Cytokinin-induced root growth involves actin filament reorganization

Abstract

Root architecture is developmentally plastic and affected by many intrinsic factors (e.g. plant hormones) and extrinsic factors (e.g. touch, gravity) in order to maximize nutrient and water acquisition. We have recently shown that asymmetrical exposure of cytokinin (CK) at the root tip causes root growth directional changes that is dependent on ethylene signaling and is potentiated by glucose signaling. Auxin homeostasis as maintained by auxin signaling and transport is also involved in CK-induced root cell elongation and differential growth. The signaling pathways eventually converge at actin filament organization since actin filament organization inhibitor latrunculin B (Lat B) can also induce similar growth. We, show that CK can actually alter actin filament organization as seen in actin binding protein 35S::GFP-ABD2-GFP transgenic lines as is also altered by auxin polar transport inhibitor 1-N-naphthylphthalamic acid (NPA) and Lat B in different manners.

Optimal root architecture not only provides mechanical support to the above ground parts but also helps in uptake of water and nutrients from the soil, and interaction with growth promoting microorganisms. Root architecture is controlled by the availability of nutrient and water gradients, thigmotropism, gravitropism and circumnutation. Root plasticity is important for the optimal positioning of plants with respect to the changing microenvironment and facilitates growth of the root via easiest route through the soil. Root architecture is influenced by a number of exogenous and endogenous factor including phytohormones. Cytokinin (CK) is one of the phytohormones which has an important role in controlling root architecture in association with auxin. CK signaling changes root architecture by modulating root growth and development. During early embryogenesis, CK antagonizes auxin signaling leading to root stem cell specification.¹ CK also controls root meristem vascular differentiation as revealed by the characterization of a wooden leg (wol) allele of CRE1/AHK4 and AHP6.² CK signaling also controls root apical meristem size by controlling cell differentiation at transition zone. CK acts antagonistically to auxin for controlling the size of root meristem via controlling expression of SHY2 (SHORT HYPOCOTYL2) gene which is a member of auxin inducible AUX/IAA gene family. CK also antagonizes auxin signaling to inhibit lateral root formation. CK inhibition of lateral root formation involves BRX (BREVIS RADIX) gene which encodes for a protein that controls root cell proliferation and elongation.³

Response of plant roots to gravity is regulated by asymmetric distribution of auxin. Aloni et al. (2004) demonstrated that CK is also asymmetrically distributed in the gravi-stimulated roots. Asymmetric CK distribution causes inhibition of elongation at the lower side and promotion of growth at the upper side of the distal elongation zone causing a downward curvature.⁴ We showed that CK leads to CK-induced root tip reorientation growth response via members of two-component system including AHKs, type-A and type-B ARRs.⁵ Ethylene signaling acts downstream to CK for transducing the signal further. Auxin signal transduction and transport mutants display highly reduced CK-induced root growth response suggesting a role for auxin homeostasis in the whole process. Differential distribution of auxin lies downstream to all the other factors mentioned above since 1-N-naphthylphthalamic acid (NPA) can evoke CK-induced root growth like response in all CK, ethylene and auxin signaling mutants. Differential auxin transport may cause differential cell elongation across the root via disrupting actin filament organization since latrunculin B (Lat B) can induce CK-induced root growth like response even in eir1 auxin polar transport defective mutant.⁵ Here we show that application of CK can actually change the organization of actin filaments in the roots of 35S::GFP-ABD2-GFP transgenic lines.⁶ In the 3% glucose containing medium the actin filaments are present in vertical orientation in the form of thin long strands. On the application of CK vertical arrangement of actin filament is lost (Fig. 1). Actin filaments are oriented in the form of tight mesh in CK treated roots. Auxin polar transport inhibitor NPA and actin filament organization inhibitor Lat B also disrupt vertical arrangement of actin filaments (Fig. 1). We have already shown that both NPA and Lat B can also evoke a response similar to CK-induced root growth response.

Figure 1.

Actin organization in roots of 7-d-old seedling stably expressing the 35S::GFP-ABD2-GFP construct. Seeds were sown on 0% glucose (G), 3% G, 3% G+1 µM BAP, 3% G+1 µM NPA and 3% G+10 µM LatB containing ½ MS medium and grown vertically in light. Roots were imaged using laser confocal scanning microscope. Images are single optical sections. Representative images from two separate imaging runs, with 6–8 roots per treatment in each run. Bar = 20 µm

Cytoskeleton plays a very important role in transmitting and transducing the signal downstream.⁷ In plants several pathways involved in cell division and differentiation, vesicular transport, membrane recycling, secretion, cell-wall formation and symbiotic interactions depend on the organization and dynamic properties of cytosketon.⁷ Auxins and CKs can modify the tension within the actin network of plant cells through activation of signaling cascades which produce lipophilic and ionic second messengers.⁷ Auxin transport inhibitors can stabilize actin. Actin stabilization interferes with endocytosis, vesicle motility, auxin transport, and plant development.⁸ There are also reports of indole acetic acid (IAA), 1-naphthalene acetic acid (NAA) and tri-iodobenzoic acid (TIBA) bundle actin filaments and slow root growth by reducing the length of the elongation zone with little or no decrease in cell production. In contrast, 2,4-dichlorophenoxy -acetic acid (2,4-D), NPA and Lat B depolymerize actin filaments and slow root growth primarily by decreasing the number of dividing cells.⁹ All these reports together suggest that actin filament organization has an important role in controlling differential cell elongation and anisotropic growth. CK modulation of actin filament organization as seen by 35S::GFP-ABD2-GFP transgenic line might also be responsible for CK-induced differential cell elongation leading to altered root directional growth.

Kushwah S, Jones AM, Laxmi A. Cytokinin interplay with ethylene, auxin, and glucose signaling controls Arabidopsis seedling root directional growth. Plant Physiol. 2011;156:1851–66. doi: 10.1104/pp.111.175794.