Cytokinin Interplay with Ethylene, Auxin, and Glucose Signaling Controls Arabidopsis Seedling Root Directional Growth

Abstract

Optimal root architecture is established by multiple intrinsic (e.g. hormones) and extrinsic (e.g. gravity and touch) signals and is established, in part, by directed root growth. We show that asymmetrical exposure of cytokinin (CK) at the root tip in Arabidopsis (Arabidopsis thaliana) promotes cell elongation that is potentiated by glucose in a hexokinase-influenced, G protein-independent manner. This mode of CK signaling requires the CK receptor, ARABIDOPSIS HISTIDINE KINASE4 and, at a minimum, its cognate type B ARABIDOPSIS RESPONSE REGULATORS ARR1, ARR10, and ARR11 for full responsiveness, while type A response regulators act redundantly to attenuate this CK response. Ethylene signaling through the ethylene receptor ETHYLENE RESISTANT1 and its downstream signaling element ETHYLENE INSENSITIVE2 are required for CK-induced root cell elongation. Negative and positive feedback loops are reinforced by CK regulation of the expression of the genes encoding these elements in both the CK and ethylene signaling pathways. Auxin transport facilitated by PIN-FORMED2 as well as auxin signaling through control of the steady-state level of transcriptional repressors INDOLE-3-ACETIC ACID7 (IAA7), IAA14, and IAA17 via TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN are involved in CK-induced root cell elongation. This action lies downstream of ethylene and CK induction. Intrinsic signaling in this response operates independently of the extrinsic signal touch, although actin filament organization, which is important in the touch response, may be important for this response, since latrunculin B can induce similar growth. This root growth response may have adaptive significance, since CK responsiveness is inversely related to root coiling and waving, two root behaviors known to be important for fitness. Root architecture is developmentally plastic (Malamy, 2005) and affected by many intrinsic and extrinsic factors (Massa and Gilroy, 2003a, 2003b) in order to maximize nutrient and water acquisition. Among the intrinsic factors, auxin has an important role in shaping root growth pattern, since mutants with altered auxin response or transport show altered root growth, lateral root primordia formation, and tropic responses such as perturbed gravitropism, coiling, waving, and skewing responses (Okada and Shimura, 1990; Garbers et al., 1996; Luschnig et al., 1998; Rutherford et al., 1998; Marchant et al., 1999; Rashotte et al., 2001; Piconese et al., 2003; Buer and Muday, 2004). Ethylene has also been shown to inhibit root waving (Buer et al., 2003), root gravitropism (Buer et al., 2006), and lateral root formation (Negi et al., 2008). Ethylene in turn can regulate auxin biosynthesis as well as transport-dependent auxin distribution (Růžička et al., 2007). Ethylene has been shown to be involved in controlling cytokinin (CK)-mediated root elongation but not meristem size (Růžička et al., 2009). CK signaling follows a multiple-step phosphorelay cascade (Heyl and Schmülling, 2003; Müller and Sheen, 2007; To and Kieber, 2008; Werner and Schmülling, 2009; Kieber and Schaller, 2010; Perilli et al., 2010; Müller, 2011) using, in this order of phosphorylation, (1) one of three hybrid His protein kinases (ARABIDOPSIS HISTIDINE KINASE2 [AHK2], AHK3, AHK4) that serve as CK receptors, (2) His phosphotransfer proteins (ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEINS [AHPs]), and (3) type A and type B response regulators (ARABIDOPSIS RESPONSE REGULATORS [ARRs]). After phosphorylation, AHPs move into the nucleus, where they phosphorylate type B and type A ARRs. Phosphorylated type B ARRs act as transcription factors and induce the transcription of type A ARRs and other CK early-responsive genes (Hwang and Sheen, 2001). CK is known to interact with other hormones so as to affect plant growth and development. Among these, ethylene and auxin are the two most important hormones with which CK exhibits extensive cross talk. CK stabilizes the ethylene biosynthetic enzyme ACS5, leading to increased ethylene production (Vogel et al., 1998a, 1998b; Chae et al., 2003; Hansen et al., 2009). CK and ethylene signaling also share in common the phosphorylation of ARR2 (Hass et al., 2004). CKs negatively regulate PIN-FORMED (PIN)-dependent auxin distribution and consequently auxin transport direction and flux (Laplaze et al., 2007; Lee et al., 2009; Pernisová et al., 2009). CK and auxin have opposite effects on root development (Dello Ioio et al., 2008) mediated by SHORT HYPOCOTYL2 (SHY2). CK activates the transcription of the SHY2 gene, while auxin degrades the SHY2 protein (Dello Ioio et al., 2008). Auxin promotes cell division, while CK controls the switch from meristematic to differentiated cell-suppressing auxin signaling and transport to the transition zone (Dello Ioio et al., 2008). This CK-auxin antagonism is also crucial for specifying the embryonic root stem cell niche during Arabidopsis (Arabidopsis thaliana) embryogenesis. It has been shown that an auxin maxima at the hypophysis activates the transcription of type A ARR7 and ARR15 genes, which are negative regulators of CK signaling. This leads to abnormal embryonic stem cell niche formation (Müller and Sheen, 2008). The different aspects of the CK-auxin interaction have been recently reviewed (Aloni et al., 2006; Zhao, 2008; Chapman and Estelle, 2009; Kuderová and Hejátko, 2009; Moubayidin et al., 2009). There are several reports that sugar can also influence plant root growth (Rolland et al., 2006). The increasing Glc concentration not only increases root length, number of lateral roots, and root hairs but also modulates the gravitropic response of the primary roots of young seedlings (Mishra et al., 2009). Increasing Glc concentrations can induce differential root length, lateral roots, gravitropism, and root hair elongation in auxin perception and signaling mutants, suggesting that auxin signaling is involved in controlling Glc-regulated root responses (Mishra et al., 2009). Glc has also been shown to act via G-protein signaling to attenuate auxin-mediated bimodality in controlling lateral root formation (Booker et al., 2010). In summary, Glc, CK, auxin, and ethylene control a number of root-related phenotypes such as root elongation, root meristem size, specification of root stem cell niche, and lateral root production. CK along with auxin is also involved in controlling root gravitropic response (Aloni et al., 2004). Here, we show that apart from gravitropism, CK has an important role in controlling other root directional/tropic responses such as coiling and waving, aspects of optimal root architecture.