Download Research Pan for next 5 years: I would like to write grant proposals

Research Pan for next 5 years:
I would like to write grant proposals and work on three research themes for the next five years.
There are three proposals or ideas I would like to follow
1) Mechanisms for increasing seed yield in model plant and crop systems at elevated CO2: Linking
whole-plant responses with gene expression
2) Genomic approach to unravel induced defense responses in Hybrid poplar (Populus) against
Melampsora spp (leaf rust) using plant activators.
3) Development of Genomic resources for abiotic stress tolerance using Salt Tolerant Populus euphratica
Mechanisms for increasing seed yield in model plant and crop systems at elevated CO2: Linking wholeplant responses with gene expression
SUMMARY: Global change factors, such as elevated CO2, extreme drought, higher temperatures, and
changing precipitation are predicted to have positive and negative effects on crops. Past studies indicate that
elevated CO2 generally enhances crop productivity. Plant responses to CO2 must be understood at the wholeplant through molecular levels in order that cultivars can be developed that most efficiently utilize elevated CO2.
I previously developed lines of Arabidopsis by selection over multiple generations that exhibit increased seed
production compared to randomly selected controls at [CO2] concentrations projected to occur 80 years into the
future. These Arabidopsis lines will provide insights as to how genetic modifications may improve crop yield in
high [CO2] environments, particularly since genes tend to be conserved between Arabidopsis and crop species. I
propose to conduct an analysis of our selected lines to identify physiological and growth responses that are
associated with increased yield. Subsequently, I will use whole genome microarrays to assess changes in the
expression of Arabidopsis genes in order to understand how responses at the molecular level influence plant
productivity in our selected lines. Finally, I will link growth responses to underlying gene expression by
identifying candidate genes for further improving crop responses to increasing [CO2] concentrations predicted
for the future. The purpose of this project is to identify plant traits at the whole-plant through molecular levels
that are associated with increased yield at elevated atmospheric [CO2] concentrations.
RESEARCH OBJECTIVES: Identify physiological and growth responses that are associated with increased
yield in Arabidopsis lines selected for high seed production at elevated [CO2]. Identify changes in global gene
expression that are associated with high yield (seed production) at elevated [CO2]. Link whole-plant responses to
underlying gene expression in order to identify candidate genes for further improving crop performance at
elevated [CO2].
APPROACH: A phenotypic analysis will be conducted to compare Arabidopsis genotypes that were selected
for high seed production at elevated [CO2] versus those that were randomly selected as controls (also at elevated
[CO2]). Measurements on these lines will include plant growth and development, physiological responses, and
biochemical changes at the leaf level. Subsequently, this research will be followed by an analysis of gene
expression differences between selected and control plants grown at current and elevated [CO2] concentrations.
This will be accomplished by hybridizing total RNA to Arabidopsis whole genome microarray chips. Candidate
genes for future study will then be identified as those whose expression was highly affected by selection for high
seed production at elevated [CO2].
Genomic approach to unravel induced defense responses in Hybrid poplar (Populus) against Melampsora spp
(leaf rust) using plant activators.
SUMMARY: We propose to modify induced defense response activity in Populus against leaf rust and follow
the consequences of this modification through multiple levels of signal molecules leading to overall fitness by
the plant. During last few decades, this pathogen causes severe damages in poplar cultivation in India. The most
important disease that limits poplar productivity in the Asian countries is leaf rust, caused by Melampsora spp. An
overarching hypothesis of this project is that specific activators used prior to or before pathogen application
lead to early response based on signal cascade such as jasmonic acid, salicylic acid or abscisic acid pathway
lead to ultimate broad spectrum resistance. A combination of laboratory scale experiments and field trials will
allow us to establish mechanistic links between resistance levels using tools of genomics, biochemistry, and
physiology. Greenhouse based experiments for application of pathogen will allow the source and concentration of
endogenous defense compounds to be explored. BABA (-aminobutyric acid), one of the popular plant activator
of resistance has been shown a significant acquired resistance against many fungal pathogen. A transcriptome
approach as well as identification of signal molecules will lead to understand detailed mechanism of systemic
acquired resistance in model tree system for the first time. A factorial study will involve infected poplar and
uninfected poplar plants prior to BABA treatment and mixtures of these to infected poplar. Measurements will
support an improved understanding of how signals are translated during poplar leaf rust spread by using chemicals.
Characterization of key signal during poplar leaf rust interaction would allow in-depth understanding of the
coordinated response, thus adding a new dimension to climate change research and providing another
important step in controlling diseases in short-rotation woody crop, poplar, which has considerable economic
impact on India croplands.
RESEARCH OBJECTIVES:
1 To study efficacy of compound BABA before and prior to pathogen spread on poplar leaf tissue under greenhouse
conditions and isolated leaf disc method to identify possible mechanism underlying induced defense response in
Populus against leaf rust using plant activators.
3. To characterize molecular event of interaction between BABA primed Populus tissue against Melampsora spp
infection (M. populini, M. medusae) and rust infected Populus tissue by transcriptional profile analysis. Microarrays
will be used to analyze global transcriptional profiles for both host and pathogen (and non-pathogens) during various
types of treatments. In addition, it will lead to identification of potential genes that can be used as targets in
biological, genetic and/or physical screens for rust resistance genes in poplar. To study tempo-spatial analyses of
candidate gene expression of BABA-primed poplar tissue against leaf rust infection. This study will provide
molecular mechanism of local as well as whole plant system resistance.
5. To investigate specific metabolic profiling leading to target signaling pathways such as SA, JA or ABA which
could be involved in BABA-induced defence response in poplar.
6. Functional characterization of candidate signaling genes of poplar using Arabidopsis mutants which are impaired
in SA, JA or ABA pathways.
Development of Genomic resources for abiotic stress tolerance using Salt Tolerant Populus euphratica
SUMMARY: High soil conductivity, primarily driven by salinity, is a major factor limiting plant establishment and
growth on more than 800 million hectares worldwide, including extensive degraded lands within the United States
and Asian Countries (FAO 2008). The goal of our research is to open these lands to energy production by
developing highly productive, salt tolerant biofuels feedstock crops. Furthermore, because highly productive crops
like trees or switchgrass can enhance carbon contents of soils, salt tolerance can simultaneously enhance the value of
these lands for carbon sequestration, thus offsetting the effects of fossil fuel combustion (Jobbagy & Jackson 2000).
Therefore salt tolerance traits of biofuels feedstocks can be used at both ends of the energy spectrum – C
production and C sequestration, so this research is an attractive investment for current energy producers as
well as for opening new energy production opportunities.
Our research is primarily focused on trees from the genus Populus (poplars, aspens, and cottonwoods), a
model system for forest biology and genomics (Yang et al. 2009; Jansson et al. 2009), and the preeminent choice for
intensive hardwood plantations for biofuels production in the northern hemisphere (Perlack et al. 2005; Rubin 2008).
Our current approach to elucidating mechanisms of salinity tolerance in Populus is focused on studying genetic
variation in species that are generally intolerant of this stress. Here we are proposing to extend this approach to
an under-studied Populus species, P. euphratica, which shows remarkable tolerance to the high levels of
salinity that are common in its native desert habitat (Chen et al., 2003; Ma et al., 1997). P. euphratica is the
subject of intensive investigation in Europe and Asia, but few genes underlying the stress tolerance response have
been identified thus far, because efforts have been hampered by a lack of genomic sequence for this species (Wang
et al., 2008; Chen et al., 2009), which is only distantly related to P. trichocarpa (Eckenwalder 1996), the species for
which a whole-genome sequence is available (Tuskan et al. 2006). This lack of genomic understanding of salt
resistance traits provides an opportunity for us to identify and clone genes associated with stress resistance
and to eventually allow creation of transgenic poplar lines for use in bioenergy production and carbon
sequestration on marginal lands.
Functional characterization of genes ultimately requires reverse genetics approaches, in which individuals
with mutated versions of a gene demonstrate informative phenotypes. This is typically accomplished in plants using
genetic transformation, in which engineered DNA constructs are introduced into the plant genome to specifically
alter expression levels of a target gene. The availability of efficient transformation protocols is one of the most
important features making Populus an attractive model plant, but the efficiency of transformation varies
tremendously among genotypes (Busov et al. 2005). Unfortunately, whole genome sequence information does not
currently exist for any of the model transformation clones (Song et al. 2006), and this has inhibited functional
genomics efforts in Populus. Here we are proposing deep transcriptome sequencing of a hybrid Populus clone,
INRA 717-1B4, P. tremula x P. alba, hereafter called 717) that has consistently shown highly efficient
transformation and propagation rates (Han et al. 2000), and which shows high sensitivity to salt stress based
on preliminary experiments in our lab.
In summary, the overall scientific goal of this proposal is to lay the groundwork for a major new
research program to enhance salt tolerance in Populus for biofuels and C sequestration applications. Our
specific objectives are to (1) sequence the transcriptomes of contrasting stress-resistant P. euphratica and saltsensitive clone 717, and (2) identify genes conferring tolerance to salt-stress, which can then be targeted in future
studies to improve salt tolerance of commercially-important Populus genotypes.
Experimental Approach:
Populus euphratica and Populus clone 717 plants are currently being propagated in our lab using shoot buds of in
vitro grown plantlets on “Woody Plant Media” following published protocols (Panetsos et al. 1987). These rooted
micropropagated plants of will be exposed to three concentrations of NaCl (0 mM, 50 mM and 150 mM) for two
different time durations (72 hr and 7 days) using a highly-efficient hydroponic growth system that we have recently
developed for Populus. These concentrations and timepoints were determined based on preliminary experiments
with clone 717, and published studies on P. euphratica (Bogeat-Triboulot et al. 2007; Wang et al. 2008). Leaf and
root tissues will be collected at each time point and frozen in liquid nitrogen, ground, and stored at -80oC.
We will perform deep sequencing on the mRNA sequences expressed during these experiments using the
Illumina mRNA-seq approach (Wang et al. 2009). In this case, we will sequence normalized transcripts from leaves
and roots for P. euphratica and clone 717 for the two salt stress treatments at both time points, resulting in a total of
8 libraries. We will mix bulk tissue across multiple ramets of the same genotype and extract mRNA using Qiagen
RNEasy Extraction kits. Complementary DNA will be generated from the total RNA using polyT primers to enrich
for 3’ sequences. The library will then be normalized to reduce the number of multi-copy sequences using
suppressive-subtractive hybridization (Diatchenko et al. 1996). The driver for the SSH normalization will be derived
from the control treatment for the same species at the same time point (e.g., clone 717 control cDNA at 72 hr will be
the driver for clone 717 cDNA drivers at the 50 and 150 mM salt levels, also at 72 hr). The mRNA will then be
fragmented to produce 250-500 bp pieces. The cDNA will be sequenced using the Illumina Genome Analyzer using
paired 75 bp reads, which typically generates 1-2 Gb of sequence per lane. We will prepare the libraries according to
Illumina specifications, and subcontract the sequencing to a core lab such as the one at UC-Riverside
(http://illumina.ucr.edu/ht/rates/rates). It is difficult to predict depth of coverage for transcriptome sequencing due to
variation in the number of transcripts expected in different tissues and conditions (Wang et al. 2009). The predicted
cumulative length of the Populus transcriptome is approximately 45 Mb (Tuskan et al. 2006). Therefore, two lanes
would provide approximately 40-fold coverage of the entire transcriptome, assuming efficient normalization.
However, only a fraction of the transcriptome will be differentially expressed in the salt tolerance experiments, so
two lanes per library should be sufficient to discover the majority of the differential transcripts.
The sequences will be assembled into contigs of candidate genes using newly-developed assembly software
(e.g., Hossain et al. 2009; Simpson et al. 2009). Reads that fail to assemble to the P. trichocarpa genome will then
be used as blast queries versus the NCBI nonredundant database, and clusters of hits will be assembled to reference
transcripts using Maq. Finally, reads within each species that fail to assemble will be assembled de novo using
Velvet (Zerbino and Birney 2008). To help with interpretation of the transcriptional responses, we will perform
analyses of Na, Ca, Fe, and K using Inductively Coupled Plasma spectrometry for leaves and roots of all samples.
Expected Results, Milestones and Timeline:
The project will take approximately 36 months to complete. The salt tolerance experiment will be performed
in the first month, and the enriched libraries will be completed by the end of month two. The sequencing will be
performed in month three, and assembly, analysis, and manuscript preparation will take place in the final 3 months.
This project will generate over 5,000,000 DNA sequences representing the total transcriptional response to salt,
thereby providing a comprehensive view of the stress-responsive transcriptomes of these contrasting species.
Furthermore, the genomic resources developed for the model transformation clone 717 will be invaluable in aiding
our interpretation of future functional genomics studies to characterize salt stress tolerance candidate genes in this
species.