Dynamic auxin transport patterns preceding vein formation revealed by live-imaging of Arabidopsis leaf primordia

Abstract

Self-regulatory patterning mechanisms capable of generating biologically meaningful, yet unpredictable cellular patterns offer unique opportunities for obtaining mathematical descriptions of underlying patterning systems properties. The networks of higher-order veins in leaf primordia constitute such a self-regulatory system. During the formation of higher-order veins, vascular precursors are selected from a homogenous field of subepidermal cells in unpredictable positions to eventually connect in complex cellular networks. Auxin transport routes have been implicated in this selection process, but understanding of their role in vascular patterning has been limited by our inability to monitor early auxin transport dynamics in vivo. Here we describe a live-imaging system in emerging Arabidopsis thaliana leaves that uses a PIN1:GFP reporter to visualize auxin transport routes and an Athb8:YFP reporter as a marker for vascular commitment. Live-imaging revealed common features initiating the formation of all higher-order veins. The formation of broad PIN1 expression domains is followed by their restriction, leading to sustained, elevated PIN1 expression in incipient procambial cells files, which then express Athb8. Higher-order PIN1 expression domains (hPEDs) are initiated as freely ending domains that extend toward each other and sometimes fuse with them, creating connected domains. During the restriction and specification phase, cells in wider hPEDs are partitioned into vascular and non-vascular fates: Central cells acquire a coordinated cell axis and express elevated PIN1 levels as well as the pre-procambial marker Athb8, while edge cells downregulate PIN1 and remain isodiametric. The dynamic nature of the early selection process is underscored by the instability of early hPEDs, which can result in dramatic changes in vascular network architecture prior to Athb8 expression, which is correlated with the promotion onto vascular cell fate.

Auxin-mediated regulation of vascular patterning in Arabidopsis thaliana leaves

“…Vascular tissues differentiate in regions where auxin concentration is elevated (Aloni 2001), and the localization of the intensive auxin flow in the developing leaf reflects the future vascular strand placement (Scarpella et al 2006; Wenzel et al 2007; Marcos and Berleth 2014). Moreover, exogenous application of auxin or its transport inhibitors drastically changes venation patterning (Sieburth 1999; Mattsson et al 1999, 2003; Scarpella et al 2006).…”

“…The theory of canalization has been confirmed in many mathematical models (Mitchison et al 1981; Rolland-Lagan and Prusinkiewicz 2005; Merks et al 2007; Bayer et al 2009; Lee et al 2014) as well as in experimental studies (Mattsson et al 1999, 2003; Sauer et al 2006; Scarpella et al 2006; Wenzel et al 2007). Main support for this theory comes from the characterization of the mechanism driving canalized polar auxin flow (Scarpella et al 2006; Wenzel et al 2007; Marcos and Berleth 2014) and the identification of some of its molecular components (Vieten et al 2007). …”

“…In the next stage of leaf development, consecutive convergence points of auxin that supply the developing secondary veins with this hormone are formed basipetally in the epidermis of the leaf blade edges (Fig. 2b III-X; Scarpella et al 2006, 2010; Wenzel et al 2007; Marcos and Berleth 2014). The convergence points for the first and usually also the second pair of loops are transient, but for subsequent loops these points turn into hydathodes.…”

Myrosin Cell Development Is Regulated by Endocytosis Machinery and PIN1 Polarity in Leaf Primordia ofArabidopsis thaliana

“…Recently, live imaging revealed that cells in PIN1 expression domains differentiate into vascular and nonvascular cells in leaf primordia (Marcos and Berleth, 2014). These nonvascular cells subsequently downregulate PIN1 expression and maintain ground meristem cell morphology (isodiametric shape) (Marcos and Berleth, 2014).…”

“…These nonvascular cells subsequently downregulate PIN1 expression and maintain ground meristem cell morphology (isodiametric shape) (Marcos and Berleth, 2014). These ground meristem cells might subsequently differentiate into myrosin cells.…”