Molecular identification of a root apical cell-specific and stress-responsive enhancer from an Arabidopsis enhancer trap line
Plant root apex is the major part to direct the root growth and development by responding to various signals/cues from internal and soil environments. To study and understand root system biology particularly at a molecular and cellular level, an Arabidopsis T-DNA insertional enhancer trap line J3411 expressing reporters (GFP) only in the root tip was adopted in this study to isolate a DNA fragment.
Using nested PCR, DNA sequencing and sequence homology search, the T-DNA insertion site(s) and its flanking genes were characterised in J3411 line. Subsequently, a 2000 bp plant DNA-fragment (Ertip1) upstream of the insert position of the coding T-DNA was in silico analysed, revealing certain putative promoter/enhancer cis-regulatory elements. Cloning and transformation of this DNA fragment and its truncated segments tagged with or without 35S minimal promoter (35Smini), all of which were fused with a GFP or GUS reporter, allowed to detect GFP and GUS expression mediated only by Ertip1 + 35mini (PErtip1+35Smini) specifically in the Arabidopsis root tip region. The PErtip1+35Smini activity was further tested to be strong and stable under many different growth conditions but suppressed by cold, salt, alkaline pH and higher ammonium and phosphorus.
This work describes a promising strategy to isolate a tissue-/cell-specific enhancer sequence from the enhancer trap lines, which are publically available. The reported synthetic promoter i.e. PErtip1+35Smini may provide a valuable and potent molecular-tool for comprehensive investigation of a gene function related to root growth and development as well as molecular engineering of root-architectural formation aiming to improve plant growth.
KeywordsEnhancer trap line Enhancer Root apex specific expression GFP and GUS Abiotic stimuli
green fluorescent protein
a transcription factor positively regulating expression of galactose-induced genes
- RB and LB
T-DNA right- and left-border
a marker for bacterial kanamycin-resistance selection
GAL4 upstream activation sequence
cetyltrimethyl ammonium bromide
One of the most important biological functions of a root system is to effectively explore soils for water and nutrients ensuring plant adaptive growth. There are three major processes involved in the development of root system: the cell division at the primary root (PR) meristem to add new cells enabling root growth; the generation of lateral roots (LR) to increase the exploratory capacity of the root system; the enlargement of the total surface of PR and LRs through root-hair growth . Such processes are known to be greatly affected by varied environmental factors including nutrients, water, pH and temperature, etc., thus conferring a high degree of morphological-plasticity of root growth in adaptive response to often changed environmental cues . Regarding the morphological plasticity, for instance, a spectrum of responses at physiological and cellular levels occurred when the roots are under water stress ; Arabidopsis LR development was strongly promoted to the soil with rich nitrate (NO3−) ; a profound effect of the combined supply of phosphorus and magnesium on the development of root system morphology was observed in Arabidopsis via auxin signaling, which regulates the elongation and directional growth of the primary root .
Morphologically and functionally, along the (primary) root axis, four zones can be divided: root cap, meristematic region, elongation-and maturation-zone. The root apex or tip represents the most critical part required for sensing and adaptively responding to environmental stimuli . A study with Arabidopsis showed that the treatment with 5 mM KNO3 could inhibit the primary root growth by 30–100% in 3 days, depending on an effect associated with a significant increase in auxin concentration at the root apex . Recently, Medici et al.  described that a reasonable molecular gate integrating phosphate (Pi)- and NO3−-signalling via AtNIGT1/HRS1 actions might exist at the Arabidopsis root apex, regulating the response of root system growth to environmental Pi and NO3−. Besides, there is also an evidence that exogenous l-glutamate at micromole concentrations can act as a highly specific signal molecule sensed by the root tip to modify root growth and branching . However, physiological and molecular determinants necessary for the architectural formation of root system, directed by the root apex in response to varied environment cues, are largely unknown.
To evaluate and/or manipulate a function of an interested genetic component (or gene) involved in the root growth at cellular and molecular levels, the application of a particular promoter driving the gene expression in root cell-type-specific organs (e.g. the root tip) should be a promising strategy. Methodologically, it has been documented that a reporter- or marker protein-based enhancer-trap line (e.g. consisting of GAL4/GFP system) would provide one of the most powerful means for the exploration of a biological event(s) associated with a gene of interest in a cell-specific manner . Basically, in the GAL4/GFP enhancer trap system, a given T-DNA is introduced randomly into a host genome; as the T-DNA integrates downstreamly near an enhancer-dependent or -activated promoter, the activity of the promoter and/or enhancer could be detected by the visualization of the green fluorescent signals derived from GFP, whose expression is GAL4-responsive . To date, GAL4/GFP enhancer-trap lines of some plant species (e.g. Arabidopsis and rice) were created and publically available [11, 12]. The use of these transgenic lines greatly favoured many excellent studies elucidating biological processes at organ/tissue/cell levels [12, 13, 14]. An additional significant contribution of such enhancer-trap lines to biological study is that some promoters with different cell-specific activities were molecularly identified based on finding of cell-type specific genes [15, 16, 17], allowing a precise assessment and manipulation of a gene function with a cell- and developmental-specificity.
The precise temporal-spatial regulation of gene expression is pivotal for the prosperous production of highly-specialized organs/cells and their abilities to respond to environmental signals . At a molecular level, this is greatly completed by the activation and/or repression of the related cis-regulatory elements (e.g. transcriptional enhancers and silencers) at the correct place and time [19, 20]. Generally, the promoter together with its up- or down-stream distal sequence (e.g. enhancer) is crucial cis-components required to control the expression of its target gene(s) , contributing to the regulation of plant growth and development. Thus, the isolation and subsequent application of the valuable promoter and/or enhancer allow a molecular manipulation of plants by miss- or over-expression of a functional gene of interest . As documented, despite the wide application of strong and constitutive promoters (e.g. ubiquitin gene promoter and CaMV 35S RNA promoter), they triggered the gene overexpression in all tissues might impair the host plant growth and development [23, 24]. In contrast, a tissue-specific promoter can accurately control the transcription within a given plant part, possibly avoiding undesirable or negative effects from expressing a foreign gene . Previously, although an approach through finding tissue-specific expressing genes was widely used to isolate related promoters from Arabidopsis, rice, sweet potato and soybean [26, 27, 28, 29], this method seems fairly tedious and inefficient because of the requirement of experimental identification and confirmation of cell-type specific expression patterns of the genes , and some tissue-specific promoters isolated based on this way might have a low activity or specificity . Regarding a promoter with its activity confined only to the root apex/tip, related publications are hitherto very limited, most probably due to people’s interest in its patent protection.
We report here a simple, direct and precise method for the isolation of a putative enhancer (Ertip1) for the root apex-specific transcription from a GAL4/GFP enhancer trap line J3411. The activity of the enhancer (fused with or without a 35S minimal promoter) was monitored by the expression and detection of reporter proteins (i.e. GFP and GUS) in Arabidopsis transgenic plants, showing its strong and specific action only in the root apex/tip zone. Furthermore, to evaluate the stability of this enhancer activity, GFP-indicated fluorescent signals were tested under varied growth conditions, revealing that the enhancer-facilitated reporter expression was strongly and rapidly suppressed by certain external stimuli. Thus, such a cell/tissue-specific enhancer and its synthetic promoter (like PErtip1+35Smini constructed in the work) should provide a valuable and potent molecular tool to favour the intensive investigation of root system biology as well as manipulation of root growth and function.
Plant materials and growth conditions
A GAL4/GFP line J3411 (Arabidopsis C24 background) was obtained from the Haseloff and Poethig collections, (http://data.plantsci.cam.ac.uk/Haseloff/tools/gal4system/page138.html). Transgenic plants harbouring putative promoters/enhancers fused with GFP or GUS were generated in this work (see “Generation of transgenic lines”).
For Arabidopsis aseptic growth, surface-sterilized seeds were germinated and cultivated vertically for 7 d on the basic medium i.e. a half-strength MS agar (0.8%)-medium (containing 1% sucrose and 0.5 mM NH4NO3) in a growth room (19–22 °C, 16 h/8 h light/dark period, 120 μmol m−2 S−1 light intensity); thereafter, seedlings were transferred to the basic medium (except for N- or P-treatment) plate for further 1 d growth under 20 different treatments as shown below: IAA (Indo-3-acetic acid, 60 nM), ABA (Abscisic acid, 200 nM), GA (Gibberellic acid or Gibberellin, 500 nM), ACC (1-Aminocyclopropane-1-carboxylic acid, 500 nM), 6-BA (6-Benzylaminopurine, 100 nM), l-Glu (0.5 mM), l-Leu (0.5 mM), l-Lys (0.5 mM), l-Met (0.5 mM), pH (4.5 and 8), P (phosphorus, high-2.5 mM, low-50 μM; in the form of KH2PO4), NH4+ (high-10 mM, low-10 µM; in the form of (NH4)2SO4), NO3− (high-10 mM, low-10 µM; in the form of KNO3), AlCl3 (50 μM), Salt (NaCl, 80 mM), and cold (4 °C). Above chemical solutions were filter-sterilized and added to the autoclaved agar-medium (at about 60 °C). 7-d-old plants were transferred to the basic medium and grown for 1 d were used as a reference (CK) in the GFP assay. Except for those treated with cold (4 °C), all plants were grown under normal growth conditions as described above. Exception of pH treatment (4.5 and 8 adjusted respectively by using HCl or KOH), the medium pH was set to 5.8 by KOH. Under conditions of cold, salt, higher-pH, -salt, -NH4+ and -P, GFP expression in the transgenic plants harbouring PErtip1+35Smini:GFP was particularly measured at different time point within 24 h (i.e. 0 h, 1 h, 6 h, 24 h).
To check the GFP/GUS expression in other upper-part tissues/organs (e.g. flower and silique), homozygous lines harbouring all individual truncated promoter/enhancer versions fused with GFP/GUS were cultivated in pot-soils for 70 d in the growth room.
Detection of the T-DNA insert position and its flanking sequence/gene
Genomic DNA was isolated from 2-week-old J3411 seedlings (around 100 mg) by using CTAB (cetyltrimethyl ammonium bromide) extraction buffer (1% CTAB, 100 mM Tris–HCl (pH 8.0), 20 mM EDTA (pH 8.0), 1.5 M NaCl and water) and was then precipitated with isopropanol and washed with 70% alcohol. The Nest PCR (according to the protocol from http://signal.salk.edu/T-DNArecovery.pdf) was conducted with degenerate primers and a set of nested primers designed from the T-DNA left border (Additional file 1: Table S1). The Nest-PCR products were subsequently cloned into pGEM-T vector (Promega) and sequenced. Sequence homology search was carried out by using BLAST in the NCBI or TAIR (www.ncbi.nlm.nih.gov; https://www.arabidopsis.org/Blast/index.jsp).
Sequence motif prediction
The sequence motifs or cis-regulatory elements in the putative promoter/enhancer Ertip1 were inspected using PLACE and PLANTCARE [30, 31]. The predicted important cis-regulatory elements are listed in Additional file 2: Table S2.
Generation of transgenic lines
The putative promoter/enhancer Ertip1, Ertip1 + 35Smini, Ertip2, Ertip2 + 35Smini and Ertip3 (Fig. 2a) were PCR-amplified using specific primers (see Additional file 1: Table S1). All primers contain the SpeI site. PCR products were digested by SpeI and cloned into a plant expression vector pBI101-GUS and pBI101-GFP using compatible XbaI/SpeI cohesive ends, yielding constructs termed here: Ertip1:GFP or :GUS, Ertip1 + 35Smini:GFP or :GUS, Ertip2:GFP or :GUS, Ertip2 + 35Smini:GFP or :GUS and Ertip3:GFP or :GUS. Arabidopsis (Col-0) was transformed by floral dipping into a cell suspension (OD600 = 0.61) of agrobacterium strain GV3101 consisting of the above constructs. Plant transformants were selected by kanamycin resistance (50 μg L−1); at least two independent homozygous lines harbouring the above individual constructs were created in the T2 or T3 generation for experimental use. Exact numbers of independent transgenic lines generated for each of the five constructs are described in Additional file 3: Table S3. Three or six lines harbouring respectively Ertip1 + 35Smini:GUS or :GFP show in fact their corresponding reporter expression only in the root tip (Additional file 3: Table S3 and Additional file 4: Fig. S1).
GUS histochemical staining and GFP microscopic observation
For the detection of β-glucuronidase (GUS) activity, plant tissues were vacuum-infiltrated (for 10 min) and incubated further for 35 h at 37 °C in a staining solution (25 mM sodium phosphate buffer at pH 7.0, 10 mM EDTA, 0.5 mM ferricyanide, 0.5 mM ferrocyanide, 0.1% Triton X-100, and 2 mM X-Gal i.e. 5-bromo-4-choro-3-indolyl-β-d-glucuronide cyclohexylamine salt). Thereafter, tissues were washed with 75% ethanol twice over a 24 h period to remove chlorophyll from leaves or flowers. Samples were visualized under a microscope (BX51, Olympus, Japan).
For the observation of GFP localization, whole seedlings or tissues were mounted in water or 30% sucrose under a glass coverslip, and GFP signals were scanned with an energy excitation at between 488 and 535 nm by confocal laser scanning microscope (OLYMPUS FluoView™ FV1000, Japan). Brightness and contrast pictures were adjusted using the Olympus FV1000 Viewer software.
The intensity of green fluorescence photographed by a fluorescence microscope-camera device (BX51, Olympus, Japan) was quantified using ImageJ (https://www.youtube.com/watch?v=nLfVSWcxMKw%26lc=UgjP3p6wpnEcOXgCoAEC). Every picture was taken at the same exposure time (i.e. 20 ms), where pixel values range from 0 to 255. The value of a non-fluorescing root image was taken as a background intensity.
The data are given in the form of a mean value with a standard deviation of replicates. Statistical test was performed using the statistical software program SPSS version 16.0 (Beijing, China). Significant differences between treatments were determined by one-way analysis of variance (ANOVA), and post hoc comparisons were done using Tukey’s multiple range test at P < 0.05.
Results and discussion
Identification of a genomic region involved in GFP specific-expression in the root apex of an enhancer trap line
Certain important cis-acting regulatory elements are predicted in the 2-kb plant intergenic region upstream of the coding T-DNA_2
Ertip1 exhibits no promoter activity but enhances a T-DNA-derived 35S minimal promoter action only in the Arabidopsis root apex
Since the activity of Ertip1 along or its truncated DNA fragments with or without the 35Smini in triggering GUS/GFP expression could not be detected in their corresponding transgenic lines (data not shown), the Ertip1 should be considered as a potent enhancer responsible for the expression limited to root-tip cells when tagged with 35Smini. This finding can be emphasized by the observation that the specific GAL4/GFP expression in J3411 was not replicated by the expression of pAt2G36360-3nGFP , because the authors, based on knowing of the same coding direction of the T-DNA and flanking gene At2G36360 (Fig. 1a, ), just took 3.9 kb fragment (not including our identified Ertip1) upstream of the At2G36360 coding start as a putative promoter to test its activity . This leads to a speculation that the GAL4/GFP specific-expression in the root apex of J3411 might be attributed to regulatory elements located in the genomic DNA immediately upstream of the T-DNA insertion . Regarding the possible target gene of the Ertip1, since it was documented that enhancers can be interacted with multiple transcription factors (TFs) to activate the transcription of genes located up to many kb or even several Mb away [41, 42], and can function in an orientation-independent manner , we propose that the Ertip1 might target to At2G36380 or At2G36360 or the both, being responsible for the specific GAL4/GFP expression pattern in J3411. Thus, it would be interesting to test the effect of the Ertip1 on specificity of the promoter in the root tips of these two genes in our future work.
The specific activity of Ertip1 in the root apex is responsive to varied growth conditions
The present work describes an uncomplicated and valuable process for the successful isolation and characterisation of a transcriptional enhancer Ertip1 specific for the root tip from an enhancer trap plant J3411. The root apex-specific activity of the Ertip1 was proven by the expression of both GFP and GUS reporters under the control of Ertip1 + 35Smini (functioning as a promoter PErtip1+35Smini) in Arabidopsis. Interestingly, among 20 different growth conditions tested, the Ertip1 activity in the root tip cells remained stable in most cases, but was rapidly reduced under cold, salt, alkaline pH, higher ammonium and phosphorus. From an application viewpoint, we deem that the identified enhancer Ertip1 and its related synthetic promoter (e.g. PErtip1+35Smini, Additional file 5: Fig. S2) should provide potent molecular means to favour comprehensive study and manipulation of any interested functional gene at the root apex, which may involve root-system formation and biological function in response to external and environmental cues.
ZL, LNQ and ZRZ performed major experiments and statistical analysis, ZL participated in the draft preparation. YYG and CZW contributed to the plasmid construction. LHL and FQC designed general study and prepared the manuscript. All authors read and approved the final manuscript.
We would like to thank professor Jim Haseloff from Department of Plant Sciences at Cambridge University (England) for gifting some valuable GAL4/GFP enhancer trap lines.
The authors state that they have no competing interests.
Availability of data and materials
The data and materials generated from this work can be offered only by the corresponding author (s) upon request.
Consent for publication
Ethics approval and consent to participate
This study was supported by the National Natural Science Foundation of China (NSFC, No. 31270881, Y333ZA1D11), and the Doctoral Research Fund Project of the Ministry of Education (No. 20134320110015).
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