How crop plants respond to stresses in their physical environment
is crucial to productivity. Of all parameters, abiotic environmental
stresses contribute most significantly to the reduction in potential
yield (USDA Statistical Yearbooks; Flowers & Yeo, 1995). We
have chosen for our studies the sensing of osmotic and ion imbalance
and the functional basis of responses to osmotic and ionic stresses.
We will compare higher plants and models with different capacities
for the expression of biochemical and physiological defense mechanisms
that counter stress.
Our results will provide the number and nature of all genes
that are implicated in stress tolerance and their response to
alterations in osmotic and ionic conditions. To achieve this,
we will identify how many and which genes provide stress responses,
which genes make essential contributions to stress tolerance,
and how many and which mechanisms serve in stress protection.
As the major result of our integrative approach, we envision characterizing
the genes that constitute the "osmome" or "xerome"
- that portion of the genome with specific functions under osmotic
challenge or water-limiting conditions. Such knowledge can eventually
be provided as a diagnostic set of microarrayed sequences. Our
One important point of this project is that the data are not
only useful to those working on salinity and osmotic stress. Stresses
targeted in our project show considerable overlap with responses
of plants to cold stress, drought, and insect, fungal, bacterial
and viral stresses. The tools and resources which we will assemble
will be available and can be used for a comparative profile for
those other stresses in addition.
The results from our program will provide important knowledge
for future plant breeding. Salinity and drought stresses constitute
a permanent and increasing agronomical problem in the arid midwest/western
growing areas. On irrigated land, with a productivity more than
three times that of rain-fed areas, long-term irrigation inevitably
increases salinity. Plant breeding has not produced varieties
suitable for use in stressful environments (Flowers & Yeo,
1995). Our work will provide the genes, their putative nature
by homology and function, comparative analysis of different stress-adaptable
organisms, and a description of gene expression over the life-time
of plants in their unstressed and stressed states.
B. Complexity and Multigenicity of Stress Responses.
1. Variations on common physiological Themes. The responses
of plants to osmotic and ionic stress have been reviewed by PIs
on this proposal and others (McCue & Hanson, 1990; Bartels
& Nelson, 1994; Niu et al., 1995; Bohnert et al., 1995; Serrano,
1996; Bray, 1997; Zhu et al., 1997; Shinozaki & Yamaguchi-Shinozaki,
1997; Nelson et al., 1998; Bohnert et al., 1998; Noctor &
Foyer, 1998), often with an emphasis on transgenic stress tolerance
engineering (Bohnert & Jensen, 1996; Jain & Selvaraj,
1997; Holmberg & Bülow, 1998).
Flowers & Yeo (1995) reviewed the complexity of salinity
stress responses in an article assigning the failure of current
breeding strategies to a lack of mechanistic understanding. Indeed,
such breeding amounts to merging two multigenic, quantitative
traits: tolerance and productivity. Our limited knowledge about
the molecular genetic basis of this complexity has been derived
largely from analysis of single to few genes and the expression
of corresponding transcripts and proteins in a wide variety of
different species. although the functional mechanisms served by
different gene products may be the same. For example, accumulation
of mannitol in celery, of pinitol in the ice plant, glycerol in
yeast/ Dunaliella and ectoine in halobacteria all seem
to assure "osmotic adjustment". Even though the importance
of osmotic adjustment is questionable, the "compatible solute"
concept appears to hold up. The accumulating solutes seem to act
in specific functions such as protein stabilization (ectoine,
glycinebetaine), and uncharged solutes (mannitol, pinitol) seem
to act as scavengers of reactive oxygen species (ROS). The latter
has recently been documented by a different strategy - over-expression
of the Fe-binding protein ferritin resulted in increased tolerance,
attributable to the removal of free iron from the cells (D. Dudits,
personal communication). Protection by the removal of iron, which
participates in a "Fenton-reaction" through which hydroxyl
radicals are produced, has already been shown (Shen et al., 1997).
Many reports suggest ROS scavenging as most essential for protection
(Allen, 1995; Roxas et al., 1997; Noctor & Foyer, 1998).
There are other biochemical mechanisms contributing to tolerance,
such as reactions that lower the NADH/NAD+-ratio thereby
decreasing reducing power. The balance, also, between nitrogen
assimilation and carbon provision requires adjustments. Uptake,
transport and compartmentation of sodium ions - while maintaining
uptake of the essential potassium - requires several genes and
pathways. Cellular integrity is maintained by changes in membrane
fluidity and protein composition of membranes. Accumulation of
stress-specific proteins, such as the late embryogenesis abundant
(LEA) proteins to high concentrations within specific tissues
is correlated with or causally related to improved tolerance under
drought, cold, or salinity stress conditions (Moons et al., 1995;
Imai et al., 1996; Close, 1996; Xu et al., 1996), although the
mechanistic basis of their effects remains elusive. Apparently,
different species have solved the problems posed by salinity and
osmotic stress by strengthening unique biochemical pathways, but
there seems to be a limited number of ways in which this can be
2. Evolutionary Conservation of Stress Responses.
Recently, substantial data from genetic transformation experiments
have established that functionally analogous tolerance determinants
exist in both unicellular organisms and plants. Largely conserved
are stress sensing, transduction of the signal and the regulation
and functioning of the downstream gene induction and repression
machinery (Frommer & Ninnemann, 1995; Serrano, 1995; Shinozaki
& Yamaguchi-Shinozaki, 1997: Zhu et al., 1997; Ishitani et
al., 1997; Nelson et al., 1998). This justifies a large-scale
genetics approach aimed at finding all genes involved in
stress responses in selected model organisms: Synechocystis
sp. PCC6803, yeast and Aspergillus nidulans. The three
models have been chosen to demonstrate and exploit the significant
evolutionary conservation of homologous adaptive strategies. Differences
exist, even among higher plants, in how signal transduction pathways
and gene expression patterns are connected via a finite set of
cis- and trans-acting elements and transcriptional activators..
For this reason, we include halophytic models, Dunaliella
and Mesembryanthemum, to compare and contrast the overlapping
and unique sets of stress-related transcripts from Arabidopsis
and rice. From a technological standpoint, determining the common
and unique portions of the genome which dictate all stress responses
will be an important prerequisite for understanding and modifying
preexisting adaptations and targeting and exploiting new adaptive
processes in the post-genomic era of biology.
C. Experimental Plan.
The nine PIs from the University of Arizona, Purdue University
and Oklahoma State University forming the "Plant Stress Consortium"
contribute strategies and technologies to a project that could
not be carried out by any individual laboratory. Our expertise
covers the entire field of study in model organisms, genome analysis
- including reverse genetics, EST and microarray analyses - and
functional studies in plant cell biology and physiology. The Consortium
project integrates our individual approaches to understanding
the genomic basis of plant stress responses in a comparative,
comprehensive way. We will use models for various overlapping
aspects of stress tolerance including cellular traits (Saccharomyces),
photosynthetic traits in a simple prokaryote (Synechocystis)
and more complex adaptive strategies in a multicellular fungus
(Aspergillus). All of these organisms are halotolerant
and have evolved related, yet unique aspects of adaptation to
ionic and osmotic stresses. For all of these organisms facile
insertional mutagenesis and complementation systems have been
worked out which will allow for the rapid functional identification
of endogenous adaptive genes as well as the isolation of gene
homologs from higher plant species.
These organisms also offer the advantage of rapid, targeted
gene disruption through homologous recombination. The use of saturation
mutagenesis in Arabidopsis by T-DNA insertions provides
a powerful tool for identifying mutations in a particular gene
of interest (Krysan et al., 1996). Targeted gene disruption is
now also feasible in the moss Physcomitrella patens (Schaefer
and Zyrd, 1997; Puchta, 1998) and in Arabidopsis using
the vacuum infiltration method (Miao & Lam, 1995; Kempin et
al., 1997). We plan to exploit each of these approaches to isolate
and functionally characterize knockout mutants. In addition to
functional assays, we plan to focus strongly on direct gene discovery
strategies (EST collections and databases) with corresponding
developments of specialized bioinformatics resources. This strategy
will bring us much closer to comprehensive characterization of
the osmome (xerome) - the set of transcripts that is stress specific
- in higher plant models where genomic sequencing remains incomplete.
Another advantage of this approach is the ability of using EST
collections to identify stress-induced genes of known or unknown
function solely on the basis of their expression profiles using
microarray hybridization technology. This will have the added
advantage of providing the tools for assessing gene family (functional)
redundancy and complexity which will facilitate targeted gene
disruption strategies in Arabidopsis and other higher plant
models. Possibly the most interesting outcome of EST microarrays
will be the ability to monitor and compare stress-induced gene
expression profiles among different related stresses and in specific
mutants. This will allow us to capture individual "transcriptome
profiles" or stress fingerprints and discern interrelationships
or connections relating stresses or mutants not immediately obvious
through the study of one or a few genes. Also available are our
unique methods for rapidly identifying genes co-transcribed with
the stress genes of interest. Applying whole genome analysis technologies
will revolutionize stress biology in short order. Because the
field of genomics is moving so rapidly, largely driven by technological
developments, we will inevitably adjust our procedures to find
those best suited for collecting pertinent data in the most efficient
way possible. This consideration also underscores the necessity
for the Consortium to provide training for pre- and post-graduate
students for working within the context of integrative, large-scale
and collaborative projects which likely will dominate future biological
research. Therefore, we propose to develop an extensive education
and training network and formal workshops and conferences.
1. Setting up Model Systems for Analysis.
a. Models for cellular aspects of stress tolerance.
(i) Saccharomyces cerevisiae.
The genome of brewers
yeast, 15 Mb, is completely sequenced (Goffeau et al., 1996).
Among the more than 5,800 potential proteins, about 70 are known
to be differentially expressed under salinity stress, but we expect
more than 200 genes that have a functional relationship to salt
stress tolerance (see: Nelson et al., 1998). According to Dr.
Goffeau, who is involved in the yeast international project, a
collection of knock-out strains will not be available for the
next three years, although individual strains may be forthcoming.
As yet there is no coordinated effort towards the isolation of
stress-related functions either for yeast itself or in a plant
context. We will generate strains with deletions in genes necessary
for our work, based on work that is ongoing in the labs of Drs.
Bressan, Hasegawa & Pardo (Purdue) and, to a
smaller degree, in the Bohnert lab (UA).
In this sub-project we will identify plant osmotic stress tolerance
determinants as (i) suppressors of stress-sensitive yeast
mutants, (ii) functional plant homologs of yeast osmotic
stress tolerance based on sequence homology and gain of function
analysis, and (iii) interacting proteins with either plant
or yeast osmotic stress tolerance. The objectives are:
Isolation of osmotic stress responsive mutants by insertional
mutagenesis. We will isolate yeast mutants with increased
or decreased sensitivity to stress; and mutants in stress signal
transduction pathways using osmotically regulated promoter-reporter
fusion traps (Purdue).
Identification of osmotically regulated transcripts by genome
array analysis. We will establish gain or loss of function
by gene deletion or overexpression (Purdue, UA, OSU).
Identification of plant suppressors of stress responsive
mutants. This will be accomplished by direct genetic suppression
of the yeast mutant phenotype by complementation and determination
of their sequence or functional homology with yeast osmotic stress
tolerance determinants. (Purdue, UA, OSU).
Protein/protein interaction determinants. We
will isolate plant proteins that interact with the previously
isolated gene products. (Purdue, UA).
Part 1. Stress Responsive Yeast Mutants by Insertional Mutagenesis.
An existing Tn3 insertion library will be used for transposon
tagging mutagenesis in yeast (Burns et al., 1994). A yeast genomic
library constructed in an E. coli plasmid is mutagenized
with a Tn3 derivative containing lacZ, yeast LEU2 and
E. coli b -lactamase. NotI
digestion releases the vector. Linear DNA is transformed into
yeast and recombinations are selected that replace homologous
genomic sequences flanking the transposon. The ORF in one of the
transposon-derived termini extends into the adjacent lacZ coding
region. Therefore, some of the Tn3 insertions in yeast DNA will
generate in-frame fusions with lacZ which result in identification
of osmotically induced genes. The large size of this library,
~20 genome equivalents, and the ease with which the tagged mutant
allele can be rescued make this system useful for identification
of osmo-sensitive mutants (Burns et al., 1994). After establishing
linkage between LEU2 and the mutation, the gene fusion
will be isolated by plasmid rescue (Burns et al., 1994) and the
interrupted gene identified by sequence analysis using the yeast
> Mutants with altered sensitivity to osmotic/salt stress.
To isolate osmotically sensitive mutants, yeast cells transformed
with the Tn3 library will be screened for growth on medium with
NaCl (in varying amount) representing a moderate salt shock (Mendoza
et al., 1994). Systematic comparisons will be made on medium supplemented
with NaCl, KCl, LiCl or polyethylene glycol/ mannitol to distinguish
between salt and osmotic sensitivity for the isolation of mutants
with altered stress signaling capacity or effector determinants.
Besides cnb, we have isolated several independent osmotic
stress sensitive strains and are presently identifying wild type
alleles (Pardo et al., unpublished).
> Mutants in stress signal transduction pathways using
osmotically regulated promoter-reporter screening. Osmotic
stress signal transduction pathways will be dissected by utilizing
screens involving promoter-reporter fusions. Stress-responsive
promoters will be used for gene fusions of tolerance determinants
such as GPD1 (glycerol-3-P DH) or ENA1/PMR2A (Na+-ATPase)
regulated by sensing or salt stress signaling pathways. GPD1
is regulated by the HOG pathway and is responsible for osmotic
adjustment by glycerol (Varela & Mager, 1996). The ENA1/PMR2A
ATPase is primarily responsible for Na+ efflux across
the plasma membrane. The gene is regulated by NaCl through calcineurin-dependent
processes (Mendoza et al., 1994, 1996; Matheos et al., 1997; Stathopoulos
& Cyert, 1997). We have used ENA1 (-1384 to +40)-lacZ
translation fusion to monitor calcineurin-dependent activation
(Mendoza et al., 1994; 1996). Since numerous components
of the HOG pathway have been identified (Valera & Mager, 1996;
Posas & Saito, 1997), a focus will be further characterization
of the salt stress signal pathway. A promoter-reporter construct
is integrated into the genome, and the strain will be transformed
with the Tn3 library to isolate mutants that are responsive to
the stress, or are constitutively expressing the promoter.
Part 2. Identification of osmotically regulated Transcripts
by Genome Arrays. Microarray analysis will be used to identify
genes differentially expressed in response to stress (details
below). We are currently using an array from Research Genetics
[http://resgen.com] which contains 6,000 yeast ORF (DeRisi et
al., 1997; Wodicka et al., 1997). Gene expression differences
are monitored in wild type in a time-dependent adaptation to salt-shock
(minutes to one generation time) by using 33P-labelled
RT-products. Signals are measured by phosphorimaging. Similar
analyses will be carried out with mutant strains, but we expect
to switch to non-radioactive detection of inexpensive yeast microarray
s soon. Mutant analysis will facilitate further categorization
of genes that are differentially expressed in response to stress.
For example, genes expressed after salt treatment in hog or
pbs2 will not include those regulated by the HOG pathway.
To date, about 70 yeast genes are known to be differentially expressed
in response to salt stress (Nelson et al., 1998).
The functional involvement in stress tolerance of differentially
expressed genes will be determined through gain or loss of function
experiments. Genes will be constructed into expression plasmids
and transformed into wild type or stress sensitive (e.g. cna1D cna2D or
cnbD ) mutant strains
(Mendoza et al., 1994; 1996). Stress tolerance sufficiency of
the wild type or suppression of the mutant phenotype will be indicative
of stress tolerance determinants. Negative regulators of stress
tolerance may mediate a stress sensitive phenotype of the wild
type. Gene inactivation mutants will be generated by replacement
with alleles disrupted with a selectable marker. These will be
evaluated for stress sensitivity (loss of function).
Part 3. Identify Plant Suppressors of Stress-responsive
Mutants: Arabidopsis, Dunaliella and Mesembryanthemum.
Plant osmotic stress tolerance determinants will be identified
based on direct functional selection as suppressors of osmotic
stress sensitive yeast mutants. Alternatively, yeast stress tolerance
will be used to identify plant genes by sequence homology. Functionality
of the plant gene will be assessed by complementation of disrupted
mutants or suppression of a stress sensitive signal pathway mutant.
We will use cDNA libraries from stress-adapted plants which are
enriched for functional determinants of tolerance.
> Direct selection of plant suppressors of osmotic stress
sensitive mutants. To date, calcineurin null mutants have
been used to isolate putative plant transcription factors STZ,
STO and TOB175 (Lippuner et al., 1996; this
laboratory, unpublished). The functions of STZ and TOB175
are dependent on ENA1and current efforts are directed
at determining if the plant transcription factors can activate
ENA1. Maize Opaque-2 can transactivate the yeast GCN4
promoter (Mauri et al., 1993). Arabidopsis SAL1 (encoding:
3(2),5-bisphosphate nucleotidase/ inositol polyphosphate
1-phosphatase) was isolated by complementation of a ena1-4
mutant (Quintero et al., 1996). Osmotically adapted Arabidopsis
thaliana, Mesembryanthemum crystallinum and Dunaliella
salina cDNA libraries will be constructed (directional cloning)
into yeast/ E. coli l-expression vectors containing the
cre/lox recombination system for excision, e.g., lYES (Invitrogen),
lPG15, lAD5 (Brunelli & Pall, 1993; Mumberg et al., 1995).
Plasmid DNA will be used to transform osmo-sensitive strains.
Presently, we are screening a 428mM NaCl-adapted Chlamydomonas
reinhardtii in pYES for cDNAs that can suppress the Na+/Li+
sensitive phenotype of W303 (ura3 cna1::TRP1 cna2::HIS3 ena2-4::LEU2).
> Plant homologs of yeast tolerance determinants. Sequence
comparisons will be made of the yeast osmotic stress tolerance
determinants with the available plant sequence databases to identify
plant homologs. Confirmation of functionality of the plant determinants
will be based on complementing the phenotype of the corresponding
yeast mutant or suppression of a stress-sensitive signal pathway
Part 4. Isolation of Plant Proteins interacting with Yeast
or Plant Stress Tolerance Determinants. Future development
of the workplan includes isolation of plant genes based on biochemical
interactions of the encoded proteins with yeast/ plant stress
tolerance determinants using the yeast two-hybrid system (Chien
et al., 1991; Fields & Sternglanz, 1994). The yeast/plant
homologs will be used as bait to screen cDNA libraries derived
from Arabidopsis, Mesembryanthemum and Dunaliella.
Functional significance of interacting proteins will be confirmed
by genetic suppression of the appropriate yeast mutant.
This strategy is feasible, because many plant genes function
in yeast, for example STO, STZ, SAL1, HKT, AtKUP1, HAK1, CAX1,
phosphate or sulfate transporters (Lippuner et al., 1996; Rubio
et al., 1995; Hirschi et al., 1996; Liang et al.,
1997; Santa-Maria et al., 1997; Fu & Luan, 1998; Kim et al.,
1998). Similarly, HKT from rice and Mesembryanthemum:
(Golldack et al., 1997; 1998) and the Mesembryanthemum
ITR1 homolog of the yeast proton/inositol symporter (Nelson &
Bohnert, 1998) function in yeast. Also, the yeast CNA/B (calcineurin)
genes affect plant salinity tolerance (Cunningham & Fink,
1996; K Hirschi, personal communication; Pardo et al., 1998).
Recently, it has been determined that vacuolar compartmentation
of Na+ is a component of ion homeostasis in yeast (Nass
et al., 1997) as it is in plants (Niu et al., 1995).
(ii) Synechocystis sp. PCC6803.
Synechocystis represents an ideal model for stress responses
in a simple autotroph - photosynthesis, ROS scavenging, respiration
and chloroplast functions. The organism can be transformed with
very high efficiency relying on homologous recombination. This
cyanobacterial genome, 3.6 Mb, is completely sequenced , and the
small, non-redundant genome allows precise DNA engineering by
PCR-mediated, not plasmid-based, manipulations .
Among the more salient findings of the sequencing project is
the detection of over 80 ORFs identified as members of two-component
signal transducers and homologs of genes involved in stress responses
in higher plants. Synechocystis sp. PCC6803 also represents
an excellent model for osmoregulation since it tolerates up to
1.2M NaCl. Tolerance is effected through the accumulation the
osmolyte glucosylglycerol, accumulating by de novo synthesis.
Following salt-shock, maximal levels of glucosylglycerol are achieved
within 3 hours. Mutant analysis has provided insight regarding
the role of two genes expressed in response to salt-shock. StpA
encodes glucosylglycerol-P phosphatase involved in the salt-induced
accumulation of glucosylglycerol . GgtA encodes a subunit
of an ABC-type transporter which permits the maintenance of high
levels of solutes in the cytoplasm by recovering leaked glucosylglycerol
. Paralleling osmolyte accumulation, changes in the expression
of photosynthesis- and respiration-related enzymes have been reported
(Jeanjean et al., 1993; Joset et al., 1996).
Using Synechocystis sp. PCC6803, options for generating
mutants include random insertion of a resistance marker and screening
for salt-sensitivity and precise knockout mutagenesis. We chose
the latter method, different from that used for yeast, because
of the efficiency of insertional mutagenesis and the low complexity
of the Synechocystis genome. The objectives are three-fold:
Generating knock-out strains for known and suggested functions.
We will target 300 to 400 functions (~100/year) that are known
or suspected elements of the stress response (OSU).
Analyzing Synechocystis microarrays. We will
utilize existing arrays and generate arrays for stress-related
ORFs which will be used for monitoring gene expression under stress
in wild type, knock-out mutants and complemented mutants (OSU,
Utilizing plant cDNA libraries for complementation.
Knock-out strains will be complemented by cloned transcripts expressed
from the higher plants (OSU, UA, Purdue).
Part 1. Stress-responsive Mutants by targeted Knockouts.
Identified in the Synechocystis genome, mostly by homology,
are 80 two-component regulatory genes (e.g., ethylene response
sensor-like), kinases and phosphatases, GTP-binding proteins,
transcriptional repressors, and approximately 100 other genes
possibly involved in stress-related functions. These are components
of photosystems (e.g., psbH), enzymes (inositol monophosphatase-like),
ROS scavenging (SOD, gluthathione peroxidase) or they seem to
be homologs of ion/ water/ and metabolite transporters (e.g.,
mipE [a Mesembryanthemum homolog], ferritin, mscL).
Targeted genes will be identified, primers synthesized and
the genes will be disrupted by a spectinomycin resistance cassette
using a two-step PCR method. After disruption is verified, we
will use, depending on which function is eliminated, a set of
informative assays to characterize the phenotype. Test reagents
include NaCl, H2O2, H2O2
and iron, methylviologen and glucose. Synechocystis provides
an additional advantage because defects that affect photosynthesis
can be monitored by fluorescence spectroscopy that highlight individual
colonies. This is routinely used in the Burnap laboratory (OSU).
The strains will be available for complementation, either with
cDNA libraries from the higher plant/ algal models, or for functional
analysis through the expression of full-length cDNAs from the
Part 2. Microarray Analysis. Building on an existing
collaboration, the Burnap laboratory will receive microarrays
on filters for the approximately 6,000 indexed clones from the
laboratory of Dr. Tabata (Kazuza, Japan). Wild type and knockout
mutants will be subjected to stress regimes (e.g., NaCl, high
light, oxidative stress) and the resulting changes in gene expression
will be monitored. We will establish a clone collection for stress-related
genes at OSU for printing arrays. RNA will be isolated,
depleted of rRNA and tRNA species and labelled as documented by
the Burnap lab for unstable Fe-stress transcripts (Al-Khaldi et
al., 1998). Signals will be detected by phosphorimaging.
This strategy will identify new stress-regulated genes (gene
discovery), and will permit the identification of stress-regulated
genes. The results can be compared with those from the higher
plant EST sub-project. In addition, the information will help
feed the Synechocystis sp. PCC6803 knock-out group with
new targets, and ultimately new phenotypes.
Part 3. Genetic Complementation. Initial targets
for complementation are the full-length cDNAs which are already
available from the Cushman/Bohnert collections from Mesembryanthemum,
the Bressan/ Hasegawa Chlamydomonas/ Dunaliella
transcripts, and the Zhu collection of Arabidopsis transcripts.
Foreign genes and libraries from Dunaliella, Mesembryanthemum
and Arabidopsis are brought into the disruption mutant
using neutral site shuttle vector. The neutral recombination site
is at a unique, separate location on the chromosome which is distinct
from the locus of the spectinomycin cassette-disrupted gene, does
not confer a phenotype when disrupted in the wild-type, and serves
a generalized recombination platform to facilitate recombination
of the test gene(s). The recombination of test genes will be forced
to coincide with the acquisition of a second antibiotic resistance
(kanamycin). Therefore, the first selection will be for conversion
of the Spr to the Spr/Kmr phenotype.
Transformants (or populations of transformants in case of an introduced
library) will then be screened for re-acquisition of the character
lost by the targeted disruption. For example, complementation
of a disrupted salt-tolerance gene would be selected on high salt
plates on which the mutant cannot grow or shows impaired photosynthesis
by fluorescence criteria. Because different organisms have evolved
unique solutions to common biochemical problems, thus we expect
that the complementing gene will encode for a non-homologous protein
performing an analogous cellular function.
(iii) Dunaliella salina. Dunaliella
species are unicellular algae that include some of the most
halotolerant plants with some species capable of growth in salt
of up to 5.5 M (Avron, 1986). The alga adjusts to osmotic perturbations
by volume regulation through a signal transduction pathway that
involves phospholipids and small G proteins (Bental et al., 1990;
Memon et al., 1993). Volume regulation is followed by osmotic
adjustment resulting mainly from glycerol synthesis and accumulation
(Sadka et al., 1989). Also, Dunaliella has great
capacity for the maintenance of ion homeostasis in saline environments
(Pick, 1992) through the plasma membrane H+-ATPase
and Na+/H+ antiporter, and other P-type
ATPases (Katz et al., 1991; Weiss & Pick, 1996). Additionally,
60 and 150 kD plasma membrane proteins have been implicated in
the control of ion homeostasis for the growth of D. salina
in high salt concentrations (Sadka et al., 1991; Fisher
et al., 1994). Currently, the Purdue group is constructing
libraries in the yeast l vectors (Brunelli & Pall, 1993;
Mumberg et al., 1995) using the GAL1 promoter with RNA
from 2 M NaCl adapted D. salina cells. The pYES2 library
will be transformed into the salt sensitive yeast strain W303
cna1-2D ena2-4D and
screened for galactose-dependent complementation of salt sensitivity.
Alternatively, subtracted cDNA libraries will be generated for
complementation of salt sensitive mutants.
cDNA libraries from salt-stressed Dunaliella will be
used for EST analysis (OSU), for complementation analysis
(OSU, Purdue) and for microarrays (UA).
(iv) Aspergillus nidulans, a simple multicellular
eukaryote includes ~8,000 genes (Kupfer et al., 1997) in
a 31 Mb genome (Brody et al., 1991), a 28% increase in
gene complexity over the unicellular yeast (Dujon, 1996). A.
nidulans is renowned for its metabolic diversity (Brown et
al., 1996), able to metabolize numerous complex food sources S.
cerevisiae is unable to utilize. Thus, this gain in genetic
complexity is likely to reflect the biological differences between
these organisms, especially with respect to genes required for
colonizing habitats in which osmotic potentials vary greatly.
A. nidulans is xero- and osmo-tolerant due to the accumulation
of glycerol/erithritol as osmoregulatory solutes (Griffin, 1981;
Beever & Laracy, 1986). In the A. nidulans EST collection
(release 1/1998), 9% of the cDNAs with known biological function
encode domains related to various stress response pathways, including
those from plants (Prade & Ayoubi, unpublished). For example,
EST entries for the heat shock and ubiquitination pathways (8.6%
of the EST-set), for ROS scavenging proteins (catalase, peroxidase,
Cu,Zn-SOD), for uptake, transport and compartmentalization of
ions (ATPases/ pumps), and for compatible solute synthesis (glycerol,
trehalose, proline, betaine, mannitol and sorbitol) were found.
Homologs for regulatory proteins are also represented (HAL2, HOG1
and calcineurin) (da-Silva et al., 1994; Kim et al., 1996; Lippuner
et al., 1996; Quintero et al., 1996; Parrou et al., 1997). Finally,
75% of the non-redundant stress-EST-subset were found to be more
homologous to genes identified in eukaryotic organisms other than
S. cerevisiae (Prade & Ayoubi, unpublished).
The Prade lab (OSU) is interested in a comparative genomics
approach towards stress biology. Recognizing the large number
of genes involved in stress adaptation in eukaryotic organisms,
the lab is interested in generating a homology-based network between
a cyanobacterium, a unicellular and a multicellular tractable
model eukaryote, and the higher plant models that will render
the complete gene complement for the salinity stress response.
The specific objectives are:
Identification of plant stress genes as A. nidulans
homologs. We will generate and examine inactivation mutants
in A. nidulans for higher plant genes involved in salinity
stress (OSU, UA).
Examination of A. nidulans salinity stress
responsive genes. We will generate A. nidulans random
insertional and overexpression mutants and select strains with
altered sensitivity to osmolytes for in-depth analysis. Novel
genes will provide the model to identify plant homologs (OSU,
Expression profile of salinity stressed A. nidulans.
We will use DNA arrays to determine stress induced gene expression
profiles in wild-type and mutant A. nidulans (OSU, Purdue,
Part 1. Identification of Plant Stress Genes as A. nidulans
Plant - fungus homologies. Fungal homologs will be identified
by one of three ways: a) direct amino acid sequence comparisons
between A. nidulans and higher plant EST databases; b)
DNA/DNA hybridization (Prade et al., 1997) using probes
derived from Dunaliella and higher plant models and, c)
functional complementation (bi-directional gene transfer) of A.
nidulans salinity stress mutants with plant genes or libraries
(Borgia et al., 1994; Lippuner et al., 1996).
Inactivation of plant stress homologs in A. nidulans.
Null strains will be constructed via gene replacements (Prade
& Timberlake, 1993; Prade & Timberlake, 1994) using pRP100B
(argB) as the homologous recombination driver and RMS11
(yA2, pabaA1, argB::trpC801, trpC801)as the recipient strain.
This simple gene replacement system is robust and efficient for
The phenotype of salinity stress response inactivation mutants.
A. nidulans mutants will be subjected to a standardized
set of osmotolerance tests, revealing loss or gain of osmotolerance
or indicating altered regulatory properties. In addition, we will
determine the effect of various mutations on radial growth and
sporulation. Alterations of radial growth or changes in conidiation
are easily detected and are criteria for functional abnormalities
in multicellular processes associated with nuclear movement (Beckwith
et al., 1995), septation (Kaminskyj & Hamer, 1998), wide-domain
gene regulation and compartmentation and metabolite traffic. The
results are likely to highlight sets of plant genes with functional
equivalence in fungi.
Part 2. Examination of A. nidulans Salinity Stress
Responsive Genes. The experiments for this section are designed
to reveal new A. nidulans stress responsive genes that
are not yet known as determinants in the plant stress response.
Isolation of salinity stress responsive genes using RIM.
We will utilize the recently developed RIM (Recombination Induced
Mutagenesis) technique to create random collections of insertional
mutants (Prade & Ayoubi, unpublished). RIM has been designed
for large-scale functional genetics experiments and is based on
optimized frequencies for disruptive and homologous random recombination
events. RIM is also useful for creating random, in frame reporter
fusions. RIM-mutants are automatically tagged and incorporate
a series of features useful in physical and genetic mapping, generation
of STSs, molecular cloning and gene characterization. Salinity
stress responsive loss-of-function mutants will be recognized
as colonies with altered radial growth rates or other phenotypic
markers in response to "high" (tolerant) or "low"
(sensitive) Na+ ion concentrations.
Isolation of stress responsive genes by overexpression.
We will utilize a nutritionally inducible (threonine) overexpression
DNA library, constructed by the Adams group (Marhoul and Adams,
1995), to identify genes that cause changes in salinity response
Cloning of stress responsive genes. Osmo-/halo-tolerant
or -sensitive mutants, obtained through both mutagenesis methods,
will be subjected to tagged-rescue, STS sequencing, gene cloning
and characterization following standard techniques. Recovery of
one-step STSs is a standard feature of our RIM libraries which
allows rapid physical and genetic mapping.
The main focus of this section is to outline the biological
functions necessary and sufficient to mediate the salinity stress
response in A. nidulans. It is anticipated that we will
find genes not yet known in higher plants, yeast or other model
systems. We will use these genes (or equivalent null mutants)
to discover homologs (or functional complements) in the other
models and in higher plants. In addition, we expect to find novel
stress-related genes, not obtainable from unicellular models such
as, for example, the ones involved in cell-cell interactions.
Part 3. Expression Profile of Salinity Stressed A. nidulans.
We will initially construct a partial but informative A. nidulans
DNA-based array that contains three well defined clusters: (a)
a partial, but comprehensive, collection of ESTs representing
major cellular and metabolic functions; (b) the complete
set of all stress related STS-tags from A. nidulans ; and
(c) all A. nidulans genes with homologies to plant
stress genes. These arrays will be utilized to produce expression
profiles of wild type A. nidulans and mutant strains under
a variety of conditions. The results will be integrated into an
expression network with other model- and plant-profiles, priming
the backbone on which comprehensive gene complements will be determined.
In summary, we hypothesize that these simple prokaryotic and
eukaryotic models exemplify, in large part, the basic adaptive
responses to salinity and osmotic stress that have evolved early
in evolution. Therefore, comparative EST analyses among these
organisms (see below) will provide a strong foundation for identifying
a common set of stress-relevant genes in higher plants. Undoubtedly,
there will be unique adaptations and signaling pathways found
in other organisms not represented in this set of models. For
future comparative studies in other organisms, the proposed work
will provide an important, definitive reference resource.
b. Higher Plant Models.
We have chosen Arabidopsis thaliana and Oryza sativa
as models for salt- and osmotic stress-sensitive species. We propose
to exploit the Arabidopsis genetic system and to extend
the analysis of rice and Arabidopsis EST accessions (Newman
et al., 1994; Sasaki et al., 1994; Cook et al., 1996; Rounsley
et al., 1996) to include salinity and osmotically challenged plants.
This work will complement functional analysis of genes using T-DNA
mutagenesis strategies carried out simultaneously in Arabidopsis.
Most ESTs have been obtained from plants or cells that had not
been subjected to any stress treatment, although several Arabidopsis
groups (e.g., Michigan State Univ. and Gyeongsang National Univ.)
are now including EST-sequencing with clones from libraries of
specifically treated plants. We know that cDNA libraries from
unstressed plants are missing a set of ionic and osmotic stress-related
transcripts (see ice plant example, below), which we estimate
comprises approximately 20% of all transcripts. For Arabidopsis,
this would amount to approximately 4,000 stress-related transcripts,
assuming approximately 21,000 genes in total (Bevan et al., 1998).
We include Mesembryanthemum in our research plan because
it is naturally salinity- and osmotic-stress-tolerant and is known
to contain genes that are absent or are not expressed in a stress
context in crop plants. Examples of such genes are documented
(Vernon & Bohnert, 1992; see: Nelson et al., 1998; Bohnert
et al., 1998). Mesembryanthemum libraries from UA and
OSU will include tissue-, cell- and developmental state-specific
cDNA libraries. In addition, Dunaliella libraries from
Purdue will be used as halophytic sources of novel genes
for (1) complementation experiments in unicellular models
and Arabidopsis, (2) EST analysis for "digital"
expression profiles, and (3) microarray and functional
analyses of stress-related transcripts.
Arabidopsis thaliana. Arabidopsis
as the most popular model for higher plants has been used successfully
for studies of plant development, phytohormones, light signaling
and disease responses. The establishment of Arabidopsis
as a model organism to study salt and drought stress tolerance
or signaling has been recent, but is proving to be extremely powerful
(Wu et al., 1996; Zhu et al., 1997; Ishitani et al., 1997). Already
(Zhu lab, UA) more than 40 salt-overly-sensitive (sos)
Arabidopsis mutants (Wu et al., 1996; Liu & Zhu, 1997)
have been identified. One of these loci, SOS3, has now
been positionally cloned and encodes a key signaling component
with homology to the B subunit of calcineurin (Liu & Zhu,
1998). cDNA libraries will be generated from ecotype Columbia
for the EST and microarray s (UA).
Oryza sativa, rice, is our model for a
crop plant for several reasons, in particular because of a small
genome size and because of availability of a large number of EST
sequences, genomic sequences, high transformation efficiency,
and information about cell biology, biochemistry and physiology
(Matsuo et al., 1993; 1995). In addition, among the more than
2,000 breeding lines several salt-tolerant indica-lines are included
to which we have access (Drs. G. Kush, D. Senadhira, J. Bennett,
IRRI, Manila). The Bohnert lab (UA) is working on salt-tolerant
(Pokkali) and -sensitive (IR29/ IR64) rice and has established
cDNA libraries for salt-stressed (150mM NaCl, 24 h) rice Pokkali.
Specific projects in the lab include the analysis of Pokkali/IR29
potassium transporters, analysis of inositol biosynthesis under
stress and transfer of multigene vectors into rice (Golldack et
al., 1997, 1998; Zhu et al., 1998). cDNA libraries for complementation
will be provided to Purdue and OSU .
Mesembryanthemum crystallinum. The halophytic
ice plant with a genome size twice that of Arabidopsis
(DeRocher et al., 1990; Meyer et al., 1990; Adams et al., 1998)
provides an excellent model for inducible responses to salinity
and drought which allow the plant to colonize extreme habitats.
The plant has been a rich source of novel genes for salinity-adaptive
responses such as the synthesis of osmoprotectants (Vernon and
Bohnert, 1992; Ishitani et al., 1996), facilitated water uptake
(Yamada et al., 1995) or ion sequestration (Tsiantis et al., 1996;
Löw et al., 1996; Su et al., 1997; 1998). The long-term adaptation
to water stress by a switch from C3 to Crassulacean acid metabolism
(CAM) is a paradigm for metabolic adaptations that improve water
use efficiency (Cushman and Bohnert, 1997). A large-scale EST
sequencing project is in progress for leaf tissues of adult plants
(OSU) and epidermal bladder cells (UA), a solute
and ion storage organ, making this the best studied higher plant
halophyte available. Large EMS- and gamma irradiation-induced
mutant populations have now been established (Adams et al., 1998;
Cushman et al., 1998). An efficient somatic embryogenesis-based
regeneration system that is now available for the ice plant (Cushman
et al., unpublished) is expected to streamline transformation
studies now in progress.
2. Collecting T-DNA tagged Arabidopsis Mutants.
a. Tagged Lines. We will generate Arabidopsis
tagged lines defective in stress tolerance and/or signaling. Gene
knock-out in Arabidopsis via T-DNA insertions provides
direct and definitive functional assessment of the gene product.
One important aspect of our genome-wide analysis of stress tolerance
is to identify and characterize all possible Arabidopsis
mutations that would lead to alterations of plant stress tolerance
and/or stress gene regulation. The Zhu lab (UA) has developed
a high throughput genetic screen for Arabidopsis mutants
defective in stress gene regulation (Ishitani et al., 1997). The
screen utilizes transgenic Arabidopsis plants expressing
the firefly luciferase marker (LUC) under control of a
salt-, drought-, cold- and ABA-responsive promoter (RD29A)
and low-light video imaging technology. Stress-induced gene expression
in hundreds of plants can be quantitatively detected by luminescence
imaging in a matter of several minutes. A Petri dish containing
up to 1,000 seedlings can be subjected to multiple stress and
hormonal treatments and imaged repeatedly. From screening 300,000
EMS-mutagenized M2 Arabidopsis seedlings containing RD29A-LUC,
the Zhu lab has recovered several hundred mutants defective in
various components of the stress response pathways (Ishitani et
al., 1997). Preliminary genetic analysis of 100 of the mutants
exhibiting strong luminescence phenotypes indicated that the screen
is still far from being saturated because most of the loci are
defined by single alleles.
We propose to expand the genetic screen for stress tolerance/signaling
mutants and to screen for T-DNA alleles. This calls for a large
scale T-DNA mutagenesis of the plants expressing the RD29A-LUC
transgene. Since the RD29A-LUC plants already contain a
NPTII gene for kanamycin resistance, we are using a construct
with the BAR gene for T-DNA insertional mutagenesis. With
the very efficient Agrobacterium-infiltration method for
Arabidopsis transformation, we expect to generate 150,000
independent transformed lines within the next two years. Our initial
transformation efficiency has ranged from 2% to 5%. Assuming an
average 1.5 inserts/per plant line, the 150,000 lines would contain
more than 200,000 inserts, which would nearly saturate the Arabidopsis
genome, i.e., one insertion per approximately 1,000 bp. We are
fully aware of the large number of T-DNA lines that have been
generated in various labs around the world. Although most of these
lines have not yet been made available to the public, our intention
is not to duplicate their effort. Rather, we will generate lines
appropriate for our goal, because the mutagenesis has to be in
the RD29A-LUC transgene background in order for us to screen
for stress-related mutants. We pledge to distribute these T-DNA
lines and the mutants freely to others as they become available.
Also, lines will be made available to others for mapping purposes.
The screen of the T-DNA populations will begin as soon as we
have T2 seeds from 5,000 or more independently transformed lines.
Therefore, most of our screening and mutant characterization work
will be carried out in parallel with mutagenesis and selection
of more transformed lines. Mutants will be characterized initially
by examining endogenous RD29A and other stress gene expression
via Northern analysis and, later microarray analysis with
the stress-responsive set of transcripts. Mutants exhibiting altered
tolerance to salt and/or drought stresses will be chosen for T-DNA
co-segregation analysis to determine whether the genes are tagged.
While it is not realistic to expect complete detailed characterization
of every mutant we isolate, we do expect to complete within the
tenure of this project the mutagenesis, mutant screening, and
tagging analysis of at least the most interesting mutants.
b. Expansions of the Concept. In addition to
the RD29A-LUC system, we have constructed Arabidopsis
plants expressing luciferase under control of other stress-activated
promoters, the osmotin promoter (in collaboration with Bressan
lab, Purdue) and a stress-regulated water channel promoter
(Bohnert lab, UA). These other stress responsive promoters
contain cis- elements different from those found in the
RD29A promoter. The use of these promoters will enable us to recover
mutants that define other stress response pathways. EMS mutagenesis,
mutant screening and characterization will be according to the
RD29A-LUC system (Ishitani et al., 1997).
These new EMS mutations, together with the more than 100 strong-EMS
mutants already identified through RD29A-LUC will be genetically
mapped. Mapping relies on inter-ecotype DNA polymorphism. We have
extensive experience using CAPS and SSLP markers (Wu et al., 1996;
Liu & Zhu, 1997). Our genetic analysis of the mutants from
the RD29A-LUC system has revealed at least 30 complementation
groups (Zhu et al., unpublished). Therefore, with new mutants
from the osmotin-LUC (UA, Purdue) and water channel-LUC
systems (UA), we will likely be looking at over a hundred
genetic loci to map with. We will initially rely on SSLP and CAPS
markers but will use SNPs (single nucleotide polymorphisms) markers
that can be of extremely high density and automated, once the
methodology is better defined (F Ausubel, personal communication).
With a large number of genetic loci placed on specific regions
of the chromosomes, we will be able to best take advantage of
the genome sequence of Arabidopsis, because candidate genes
can be identified and confirmed through functional complementation
of our mutants.
3. Generating Libraries of stress-adaptation Transcripts.
Estimating 20% or 4,000 transcripts to be stress-relevant in
Arabidopsis (and a slightly higher, but similar number
in Mesembryanthemum and rice), we propose to sequence at
least 10,000 ESTs from these species to ensure good representation
of stress-regulated transcripts in the EST databases. At OSU
(and UA), the M. crystallinum EST database and clone
collection for the ice plant now includes 1,600 accessions representing
randomly selected cDNAs from libraries constructed in l
-Uni-Zap-XR (Stratagene) using mRNA isolated from shoots of six-week
old, hydroponically grown plants either well-watered or osmotically
stressed (30 h; 0.4 M NaCl). Approximately 15% of the EST represent
genes characterized previously in the ice plant. On average, more
than 42% of the clones show no significant homology to the non-redundant
databases and presumably represent unidentified or novel genes.
Importantly, 31% of the clones isolated from well-watered
plants are unknown, whereas 54% of the clones from salinity-stressed
plants represent unidentified transcripts, with greater than 50%
of these transcripts having no significant homology to EST accessions
derived from other higher plants (e.g., rice, Arabidopsis).
The high proportion of unidentified and novel genes in stressed
plants underscores how little we know about gene function and
plant gene expression patterns under environmental stress conditions.
Characterization of expression patterns of unknown genes using
microarrays will facilitate targeted functional and comparative
analyses of these genes. Dramatic changes in expression patterns
are apparent when comparing unstressed and salinity stressed plants.
For example, transcripts for photosynthetic functions (RUBISCO,
photosystem and light harvesting complexes) decrease 5-7 -fold
in abundance, whereas transcripts encoding ion transport and compartmentation,
osmotic adaptation, and CAM (H+-translocating vacuolar ATPase,
glycolytic/gluconeogenic, malate synthesis and decarboxylation,
inositol biosynthesis) increase 5-6-fold in abundance. ESTs representing
proteolytic enzymes (ubiquitin hydrolase, proteosome components,
cysteine proteases) increase 12-fold in abundance (Cushman et
al., unpublished). We propose to expand the scope of our current
EST sequencing efforts to include the following additional libraries:
(i) From Arabidopsis (ecotype Columbia) one library
each from stressed (100mM NaCl) seedlings and mature early-bolting
plants (generated at UA).
(ii) From rice (Pokkali) one library for plants approximately
three weeks of age (three to four leaf stage) stressed for 12
h by the addition of 125 mM NaCl (generated at UA).
(iii) From Dunaliella one library from an upshift
experiment (e.g., 300mM to 2M NaCl) within less than one hour
after the upshift (generated at Purdue).
(iv) From Mesembryanthemum we will utilize four
types of libraries. One library will be for seedlings (250 mM
NaCl) and one for plants between the juvenile and reproductive
phase (5 weeks old, 500 mM NaCl) (Adams et al., 1998, for a description
of growth phases). The third library will be for epidermal bladder
cells, a salt-storage organ (Adams et al., 1998), after approximately
24 hours of stress when these cells begin to develop (UA).
A fourth library of salt-stressed roots (6 weeks old, 400 mM NaCl)
will be included (provided by OSU) to define adaptive changes
occurring at the primary site of ionic contact. At present all
cDNA libraries, after mass excision, are available as double-stranded
plasmids. For the complementation projects at OSU and Purdue,
vectors compatible with the models will be used. At UA,
we will make new libraries for this purpose, while using existing
libraries for the EST sequencing, generating full-length cDNAs
by 5 RACE (see below) and microarrays at present. To reduce
the probability of redundant sequencing of abundant stress-related
transcripts, libraries will be prescreened by colony hybridization.
We will also limit the number of non-stress-related transcripts
by using subtracted libraries. Current estimates are based on
manual preparation of templates and sequencing reactions. However,
we expect projected cost to be lower in the future as we implement
operation of robotic workstations requested for UA and
OSU. With these capabilities, we will be able to accelerate
the rate of finding new transcripts. Although limited EST sequencing
will be done at UA, most ESTs information will be generated
at OSU which will also serve as the project-hub for bioinformatics,
including sequence data processing, polishing, annotation, submission,
4. Constructing Diagnostic Micro-arrays for Stress Parameters.
a. Microarray Analysis of Wildtype Plants under Stress Conditions
and of Mutants with Defects in Different Stress Signaling Components.
Microarray technologies have greatly facilitated the analysis
of perturbations of gene expression (DeRisi et al. 1996, 1997;
Heller et al., 1997; Schena, 1996; Schena et al., 1995, 1996;
Shalon et al., 1996). Microarrays are available in two different
forms: oligonucleotides photolithographically synthesized in
situ on "chips" (Affymetrix, InCyte), or DNAs mechanically
deposited onto microscope slides (Synteni). Information is provided
using mRNA from the tissue under control and test conditions.
cDNAs are then synthesized in the presence of two fluorescent
NTPs, typically Cy3 and Cy5-dUTP, mixed and hybridized to the
arrays, and the intensities of the two fluorescent signals recorded.
We will not discuss photolithographic production of oligonucleotide
chips, because of the presently high cost, although this technology
has advantages which could be exploited in our project. Deposition
of DNA for microarrays is done using robotic spotters. They typically
employ a quill-type tool for sampling/depositing the DNA; other
approaches are under development. Element densities are determined
by drop size and positional accuracy of the robot. A typical array
spacing of 500 µm results in 2x103 elements on
a 22 x 22 mm microscope slide; forthcoming instruments aim at
a spacing of 200 µm, resulting in 12 x 103 elements/slide.
Following hybridization, arrays are read using fluorescent scanners,
which are either custom fabricated or, recently, have become commercially
available. Software to quantitate, compare, and store hybridization
signals is available in both the public and private sectors.
Technical Details. The Galbraith lab (UA) has
adapted a Biomek 2000® Laboratory Automation Workstation
for first-generation robotic cDNA spotting (Macas et al. 1998).
The resultant arrays have cDNA elements of ~125 m
m diameter, and a spacing of 500 m
m. We wrote software in Tcl to control the robot, and have made
this software freely available. Arraying involves dipping the
pins into the DNA samples, followed by consecutive spotting of
the samples onto 28 slides. Pins are cleaned by two washing steps
and one drying step before reloading. Using four pins, the Biomek
will spot the samples of a 96-well microtiter plate onto 28 slides
in 1 h (~one-half the speed described for the NIH robot (http://www.
nhgri.nih.gov/DIR/LCG/15K/HTML/aboutreader.html); spotting 104
elements on 28 slides using 16 pins would take about 24 h.
As part of this proposal, the Galbraith lab wishes to continue
development of arraying technologies by constructing a second-generation
arraying instrument with an increased array density (center-to-center
spacing of 200 µm), permitting arrays of 12,100 DNAs within
a 22 x 22 mm area. It should also have an increased print speed
to permit arraying of elements within a shorter time. Unpublished
and web-based data of other groups suggest these increases are
possible. Short of purchasing an arrayer, should instruments become
available, there are two ways that a second-generation instrument
can be developed for use in this project. They involve custom
fabrication of the instrument either in house, or by a third party,
using available or modified design specifications. A final decision
on which direction to take will depend on the developments over
the next few months in this extremely rapidly-changing field.
Mechanical deposition typically employs cDNA samples that have
been PCR amplified from library clones. We employ 5 amino-modified
primers, which produce amplified DNA that can be attached to silylated
slides using aldehyde chemistry. For array scanning, the Galbraith
lab (UA) has obtained a General Scanning ScanArray®
3000 DNA array hybridization scanner. With a pixel size of 10x10
µm the instrument provides spatially accurate and sensitive
analyses of the hybridization patterns of the DNA arrays for analyzing
first and second generation arrays.
For microarray-based analysis of stress-specific transcription,
our experimental approach involves first placing wild type DNA
from EST/clones of the higher plant models, derived mostly from
"stressed libraries", in the array analysis. Arrays
from an increasing number of elements will be probed using the
mixed fluorescent cDNAs prepared from these plants. In the second
set of experiments, comparisons will be made between different
mutants and wild type, under control and stressed conditions.
Array capacity is sufficient to gradually accumulate samples from
the unicellular models and their mutants. Arraying is established
at the UA where we have begun to analyze ESTs (see the dBEST database)
from Mesembryanthemum (provided by OSU and UA).
All participant laboratories will be contributing to array
analyses, mailing 96-well plates to UA; a second arrayer/reader
will be set up at OSU.
b. Direct Identification of Stress-affected Transcripts.
At UA, the Galbraith lab has recently developed techniques
that directly characterize changes in nuclear transcripts that
occur in response to alterations to a stimulus applied in vivo
(Macas et al., 1998b). Termed Nuclear Expressed Sequence Tag (NEST)
analysis, this technique starts with control and test plants that
are subjected to the stimulus of interest. Next, tissue homogenates
are prepared and nuclei rapidly isolated through flow sorting,
after staining with a DNA-specific fluorochrome. RNA is extracted
from the sorted nuclei, and is captured on magnetic beads carrying
oligo-dT. cDNA is synthesized, and is restricted using an enzyme
that cuts frequently (NlaI). Linkers are then ligated onto
the 5 ends of the bead-linked cDNA tags, and PCR is employed
for amplification. Tags are then displayed on large, diagnostic
polyacrylamide gels. Differences in transcription produce altered
profiles in the different lanes, which are visualized using silver
staining. Individual bands are excised, reamplified and directly
NEST analysis is conceptually similar to differential display,
but has important advantages. First, since a single linker is
ligated onto the cDNA tags, PCR amplification is done under stringent
conditions, which eliminates low stringency artifacts (c.f. differential
display). Second, NEST analysis is highly sensitive, employing
few nuclei and correspondingly requiring small amounts of RNA.
Finally, this approach samples the nuclear RNA transcriptional
spectrum, rather than the steady-state cytoplasmic mRNA pool.
It therefore is not biased against transcripts having short cytoplasmic
half-lives and, therefore, represents faithfully transcriptional
activities of cells.
c. Analysis of Gene Expression Coordinated with Promoters
of Interest. For the various stress regulated transcripts
that are identified, a critical next step is to define those genes
that are coordinately up- and down-regulated. This is done as
follows: first, the promoter of interest is placed upstream from
a chimeric coding region comprising an effective nuclear localization
signal, GFP, and b -glucuronidase (GUS).
We have previously described the characteristics of this recombinant
construction (Grebenok et al., 1997a), and the properties of transgenic
plants constitutively expressing the chimeric protein (Grebenok
et al., 1997b). Briefly, transgenic plants exhibit green-fluorescent
nuclei within all the cells that are expressing NLS-GFP-GUS. These
nuclei can be isolated from homogenates and flow-sorted based
on green fluorescence. For transgenic plants expressing NLS-GFP-GUS
under the control of tissue specific promoters, only these particular
tissues have green nuclei; therefore, flow cytometry can be used
to purify them away from the remaining, non-fluorescent nuclei.
The second part of the strategy involves NEST analysis as described
above (UA, with sub-sets of cDNAs provided by Purdue,
d. Subcellular localization of the protein products of
stress-induced genes. A large proportion of the genes
identified as being stress-induced will bear no homology to known
genes. For this reason alone, systematic approaches are needed
to provide information about the potential roles of the encoded
protein products. We propose construction of translational gene
fusions between these proteins and GFP. The chimeric coding sequence
is then placed under the control of a suitable promoter, and the
subcellular distributions of the chimeric protein product examined
in transfected protoplasts or transgenic plants using confocal
microscopy, techniques for which considerable experience and equipment
is available at UA (Sheen et al., 1995; Yamada et al., 1995; Galbraith
et al., 1995; Grebenok et al., 1997a; 1997b). For GFP fusions,
variants are available that improve protein expression and enhance
fluorescence emission through altered fluorescence yield, photostability,
and emission/excitation spectra (Haseloff et al., 1997;
reviewed by Galbraith et al., 1998). For construction of translational
fusions, Clontech provides several vectors (pEGFP-N & pEGFP-C
series) whose coding regions are identical to ours (Grebenok et
al., 1997a, 1997b).
An additional advantage of a transgenic approach will be that
subcellular distributions of chimeric proteins can be examined
as a function of the imposition of stress. Stress-induced alterations
in subcellular location, e.g. membrane cycling of water channels,
will provide information about functions. We propose this as a
systematic approach in the sense that all genes that are identified
as being stress-relevant will be candidates for analysis, but
considering the high number of expected transcripts, only a subset
of these will be selected for study as translational GFP fusions.
The subset will be chosen based on whether the predicted coding
sequence contains oligopeptide motifs or structural domains of
interest, such as putative transmembrane domains, nuclear targeting
signals, or transcription-relevant motifs (zinc-fingers, activation
domains), or motifs that imply protein-protein interactions (zinc-fingers,
leucine zippers). The approach is highly sensitive and measurements
can be made with a high degree of spatial resolution in vivo.
We anticipate that some gene products will be recalcitrant to
over-expression in plants as fusion proteins. Thus, for all proteins
displaying specific subcellular patterns of targeting, these patterns
will be confirmed through independent methods (biochemical fractionation;
western blotting; immunolocalization). GFP spectral variants also
provide the possibility of co-localization experiments. Considering
the finite number of different intracellular locations to which
proteins can be targeted, it will be possible to categorize the
targeting patterns of the different proteins.
5. Defining the Master Set of Stress-relevant Genes.
Generating stress-responsive EST collections represents the
first step in our complete characterization of genes involved
in stress adaptation. We propose to generate and sequence a collection
of full-length cDNA clones representing the master set of stress-responsive
genes in Arabidopsis, Rice, Mesembryanthemum and
Dunaliella which will serve as an adjunct to chromosome sequencing
projects and molecular marker development in, for example, corn
and Arabidopsis. This master set of stress genes will be
extremely useful in guiding the choice of future, directed mutagenesis
strategies and in identifying open reading frames in genomic DNA
sequences of genes identified by random insertional mutagenesis.
Full-length cDNAs will also be needed for systematic, functional
analysis of the potential roles of unknown protein products using
gene fusions between these proteins and GFP (see previous section).
cDNAs will provide essential reagents for systematic microarray
analysis for comparing expression patterns in response to different
stresses and in specific mutants analogous to the commercially
available yeast ORF collections. Complete cDNA sequences will
also be important for correct categorization and selection of
clones based on predicted structural motifs and domains, as well
as comparative informatics studies across phyla. Selection criteria
for inclusion in this set of genes will not be limited to stress-inducible
expression patterns discerned from microarray analyses. Since
not all genes encoding stress-relevant functions will be inducible,
we will include additional genes defined by genetic and reverse
genetic approaches. In yeast, for example, constitutively expressed
components of the HOG signaling pathway are known (Posas &
Saito, 1997). This fact justifies the emphasis we place on transcripts
present in libraries from long-term stressed plants, as well as
the emphasis on three different unicellular models for complementation
and the mutant screens using T-DNA tagged Arabidopsis lines.
Technical Aspects. Standard cDNA libraries contain truncated
versions of the original mRNA. Rather than repeating all analyses
with the new cDNA libraries, we will capitalize on what is already
available in the current EST collections in Arabidopsis
and Mesembryanthemum and conduct 5' RACE for the master
set of ESTs. Similarly, all new stress-responsive ESTs will be
converted into full-length cDNAs for the functional analyses (UA,
Purdue, OSU). To favor production of full-length cDNAs, double
stranded cDNA will be made from different tissue types (e.g.,
leaves and roots) using "lock-docking" oligo(dT) primers
to eliminate 3' heterogeneity during first strand synthesis reactions
and Marathon cDNA adaptors which block 3' end extension of ligated
adaptor primers during double-stranded cDNA synthesis (Chenchik
et al., 1996). 5' RACE reactions will be primed with gene-specific
primers designed from EST information. 5' RACE amplifications
will be performed using KlenTaq polymerase mix (Clontech, Inc.)
to ensure recovery of large cDNAs. RACE products will be cloned
and sequenced. Using the 5' sequence information, full-length
cDNAs will be recovered by "end-to-end" PCR. Rapid sequencing
of longer cDNA inserts will be accomplished at the OSU sequencing
facility which currently houses an AB 373 automated DNA sequencing
systems with the stretch liner and 64 well upgrades (Perkin-Elmer).
Recent technological improvements in automated sequencing systems
including more sensitive fluorescent dyes (e.g., BigDye™,
Perkin-Elmer), more accurate base-calling algorithms, and higher
resolution long (48 cm) gels will allow relatively good coverage
of cDNA less than 2 kb by just two overlapping reads on opposite
DNA strands (OSU will be the lead institution, also UA).
A second sequencer (AB 377) is requested which will be housed
at OSU for EST and full-length cDNA clone sequencing. Contig assembly
of ESTs and full-length cDNA sequences will be conducted using
bioinformatics resources at OSU. All sequence information generated
by this project will be curated at the proposed Stress Site
Database (SsDb) (see Appendix 2).
6. Testing of identified Halotolerance Genes.
a. Transgenic Plant Analysis. Genes which are
identified in various screens (complementation, microarray, etc.)
with the greatest promise for affecting salinity tolerance in
plants will be tested for sufficiency in model plant transformations
using Arabidopsis and tobacco. Either overexpression or
permanent activation (if a mechanism such as the presence of an
inhibitor domain is known) will be achieved using constitutive
promoter expression systems. In addition, we will use our T-DNA
mutation library of Arabidopsis to identify by reverse
genetics knockouts of identified halotolerance genes and subsequently
test salinity tolerance. Labs at the UA and Purdue
have considerable experience transforming and testing in these
two species for salinity tolerance. Recently a new $7.8 million
environmental-controlled growth facility has been added to the
Horticulture Dept at Purdue which will be available. Purdue
will be our hub for transgenics.
b. Reverse Genetics. Based on the preliminary
statistics from the ice plant ESTs, and extension to Arabidopsis
would mean that there may be 4,000 salt/osmotic stress-regulated
genes. The functionality of some of these genes will become clear
if they correspond to those identified through yeast-, Synechocystis-
or Aspergillus-complementation, or those defined by Arabidopsis
stress tolerance/signaling mutants. For others, we will establish
their role in stress responses by two approaches: (1) identifying
knock-out mutants with T-DNA or transposon insertions in the genes
of interest. This can be done by PCR screening of DNA from T-DNA
populations (Krysan et al., 1996), or more efficiently by searching
databases for sequences which flank T-DNA or transposon insertions,
which are being built in various labs (Klimyuk et al., 1997);
and (2) targeted gene disruption via homologous
recombination (Kempin et al., 1997). These powerful reverse genetics
approaches are presently in their infancy, but are expected to
mature during the next several years, and will lead to the disruption
and functional evaluation of every one of the stress-relevant
genes we find (UA leading, OSU, Purdue).
D. Undergraduate/Graduate Training/ Outreach Programs.
OSU (enrollment 26,000) provides valuable research opportunities
to undergraduate students in a variety of science and engineering
disciplines (currently >50% are from underrepresented groups).
OSU consortium faculty will recruit students for research training
through several different outreach programs such as the Oklahoma
Partners for Biological Sciences (OPBS) program (HHMI), the
Native Americans in Biological Sciences (NABS) program,
and the Oklahoma Alliance for Minority Participation in Science,
Mathematics, Engineering and Technology (OKAMP-SMET) both
NSF-funded programs. The OPBS program enhances training and research
opportunities for undergraduates as freshman or upperclassmen
transferring to OSU from regional two-year colleges. The NABS
and OKAMP-SMET programs target minority and native American student
training, recruitment and retention in scientific disciplines.
NABS and OKAMP-SMET, comprising 27 colleges, offer summer research
internship programs to over 2,000 minority students making the
transition from high school-to-college, 2-year to 4-year college,
or undergraduate-to-graduate study (~150 students).
Programs at UA (enrollment 38,000) are similar in breath
to those at OSU for underrepresented groups, including NABS. The
UA has a nationally recognized Undergraduate Biology Research
Program (funding: NSF, HHMI, NIH, and Am. Soc. Pharmaceut.
& Exptl. Therapeutics) providing research experience to students
working in biology. The program has served more than 750 students
(84% graduating with bachelors degrees) and now supports 140 students
annually. More than 200 students have published their work. Our
alumni are in medical school or practicing medicine (33%), in
biology graduate programs (21%), working in university/ governmental/
private sector research (16%), and 15% are either teachers, working
in regulatory agencies, or went to law school. Also funded by
NSF and HHMI is a program in which (presently 37) high school
teachers work towards a M.S. degree in Biochem. & Mol. Cell.
The Purdue University (enrollment 36,000) campus includes
nearly 6,000 students in graduate programs of 57 departments.
A large number of ethnic minorities have received Ph.D. degrees
in science at Purdue. Competitive fellowships (30 to 35/year)
are offered to Ph.D. candidates from underrepresented groups.
Purdue participates in the federal Minority Access to Research
Centers (MARC) program which provides funds for student participation
in the sciences. Undergraduate participants in this project may
receive academic credit through a Special Assignments or a Honors
Thesis Research course. Graduate students will have access to
the Horticulture, Plant Biology or Genetics interdisciplinary
Hans Bohnert (Dept. of Biochemistry, The University
of Arizona, Tucson, AZ)
The main activity in the laboratory is closely related to this
proposal - working on (1) stress responses in the ice plant
(Mesembryanthemum crystallinum) for ten years [1-5, 8];
(2) gene transfer into tobacco/ rice to study mechanisms
of ionic and osmotic stress tolerance [many of the concepts have
come from work with the ice plant] [2, 6-7, 11]; and (3)
modifications of enzymes of carbohydrate metabolism [3, 9-10].
The halophytic ice plant switches during water/ion stress from
C3 to Crassulacean acid metabolism, CAM. While Dr. Cushman, OSU,
is focusing on the molecular induction of CAM, which began in
my lab, continued collaboration includes signaling and signal
transduction under stress and CAM switching .
I consider all we do as one project, different portions of
which are funded by different agencies. NSF (IBN or, maybe, MCB)
funds tracing inositol biosynthesis and its relationship
to stress tolerance in the ice plant (1-2, 4-5). Funding by NSF-INT
is for analyzing ion transport proteins expressed in insect cells
and oocytes (with Dr. Pantoja, Mexico). Funding by DOE is for
analysis of transgenic tobacco expressing proteins that
may provide enhanced stress tolerance (2, 6-7, 11). USDA-NRI has
just begun funding work on water channel protein turnover
during stress. Additionally, people with their own funding by
DFG (Germany), NEDO/RITE (Japan), the Rockefeller Fndtn, and JSPS
(Japan) have worked or are presently in the lab.
Our work is inspired by physiological and biochemical approaches
to study mechanisms of osmotic and ionic stress tolerance.
The relationship to the proposed project is that we bring the
accumulated knowledge of the laboratory on stress responses in
halophyte models to the center. I expect that the characterization
of the core set of stress-related transcripts will provide a better
understanding of the mechanisms of plant stress tolerance and
shed light on the reasons for the lack of tolerance in crop plants.
We have already begun working on multi-gene vectors, up to nine
genes at present, but I imagine that we will make mini-chromosomes
in the near future for the transfer of an even larger number of
genes into rice.
Robert Burnap (Dept. Microbiology & Molecular
Genetics, Oklahoma State University, Stillwater, OK)
Research in this laboratory aims at an understanding the molecular
basis of oxygenic photosynthesis. The PI investigates two major
aspects of the oxygenic photosynthetic mechanism: 1.) assembly
and function of the PSII H2O oxidation complex and
2.) adaptation of the photosynthetic apparatus to environmental
stress. The scientific approach involves isolation and directed
modification of photosynthetic genes combined with biochemical
and biophysical characterization. The goal is to deduce mechanism
by analyzing the phenotypic consequences of defined genetic changes.
Targeted deletion strains are constructed in the experimental
model, the transformable, genomically determined cyanobacterium
Synechocystis sp. PCC6803, by replacing target genes with
antibiotic resistance cassettes. Site-directed and random mutations
are introduced into the gene in vitro and the modified
gene returned to the cognate deletion strain, such that the transformed
strains only express the mutant forms of the gene. The proposed
project builds on the P.I.s expertise in engineering specific
mutations into the cyanobacterial genome , but represents a distinct
and new line of experimentation. The PI. studied the function
and expression of Fe-responsive genes that are involved in a major
reorganization of the photosynthetic and respiratory complexes
during episodes of Fe-limitation . The most recent contributions
from the P.I.s lab concern the photoassembly and catalytic
properties of PSII complex and studies on the interaction of the
respiratory and photosynthetic pathways . Studies of the H2O
oxidation complex have led to an investigation into the source
and activity of reactive oxygen species (ROS) that appear to increase
as a consequence of environmental or mutational perturbation of
the H2O oxidation complex. These studies are connected
with the recent characterization of a novel heme-containing component
of the cyanobacterial H2O oxidation enzyme, that is
not found in higher plant PSII . This protein is hypothesized
to cope with oxidative stress. As part of this effort, the laboratory
is currently developing bacterial luciferase constructs in Synechocystis
to monitor changes in the expression of genes induced to cope
with these stress conditions and to deconvolute the regulatory
John C. Cushman (Dept. of Biochemistry &
Molecular Biology, Oklahoma State University, Stillwater, OK)
Current research in the Cushman laboratory is directed towards
understanding the genetic and molecular genetic basis of Crassulacean
acid metabolism (CAM) in the facultative CAM plant M. crystallinum
. We maintain an active and continuing collaboration with the
Bohnert lab on various aspects of ice plant biology. Major projects
include 1) large-scale EST sequencing of ice plant leaf
cDNAs 2) establishing fast neutron and EMS mutagenized
plant populations to isolate and characterize mutants defective
in CAM, 3) characterization of the stress-inducible enzymatic
machinery of the CAM pathway [2, 3, 4], 4) understanding
the mechanisms of transcriptional activation of CAM-specific enzymes
, and 5) elucidation of the signal transduction
pathways leading to CAM induction and other adaptive responses
brought about by environmental stress [6, 7]. The EST and mutant
projects are currently funded by NSF (Integrative Plant Biology)
whereas the transcriptional activation and signal transduction
aspects of CAM are funded by USDA-NRI (Plant Responses to the
The proposed EST sequencing and microarray characterization
of a master set of stress-inducible genes represent a natural
extension of ongoing research in the Cushman laboratory. To date,
over 1600 cDNA randomly selected cDNA clones derived from mRNA
isolated from leaves well-watered or NaCl stressed plants. The
long-term goal of this project is to sequence all of the expressed
genes in ice plant leaves to fully understand the molecular aspects
of CAM and to lay the foundation for physical mapping and sequencing
of the ice plant genome. Once established, mutant populations
can also be screened to identify mutants defective in stress tolerance.
The current proposal would allow us to expand our current EST
project to include cDNA clones derived from other tissues (e.g.,
roots, floral tissues, epidermal bladder cells, etc.) of the ice
plant and from other halophytic and glycophytic species under
study within the consortium. My laboratory is adjacent to the
OSU Recombinant DNA/Protein Resource Facility where I oversee
the automated DNA synthesis and sequencing services. With an additional
automated DNA sequencing system and technical support, we would
be able to meet the DNA sequencing needs of the entire stress
consortium. My lab, in collaboration with other OSU researchers,
has received funding from the Oklahoma Agricultural Experiment
Station to build and operate a microarrayer within the Recombinant
DNA/Protein Resource Facility to monitor global gene expression
patterns. Analysis of gene expression patterns using microarray
technology will allow us to rapidly identify a master set of salt-responsive
genes implicated in stress-adaptation responses including CAM.
David W. Galbraith (Dept. of Plant Sciences,
The University of Arizona, Tucson, AZ)
My research program has historically combined interests in
the development of biological instrumentation and reagents with
the investigation of specific biological questions concerning
plant cell, molecular, and developmental biology [1-3]. The specific
aims of our funded research projects are: (I) Flow cytometry:
digital processing of molecular information (NSF Instrument Development
Program). This involves the development of hardware and software
for the analysis of flow cytometric pulse waveforms through digital
signal processing, and the real-time sorting of cells based on
waveform shapes [4,6,7]. This aims at providing new ways to examine
cell structure and function. (II). Novel techniques for
gene characterization in higher plants (USDA-NRICGP, Plant Genome).
This involves developing flow sorting methods for the identification
of genes expressed within specific cell types, and is achieved
via cell-specific expression of GFP, targeting of this molecule
to the nucleus, recovery of labeled nuclei through flow sorting,
and analysis of the nuclear transcripts [5,8,9]. We are also developing
microarray techniques for analyzing the expression of these transcripts
. (III). Systematic analysis of the P450 superfamily
in Arabidopsis (USDA-NRICGP, Plant Genome). This project involves
PCR-based reverse genetics, using pooled, T-DNA tagged Arabidopsis
lines, and primers based on P450 and T-DNA gene sequences, to
identify mutants carrying specific P450 gene insertions. We are
analyzing the mutant phenotypes to gain an understanding of the
enzymatic activities encoded by the different P450s. We also employ
microarray technologies to systematically analyze global P450
gene expression, as a function of development and in response
to applied stimuli . (IV). Flow karyotyping and sorting
of translocations in maize (NSF, International Programs). This
project involves use of flow cytometry for the production of chromosome-specific
genomic libraries. (V). Interdisciplinary research training
group on plant-insect interactions (NSF, Biological Infrastructure).
This program supports postdoctoral research associates and graduate
students in various laboratories within three UA Departments.
My research activities as a whole emphasize technology development
and implementation. My contribution to this proposal involves
the development and application of novel technologies to examine
stress responses at the molecular level.
Paul M. Hasegawa/Ray A. Bressan (Dept. of Horticulture
and Landscape Architecture, Purdue University, W. Lafayette, IN)
Our laboratories are organized for everyone in both groups
to work together in completely unhindered collaboration. We currently
focus on 3 (surprisingly related) areas: 1) Insect resistance,
2) fungal disease resistance, and 3) osmotic stress resistance.
All projects have basic science components seeking to elucidate
molecular mechanisms of tolerance. In addition, we conduct work
aimed at the more practical application of genomics technology
mainly by the production of transgenic plants with altered expression
of "tolerance" genes. Our recent collaboration with
José Pardo's group in Spain has led to the extensive use
of yeast as a model system to dissect the functional genomics
of both resistance to osmotic stress and plant antifungal toxins.
We are in a particularly unique position with these collaborations
to unify the powerful molecular genetics of yeast with plant molecular
biology and gene manipulation approaches toward salinity tolerance.
For this project, our experience and expertise with the yeast
model system and with plant molecular biology allows us to interface
extremely well with the even more developed projects at the University
of Arizona designed to allow multi-gene transferes to plants.
José M. Pardo (Department of Horticulture
and Landscape Architecture, and Instituto de Recursos Naturales
y Agrobiologia in Seville, Spain.
Dr. Pardo has an active lab at the Instituto de Recursos Naturales
y Agrobiologia in Seville, Spain. Working with colleagues on a
European Union Sponsored project, he has developed an extensive
set of salinity sensitive mutants of yeast. By exploiting the
many important advantages of the yeast model (genomic sequence
completed saturation transposon tagged mutagenesis/gene knockout
replacement/haploid genome with sexual cycle/efficient transformation/etc.).
The Pardo lab has been able to characterize some of these mutants
extensively and isolate the genes responsible for altered salinity
tolerance. Dr. Pardo has spent three of the last five years as
a visiting professor at Purdue University, where his collaboration
with the Purdue group has led to the isolation of plant genes
that complement salt sensitive yeast mutants and the transformation
of plants with some of these genes. He will continue to spend
part of his time at Purdue. His participation on this project
will greatly facilitate the exhaustive isolation of plant genes
involved in salinity tolerance using yeast as a model for mutant
complementation and micro-assay analysis.
Rolf A. Prade (Dept. Microbiology & Molecular
Genetics, Oklahoma State University, Stillwater, OK) (In collaboration
with Dr. P. Ayoubi).
Current research in the Prade laboratory is focused towards
the understanding of fungal molecular sensing mechanisms involved
in environmental (e.g., stress and fungal plant infections) and
developmental [1, 2] adaptations. Particularly, we are interested
in the genetics of how and why fungi, such as Cochliobolus
sativus (plant pathogen) and Aspergillus nidulans (model
organism), sense and respond to the presence of complex carbon
sources (e.g., polysaccharides). In addition, numerous experimental
clues strongly point to a direct relationship between various
stress response pathways and carbon sensing. Our current research
activities related to carbon sensing are funded by grants from
the USDA-NRI (Plant Pathology) and the Oklahoma Agricultural Experiment
Station (in 1997).
Several of the large-scale genetics resources that will be
extensively utilized during this project have been developed in
the last few years in collaboration with several groups. Some
of these resources include: the A. nidulans physical map
(Prade RA, Arnold JA et.al ) and the A. nidulans
EST database (Prade RA, Roe BA et.al ). Both resources
are publicly available (www.aspergillus.okstate.edu/genome/) and
many of the related technical aspects have also been published
[3-5]. In addition, construction of A. nidulans recombinant
strains, mycological techniques, classical Mendelian and bacterial
mobilization genetics, manipulation of proteins, eukaryotic and
prokaryotic nucleic acids are routinely employed in our laboratory
Our current limitation in providing clear answers to key questions
on how, when and why living cells need to sense and respond to
specific carbon driven cues are twofold: i) we realize
that carbon sensing affects the expression of a large number of
genes. Thus, the answers to basic questions require the analysis
of many genes (genomics), and ii) carbon sensing is not
only directly linked to development and the cell-cycle but is
also strongly associated with various stress conditions as well.
We recently determined that 9.2% of all genes expressed in A.
nidulans during asexual development are associated with known
stress responses encompassing; heat- and cold-shock proteins (7.1%
and 0.1% of all ESTs, respectively), ubiquitination (1.5% ESTs),
ROS scavenging (catalase, peroxidase, Cu-, Zn-superoxide dismutase),
uptake, transport and compartmentalization of sodium and other
compounds (8 ATPase proton efflux pumps), compatible solutes (0.4%
ESTs and comprising, glycerol-, trehalose-, proline-, betaine-,
mannitol- and sorbitol-synthesis genes) and various regulatory
homologs such as, HAL2, HOG1, and calcineurin (R.A. Prade and
P. Ayoubi, unpublished results).
Jian-Kang Zhu (Dept. of Plant Sciences, The University
of Arizona, Tucson, AZ)
Efforts in the laboratory are focused on the genetic analysis
of salt tolerance and osmotic stress signal transduction using
Arabidopsis thaliana as the model organism. Our goal is
to identify genes that are essential for plant salt tolerance
and osmotic stress signaling, no matter they are induced by stresses
or not. To this end, we have been working to isolate and characterize
Arabidopsis mutants defective in NaCl tolerance (1-5) or
stress gene regulation (6).
Work on the isolation and characterization of salt-hypersensitive
Arabidopsis mutants, and positional cloning of the salt tolerance
loci is supported by USDA-NRI. Funding by Southwest Consortium
on Plant Genetics and Water Resources is for genetic analysis
of osmotic and cold stress signal transduction. We also work on
cell surface proteins that mediate plasma membrane-cell wall interaction,
which is funded by NSF (IBN).
Our current work firmly establishes Arabidopsis as a
powerful model to study salt stress tolerance and signaling. The
proposed work builds on our expertise and knowledge of T-DNA mutagenesis
of Arabidopsis (5), mutant screening (1, 2, 5), genetic mapping
and positional cloning (7) to achieve genome-wide genetic analysis
of salt stress responses.