The mechanism of auxin transport in lateral root spacing

Abstract

Transport of the plant hormone auxin (Indole-3-acetic acid; IAA) is crucial to ensure proper growth patterns. In particular, the regular spacing of lateral roots (LRs) is governed by intricate auxin movements and an internal oscillator, called the “root clock” (Xuan et al., 2020Xuan W. De Gernier H. Beeckman T. The dynamic nature and regulation of the root clock.Development. 2020; 147: dev181446Crossref PubMed Scopus (15) Google Scholar). Multiple auxin influx and efflux carriers have been characterized in the broad context of root development. However, one of the remaining challenges is to puzzle out the auxin transport machinery that regulates the root clock and LR spacing. One of the first indications of the central role of auxin transport in LR spacing came from the observation that the auxin transport inhibitor 1-Naphthylphthalamic acid (NPA) inhibits LR initiation in Arabidopsis (Casimiro et al., 2001Casimiro I. Marchant A. Bhalerao R.P. Beeckman T. Dhooge S. Swarup R. Graham N. Inze D. Sandberg G. Casero P.J. et al.Auxin transport promotes Arabidopsis lateral root initiation.Plant Cell. 2001; 13: 843-852Crossref PubMed Scopus (758) Google Scholar). Although this inhibitor indiscriminately inhibits shootward and rootward auxin transport, it was at that time already proposed that rootward auxin transport is involved LR spacing. Subsequent research led to the concept of a root clock that instructs the spatiotemporal LR distribution (Xuan et al., 2020Xuan W. De Gernier H. Beeckman T. The dynamic nature and regulation of the root clock.Development. 2020; 147: dev181446Crossref PubMed Scopus (15) Google Scholar). The output of such oscillator is the periodic formation of auxin-response maxima shootward of the apical meristem, followed by LR organogenesis when a critical amplitude is reached (Figure 1). Interestingly, the amplitude and periodicity of the oscillatory auxin response depend on auxin derived from the LR cap (LRC) via an NPA-sensitive mechanism (Xuan et al., 2020Xuan W. De Gernier H. Beeckman T. The dynamic nature and regulation of the root clock.Development. 2020; 147: dev181446Crossref PubMed Scopus (15) Google Scholar). This therefore suggests that the root clock is controlled by auxin transport. In addition, mutant analyses and tissue-specific complementation studies show that the oscillatory auxin-response amplitude most likely reflects auxin signaling in the stele, rather than in the outer tissues (Xuan et al., 2020Xuan W. De Gernier H. Beeckman T. The dynamic nature and regulation of the root clock.Development. 2020; 147: dev181446Crossref PubMed Scopus (15) Google Scholar). The spatial separation between the auxin source (LRC) and the site of auxin perception (stele) thus suggests the additional involvement of radial inward auxin transport across the epidermis, cortex, and endodermis in tuning the root clock (Figure 1). Genetic and molecular analyses have allowed the development of in silico models that reproduce realistic auxin accumulation patterns in the root, suggesting that they capture the main auxin transport routes in the root. This thus allows interrogation of the individual contributions of different auxin influx and efflux transporters in the model to the root clock. Consistent with the model, the auxin-uptake activity of the IAA−/H+ symporter AUX1 in the LRC could be experimentally validated as important for LR spacing (Xuan et al., 2020Xuan W. De Gernier H. Beeckman T. The dynamic nature and regulation of the root clock.Development. 2020; 147: dev181446Crossref PubMed Scopus (15) Google Scholar). In contrast, none of the mutants in ABCB- and PIN-type auxin transporters with predicted key roles in auxin efflux in the model display reduced LR formation. This illustrates an important gap in our understanding about how auxin transport dictates LR positioning via the root clock. To date, the strongest experimental evidence for the involvement of auxin efflux transporters in regulating the root clock derives from the chemical auxin transport inhibitors NPA and BUM (2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid) (Casimiro et al., 2001Casimiro I. Marchant A. Bhalerao R.P. Beeckman T. Dhooge S. Swarup R. Graham N. Inze D. Sandberg G. Casero P.J. et al.Auxin transport promotes Arabidopsis lateral root initiation.Plant Cell. 2001; 13: 843-852Crossref PubMed Scopus (758) Google Scholar; Xuan et al., 2020Xuan W. De Gernier H. Beeckman T. The dynamic nature and regulation of the root clock.Development. 2020; 147: dev181446Crossref PubMed Scopus (15) Google Scholar). Therefore, these chemicals can help in identifying the auxin transporter mechanism that controls the root clock. Both NPA and BUM likely interfere with ABCB-mediated auxin transport via disruption of the association of ABCBs with the regulatory immunophilin-like FKBP42, TWISTED DWARF1 (Bailly et al., 2008Bailly A. Sovero V. Vincenzetti V. Santelia D. Bartnik D. Koenig B.W. Mancuso S. Martinoia E. Geisler M. Modulation of P-glycoproteins by auxin transport inhibitors is mediated by interaction with immunophilins.J. Biol. Chem. 2008; 283: 21817-21826Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Recently, NPA was also proposed to inhibit PIN-mediated auxin transport via direct interaction (Abas et al., 2021Abas L. Kolb M. Stadlmann J. Janacek P.D. Lukic K. Schwechheimer C. Sazanov L.A. Mach L. Friml J. Hammes U.Z. Naphtylphtalamic acid associates with and inhibits PIN auxin transporters.Proc. Natl. Acad. Sci. U S A. 2021; 118 (e2020857118)Crossref PubMed Scopus (18) Google Scholar) and altered PIN dimerization (Abas et al., 2021Abas L. Kolb M. Stadlmann J. Janacek P.D. Lukic K. Schwechheimer C. Sazanov L.A. Mach L. Friml J. Hammes U.Z. Naphtylphtalamic acid associates with and inhibits PIN auxin transporters.Proc. Natl. Acad. Sci. U S A. 2021; 118 (e2020857118)Crossref PubMed Scopus (18) Google Scholar; Teale et al., 2021Teale W.D. Pasternak T. Dal Bosco C. Dovzhenko A. Kratzat K. Bildl W. Schworer M. Falk T. Ruperti B. J V.S. et al.Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport.EMBO J. 2021; 40: e104416Crossref PubMed Scopus (16) Google Scholar). However, no mutants in PINs or ABCBs phenocopy the NPA root clock phenotype. Higher-order mutants of members of a group of AGC kinases (D6PK) regulating PIN-mediated auxin transport lack LRs (Barbosa et al., 2018Barbosa I.C.R. Hammes U.Z. Schwechheimer C. Activation and polarity control of PIN-FORMED auxin transporters by phosphorylation.Trends Plant Sci. 2018; 23: 523-538Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The central role of D6PK activity in LR development is corroborated by the characterization of D6PK-regulatory PDK1/2 kinases, whose double mutant is also defective in LR development (Tan et al., 2020Tan S. Zhang X. Kong W. Yang X.L. Molnar G. Vondrakova Z. Filepova R. Petrasek J. Friml J. Xue H.W. The lipid code-dependent phosphoswitch PDK1-D6PK activates PIN-mediated auxin efflux in Arabidopsis.Nat. Plants. 2020; 6: 556-569Crossref PubMed Scopus (17) Google Scholar). Notably, the expression patterns of the synthetic auxin-response reporter DR5::GUS in d6pk and pdk1/2 mutant roots correspond to those seen after NPA treatment (Xiao and Offringa, 2020Xiao Y. Offringa R. PDK1 regulates auxin transport and Arabidopsis vascular development through AGC1 kinase PAX.Nat. Plants. 2020; 6: 544-555Crossref PubMed Scopus (17) Google Scholar). Moreover, d6pk mutant root elongation is hypersensitive to NPA, suggesting that D6PK also affects the root clock. An indirect link between D6PK activity and root clock regulation can be inferred from the rapid plasma membrane dissociation and inactivation of D6PKs in response to the fungal toxin Brefeldin A (BFA; Barbosa et al., 2018Barbosa I.C.R. Hammes U.Z. Schwechheimer C. Activation and polarity control of PIN-FORMED auxin transporters by phosphorylation.Trends Plant Sci. 2018; 23: 523-538Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). This response depends on BFA's ability to inhibit the ADP-ribosylation factor–guanine exchange factor GNOM (Barbosa et al., 2018Barbosa I.C.R. Hammes U.Z. Schwechheimer C. Activation and polarity control of PIN-FORMED auxin transporters by phosphorylation.Trends Plant Sci. 2018; 23: 523-538Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), which has recently been shown to be essential for the root clock function (Wachsman et al., 2020Wachsman G. Zhang J. Moreno-Risueno M.A. Anderson C.T. Benfey P.N. Cell wall remodeling and vesicle trafficking mediate the root clock in Arabidopsis.Science. 2020; 370: 819-823Crossref PubMed Scopus (24) Google Scholar). D6PKs can activate PIN-mediated auxin transport via direct phosphorylation (Barbosa et al., 2018Barbosa I.C.R. Hammes U.Z. Schwechheimer C. Activation and polarity control of PIN-FORMED auxin transporters by phosphorylation.Trends Plant Sci. 2018; 23: 523-538Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Interestingly, phosphomimetic substitutions that overlap with a D6PK-phosphorylation site seem to modify PIN1's capacity to dimerize (Teale et al., 2021Teale W.D. Pasternak T. Dal Bosco C. Dovzhenko A. Kratzat K. Bildl W. Schworer M. Falk T. Ruperti B. J V.S. et al.Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport.EMBO J. 2021; 40: e104416Crossref PubMed Scopus (16) Google Scholar). This suggest that NPA and D6PK antagonize each other at the level of PIN-mediated auxin transport, highlighting a potential involvement of PINs in the root clock (Figure 1). However, the function of PINs in the root clock could not yet be determined due to functional redundancy and pleiotropic developmental defects in higher-order pin mutants. Alternatively, it is possible that other joint targets control the root clock (Figure 1). LR initiation is not universally sensitive to NPA. In late-diverged ferns, such as Ceratopteris richardii, NPA has no impact on LR initiation, nor on its morphogenesis (Ilina et al., 2018Ilina E.L. Kiryushkin A.S. Semenova V.A. Demchenko N.P. Pawlowski K. Demchenko K.N. Lateral root initiation and formation within the parental root meristem of Cucurbita pepo: is auxin a key player?.Ann. Bot. 2018; 122: 873-888PubMed Google Scholar). What is more, the lack of NPA binding in PINs of green algae (Klebsormidium flaccidum) (Abas et al., 2021Abas L. Kolb M. Stadlmann J. Janacek P.D. Lukic K. Schwechheimer C. Sazanov L.A. Mach L. Friml J. Hammes U.Z. Naphtylphtalamic acid associates with and inhibits PIN auxin transporters.Proc. Natl. Acad. Sci. U S A. 2021; 118 (e2020857118)Crossref PubMed Scopus (18) Google Scholar), and the shootward auxin transport in C. richardii roots being largely NPA insensitive (Zhang et al., 2019Zhang Y. Xiao G. Wang X. Zhang X. Friml J. Evolution of fast root gravitropism in seed plants.Nat. Commun. 2019; 10: 3480Crossref PubMed Scopus (41) Google Scholar), is consistent with an independent evolutionary origin of LR branching in ferns (Hetherington et al., 2020Hetherington A.J. Berry C.M. Dolan L. Multiple origins of dichotomous and lateral branching during root evolution.Nat. Plants. 2020; 6: 454-459Crossref PubMed Scopus (6) Google Scholar). Despite the putative single evolutionary origin of LR branching in the seed plant lineage (Hetherington et al., 2020Hetherington A.J. Berry C.M. Dolan L. Multiple origins of dichotomous and lateral branching during root evolution.Nat. Plants. 2020; 6: 454-459Crossref PubMed Scopus (6) Google Scholar), not all seed plants display NPA-sensitive LR initiation. An example of this is pumpkin (Cucurbita pepo), in which NPA does not abolish LR initiation, but impairs LR morphogenesis (Ilina et al., 2018Ilina E.L. Kiryushkin A.S. Semenova V.A. Demchenko N.P. Pawlowski K. Demchenko K.N. Lateral root initiation and formation within the parental root meristem of Cucurbita pepo: is auxin a key player?.Ann. Bot. 2018; 122: 873-888PubMed Google Scholar), suggesting auxin transport dependence of the latter, but not the former. Interestingly, LR initiation in C. pepo occurs in close proximity to the root stem cell region, while LR initiation in Arabidopsis and the bulk of other seed plants occurs distal to the meristem, explaining a differential dependence for auxin transport in both types of LR spacing mechanisms. Remarkably, the most primitive root branching mechanism that could be detected in the extinct lignophytes (seed plants and their seedless ancestors that produced the wood lineage) is root bifurcation, a process that occurs at the very tip of the meristem (stem cell region). This is consistent with the proposal that LR formation as seen in C. pepo corresponds to an evolutionary intermediate between bifurcation and Arabidopsis-type LR formation (Ilina et al., 2018Ilina E.L. Kiryushkin A.S. Semenova V.A. Demchenko N.P. Pawlowski K. Demchenko K.N. Lateral root initiation and formation within the parental root meristem of Cucurbita pepo: is auxin a key player?.Ann. Bot. 2018; 122: 873-888PubMed Google Scholar). During this evolution, NPA-sensitive auxin transport seems to be recruited to maintain communication between the meristem and the more distal site of LR initiation. Even after 20 years of research, the auxin transport mechanism that controls LR spacing still remains to be fully elucidated. Recent findings on NPA targets and PDK–D6PK activity strongly point to the involvement of PINs. However, their involvement in the root clock has been difficult to assess. We anticipate that the advent of tissue-specific and inducible genome-editing approaches (Wang et al., 2020Wang X. Ye L. Lyu M. Ursache R. Loytynoja A. Mahonen A.P. An inducible genome editing system for plants.Nat. Plants. 2020; 6: 766-772Crossref PubMed Scopus (33) Google Scholar) will allow us to overcome pleiotropic developmental defects in higher-order pin mutants and finally answer this open question in developmental biology.