Boronic acids as tools to study (plant) developmental processes?

ABSTRACT

Boron (B) is an essential micronutrient for organisms. In plants, B is known to stabilize the cell wall by crosslinking Rhamnogalacturonan II through ester bonds formed with cis-diols of sugar moieties. However, B is believed to be required for additional functions such as stability and function of (plasma membrane) proteins involved in signal transduction pathways. We have recently shown that boronic acids, competitors of B, efficiently induce perfect phenocopies of monopteros mutants. This effect is enigmatic because like B, boronic acids should find numerous cellular targets and thus disturb many biologic processes ending in a spectrum of unspecific embryo phenotypes. Based on chemical characteristics of boronic acids and their derivatives we discuss reasons that could explain this unusual specificity. The peculiarities of this class of compounds could provide new tools for studying developmental processes.

The essential role of boron (B) for plant development was established in the early 20^th century.¹ One proven function of B in plants is the stabilization of cell walls by cross-linking Rhamnogalacturonan II pectic polysaccharides.^2,3 In fact B's capability to form (ester) cross-links between neighbored molecules through their cis-diol moieties is key for understanding its molecular functions.^4,5

Although boric acid is capable to enter and exit cells by diffusion, plasma membrane (PM) B transporters are required to adjust B´s homeostasis in plants when its concentration is too high or too low in the soil.⁶ Notably, active B transport is also necessary in mammals as shown by the electrogenic Na⁺-coupled B transporter NaBC1.⁷ Consequently, B´s significance for human and animal nutrition is discussed⁸ and various observations in animals^9-11 support stabilizing roles for B in PM protein function - one possible target moiety being cis-diols of glycosyl residues. Interestingly, comparable roles for B in plant PM protein stability and in phytohormone signaling, specifically auxin (= IAA, indole-3-acetic acid), have been proposed.¹²

Our work places one possible role of B very near to these ideas.¹³ It was prompted by an earlier report, where phenylboronic acid (PBA) was shown to induce monocotyly in Eranthis hyemalis.¹⁴ Boronic acids such as PBA are specific B competitors with the same binding specificity as boric acid but cannot serve to cross-link two molecules.¹⁵ PBA treatment of Arabidopsis thaliana embryos phenocopied monopteros mutants (mp-phenocopies) by specifically affecting embryonic root development. This process depends on two essential signals - the presence of an auxin maximum and of the transcription factor (TF) TARGET OF MONOPTEROS 7 (TMO7) in the hypophysis cell.¹⁶ Both signals are in-/directly controlled by the TF MONOPTEROS (MP), which becomes active when its repressor BODENLOS (BDL) is degraded. This in turn depends - on the presence of auxin, which is mainly transported by the efflux carrier PINFORMED1 (PIN1). With this element a feedback loop regulation is established because MP itself activates PIN1 and TMO7. Our analysis of mp-phenocopies detected two possible points where PBA affects this system: the degradation of BDL and/or the stability of PIN1 at the PM.

The induction of the mp phenocopies by boronic acids is intriguing for several reasons. First, the effect is highly precise because mp-phenocopies cannot be distinguished on a morphological level from mp mutants. Second, the effect is highly efficient because it performs to completion - a silique harbouring 50 ovules might provide 50 mp-phencopies. Third, the timing is highly specific - mp phenocopies are only induced during the formative division of the hypophysis.

With the cis-diol binding capacity of PBA in mind one would expect to find a plethora of possible targets causing general, stochastic disturbances of disparate cellular processes. This in turn should produce a spectrum of unspecific phenotypes scattered along the complete embryo developmental time scale like numerous mutations found in saturated mutant embryo screens.¹⁷ At present, we can only speculate about some possibilities, which might explain this exceptional specificity.

One important feature of boronic acids is that their affinity to possible targets varies. Chemical analysis has revealed that the substituents of PBA might alter its affinities.¹⁸ Moreover, the complexation of PBA itself with different diol containing compounds, in particular sugars, differs significantly^19,20 sometimes by orders of magnitude.²¹ This is also the case for other simple and more complex compounds such as boronic acid-functionalized rhodamine derivatives.^19,22 Affinities to sugars can also be refined combining two different boronic acids.²²

Varying affinities were also observed in cases where boronic acids inhibit hydrolytic enzymes such as fatty acid amide hydrolase²³ (FAAH). Therefore, specificity could also result from a restricted affinity to a single protein. Analysis of auxin vs. auxin+PBA treatments of roots with the auxin reporter DII opened the possibility for a targeted impact on the BDL repressor of MP.¹³ Another report demonstrated a particularly strong inhibition of root growth by two PBA derivatives (4-biphenylboronic acid and 4-phenoxyphenylboronic acid). In vitro analysis showed inhibition of the YUCCA monooxygenase enzyme activity required for the synthesis of auxin from indole-3-pyruvate.²⁴ Not only are inhibitory effects of boronic acids on enzymes known. A rhodamine-derived bis-boronic acid (RhoBo) compound has been shown to function as a highly specific, cell-permeable turn-on fluorescent sensor for the tetraserine motif SSPGSS²⁵ whereas naphthorhodamine derivatives of the same RhoBo have been developed as fluorescence carbohydrate detectors with large stoke shifts.²⁶ Thus, the peculiarities of boronic acids could indeed significantly shorten the list of their possible cellular targets and restrict them to few sugar moieties or particular proteins among a plethora of compounds including monosaccharides with hydroxyl-rich functionalities. Further restrictions might also result from different physiologic parameters such as the pH, which has substantial impact on sugar-boronic acid complexation.¹⁹

Several aspects linked to B´s/boronic acid´s impact on plant development are currently under investigation such as its impact on organogenesis in general. This includes (postembryonic) leaf development as shown for Solanum lycopersicum where the complex leaves were reduced to simple lanceolate leaves by PBA.²⁷ It is conceivable that these reductions also involve internalisation of auxin-transport related proteins like PIN1 and ENHANCER OF PINOID,¹³ whose functions and correct cellular polarisations are essential for cotyledon (i. e. embryonic leaf) development.^28,29 In addition, B depletion has been shown to induce PIN1 internalisation and interrupted growth in postembryonic Arabidopsis root development.³⁰ The identification of the corresponding B/boronic acid targets by analytical techniques is another important aim. These analyses might be stimulated by numerous boronic acid variants offered on the market and promise a bright future for these compounds as tools for studying developmental processes.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

We thank the Deutsche Forschungsgemeinschaft [To 134/8–1] and Technische Universität München (Equal Opportunity Program Fellowship to M.M.) for financial support.