Salomon Garcia: Week 11

Preparation for Journal Club 3
[[Media:Intro Methods.ppt|DNA Microarray Journal club]] Paper7 pmid=16998091 //van de Mortel et al. (2006) link to full text
 * This is a link to the DNA Microarray journal club that was done on April 13, 2010:
 * Link to the Journal Article:
 * Link to the Powerpoint presentation for Journal Club 3:
 * Link to the Data Set: [[Media:VanDeMortel_Raw_Compiled_Data_Arabidopsis.xls|Data Set]]

Ten New Words from the van de Mortel paper


 * 1) Hydroponic- the cultivation of plants by placing the roots in liquid nutrient solutions rather than in soil; soilless growth of plants.http://dictionary.reference.com/browse/hydroponic
 * 2) Hyperaccumulator-A plant is said to be a hyperaccumulator if it can concentrate the pollutants in a minimum percentage which varies according to the pollutant involved.http://en.wikipedia.org/wiki/Hyperaccumulator#Hyperaccumulators_and_biotic_interactions
 * 3) putative genes-a putative gene is a piece of DNA thought to be a gene based on sequence (ex. Open Reading Frame) but either the protein produced or the function of what is thought to be the expressed protein is not known.http://wiki.answers.com/Q/What_is_putative_gene
 * 4) ethylene biosynthesis- Protein involved in the synthesis of ethylene (C2H4), an unsaturated hydrocarbon gas mainly produced in plants. It has developmental effects as a hormone, including growth inhibition, regulation of fruit development, leaf abscission and aging.http://www.uniprot.org/keywords/KW-0266
 * 5) lignin biosynthesis-begins in the cytosol with the synthesis of glycosylated monolignols from the amino acid phenylalanine. http://en.wikipedia.org/wiki/Lignin#Biosynthesis
 * 6) nonaccumulator- is a plant that tends to not accumulate high amounts of a metal. (from the van de Mortel et. al. paper)
 * 7) defensin genes-defensin, alpha 1, is found in the microbicidal granules of neutrophils and likely plays a role in phagocyte-mediated host defense.http://en.wikipedia.org/wiki/Defensin,_alpha_1
 * 8) vacuolar transporter-
 * 9) suberin biosynthesis- The biosynthesis of the aliphatic monomers shares the same upstream reactions with cutin biosynthesis, and the biosynthesis of aromatics shares the same upstream reactions with lignin biosynthesis. Lignin and suberin are the only known biological polymers that are irregular. http://en.wikipedia.org/wiki/Suberin
 * 10) lignification-  a change in the character of a cell wall, by which it becomes harder. It is supposed to be due to an incrustation of lignin.http://www.biology-online.org/dictionary/Lignification

Introduction

 * Micronutrients essential for plants
 * Zinc plays important role in biological process
 * Toxic in high amounts
 * Plant keeps tight regulation in zinc homeostasis
 * Zinc homeostasis mechanism is universal except for few exception


 * Accumulation of large amount of zinc by zinc hyperaccumulators
 * more than 1% of dry weight
 * 10000 micro gram of zinc per gram in hyperaccumulators
 * 30-100 micro gram per gram in most other plants
 * Over 300 is usually toxic
 * T.caerulescens is a zinc hyperaccumulator
 * Closely related to A. thalina
 * Zinc concentration in shoots often found to be higher in roots of hyperaccumulators


 * Homeostatic mechanisms have evolved in plants
 * control the uptake, accumulation, trafficking, and detoxification of metals
 * Physiology of metal hyperaccumulation is understood, the molecular genetics has not been explored in great detail


 * Genes that are more likely to be relevant in adaptation to high zinc exposure in T. caerulescens
 * root response to zinc was examined in deficient, sufficient and excess amounts.
 * comparison between Arabidopsis and T. caerulescens

Materials and Methods

 * Preparation of plants for root and shoot metal accumulation assay
 * Germinated in garden peat soil
 * 3 week old seedlings were transferred to pots containing Hoagland solution
 * pH buffer was added and was set to a 5.5 pH
 * After 3 weeks both species were transferred to the modified solution containing:
 * Deficient (0 micro meters) ZnSO4
 * Sufficient (10 micro meters) ZnSO4
 * Excess (1000 micro meters) ZnSO4


 * 4 weeks later plants were harvested
 * Root system was desorbed with cold 5mM PbNO3
 * Shoots and roots dried overnight
 * Analysis for zinc, iron, and manganese was done using atomic absorption spectrometry


 * cDNA from T. caerulescens roots was exposed to sufficient zinc and was used as the common reference
 * Common reference was labeled with Cy3, treatment samples were labeled with Cy5
 * Roots of one pot were pooled and homogenized in liquid nitrogen
 * Each pool considered to be one biological replicate


 * RNA was extracted, purified, and hybridized on slides for the microarray
 * 27,000+ annotated genes and 10000+ nonannotated genes
 * After hybridization slides were scanned, analyzed and normalized
 * Agilent Feature Extraction software was used
 * T-test was done in order to find differentially expressed genes
 * Differentiated genes were clustered in tree diagram
 * Dye Swap hybridization was performed for QC


 * RNA extracted from both species in order to run semiquantitative RT-PCR
 * Primers were created in order to ensure correct amplification of T. caerulescens genes
 * Care was taken in order to ensure comparable positions and lengths between the two
 * 25-35 PCR cycles were done


 * Three conditions were done to expose zinc in Arabidopsis
 * Sufficient condition (2 micro meters ZnSO4)
 * No phenotypic difference
 * Deficient Condition ( 0 micro meters ZnSO4)
 * No phenotypic difference
 * Excess condition (25 micro meters ZnSO4)
 * Little growth inhibition was found in the roots


 * Three conditions were different in T. caerulescens
 * Sufficient condition (100 micro meters ZnSO4)
 * Deficient condition (0 micro meters ZnSO4)
 * Excess condition (1 micro meter ZnSO4)
 * No altered phenotype in all conditions

Discussion

 * Zinc homeostasis found differential along with iron accumulation
 * 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.


 * Genes involved in zinc homeostasis are highly expressed in deficiency than other conditions
 * 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.


 * NAS and YSL genes and their ability to be induced by zinc deficiency
 * NAS2 and NAS4 genes are highly expressed in roots under deficiency rather than sufficiency.
 * YSL genes are also induced by zinc deficiency.
 * YSL genes are implicated in the transport of NA metal chelates within the plant and the entry of metals to the phloem and xylem.
 * YSL2 and YSL3 are slightly affected by different zinc treatments, which in turn lead them to find that genes were slightly induced by lower zinc concentrations.


 * High zinc deficiency- induced expression of FRD3, FRO4, and FRO5
 * Arabidopsis was grown under zinc deficient conditions
 * This was a significant observation because FRD3 has been known to be implicated for the most part with iron homeostasis.
 * FRO4 and FRO5 approximate the ferric chelate reductase gene FRO2, in the contrary to FRO2 their expression was not induced in the root of Arabidopsis upon iron deficiency.


 * 128 genes of Arabidopsis was more highly expressed in excess
 * The expression of these genes is related to a defense against oxidative stress caused by the treatment of zinc.
 * Large fraction of these genes was found to have an expressed comparison between wild type Arabidopsis and fit1 mutant.


 * T. caerulescens has a smaller differential in genes
 * For this species the response to zinc deficiency and zinc excess is quite different from Arabidopsis.
 * Unlike Arabidopsis, T. caerulescens expresses the ZIP family (ZIP3, 4, and 9) under zinc sufficient conditions
 * 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.


 * NAS genes and their importance to T. caerulescens
 * The expression of these three NAS genes in T. caerulescens suggest that they are a major function for metal adaptation.
 * The presence of these genes indicates that there will be flexibility when it comes to NAS gene expression


 * Genes expressed differently from T. caerulescens and Arabidopsis
 * There are more than 2200 genes which are quite significant and differentially expressed in the three zinc treatments
 * 50% of the genes found in T. caerulescens are of unknown function.
 * Stress respond genes expressed in T. caerulescens are different from Arabidopsis.


 * Expressed genes that were found and biological role is unclear
 * Several expressed genes were 100-fold on T. caerulescens. These genes were defensin genes or PDF genes
 * 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.

Potassium transporter genes HAK5, KUP3, and KAT, were found to be highly expressed in T. caerulescens.
 * Arabidopsis had low expression of PDF genes
 * Jasmonic acid (stress hormone) is responsible for the expression of PDF1.2 in Arabidopsis.
 * Heavy metal stress is involved in the accumulation of jasmonic acid.
 * PDF1.2a, 1.2b, 1,2c, and 1.3 are induced upon potassium starvation. These genes suggest a relation between jasmonic signaling and potassium starvation.
 * 46 genes were found to be more highly expressed in T. caerulescens than in Arabidopsis

HMA3 is similar to HMA4, and in Arabidopsis the expression of this gene is not affected by exposure to zinc
 * Zinc hyperaccumulation trait in T. caerulescens and the required expression of metal hyperaccumulation genes
 * Considered 16 highly expressed genes at 100 micrometer ZnSO4, of which 4 were known to be Zinc transporters HMA4, MTP1, ZIP1, and IRT3
 * In the study conducted by Papoyan and Kochian HMA4 was identified to being involved in zinc hyperaccumulation, particular loading of zinc into the xylem.
 * Other Zinc transporters that were highly expressed in T. caerulescens included HMA3, MTP8, and NRAMP3.

This gene was found to be highly expressed in T. caerulescens at sufficient and deficient conditions compared to Arabidopsis.
 * Genes found to contribute in the movement of zinc
 * The MTP8 gene is a member of the cation diffusion facilitator family.
 * AtNRAMP2 is a vacuolar transporter is able to transport cadmium and iron. The induction of TcNRAMP3 gene expression by deficiency of zinc suggests it has an important role in the movement of zinc and iron in T. caerulescens and Arabidopsis.


 * Iron homeostasis genes and the unexpected outcome that resulted between T. caerulescens and Arabidopsis
 * IRT1, IRT2, and FRO2 (iron homeostasis genes) were not induced in T. caerulescens upon excess treatment of zinc.
 * Testing with RT-PCR did not detect the expression of TCIRT1 except in roots at lower levels of zinc.
 * This suggests that T. caerulescens is able to regulate its iron and zinc homeostasis independently, unlike Arabidopsis, or that continued expression of zinc transporters enables low efficiency but enough iron uptake in T. caerulescens.


 * Expression of similar genes and the different outcome between T. caeulescens and Arabidopsis
 * 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.
 * This U-shape apearence is uncommon in plants, this usually occurs at older sections of the root hairs when they are no longer active.
 * Suggesting that this layer helps to prevent the eflux of metals from the vascular cylinder.


 * Genes differentially expressed certain alterations in transcript levels may occur
 * 131 transcriptional regulators with more than 5-fold higher expression were found in T. caerulescens
 * Of the 19 genes that were more than 10-fold higher expressed in zinc deficient conditions 2 genes (INO and SPL) are said to be involved with the development of flowers in Arabidopsis but expression is irregular.
 * Similar to this irregular expression it was also found that FIS2 gene is more highly expressed in the roots of T.caerulescens.
 * In Arabidopsis, this gene is expressed in the development of seeds.
 * This gene is found in A. halleri and is induced in the response to the exposure of zinc.


 * Comparative analysis show T. caerulescens and Arabidopsis sshow role in zinc hoeostasis genes and there adaptation to high zinc adaptation
 * The analysis also suggests that there are many uncharacterized genes with similar functions and that there are many that are unaccounted for and their functions are unknown.
 * While some of the genes were differentially expressed between A. halleri and Arabidopsis:
 * Many of them were not at different levels
 * Thus, suggesting an overlap in mechanisms of metal accumulation and tolerance.