Kristoffer Chin: Week 11

Entry
[[Media:Intro Methods.ppt|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.