Arabidopsis FIM4 and FIM5 regulates the growth of root hairs in an auxin-insensitive way

ABSTRACT

Tip-growing cells provide a useful model system for studying the underlying mechanisms of plant cell growth. The apical growth of root hairs is dependent on the microfilament skeleton, and auxin is an important regulator of root hair development. We functionally characterized actin bundling proteins AtFIM4 and AtFIM5, which were preferentially expressed in tip-growing cells such as pollen tubes and root hairs. The morphology and length of root hairs in atfim4/atfim5 double mutant line had obvious defects. In addition, we found the growth of root hairs of atfim4/atfim5 double mutant was insensitive to exogenous IAA (indole-3-acetic acid) treatment. So we consider that AtFIM4 and AtFIM5 act together to regulate the growth of root hair in an auxin-insensitive way.

Root is an important part for plant which can absorb all kinds of nutrient elements. It contains the following four parts: the root cap, meristematic zone, elongation zone and root hair zone.^1–3 Similar to the growth of pollen tubes, the growth pattern of root hairs belongs to polar growth.⁴ In the early stage of root hair formation, the root epidermal cells differentiate into hair cells and non hair cells through a complex mechanism. When the fate of the root hair cells is determined, the surface of the cell begins to form the protrusion, and after the root hairs form the protrusion structure, the root hair enters the tip growth stage. In the course of this period, the secretory vesicles which are synthesized in the endoplasmic reticulum and golgi bodies transport the cell wall substances and other proteins needed to the tip of the root hair along the cytoskeleton system to complete the growth of the root hair.⁴ A large number of genes have been identified to affect the apical growth of root hair, including genes involved in cell wall synthesis and modification, cytoskeleton protein genes, inositol signaling pathway related genes, small G protein genes and so on.^1,5,6 When the root hair grows to a certain length and enters the mature stage, the root hair stops growing.

The microfilament skeleton, which is arranged in a parallel pattern in the shank but highly dynamic in the apex, plays an important role during the process of tip growth. ⁷ It guide the cytoplasmic circulation and transport the vesicles to the tip of the root hair together with myosin.⁸ The presence of a small filamentous network at the tip of the root hair prevents large organelles from entering the apex, and ensuring vesicles quickly fuse with the plasma membrane.

In all eukaryotes, actin is highly conserved, and microfilaments perform pivotal roles in different types of cells, together with hundreds of actin-binding proteins (ABPs). Studies have shown that a variety of ABPs are involved in the growth and development of root hair. Profilin is a common G-actin binding protein in eukaryotes, which inhibits nucleation and prevents the growth of negative ends of microfilaments. ^9,10 Overexpression of PFN1 produces longer root hair. ¹¹Formin is a kind of microfilament nucleation factor, which can accelerate the nucleation of oligomer by G-actin, promote the polymerization of microfilament, and play an important role in many polar growth cells.^12–14 AtFH8 is the homologous gene of Formin, which participates in the polar growth of root hair by regulating actin cytoskeleton.¹⁵ The Villin proteins in plants have many functions: bundles of microfilaments, cutting of microfilaments, and capping of microfilaments. Studies on Villin protein in Arabidopsis thaliana show that it can regulate the dynamics of microfilaments in apical growth cells such as pollen tube and root hair.^16–19

Auxin is the earliest plant hormone that plays an important role in the process of plant growth and development. It is also an important regulator of root hair formation and development.²⁰ Both auxin signaling and homeostatic change can directly affect root hair development.^21,22

Multiple studies have revealed that auxin is closely related to actin cytoskeleton. the auxin concentration in the cell has profound effect on actin arrays, and conversely, the actin dynamics can regulate the polar transport of auxin in plants. The membrane localization of auxin influx or efflux carriers AUX1, PINs and PGPs is regulated by actin cytoskeleton.^23,24

It is revealed that the exogenous application of IAA (indole-3-acetic acid) or NAA (1-naphthlcetic acid) could affect the actin arrays in plant cells. For example, Exogenous auxin triggers a loosening of the actin bundles in the coleoptile of rice and maize.^25–27 Similarly, exogenous auxin treatment lead to actin bundles reduction in Arabidopsis roots.²⁸ Then how the auxin signaling pass to actin cytoskeleton? In rice, RMD (Rice Morphology Determinant) is an actin bundler, which belongs to the Formin homolog family. It is revealed that AUXIN RESPONSIVE FACTORS (ARFs), OsARF23 and OsARF24 regulate the expression level of RMD, then further affect the actin arrays.²⁹

On the other hand, the actin arrays can affect auxin polar transport in plants. It is demonstrated that auxin transporters reach their final destination by active movement of secretory vesicles along F-actin tracks. The membrane localization of auxin influx carrier AUX1 and efflux carriers including PIN1, PIN2 and PIN3 was impaired when the actin filaments was disrupted by cytochalasin D or latrunculin B.^30–32

The actin arrays is regulated by a series of actin binding proteins including severing proteins, capping proteins, bundling proteins,etc.³³ Among them, ARP3 (Actin-Related Protein 3, an actin nucleator), RMD (an actin bundler), and VLN2 (VILLIN2, an actin bundler) have been verified to affect auxin polar transport in plants by disturbing the localization of efflux carriers.^29,34,35

Fimbrin, also known as plastin, is the only CH-domain actin binding protein identified in plants. The Arabidopsis contains five genes encoding fimbrin homologous, named FIM1-FIM5,³³ in which AtFIM5 is extensively studied so far. It is necessary for pollen germination and pollen tube growth.^36–38 Except AtFIM5, chip data showed that AtFIM4 was also expressed in pollen tubes. Biochemistry data showed AtFIM4 made microfilaments bundle and branch, while AtFIM5 could only make microfilament bundle.³⁸ In vivo, AtFIM4 acts coordinately with AtFIM5 to organize and maintain the longitudinal actin bundles in pollen tubes.³⁹

In this study, the wild type (WT) and mutant seeds (atfim4-1/atfim5-2) were spread on 1/2MS medium with 250 nM indole-3-acetic acid (IAA), and after seven days germination, the root hair length was measured. we found that wild type root hairs treated by exogenous IAA were much longer, whereas the double mutant root hairs had no significant difference, that is to say, double mutants is insensitive to exogenous IAA (Figure 1). Therefore, we speculated that the function of AtFIM4 and AtFIM5 in root hair growth may depend on auxin signal. The detail mechanism about how AtFIM4 and AtFIM5 regulate the process of root hair growth and why the root hairs of atfim4-1/atfim5-2 double mutant are insensitive to the auxin still needs further study.

Figure 1.

Loss of function of both AtFIM4 and AtFIM5 results in short root hairs and low sensitivity to IAA.

1. Phenotypic analysis of root hair response to auxin in wild type and the double mutants. Scale bars = 100 μm. (B) Length of root hairs in WT and the double mutant atfim4-1/atfim5-2.(n ≥ 40,**P < 0.01).

Funding Statement

This work was supported by the Scientific Research Program Funded by Shaanxi Provincial Education Department [Program No. 17JK0774].

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.