Arabidopsis JAGGED LATERAL ORGANS Acts with ASYMMETRIC LEAVES2 to Coordinate KNOX and PIN Expression in Shoot and Root Meristems

Organ initiation requires the specification of a group of founder cells at the flanks of the shoot apical meristem and the creation of a functional boundary that separates the incipient primordia from the remainder of the meristem. Organ development is closely linked to the downregulation of class I KNOTTED1 LIKE HOMEOBOX (KNOX) genes and accumulation of auxin at sites of primordia initiation. Here, we show that Arabidopsis thaliana JAGGED LATERAL ORGANS (JLO), a member of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) gene family, is required for coordinated organ development in shoot and floral meristems. Loss of JLO function results in ectopic expression of the KNOX genes SHOOT MERISTEMLESS and BREVIPEDICELLUS (BP), indicating that JLO acts to restrict KNOX expression. JLO acts in a trimeric protein complex with ASYMMETRIC LEAVES2 (AS2), another LBD protein, and AS1 to suppress BP expression in lateral organs. In addition to its role in KNOX regulation, we identified a role for AS2 in regulating PINFORMED (PIN) expression and auxin transport from embryogenesis onwards together with JLO. We propose that different JLO and AS2 protein complexes, possibly also comprising other LBD proteins, coordinate auxin distribution and meristem function through the regulation of KNOX and PIN expression during Arabidopsis development.     

The Arabidopsis LATERAL ORGAN BOUNDARIES-domain gene ASYMMETRIC LEAVES2 functions in the repression of KNOX gene expression and in adaxial-abaxial patterning

The normal development of lateral organs of the shoot requires the simultaneous repression of meristem-specific genes and the activation of organ-specific genes. ASYMMETRIC LEAVES2 (AS2) is required for the development of normal leaf shape and for the repression of KNOX genes in the leaf. AS2 is a member of the recently identified, plant-specific LATERAL ORGAN BOUNDARIES (LOB)–domain gene family. Expression of AS2 at high levels resulted in repression of the KNOX homeobox genes BREVIPEDICELLUS, KNAT2, and KNAT6 but not of the related SHOOT MERISTEMLESS gene. Overexpression of AS2 also led to a perturbation of normal adaxial-abaxial asymmetry in lateral organs, resulting in the replacement of abaxial cell types with adaxial cell types. These results indicate that AS2 is sufficient to induce adaxial cell fate and repress KNOX gene expression.     

Developmental regulation and significance of KNOX protein trafficking in Arabidopsis

Intercellular communication delivers critical information for position-dependent specification of cell fate. In plants, a novel mechanism for cell-to-cell communication involves the intercellular trafficking of regulatory proteins and mRNAs. The maize KNOTTED1 (KN1) gene acts non cell-autonomously in the maize leaf, and KN1 was the first plant protein shown to traffic cell-to-cell, presumably through plasmodesmata. We have compared the intercellular trafficking of green fluorescent protein (GFP) fusions of KN1 and Arabidopsis KN1-related homeobox proteins to that of the viral movement protein from turnip vein clearing tobamovirus. We show that there is specific developmental regulation of GFP approximately KN1 trafficking. GFP -- KN1 was able to traffic from the inner layers of the leaf to the epidermis, but not in the opposite direction, from epidermis to mesophyll. However, GFP or the GFP -- movement protein fusion moved readily out of the epidermis. GFP -- KN1 was however able to traffic out of the epidermal (L1) layer in the shoot apical meristem, indicating that KN1 movement out of the L1 was developmentally regulated. GFP -- KNAT1/BREVIPEDICELLUS and GFP -- SHOOTMERISTEMLESS fusions could also traffic from the L1 to the L2/L3 layers of the meristem. In a test for the functional significance of trafficking, we showed that L1-specific expression of KN1 or of KNAT1 was able to partially complement the strong shootmeristemless-11 (stm-11) mutant. However, a cell-autonomous GUS fusion to KN1 showed neither trafficking ability nor complementation of stm-11 when expressed in the L1. These results suggest that the activity of KN1 and related homeobox proteins is maintained following intercellular trafficking, and that trafficking may be required for their normal developmental function.     

PIN1-Independent Leaf Initiation in Arabidopsis

Phyllotaxis, the regular arrangement of leaves and flowers around the stem, is a key feature of plant architecture. Current models propose that the spatiotemporal regulation of organ initiation is controlled by a positive feedback loop between the plant hormone auxin and its efflux carrier PIN-FORMED1 (PIN1). Consequently, pin1 mutants give rise to naked inflorescence stalks with few or no flowers, indicating that PIN1 plays a crucial role in organ initiation. However, pin1 mutants do produce leaves. In order to understand the regulatory mechanisms controlling leaf initiation in Arabidopsis (Arabidopsis thaliana) rosettes, we have characterized the vegetative pin1 phenotype in detail. We show that although the timing of leaf initiation in vegetative pin1 mutants is variable and divergence angles clearly deviate from the canonical 137° value, leaves are not positioned at random during early developmental stages. Our data further indicate that other PIN proteins are unlikely to explain the persistence of leaf initiation and positioning during pin1 vegetative development. Thus, phyllotaxis appears to be more complex than suggested by current mechanistic models.     

Phosphorylation of Conserved PIN Motifs Directs Arabidopsis PIN1 Polarity and Auxin Transport

Polar cell-to-cell transport of auxin by plasma membrane–localized PIN-FORMED (PIN) auxin efflux carriers generates auxin gradients that provide positional information for various plant developmental processes. The apical-basal polar localization of the PIN proteins that determines the direction of auxin flow is controlled by reversible phosphorylation of the PIN hydrophilic loop (PINHL). Here, we identified three evolutionarily conserved TPRXS(N/S) motifs within the PIN1HL and proved that the central Ser residues were phosphorylated by the PINOID (PID) kinase. Loss-of-phosphorylation PIN1:green fluorescent protein (GFP) (Ser to Ala) induced inflorescence defects, correlating with their basal localization in the shoot apex, and induced internalization of PIN1:GFP during embryogenesis, leading to strong embryo defects. Conversely, phosphomimic PIN1:GFP (Ser to Glu) showed apical localization in the shoot apex but did not rescue pin1 inflorescence defects. Both loss-of-phosphorylation and phosphomimic PIN1:GFP proteins were insensitive to PID overexpression. The basal localization of loss-of-phosphorylation PIN1:GFP increased auxin accumulation in the root tips, partially rescuing PID overexpression-induced root collapse. Collectively, our data indicate that reversible phosphorylation of the conserved Ser residues in the PIN1HL by PID (and possibly by other AGC kinases) is required and sufficient for proper PIN1 localization and is thus essential for generating the differential auxin distribution that directs plant development.     

PIN polarity maintenance by the cell wall in Arabidopsis

A central question in developmental biology concerns the mechanism of generation and maintenance of cell polarity, because these processes are essential for many cellular functions and multicellular development. In plants, cell polarity has an additional role in mediating directional transport of the plant hormone auxin that is crucial for multiple developmental processes. In addition, plant cells have a complex extracellular matrix, the cell wall, whose role in regulating cellular processes, including cell polarity, is unexplored. We have found that polar distribution of PIN auxin transporters in plant cells is maintained by connections between polar domains at the plasma membrane and the cell wall. Genetic and pharmacological interference with cellulose, the major component of the cell wall, or mechanical interference with the cell wall disrupts these connections and leads to increased lateral diffusion and loss of polar distribution of PIN transporters for the phytohormone auxin. Our results reveal a plant-specific mechanism for cell polarity maintenance and provide a conceptual framework for modulating cell polarity and plant development via endogenous and environmental manipulations of the cellulose-based extracellular matrix.     

YABBY polarity genes mediate the repression of KNOX homeobox genes in Arabidopsis

The YABBY (YAB) genes specify abaxial cell fate in lateral organs in Arabidopsis. Loss-of-function mutants in two early-expressing YAB genes, FILAMENTOUS FLOWER (FIL) and YAB3, do not exhibit vegetative phenotypes as a result of redundancy. Mutations in these genes result in the derepression of the KNOX homeobox genes SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS, and KNAT2 in the leaves and in the partial rescue of stm mutants. Here, we show that fil yab3 double mutants exhibit ectopic meristem formation on the adaxial surfaces of cotyledons and leaf blades. We propose that in addition to abaxial specification, lateral organ development requires YAB function to downregulate KNOTTED homeobox genes so that meristem initiation and growth are restricted to the apex.     

Auxin redistribution and shifts in PIN gene expression during Arabidopsis grafting

Auxin is important in the development of plant vascular tissues. Reconnection of vascular bundles between scion and stock is a primary aim of grafting, and polar auxin transport greatly affects the formation of a continuous vascular model. The role of auxin in the process of graft-union development was studied by grafting the seedlings of Arabidopsis thaliana (L.) Heynh. DR5:GUS marker plants, which exert the auxinspecific responses. Auxin induced the DR5:GUS expression in the vascular bundles around graft surface and stimulated the formation of multiple vascular bundle reconnections on the third day after grafting (DAG). DR5:GUS expression was delayed for one day in both scion and stock and dramatically declined by the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Vascular bundle reconnection was observed only on the 4th DAG. These results suggest that auxin stimulates the reconnection of the vascular bundles, whereas NPA inhibits it. We studied the role of PIN proteins in graft development by grafting seedlings of PIN:GUS plants. PIN had different expression patterns in the graft process. Expression levels of PIN genes were analyzed by real-time PCR. All PIN genes had the higher expression level at the third DAG. We conclude that auxin stimulates the development of graft unions, and the patterns of expressions of PIN family genes can affect the development of graft-union by controlling the auxin flow.     

Dynamics in PIN2 auxin carrier ubiquitylation in gravity-responding Arabidopsis roots

Plant tropisms are decisively influenced by dynamic adjustments in spatiotemporal distribution of the growth regulators auxin. Polar auxin transport requires activity of PIN-type auxin carrier proteins, with their distribution at the plasma membrane significantly contributing to the directionality of auxin flow. Control of PIN protein distribution involves regulation of their endocytosis and further sorting into the lytic vacuole for degradation and recently, protein ubiquitylation has been demonstrated to control degradative sorting of plasma membrane proteins in plants.1-6 Here we show dynamic adjustments in PIN2 ubiquitylation in gravity-stimulated roots, a response that coincides with establishment of a lateral PIN2 expression gradient. Our results imply that perception and transduction of gravity signals triggers differential ubiquitylation of PIN2, which might feed back on the coordination of auxin distribution in root meristems.