Kristoffer Chin: Week 11
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Entry
Definitions
- lignin - organic substance that adds strength and rigidity to cell wall http://www.biology-online.org/dictionary/Lignin
- hydroponic - method of growing plants with minerals without the use of soil, only water http://en.wikipedia.org/wiki/Hydroponics
- MADS box - conserves sequence motif which contain many transcription factors http://en.wikipedia.org/wiki/MADS-box
- Superoxide dismutase - enzymes that causes superoxide to form into oxygen and hydrogen perozide http://en.wikipedia.org/wiki/Superoxide_dismutase
- PDF gene - peptide feformylase and enzyme http://en.wikipedia.org/wiki/PDF_(gene)
- Ferric-chelate reductase - a family of enzymes belonging to reductases which uses oxidized metal ions as acceptors http://en.wikipedia.org/wiki/Ferric-chelate_reductase
- oxidoreductase - enzyme that transfers electron from a molecule to another oxidant
- defensin - proteins that act against viruses, bacteria, and other would be pathogenic organisms. http://en.wikipedia.org/wiki/Defensin
- suberin - wazy substance in plant preventing water to escape http://en.wikipedia.org/wiki/Suberin
- prolyl oligopeptidase - enzyme involved in the maturation of peptides http://en.wikipedia.org/wiki/Prolyl_endopeptidase
Outline
- Introduction
- Zinc is an important micronutrient for plants as a cofactor
- Essential, but toxic in large amounts
- Zinc homeostasis in plants prevent over accumulation
- There are some species of plants that are able to live with a large amounts of zinc without having toxic consequences called hyperacculmulators
- Thlapsi caerulescens is a hyperaccumulator of zinc, cadmium, and nickel
- Concentrations of zinc are higher in the shoot than root
- T. caerulescens is similarly related to Arabidopsis thaliana
- These plants are used to understand the molecular genetics of hyperaccumulators through comparison
- Main aim is to find out which genes are responsible for adaptation to zinc exposures in T. caerulescens
- Use of DNA microarray to cover the A. thaliana species
- Compare plants when moved from low or high supply of zinc intraspecifically
- Compare transcription in zinc deficiency, sufficiency, and excess
- Examine data to identify processes, biochemical pathway, or gene class that works with the zinc accumulation
- Zinc is an important micronutrient for plants as a cofactor
- Materials and Methods
- Plant Material and conditions
- Arabidopsis thaliana Columbia-0
- Thlaspi caerulescens J. & C. Presl accession La Calamine
- Germinated on garden peat soil
- 3 week old seedlings transferred to pots containing half strength Hoagland solution
- pH buffer was added and the pH was set at 5.5
- After 3 weeks, both species were transferred to the modified Hoagland solution containing:
- Deficient (0 µM) ZnSO
- Sufficient (100 µM) ZnSO
- Excess (1,000 µM) ZnSO
- Root and shoot metal accumulation assay
- Root system was desorbed with cold 5mM PbNO
- Roots and shoots were dried overnight
- Wet-ashed
- Mixture of HNO and HCL
- Analyzed for zinc, iron, and manganese using flame atomic absorption spectrometry
- Microarray
- The common reference was labeled with Cy3, treatment samples were labeled with Cy5
- Dye-swap used for quality control (QC)
- Roots of one pot were pooled and homogenized in liquid nitrogen
- Each pool (3 plants of either species) was considered as 1 biological replicate
- 2 biological replicates were used
- Semiquantitative Reverse Transcription –PCR
- New primers were created to ensure the correct amplification for T. caerulescens genes
- MMLV reverse transcriptase
- Care was taken in creating primers for Arabidopsis to ensure comparable positions and lengths as T. caerulescens
- 25-35 PCR cycles
- Microscopic analysis of T. caerulescens
- Plant Material and conditions
- Results
- Design
- A. thaliana
- Used hydroponic culturing system
- Three conditions made and exposed for only one week then transferred after three weeks
- Sufficient condition had 2 µM ZnSO4 which showed no phenotypic differences with deficient condition
- Deficient condition had 0 µM ZnSO4 which showed no phenotypic differences with sufficient condition
- Excess condition had 25 µM ZnSO4 which showed little growth inhibition in the roots
- One third of plants stayed in sufficient to act as control
- None of the plants were flowering
- T. caerulescens
- Hydroponic culturing system and three conditions made and exposed for only one week and transferred after three weeks
- Sufficient condition had 100 µM ZnSO4 which showed no phenotypic differences with deficient condition
- Deficient condition had 0 µM ZnSO4 which showed no phenotypic differences with sufficient condition
- Excess condition had 1 mM ZnSO4 which showed no differences in conditions
- No differences was found among these exposures
- Hydroponic culturing system and three conditions made and exposed for only one week and transferred after three weeks
- A. thaliana
- Minerals found in the plants
- Three mineral concentrations were found in the plants: Zinc, Iron, and Manganese
- Zinc
- No difference found in zinc deficiency except same amount of zinc levels found in roots
- 3 times more zinc found in T. caerulescens at sufficient level in roots and shoots
- 4.5-fold higher zinc in roots and 9-fold lower in leaves of A. thaliana in excess compared to T. caerulescens. T. caerulescens was the same result with sufficient
- Iron
- Iron increased in the roots with the increase of zinc in both plants
- 2-3-fold higher iron in T. caerulescens.
- T. caerulescens had same concentration in iron in all three conditions
- A. thaliana leaves show decrease in iron with increase of zinc
- Manganese
- Manganese decreases with the increase of zinc in roots of T. caerulescens
- A. thaliana gets a decrease of manganese with the excess zinc in roots
- The same works with the concentration in leaves
- Zinc effect on A. thaliana
- Microarray analysis was used to find the genes responding to the zinc exposures in the three conditions
- 608 genes were found in the comparison
- Most of the differences were found between the deficient and excess zinc
- Four clusters were distinguished
- Cluster I had 98 genes
- Most of the genes found in the deficiency
- Genes dealt with stress response, metabolism, heat shop proteins, and some unknown
- Cluster II had 128 genes
- Most genes found in the excess
- Genes dealt with metal homeostasis with iron than zinc, stress response by disease, and metabolic genes
- Cluster III had 347 genes
- Most genes found in deficiency
- Genes dealt with meal homeostasis, transporter proteins, and 164 genes encoding for proteins with unknown function
- There are also many genes that dealt with transcription regulation and protein stability
- Cluster IV had 35 genes
- Expressed genes found in deficient and sufficient
- Genes dealt with secondary metabolism and some unknown
- Cluster I had 98 genes
- Microarray analysis was used to find the genes responding to the zinc exposures in the three conditions
- Microarray hybridization
- Hybridization with cDNA from A. thaliana and T. caerulescens roots in sufficient group
- Only small amount of differences found with the hybridization of the two plants
- 220 genes did not hybridize with T. caerulescens
- Zinc effect on T. caerulescens
- 350 genes were identified as significantly expressed and 50 were differentially expressed in the three different conditions
- Six clusters were made
- Cluster I and II had 38 genes
- Mostly found in deficiency
- ZIP like genes were found in these clusters along with metal homeostasis and proteins dealing with lignin biosynthesis
- Metal homeostasis proteins NAS4 and FRO5 were found expressed more in the roots of A. thaliana
- Cluster IIIA and IIIB had 74 and 16 genes
- IIIA genes expressed mostly in zinc deficiency
- IIIB genes expressed mostly in excess zinc
- Genes dealt with oxidative stress response, senescence, ethylene biosynthesis, and plant defense
- Clusters IVA and IVB had 19 and 14 genes
- Found mostly in excess zinc
- Lacks iron homeostasis genes compared to A. thaliana
- Other genes are in two different clusters found in sufficient conditions and had unknown function or metabolic and stress response
- Cluster I and II had 38 genes
- Comparison of zinc response
- 2272 genes found to have a significant expression in T. careulescens compared to A. thaliana at least five time more
- 420 were not found in the roots of A. thaliana
- 929 genes found minimum variation among the conditions
- 121 genes differentially expressed with different zinc exposures in T. careulescens
- PDF genes were expressed in deficient and excess conditions in T. careulescens compared to A. thaliana
- Metal homeostasis genes, stress response, and lignin biosynthesis genes were greatly found in T. careulescens
- higher expressions should find phenotypical difference in the roots of the two plants
- They grew both plants to find the difference using autofluorescence
- T. careulescens showed more staining in the endodermis
- 2272 genes found to have a significant expression in T. careulescens compared to A. thaliana at least five time more
- Semi quantitative Reverse Transcription-PCR
- Used to confirm findings of microarray expressions
- Target genes were from root and leaf tissues of each plant in each conditions
- atNAS1 found in A. thaliana in zinc deficiency in roots and leaces
- TcNAS1 found in leaves of T. careulescens at deficient level
- TcAPX2, TcHMA4, and TcZIP4 found only in T. careulescens leaves
- TcFER1 found in excess, AtFER1found in deficient
- Design
- Discussion
- Zinc homeostasis found to be the differential gene
- T. caerulescens is able to maintain nontoxic zinc levels while translocating high amounts of zinc to the leaves
- An unexpected event occurs and that is that iron accumulates in the roots of Arabidopsis and T. caerulescens at increasing zinc concentrations
- The effect found in both species suggests that the increase in iron uptake is due to prevent possible risks of iron deficiency in leaves.
- Some genes known to be involved in zinc homeostasis are ZIP2, 4, 5 and 9, NAS2 and HMA2 genes
- Highly expressed in zinc deficiency include ZIP1, 3, and 10, IRT3, MTP2, and NAS4
- These transporters are involved in the transport of cations across plasma membrane. Not all of them are involved in the uptake of zinc in the same tissue.
- It is likely that these transporters do similar functions in different parts of the roots or are found in intracellular membrane.
- T. caerulescens has a smaller differential in genes
- Similar to Arabidopsis, T. caerulescens also expresses a cluster of genes in zinc deficient conditions, but this cluster is quite smaller. The probable cause for this is differences in hybridization efficiency
- Many unknown genes between the two plants
- These genes included 15 genes which 4 were PDF genes. One of these PDF genes included one that was close to being 1000-fold which was expressed in both deficient and excess zinc. (PDF1.1)
- The biological role of defensin is unclear
- Lignin biosynthesis was also expressed differently between the plants
- High expression of 24 genes suggested a function in lignin biosynthesis, and 13 genes are involved in suberin biosynthesis in T. caerulescens.
- These genes included (CER3, CER6,and 11 LTP genes)
- CER3 is known to be expressed in the roots of Arabidopsis, but the expression of similar gene CER6 in the roots of T.caerulescens is quite different.
- High expression of lignin and suberin biosynthesis concurs well with the U-shaped lignification and suberinization of the endodermis cells and the occasional presence of second endodermal layer found in the roots of T.caerulescens.
- Zinc homeostasis found to be the differential gene
