“CLASPing” tungsten's effects on microtubules with “PINs”

Abstract

Tungsten, supplied as sodium tungstate, inhibits root elongation in Arabidopsis thaliana, which has been attributed to a diminishing of PIN2 and PIN3 auxin efflux carriers. In this work, we sought to analyze the effect of tungsten on cortical microtubules and CLASP (Cytoplasmic Linker Associated Protein), which are also involved in the anisotropic cell expansion of root cells. Seedlings grown in a tungsten-free substrate for 4 d and then transplanted into a tungsten-containing substrate exhibited randomly oriented microtubules in a time-dependent manner. While tungsten had no effect on roots treated for 3 h, microtubule alignment was obviously affected in the transition and elongation zones after a 6, 12, 24, 48 h tungsten treatment, at prolonged tungsten administrations and in seedlings grown directly in the presence of tungsten. This change in microtubule orientation may be associated with the reduction of CLASP protein expression induced by tungsten, as evidenced in experiments with plants expressing the CLASP-GFP protein. A possible mechanism, by which the coordinated functions of CLASP, PIN2 and microtubules are affected, as revealed by inhibited root growth, is discussed.

The toxicological profile of tungsten has recently become a matter of increasing concern, due to its inspected enrolment in a children's leukemia cluster, its potential toxicity to organisms, and its increasing presence in the environment as a result of human activity.¹ Tungsten is taken-up actively by animals and plants, raising concerns about its biological action.² Tungsten uptake by plants, especially by agricultural crops, is at present investigated as it may enter the food supply chain³ but also because tungsten-polluted sites could be remediated by phytoextraction⁴ to protect the environment from its latent toxicity.

The response of Arabidopsis thaliana to tungsten's presumed toxicity was so far restricted to a hindering of root elongation, due to the strong inhibition of nitrate reductase-mediated nitric oxide production.^5,6 Any detailed effects of the metal on A. thaliana were hitherto unknown. Recently, Adamakis et al. (2014)⁷ reported that reduced root growth of A. thaliana is probably mediated through a nitric oxide-independent mechanism, involving auxin transport disruption.

Tungsten Induces Microtubule Reorientation in Arabidopsis thaliana Root

Tungsten toxicity has been found to affect cortical microtubules in various plants.⁸ The specific effect of tungsten varied among different species and cell types, from depolymerization in Pisum sativum roots⁹ to even no effect at all in A. thaliana hypocotyls and leaves.⁸ However, the effects of tungsten on A. thaliana root microtubules have not been thoroughly investigated. It has been stated that reduced root growth of A. thaliana is probably due to defective cell expansion in the elongation zone, resulting in the dwarfish appearance of tungsten-treated plants.⁷ As cell expansion and cortical microtubule orientation are interconnected,^10,11 tungsten's effects on cortical microtubule orientation in A. thaliana roots were investigated in further detail. In untreated roots, cortical microtubules followed distinct orientation patterns, as already described.¹⁰ In brief, at the external cell faces, the early protodermal cells of control roots in the meristematic zone exhibited cortical microtubules in a loose longitudinal arrangement, which in the transition zone became uniformly transverse, relatively to the longitudinal root axis (Fig. 1A). Transverse orientation persisted in the cells of the elongation zone (Fig. 1A, B, and C), a pattern altered in the cells of the differentiation zone (Fig. 1D).

Figure 1.

Tubulin immunostaining in control roots of A. thaliana (ecotype Columbia-0), conducted according to previously described protocols.^7,10 In this study the terms “rootward” and “shootward” were adopted to determine cell polarity.¹⁹ In accordance with Baluška et al. (2010),¹⁷ A. thaliana growing region of the root tip was considered to consist of 3 zones: the meristematic, the transition and the elongation zone, all of which constituted the undifferentiated zone. The region where root hair formation took place was considered as the maturation zone. Projections of serial CLSM optical sections through the protodermal cell files at different distances from the root apex and at successive developmental stages. (A) Meristematic, transition and early elongation zone. (B, C) Elongation zone. (D) Differentiation zone. Microtubule orientation shifts from random in the meristematic zone (bracket in A) to transverse in the early transition zone (arrows in A), which persists throughout the elongation zone (B, C), but changes to rather loose in the early differentiation zone (D). Scale bars: 20 μm.

Seedlings germinated on a tungsten-free medium for 4 d and then transferred to medium with tungsten displayed disrupted microtubule orientation in a time-dependent manner (Fig. 2). In particular, a 3 h treatment with tungsten had no effect (data not shown). The transition zone cells of roots treated with tungsten for 6 h exhibited randomly oriented microtubules indicating that the transition zone cells were firstly affected, while those of the elongation zone bore transversely aligned microtubules (Fig. 2A). Cells with randomly arranged microtubules were found in both the transition and elongation zones of roots treated with tungsten for 12, 24 and 48 h (Fig. 2B, C, and D, respectively). At prolonged exposures (10 days; Fig. 2E) and in seedlings directly grown in the presence of tungsten (data not shown), the effect was intensified and trichoblasts with randomly oriented microtubules seemed to develop just above the cells of the transition zone.

Figure 2.

Projections of serial CLSM optical sections through the protodermal cell files after tubulin immunostaining at the transition and early elongation zones of tungsten-treated roots of A. thaliana. Four-day-old seedlings were transplanted in Petri dishes in culture media supplemented with 100 mg/L sodium tungstate (Na₂WO₄). This concentration was used considering that in some cases of tungsten pollution its concentration was found to vary between 135 and 337 mg/L²⁰. Treatments lasted for 3, 6, 12, 24 and 48 h, while prolonged exposures of 4, 6 and 10 d were also conducted. Moreover, seedlings directly grown in the presence of tungsten for 9 consecutive days were studied as well. (A) After 6 h of tungsten treatment, microtubule orientation is random in both the meristematic (bracket) and the transition (arrowheads) zones and becomes transverse (arrows) in the early elongation zone. (B-D) After 12, 24 and 48 h of tungsten treatment, microtubule orientation appears severely affected, as cells in the transition and early elongation zones bear randomly oriented microtubules (arrowheads). (E) At prolonged tungsten treatments (10 days), cells with transversely oriented microtubules (arrows) co-exist with cells exhibiting randomly aligned microtubules (arrowheads), while root hairs (asterisk) are found close to the root tip. Scale bar: 50 μm.

Tungsten severely reduces CLASP expression

CLASP (Cytoplasmic Linker Associated Protein) belongs to a family of microtubule associated proteins (MAPs) involved in the attachment of microtubules to the cell cortex in both animal¹² and plant¹³ cells. Previous reports stated that mutations in the A. thaliana CLASP gene resulted in characteristic cell expansion-defective phenotypes, as the roots of clasp-1 mutant had significantly short elongation zone.¹⁴ Importantly, apart from any direct involvement of CLASP in cortical microtubule orientation¹⁵, defective CLASP expression was correlated with a reduction of the auxin transporter PIN2.¹⁶

Taking into account the similarity between the phenotype of tungsten-treated roots⁷ and those of clasp-1,¹⁶ as well as that both were related with a defect in PIN2 abundance, the expression of CLASP-GFP was followed in untreated and tungsten-treated A. thaliana roots. In untreated CLASP-GFP expressing seedlings the CLASP-GFP signal was intense (Fig. 3A), in 3 or 6 h treatments it resembled that of the untreated seedlings (data not shown), but after 24 h and in seedlings directly grown in tungsten-containing media, the CLASP-GFP signal was severely reduced (Fig. 3B, C). Accordingly, it appears that the effect of tungsten on A. thaliana root growth involves 3 players: cortical microtubule orientation, CLASP expression and PIN2 abundance.

Figure 3.

Projections of serial CLSM sections through the root meristem of CLASP-GFP expressing A. thaliana seedlings. Seeds of A. thaliana L. (Heynh) (ecotype Columbia-0) expressing CLASP-GFP were purchased from the NASC (European Arabidopsis Stock Center). Control (A) and tungsten-treated (B, C) as indicated on image. In the presence of 100 mg/L tungsten a reduction in CLASP signal is noticed. Scale bar: 50 μm.

CLASP, PIN2, microtubules: “Tres faciunt collegium”

Adamakis et al. (2014)⁷ found that the reduced root growth of A. thaliana due to tungsten is probably mediated through an auxin transport disruption mechanism. In particular, the polar basipetal “outside-in” auxin transport, normally generated by the concerted action of PIN2 and PIN3,¹⁷ was disrupted by tungsten since the expression of PIN2 and PIN3 decreased. Consequently, cell expansion in the elongation zone was inhibited, thus explaining the dwarfish appearance of tungsten-treated plants.

The very first players affected by tungsten are PIN2 expression⁷ and microtubule orientation (this study). The latter may be considered as an indicator of inhibition of expansion, as it is gradually altered during tungsten treatment in the roots. Firstly, cortical microtubule orientation is initially altered in the transition zone only (Fig. 2A), which could be related to the diminishing of PIN2 expression, since transition zone cells rely upon auxin signaling to enter the elongation zone.¹⁸ Arrest of elongation in transition zone cells may result in microtubule reorientation, as generally occurs by cessation of expansion.^10,11 As a second step, tungsten affects CLASP expression (Fig. 3B and C). This may result in a further decrease in PIN2 expression, extending the inhibitory effect shootward to the elongation zone, inducing extensive microtubule reorientation. According to this view, the phenotype similarity between clasp-1 and tungsten-treated wild-type roots can be attributed to CLASP deficiency. Consequently, tungsten seems to affect root elongation in 2 phases, one early, without the involvement of CLASP, and another, enhancing the inhibitory effect, including the effect of CLASP on PIN2.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) – Research Funding Program: Heracleitus II.