February 20, 2005 Sharp – what and why Davis – mapping genes Clark - maize rootworm Bohnert - get genes/transcripts Tao – dynamics of genes Springer –

February 20, 2005

Sharp – what and why

Davis – mapping genes

Clark - maize rootworm

Bohnert - get genes/transcripts

Tao – dynamics of genes

Springer – making sense of all

Plant Genome &

Gene Complexity,

Gene Regulation,

Place & Timing,

and how to sort the

PlayersNSF DBI #0211842

The Plant Genome DiploidPolyploid

Chrysanthemum species illustrate the phenomenon.

Monoploid number (the basic set) = 9 chromosomes

In Chrysanthemum species, the number of chromosomes fall into 5 categories: 18 chromosomes = diploid (2 copies of the monoploid) 36 chromosomes = tetrapoid (4 copies of the monoploid) 54 chromosomes = hexapoid (6 copies of the monoploid) 72 chromosomes = octaploid (8 copies of the monploid) 90 chromosomes = decaploid (10 copies of the monoploid)

50% of all flowering plants are polyploid.

• Ploidy changes - a recurring process• Many ‘diploid’ species have gone through ploidy changes• Fusions of related species new species

See: Arabidopsis

PIP2;2

Ch-1

PIP1;3 TIP3;2 NIP3;1 TIP2;xpseudo TIP3;1 NIP6;1

Ch-5

SIP1;2 NIP4;1 NIP4;2 TIP2;3 PIP2;4

5 10 20 30Mb

Ch-4

PIP1;4 TIP1;3 NIP5;1 TIP2;2NIP1;1NIP1;2 PIP1;5

Ch-3

TIP1;2TIP2;1NIP7;1SIP1;1 TIP5;1

PIP1;1

PIP2;1

PIP2;5

SIP2;1

Ch-2

PIP1;2TIP4;1NIP2;1pseudoNIP3;1pseudo NIP2;1

TIP1;1

PIP2;6

PIP2;3

PIP2;8

PIP2;7

(15)

(4)

(14)

(3)

(12)

- duplicated regions that include AQPs.

rDNA

AQP are distributed over all Chromosomes - a few clusters, many duplications

Figure 3

control

O3

CO2

Columbia grown in Soy-FACE

Field ona dish!

Arabidopsis – model plant

small, fast, prolific,mutants, lines, ecotypes,genome sequence

Arabidopsisgrowing

in the fieldin high

CO2 and/or ozone

downthere

concept

plant performance inthe future earth’

atmosphere (~2040)-

also: soy, corn, weeds

FACE-rings

Plants in silico? Sure! And then: Plant Design from Scratch

Ecosystem – population – species – ecotype (breeding line)

Organism – organ – tissue – cell – compartment

Nucleus – envelope & pore – nucleoplasm, nucleolus & chromosomes

Euchromatin & heterochromatin – gene islands – gene

Promoters – 5’-regulatory –

introns & exons - coding region –

3’-regulatory regions

The Plant Genome

The Plant Genome ControlsControls for Gene Expression – many Switchboards

• Chromatin condensation state

• Local chromatin environment• Transcription initiation• Transcript elongation• mRNA splicing • mRNA export• mRNA place in the cell• RNA half-life• Killer microRNAs• Ribosome loading• Protein transport/targeting• Protein modifications• Protein turnover

Levels of regulation that

affect what we call

“gene expression”

The Plant Transcriptome

Killer RNAs(there are micro-genes)

no protein-

gene isessentially“silenced”

5 years ago, we did not know that

such a control system existed!

microRNAs

The Plant Transcriptome

How to sample the transcriptome?

Morphological dissection remember Bob Sharp’s talk! (root, leaf, flower - epidermis, guard cell, etc.)

Cell sorting make single cells, send through cell sorter (size, color, reporter gene)

Laser ablation micromanipulation of laser to cut individual cells

Biochemical dissection chloroplasts, mitochondria, ribosomes, other membranes

Painting cellswith a

reporter gene-

here this isGFP

GreenFluorescence

Protein

The Endodermis of the root tip

is highlighted in transgenic

plants using pSCR::mGFP5.

Painting tissuesthen isolating desired cells

Enzymatic staining

The Plant Transcriptome

Cell-Specific Cell-Specific GFP ExpressionGFP Expression

• Catalog of available transgenic Arabidopsis lines.

• Lines are available from the stock centers.

• However, the molecular basis for the observed phenotype is usually uncharacterized.

The Plant Transcriptome

> cDNA libraries

• “neat”

• normalized

• subtracted

> SAGE libraries

cDNA – complementary DNA

converts messenger RNA into

double-stranded DNA

“Normalization” removes mRNAs

for which there are many copies

in a cell – thus enriching for

“rare mRNAs” (not so much sequencing to do)

Subtraction removes cDNAs which you already know

(less sequencing)

Total RNA

Poly(A)+ RNA

1st strand cDNA

ds-cDNA

Size-selected double stranded cDNA (>500 bp)

Ligate to EcoRI adapters/digest NotI

Clone (EcoRI/NotI) digested pBSII/SK+ & adaptored cDNA

Primary cDNA Library

Primary (neat) library may be used for “normalization”

Library Normalization

primary cDNA library

ss-DNA

DNA “tracer”

PCR inserts by T7 and T3

standard primers

DNA “driver”tracer/driverhybridization

column chromatogr.(double-strands stick)

Non-hybridized DNA from flow-through = normalized clones

make ss-DNA out of primary

library

cDNA Libraries

Cloning ofroot RNAs

from segmentsS1 – S4root tip

(Sharp lab)

sequenced~18,000 clones

found~8,000 unique

and~130 novel genes

How many genesmake a root?

The Plant Transcriptome

Isolate total RNA from cells or tissue

Isolate small regions (SAGE tags) of each

mRNA transcript in a cell

Digest tags and ligate into concatamers for sequencing

Reference sequencing results against public databases

SAGE – Serial Analysis of Gene Expression – an Overview

The Plant Transcriptome

Quantitative PCRin 384-well plates

(96 primer pairs,3 repeats each)

Taking SAGE & cDNA

sequences together-

corn roots

“express”

20-23,000 genes(i.e., mRNA is made)

-

The entire corn genomeis expected to include

~50,000 genes

The Plant Transcriptome

Why are we doing this?• Genes expressed in well-watered conditions,

how many, where and which?

• Changes during drought episodes?

• Variation in different lines or land races?

• Breeders to cross and select for tolerance!

• Proteins and substances (metabolites) made?(how to make a cell wall, how to defend against rootworms)

• Make corn with thicker (modified) cell walls!

Array onto glass slides using Robotic Gridder

Block reactive groups Fix & denature DNA

mRNA 1 mRNA 2

Reverse Transcription labelling using Cy5 and Cy3 dyes

cDNA 2-cy5

cDNA 1-cy3

Hybridize

DNA

Measuringthe ratio of expression(control to testPopulation)

Transcript Dynamics –

Wenjing Tao is next