the function of
SOS1 (Ref. 6) in cells surrounding vascular
elements is shown in Fig. 1. It is also conceivable that the specific cells, and particular
SOS family members that are most essential
for salt tolerance vary during ontogeny, to
establish a correct pattern of physiology for
adaptation. Systematic reverse genetics and
knockout studies should also help to determine if SOS family members participate in
response to other environmental stresses such
as temperature or nutrient availability.
Other possible osmotic stress signaling
pathways in plants
Other protein kinases, that are presumed to
be focal intermediates in signal cascades,
have been implicated in the salt stress response of plants, including Ca21-dependent
protein kinases (CDPK), mitogen-activated
protein kinases (MAPK), and SNF-like and
Dbf2-like kinases. Although none of these
protein kinases seems to regulate ion homeostasis specifically, their involvement in some
aspect of the salt stress response is probable.
Specific CDPKs transcriptionally regulate an
osmotic stress responsive LEA (late embryogenesis abundant) gene (HVA1) that is an
osmotic stress tolerance determinant18. An
oxidative stress-activated MAPK [(NPK/
ANP) Nicotiana protein kinase/Arabidopsis
homolog of NPK] pathway is implicated in
enhanced abiotic stress tolerance because
expression of the activated MAPKKK (mitogen-activated protein kinase kinase), NPK1,
increases survival of seedlings in response to
freezing, heat shock and hyperosmolarity or
salt19. Apparently, the NPK/ANP pathway
promotes stress tolerance independently of
known cold- or osmotic-induced-stress signaling pathways, because activation does
not lead to transcription of signature stressinduced genes. NPK1 has been implicated in
cell cycle regulation. Salicylic acid-induced
protein kinase (a MAP kinase) and an
SNF-like Arabidopsis ASK1 ortholog are activated by hyperosmolarity20. The putative cell
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cycle-regulated protein kinase At-DBF2
mediates salt and osmotic sufficiency, as well
as heat and low temperature sufficiency, but
does not mitigate oxidative stress effects21.
DBF2 might be a component of the general
transcription complex, analogous to CCR4 of
yeast, which regulates expression of genes
involved in osmotic and temperature tolerances. ATHK1 resembles the yeast osmosensor SLN1 that functions both as the sensor and
receiver of the phospho-relay system that initiates the two-component HOG MAPK pathway that mediates hyperosmotic stress
tolerance22. Yeast genetic suppression data
indicate that ATHK1 can function as an
osmosensor, although its role in plants has
not yet been determined, and other components of the phospho-relay system or the
signal cascade have not been identified.
As genetic analysis of stress tolerance progresses, unexpected links between regulatory
network(s) might come to light and further
the knowledge base about the intersection
of regulatory pathways. For example, the
Arabidopsis mutant uvs66 is affected in the
perception of signals triggered by genotoxic
treatments (UV light and DNA-damaging
chemicals)23. The uvs66 mutant is also hypersensitive to abscisic acid (ABA) and salinity.
NaCl sensitivity is specific to Na1 because the
mutant is resistant to high KCl and hyperosmolarity. Both SOS1 and UVS66 map to
chromosome 2 but are not allelic because sos1
mutants are tolerant to UV light, DNAdamaging chemicals and ABA. The uvs66
mutant reveals crosstalk between ABA, Na1
homeostasis and response to genotoxic stress.
Salt tolerance sufficiency
The primary explanation for the slow inadequate progress towards understanding salinity tolerance has always been the complexity
of this trait24. The research by Zhu and coworkers is an exemplary illustration of how
genetics can lead to seminal dissection of a
mechanistically complex phenotype. Several
groups now use forward and reverse genetic
approaches to identify determinants that mediate tolerance to salt, as well as other abiotic
stresses. Soon, lists of stress tolerance determinants, both effectors and signal molecules,
will be compiled on the basis of functional
rather than correlative evidence. The genetics
approach and application of genomics concepts is bringing the physiology of salt tolerance back from a cellular to an organismal
focus. It is likely that the understanding of salt
stress perception and adaptive responses of
plants that lead to tolerance will be elucidated,
rather than inferred, based on comparisons
with unicellular models. However, it is also
likely that another phase of the genetics
approach will involve determining how the
specific genes that control salt tolerance in
model systems, such as Arabidopsis, differ
from their orthologs in halophytic species. The
identification of the halophytic versions of
salt tolerance determinants will be facilitated
greatly by the use of halophytes closely related
to Arabidopsis thaliana, such as Arabidopsis
(Thellungiella) halophila (Ref. 2). This species has considerable genetic orthology and
will probably exhibit high synteny with
Arabidopsis. At this point it is clear that
genetic manipulation of crop plants for salt
sufficiency should be possible.
Acknowledgements
We thank colleagues for informative discussion that helped in the preparation of this
manuscript. We request the understanding of
scientific peers whose papers were not cited
owing to space constraints.
Mike Hasegawa* and Ray Bressan
1165 Horticulture Building, Purdue University,
West Lafayette, IN 47907-1165, USA
Jose M. Pardo
Consejo Superior de Investigaciones Cientificas,
Instituto de Recursos Naturales y Agrobiologia
de Sevilla, Campus de Reina Mercedes,
Apartado 1052, Sevilla, Spain 41080
*Author for correspondence
(e-mail [email protected])
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