Jan. 13, 2011B4730/5730

Plant Physiological EcologyIntroduction 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 OverviewPlasma membrane

Nucleus

Smooth ERRough ER

Ribosomes

Golgi Apparatus

Mitochondrion

Centrosome

Microtubules

Lysosome

Chloroplast

Vacuole

Cell Wall

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

Nucleus

Ribosomes

A. Genes on DNA in Chromatin or ChromosomesB. PremRNA transcribed, rRNA & tRNA synthesized

D. mRNA translated into polypeptides by ribosomesC. mRNA, rRNA & tRNA transported

A.B.

C.

D.

DNA

premRNA; RNA

mRNA and tRNA

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 inducedGreen-cold repressedYellow-equal expressionBar is 300 micrometers

Identification of Genes