LAZY1-LIKE-mediated gravity signaling pathway in root gravitropic set-point angle control

Abstract

Gravity signaling components contribute to the control of root gravitropic set-point angle through protein polarization relay within columella.

Root systems anchor plants to the substrate in addition to transporting nutrients and water, playing a fundamental role in plant survival. After seed germination, the growth of the primary root penetrates the soil in relation to gravity (Morita, 2010). In general, plant tropisms are directional movements in response to an external factor, such as gravity (gravitropism) or light (phototropism). The maintenance of vertical primary root growth is called positive orthogravitropism (Rosquete et al., 2013). Lateral root branches emerge from the primary root or node orthogonally from the primary stem–root axis. The growth of root branches tends to incline downward from the emergence axis in response to gravity (Digby and Firn, 1995; Kiss et al., 2002). Interestingly, the inclination of the growth of root branches is set and maintained relative to gravity, which is called plagiogravitropism (Rufelt, 1962). The angles created by gravitropism are known as gravitropic set-point angles (GSAs). Primary roots exhibit vertical GSAs (orthogravitropism; GSA = 0°), whereas young root branches maintain nonvertical GSAs (plagiogravitropism; GSA>0°) (Figure 1). The GSA of lateral root branches is 90° just after they emerge, and then decreases to a particular angle while inclining. The GSAs of root branches are genetically determined in accordance with developmental cues. By regulating GSAs, root branches expand radially away from the primary root or deeply along with the primary root. GSA-mediated root distribution is also affected by environmental cues, such as water, nutrients, and physical soil conditions (Braam, 2005; Bai et al., 2013; Moriwaki et al., 2013; Roychoudhry et al., 2017; Huang et al., 2018; Su et al., 2020; Yamazaki et al., 2020). Because of these root behaviors, plants exhibit a wide variety of root distribution patterns that are adaptable to diverse natural environments. It has been proposed that the GSA is determined by a balance between gravitropism and anti-gravitropic offset that counteracts gravitropic growth (Roychoudhry et al., 2013; Roychoudhry and Kepinski, 2015). Accordingly, it is conceivable that gravitropism predominates in the primary root, whereas in lateral root branches, gravitropism initially predominates, leading to GSA decrease in accordance with downward curvature, and is subsequently equilibrated with anti-gravitropic offset, resulting in GSA setup (Figure 1). However, until recently, the mechanisms underlying GSA regulation were not well understood. Here, we focus on the recent advances in our understanding of root GSA regulation with a particular focus on the role of LAZY1 family genes.

Figure 1.

LZY-mediated gravity signaling in root GSA regulation. The growth angle of roots is set and maintained related to gravity, known as root GSA. Primary roots display vertical GSAs (GSA = 0°), whereas lateral root branches maintain non-vertical GSAs (GSA>0°). g represents the direction of gravity. LZY regulates the root branch GSA by controlling gravitropism. After amyloplast sedimentation, the gravity signal is mediated by LZY-RLD-PIN polarization relay. GSAs are proposed to be determined by the balance of two opposite direction components: root gravitropism (blue) and anti-gravitropic offset (red). In lateral roots, gravitropism initially predominates over anti-gravitropic offset, and then is equilibrated with anti-gravitropic offset. Blue arrows indicate the direction of auxin transport to the lower part of the root, driven by gravitropism, and red arrows indicates the direction of auxin transport to the upper region of the root, likely to be driven by anti-gravitropic offset. Although a lateral root is maintained at a given GSA, the upper and lower auxin transport fluxes should be balanced.

Advances

• The LAZY1 family mediates gravitropism gravity signaling, contributing to root GSA control in various plant species.

• The highly conserved CCL domain of LAZY1-LIKE proteins interacts with the BRX domain of the RLD family.

• Following amyloplast sedimentation, the polarization relay of LZY-RLD-PIN in the plasma membrane of gravity-sensing columella induces lateral auxin transport to the lower part of the root tip, bending the root toward the direction of gravity.

• Anti-gravitropic offset involves gravity sensing via amyloplast sedimentation within columella; however, auxin is transported in the direction opposite to gravity, producing an upward bending of the root.

• Complete loss of the LAZY1 family function produces inverse gravitropic phenotypes, such as upward root growth, which are considered to be the result of the appearance of anti-gravitropic offset.

Gravity-signaling pathway in roots

The LAZY1 genes: key gravity-signaling players in GSA regulation

The recent identification and characterization of the LAZY1 family of genes have advanced our understanding of gravity signaling, the most poorly understood process in gravitropism (Box 1) (Morita and Tasaka, 2004; Morita, 2010; Toyota and Gilroy, 2013; Su et al., 2017; Nakamura et al., 2019a, 2019b; Su et al., 2020). LAZY1 was originally identified in rice (Oryza sativa) over 20 years ago as the gene responsible for a mutation that produces a prostrate shoot and a gravitropic defect in the leaf sheath and coleoptile without affecting amyloplast sedimentation in the statocytes of vertically grown shoots (Abe et al., 1994; Li et al., 2007; Yoshihara and Iino, 2007). Upon reorientation, the mutant fails to establish a lateral auxin gradient in coleoptiles. The gene responsible for these phenotypes was found to encode a plant-specific protein of unknown function. LAZY1 orthologs have since been identified in most land plants. Loss-of-function mutants of Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and silver birch (Betula pendula) LAZY1 orthologs showed defective vertical shoot branch growth, similar to those observed in rice lazy1 mutants (Dong et al., 2013 ; Yoshihara et al., 2013; Salojarvi et al., 2017). In addition, a family of six LAZY1 homologs, LAZY1–LIKEs (LZYs: LZY1/At5g14090, LZY2/At1g17400, LZY3/At1g72490, LZY4/At1g19115, LZY5/At3g24750, LZY6/At3g27025), has been identified in Arabidopsis (Table 1; Yoshihara et al., 2013; Ge and Chen, 2016; Guseman et al., 2017; Taniguchi et al., 2017; Yoshihara and Spalding, 2017). Four Arabidopsis LZY genes (LZY1–4) are expressed in the statocytes (LZY1–3 in shoot statocytes; LZY2–4 in root statocytes). Detailed phenotypic analyses of lzy mutants revealed that the four LZY genes share redundant functions in Arabidopsis gravitropism. The statocyte-specific expression of LZY is sufficient to attenuate the gravitropic defects of lzy mutants, indicating that LZY genes function in the statocytes, contributing to gravitropism (Taniguchi et al., 2017). The gravitropic defects in lzy mutants were associated with defective lateral auxin transport, whereas lzy mutations did not affect amyloplast sedimentation in the statocytes upon reorientation. Together, these results demonstrate the role of the Arabidopsis LAZY1 family genes in gravity-signal transduction within the statocytes, linking between gravity-induced amyloplast sedimentation and lateral auxin transport. Recently, several genes involved in root gravitropism in rice, Medicago (Medicago truncatula), and Lotus (Lotus japonicus), namely, DEEPER ROOTING 1 (DRO1), DRO1-like (DRL), NEGATIVE GRAVITROPIC RESPONSE OF ROOTS (NGR), and L. japonicus LAZY3 (LjLAZY3), were reported to be members of the LAZY1 family of genes (Uga et al., 2013; Ge and Chen, 2016; Chen et al., 2020; Kitomi et al., 2020).

Box 1.

Root gravitropism: from gravity sensing to root bending

When a plant body is inclined horizontally, the inflorescence stem grows upward in the opposite direction of gravity, which is called negative gravitropism. Conversely, the downward growth of a plant root toward the direction of gravity is referred to as positive gravitropism (Morita, 2010). This physiological response can be subdivided into four components as follows: gravity sensing, gravity signaling, intercellular signal transmission, and asymmetric organ growth (Morita and Tasaka, 2004; Morita, 2010; Toyota and Gilroy, 2013; Su et al., 2017; Nakamura et al., 2019a, 2019b; Su et al., 2020). Gravity sensing involves statocytes, the specialized gravity-sensing cells located in the root tips and shoot, which perceive the inclination of the plant body (Haberlandt, 1900; Nemec, 1900). Statocytes contain high-density starch-accumulating plastids called amyloplasts that sediment according to the direction of gravity (Kiss et al., 1989, 1996; MacCleery and Kiss, 1999). Upon the inclination of the plant, amyloplasts relocate according to the gravity vector. Gravity signaling involves the transformation of this physical information into a biochemical signal within the statocytes, which contribute to the directional transport of auxin, a plant hormone. Gravity signaling within the root statocytes (columella cells) triggers the lateral intercellular transport of auxin via polarly localized auxin transporters, leading to auxin accumulation at the bottom of the root cap (Friml et al., 2002; Kleine-Vehn et al., 2010; Band et al., 2012). Intercellular signal transmission occurs as the auxin gradient is transported laterally by another auxin transporter through the root epidermis to the root elongation site (Swarup et al., 2005; Abas et al., 2006).

Here, auxin creates gravitropic curvature by inhibiting cell elongation at the bottom of the root, which induces root bending downward to the vertical, that is asymmetric organ growth. Although gravitropism has been widely studied from the aspect of auxin and its transporter, the molecular mechanism of gravity signaling among the four steps has not been fully elucidated.

Table 1.

Nomenclature of LAZY1 family genes in A. thaliana

Gene ID	Nomenclature	Gravitropism	References
At5g14090	AtLAZY1 LZY1 a	Shoot	Yoshihara et al. (2013); Taniguchi et al. (2017)
At1g17400	AtNGR1 AtDRO3 LZY2 a AtLAZY2	Shoot, root	Ge and Chen (2016); Guseman et al. (2017); Taniguchi et al. (2017); Yoshihara and Spalding (2017)
At1g72490	AtNGR2 AtDRO1 LZY3 a AtLAZY4	Shoot, root	Ge and Chen (2016); Guseman et al. (2017); Taniguchi et al. (2017); Yoshihara and Spalding (2017)
At1g19115	AtNGR3 AtDRO2 LZY4 a AtLAZY3	Root	Ge and Chen (2016); Guseman et al. (2017); Taniguchi et al. (2017); Yoshihara and Spalding (2017)
At3g24750	LZY5 a AtLAZY5	Unknown	Taniguchi et al. (2017); Yoshihara and Spalding (2017)
At3g27025	LZY6 a AtLAZY6	Unknown	Taniguchi et al. (2017); Yoshihara and Spalding (2017)

^a LZY is adopted as the representative nomenclature of LAZY1 gene family in this review for simplicity.

Phenotypes of lzy mutants suggest that significant associations exist between LZY genes and GSA regulation (Figure 1; Guseman et al., 2017; Taniguchi et al., 2017; Yoshihara and Spalding, 2017; Ge and Chen, 2019). The lateral roots of lzy3 single mutants exhibited wider lateral root angles (i.e. larger GSA) than those of the wild-type. Additional lzy2 mutations enhanced the lzy3 phenotypes, leading to much larger GSAs, upward growth of lateral roots, and mild gravitropic defects in the primary root. Furthermore, when combined with lzy4, the primary root GSA in lzy2 lzy3 lzy4 triple mutants was enlarged further, resulting in upward growth of all roots. The stepwise genetic reduction of LZY activity causes a graded increase in the root GSA, representing an inverse relationship. Interestingly, a linear relationship between LZY activity and shoot branch GSA was observed in Arabidopsis. In fact, the upregulation of LZY3 activity was reported to narrow the lateral root and shoot branch growth angles, reducing the lateral root GSA and increasing the shoot branch GSA (Guseman et al., 2017). These results indicate that LZY genes regulate lateral branch GSAs by controlling gravitropism. LZY loss-of-function mutants appear to display reversed gravitropism; however, considering the hypothesis that the GSA is determined by balancing gravitropism and anti-gravitropic offset (Roychoudhry et al., 2013; Roychoudhry and Kepinski, 2015), anti-gravitropic offset emerges as an inverse growth phenotype caused by the loss of gravitropism in the mutants (Figure 1). Thus, the LAZY1 family genes could increase or decrease the GSAs of shoots or roots, respectively, by controlling gravitropism.

The critical role of the CCL domain in GSA regulation

Although no functional domains of the LAZY1 family proteins have been characterized, amino acid sequence comparisons revealed the existence of five conserved regions (I–V) (Yoshihara et al., 2013). A highly conserved sequence of 14 amino acids, referred to as the conserved C terminus in LAZY1 family proteins (CCL) domain, was identified in region V (Taniguchi et al., 2017). The functional role of the CCL domain in the regulation of GSA in Arabidopsis has been investigated. Truncated versions of LZY2 and LZY3 that lacked the CCL domain failed to rescue the GSA defects in lzy1 lzy2 lzy3 mutants. In addition, mutated forms of LZY1 and LZY3 in the CCL domain also did not prevent the GSA defects of lzy1 and lzy1 lzy2 lzy3 mutants, respectively (Taniguchi et al., 2017; Yoshihara and Spalding, 2020). Furthermore, overexpression of the CCL domain alone in columella cells increased the GSA of the primary roots of the wild-type and lzy1 lzy2 lzy3 mutants, perturbing root gravitropism in the wild-type and producing upward-growing primary roots in lzy1 lzy2 lzy3 mutants, similar to the lzy2 lzy3 lzy4 mutant phenotype (Taniguchi et al., 2017; Yoshihara and Spalding, 2020). These results are interpreted to suggest that overexpression of the CCL domain alone perturbs gravity signaling by competing with LZY family proteins for interacting partners that are essential to GSA regulation.

RCC1-LIKE DOMAIN (RLD) proteins were identified as LZY CCL-domain-interacting partners (Furutani et al., 2020). RLD proteins are conserved among land plants and share four domains as follows: a Pleckstrin Homology (PH) domain, Regulator of Chromosome Condensation 1 (RCC1)-like repeats, a Fab1/YGL023/Vps27/EEA1 (FYVE) domain, and a Brevis Radix (BRX) domain (Jensen et al., 2001; Briggs et al., 2006; Furutani et al., 2020). Among these domains, the PH and FYVE domains of RLD1 have been reported to bind to phospholipids (Jensen et al., 2001; Heras and Drøbak, 2002). The RLD1 fragment containing RCC1-like repeats has been shown to catalyze guanine nucleotide exchange on Rab8a in vitro; Rab8a belongs to the RAB-E subclass known to contribute to post-Golgi transport to the plasma membrane. The BRX domain of RLD proteins was found to directly interact with the CCL domains of LZY proteins (Furutani et al., 2020). In Arabidopsis, four RLD genes (RLD1–4) are expressed in the columella and vascular tissues of the primary and lateral root caps. rld1 rld4 double mutants displayed defective gravitropism in the primary root and increased the lateral root GSAs. Furthermore, rld1 rld2 rld3 rld4 quadruple mutants displayed severe embryo-development defects associated with altered vasculature development. Gravitropic and embryonic defects in rld multiple mutants are associated with defective lateral auxin transport in the columella and defective basipetal auxin transport through embryonic vascular tissues, respectively. Together, these data suggest that RLD genes play a fundamental role in polar auxin transport during GSA regulation and plant development. In addition, Rice BRX-like 4 (OsBRXL4) was identified as a rice LAZY1-interactor (Li et al., 2019). A member of the BRX family, OsBRXL4 contains three tandem repeat BRX domains. The BRX domain of OsBRXL4 interacts with the C-terminal region of LAZY1 that contains the CCL domain. RNAi knockdown of OsBRXL4 led to narrowed tiller angles, resulting in a compact shoot phenotype. Conversely, the OsBRXL4 overexpression line exhibited a prostrate growth phenotype due to wider tiller angles, similar to that of rice lazy1 mutants. These observations suggest that OsBRXL4 has an antagonistic effect on LAZY1 via direct interaction, possibly by competing with OsRLD for LAZY1, to control the shoot GSA. Similarly, statocyte-specific expression of the RLD2 BRX domain increased the GSA of lateral roots in Arabidopsis (Furutani et al., 2020). In addition, the CCL domain of LAZY1 family proteins includes a small motif similar to the Ethylene-Responsive Element Binding Factor-Associated Amphiphilic Repression (EAR) motif that interacts with a transcriptional corepressor (Dardick et al., 2013; Yoshihara et al., 2013; Guseman et al., 2017). In fact, the wheat (Triticum aestivum L.) LAZY1 family protein, TaDRO1, was found to interact with the wheat ortholog of TOPLESS, the Arabidopsis transcriptional corepressor (Ashraf et al., 2019). LZY1 and rice LAZY1 are localized in the plasma membrane and nucleus (Yoshihara et al., 2013; Li et al., 2019); however, mutations that prevent nucleus localization of LZY1 have no functional effect on the LZY1 control of the shoot branch GSA (Yoshihara et al., 2013; Yoshihara and Spalding, 2020). In addition, LAZY1 family protein localization analyses have shown that LZY2, LZY3, and LjLZY3 are localized only within the plasma membrane; thus, it is unlikely that LAZY1 family proteins are involved in transcriptional repression at the nucleus, at least, in root branch GSA regulation (Taniguchi et al., 2017; Chen et al., 2020; Furutani et al., 2020).

Gravity-dependent polarization in columella

During gravitropism, amyloplast sedimentation is the primary mechanism by which columella cells sense the inclination of roots toward the direction of gravity (Haberlandt, 1900; Nemec, 1900; Kiss et al., 1989; Kiss et al., 1996; MacCleery and Kiss, 1999). The physical stimulus is assumed to trigger the gravity-signaling pathway, changing the direction of auxin transport in the columella. In vertical roots, PIN-FORMED3 (PIN3), an auxin efflux carrier, is uniformly distributed over the plasma membranes of columella cells, establishing a symmetrical auxin distribution in the root cap (Friml et al., 2002; Kleine-Vehn et al., 2010). Upon reorientation, PIN3 becomes localized on the bottom of the plasma membrane, contributing to the directional transport of auxin toward the lower portion of the root cap (Swarup et al., 2005; Abas et al., 2006; Band et al., 2012). In young lateral root tips, asymmetric expression and polarized localization of PIN3 contribute to the establishment of asymmetric auxin distribution, facilitating inclined downward growth of the roots. Reorientation-induced PIN3 localization requires the activation of guanine nucleotide exchange factors for the ARF GTPase (ARF-GEF) GNOM-dependent polar-targeting pathway (Geldner et al., 2003; Kleine-Vehn et al., 2010). However, the molecular mechanism of PIN3 polar targeting within columella cells is largely unknown.

Studies on LZY and RLD provide insights into the gravity-induced PIN3 polarization mechanism within columella cells (Taniguchi et al., 2017; Ge and Chen, 2019; Furutani et al., 2020). Asymmetrical expression and polarized localization of PIN3 proteins were reversed in the columella cells of the lzy2 lzy3 lzy4 primary root and lzy1 lzy2 lzy3 lateral roots with larger GSAs (Taniguchi et al., 2017; Ge and Chen, 2019). The combinations of rld mutations that increased root GSA and caused defective embryo development also reduced the abundance of PIN proteins in the plasma membrane of young lateral root columella and embryos (Furutani et al., 2020). These results indicate that LZY genes control the asymmetric PIN3 distribution in columella and RLD genes regulate the targeting of PIN3 to the plasma membrane, possibly via Rab-GEF activity. Recently, LZY localization within columella was confirmed using a tissue-clearing method (Furutani et al., 2020). In young lateral root columella, LZY3 was localized along the bottom of the plasma membranes. Upon reorientation, LZY3 relocalized to the new bottom of the plasma membrane within 30 min, when the amyloplasts had sufficiently sedimented toward the new bottom. The rapid relocalization of LZY3 to the lower side of the root columella plasma membrane in response to reorientation suggests that LZY3 plays a role in the PIN3 polar-targeting pathway. In addition, the LZY-interacting partner RLD1 was recruited to the lower side of the plasma membrane in a LZY-dependent manner via a direct interaction (Figure 1). When LZY3 is statocyte-specifically overexpressed, LZY3 is localized all over the columella plasma membrane; LZY3 recruits RLD1, resulting in aberrant lateral root GSAs. This demonstrates a strong association between LZY3 polarization and lateral root GSA. Considering the GEF activity of the RLD1 RCC1-like repeat for small GTPase in vitro, this might trigger PIN3 targeting to the lower side of the plasma membrane via vesicle trafficking, leading to lateral auxin transport and a decrease in lateral root GSA. ARF-GEF GNOM was also identified as a LZY-interactor, and rld1 rld4 double mutants displayed hypersensitivity to the ARF-GEF inhibitor brefeldin A (BFA), suggesting that LZY-RLD could regulate PIN3 polar-targeting to the plasma membrane in the same pathway as GNOM. Future research to investigate the relationship between RLD and GNOM and identify the small GTPases activated by RLD and GNOM within columella is warranted.

Polar localization and auxin transport activity of PIN proteins could be controlled by PIN phosphorylation in conserved Ser residues via the plasma membrane-associated AGCVIII Ser/Thr kinase family, including PINOID (PID), WAVING AGRAVITROPIC ROOT (WAG), and D6 PROTEIN KINASEs (D6PKs) (Friml et al., 2004; Michniewicz et al., 2007; Kleine-Vehn et al., 2009; Zourelidou et al., 2009, 2014; Barbosa and Schwechheimer, 2014; Barbosa et al., 2014, 2016, 2018; Marhava et al., 2018). PID/WAG-dependent PIN3 phosphorylation was shown to be involved in gravity-induced PIN3 relocalization within columella cells (Grones et al., 2018). A member of the D6PK family, PROTEIN KINASE ASSOCIATED WITH BRX (PAX) is involved in protophloem sieve element differentiation together with AtBRX, which is composed of double BRX domains. PAX and AtBRX are polarly localized in the plasma membrane and colocalized with PIN proteins (Marhava et al., 2018). PAX activates PIN-mediated auxin efflux activity through phosphorylation, whereas AtBRX inhibits PAX kinase activity. In addition, AtBRX localization in the plasma membrane is PAX-dependent; auxin negatively regulates AtBRX association with the plasma membrane leading to the activation of PAX. Analogous with the AtBRX-AGC kinase-PIN pathway, RLD (with the BRX domain) recruitment to the plasma membrane by LZY within columella might negatively control the activity of PID/WAG kinases, leading to gravity-induced PIN3 relocalization.

The molecular mechanism of polarized localization of LZY3 in the plasma membrane of columella cells also remains unclear. LAZY1 family proteins have no transmembrane regions; however, K/R-rich regions could contribute to the association with positive-charged anionic phospholipids (Barbosa et al., 2016; Platre and Jaillais, 2017). Whether phospholipids play a role in the polarized association of LZY3 with the plasma membrane remains unknown. Previously, ALTERED RESPONSE TO GRAVITY1 (ARG1) and ARG1-LIKE2 (ARL2) were identified as factors involved in the gravity-signaling pathway (Harrison and Masson, 2008). These mutants showed aberrant gravitropism associated with the absence of PIN3 relocalization and normal amyloplast sedimentation upon reorientation. ARG1 and ARL2 encode DNAJ-like proteins, which are localized in the plasma membrane and intracellular compartments of columella cells. ARG1 and ARL2 might contribute to polarized LZY3 localization in the plasma membrane or LZY-RLD-dependent PIN3 polar targeting to the plasma membrane. More studies are needed to confirm these hypotheses.

Anti-gravitropic offset pathway in roots

Characterization of “enigmatic” anti-gravitropic offset

The GSA is thought to be determined by the balance between gravitropism and anti-gravitropic offset (Roychoudhry et al., 2013). The mechanisms underlying gravitropism have been well-studied; however, little is known about the mechanism of anti-gravitropic offset. As mentioned above, LZY genes regulate the lateral branch GSAs through gravitropism. Mutants of LAZY1 family genes appear to exhibit anti-gravitropic growth, which could be described as an “inverse gravitropic phenotype” (Ge and Chen, 2016; Taniguchi et al., 2017; Yoshihara and Spalding, 2017). However, it is assumed that this phenotype is the result of the emergence of anti-gravitropic offset due to the loss of gravitropism in mutants. Thus, the characterization of an inverse gravitropic phenotype in the lzy null mutants (lzy1 lzy2 lzy3 shoot branches and lzy2 lzy3 lzy4 roots) may elucidate the nature of anti-gravitropic offset. In gravitropism, amyloplast sedimentation provides a physical stimulus to sense the inclination toward the direction of gravity. Recently, suppressor screening of lzy null mutants was performed in a genetic approach to identify factors involved in the control of anti-gravitropic offset (Kawamoto et al., 2020). The starchless mutation phosphoglucomutase (pgm), which causes mild gravitropic defects and reduces amyloplast sedimentation, was introduced into lzy null mutants. In the resulting mutants, anti-gravitropic growth was reduced, indicating that amyloplast starch accumulation is required to produce the inverse gravitropic phenotype of lzy null mutants. In addition, mutations that prevented statocyte development in shoots also eliminated anti-gravitropic shoot growth. These results suggest that gravitropism and anti-gravitropic offset share a gravity-sensing mechanism within the statocytes. In addition, genetic and pharmacological disruption of auxin transport, PIN gene mutations, and treatment with an auxin transport inhibitor also reduced the directional root growth of lzy null mutants against and toward gravity (Ge and Chen, 2019). Thus, PIN-dependent auxin transport may play a common role in directional root growth among gravitropism and anti-gravitropic offset. In addition, an inverse asymmetry of PIN3 localization and auxin accumulation was established in roots of lzy null mutants without producing defective amyloplast sedimentation (Taniguchi et al., 2017; Ge and Chen, 2019). In the process of anti-gravitropic offset, auxin is transported in the opposite direction of amyloplast sedimentation in root tips (Figure 1). Gravity signaling of anti-gravitropic offset remains an enigmatic process. What happens after amyloplast sedimentation? What mechanism is responsible for PIN relocalization without LZY activity in columella cells? Are RLD proteins involved in anti-gravitropic offset? These questions should be addressed to gain a better understanding of the molecular mechanism of GSA regulation.

Balancing between gravitropism and anti-gravitropic offset in roots

After orthogonal emergence from the primary root or node, lateral root branches incline and grow in a nonvertical direction. As gravitropism is always the predominant force contributing to vertical GSAs, the existence of mechanisms to balance gravitropism and counteracting anti-gravitropic offset has been assumed in nonvertical root branch GSA formation. Recently, several factors involved in balancing gravitropism and anti-gravitropic offset have been identified. First, auxin may regulate the balance, controlling the root branch GSA. Considering that lateral auxin transport contributes to the processes of gravitropism and anti-gravitropic offset, the control of auxin transport in the columella located in the center of the root cap could play a key role in balancing these processes (Rosquete et al., 2013). In the columella cells of lateral root branches, PIN3 is expressed just after the emergence of root branches, while the expression of PIN4 and PIN7 is delayed. However, in the columella of the primary root—which exhibits vertical GSA–PIN3, PIN4, and PIN7 are all expressed, suggesting that the repression of PIN4 and PIN7 expression in young root branch columella increases the root branch GSA by reducing gravitropism (Rosquete et al., 2013). In addition, EXOCYST70A3 was recently identified as a modulator of root GSAs by controlling PIN4 expression in columella cells (Ogura et al., 2019). Interestingly, when auxin signaling was blocked in the root branch root cap cells, including columella, the root branch GSA decreased significantly, suggesting that auxin signaling controls anti-gravitropic offset. Second, cytokinin was shown to function as an anti-gravitropic offset component in root branches (Waidmann et al., 2019). Asymmetric cytokinin signaling was observed in the root caps of root branches, with a stronger cytokinin signal in the upper portion of root caps, inhibiting the growth of the upper part of the root branches and preventing downward root bending. The regulation of lateral auxin transport by the cytokinin signaling pathway has not been confirmed, but analyses using the lzy null mutant background that lacks a gravitropism component could elucidate the effect of cytokinin signaling on lateral auxin transport. Finally, environmental cues, such as soil and light conditions, function as modulators of root branch GSAs, conceivably by controlling the balance between gravitropism and anti-gravitropic offset. The cytokinin signaling pathway integrates environmental signals, such as hypoxia into root branch GSA control. In addition, nitrogen and phosphorous deficiency produce auxin-dependent effects on root branch GSAs (Roychoudhry et al., 2017). Low nitrogen has been shown to reduce vertical root branch GSAs in Arabidopsis, and local nitrogen stimulates positive tropic responses (Yamazaki et al., 2020), possibly by altering the balance between gravitropism and anti-gravitropic offset. Furthermore, the growth direction of adventitious roots in flooded rice is controlled by light (Lin and Sauter, 2018). Adventitious roots grow upward in the dark (large GSA) and downward in the light (small GSA). Phytochrome and blue light-signaling pathways could control adventitious root GSA by modifying the gravitropism and anti-gravitropic offset balance in rice. Future works investigating two separate aspects of the LZY-mediated gravitropism pathway and the anti-gravitropic pathway manifested by the lzy null mutants would provide important insights into the balancing mechanisms between gravitropism and anti-gravitropic offset in root GSA regulation.

Concluding remarks

The root GSA is likely determined by the balance between gravitropism and anti-gravitropic offset. Over the last decade, examinations of gravity-signaling pathways have uncovered several aspects of GSA control through functional analyses of the LAZY1 family protein, a key player in the control of lateral auxin transport. From these data, it appears that the polarization relay of LZY-RLD-PIN induces lateral auxin transport from columella to the lower portion of the root tip in gravitropism. The gravity-signal passing between LZY and RLD is a direct protein–protein interaction (via LZY CCL and RLD BRX domains), whereas the signal passing of RLD-PIN may be mediated through vesicle trafficking. Although the molecular function of the RLD family remains unknown, a functional relationship between RLD and GNOM, involved in PIN recycling from endosomes to the plasma membrane, has been identified. Future studies of the function of RLD in PIN trafficking should advance our understanding of the auxin transport mechanism. One key unanswered question is how LZY3 is polarly localized in the plasma membrane after amyloplast sedimentation (see “Outstanding questions”). The functional importance of the LZY protein CCL domain in gravitropism has been revealed; however, the functions of four other conserved regions (I–IV) are still unknown. Therefore, the functional characterization of these regions could reveal the link between amyloplast sedimentation and LZY polarization as well as the mechanism of LZY association with the plasma membrane. However, the characterization of the anti-gravitropic offset process has just started using lzy null mutants that lack a gravitropism component. From these genetic analyses, it is apparent that amyloplast sedimentation plays a key role in anti-gravitropic offset gravity-sensing. In addition, lateral auxin flow in the opposite direction to gravity generates asymmetric root elongation, resulting in upward root bending. However, the physiological processes of anti-gravitropic offset gravity-signaling remain unclear (see “Outstanding questions”). Genetic identification of the factors involved in anti-gravitropic offset in lzy null mutants will provide a comprehensive understanding of anti-gravity-signal passing. Further elucidation of the mechanisms by which modulators of root GSAs (e.g. auxin, cytokinin, nutrients) affect gravitropism and/or anti-gravitropic offset will lead to a greater understanding of plant root systems.

Outstanding questions

• How is the LZY3 protein associated with the plasma membrane of columella cells and polarized in response to amyloplast sedimentation?

• Do RLD family proteins work together with GNOM to regulate PIN targeting to the plasma membrane?

• What gravity-signaling mechanisms control lateral auxin transport in the direction opposite to gravity in the anti-gravitropic offset of lzy null mutants?

• Does the balance between gravitropism and anti-gravitropic offset accurately determine the root GSAs, shaping the whole root system?

• Do previously reported root GSA modifiers affect the balance between gravitropism and anti-gravitropic offset?

Funding

This work was supported by a Core Research for Evolutionary Science and Technology (CREST) award from the Japan Science and Technology Agency (JST) (grant no. JPMJCR14M5 to M.T.M.), a MEXT Grant-in-Aid for Scientific Research on Innovative Areas (grant nos. 18H05488 to M.T.M., 26113513 and 16H01244 to M.F.), the National Natural Science Foundation of China (NSFC) (grant no. 31970203, “General Program”) to M.F., and the Natural Science Foundation of Fujian Province, China (grant no. 2019J01382, “Natural Science Foundation Program”) to M.F.

Conflict of interest statement. We have no conflict of interest to declare.

 

M.F. and M.T.M. wrote the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Miyo Terao Morita (mimorita@nibb.ac.jp).