Download May 4, 2004 B4730/5730 Plant Physiological Ecology

Jan. 13, 2011
B4730/5730
Plant Physiological Ecology
Introduction to Physiology and
Genetics
Membranes
• Keep internal and external environments
different
– Set up gradients
• Fluid Mosaic model
– Hydrophobic vs. hydrophilic zones
– Phosphorylation and pumping
• Electrochemical gradients
– Redox and electron transport
– pH and ATPase
Cell Overview
Plasma membrane
Nucleus
Cell Wall
Lysosome
Microtubules
Smooth ER
Rough ER
Ribosomes
Vacuole
Centrosome
Mitochondrion
Golgi Apparatus
Chloroplast
Mendelian Genetics
• Laws of segregation and independent
assortment explain randomness of alleles
passed to offspring
• Chromosome shuffling in
meiosis/fertilization results in offspring
traits
• Two copies of alleles determines traits
– Dominant, recessive, pleiotropy, epistasis,
quantitative traits, norm of reaction
Overview of Genes to Proteins
• Eukaryotes have complex packing of DNA
– Amount of packing influences gene expression
• Much of the DNA in eukaryotes does not code
for genes
– Repetitive DNA and introns
• DNA sequences can be modified to alter gene
expression
• Gene expression can be modified at any point
between DNA and final protein
• Control of gene expression allows development
and response to environment
Fig. 12.7
Brooker Biol. 2007
From DNA to Protein
A. Genes on DNA in Chromatin or Chromosomes
B. PremRNA transcribed, rRNA & tRNA synthesized
C. mRNA, rRNA & tRNA transported
D. mRNA translated into polypeptides by ribosomes
Nucleus
DNA
A.
B
.
mRNA and tRNA
premRNA; RNA
Ribosomes
C.
D.
http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a3.html
Gene Libraries
• cDNA libraries
– track gene expression
• EST libraries
– Track changes in gene expression
– promoters or RNA splicing sites
• New genes compared with gene libraries
– 1) exactly match gene from some organism
– 2) partially match known gene suggesting a
function
– 3) partially match sequence of unknown
function
– 4) entirely new sequence
Studying Gene Expression
• DNA microarray assays (DNA chips)
– Study thousands of genes at once (genomics)
– Glass slide contains single strand gene fragments
– Fragments tested for hybridization with cDNA molecules
• In vitro mutagenesis
– Phenotype of expressed gene in mutant organism
• RNA interference (RNAi)
– Double stranded RNA stops gene’s messenger RNA
– Mechanism?
• Proteomics
– Full protein sets (proteomes) encoded by genomes
• Proteomics/genomics/metabolomics
– Holistic approaches to organisms without excessive
reductionism
– Epistasis problems
– Key developments will require advances in bioinformatics
Use of Arabidopsis for study of
drought gene expression
• Dehydration-responsive element/C-repeat (DRE/CRT) is
a cis-acting element in drought, salt, and cold stress
responses
– Transcription factors for cold and drought responsive genes
(DREB/CBF) have been cloned; DREB1 is cold responsive,
DREB2 is drought responsive
– Promoter is 35S from cauliflower mosaic virus
• Study used cDNA microarray analysis to identify new
DREB1A target genes
– full length cDNA libraries were constructed from drought and
cold treated plants
– 1300 full length independent cDNAs were isolated
– Genes identified were responsive to dehydration (rd) and early
responsive to dehydration (erd); used as positive controls
– α-tubulin gene as same expression level used as internal control
and nicotinic acetylcholine receptor epsilon-subunit (nAChRE)
gene from mouse as negative control (no homology to any
sequence in Arabidopsis database)
Seki et al. 2001
Isolation of Arabidopsis cold and
drought responsive genes
• cDNA microarray was prepared from mRNA
which was hybridized with flourescent probes
Cy3 in stressed and Cy5 in unstressed plants by
reverse transcription
Red-cold induced
Green-cold repressed
Yellow-equal expression
Bar is 300 micrometers
Identification of
Genes