Kristoffer Chin: Week 11

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Entry

DNA microarray journal club

Definitions

  1. lignin - organic substance that adds strength and rigidity to cell wall http://www.biology-online.org/dictionary/Lignin
  2. hydroponic - method of growing plants with minerals without the use of soil, only water http://en.wikipedia.org/wiki/Hydroponics
  3. MADS box - conserves sequence motif which contain many transcription factors http://en.wikipedia.org/wiki/MADS-box
  4. Superoxide dismutase - enzymes that causes superoxide to form into oxygen and hydrogen perozide http://en.wikipedia.org/wiki/Superoxide_dismutase
  5. PDF gene - peptide feformylase and enzyme http://en.wikipedia.org/wiki/PDF_(gene)
  6. 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
  7. oxidoreductase - enzyme that transfers electron from a molecule to another oxidant
  8. defensin - proteins that act against viruses, bacteria, and other would be pathogenic organisms. http://en.wikipedia.org/wiki/Defensin
  9. suberin - wazy substance in plant preventing water to escape http://en.wikipedia.org/wiki/Suberin
  10. 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
  • 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
  • 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
    • 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
    • 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
    • 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
    • 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
  • 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.
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