The plant biologists’ goals are to…The plant biologists’ goals are to…

> communicate desired outcomes of visualization > generate tools that unite data sets > address the genoytpe<>phenotype conundrum - G2P enable new insights into processes that are rendered in visually intuitively accessible form

Investing Images of Biological Processes with Added MeaningInvesting Images of Biological

Processes with Added Meaning• Events occurring in time and space, in three

dimensions.• Relationships between different levels of

biological organization/data types.• Transforming images by populating them with

quantitative, dynamic, data.

Questions Plant Biologists AskQuestions Plant Biologists Ask

• 1. What chains of changes occur in a plant over time and space in response to alterations in the environment or “signals”? Drought, Cold, Light

• 2. What internally “programmed” chains of changes in a plant occur over time and space ? Flowering, getting old, beginning photosynthesis.

• 3. How are these events regulated and what interactions occur between 1 and 2? That’s why we need more sophisticated, and quantifiable, images.

Overarching Goal and DifficultyOverarching Goal and Difficulty

• Goal: To find dependable ways to unite the data available at various scales into coherent pictures for particular plants in particular contexts. Generalizations/hypotheses may follow.

• Difficulty: There are no complete data sets, and the mechanistic bases for the data that are available are not fully understood. Intelligent guesses are the norm! We want to create a context in which those guesses can become more accurate.

The Current TerminologyThe Current Terminology

• The term “plant biology” is used to avoid categorization into scales and modes of measuring the data.

• A unifying goal of the plant biology community is to reveal and make clear the underlying cellular events (“mechanisms”) that give rise to a particular outcome (a “phenotype”).

• This is ultimately proved by establishing a causal connection between “genotype” and “phenotype”. Not easy.

Genes and GenomesGenes and Genomes

• Genes contain information. They are encoded in the molecule DNA (deoxyribonucleic acid).

• Genomes contain all the DNA in an organism. Different information is accessed under different circumstances, “readout”.

• Not all of that DNA is in the form of genes.

• The ancestral relationships of organisms are manifest in the relatedness of their genomes.

Cells and GenomesCells and Genomes• All organisms are composed entirely of cells, or the products of cellular activity.

• With some exceptions, all cells contain a complete copy of that organism’s genome.

The plant genome is divided among the nucleus, the chloroplast and the mitochondrion. Readouts from the three organelles are coordinated.

Each organelle serves a distinct function and is surrounded by a barrier or membrane

Diagram of a Plant CellDiagram of a Plant Cell

Nucleus – majority of the plant genome

Chloroplast - site of photosynthesis. (Tom will show data about photosynthesis.)

Cytoplasm- site of translation

Plant cells have internal compartments or organelles.

Genes and Genomes 2Genes and Genomes 2

No two genomes are identical, even within a species. (Genetic variation)• Each genome is also a historical document, illustrating past evolutionary processes. We cannot completely read the document, however.

Genotype, Phenotype? Genotype, Phenotype?

• A fundamental distinction has to be made between all the genes possessed by an organism (Mendel’s “heritable units”), its “genotype” AND• The characteristics that it exhibits, or its appearance, its “phenotype”. Phenotype is the manifestation of a particular readout of the genome.

“Phenotype”“Phenotype”

“Phenotype” means appearance. It originally meant “appearance” as it appears to the human eye of the observer e.g. the surface of Mendel’s pea seeds or fruit fly eye color.

Nowadays it can also mean any manifestation of genome readout, many of which are observed with the help of contemporary, high-throughput technology, as “read” by computational tools.

Plants Have A Special ProblemPlants Have A Special Problem

Running away is not an optionRunning away is not an optionI’m out of here!I’m out of here!

VersusVersus

External and Internal SignalsExternal and Internal Signals

Buchanan, Gruissem, Jones, 2000, Biochem,. Mol. Biol. Of Plants (BGJ)

Internal Developmental SignalsInternal Developmental Signals

Signal is perceived at the cellular level .

Signal is perceived at the cellular level .

Signals

Genome respondsGenome responds

One kind of readout One kind of readout

Light Light

Sorry, Tom, you’ll have to pretend it’s a corn plant!

Scales Over Which Data Are ReadScales Over Which Data Are Read• 1. Whole fields of plants, or whole plant behavior (Crop

Science). (Steve W., Jeff White, Jim Jones)• 2. Individual tissues, e.g. roots or leaves, (Plant Physiology).

(Steve W., Siobhan Brady).• 3. Cell types, e.g. mesophyll cells or bundle sheath cells. (Tom

B.) • 4 The genome, transcriptome, proteome, metabolome.

Language of “omics”). (Nick P., Tom B., RG, Siobhan B., All of us, ultimately)

• Note: Individual genes come in different “flavors”, called alleles, giving rise to slightly different mRNAs, and slightly different proteins, but still with the same function

“omics” alludes to the study of the totality of information associated with defined levels of biological organization. (This classification is way over-simplified).

“Genomics” – genes, structure/”landscape” of the genome.. A constant for any given organism.

“Transcriptomics”- transcripts. Variable

“Proteomics” – proteins. Variable

“Metabolomics”- metabolites, Variable

“Phenomics”- phenotypes. Variable

Different Views of GenomesDifferent Views of Genomes

• 1. Some people want to know about the history of genomes, how they came to have a particular structure, and, from that, how different organisms are related. (Eric L.)

• 2. Others want to know how the information in the genome (”genotype”) is variously read so as to produce a specific organism that grows, reproduces, and responds to internal and external signals (“phenotypic plasticity”). (RG, TB, SW, NP, BU).

Genome FunctionGenome FunctionWould that it were that simple!Would that it were that simple!

Different proteins are present under different conditions

Different proteins are present under different conditions

Complete informationComplete information

The readout cannot really be described as simply proceeding from the first level of transcription because there are many other points of control.

Proteins: the workhorses of the cell

Proteins: the workhorses of the cell

Readout from the genomeReadout from the genome

Translation from the language of DNA/RNA into protein

Translation from the language of DNA/RNA into protein

Harder SolutionsHarder Solutions

• The era of “blame it all on one gene/allele” is coming to an end, although it still holds in some cases.

• We now know that any particular phenotype may result as the outcome of a network of interacting genes. (Actually, the readout from those genes.)

• That interaction may be manifested between genes or protein molecules, and always at a specific location and under a specific condition within the plant cell.

Different Levels of Genome RegulationDifferent Levels of Genome Regulation

The amount of mRNA available for translation can be varied

The amount of mRNA available for translation can be varied

The amount of protein can be varied

The amount of protein can be varied

The protein can be chemically modified in different tissues.

The protein can be chemically modified in different tissues.

mRNAmRNA

Functions of ProteinsFunctions of Proteins

• The proteins that are produced perform functions such as– Catalyzing chemical reactions that would not otherwise go on in

the cell, producing “metabolites”.– Acting as structural components of the specialized membranes

in the cell.– Causing the activation of particular genes by binding to their

promoter region, “transcription factors”.– Interacting with each other in a “signal cascade”.

• For each of these categories, “There’s a database for that”, but they are not yet fully integrated, nor fully visualized.

Nick will show us a tool that integrates databases.

Transcription Factors (TFs)Transcription Factors (TFs)

/ TFs/ TFs

Gene is transcribed to mRNA after proteins have bound to adjacent DNA, “promoter”.

Gene is transcribed to mRNA after proteins have bound to adjacent DNA, “promoter”.

DNA binding proteins often interact with each other.

The TFs bind to sequences that are specific for the gene

The TFs bind to sequences that are specific for the gene

The expression of groups of genes is often coordinated.

Much active work in analyzing patterns of gene expression in response to given stimuli (Nick, RG, and many others).

“Promoter region”“Promoter region”

External SignalsExternal Signals

Signal acts on the genome though TFs

Signal acts on the genome though TFs

A “Signal Cascade”A “Signal Cascade”

Transmission of a signal, from, say, the periphery of a cell to the nucleus, where a particular gene (s) is then activated.

This mechanism involves the physical interaction of proteins with each other.

Databases of known protein-protein interactions are available. (Nick’s work on “The Interactome”)

Published by AAAS

C. E. Harjes et al., Science 319, 330 -333 (2008)

Simplified carotenoid biosynthetic pathway in plants

Simplified carotenoid biosynthetic pathway in plants

Example of a metabolic pathway

Tom and his colleagues studied the effect of genetic variation in maize on the levels of the nutrient compounds in red.

The variation is due to small differences in the genes that encode these enzymes.

Caps and bold: enzymes

Caps and bold: enzymes

Stress hormone

Metabolic PathwaysMetabolic Pathways

Many metabolic pathways involve physical interactions among enzymes.

Metabolic Pathways: “MapMan”Metabolic Pathways: “MapMan”

• Bjoern will show us a tool for the depiction of metabolic pathways in plants, and for pasting gene expression values on to those pathways. In MapMan, genes are “binned” into pathways.

What is MissingWhat is Missing

• A way to integrate these phenomena so that the user can visually follow known events from, say, the appearance of the signal all the way to the phenotype passing through the corresponding chain of events.

A Crossroads for Plant Biology.. There are models and then

there are models

A Crossroads for Plant Biology.. There are models and then

there are models“…….models bring together two of biologists’ favorite tools: abstraction and a love of drawing. Often they indulge in both of these passions by sketching down simplistic models, which may include five arrows, a factor X, and two question marks.

What has worked for decades to illustrate interactions among a few genes, has clearly reached its limits in the post-genomic era where research is no longer data-limited.”

AAVVOOIIDDAANNCCEE

or

* protein unfolding, membrane leakage, water/ion imbalance; ** distinct pathway, not specific for a particular stress; *** overlapping, pathways

SalinityDrought

Temperature

Ionic

Osmotic

Temperature

SSignaling ignaling SSensingensing RResponsesesponses

Stress-specificHomeostaticAdjustment

InjuryStatus

Cell Death

Growth Control/ Development

Detoxification** Damage Control

& Repair

TTOOLLEERRAANNCCEE

*

***

SOSs, DREBs, CBFs, NACs SOSs, DREBs, CBFs, NACs [important stress response transcription factors]

are affected by and themselves affectROS- and redox-dependent

pathwaysacc. Zhu, 2001, Bohnert & Bressan, 2001; Grene & Bohnert, 2009

AAVVOOIIDDAANNCCEE

• 1. We only know the full sequence of a few genomes.• 2. Even for those in which the sequence is known, we do

not have a full picture of how the genome is “read”.• 3. Valid generalizations about genome structure and

genome readout can be made, but, mostly, we cannot identify all the “working parts” for any organism.

• 4. Some biologists draw “models”, which usually means a static pictorial summation of what we know about a given phenomenon. No ODEs or stats involved, unless you are Steve Welch and the like.

Seo, P. J., et al. Plant Physiol. 2009;151:275-289

MYB96 mediates ABA signals via RD22 in regulating drought stress response

MYB96 mediates ABA signals via RD22 in regulating drought stress response

PhenotypePhenotype

PhenotypePhenotype

Expression of this TFExpression of this TF

When drought occurs, ABA is made, causing the TF gene to be expressed

When drought occurs, ABA is made, causing the TF gene to be expressed

The TF activates this geneThe TF activates this gene

ABA is a plant hormone, produced under stressABA is a plant hormone, produced under stress

An early response to drought is the synthesis of ABAAn early response to drought is the synthesis of ABA

A “Model” of ABA-Dependent and Independent Stress Responses in Arabidopsis

A “Model” of ABA-Dependent and Independent Stress Responses in Arabidopsis

Everything beyond expression of this gene is a black box.

Everything beyond expression of this gene is a black box.

Inhibition by these proteinsInhibition by these proteins

TFs binding to promoter of this genes

TFs binding to promoter of this genes

A “Model” for Temperature Responses in Arabidopsis

A “Model” for Temperature Responses in Arabidopsis

The rectangles contain generalized allusions to phenotypes The arrow leading to each rectangle is a type of hand-waving.. Time and space are hard to decipher here. Can we improve on this?

(ABA, GA, auxin, ethylene are plant hormones).(ABA, GA, auxin, ethylene are plant hormones).

TFsTFs

Grey ovals contain TFs that respond to more then one stressGrey ovals contain TFs that respond to more then one stress

Wish List: LayeringWish List: Layering• A system of layers, where the user can mine a given set of

data/”layer” (repositories of gene expression, metabolite data, protein-protein interactions), and view those data in a cellular context in 3D. A suite of genes/proteins/metabolites could be visualized dynamically, showing changes over time. Layers could be fused at will, within a temporal or spatial context.

• The user could upload his/her own data.• All data would be linked to other available information, for

example, orthologs in other species, published diagrams of signaling pathways, what is known about regulation of the genes, proteins or pathways of interest…..

Each Sub-Discipline has its Own Manhattan, NY

Each Sub-Discipline has its Own Manhattan, NY

The Flyover- everybody elseThe Flyover- everybody else

“Center of the Universe”, e.g. Fields, Tissues, Cells, Genomes

“Center of the Universe”, e.g. Fields, Tissues, Cells, Genomes

Unite the languages, enable a view that yields quantitative data from fields of chilly plants to cells where signaling cascades operate to activate defense genes, to altered metabolism to chilling-tolerant plants.

Unite the languages, enable a view that yields quantitative data from fields of chilly plants to cells where signaling cascades operate to activate defense genes, to altered metabolism to chilling-tolerant plants.

Rallying CryRallying Cry

Bjoern Presents His DataBjoern Presents His Data

Fujita, Y., et al. Plant Cell 2005;17:3470-3488

A “Model” of the Regulation of ABA Signaling by AREB1 (a TF)

A “Model” of the Regulation of ABA Signaling by AREB1 (a TF)

Stress ResponseStress Response

Genome Readout 3Genome Readout 3

• Production of mRNA and its regulation• Production of protein and its regulation.• Production of functionally active protein in a

specific subcellular location.• Mechanisms for providing other factors

needed for function (e.g. substrates for enzymes).

• Effect of the functioning of proteins on cell/tissue/ plant function.

Making the G2P ConnectionMaking the G2P Connection

Connecting a particular allele to a particular phenotype. How is gene expression regulated?

Connecting a substring within a given chromosome to a given phenotype. Stats.

Connecting a gene network to a given phenotype. How is the network regulated?

Connecting a metabolic pathway to a given phenotype. How is the pathway regulated?