Inducible Gene Expression Systems for Plants
Jeff Leung, personal communication; H. Betts and I. It has also been hpRNA in Arabidopsis seedlings, found that induced expres- used for activation tagging Patrick Gallois, University of sion returned to background levels within 24 h of removal Manchester, UK, personal communication. The latter authors binding to HSP90 in the uninduced state is predicted to showed that another glucocorticoid, triamcinolone aceto- prevent operator binding by LhGR.
In keeping with this, ipt nide, was as efficient an inducer as dexamethasone but was expression was stringently regulated in Arabidopsis and turned over with a half-life of only 24 h in tobacco. We are tobacco Craft et al.
By growing Arabid- ing medium may further increase the rate at which the opsis plants continually on dexamethasone from germina- system is switched off Baroux et al. In greenhouse studies involving long-term mum rates of transcription Wielopolska et al. Under despite its increased toxicity, as it is more volatile than greenhouse conditions, less thorough tests of dexametha- DMSO, which accumulates in the soil over several weeks of sone stability have been conducted, but we typically add watering.
If a gene of to sustain activities of a stable protein like GUS for several interest is placed under control of the pOp6 promoter to al- weeks M.
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Samalova and I. We routinely make fresh stocks of also be used in conjunction with the growing collections of dexamethasone every 2 weeks or so and store it in DMSO tissue-specific LhG4 activator lines described above. The CaMV specific patterns of inducible pOp6 expression e. In these studies, stable inducible expression of GUS, et al.
This indicates that the inducible R2-compatible entry clones that can be used in conjunction system was still active and that silencing occurred through with this vector.
We are currently validating the expression sequences outside the operator array. It is consistent with patterns driven by a subset of these promoters Table 6 and the idea of sequence-specific PTGS thresholds proposed by Renate Schmidt and colleagues Schubert et al. Chris reports have used 10—25 lM and Tornero et al. Oestrogens such as b-oestradiol are efficiently below. Accumulation appears to be slower than Chemically inducible expression from the OlexA promoter with other systems as transcripts are most abundant only This promoter is the target for the XVE transcription factor, 24 h after induction and they decrease again over the next which is activated by oestrogens Zuo et al.
It has 3 days indicating that b-oestradiol is unstable under been used in a dozen different studies in Arabidopsis to culture conditions and in planta Guo et al. It also implies that repeated application of oestrogen molecules and for inducible activation tagging. The tech- every 2—3 days may be required to sustain transcript nology has been incorporated into vectors that drive indu- abundance.
Long-term localized Guo et al. The XVE transcrip- medium, but it is not clear how efficiently b-oestradiol is tion factor is expressed from the strong synthetic promoter transported in older plants. We are not aware of any reports G that exhibits eightfold higher activity than CaMV 35S of b-oestradiol being applied via the soil and most Ishige et al. However, these methods suggest from the treated leaves as newly developing leaves did not that genes of interest can be induced significantly more than exhibit the hypersensitive response induced by transgene fold from undetectable or low levels Carranco et al.
The ; Zuo et al. For similar reasons, it is difficult to relative immobility of b-oestradiol was important for this evaluate the stringency with which XVE is regulated as we study and may be useful in other cases where highly are not aware of the system being used with sensitive localized expression is required.
In contrast, although physiological reporters such as bacterial ipt or avr genes. Some plants, notably soybean, observed to elicit physiological effects on plants and that accumulate significant quantities of phytoestrogens, which there is no evidence that it disturbs endogenous gene can activate the system Zuo and Chua, , but this has expression or development in Arabidopsis. They recom- not presented a problem in Arabidopsis or several other mend that b-oestradiol should be dissolved in DMSO as plant species see below.
Most gene expression. Furthermore, Zuo et al. Indeed, Sun et al. Nevertheless, it is possible to identify lines that A dexamethasone-inducible derivative of XVE has been deliver induced expression levels that are typical of direct used successfully in Arabidopsis protoplasts Yanagisawa promoter::uidA fusions Deveaux et al.
Suicide seeds. An application of inducible gene expression in plants and its possible implications
Mark Curtis Weigel, As the palcA XVE to exploit their patterns Mark Curtis, University promoter is not active in wild-type plants, this increased of Zurich, Zurich, Switzerland, personal communication. Anoxia and similar to XVE but incorporates the spruce budworm EcR tissue culture conditions are thought to promote the accu- ligand-binding domain in place of the oestrogen receptor mulation of these metabolites precluding the use of this sequences and can potentially be used with methoxyfe- system in tissue culture Roberts et al.
It may be for nozide in the field see below. Methoxyfenozide may also this reason that we did not find any published study in which have better uptake and distribution characteristics than the alc system has been used to regulate the activity of b-oestradiol, though a detailed analysis has not been genes that affect physiology or development at very low reported and foliar application is apparently ineffective expression levels, though Howles et al. Leaky expression in vitro may not compromise many Ethanol-inducible expression from the AlcA promoter overexpression or RNAi experiments that depend on strong In contrast to the steroid-induced systems that utilize syn- induction to generate phenotypes.
Laufs et al. Genes of interest are cloned in the meristem was still insufficient to complement the ufo under control of the palcA promoter, which comprises the phenotype. Deveaux et al. In the prevented from penetrating the medium, for example by absence of ALCR, palcA is silent in Arabidopsis even under increasing the agar concentration or inclining the plates. A conditions that generate endogenous ethanol Roslan et al. In this way, it has 0. Ethanol can also be episodes of induction as it maximizes the efficiency of applied effectively as a vapour if a tube containing a 50— induction yet minimizes the toxicity of ethanol Roslan et al.
However, the requirement to keep plants enclosed or in a plastic bag or closed propagator containing soil- can affect their biology if treatment is extended beyond a grown plants Deveaux et al. Enclosure increases the efficiency of the induction and It is possible to maintain lines that exhibit sustained prevents ethanol vapour from inducing expression in neigh- expression from the alc system but silencing was seen in bouring plants. The effect of ethanol vapour on neighbours some of the selected lines studied by Roslan et al.
In must be taken into consideration when applying ethanol by most instances, this was correlated with loss of ALCR soil drenching, particularly in confined growth chambers expression from the CaMV promoter suggesting that palcA Salter et al. The volatility of remains accessible to activation over many generations ethanol means that it is readily applied to whole plants, be Roslan et al.
Focusing on tematic cells Deveaux et al. These communication. The rapid uptake and metabolism of studies indicate that the half-life of ALCR is sufficiently low ethanol means that the induction period can be limited. In solanaceous The alcR ORF has been placed under control of the pOp species, bagging organs and the use of acetaldehyde rather promoter in a vector that allows a gene of interest to be than ethanol have been used to restrict palcA induction, but inserted downstream of palcA, thereby generating what we have not found similar studies with Arabidopsis.
Addi- promises to be a particularly versatile expression vector tional consequences of the physical and metabolic proper- Daniel Couch and Christine Perrot-Rechenman, ISV, Gif-sur- ties of ethanol are that soil drenching needs to be repeated at Yvette, France, personal communication. With this vector, 0. Owing to the stringent regulation of As ethanol can be toxic, Roslan et al. Consistent with this, inclusion of 0.
The use of pOp6 see above in a similar vector may increase its efficiency. It has also been used successfully in the widely used BY-2 cell culture system Nara et al. Induc- Soil drenching Soil drenching Soil drenching Liquid media Hydroponics Hydroponics tion is rapid, with luciferase being first detected 30 min after Agar media Agar media Agar media Induction Spraying Spraying application of 10 lM dexamethasone and transcripts accu- Lowest concentrations known to induce the target promoter? Painting Switch off Gene tested method Vapour mulating to maximum after 4 h.
However, fully induced activities were only fold over background owing per- haps to relatively high basal expression levels Aoyama, These concentrations are fold higher than the concen- trations required to induce GVG in Arabidopsis and to fold higher than the concentrations necessary to in- 4 h CAT activity max 96 h duce other GR-based systems in tobacco Roberts et al.
It may be that the original characterization underestimated the sensitivity of GVG as Induction Table 8 Characteristic of four inducible expression systems in tobacco studies with liquid-grown seedlings or suspension cultures speed found that 0. Alternatively GVG may have very different sensitivities in liquid culture and Ethanol Acetaldehyde agar plates.
Dexamethasone is applied to seedlings by transfer to LhGR Samalova et al.
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Mature plants are induced by spraying, painting, or infiltrating leaves. Uptake Inducer into tobacco from soil via the roots seems not to have been reported. Local application of dexamethasone to one-half of a leaf resulted in induction only in that half after 24 h. Transport of dexamethasone within tobacco plants was GVE Padidam et al. Lucif- Alc Salter et al.
As with been reported in any of the tobacco studies. In addition, the Arabidopsis, ethanol should be avoided as a solvent for Gal4-UAS array in the target promoter does not become dexamethasone in tissue culture as it resulted in marked rapidly silenced by methylation as reported for another inhibition of seedling development at 0. Dexamethasone does not affect tobacco growth The GVE system has been used in tobacco to express up to at least 60 lM in soil water Samalova et al. Induction Figure 5a. It was possible to apply the 24 h after watering the soil with 50 ml of 20 lM dexameth- inducer in vitro and via the soil water but foliar application is asone or painting the leaves with the same concentration of not effective so local induction may not be possible Padi- inducer Samalova et al.
Earlier time points were not dam, ; Padidam et al. Although promising, the tested but, in other studies, induced YFP expression has system has yet to be tested with physiologically active been detected 1 h after dexamethasone was applied to transgenes, the effect of GVE induction on tobacco growth leaves of a CaMV::LhGR plant that were previously infiltrated has not been documented and a detailed histochemical with Agrobacterium harbouring a reporter construct Ann analysis of the induced activity has yet to be reported.
Although dexameth- asone was transported efficiently from roots to shoots in pOp6 promoter soil-grown tobacco, it did not spread efficiently from the Samalova et al. Thus even 7 days constructs. As with Arabidopsis, the best induction charac- after treatment, staining was confined principally to the teristics were obtained with the newer pOp6 promoters see veins Samalova et al.
However, uniform induction above.
After painting, movement of dexamethasone min 1 lg 1 were similar to the best CaMV 35S::GUS values between two halves of the same leaf was minimal within in tobacco Martinez et al. Transport to some individuals exceeding these values threefold. In the neighbouring leaves was weak, sporadic and confined to the absence of dexamethasone, GUS activity was indistin- main veins.
With palcA promoter seedlings in liquid medium, induction is rapid, as transcripts are detectable within 1 h and are maximally abundant at 8 h, The alc system was first described in tobacco where it was while reporter protein activity is detectable throughout the used for expression of the CAT reporter gene and a cytosolic seedling within 1 h Samalova et al. Dexamethasone transport and activation in tobacco. Shown are plants after 4 weeks of treatment. Plants were additionally watered with water whenever necessary, however not directly prior to any of the dexamethasone treatments.
No differences in plant development were measured in dexamethasone treated plants. The reaction was stopped after 16 h of incubation with the substrate. Samples were taken from the painted areas 24 h after induction and histochemically stained for GUS activity. Expression is detected homogeneously throughout the leaf sample. Samples from leaves painted with a control solution lacking dexamethasone are shown as a control.