Floriculture Genomics - Basic Tools for Crop Improvement Through Biotechnology
Floriculture Genomics - Basic
Tools for
Crop Improvement Through Biotechnology
Dr. David Clark
University of Florida
Dr. Michelle Jones
Ohio State University
Executive Summary
This project will produce an unprecedented number
of approaches to studying the regulation and development of all aspects of the
flowering process. By the end of the project, we hope to have sequenced and
characterized a majority of the genes that are expressed in Petunia flowers. By
characterizing the genes involved in flowering and senescence, we will be able
to understand their regulation and manipulate them for commercial utility As a
result we will continually develop new technologies that will be useful in
developing important research collaborations, and in providing the floriculture
industry the genetic tools needed to get around some of the basic problems that
have plagued flower breeders for years. it is our ultimate goal to take
promising new technologies discovered during this research into other
floriculture crops in the not too distant future.
Introduction and Literature Review
One of the major limitations in the marketing of
cut flowers and flowering plants is their short lifespan. A flower’s lifespan
and post-production quality is determined by senescence, a genetically
controlled stage of flower development that culminates in the wilting or
abscission of the corolla (Borochov and Woodson, 1989). Although flower
senescence has received much research attention in recent years, the molecular
mechanisms by which plants regulate flower senescence are not well understood.
In many flowers the signal that initiates and coordinates senescence is the
plant hormone ethylene. Current postharvest treatments used to prolong flower
longevity utilize chemical inhibitors of ethylene biosynthesis or action. The
genetic manipulation of ethylene biosynthetic and signal transduction pathways
has also resulted in flowers with delayed senescence (Savin et al., 1995;
Wilkinson et al.. 1997; Bovv et al.. 2000). Although both of these methods are
effective at delaying senescence in ethylene-sensitive flowers, there are
limitations to their commercial use. Limitations with chemical controls of
ethylene include environmental contamination (STS). mode of product delivery (MCP
gas), and efficacy problems over the complete
postharvest time period. The production of transgenic plants that are
ethylene-insensitive is still not considered to he commercially viable because
ethylene is involved in other aspects of growth and development, like seed
germination and adventitious rooting. Although the manipulation of plants for
ethylene-insensitivity has resulted in production of plants with long-lasting
flowers. it has been shown to produce negative side effects on other whole plant
characters (Clark et al.. 1999: Gubrium et al.. 2000). In order to more
precisely alter a single trait, like flower longevity, we need to have a
detailed understanding of the molecular mechanisms regulating that process
within the plant before any technology will be commercially viable.
To date, cultivar improvement in floriculture
crops has been conducted using a traditional breeding and genetics approach. In
recent years. new experimental approaches facilitated by the use of molecular
biology tools have shown a great deal of promise in producing floriculture crops
with unique genetic features not previously available to breeders. Petunia
plants have been engineered to be insensitive to ethylene for.the practical
purposes of extending postharvest life (Wilkinson et al.. 1997; Clark et al..
1999: Gubrium et al.. 2001). Petunias have also been engineered for delayed leaf
senescence. environmental stress tolerance (Dervinis et at. 1999). and fungal
tolerance (Clark unpub.).
Within the last 5 years there has been an
enormous amount of research focused on unraveling the genomes of numerous
organisms ranging from plants (Arabidopsis) and yeast (Sacchromyces) to
insects (Drosophila) and humans (Horno sapiens). With such technological
advancements, the cost of related supporting technologies (especially DNA
sequencing and microarray analysis) has declined while the efficiency at which
scientists can obtain genetic data has increased. This area of research. termed
functional genomics. can he utilized on the grandest scale to determine
the function and regulation of all the genes in any given organism. Ultimately
experiments utilizing functional genomics tools will lead to the development of
critical hiotechnologies that can be utilized to understand the mechanisms that
control complex processes like senescence, and to provide flower breeders with
previously unobtainable traits.
Simply stated. functional genomics is a series of
experimental processes used to determine the location, sequence. function, and
interrelationships of all or most of the genes in an organism (Schena et al.,
1995; 1998). Genes that are expressed produce messenger RNAs (mRNA) that can
be extracted from the plant tissue of interest. These mRNAs arc cloned to
produce a cDNA library a pool of complementary DNA
representing all genes being expressed in that tissue. Each individual eDNA is
then DNA sequenced to decode the genetic information. and the information is
cataloged in a computer database. This database can then be used by any
researcher to search for any gene of interest that is represented in the pools
of expressed genes. Using extremely precise robots, each eDNA can then
physically deposited onto glass-based microchips in an array that allows for the
representation of as many as 10.000 genes per 1 cm2 of chip (Lemieux et al..
1998). Once these chips are produced they can be screened to determine which
genes are induced by specific stimuli or developmental events (see methods,
objective 2). This technology will allow us to determine the expression patterns
of literally thousands of genes at a time, under an infinite number of screening
conditions. It ill also enable us to identify large groups of genes that are
responsible for the regulation of many different aspects of growth and
development like flower senescence. The ability to have a computer-driven
floriculture genetics database combined with the ability to determine the
genetic expression patterns of thousands of genes at a time will provide
floriculture geneticists with unprecedented numbers of options for understanding
their genetic systems.
Preliminary Data:
Petunia cDNA libraries
In order to create DNA microarrays we had to
produce a pool of genes expressed in flowers that contained the genes involved
in the processes we are interested in studying. Three high quality eDNA
libraries were constructed from petunia flowers at 1) various stages of
development and 2) following pollination and 3 following treatment with
ethylene. The developmental library contains genes expressed from early flower
initiation to anthesis and can be used to identify both temporally and spatially
regulated genes. The post pollination and ethylene-treated flower libraries
contain genes expressed in petunia floral tissues during all aspects of
programmed senescence, and can he used to find genes that are differentially
regulated by pollination and ethylene. These 3 libraries were constructed in Dr.
Clark’s lab at UF with the assistance of a post doe from the Jones’ lab.
Gene Sequencing
Once the petunia eDNA libraries were produced. we
worked in cooperation with the UFICBR Genomics Sequencing Core (GSC) to decode
and identify our expressed genes using high throughput
DNA sequencing. So far, we have obtained the DNA sequence of approximately 3500
unique genes from the three libraries we constructed (described above). After
analyzing the initial sequence from these genes, we are confident that we have
excellent representation of a large number of the genes expressed in petunia
flowers. The reason we believe this is because we have found many genes involved
in ethylene biosynthesis and perception. as well as genes involved in
transduction of the ethylene regulated signals leading to senescence ¬ó ie. all
of the genes we expected to find have been present.
Objectives and Anticipated Benefit
The main benefit of this research is the
production of’ functional genomics tools that can be utilized by US
floriculture researchers and industry to genetically engineer important
commercial traits. Another chief benefit is that academic and industry
researchers can utilize these newly developed technologies to establish
interdisciplinary collaborations in floriculture biotechnology.
The specific objectives outlined in this
proposal are as follows:
# 1: To analyze DNA sequence data and
establish a database of genes expressed in flowers.
#2: To create DNA microarrays to
identify and characterize groups of genes involved in flower senescence.
# 3: To identify components of the
senescence signaling pathways and engineer the most appropriate genetic targets
for the manipulation of senescence.
This project will ill produce an unprecedented
number of approaches to studying the regulation and development of all aspects
of the flowering process. We have already proven that genetic engineering of’
value-added commericals traits can be achieved in petunia. We have also proven
that these plants are highly desirable models that can be used by the scientific
community to study fundamental physiological processes. By characterizing a
majority of the genes involved in flowering and senescence, we will he able to
understand their regulation and manipulate them for commercial utility. It is
our ultimate goal to take promising new technologies discovered during this
research into other floriculture crops of commercial importance.
Materials and Methods
We have chosen to work with Petunia x hybrida
Michell Diploid’ as our model flower system for several reasons. It is a
colchidiploid (doubled haploid). which allows us to have a clean inbred with
minimal heterozygosity and a simpler genome. Petunia has also served as a model
system for ethylene and pollination-regulated flower senescence, and much
physiological research has been conducted with it. For application of the
technologies developed by this research. Petunia is attractive because it
can be genetically transformed with minimal effort and has a short crop cycle.
Objective 1: To analyze DNA sequence data and
establish a database of genes expressed in flowers.
The clones available from the 3 libraries
represent all stages of flower development including senescence and as such
would be useful for scientists in many areas of research including those
studying flower color, fragrance, flower form and organ formation, pollination
responses and compatibility, fruit ripening and seed germination, in addition
they will be useful for identifying genes specific to individual flower tissues
and identifying flower specific promoters. It is one of the objectives of this
research to develop this database as a Floriculture resource around which to
build future collaborative research.
After the genes are sequenced their sequence data
is compared to sequences in the NCBI databases to determine their putative
function. These sequence comparisons can also be used to identify gene families
and to weed out duplicated genes. so that a pool of unique genes can be
compiled. This pooi of unique genes will be cataloged and archived for long-term
storage as glycerol stocks in an ultra low freezer (¬ó80¬? C). These
clones will then be available to other floriculture researchers interested in
any aspect of flower development. The sequence data for the unique clones will
also be entered into the national databases to create a Petunia EST database.
The creation of the petunia genomics resource will occur solely at the
University of Florida in the lab of Dr. Clark.
Objective 2:
To
create DNA microarrays to identify and characterize groups of genes involved in
flower senescence.
Most plant cells contain all of’ the same
genes, but not all genes are used in each cell all the time. Some genes are
turned on or expressed” only when needed. To understand the genetic or
molecular regulation of a biological process like senescence. we need to
identify which genes are expressed in senescing tissues. Where previously we
could identify and characterize only a few genes at a time, mieroarray
technology now allows us to look at many hundreds or thousands of genes at once
and to determine which are expressed in a particular tissue (i.e. petal
specific) or during a particular developmental stage (i.e. senescence specific).
Microarrays are simply ordered sets of DNA
molecules of known sequence identity (Lemieux et al., 1998). DNA molecules
representing thousands of genes can be spotted on a single glass microscope
slide or microarray (also called a DNA chip). In order to create these arrays we
produced a pooi of petunia genes that contains the genes involved in every
aspect of flower development. These genes have been isolated and will be spotted
onto the glass arrays by a robot (arrayer). Once microarrays are made. they can
he screened with comparative populations of mRNAs isolated from our tissues of
interest in order to identify those genes involved in senescence. In our first
experiments we will compare populations of mRNAs from nonsenescing and senescing
petals. We will use ethylene treatment as a means of accelerating and
coordinating the senescence process among many flowers. mRNA molecules isolated
from ethylene-treated and air-treated flowers can be differentially labeled with
fluorescent dyes and then visualized under a fluorescent microscope. For
example. we can tag our ethylene-treated mRNA with red dye and our air-treated
mRNA with green dye. The labeled RNA will stick only to its complementary DNA on
the chip (the gene that encodes it) and we will identify (by fluorescence) which
genes are turned on (or off) in senescing or nonsenescing petals. In our first
experiments we will look for: 1) Genes that are expressed in senescing petals
and not in nonsenescing; these are the genes that are involved in initiating
and! or coordinating senescence: 2) Genes that are on in non-senescing and off
in senescing petals; these genes could function as senescence repressors.
In many flowers including daylilies. senescence
is not regulated by ethylene and there is evidence that it is controlled by
other plant hormones like ABA (Panavas et al,. 1998). While petunias and
daylilies may have different hormone signals initiating senescence. the
senescence process is likely to be similar. For example, senescence-related
genes like the cysteine proteases have been found to be involved in the
senescence of ethylene sensitive and insensitive flowers
(Valpuesta et al.. 1995; Jones et al. 1995). In
order to determine which genes identified as senescence-related are regulated by
ethylene (ethylene response genes) and which ones are turned on during
senescence independent of ethylene. RNA from ethylene insensitive petunias (etrl-l)
will also be used to screen the microarrays. Genes expressed in senescing etrl-1
flowers will be compared to the genes expressed in ethylene-sensitive wild
type flowers, and senescence-regulated genes that are regulated by ethylene will
be separated from genes not regulated by ethylene.
Objective 3: To identify components of the
senescence signaling pathways and engineer the most appropriate genetic targets
for the manipulation of senescence.
The screening of DNA microarrays will help to
identify 10’s or 100’s of senescence-related genes, but this is only the
first step. These genes must be further characterized to determine more precise
patterns of temporal and spatial expression during the progression of
senescence. This will be done using RNA gel blots (northerns) or real-time
quantitative PCR (polymerase chain reaction). Temporal patterns of gene
expression will help to order these genes into a senescence signaling pathway
and help us determine which genes might be involved in the initiation versus the
execution of the senescence program. Spatial patterns of gene expression will
help identify which genes are flower or petal specific and which might be
involved in the senescence of vegetative as well as floral tissues. Ultimately
we will be able to use this knowledge to identify the best genetic targets to
delay senescence without affecting other developmental events.
The role of these genes in the regulation of
senescence will be confirmed by overexpressing them in transgenic petunia
plants. Genes identified as putative regulators or initiators of the senescence
process will be “knocked out” in the flower by ectopic expression of
an antisense copy of the gene, while those identified as putative senescence
repressors will be overexpressed in the sense orientation to create plants that
overproduce the repressor protein. Petunia transformation with Agrobacterium
tumefaciens is already a routine process in both of our labs, and will be
used to produce 30 to 50 independent T0 lines of all genetic
constructs. These plants will be grown to flowering and screened for a delayed
flower senescence phenotype. After selfing the T0 lines, the T1 plants will also
be screened for the phenotype of increased flower longevity to show that the
trait is heritable, thus proving that a given technology flower
senescence will be evaluated to determine their true commercial utility To
confirm that other important characteristics are not negatively impacted by the
genetic transformation event, time to flowering, flower number and size, leaf
senescence. fruit maturation. seed set, and adventitious rooting of cuttings
will be evaluated on all selected genetic lines.
Timetable:
The microarrays will be constructed and screened
in facilities at the UF. Microarrav data analysis. gene characterization and
transgenic plant production and evaluation will take place in Florida and Ohio.
Year 1: Develop clone and sequence
database. Construct DNA microarrays and screen microarrays for
senescence-related genes. Begin analysis of data from microarray screens and
characterization of genes.
Year 2: Continue to analyze data from
microarrays and finish characterization of genes of interest. Begin construction
of transgenic plants to confirm function of genes in senescence.
Year 3: Continue transforming petunias
with selected genes of interest and analyze transgenic plants for delayed flower
senescence and other aspects of horticultural performance.
Literature Cited
Borochov and Woodson, 1989. Hortic Rev. 11:1
5-43.
Clark et al., 1999. Plant Physiology 121:53-59.
Dervinis et al..1999. Proceed. Flor. State Hort
Soc. Annual Mtg.
Gubrium et al.. 2000. J. Amer. Soc. Hort. Sei.
125:277-28 1.
Jones et al.. 1995. Plant Mo!. Biol. 28:505-5 12.
Lemieux et al,. 1998. Mol Breeding 4:277-289.
Panavas et al., 1998. J. Exp Bot. 49:1987-1997.
Savin et al., 1995. Hortscience 30:970-972.
Schena et al.. 1995. Science 270:467-470.
Schena et al.. 1998. Trends Biotech. 16:301-306.
Valpuesta et al.. 1995. Plant Mol. Biol,
28:575-582.
Wilkinson et a!,. 1997. Nature Biotech.
15:444-447.
List of Materials and Supplies
PCR reagents for amplifying DNA to spot on
microarrays
Reagents for extracting mRNA from plant tissues
Fluorescent Dyes for labeling mRNAs for
microarray screening
Radioisotopes for screening northern blots
RealTime PCR reagents (kits) for gene expression
analysis
General molecular biology reagents for
cryostorage ostorage preservation (cloning supplies)
Plasticware for conducting high throughput
research (eg. 96-well plates. 384-well plates. etc.)
Maintenance contracts for microarrayer and
microarray reader
Multi-channel pipettors
