Transcriptome analysis and crop improvement
(A review)

JIM M DUNWELL¹, MARIA A MOYA-LEÓN², RAUL HERRERA^2*

1. School of Plant Sciences, The University of Reading, Reading RG6 6AS, U.K.
2. Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, 2 Norte 685, Talca, Chile

Corresponding Author: Raúl Herrera. Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, 2 Norte 685, Talca, Chile Tel: (56-71) 200280 Fax: (56-71) 200276 e-mail: raherre@pehuenche.utalca.cl

Received: April 17, 2001. In Revised form : October 9, 2001. Accepted : October 10, 2001

ABSTRACT

The identification and characterization of differential gene expression from tissues subjected to stress has gained much attention in plant research. The recognition of elements involved in the response to a particular stress enhances the possibility of promoting crop improvement through direct genetic modification. However, the performance of some of the `first generation' of transgenic plants with the incorporation of a single gene has not always been as expected. These results have stimulated the development of new transgenic constructions introducing more than one gene and capable of modifying complex pathways. Several techniques are available to conduct the analysis of gene regulation, with such information providing the basis for novel constructs specifically designed to modify metabolism. This review deals with techniques that allow the identification and characterization of differentially-expressed genes and the use of molecular pathway information to produce transgenic plants. (Biol Res 2001; 34 3-4: 153-164)

INTRODUCTION

In 1995, when the entire DNA genome sequence of the first self-replicating organism, Haemophilus influenzae, was described, the age of genomics was initiated. In the coming months and years the DNA sequences for different plant species will be delivered. In fact, within the last year the complete sequence of Arabidopsis thaliana has been published (Arabidopsis Genome Initiative, 2000), and that of rice has been reported by the commercial sector (see Butler and Pockley, 2000; Davenport, 2001; Dickson and Cyranoski, 2001; Dunwell, 2000a; Eckardt, 2000). In Latin America the first successfully sequenced organism has been the prokaryote Xylella fastidiosa (Simpson et al, 2000). This extensive knowledge, often generated from large-scale international projects, can be used in several ways, both directly and indirectly. Molecular biology provides several techniques to assess gene function, and indeed most efforts have been applied to the development of novel strategies to demonstrate the expression and translation of a particular gene. Transcript analysis (transcriptomics) and protein profiling (proteomics) are the most advanced strategies to provide an answer to the question:- How does the plasticity of gene expression respond to any particular external or developmental stimulus? The identification of genes that control economically important traits provides the basis for new progress in genetic improvement of crop species, complementing traditional methods based on assisted crosses. This issue has gained particular importance, since molecular biologists first used transgenic methods in crop improvement. Initially, single genes were introduced, but after a few generations some transgenics no longer expressed the acquired characteristic, thus indicating either a physical loss of the introduced gene or a loss in its expression. Similarly, even when transgenic plants carried an exogenous gene designed to provide protection against a particular pathogen, they remained susceptible because the response was not monogenic, as was initially believed. It was therefore essential in these cases to more fully determine the plant response to a particular biotic or abiotic stress.

The identification of candidate genes influencing any important trait can be approached through an analysis of complementary DNA (cDNA), copies of messenger RNA (mRNA). Devoid of intronic and intergenic sequences, whose biological significance is still obscure, these mRNAs represent only a small percentage of the total genome (about 1-3% in eukaryotes). However, they do contain valuable information on gene activity since they correspond to the proteins expressed in a specific tissue and responsible for the identity of that tissue. The formal identification of genes proceeds first by sequencing the expressed sequences tags (ESTs), partial sequences from either end of the complementary cDNA, and then by cloning the complete gene. Sequence information from ESTs can be used for deciphering the function and the organization of the genome, particularly if the strategies used have the goal of determining the order and time of gene expression, which represents another level of complexity in the analysis of biological organisms.

Analysis of Protein Profiles (Proteomics)

The study of the proteome (proteomics, Table I) has been extensively used in the analysis of mammalian tissues and to a lesser extent in plants (Rossignol, 2001). The use of this technique has provided evidence of a particular expression pattern within a specific cell or tissue and has made a major contribution in human and microbial functional genomics (Anderson and Anderson, 1998; Dutt and Lee, 2000). For example, differentially expressed proteins in melanoma cell lines and cancer cells have been described using 2-D protein gels (Robinson et al, 1998; McKerrow et al, 2000; Voss and Harberl, 2000); these protein profiles have been used to establish a tumor classification (Alaiya et al, 2000). Following the extensive development in mammals and bacteria, there has been an increasing analysis of protein profiles in plants (Thiellement et al, 1999; Van Wijk, 2000). The small number of studies include those on legume root nodules associated with nitrogen fixing bacteria (Natera et al, 2000; Panter et al, 2000); the lumenal and peripheral thylakoid membrane in pea (Peltier et al, 2000); organelles of Arabidopsis (Prime et al, 2000); the interconversion through a phosphorylation mechanism of two type of chloroplast RNA polymerase subunits (Pfannschmidt et al, 2000); leaf development (Komatsu et al, 1999) and defense responses in rice (Rakwal and Komatsu, 2000). In addition, the protein pattern from xylem subject to abiotic stress has also been characterized, and this fingerprinting is being used to establish a QTL map from a maritime pine species (Plomion et al, 2000). Apart from the specific advantage pointed out by Plomion et al. (2000) describing the presence of proteins not detected through transcript analysis, certain disadvantages are limiting the use of proteomics. These include the need for a more sensitive analytical system and the absence of an effective method for large-scale data comparison (Pandey and Mann, 2000). Despite these limitations, there have been significant advances in the application of proteome analysis to genome analysis, particularly in describing the expression products in human and bacteria (Banks et al, 2000; Legrain, et al, 2000; Asher, 2000; Parsons and Rodriguez-Tome, 2000).

Analysis of Transcript Profiles (Transcriptomics)

The analysis of transcripts expressed in different cell (transcriptomics) and tissues has been enormously facilitated by the development of Polymerase Chain Reaction (PCR). Several different strategies have been established to reveal expressed sequence tags (ESTs); their principles and applications will be discussed below. In general, the identification of a DNA encoded product is based on the synthesis of a copy DNA followed by PCR amplification.

A. Differential Display

Differential Display technique is based on the synthesis of cDNA, using an oligo dT primer (3') and an arbitrary oligonucleotide primer for the 5'-end (Liang and Pardee, 1992). This specific condition makes it difficult to conduct analysis using the available databases (Bachem et al, 1996). In an alternative protocol, arbitrary primers are used both for cDNA synthesis and PCR amplification (Welsh et al, 1992). Both variants of RNA fingerprinting require a low annealing temperature during PCR amplification in order to achieve visualizable products. Consequently, the quantity of individual amplification products is not only a function of the initial concentration of that cDNA, but also is dependent upon the quality of a particular match between primer and template (McClelland et al, 1995). As a result, abundant cDNAs with poor matches to the primers used are likely to outperform rare species with perfect matches during the course of PCR amplification. Despite this disadvantage, there are several reports of the description of genes expressed differentially in plants. These include the cloning of: the last enzyme (1 aminocyclopropane-1-carboxylate oxidase) involved in the synthesis of ethylene, a hormone that plays a role in fruit ripening and senescence in tomato (Barry et al, 1996); specific genes involved in the synthesis of flavonoids (Saito et al, 1999; Yamazaki et al, 1999); genes differentially expressed in carpel development (Yung et al, 1999) or in floral transition (Yu and Goh, 2000); modulators such as nitrate transporters concerned with plant/environment interaction (Filleur and Daniel-Vedele, 1999); genes regulated by the light photoreceptor, phytochrome (Kuno et al, 2000); and proteins involved in the response to abiotic stress (Brosche and Strid, 1999; Baldi et al, 1999; Kim et al, 2000). The technique has also been used to discriminate between individuals within a species (Lapopin et al, 1999; Ni et al, 2000).

B. cDNA-AFLP

This fingerprinting method relies on the selective amplification of a subset of DNA molecules from a more complex pool. The development of an amplified fragment length polymorphism (AFLP) provides a reliable tool to reveal the few differences between two close individuals (Vos et al, 1995). The system is based on the use of high stringency conditions, facilitated by adding double-stranded adaptors on the ends of restriction fragments, which serve as primer sites during amplification. Selective fragment amplification is achieved by adding one or more bases to the PCR primers which will only then be successfully extended if the complementary sequence is present in the fragment flanking the restriction site, thereby reducing the number of visualized bands. The advantage of this strategy to reveal DNA fingerprinting has been presented in numerous reports _ more than 260 in the last five years (PubMed database). Because of the advantage of this technique in generating a good individual polymorphism, cDNA has been used as template to amplify transcripts in plants. The use of any pair, of four and six base restriction enzymes, has been proposed as a method to generate differentially expressed products. In fact, a combination of Pst I/Mse I or Ase I/Taq I has been successfully applied (Bachem et al, 1996; Money et al, 1996, Habu et al, 1997). This method seems to be more efficient as a way to discriminate and identify a particular RNA fingerprint; the cloning and subsequent characterization of a specific band can easily be done, particularly if silver stain is used to develop the fingerprinting pattern (Dubos unpublished results). The description of several ESTs have been reported (Suarez et al, 2000; Durrant et al, 2000; Qin et al, 2000; Bachem et al, 2000). All these reports describe the modulation of biochemical pathways responding to different stimuli.

C. Microarray

Recently a DNA chip technology has been developed to study gene expression profiles (Schena et al, 1995). Such techniques are based on the capacity to bind either DNA fragments or previously characterized oligonucleotides on a microscope slide. The plates are built by depositing specific DNA fragments at indexed positions using a computer controlled high speed robot with the specific DNA fragment sequences determined from any database originated including: cDNA clones, EST clones, anonymous genomic clones or DNA amplified from open reading frames (ORFs). Today's technology can display 409,000 spots in an area of 1.28 cm² (Fodor, 1997). In theory, all 20,000-25,000 Arabidopsis genes could be displayed on a single slide (Kehoe et al, 1999). In fact, a slide with the Arabidopsis cDNA has been prepared for a large transcript analysis (Deprez et al, 1998). The disposition of genes from the conifer Pinus taeda has also been prepared for use in physiological and molecular studies for wood formation (Sederoff R., personal communication). The technique is very sensitive, detecting mRNAs at level of 1/100,000 or 1/500,000 (Gerhold et al, 1999). Despite the advantage of this precise technique, several problems have arisen and are expected to be resolved in the near future, including the high cost, identifying appropriate software for analysis of results, and standardizing methods to allow comparison between results from different labs in light of the fact that in some systems the slide can be used only once (for review, see Richmond and Somerville, 2000; Van Hal et al, 2000). Studies of genetic expression patterns have been done principally in humans and yeast. The determination of differentially expressed genes in cancer cells has been the primary goal of array efforts ("Transcriptome 2000: From functional genomics to systems biology," a conference held in the Pasteur Institute, Paris). Results from Saccharomyces cerevisiae include a large study on the modulation of differentially expressed genes involved in carbohydrate metabolism; 6,400 different genes were analyzed simultaneously on a slide with the data providing valuable conclusions about the method of regulation of anaerobic respiration (DeRisi et al, 1997). Recent examples of such technology applied to plants include those on Arabidopsis (Ruan et al. 1998; Maleck et al. 2000; Petersen et al, 2000; Reymond et al, 2000; Seki et al, 2001), soybean and maize (McGonigle et al. 2000), lima bean (Arimura et al. 2000), and strawberry (Aharoni et al. 2000).

D. Serial Analysis of Gene Expression (SAGE)

Among the various techniques used to assess transcript abundance, the most powerful is probably SAGE. This technique (Powell 2000; Velculescu et al, 2000), invented first to quantify gene expression in yeast (Velculescu et al. 1995;1997), comprises the production of a short 10-14 nucleotide tag, with each tag representing a unique transcript present in a cell (all possible combination of 4 bases in a 10 bp sequence, 4¹⁰ , gives more than 1 million different sequences). Determination of the sequence of a tag allows identification of the corresponding gene (Chen et al, 2000; Van den Berg et al, 1999), and the frequency of a tag represents the steady state level of the mRNA from which it was derived. The unique advantages of SAGE over alternative techniques for transcript analysis are: high sensitivity, threshold detection is one transcript in three cells (Ishii et al, 2000); scalability, the technique can be used on any size of sample, from a few cells upwards; detection of all genes, including those of unknown function; avoidance of amplification bias; the data are digital and not derived (eg from an analogue fluorescent signal); the data are immortal and can be used at any time in a comparative study; data sets generated by one lab can be related directly to those produced by another.

These advantages have led to a rapid expansion of interest in using the method in a wide range of disease (Zhang et al. 1997) and developmental studies, almost all in humans (Caron et al, 2001; Lal et al, 1999; Lash et al, 2000). For example, in the last 18 months alone there have been approximately 50 published studies dealing with gene expression in cancers of the ovary, thyroid, skin, brain, bladder, esophagus, stomach, colon, breast (Nacht et al, 1999) and prostate, together with developmental studies on liver, kidney, muscle, skin, blood, nerve, bone, retina, and oocytes. Such digital data sets are amenable to statistical analysis (Larsson et al, 2000; Lee et al, 2000; Man et al, 2000; Margulies and Innis, 2000; Van Kampen et al, 2000), and much of the data from these and other studies are now available in a SAGE site at the NCBI (see http://www.ncbi.nlm.nih.gov/SAGE/) sponsored by the US National Cancer Institute (see also http://www.sagenet.org/). It is estimated that more than 200 medical research groups are now using SAGE methods for transcript profiling. To date, however, this expansion of activity has not been reflected in the plant research community; with the exception of a single limited study (Matsumura et al, 1999) there are no published SAGE data available for any plant species. The vast majority of plant scientists interested in large-scale gene expression studies (reviewed in Schaffer et al, 2000) seem to have only considered array methodology (see above).

Applications to Transgenic Crop Improvement

All the information provided by the techniques described above can be used to investigate appropriate targets for modification by transgenic methods. The main objective of this strategy is to improve the quality traits of a particular crop intended to provide direct consumer benefit. The public resistance against using `first generation' genetically modified organisms (GMO) has stimulated the development of GMOs that avoid the use of antibiotic markers. The so-called `second generation' GM products (Dunwell, 1999, 2000b) are currently in the research and development stage and may follow the first generation examples, which are primarily herbicides and insect-resistant products. Many such products are already undergoing field-testing in the USA, and there seems little doubt that in the future, many GM crops will include changes identified from transcriptome analysis. As an example of progress in this field, we focus on the new approaches to improve the resistance of crops to biotic and abiotic stress.

Response to stress

There are many proposed transgenic routes for the improvement of stress-related responses in plants (Winicov, 1998; Chrispeels et al, 1999; Duncan and Carrow, 1999; Bajaj et al, 1999; Barkla et al, 1999; Nuccio et al, 1999; Cushman and Bohnert, 2000; Hasegawa et al, 2000; Zhang et al, 2000). Of the stress-related osmoprotectant compounds (Rathinasabapathi, 2000), the three most extensively studied are probably glycinebetaine (Huang et al, 2000; McNeil et al, 2000; Sakamoto and Murata, 2000), the sugar alcohol mannitol (Adams et al, 1998; Loescher et al, 2000) and trehalose (Londesborough et al, 2000), a sugar known to play a role in drought resistance of many organisms including the `resurrection' plant. In bacteria this sugar is produced by the action of the two enzymes, trehalose phosphate synthase, which produces trehalose phosphate, and trehalose phosphatase, which degrades T-6-P into trehalose. When one or both of these two enzymes are expressed in plants, the transgenics have larger leaves, altered stem growth, and improved response to stress (Goodijn et al, 1997; Pilon-Smits et al, 1998; Yeo et al, 2000). For example, when two selected transgenic tobacco plants grew under drought stress, their total dry weights were 28% and 39% higher than the controls. Chlorophyll fluorescence measurements showed a more efficient photosynthesis in the transgenic under these stress conditions. More recently (Goodijn et al, 1998), it has been claimed that similarly beneficial results can be achieved by modifying T-6-P via inhibition of endogenous trehalase _ an enzyme that hydrolyses trehalose into two glucose moieties. Transgenic tomato lines with various levels of trehalose are now being field tested.

Additionally, overexpression of the yeast HAL1 gene in tomato has been shown to have a positive effect on salt tolerance by maintaining a high internal K⁺ concentration and decreasing intracellular Na⁺ during salt stress (Gisbert et al, 2000), and production of fructans in transgenic plants has also improved their stress resistance (Park et al, 1999; Pilon-Smits et al, 1999).

Overexpression of various glutamine synthases (Hoshida et al, 2000) or glutamate dehydrogenase (GDH) is also claimed to improve growth and stress tolerance. Specifically, plants have been transformed with genes encoding the a- and ß-subunits of the chloroplast-located GDH from the alga Chlorella sorokiniana (Schmidt and Miller, 1999). An alternative approach concerns the introduction of the uridine diphosphate glucose pyrophosphorylase gene from the bacterium Acetobacter xylinum (Ellis et al, 1998). These transgenics, which have modified concentrations of cellulose precursors, are claimed to have increased growth rates and yield and improved response to stress conditions. Similar improvements in performance are reported for tobacco plants overexpressing a cell wall peroxidase (Amaya et al, 1999), and wheat (Sivamani et al, 2000) or rice (Wu and Ho, 1999) plants transformed with the barley late embryogenesis (LEA) gene. Modification of calcium related proteins is another method claimed to improve crop performance. For example, the introduction of a protein kinase domain-containing gene, a calcium dependent protein kinase gene (Saijo et al, 2000), or a calcium/calmodulin-dependent gene, are all reported to be beneficial (Sheen, 1998). The most recent approach of this type is that involving the introduction of functional calcineurin activity as means of improving a salinity tolerant plant (Pardo et al, 1999).

Another recently published claim (McCourt et al, 1999) is that introducing a gene-encoding plant farnesyltransferase (Pei et al, 1998) and enzyme inhibitors will enhance drought tolerance, improve resistance to senescence and modify growth habit when expressed in plants.

Two other related strategies are based on the premise that many of the deleterious effects of stress are mediated through the accumulation of reactive oxygen species (Tsugane et al, 1999). First, it has been suggested that the accumulation of these radicals is dependent on the presence of free iron in the cell, and consequently it has been proposed that controlling the amount of free iron could reduce oxidative damage. Convincing evidence of the value of this strategy was shown recently in a study of transgenic tobacco expressing the alfalfa ferritin, an iron-binding protein (Deák et al, 1999). The second approach of this type is the recent claim (Altier et al, 1999) that plants may be protected from the effects of stress-induced reactive oxygen species by the introduction of a transgene encoding an enzyme such as barley germin, an enzyme with oxalate oxidase (Dunwell et al, 2000c) and superoxide dismutase (SOD) activity (Woo et al, 2000) that generates hydrogen peroxide. This molecule is known to stimulate the plant's endogenous defenses, for example by the induction of PR proteins. Similar beneficial effects from modifying the levels of various SOD enzymes have been reported from other species (Van Breusegem et al, 1999; Kwak et al, 2000; McKersie et al, 2000). A specific heat shock protein (HSP 101) has recently been implicated in thermal stress responses in Arabidopsis (Queitsch et al, 2000), whereas various proteins including choline oxidase (Sakamoto et al, 2000) and antifreeze proteins (Griffith, 1999) are claimed to be useful in protecting against low temperature stress.

An additional recent method for providing general non-specific protection (Winicov and Bastola, 1999) by up-regulating or pre-activating an existing defense pathway is exemplified by the introduction of a gene encoding the DREB1A (dehydration response element B 1A) transcription factor from Arabidopsis (Kasuga et al, 1999). This factor is induced by a range of stresses and its introduction into transgenic Arabidopsis under the control of various promoters improved the tolerance to stress. The best results were achieved with transgenic expressing the gene under the control of the stress inducible promoter rd29A. Such transgenics showed better survival than the controls when exposed to salt, freezing and drought. The only field trial of material claiming to have salt tolerance is that conducted on Agrostis containing a betaine aldehyde dehydrogenase gene (Application number 98-103-24N).

Apart from the transgenic approach used in salt tolerance, the recognition of a particular marker can be used in direct selection on a specific progeny. Both strategies require basic information before selecting the way to make an improved crop.

ACKNOWLEDGEMENTS

The authors would like to thank the British Council, the Biotechnology and Biological Sciences Research Council, Syngenta and CONICYT (International Liaison Program) for their support.

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