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. 2011 Jun 1;6(6):815–820. doi: 10.4161/psb.6.6.14719

Induction of karyopherin α1 expression by indole-3-acetic acid in auxin-treated or overproducing tobacco plants

Karin Rand 1,2, Irina Kobrinsky-Aaronowitz 1,3, Yael Levy 1, Orit Shaul 3, Roni Aloni 2, Yedidya Gafni 1,3,✉
PMCID: PMC3218478  PMID: 21646869

Abstract

Macromolecules may transfer between the cytoplasm and the nucleus only through specific gates—the nuclear pore complexes (NPCs). Translocation of nucleic acids and large proteins requires the presence of a nuclear localization signal (NLS) within the transported molecule. This NLS is recognized by a class of soluble transport receptors termed karyopherins α and β. We previously characterized the expression pattern of the tomato karyopherin α1 (LeKAPα1) promoter in transformed tobacco plants. Expression of LeKAPα1 was mainly observed in growing tissues where cell division and extension is rapid. The expression pattern of LeKAPα1 resembled that of auxin-responsive genes. This led us to suggest that auxin participates in the regulation of LeKAPα1 expression. Here we characterized the correlation between auxin level and the activity of the LeKAPα1 promoter. To this end, transgenic tobacco plants carrying the GUS reporter gene under the control of the LeKAPα1 promoter were treated with various levels of exogenous auxin. We also studied transgenic plants in which we increased the endogenous levels of auxin. For this, we expressed in plants both the LeKAPα1 promoter-GUS reporter and the Agrobacterium tumefaciens iaaM gene, which increases the endogenous levels of auxin. The results indicate that the auxin indole-3-acetic acid (IAA) can induce LeKAPα1 expression. We also identified that the sites and levels of LeKAPα1 expression correlated with the endogenous pathways of polar auxin transport.

Key words: auxin, karyopherin α1, nuclear pore complex, TYLCV, plant virus

Introduction

Nucleocytoplasmic transport is an essential activity in all eukaryotic cells. This transport occurs through the nuclear pore complex (NPC), which serves as a gateway for entry and exit from the nucleus.1,2 While small molecules diffuse freely through the nuclear pores, translocation of nucleic acids and proteins requires the presence of nuclear localization signals (NLSs) within the transported molecule.3 One of the best characterized types of NLSs is a classical NLS that contains a high proportion of positively charged, basic amino acid residues. These basic NLSs are recognized by a class of soluble transport receptors, termed karyopherins or importins α and β.3–5 Karyopherin α directly binds the basic NLS sequence and interacts with karyopherin β via its N-terminal region. The resultant heterotrimeric complex is targeted to the NPC through the direct interaction of karyopherin β with specific nucleoproteins. The heterotrimer is then translocated into the nucleus in an energy-dependent manner.6 After the translocation of the trimeric complex into the nucleus, the karyophilic cargo, i.e., the NLS-containing protein, is released inside the nucleus.7 Nuclear import, which has been well studied in animal cells and yeast, is now being investigated in plant cells and appears to be a highly conserved process with very similar import machinery components.8—11 Furthermore, proper localization of proteins in plant cells depends on the same type of NLS found in other organisms.12,13

Karyopherin α is a 58.6 kDa protein found in every eukaryote studied so far. In tomato (Solanum lycopersicum), one isoform [karyopherin α1, (LeKAPα1), GenBank accession no. AF017252] has been cloned and characterized14 and shown to interact with the Tomato yellow leaf curl virus (TYLCV) coat protein. This virus, the cause of the most severe tomato disease in many countries, probably uses this member of the karyopherins to insert its genome into the nucleus for its transcription and replication during infection.15 Previous studies in mammalian cells have shown that the expression of various NLS receptors is controlled in both tissue-specific and cell type-specific manners at the mRNA level.16–19 However, the spatial regulation of karyopherin α gene expression was hardly investigated in the plant kingdom. In our previous study of the LeKAPα1 gene regulation, we characterized the expression pattern of its promoter in transgenic tobacco plants.20 The 2,170 bp fragment of the LeKAPα1 promoter (GenBank accession no. AY189742) was named LM1.20 LeKAPα1 expression mainly occurred in specific leaf zones, such as the tips and expending blade margins of growing leaves, but not in resting tissues. These regions are known to contain high levels of the plant hormone auxin.21 Hence, it was suggested that auxin may be involved in the regulation of LeKAPα1 expression. Auxin is a name given to a class of plant growth substances which plays an essential role in the coordination of many growth and physiological processes in the plant life cycle. It controls diverse cellular processes including gene expression.22–25

Here we investigated the induction of the LeKAPα1 promoter by high levels of auxin in two approaches. First, we studied transgenic tobacco plants that had elevated levels of endogenous auxin. For this, we expressed in the same tobacco plant two transgenes. The first transgene included the coding region of the Agrobacterium tumefaciens iaaM gene under the control of the auxin-inducible promoter of the Arabidopsis thaliana Cel1 gene (GenBank accession no. X98543).26 The iaaM gene codes for a Trp-2-monoxygenase enzyme that converts the amino acid tryptophan to indole-3-acetamide, which is then hydrolyzed to the auxin indole-3-acetic acid (IAA).27,28 Therefore, production of auxin should be amplified in endogenously high-auxin regions. The second transgene included the reporter gene GUS fused to the promoter region, termed LM1, of LeKAPα1.20 Studies of these plants indicated that LeKAPα1 expression occurred largely in plant areas where auxin accumulates. In the second approach, we challenged transgenic tobacco plants containing the LM1-GUS construct with different concentrations of exogenous auxin. Our findings indicated that the expression of LeKAPα1 is regulated by the level of auxin in the tissue.

Results

The effect of exogenous auxin application on pattern of expression of LeKAPα1 promoter in LM1-GUS transgenic tobacco plants.

To study the effect of exogenous auxin on LeKAPα1 expression, we utilized the LM1-GUS transgenic plants.20 The pattern of LeKAPα1 promoter expression was examined in LM1-GUS expressing tobacco seedlings exposed to different concentration of exogenous auxin. As shown in Figure 2, treatment of tobacco seedlings with two different auxin solutions (1 and 3 µg mL−1) led to a clear induction of GUS activity, indicating the expression of the LeKAPα1 promoter only in the presence of high auxin levels. Expression of the LeKAPα1 promoter was mainly observed in roots and in leaf veins, but was hardly visible in hypocotyls. These results, which directly show the induction of LeKAPα1 promoter by auxin, provide the molecular basis for our previous observation20 that this promoter was expressed in plant tissues known to contain high auxin levels.

Figure 2.

Figure 2

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Auxin induced LeKAPα1 expression in two week old tobacco shoots. Plants were exposed to 0 µg mL−1 (A), 1 µg mL−1 (B) or 3 µg mL−1 (C) IAA. All three plants shown are 4–5 cm in size.

Production of pKAPGUSiaaM transgenic plants.

To further investigate the regulation of the LeKAPα1 promoter by auxin, its expression pattern was analyzed in the presence of endogenous high levels of IAA. The chimeric construct harboring both a gene for overproducing IAA in endogenous high-auxin tissues (cel1-iaaM) and a reporter gene driven by the LeKAPα1 promoter (LM1-GUS), was introduced into tobacco plants by Agrobacterium. The binary vector pKAPGUSiaaM contained both chimeric constructs as well as the kanamycin resistance gene nptII. Twelve independent T0 lines of kanamycin-resistant plants, generated from independent transformation events, were selected for further analyses. All twelve plants were confirmed to be transgenic for both constructs by PCR (data not shown).

One line called KR1, which expressed both the gene for overproducing IAA and LM1-GUS, was chosen as a representative line based on its similarity to nine out of the twelve plants obtained. In all parallel analyses performed with control non-transformed plants, no non-specific endogenous GUS activity was visualized.

Characterization of leaf morphology in IAA-overproducing plants.

To further establish the induction of LeKAPα1 promoter by auxin we used eight vegetatively propagated KR1 plants expressing both iaaM for endogenous production of high auxin levels and LeKAPα1 promoter driving the GUS reporter (LM1-GUS). We first studied the effect of auxin overproduction on plant phenotype. It was previously shown that overproduction of auxin results in epinastic or curled leaves.35,36

In our study, the KR1 plants showed typical epinasty and curling of leaves (Fig. 3A), indicating excessive expansion of the adaxial cells of the leaf relative to the abaxial cells. This phenomenon indicated that overexpression of the iaaM gene resulted in high levels of endogenous auxin. When free IAA levels were directly measured, it was found that the KR1 plants had twice as much auxin as wild-type plants (Fig. 4). At the same time, the LM1-GUS transgenic plants showed normal leaf morphology (Fig. 3B) and no increase in auxin content as compared to wild-type plants.

Figure 3.

Figure 3

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Auxin-induced epinastic response in two-month-old tobacco plants. Note epinasty of leaves in pKAPGUSiaaM transgenic (A) but not wild-type (B) plants.

Figure 4.

Figure 4

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IAA content of leaves of two-month-old LM1-GUS plants (LM1) and KR1 plants (KR1) as measured by radioimmunoassay.

Induction of the LeKAPα1 promoter in IAA-overproducing plants.

To further investigate the involvement of auxin in the induction of the LeKAPα1 promoter, GUS expression was studied in leaves of the transgenic KR1 plants that contained the LM1-GUS construct and also over-produced IAA (Fig. 5). The LM1-GUS plants, which did not over-produce IAA, were used as a control (Fig. 5A). The induction of GUS expression in plants having endogenously-increased levels of IAA (Fig. 5B) confirmed the induction of the LeKAPα1 promoter by auxin.

Figure 5.

Figure 5

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Induction of GUS expression in leaves of transgenic KR1 plants. The figure shows histochemical GUS staining of leaves of LM1-GUS plants (A) and KR1 transgenic plants expressing both the LM1-GUS construct and the construct for over-producing IAA (B).

Auxin flows in young leaves from the tip towards the base through the vascular region.21 We exploited this to artificially concentrate the endogenous auxin by incision made in young leaves of KR1 tobacco plants (Fig. 6). A diagonal incision in the leaf obstructed polar auxin flow, resulting in higher auxin concentration above the incision, which consequently induces higher expression of the LeKAPα1 promoter (indicated by GUS staining). Figure 6 shows that expression of the LeKAPα1 promoter was mainly observed in those parts of the leaf where auxin accumulates, particularly in the vascular region, and almost no GUS expression could be detected directly below the incision line. Intact KR1 transgenic tobacco leaves did not show GUS staining in their veins or other blade tissues. Incisions made in leaves of LM1-GUS transgenic tobacco showed no GUS staining, thus ruling out the possible effect of wounding per se on GUS expression.20

Figure 6.

Figure 6

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Polarity assay for auxin in leaf of KR1 transgenic tobacco. (A) The arrows in the cartoon show the effect of an auxin-concentrating diagonal cut on polar auxin flow, asterisks indicate the site of free-IAA production. (B) Higher levels of GUS staining are evident above an auxin-concentrating diagonal cut (arrow) than below it.

LeKAPα1 promoter is induced in the stem vascular tissue of KR1 plants.

It is well known that the sieve elements of the plant provide one pathway for auxin transport (reviewed by Aloni, 2003).21 We therefore studied the expression of the LeKAPα1 promoter in stems of KR1 tobacco plants. Transverse sections were made in stems of seven-week-old plants and GUS expression was analyzed (Fig. 7). GUS expression was not detected in wild-type non-transgenic plants (A), while it was clearly seen in the transgenic KR1 plants (B). The expression was observed in the cells layers through which auxin preferably moves (Aloni 2003): the cambium (a), epidermis (b) and sieve tubes (c), in both the inner and outer phloem.21

Figure 7.

Figure 7

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Light micrographs of transverse sections of stems of seven-week-old tobacco plants. (A) wild-type plants (B) KR1 plants. (a) cambium; (b) epidermis; (c) sieve tubes. Bar = 12 µm (A), 20 µm (B).

Discussion

We previously characterized the expression pattern of the karyopherin α1 promoter, and observed that it was associated with plant tissues known to accumulate the hormone auxin.20 The possibility that auxin is involved in regulating the expression of karyopherin α1 promoter was intriguing. To explore this possibility, it was necessary to directly examine if this promoter is induced by auxin.

In this work we describe the expression pattern of the LeKAPα1 promoter in transgenic tobacco plants exogenously induced by or overproducing auxin. In both experimental approaches we observed a clear induction of LeKAPα1 promoter by auxin. These findings suggest, for the first time, that auxin is involved in the regulation of expression of a member of the karyopherin family, namely karyopherin α1. Auxin is an essential plant hormone known for almost 80 years and implicated in many processes.37 It was suggested that regulation of nuclear transport of proteins by auxin is another mechanism by which auxin-dependent signal transduction is mediated.38 Auxin responses are mediated by successful nuclear transport of signaling regulators and transcription factors.38 It is currently unknown how auxin regulates the nuclear transport of specific proteins. Based on the findings presented in this work, we suggest that induction of karyopherin α1 by auxin plays a role in this regulation.

Thus, at least in those cases where nuclear transport depends on karyopherin α1, auxin apparently plays a role in the regulation of this transport. One such case is the nuclear transport of the capsid protein (CP) of TYLCV. Similar to all geminiviruses known thus far, TYLCV employs the cellular nuclear import machinery to actively transport its genome into the nucleus for its replication and transcription. For this, the capsid protein of the virus interacts with one member of the nuclear shuttling receptors, the karyopherin α1.14 The CP protein is not transported to the nucleus alone but rather as a CP-viral DNA complex, in which the CP serves as a shuttle protein for the viral genome.10,13 Karyopherin α1 thus serves the shuttling of the TYLCV CP-viral DNA to the nucleus of the host cell, thereby enabling viral replication and spread.14 We suggest that due to auxin induction of karyopherin α1 expression, TYLCV can propagate more easily in tissues with high auxin content.

TYLCV is a phloem-restricted virus. The sieve elements of the plant provide one pathway for auxin transport (reviewed by Aloni 2003).21 Cross sections in stems of KR1 tobacco plants, which have increased levels of auxin in its endogenous accumulation sites and also express the LeKAPα1::GUS reporter, showed that the LeKAPα1 promoter is highly expressed in the phloem (Fig. 7). This supports a model in which auxin induction of the LeKAPα1 promoter in the phloem, where TYLCV resided, results in enhanced shuttling of the TYLCV genome into the nucleus, and, thereby, in more efficient viral propagation. This work is the first to suggest the possible involvement of auxin in the nuclear shuttling of a CP-viral DNA. Our findings may therefore lead to a new understanding of the regulation of TYLCV infectivity. It will be also interesting to identify the plant proteins whose nuclear shuttling is enhanced due to auxin induction of karyopherin α1 expression.

In addition, one of the well known phenomenon associated with TYLCV disease is leaf curling, which resembles the curling observed in auxin over producing plants (Fig. 3). It will be interesting to find if TYLCV is able, perhaps through some signal transduction pathway, to enhance auxin production and thereby to increase the expression of the karyopherin α1 protein necessary for its propagation.

Materials and Methods

Plasmid construction.

To create the plant transformation constructs, plasmid pBINPLUS29 was digested with HindIII and BamHI, and the iaaM gene from Agrobacterium tumefaciens, driven by the cel1 promoter,30 was introduced into it as one piece flanked by the HindIII and BamHI sites. Then, plasmid pLM1-GUS20 was digested with BglII and HindIII to release the LeKAPα1-GUS fragment. AscI adaptors were added to the resultant fragment, which was then inserted into the AscI site of the above mentioned pBINPLUS derivative which harbored the iaaM gene driven by the cel1 promoter. The resultant plasmid, pKAPGUSiaaM (Fig. 1), bearing both a gene for IAA production and a GUS reporter gene under the control of the LeKAPα1 promoter, was used for tobacco plant transformation.

Figure 1.

Figure 1

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A schematic representation of the plasmid pKAPGUSiaaM.

Transgenic plant material.

The production of the LM1-GUS transgenic plants was previously described in reference 20. These plants were used here to study the effect of exogenous auxin on karyopherin α1 gene expression. In order to study the effect of endogenous overproduction of auxin on LeKAPα1 promoter activity, the binary vector pKAPGUSiaaM was introduced into Agrobacterium tumefaciens EHA105 by the freeze-thaw method. A. tumefaciens containing the pKAPGUSiaaM plasmid were used for transformation of tobacco (Nicotiana tabacum cv. Samsun NN), which was performed as described previously in reference 20. Kanamycin-resistant transgenic tobacco plants were selected and continued their development in Magenta boxes in a growth chamber at 24°C under fluorescent white light in 16/8 h light/dark cycle. At the age of 75 days, plants were screened for GUS activity.

Auxin treatment.

To prepare aqueous solution of IAA (Sigma Chemicals), a stock solution of 1 mg mL−1 of IAA was made in ethanol, and then warm deionized water were added to make the indicated final concentrations. To test the effect of exogenous auxin on LeKAPα1 promoter, wild-type and LM-GUS transgenic tobacco seedlings were grown in Magenta boxes in sterile conditions on half MS media (0.3% agar) including kanamycin 100 mgL−1. Ten-day-old seedlings were transferred to Petri dishes, containing 10 seedlings each, and incubated 48 h in liquid half MS medium with 0 to 3 µg mL−1 IAA. This was followed by GUS expression analysis.

Histochemical GUS staining.

Localization of reporter gene expression was visualized by in situ histochemical staining. Seedlings were vacuum-infiltrated for 30 min with a staining solution containing 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc, Molecular Probes, Eugene, OR USA) at pH 7.0 and 0.1% Tween 20, and were incubated at 37°C for 2 h according to Jefferson31 and Stomp.32 After histochemical staining for GUS activity the plants were vacuum-infiltrated for at least 4 h with a clearing solution of 100% chloral hydrate: 90% lactic acid (2:1 v/v) and were kept at 4°C over night. Cross-sections of ten-week-old stems were made with sharp razor blades and slices were stained for GUS as described, and visualized by light microscopy. GUS staining was visualized using an MZFIII stereoscope (Leica, Heerbrugg, Switzerland) or a BMLB light microscope (Leica). In all histochemical assays, the pattern of GUS staining was confirmed by observations in at least five different transgenic lines. Control non-transformed plants were analyzed for GUS activity in order to exclude non-specific staining resulting from endogenous activity.

Radioimmunoassay for IAA.

For detection and quantification of IAA, we used a standard radioimmunoassay (RIA) procedure,33 which allows the measurement of physiological levels of IAA in plant tissues. One gram of leaf tissue was homogenized in 5 mL of 80% methanol containing 100 mM ammonium acetate and 45 µM butylated hydroxytoluene. Homogenization was performed for 1 min in an ice bath. The homogenized samples were kept in the dark at 4°C. After 30 min the samples were vigorously mixed, centrifuged at 10,000 rpm for 15 min, and the supernatant was saved. The pellet was resuspended in 5 mL of distilled water and centrifuged as above. Both supernatants were pooled, and IAA was subjected to three open-column liquid chromatography steps. The extract was loaded onto polyvinylpolypyrrolidone column, and IAA was eluted with 10 mM ammonium acetate. The elute was then loaded directly onto DEAE-Sephadex column in the acetate form equilibrated with the same solution. The IAA was eluted from the DEAE-Sephadex column with 1 M acetic acid and applied to a C18 Sep-Pak column. The column was washed with distilled water to remove acetic acid and IAA was eluted with methanol. The methanol solution containing the IAA was evaporated to a small volume and dried under a stream of nitrogen. The IAA was dissolved in 100 µL methanol and methylated with 900 µL diazomethane for 20 min with gentle shaking. The methanol and diazomethane were evaporated under N2, and redissolved in 50 µL of methanol, and 950 µL RIA buffer. IAA was quantified by RIA according to Weiler.34

Acknowledgements

This article is dedicated to the memory of Dr. Aaron Zelcer (1943–2010), to whom we are also thankful for many helpful discussions. This work was supported by a grant from the US-Israel Binational Agricultural Research and Development Fund (BARD) to Y.G. This paper is a contribution from the Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel, no. 134/2010.

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