BACKGROUND:

Amyloplasts – Plant gravisensors

Plants orient themselves along the gravitational vector – the roots grow with it, the shoots against it. After first experiments on this phenomenon were conducted in the 19^th century, two seminal papers were published in 1900 which describe a role for non-photosynthetic chloroplasts in the very tip of the root, the columella, as root gravisensors (Haberlandt, 1900; Němec, 1900). These plastids, called amyloplasts due to their starch content, were thus considered statoliths – gravity-sensing organelles.

Auxin – Mediator of gravitropic growth

The next major breakthrough in understanding plant gravitropism was driven by research on the first-described phytohormone, auxin, in the 1920s (Went and Thimann, 1937). Two scientists, Nikolai Cholodny and Frits Went, spearheaded the work on the role of auxin in gravitropism, and eventually it led to the formulation of the Cholodny-Went theory (Cholodny, 1929; as an interesting side-note: Despite this theory being arguably one of the most widely studied and discussed in 20^th century plant science, there was never a specific publication dedicated to it. It was simply the result of Cholodny and Went working in parallel on the same topic, while staying in constant exchange and discussion with one another and their peers. Through this constant exchange, the theory just emerged and found its way into the literature). It postulates that growth changes in response to gravity are the result of an unequal distribution of auxin in the growing organ, resulting in different elongation rates on the down- and upward facing side, and thus organ bending. With the advent of plant molecular biology and the availability of several new Arabidopsis thaliana mutants to study gravitropism, the Cholodny-Went theory has been pronounced dead several times due to new, seemingly conflicting results. However, it appears to have even more lives than a cat; because despite all the obituaries published over the past 35 years, its core ideas still hold up pretty well (Sato et al., 2015).

PINs & AUX – Facilitators of auxin-(re)distribution

In the late 1990s, a revival of auxin-research was initiated in part by the description of the first auxin transporters; the PIN efflux, and AUX influx carriers (Gälweiler et al., 1998; Marchant et al., 1999; Sauer and Kleine-Vehn, 2019). Work on the transporters resulted in a model of directional auxin flux within the root, which postulates that shoot-produced auxin is transported through the vasculature toward the root tip. From there, the auxin carriers then redirect auxin from the center toward the periphery of the root and back up toward the shoot. This inverse fountain produces an auxin concentration maximum in the center of the root tip. As a result, the root grows straight down. In response to changes in graviperception, the carriers mediate an unequal redistribution of auxin according to the Cholodny-Went theory, and thus, the root bends toward the ‘new down’.

LAZYs – Bringing it all together?

This now brings up one major question that has occupied the field for years: How do the amyloplasts relay their positional information to the plasma membrane-localized auxin carriers? This is where the LAZY family proteins come in. LAZY mutations were first described in rice and maize in the 1930s, but only in the 1990s, the function of these proteins could be studied in closer detail (van Overbeek, 1936; Jones and Adair, 1938; Nakamura et al., 2019). lazy234 triple mutants are agravitropic, thus they do not respond to changes in the gravitropic vector (Yoshihara and Spalding, 2017). Their amyloplasts still move and relocate according to changes in the vector, but auxin is no longer redistributed upon gravistimulus (Yoshihara and Spalding, 2017; Abe et al., 1994). Thus, these proteins may be involved in the information transfer from amyloplasts to plasma membrane.

MAIN FINDINGS:

In the two preprints discussed here, Nishimura et al. and Chen et al. (2023) have both independently described the principal mechanisms of how LAZY proteins can transfer the gravitropic signal from the amyloplast to the plasma membrane, and thus the compartment in which the auxin carriers can facilitate auxin redistribution.

LAZY proteins localize to downward-facing membranes and amyloplasts

Both groups first show that LAZY2, 3 and 4 localize predominantly to the downward-facing plasma membrane of amyloplast-containing columella cells, but more importantly, they also observed them on the outer membrane of the amyloplasts. Since the proteins don’t have any membrane-targeting motifs and sequences, both groups investigated the possibility that hydrophobic regions in the proteins allow them to bind to lipids in the plasma membrane. To this end, Chen et al. used a lipid overlay assay to demonstrate that the LAZYs can indeed bind to phosphatidylinositolphosphates (PIPs). Similarly, Nishimura et al. showed that mutating the hydrophobic regions reduced membrane-localization, indicating that it is these regions that aid in PIP-binding. Furthermore, using a fluorescent biosensor for PIPs, they showed that while these lipids might be involved in localizing the LAZYs to the membrane, they do not function in the relocalization of the proteins following gravistimulation.

Following gravistimulation by a 90° rotation, the LAZY proteins also changed their position from the formerly downward-facing membrane to the newly downward-facing membrane. And this relocalization was dependent on the concurrent relocalization of the amyloplasts, since impaired amyloplastic relocalization in the phosphoglucomutase 1 (pgm) mutant also resulted in impaired LAZY relocalization.

LAZY proteins bind to amyloplasts and move with them to the new downward-facing membrane

Given the localization of LAZYs on the surface of the amyloplasts, both groups speculated that the LAZYs may be literally transported to the membrane by the moving amyloplasts. Nishimura et al. tested this hypothesis using the photoconvertible mEos2 fluorophore. After converting the green fluorescence of amyloplast-localized LAZY4-mEos2 to red, they observed accumulation of red fluorescent LAZY4-mEos2 at the newly downward-facing plasma membrane, indicating that the photoconverted LAZY4s were indeed transferred from the amyloplasts to the plasma membrane. As confirmation, they also employed optical tweezers to trap amyloplasts with photoconverted LAZY4-mEos2, which they then moved into contact with a different membrane. This contact resulted in transfer of photoconverted LAZY4-mEos2 to this new membrane.

LAZYs are bound to amyloplasts by TOC-receptor proteins

Chen et al. used a different approach to show that the LAZYs are indeed hitchhiking with the amyloplasts. They showed that MKK5/MPK3 phosphorylated the LAZY proteins in response to gravistimulation. This phosphorylation induced an interaction of the LAZYs with TRANSLOCON OF OUTER MEMBRANE OF CHLOROPLASTS (TOC) complexes, particularly TOC34, 120 and 132, all of which are receptors on the surface of the amyloplasts. Mutating these TOCs resulted in impairment of LAZY localization to the amyloplasts, relocalization to the plasma membrane, and subsequent auxin redistribution. Thus, the authors concluded that the TOCs bind the LAZYs, once they are phosphorylated in response to gravistimulation, allowing them to move with the amyloplasts to the newly downward-facing membrane (Fig. 1).

SIGNIFICANCE:

While root gravitropism is now a pretty well understood process, and early hypotheses such as the amyloplast-sedimentation and Cholodny-Went models have stood the test of time, there was still a long-standing question: How is the physical stimulus of moving amyloplasts translated into the physiological response of altered auxin distribution (Kawamoto and Morita, 2022)?

The predominant idea of how this could be achieved is mechanotransduction (Häder et al., 2017). With their movement, the starch-filled amyloplasts would put pressure on the actin-cytoskeleton, as well as the membranes of the endoplasmic reticulum and the plasma membrane itself. This force could be perceived by mechanosensitive receptors, which then may indirectly relay the signal to the auxin carriers (Perbal and Driss-Ecole, 2003; Häder et al., 2017). This idea is supported by several experiments and observations, but no such receptor could so far be identified. On the other hand, one very interesting study that used flight-induced weightlessness to investigate this hypothesized role of pressure force in rhizoids of green algae, came to a different conclusion (Limbach et al., 2005). Parabolic flight in a zero-G aircraft or sounding rockets caused microgravity conditions, in which the statoliths were basically weightless. Under these conditions, statolith relocalization to a membrane was still sufficient to induce a gravitropic change in the rhizoids, indicating that contact with the membrane, even without any mechanical pressure, was sufficient for signal transduction (Limbach et al., 2005). With this observation not fitting with the predominant mechanotransduction model, it maybe didn’t receive the attention it should have attracted. However, this observation could now be proven correct, 20 years later, by the results discussed here. With the LAZY proteins being transferred from the surface of the amyloplast to the plasma membrane, contact alone would indeed suffice, and no mechanotransduction would be necessary. Who would have guessed that one of the keys to solve this plant science question would require the deployment of zero-G aircrafts and sounding rockets?

OPEN QUESTIONS:

While the results reported here are a major step forward, there are, of course, still more interesting questions to answer:

One obvious question is what triggers the MKK5/MPK3-mediated phosphorylation of the LAZYs, since this would now pose an early step in the plant’s graviperception.

Then, once the LAZYs arrive at their new destination, how do they relay their signal to the auxin carriers? This is unlikely to be a direct interaction, but this link may be coming by way of RCC1-like domain (RLD) proteins, which co-localize with the LAZYs and may function in PIN-localization (Furutani et al., 2020).

With both LAZY and PIN proteins being localized to phosphatidylinositols and/or membrane nanodomains, it is possible that these proteins are sequestered in membrane domains with distinct lipid signatures (Somssich, 2018; McKenna et al., 2019). In that case, signalling intermediates like RLDs should be present in those same nanodomains, and this could lead to the identification of other proteins involved in this process.

And finally, it will be important to investigate if and how this also affects the gravitropic set-point angle, in which gravitropic responses are not triggered by a certain stimulus but are a constant readout of the gravitropical vector (Kawamoto and Morita, 2022). In lateral roots of Arabidopsis, which grow in a more horizontal angle than the primary roots, this process is at least partially dependent on auxin transport and asymmetric auxin signaling (Rosquete et al., 2013). Therefore, it would be interesting to analyze if the LAZY dependent localization of auxin transporters differs from the primary root, or if it is the readout in later signaling steps that makes the root grow not completely vertically, but at a set angle.

FINAL WORD (Why we chose this preprint):

The root graviresponse is a textbook topic for biology students, and the actual experiment, in which a change in root growth direction can readily be observed within a couple of hours after rotating the plant by 90°, is typically part of basic hands-on plant biology courses. The question of how the tumbling of amyloplasts onto a membrane can trigger a physiological response has thus been a key question discussed in these courses, and we still remember this being the case when we were students 15 and 20 years ago, respectively. To now read a solution to a textbook problem feels like a real milestone, and it is exciting to read about this and preLight it here.

 

Correction (May 7th, 2023):

In the first version of this article we incorrectly stated that “Nishimura et al. couldn’t detect binding [of LAZY proteins] to lipids”. This has been corrected.

 

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Tags: amyloplast, auxin, graviperception, gravitropism, plant biology, plant science, root development, statoliths

doi: https://doi.org/10.1242/prelights.34535

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