Using transgenic modulation of protein synthesis and accumulation to probe protein signaling networks in Arabidopsis thaliana

Abstract

Deployment of new model species in the plant biology community requires the development and/or improvement of numerous genetic tools. Sequencing of the Arabidopsis thaliana genome opened up a new challenge of assigning biological function to each gene. As many genes exhibit spatiotemporal or other conditional regulation of biological processes, probing for gene function necessitates applications that can be geared toward temporal, spatial and quantitative functional analysis in vivo. The continuing quest to establish new platforms to examine plant gene function has resulted in the availability of numerous genomic and proteomic tools. Classical and more recent genome-wide experimental approaches include conventional mutagenesis, tagged DNA insertional mutagenesis, ectopic expression of transgenes, activation tagging, RNA interference and two-component transactivation systems. The utilization of these molecular tools has resulted in conclusive evidence for the existence of many genes, and expanded knowledge on gene structure and function. This review covers several molecular tools that have become increasingly useful in basic plant research. We discuss their advantages and limitations for probing cellular protein function while emphasizing the contributions made to lay the fundamental groundwork for genetic manipulation of crops using plant biotechnology.

Overview

Analyses of numerous genetically modified plants generated in recent years have greatly expanded our knowledge of physiological processes and gene regulation mechanisms. Efficient tools for forward and reverse genetics are invaluable for determining protein function. One vital module in these molecular tools is the ability to carry out targeted analyses of gene function. Emergence of a new model species for basic research requires the development and/or improvement of a myriad of genetic and genomic tools and one model plant, Arabidopsis thaliana, has been utilized widely in basic research towards understanding biological and biochemical functions. In the past three decades, the Arabidopsis genetic system has undergone extensive manipulation to aid researchers in better understanding protein function and dissecting complex signaling networks. Arabidopsis has proven to be an exceptional model plant to study gene and/or protein function. A tractable genome and short generation time are two of the features that make it useful for basic plant research. Considering the ongoing quest to identify the biological function of all of the genes of Arabidopsis, in this review we collectively assess some of the molecular tools that are commonly used in determining functions of effectors in complex protein signaling networks.

Approaches to study plant gene function.

The availability of efficient and cost-effective genetic tools that are applicable to different plant species is essential for functional gene analysis. As a result of the Arabidopsis Genome Sequencing Project, the daunting task of determining the functions of all genes in Arabidopsis was initiated. Genomic and proteomic tools are useful for the initial step in genome-wide screening for mutants and selection of genes with desirable traits for subsequent application of knowledge to other plant species. Therefore, mutant analyses, ectopic expression, mis-expression, overexpression and RNA interference (RNAi), have become effective tools to study numerous biological functions of proteins in planta.

A common genetic approach to the study of protein function is mutational analysis. Classic loss-of-function mutations generated through random mutagenesis mediated by chemical mutagens and radiation aid in dissecting gene functions in genetic pathways. However, a handicap of loss-of-function mutant screens is that they are not effective in identifying functionally redundant genes or genes whose loss of function results in early embryonic or gametophytic lethality.¹ A more recent tool in mutational analysis is the construction of DNA insertion mutants. This approach requires transgenic generation of populations of DNA insertion mutants, mapping the disrupted genes through screening, characterizing the mutations and cloning the target genes.² A limitation of classic and DNA insertion loss-of-function mutants is that they can reveal only global effects of gene function.

Constitutive mis-expression or overexpression of transgenes has been successful for elucidating gene function in many species, but not all species can be transformed, nor can all genes be expressed in a constitutive fashion in planta. Examples are genes encoding proteins with highly detrimental or lethal dominant-negative phenotypes that lead to drastic changes in crucial metabolic pathways leading to growth and developmental defects (reviewed in ref. 3). Transfer-DNA (T-DNA) mutagenesis is frequently associated with loss-of-function or hypomorphic mutations. However, T-DNA mutagenesis can be manipulated to generate gain-of-function alleles by activation tagging. Commonly, T-DNA vectors that contain multiple copies of transcriptional enhancers from the cauliflower mosaic virus (CaMV) 35S gene have been constructed to transform Arabidopsis plants through Agrobacterium-mediated transformation.⁴ Upon integration into the genome, the enhancers stimulate the transcription of a gene and cause its ectopic expression.¹ Through activation tagging, novel gene functions have been discovered, even though constitutive mis-expression has the drawback of not being able to limit transgene expression to discrete tissues, organs or developmental stages.⁵ In particular, the mis-expression of genes that cause lethality or strong pleiotropic effects cannot be accomplished through activation tagging.⁶ Another concern of activation tagging is whether the gain-of-function phenotype of a mutant reflects the normal biological role of a gene. Notably, the CaMV 35S enhancers can lead to enhancement of endogenous gene expression and the resultant phenotype, as opposed to ectopic overexpression or mis-expression driven by native promoters, which would be more likely to reflect the normal role of the activated gene.¹

Transgenic approaches such as overexpression, ectopic expression, mis-expression and RNAi have become effective strategies in functional genomics and proteomics in Arabidopsis. These technologies can be implemented easily for a single gene using specific constructs that direct its down or upregulation under constitutive, tissue-specific or inducible conditions.⁶ Developmental processes and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Most systems that manipulate gene expression allow control in one dimension, space or time. Molecular tools that allow spatial and/or temporal control of gene expression are vital for the accurate elucidation of gene and protein function. Furthermore, to understand the functions of proteins encoded by critical genes, which if mutated could be severely detrimental to plant growth and survival, one solution has been to use tissue- and/or organ-specific promoters to limit the expression and the activity of transgenes to particular tissues or organs. Such systems that are currently available have advantages as well as limitations. One limitation is that tissue-and organ-specific promoters are active during the process of regenerating transgenic plants. Tissue-specific expression also restricts the scope of the analysis to a few cell types and does not always allow the appropriate tissues to be studied.⁷ In cases where promoters respond to external inducers (i.e., inducible promoters) the expression of the transgenes can be controlled temporally and/or spatially. Techniques such as those described above require establishment of stable and independent transgenic lines whose growth and propagation impose extensive time, labor and space requirements. Development of enhancer trap and two-component transactivation systems has allowed more carefully controlled expression and has been used to overcome some of the limitations of spatial-specific, promoter-driven mis-expression strategies.

Advances in inducible mis-expression technologies, spatial and temporal regulation of transgenes and RNAi are anticipated to facilitate more fine-tuned functional analyses of endogenous and exogenous genes, revealing new roles for genes that act at multiple stages in the plant life cycle. Such analyses are expected to assist the development of new, improved crop varieties, as plant biotechnology depends on the manipulation of genomes to induce desirable traits, while mitigating detrimental traits, in commercial crops. Basic research on Arabidopsis promotes our understanding of the molecular and cellular bases of complex protein signaling networks; this review covers some examples, advantages and limitations of spatial-specific and inducible mis-expression systems, RNAi, enhancer trap-induced gene expression and twocomponent transactivation systems that have increasingly gained popularity and efficacy in recent years (Table 1).

Table 1.

Experimental approaches for modulating gene expression and protein accumulation in planta

Approach	Utility	Advantages	Disadvantages
Mutagen-induced mutagenesis	Induced mutations using mutagens, Including chemicals or radiation	Allows recovery of point mutations (i.e., missense or nonsense), which can provide important insight into gene function; Can result in dominant-negative mutations, which have proven useful for novel insight into gene function; Useful for all species, including those that cannot be transformed.	Mapping untagged mutations is laborious; Can result in multiple genetic mutations in a single individual making it difficult to definitively associate disrupted gene function with specific phenotypes.
T-DNA insertional mutagenesis	Random insertion of transfer DNA (T-DNA) resulting in tagged insertional mutants	Gene tagging with known insertion sequence allows easier recovery of disrupted genes; If insertion sequence has a selectable marker, single insertion mutants can be identified.	Not useful for inactivating multiple, tandemly arrayed genes; Not useful for studying genes of essential function; Cannot be used for species that cannot be transformed.
Activation tagging	Randomized genomic insertion of transcriptional enhancers resulting in ectopic gene expression	Allows analyses of gain-of-function phenotypes; Genes are tagged and thus easier to identify.	Gain-of-function phenotypes associated with activation tagging may not reflect normal biological gene function; Not useful for lethal genes or those associated with strong pleiotropic effects; Cannot be used for species that cannot be transformed.
Constitutive gene expression	Constitutive mis- or overexpression of genes of interest	Allows complementation studies using null mutants; Allows gain-of-function analyses.	May not reflect normal biological function; Not useful for lethal genes or those associated with strong pleiotropic effects; Cannot be used for species that cannot be transformed; Can result in co-suppression of some genes.
Promoter-driven gene expression	Using tissue- or organ-specific promoters, or inducible promoters, selective expression of transgenes can be accomplished	Allows spatial- or temporal-specific expression or expression at specific developmental stages; Allows inducible expression, which can be used for the study of lethal genes.	Use of specific promoters is limited to isolated and characterized promoters; Requires generation, isolation and characterization of several independent transformants; Cannot be used for species that cannot be transformed.
RNA Interference (RNAi)-mediated inhibition of gene expression	Constitutive or selective suppression of gene expression based on sequence-specific RNA targeting and turnover	Allows robust suppression of gene expression in cell culture or in whole organisms; Can be useful for suppression of multiple, tandemly arrayed genes or genes closely related in sequence.	Can result in suppression of closely related genes; Cannot be used for species that cannot be transformed.
Enhancer trap-driven gene expression	Random insertion of a transgene with a minimal promoter results in expression of a gene of interest driven by endogenous genomic enhancers	Allows spatial- or temporal-specific gene expression that depends on genomic enhancers rather than isolated, characterized promoters.	Cannot be used for species that cannot be transformed.
Two-component transactivation systems (e.g., GAL4-UAS or pOp6/LhG4 systems)	Random insertion of a promoter-driven transgene or a transgene with a minimal promoter driven by endogenous genomic enhancers results in expression of a transcriptional activator that in turn drives expression of a target gene in progeny resulting from a cross between activator line and a line carrying a transactivatable target gene	Allows silent maintenance of a target transgene until expression is activated by the combined action of two distinct components; Can be used for temporal- and/or spatial-specific or inducible control of gene expression.	Cannot be used for species that cannot be transformed.

Tissue- and Organ-Specific, Promoter-Driven Gene Expression

Tissue- and organ-specific promoters enable researchers to drive the expression of a transgene in a distinct subset of cells, tissues or organs. Spatial-specific regulation of transgene expression has been successfully utilized in many studies that were aimed at further understanding the functions of proteins during the life cycle of Arabidopsis. Proper onset and fine tuning of developmental transitions and adaptive processes requires detection of external, environmental cues. For example, plants detect the presence or absence of light, but also the spectral quality, quantity, directionality and periodicity (reviewed in ref. 8). Plants sense and adapt to light through light-absorbing molecules called photoreceptors (reviewed in ref. 8–11). Through photoreceptor mutant analyses, it is known that far red-light-absorbing phytochrome A and blue light-absorbing cryptochrome 2 (cry2) are involved in perceiving long-day photoperiods and thus control flowering (reviewed in ref. 8). Although promoter-fusion studies indicate that these photoreceptors are found in tissues and organs throughout plants,¹² a wealth of prior studies has confirmed that light perception in leaves is associated with the photoperiodic induction of flowering (reviewed in ref. 13). Transgenic Arabidopsis lines where the CRY2-GFP gene fusion was expressed under the control of organor tissue-specific promoters (e.g., CAB, mesophyll specific; SUC2, vascular bundle specific; ML1 and CER6, epidermis specific) in a cry2-deficient mutant background have been used to study the site of light perception by cry2 at the molecular level in the regulation of flowering.¹⁴ Only cry2-GFP accumulation in vascular bundles was able to rescue the late flowering phenotype of a cry2 mutant, suggesting that the site of cry2 photoperception that regulates flowering is localized in vascular bundles. Phytochromes are red- and far red-light-absorbing photoreceptors.¹⁵ Studies with stable transgenic lines displaying mesophyll-specific and meristem-specific phytochrome-chromophore deficiencies have revealed that localized pools of phytochromes can regulate distinct physiological responses and established the efficacy of a novel tissue- and organ-specific, promoter-based molecular technique to investigate sites of light perception.¹⁶

Additional studies investigating the tissue-specific function of specific proteins that impact light-dependent growth and development in Arabidopsis have also emerged.¹⁷ Tissue-specific expression of SPA1, a gene that encodes a repressor of photomorphogenesis, indicated that SPA1 has distinct functions in discrete tissues.¹⁷ In Arabidopsis, phloem-specific SPA1 functions in the regulation of etiolation in dark-grown seedlings and in the regulation of photoperiodic flowering in light-grown plants.¹⁷ By comparison, both phloem- and mesophyll-localized expression of SPA1 is required to regulate light-dependent leaf expansion.¹⁷

During development, leaves of dicotyledonous plants undergo a gradual transition from sink to source status; therefore, establishing and maintaining phloem pressure is central to plant growth.¹⁸ AtSUC2 in Arabidopsis (1) encodes a phloem-localized sucrose/proton symporter involved in photoassimilate transport from source to sink tissues, (2) plays a central role in coordinating the demands of sink tissues with the output capacity of source leaves and (3) is active in maintaining phloem hydrostatic pressure during changes in plant-water balance.^18,19 To elucidate the role of AtSUC2 in whole plant carbon partitioning, the promoter of the GALACTINOL SYNTHASE gene from Cucumis melo was fused to AtSUC2 cDNA to target its expression to collection phloem (allows the entry of nutrients to the phloem network in source tissues) in an Atsuc2 mutant background.¹⁹ The results from this report suggested that AtSUC2 is not required for efflux in the transport and release of phloem, but its retrieval function (acceptance of nutrients by recipient cells) likely participates in fine tuning whole-plant carbon partitioning.¹⁹ Because the expression of AtSUC2 and activity of AtSUC2 are regulated, both positively and negatively, by developmental (sink to source transition) and environmental cues (i.e., light, diurnal changes, photoassimilate levels, turgor pressure, drought and osmotic stress and hormones), in a follow-up study to further understand its regulation, AtSUC2 was expressed under the regulatory control of two phloem-specific promoters [a promoter element from Commelina Yellow Mottle Virus (CoYMV) and rolC promoter from Agrobacterium rhizogenes] in an Atsuc2 mutant background.¹⁸ Conclusions from this study suggest that (1) strong, phloem-localized AtSUC2 expression is sufficient for efficient photoassimilate transport and (2) expressing AtSUC2 from promoters that aid in efficient phloem transport but are subject to regulatory cascades different from the endogenous sucrose/proton symporter genes has implications for biotechnology, especially in manipulating carbon partitioning as desired.¹⁸

Even though targeted expression of transgenes using tissue- and organ-specific promoters has been promising in providing additional insight into protein function, a current limitation is the availability of cloned and characterized promoters that can direct gene expression in a targeted manner. The use of tissue-specific promoters across species, even those that are closely related, can impose changes or loss of transgene expression due to the lack of essential trans-acting factors in the recipient plant species. The specificity of transgene expression also could be lost due to the presence of novel trans-acting factors that interact with the promoter cis-elements.² Moreover, this tool requires the establishment of stable, independent transgenic lines whose growth, propagation and analyses impose extensive time, labor and space requirements. Some of the aforementioned limitations can be overcome through inducible systems, which add another dimension to the regulation of transgene expression, allowing temporal and quantitative control of transferred genes in vivo. Induction systems with spatial, temporal and quantitative control of transgenes are applicable in many areas of basic and applied biology, including the study of gene function, cell lineage ablation experiments, enhanced synthesis of recombinant proteins and expression of commercially valuable traits.²⁰

Inducible Promoter-Driven Gene Expression

Use of promoters that respond to inducers allows fine-tuned manipulation of transgene expression. This molecular technique has become an attractive tool to maintain a transgene silently in the absence of the inducer, allowing transgene expression to be regulated by the presence of a specific, inductive signal under defined conditions. With appropriate chemical inducers (i.e., tetracycline, dexamethasone, 17-β-oestradiol and ethanol) and/or physiological cues (i.e., heat or cold), transgene activity can be controlled spatially, limiting the expression to particular cells, tissues or organs of interest; temporally, determining the effects at distinct developmental stages; and quantitatively, analyzing gene dosage responses in complementation experiments (reviewed in ref. 3).

Inducible expression systems rely on either endogenous or exogenous elements to control transgene activity; the ideal system should have very low activity in the absence of the inducer. Activity of the system should increase and decrease quickly and significantly in direct correlation with the amount of inducer to which the system is exposed, in vivo as well as in vitro, and with neither pleiotropic effects on endogenous gene expression nor toxic effects on plant metabolism.²⁰ Because most if not all genes in plants respond to external (i.e., heat, cold, light, wounding) and internal (i.e., hormones) cues that affect gene expression, such cues may not serve as the best candidates in vivo. Regulatory sequences that are responsive to chemical treatment, such as promoters and enhancers, are attractive because techniques based on such sequences depend upon the cloning of the responsive promoter upstream of the coding region of the gene of interest. However, a disadvantage of this approach is that native genes controlled by these regulatory sequences may also be induced upon addition of the chemical regulator.²⁰ Thus, it is important to choose an inducer that affects a set of genes that does not interfere with normal growth and development. Notably, four such chemical types—elicitors, safeners, wound signals and compounds that activate genes in systemic acquired resistance—have been widely used in the construction of inducible expression systems in planta.²⁰ Promoters that respond to otherwise inactive chemical inducers²⁰ have added to the molecular tools available for analyzing gene and protein function in vivo and manipulating desirable traits in plants, including crops.

In plant biotechnological applications, most commercially important crops, as well as non-crop plants, are regenerated via somatic embryogenesis,⁵ which is useful for mass asexual propagation or somatic cloning in plants. The underlying signal transduction pathway—i.e., the molecular mechanism involved in the transition of a vegetative cell to an embryogenic competent cell—remains largely unexplored.²¹ A study designed to gain understanding of the complex molecular process of induction of somatic embryogenesis in Arabidopsis is based on an estradiol-inducible system. WUSCHEL (WUS) is essential for meristem identity of shoot and floral meristems, i.e., to maintain their structural and functional integrity,²² and also induces shoot stem cell activity and developmental plasticity in the root meristem.²¹ To dissect the signaling pathway active during somatic embryogenesis, an estradiol-inducible XVE-WUS transgene was expressed in wildtype Arabidopsis plants. These transgenic plants are phenocopies of pga6-1 and pga6-2 gain-of-function mutant phenotypes having a high vegetative-to-embryogenic transition, which suggests that WUS and newly identified Plant Growth Activator 6 (PGA6) play a key role during embryogenesis, presumably by promoting the vegetative-to-embryogenic transition and/or maintaining the identity of the embryonic stem cells.⁵ In the event that a gain-of-function mutation causes growth defects or seedling lethality, the use of an inducible promoter-driven mis-expression system has an added advantage, i.e., allowing transgene activation upon application of the inducer at a specific time and elimination of its activity once the inducer is removed.²³

An ethanol-inducible gene switch system has been used to study carbon metabolism²³ and stomatal development²⁴ based on the alc regulon of the fungus Aspergillus nidulans. Several factors made this system promising: no plant transcription factor was known to interfere at the alcA promoter, levels of natural ethanol in the plants are extremely low, and ethanol is a small organic inducer with relatively low phytotoxicity.²³ These attributes have made the use of this inducible system favorable for many different plant species, including Arabidopsis,²⁵ Tobacco,²³ tomato,²⁶ Populus²⁷ and Catharanthus roseus.²⁸ However, the ethanol-inducible system is limited by the volatile nature of the inducer, which can cause unwanted gene activation in adjacent plants, phytotoxicity on the induced plant, and activation by endogenous inducers under low-oxygen conditions.²⁹ These drawbacks complicate the use of an ethanol-inducible system to produce distinct sectors of induced and uninduced gene expression that are essential to determine the cell autonomy of a phenotype. Some other inducible systems, based on nonvolatile inducers, have proven to be leaky²³ or are unable to activate accurately inducer-dependent T-DNAs randomly inserted in the genome.⁶

Another option to overcome the above limitations is the use of heat-shock promoters that can achieve relatively high-level expression in many cell types. In a study of gene function in epidermal cell polarity, a heat shock-inducible promoter from the Arabidopsis HSP18.2 gene was fused to a gene encoding ADP-ribosylation factor1 (ARF1).³⁰ Six identical ARF1 genes are ubiquitously expressed and single loss-of-function mutants in these genes reveal no obvious developmental phenotypes due to gene redundancy, thereby precluding conventional genetic dissection of ARF1 functions.³⁰ However, a study using transgenic Arabidopsis lines expressing heat shock-inducible, promoter-driven ARF1 reported a successful dissection of downstream signaling networks involved in local and cell-specific aspects of epidermal cell polarity.³⁰ Another study reported the use of a heat shock-inducible construct to study the function of the ACL gene in internode elongation in Arabidopsis.³¹ Even though heatshock promoters have been successful in many studies, one of the disadvantages is that subsequent analyses have to be performed at a temperature around 37°C and plants can be subjected to heat stress. Therefore, carefully controlled growth conditions become a necessity. Another general concern of heat-shock inducible systems is induction of endogenous heat shock proteins or related endogenous responses that could potentially complicate analysis of results.

The best-characterized, plant-based inducible system involves a tetracycline-inducible promoter developed for tobacco (that provides temporal regulation of gene expression in tobacco cell cultures³²). This system has been successfully utilized with cell culture, though its usefulness and reliability for spatial control of gene expression in the various tissues of whole plants is less clear.⁷ It appears that the tetracycline chemical switch is not effective in Arabidopsis and is leaky when the genes of interest are lethal in planta.²³ A few studies have combined the benefits of tissuespecific promoters with the use of inducers to drive inducible, tissue-specific gene expression (reviewed in ref. 33). Despite the efficient use of targeted transgene expression with spatial-specific and inducible promoters in Arabidopsis, they are not entirely adequate in some studies. The reason is that the tool is relatively inefficient (high background and/or only modest induction), depends on sustained gene repression or relies on the application of chemical inducers at concentrations that may be toxic to plants.²⁰ In addition to mutational analyses and expression of transgenes, an RNA-based reverse genetics approach is currently in use for studies of protein and gene function in plants.

RNAi Interference

RNAi is a remarkable experimental tool that regulates gene expression based on sequence-specific targeting and turnover of RNA molecules, which would otherwise be translated to produce protein. RNA silencing was first observed in transgenic Petunia plants as cosuppression,³⁴ leading to silencing of expression from integrated genes and restricting molecular applications that necessitated high-level transgene expression. With an increased understanding of the molecular basis of RNAi, it has become a plant engineering tool with enormous potential to control gene expression. Several mechanisms result in RNAi, and sequence specificity of RNAi-based gene activation allows silencing of individual genes as well as several genes simultaneously.^35,36 These properties make it an invaluable technique in functional genomics and in gene function validation. Silencing can be achieved constitutively or in a targeted manner, with the latter being very useful in determining gene function. Successful reports on tissue-specific and/or cell type-specific RNAi have been presented in many model organisms, namely, in Caenorhabditis elegans,³⁷ mice,³⁸ Drosophila,³⁹ Arabidopsis,^40,41 and Brassica napus.⁴⁰

Some recent studies have been focused on manipulating floral organs to improve photosynthetic efficiency.⁴² During analyses of flowering time in B. napus, researchers determined that bright-yellow flowers significantly reduce the amount of photosynthetically active radiation that reaches the leaf canopy, thereby accelerating leaf and bract senescence.⁴⁰ To gain better understanding of genes associated with floral organ identity and heritability of floral organ variants, gene silencing technology was implemented to silence B-type MADS-box floral identity genes in a second whorl-specific manner in Arabidopsis and B. napus.⁴⁰ The results from this study indicate that transgenic plants have the ability to produce male fertile flowers, which exhibit conversion of petals to sepals in Arabidopsis and petals to sepaloid petals in B. napus.⁴⁰ Because organ conversion is preferable over the removal of petals to avoid interfering with insect pollination, this study confirmed that organ-specific silencing of B-type MADS-box genes can result in novel floral organ variants in both species that were stable in subsequent generations,⁴⁰ revealing a strategy to manipulate organ identity in a desirable manner.

To achieve temporal and quantitative regulation of RNAi, RNA-mediated silencing can be chemically induced. Inducible RNAi systems maintain the silencing construct inactively in the absence of the inducer to avoid spurious gene silencing and, upon inducer addition, silencing is induced rapidly across the whole plant or tissues of interest to achieve repression of target genes. An important feature of such a system is reversibility after the removal of the inducer.⁴¹ An inducible RNAi system has been constructed using a dexamethasone-inducible pOp6/LhG4 promoter⁴³ and pHELLSGATE GATEWAY™ gene silencing vectors.⁴¹ In the latter report, stable transgenic lines with the inducible gene silencing system have displayed efficient silencing of two genes encoding phytoene desaturase and luciferase, and recovery of transcript levels was observed upon removal of the inducers. Heat shock-⁴⁴ and ethanol-inducible⁴⁵ RNAi technologies have also been described in plants. Inducible RNA-based silencing opens the possibility for knocking down the expression of gene products at specific developmental stages or in specific tissues. The capacity to manipulate gene expression through inducible RNAi allows the dissection of gene functions related to complex pleiotropic phenotypes in loss-of function mutants or stable RNAi lines.

RNAi can be engineered as an efficient method for silencing genes in cultured cells³⁸ and can be manipulated to study protein function in isolated protoplasts,⁴⁶ thereby allowing rapid functional genetic screens and circumventing the maintenance of stable RNAi transgenic lines. Protoplasts can be isolated from various plant tissues, making the study of cell type-specific processes feasible. Moreover, cellular transport mechanisms and sub-cellular localization of proteins of interest can be determined through the implementation of RNAi in protoplasts. An example for efficient use of RNAi in protoplasts is the reduction of transcript and polypeptide levels of γ-glutamylcysteine synthase, a key enzyme in glutathione biosynthesis, by transient double-stranded (ds) RNAi, causing a drastic reduction in glutathione content in Arabidopsis protoplasts.⁴⁶ The reduction of γ-glutamylcysteine synthase activity through RNAi was comparable to cad2-1 (a mutant in the gene encoding γ-glutamylcysteine synthase, ECS1).⁴⁷ Protoplasts can be isolated from tissues as young as 14 days old instead of 1 month old,⁴⁶ thus avoiding the need to maintain stable transgenic RNAi lines. Additionally, the use of RNAi in isolated protoplasts complements current genetic tools by allowing fast, cost- and space-effective initial screening and selection of genes for later in planta studies. In summary, RNAi at the whole-plant level and/or in isolated protoplasts is useful to silence a single gene or a family of related genes. In cases where genes within a gene family are functionally redundant, RNA-based silencing can effectively knock down the transcripts encoding the protein of all family members, which is otherwise difficult using classic mutational analysis. Silencing mechanisms can be experimentally induced with chemicals and unwanted RNA silencing can be alleviated using viral suppressor technology or mutants harboring mutations in the gene encoding key players in the RNAi mechanism.³⁶ Because the silencing mechanism can be manipulated, RNAi has a wide range of practical applications in biotechnology.

Despite a plethora of molecular tools available for use in plants, many of these molecular genetic approaches are not efficiently applied to genes that regulate primary growth and developmental processes and reproduction. The main drawback is that changing the expression of such vital genes can negatively impact plant growth, propagation and survival. In such cases, the generation and maintenance of useful transgenic plants may not be cost-effective. Additionally, numerous environmental factors such as light and molecules such as phytohormones have distinct effects on growth and development in different tissues and organs.^13,14,16,48 Thus, it is vital to establish an applicable system for successful spatial regulation of transgene expression that is not hampered by intervention or imposition of abiotic stress.

Enhancer-Trap and Two-Component Transactivation Systems

The limited number of well-characterized promoters and corresponding expression patterns restricts the cell types and developmental processes that can be targeted. Limitations of tissue-, organ-specific or inducible promoter-driven transgene expression and RNAi can be overcome through the utilization of enhancertrap-based gene induction or inactivation or two-component transcription activation systems based on promoter- or enhancertrap strategies.

Enhancer-trap systems.

Through the utilization of enhancer-trap systems, distinct expression patterns can be achieved in a localized manner, which makes it an effective tool to determine sites of signal perception in physiological processes (e.g., photoperception) that have spatial-specific aspects. Use of the Cauliflower mosaic virus (CaMV) 35S minimal promoter-based enhancer-trap system to express phyB-GFP in a phytochrome B (phyB) mutant of Arabidopsis revealed that phyB expression in the mesophyll, but not in vascular bundles, suppresses the expression of a key flowering regulator, FLOWERING LOCUS T (FT), in vascular bundles.⁴⁹ This finding indicates that a novel inter-tissue signaling mechanism occurs between mesophyll and vascular bundles, making it a critical step in the regulation of flowering by the photoreceptor phyB.⁴⁹

Enhancer-trap approaches have also been used to identify expression patterns of genes. In this approach, reporter genes under the control of minimal promoters are transformed into host plants to identify genes regulated by external cues, e.g., circadian-regulated genes⁵⁰ or pathogen infection-induced genes.⁵¹ Such systems have also been used to identify tissue- or organspecific expression of genes in a range of plant species.^52–55

Two-component transactivation systems.

For two-component transactivation systems, genes of particular interest can be introduced into the Arabidopsis genome under the control of a heterologous upstream-activating sequence (UAS).⁵⁶ The parental UAS-transgenic line is maintained independently and the gene of interest will not be expressed until it is crossed with an activator line that expresses the corresponding transcription factor or transcriptional activator. The activator line contains a gene encoding a transcriptional activator under control of a minimal promoter. In this system, the gene of interest is expressed only in a subset of cells in which the transcription factor is expressed, and this depends on native genomic enhancers that drive the expression of the transcription factor. The effect of transgene expression on plant growth and development can be analyzed in the F3 progeny. Two-component transactivation systems circumvent the necessity to maintain and genotype multiple stable transgenic lines, which is tedious and labor intensive.

GAL4/UAS two component transactivation. A two-component, enhancer-trap mis-expression system based on the yeast Gal4 transcription factor has been used successfully in Drosophila to study regulatory mechanisms during embryonic development,⁵⁷ and in both Arabidopsis⁵⁸ and rice.⁵⁹ A more stringent plant regulatory system based on promoter/enhancer-trap activation can be achieved through the use of transcription factors with sequence-specific DNA-binding activities that are not normally found in plants.⁷ Transgenic GAL4-enhancer trap lines are T-DNA insertion lines with diverse expression patterns of the yeast GAL4 transcription factor and its expression depends on the presence of native genomic enhancer sequences. The GAL4- responsive mGFP5 gene marks the expression pattern mediated by genomic enhancers in green fluorescence.^58,60 The GAL4-enhancer trapping system overcomes the limited availability of cloned and characterized promoters by using native genomic enhancers within a host genome.²

The mechanisms of lateral root development have been studied using an effective GAL4/UAS two-component system. Laplaze et al.⁵⁸ screened a population of Arabidopsis GAL4-GFP enhancer trap lines and selected lines with GAL4 expression in root xylem pole pericycle cells (i.e., cells competent to become lateral root founder cells, line J0121). These authors initiated a study to investigate the molecular and cellular bases of lateral root development in genetic ablation experiments by targeting the expression of a toxin-encoding gene (i.e., Diphtheria toxin chain A, DTA).⁵⁸ J0121-directed transactivation of the toxin gene indicated that only xylem-pole pericycle cells can form lateral roots.⁵⁸ In a follow-up study, Parizot et al.⁶¹ reported that there are two distinct pericycle cell types and confirmed that the cell specification between them occurs in early development with vascular tissue determination. Genetic crosses between transgenic lines with a UAS-linked IPT (isopentenyltransferase) transgene and xylem-pole pericycle cell or lateral root primordia-specific GAL4-GFP expression demonstrated that xylem pole pericycle cells are sensitive to cytokinins, whereas early lateral root primordia are not.⁶² The physiological consequences of this difference were that endogenous levels of cytokinins in lateral root founder cells limit lateral root formation and, in xylem pole pericycle cells, cytokinins are able to disrupt lateral root formation.⁶²

An ability to perform non-invasive measurements of cell-specific changes in molecules in vivo is an additional advantage of the GAL4/UAS enhancer trap system. To gain insight into low-temperature-induced calcium ion signaling in guard cells, an enhancer-trap line with guard cell-specific GFP expression was utilized to obtain cell-specific aequorin expression.⁶³ Temperature-dependent accumulation of aequorin, a recombinant bioluminescent reporter of [Ca²⁺], in guard cells of Arabidopsis indicated that the circadian clock can modulate cold-induced Ca²⁺ signals.⁶³ The GAL4/UAS system has been combined with RNAi technology to investigate cell-specific inactivation of particular genes as well. In an investigation of the chlorophyll synthase gene (CS) in Arabidopsis, an enhancer-trap approach was taken to express an RNAi construct specifically in cells surrounding the plant veins.⁶⁴ In this study, a cell-specific reduction of CS expression in veins and vein-proximal cells was associated with lower photosynthetic capacity and reduced growth and leaf senescence.⁶⁴ The GAL4-GFP system has also been combined with an inducible system for spatial and temporal control of gene expression.⁶⁵ In this system, researchers transformed GAL4/UAS enhancer-trap lines with an ethanol-inducible system under UAS control.⁶⁵ Thus, in the cells producing GAL4 protein, the UAS-driven, ethanol responsive gene could be temporally turned on by ethanol treatment.⁶⁵ Despite many reports of successful utilization of GAL4/UAS transactivation systems in planta,^58,59 a limitation is that the DNA-binding domain of GAL4 possesses intrinsic sensitivity to UAS methylation, especially in plants, thereby making GAL4-based expression systems less effective in some cases.⁶⁶

LhG4/pOp two-component transactivation. Moore et al.⁷ initiated work to combine the high-affinity DNA-binding activity of a mutant form of the lac repressor from Escherichia coli with a transcription activation domain from the yeast GAL4 protein to produce an effective chimeric transcriptional activator for plants called LhG4. The LhG4/pOp two-component transactivation system is based on the lac operon and pOp promoter that consists of two lac operators cloned upstream of a CaMV 35S minimal promoter.⁶ The LhG4/pOp system may be used to study toxic or deleterious gene products, to coordinate the expression of multiple gene products, or to restrict transgene phenotypes to the F1 generation.⁶ The LhG4 system complements inducible expression systems that offer temporal control of gene expression in tissues that can be treated with inducing chemicals. Furthermore, as the lac repressor is derived from Escherichia coli, it is unlikely that plants harbor genes that are regulated by a similar DNA-binding activity. It also seems unlikely that LhG4 will bind fortuitously near an endogenous plant gene and cause its ectopic expression, a potential problem with other systems.⁷ A chemically inducible and spatially controlled expression system may be obtained by regulating LhG4 by fusing it to the ligand-binding domain of the rat glucocorticoid receptor, allowing an additional level of stringency to mediate transgene expression.⁷ The LhG4/pOp system has been used successfully to regulate the activity of genes involved in meristem maintenance,⁶⁷ organ polarity,⁶⁸ cytokinin metabolism,^69,70 and to study embryogenesis in Arabidopsis.⁷¹

Prior studies with spatial-specific promoters have added to our understanding of meristem development in Arabidopsis.^5,21,22 As previously mentioned, WUS protein is required for stem cell identity, whereas the CLAVATA 1, 2 and 3 (CLV1, 2, 3) genes promote organ initiation.⁶⁸ The LhG4 transcription factor was expressed under control of the CLV1 promoter in the activator line and was crossed to the target line with a pOp-WUS::GUS construct, resulting in WUS expression in apical, lateral cells and in cells at the periphery of the shoot meristem. Analysis of progeny resulting from the genetic cross aided in revealing a regulatory feedback loop between WUS and CLV activities in late stages of embryogenesis in Arabidopsis.⁶⁸

Kuderova et al. employed the pOp/LhGR system to induce ectopic ipt (isopentenyltransferase) overexpression with a glucocorticoid, dexamethasone, to obtain temporal regulation of ipt and study the inhibitory effect of ipt-dependent cytokinin enhancement on root growth. This study showed that mechanisms involved in the maintenance of cytokinin homeostasis by cytokinin glycosylation might differ in a temporal manner. The group learned how differences in the specificity of inactivation of cytokinins by glucosylation might influence the developmental-specific sensitivity of the immature root meristems of Arabidopsis.⁷⁰ Mis-expression of the homeodomain gene BEL1-like homeodomain 1 (BLH1) in the embryo sac by the pOp/LhGR transactivation system showed that the cell-fate switch of synergid to egg cell in Arabidopsis eostre mutant embryo sacs is mediated by BLH1.⁷¹

Two-component transactivation systems can be further manipulated to achieve conditional transactivation, allowing temporal as well as spatial ectopic gene expression. Developing a system that allows localized, conditional gene induction within sectors of the plant exposed to the inducer would make possible an investigation into protein function in two dimensions. One limitation of two-component transactivation systems that could occur in very rare cases would be the integration of the transgene at the same locus in the enhancer-trap parent and the UAS parent. However, the occurrence of transgene integration to the same locus within the 120-Mb genome of Arabidopsis is very low, making two-component systems efficient for study of protein function.

Summary and Perspectives

Genomic and proteomic tools are fundamental in genomewide assessment of gene and protein function. Widely accepted and extensively used plant biology molecular tools are mutant analyses, ectopic expression, mis-expression, overexpression and RNAi. Even though each technique has its own advantages and limitations, a suitable strategy or system must be selected to address the specific goals of individual projects. Whereas a number of tools allow tissue-, organ- or temporal-specific regulation of gene expression that aids investigations into the roles of specific genes in distinct aspects of biological or metabolic processes, additional methods for such analyses are emerging. The use of laser-capture microdissection (LCM), which results in the isolation of relatively pure pools of cells⁷² and can be used in combination with genomic (e.g., microarray) and proteomic methodologies, is gaining in popularity and providing novel insight into cell- and tissue-specific aspects of biological processes in plants (reviewed in ref. 73–79). Furthermore, tested and reproducible methods for regulating tandemly arrayed genes, members of gene families that exhibit closely related sequences and targeted gene deletion or modification (e.g., homologous recombination) are still atypical and infrequently used in plants. Improvements to RNAi will contribute to the analyses of tandemly arrayed genes,⁸⁰ a notoriously difficult biological problem. Improved methods for gene targeting via homologous recombination in plants are emerging,^81–85 as are new methods for gene targeting, including the use of zinc-finger nucleases to modify genes in a directed fashion.^86–88 In the latter protocol, zinc-finger nucleases are used to modify genes based on their ability to create double-strand DNA breaks at sequence-specific sites.^86–88 The continued development of genetic tools that will add to our comprehensive understanding of biological, metabolic, growth and developmental processes in Arabidopsis thaliana is essential for improving our general knowledge of plants, as well as aiding in subsequent application of knowledge to crop plants for successful manipulation of their genomes in order to increase crop yield and quality.

Abbreviations

RNAi

RNA interference

T-DNA

transfer-DNA

UAS

upstream-activating sequence