Coordination of Tissue Cell Polarity by Auxin Transport and Signaling

Background: Auxin transport shapes the Arabidopsis vein network

The plant vascular system, which transports water, nutrients and assimilates, represents a network of cell files extending through all organs. In leaves, these vascular strands are referred to as veins or nerves and are arranged in a ramified pattern with a central midvein, lateral loops and higher order terminating and connecting veins¹. They are inherently polar structures as their cells are elongated along the vein axis and the vein network connects the leaf with stem vasculature. In agreement with this, vein formation requires coordination of cell polarity, and the plant hormone auxin has long been established as a central player in this process².

In particular, polar auxin transport defines the site of vein formation: from a local maximum, auxin flows towards the basal part of the plant, and this flow determines where vascular strands are formed¹. This polarity in auxin flux is controlled by auxin efflux carriers of the PIN-FORMED (PIN) family, especially PIN1, and directed auxin flux feeds back on PIN1 asymmetrical distribution^1,3. How polar PIN1 distribution is established is not fully understood, but the guanine-nucleotide exchange factor GNOM (GN), which regulates vesicle formation in membrane trafficking, is thought to play a pivotal role in this process. gn mutants display altered venation patterns as well as changes in PIN1 subcellular trafficking, suggesting that GN controls PIN1 distribution during vein pattern formation⁴.

Key findings: Not only auxin transport, but also auxin signalling contributes to vein patterning downstream of GN.

The authors decided to test the prevailing hypothesis that GN controls vein patterning via polar distribution of PIN proteins. They first confirmed that vein patterning is severely compromised in gn mutants. Intermediate gn alleles displayed generally thicker veins, lateral veins running parallel to instead of joining the midvein and narrow clusters of vascular elements forming near the leaf margin. In several strong gn mutant alleles the vein network was reduced to a central, shapeless cluster of vascular elements (Fig. 1). Furthermore, a PIN1::PIN1-GFP reporter showed a broader expression domain and displayed strongly reduced polarity in gn compared to the wild-type background. These observations agree with previous reports and with the idea that GN controls venation via PIN1 polar localisation.

PIN1 is part of an eight-member family, and plasma membrane-localised PIN3, PIN4 and PIN7 as well as ER-localised PIN6 and PIN8 contribute to vein patterning. Simultaneous mutation of these six PIN genes causes strong vein patterning defects, with many lateral veins failing to connect to the midvein and running parallel instead; this phenotype is also observed when plants are treated with the PIN auxin transport inhibitor NPA (Fig. 1). However, neither pin1,3,4,6,7,8 mutants nor NPA-treated wild type fully phenocopied the vein patterning defects of gn mutants, suggesting that additional factors act downstream of GN.

Figure 1: Dark-field illumination of mature first leaves of wild type (A), the pin1,3,4,6,7,8 mutant (B), wild type treated with the auxin transport inhibitor NPA (C) and the severe gn-13 mutant (D). Scale bars represent 0.5 mm (reproduced with altered numbering from Verna et al., Fig. 5A, C, D and Fig. 8A).

Besides PINs, transport proteins of the ATP BINDING CASSETTE B (ABCB) and AUXIN1/LIKE AUX1 (AUX1/LAX) families are known to control polar auxin efflux and influx, respectively. To test whether these transporters contribute to GN-mediated vein patterning, Verna et al. analysed the phenotype of various combinations of pin, abcb and aux1/lax mutants as well as of NPA-treated abcb and aux1/lax mutants. None of the respective mutants displayed gn-like vein pattern defects, suggesting that ABCBs and AUX1/LAX transporters are not the missing component regulating venation downstream of GN.

Local auxin application can induce the formation of additional veins in the wild type, but intriguingly, Verna et al. also observed this effect in pin1,3,4,6,7,8 mutants. This implies that in the absence of PINs, there is still an auxin-dependent, yet apparently auxin transport-independent, mechanism inducing vein formation. Double mutants of TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and AUXIN SIGNALING F-BOX 2 (AFB2) as well as mutants of AUXIN RESISTANT 1 (AXR1), which are both severely compromised in auxin signalling, display vein pattern defects such as open loops and fragmented veins. Treatment of these mutants with NPA to inhibit PIN function generated phenotypes similar to those of intermediate gn alleles. These phenotypes were also observed in axr1 pin1,3,4,6,7,8 and tir1 afb2 pin1,3,4,6,7,8 mutants, while combining gn and axr1 mutations did not further enhance the vein pattern defects. Finally, PIN1-GFP distribution is similarly altered in NPA-treated tir1 afb2 mutants and in intermediate gn mutants. Taken together, these observations suggest that AXR1- and TIR1/AFB2-mediated auxin signalling contributes to vein patterning and establishment of polarity downstream of GN.

What I like about this preprint

There is one word that came to mind after reading this preprint: thorough. The authors have performed an extremely comprehensive genetic and pharmacological analysis of vein patterning and thereby revealed that our knowledge about this process does not stretch as far as we previously thought it does.

Open questions/future directions

This study has raised many more questions about vein pattern formation since it has shown us that our previous concepts, while not incorrect, were clearly incomplete. As the authors state, the obvious follow-up question will be how auxin signalling, being inherently unpolar, can promote polarity in vein patterning, but this will potentially require years of investigation. Other questions more directly related to the present preprint are:

• Compromising PIN-mediated auxin transport and auxin signalling phenocopies intermediate, but not strong, gn mutant alleles. Is this difference due to residual auxin signalling in the tested mutant backgrounds, or could there be an auxin-independent component to GN function as well? In this regard, have the authors tested whether auxin application can still induce vein formation in NPA-treated tir1 afb2 or axr1 mutants?
• PIN1-GFP localisation is one read-out for polarity during the vein patterning process. It would be interesting to know whether the authors have looked at the subcellular localisation of any other PIN protein in a gn mutant background.

References/Further reading

1. Scarpella E, Barkoulas M, Miltos Tsiantis M (2010) Control of Leaf and Vein Development by Auxin. Cold Spring Harb Perspect Biol 2:a001511.
2. Sachs T (1981) The control of the patterned differentiation of vascular tissues. Adv Bot Res 9:151–262
3. Linh NM, Verna C, Scarpella E (2018) Coordination of cell polarity and the patterning of leaf vein networks Curr Opin Plant Biol 41:116–124
4. Richter S, Anders N, Wolters H, Beckmann H, Thomann A, Heinrich R, Schrader J, Singh MK, Geldner N, Mayer U, Jürgens G (2010) Role of the GNOM gene in Arabidopsis apical-basal patterning – From mutant phenotype to cellular mechanism of protein action. Eur J Cell Biol 89:138-144

 

Tags: arabidopsis, auxin, polarity, vein patterning

doi: https://doi.org/10.1242/prelights.12862

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