Salinity is one of the major abiotic stresses that causes damage of the productive land. Approximately 6% of the Earth's total landmass is affected by salt (Munns, 2005). More than 5% of the total 1.5 billion hectares of cultivated landmass is associated with salinity (Tester and Davenport, 2003).
In Australia, saline soils are estimated to cost on average $130 million each year in crop loss. Moreover, a rapport from The National Land and Water Resources Audit presented an expected increase of saline soil in the next 40 years from 5.7M to 17M ha in Australia (NLWRA, National Land and Water Resources Audit 2001). Soils management, water balance (land clearing, irrigation) are the main causes for an expected increase of drylands.
Around the world, there are different types of salts. Among salts like magnesium chloride, sulphate chloride, calcium chloride, the most common and soluble in soils is the sodium chloride (NaCl). Land is considered to be saline when the electrical conductivity of the saturated paste extract (ECe) is 4dS/m or more, which corresponds to a concentration of 40mM of NaCl (Munns and Tester, 2008).
Plants have colonized the planet 480 million years ago and are still found in a wide range of habitats. Plants are composed of 2 main parts: one related to photosynthesis via the shoot area and the other one, the root system to extract water and nutrients from the soil. Water and nutrients are transported to the leaf area via the vascular system. Plants are not mobile and will grow where they germinated even in unfavourable environments. As a result, plants have developed different mechanisms to survive or tolerate extreme conditions (such as drought, high salinity etc...).
According to Munns and Tester (2008), 3 main salinity tolerance mechanisms have been identified in the flowering plants: osmotic tolerance, tissue tolerance and sodium exclusion.
Osmotic stress is not specific to salinity. The results are a reduction in growth of the young leaves and stomatal closure. The complex mechanisms controlling osmotic tolerance are still unknown but reduce the entry of salt into the cells (Munns, 2005).
The two others mechanisms follow the same principle: to keep sodium out of the cytoplasm because high concentration is toxic. The sodium (Na+) and chloride (Cl-) ions can interfere with enzyme activities and lead to the death of the cell.
The second mechanism, the tissue tolerance, mainly results in a sequestration of the Na+ and Cl- ions into a compartment like the vacuole. The third mechanism is the sodium exclusion. Plants have developed diverse transporter systems to minimize the loading of the sodium into the xylem. Along with
the discovery of Na+ transporters, a gene family named HKT (high affinity K+ transporter) has been identified in different species (Platten et al., 2006). One member of the family; HKT1;5 has been well characterised. Its function is to retrieve sodium from the xylem sap and thereby limit the accumulation of ions in the leaf area. (Ren et al., 2005)
A lot of research focussed on crops because of their economical importance. Nevertheless, cultivated plants represent a very diverse range of species across seed plants. To make the research progress faster, a model plant have been selected for each botanical groups of plants. One group the Angiosperm, the flowering plants, is divided in 2 major subclasses: the dicotyledons and monocotyledons. The seedlings of the dicotyledons have 2 cotyledons (seed-leaves) in contrast with the monocotyledons which only have one. A model plant for each group has been chosen by the scientific plant biology community: Arabidopsis thaliana for the dicotyledons and the rice japonica cultivar (Nipponbare) for the monocotyledons.
The 120Mb genome of Arabidopsis thaliana was sequenced in 2000 by the 3 research laboratories: Salk, Stanford and Plant Gene Expression Center (PGEC). The plant belongs to the mustard family but is of no agronomic interest. However, its short life cycle and availability of knockout mutants as well as the easy routine transformation via Agrobacterium tumefaciens have made this model a very useful tool in genetic and plant molecular biology.
As a model plant for the monocotyledons, the 430Mb genome rice japonica cultivar (Nipponbare) was sequenced in 2005 by the International Rice Genome Sequencing Project (IRGSP). Rice is one of the main cereals cultivated around the world and also a source of food for the world's population. It is mainly cultivated and consumed in Asia. Regarding the resistance to abiotic stresses different cultivars from different plant species show different sensitivity. For example, rice has been reported to be very sensitive to salt at the vegetative stage (Munns and Tester, 2008). Rice is also affected by salt during grain development (Flowers, 2004).
During the past two decades, a lot of research has focussed on improving salinity tolerance by manipulating genes. Flowers claimed that, between 1993 and 2003, 63 papers have been published about increasing salt tolerance in plants. Believing that this abiotic stress was a simple trait, scientists tried to over express one or two keys genes at a time. Unfortunately, these experiments did not provide the expected results: even if resistance to salt was seen in some cases, the retardation of plant growth and sterility were a major disadvantage for the agriculture (Gao et al., 2007; Nakashima et al., 2009). As an example, the constitutive expression of HKT1;1 in Arabidopsis thaliana showed an accumulation of sodium in the shoots, whereas expression under a stele specific promoter showed an enhancement in salinity tolerance (Mller et al., 2009). Those experiments lead to the conclusion that salinity is a multigenic trait and it is physiologically difficult to dissect. Moreover, in those same studies, plants were submitted to very high salt stress (150mM to 300mM) which corresponds to an osmotic stress than really measuring tolerance to salt (Flowers, 2004). Engineering genes may be a good way of dissecting the salinity traits, however it is good to keep in mind that a plant is a multicellular organism; each cell has a different function. To understand how a plant can respond to an abiotic stress like salt, it is important to understand what is regulated and specific to each cell type.
In order to better understand the genetic mechanisms of salinity tolerance at the cell type specific level, this research will focus on the comparison of the transcriptional profile under salt and control conditions in 2 cell types of the root: cortex and stele. This research is likely to reveal new cell type specific candidate genes implicated in salinity tolerance. The manipulation of those genes and/or their promoters will improve knowledge in the complex salt resistant pathway. Moreover, the study will use expression data of the two sequenced model plants Arabidopsis thaliana and rice to enable a comparison between these 2 types of Angiosperms. This research will provide a good overview of the gene expression and the regulation of the mechanisms to salinity in both models plants at the cell type specific level.
Literature review
Global transcription profile analysis
In responses to abiotic stresses plant have developed divers mechanisms to adapt. Many genes responses will be involved at different time points in those mechanisms. Researchers are interested in genes up or down regulated under salt stress: global transcription profile.
There are many techniques available to visualize the RNA expression from one sample.
Northern blot : no quantification no measurement of level expression
Rt PCR : quantification of the gene expression relative to one control.
Problem how to visualize the expression for many genes at the same time point ?
For this purpose, scientists used a technology called microarray.
The microarrays
A microarray is a solid support on which you spot some DNA sequences also called probes. The complementarity DNA or RNA sequences marqued will bind the probe.
There are different types of microarrays :
One type is the 'classical' microarray. The DNA sequences are amplified via PCR and then spotted on the chip. The template of the PCR can be an EST or genomic DNA. Different research group have created their own chip to answer their scientific questions: chip with on gene linked to the cell wall synthesis. With this type of array two samples will be compared (one control and one treated) marked with 2 different fluorochromes (Cy3 and Cy5 dye) to quantified the changes in expression. Used of a swap.
The second type of chip is the in silico oligo synthetised type. It is mostly a commercialized chip where the DNA sequences , generally shorter than the classical array, are synthetised straight on the support. A one labeling dye is requirement because only one sample is hybridise by chip. Those chips gave a comprehensive coverage because they are based on the genome sequencing. They are commonly used now in labs for genome wide analysis of gene expression.(Zhu, 2003)
Also this chip allow a lot of flexibility with the printed sequences it is also very expensive.
GeneChip from Affymetrix
One of the most used oligo microarray is from a commercial company : Affymetrix (http://www.affymetrix.com). The company sells some microarrays called GeneChip probe array in a various range of GeneChip from humans to Escherichia Coli chip.
Many Genechip are also available for the plant organsm like Soybean, Ath, rice and maize and medicago. (Zhu, 2003).
The Affymetrix Rice GeneChip is the one I will be using in the project. It concern 51279 transcripts from 2 cultivars indica and japonica.
It contains Short sequences probes synthetised , those sequences are based entirely on sequence information of rice genome , which make them highly specific. To obtain a good survey of the genome expression, it is required a good coverage of teh genome which affym genechip gives.
To detect a gene transcripts to genechip contain 2 or more sets probes.
A probe set is a group of probes specific to one genomic sequence (figure 1). It contains 11 probes (25 mer each) representing one gene or expressed seq.(Galbraith and Birnbaum, 2006).
Affymetrix rice (describe it 510000 genes)
A lot of data available on web can be compared but limits cause reproducibility not good: change in experimentor
Normalization and statistical tests to confirm the changing in expression level is real.
Global transcription profile under salt stress (using microarrays)
Salt responsive gene /Novel TF identify via arrays (Chao et al., 2005; Wu et al., 2006; Gao et al., 2007)Transcription profile studies identification genes and TF under high salinity (Wang et al., 2003; Seki and Shinozaki, 2009)
Tf regulated in under s alt stress classification (Nakashima et al., 2009)
Limits for crop yield
high salt stress
do it correspond to a physiologic stress ? (review Munns and tester )
characterise molecular functions of genes via mutants
overexpression OSABI5 genes in whole plant high salinity resistance (Nakashima et al., 2009)
(decrease the yield rice AP37 )(Kim and Kim, 2009)
Profile expression in whole root / tissue and now in cell type specific
Cell specific transcription profile
Def : study the expression of one cell type in one environment.
Try to discover genes that are only expressed in one cell type.
Example pollen between 650 to 850 genes expressed at the 4 stages of the pollen development massive difference in correlation between those genes in early and late stage of development .(Galbraith and Birnbaum, 2006)
Categorize :
15 localized expression domains (LED) or zones of the arabidpsis root mapped and correspond to .
(Birnbaum et al., 2003)
To do that need to isolate the cells : 2 methods
Meca : dissection of cells in root or one organ under microscope but may be difficult for some cells like pollen, or cells emballed in a organ.
2d methods using laser : fluorescent activated sorting : use of reporter gene to select the cells (GFP).(Johnson et al., 2005)
And stage of protoplasting, eliminate the wall
Organization of roots in monocotyledons and dicotyledons
What is the advantage ? Discover genes cell type specific, manipulation of 1 gene in 1 cell type (not GMOs anymore)
Disadvantage Arabidopsis thaliana root expression profile (Birnbaum et al., 2003) good base for comparative study
Identification of promoters
( control spatial and developmental stage)
rice system GAL4 enhancer trap (Johnson et al., 2005) and his double advantage to control gene of interest expressed in cell type specific (Mller et al.)
Expression of one gene of interest in 1 cell type
Identification of group genes specific to a cell type and comparative studies (Nawy et al., 2005)
Example of Inge HKT1.1 (example for salt) (Mller et al., 2009)
"A transcriptome atlas of rice cell types uncovers cellular,
functional and developmental hierarchies" (Jiao et al., 2009)
Example calcium cell type specific in Arabidopsis thaliana roots endo and peri (Kiegle et al., 2000)
Compartimentation section in roots (Galbraith and Birnbaum, 2006) REVIEW
Profile gene expression in rice shoot (Zhou et al., 2007)
Research questions
In order to identify the candidates genes specifically up regulated or down regulated expressed in the stele and cortex of the rice and Arabidopsis thaliana under mild salt stress; this research has the following aims.
Aims of the project
The first step of this study will be to explore the transcription profile of cell type specific under mild salt stress in the rice.
The second step will be to identify new genes implicated in salt responses in rice in cell type specific: stele and cortex.
In order to characterize the function of those candidates' genes, the seeds corresponding to the selected mutated genes will be order. The seeds will be growth to produce more descendants.
The new generation of rice plants will be tested in salt and control conditions to characterize the molecular functions of those genes and their implications in salt pathway resistance.
The complete transcription profile analysis of Arabidopsis thaliana under mild salt stress will also be performed.
The results will help to identify Arabidopsis thaliana's genes from cortex and stele specifically up or down regulated under salt stress.
Bioinformatics will be use to categorize the major classes of genes implicated in mild salt tolerance in the 2 model species rice and Arabidopsis thaliana.
Theoretical framework and methods
Microarray experiments
Growth conditions and salt treatment
Generating the protoplasts
Sorting the protoplasts via FACS
RNA extraction
cDNA amplification and shearing
Hybridization, washing and scanning
Bioinformatic analysis
Normalization
Statistical test
Contribution to the discipline
Abiotic stress mecha (Gao et al., 2007)
"A transcriptome atlas of rice cell types uncovers cellular,
functional and developmental hierarchies" (Jiao et al., 2009)
Arabidopsis thaliana root expression profile (Birnbaum et al., 2003) good base for comparative study
Example calcium cell type specific in Arabidopsis thaliana roots endo and peri (Kiegle et al., 2000)
Compartimentation section in roots (Galbraith and Birnbaum, 2006) REVIEW
Profile gene expression in rice shoot (Zhou et al., 2007)
Transcriptome analysis of root transporter under salt stress (Maathuis et al., 2003)
"OsHKT2;1 was mainly expressed in the cortex and endodermis of roots" (Horie et al., 2007)
"we determined that the expression of OsRPK1 was localized in the plasma membrane of cortex cells in roots. The results suggest that different rice cultivars might have different salt stress response mechanisms." (Cheng et al., 2009)
'Stress-induced changes in the Arabidopsis thaliana
transcriptome analyzed using whole-genome tiling arrays' (Zeller et al., 2009)
'global identification of genes that are expressed within specific cell types
within complex tissues" (Zhang et al., 2008)
Expression profile under salt stress in cell type specific (Dinneny et al., 2008) find limits and not done What else can I bring with my study ? publi difficult to understand and read !!!!
Root architecture dicot / mono
Question11: what specific genes are activated in response to salt treatment in the stele and cortex cells?
Hypothesis: A combination of transporters, osmotic regulators, enzymes. Discovery of new proteins implicated in salt tolerance?
Experiments: affymetrix chips from enriched stellar or cortex protoplasts.
Question1: what specific transcription factors activate HKT1.5 in response to salt treatment in the stele and cortex cells?
Hypothesis: A combination of TF should bind different promoter sites. There will be a specific combination of TF under salt stress to over expressed HKT1.5. Expecting different TF in the 2 type's cells?
Experiments:
Microarray experiment : cell specific expression
Rt PCR to confirm the genes up or down regulated.
Yeast one hybrid : find the combination of TF
Link to the proteomic?
http://nlwra.gov.au/files/products/national-land-and-water-resources-audit/pn21442/pn21442.pdf
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