Actin-Dependent and -Independent Functions of Cortical Microtubules in the Differentiation of Arabidopsis Leaf Trichomes

Arabidopsis thaliana tortifolía2 carries a point mutation in α-tubulin 4 and shows aberrant cortical microtubule dynamics. The microtubule defect of tortifolia2 leads to overbranching and right-handed helical growth in the single-celled leaf trichomes. Here, we use tortifolia2 to further our understanding of microtubules in plant cell differentiation. Trichomes at the branching stage show an apical ring of cortical microtubules, and our analyses support that this ring is involved in marking the prospective branch site. tortifolia2 showed ectopic microtubule bundles at this stage, consistent with a function for microtubules in selecting new branch sites. Overbranching of tortifolia2 required the C-terminal binding protein/brefeldin A-ADP ribosylated substrate protein ANGUSTIFOLIA1, and our results indicate that the angustifolia1 mutant is hypersensitive to alterations in microtubule dynamics. To analyze whether actin and microtubules cooperate in the trichome cell expansion process, we generated double mutants of tortifolia2 with distorted1, a mutant that is defective in the actin-related ARP2/3 complex. The double mutant trichomes showed a complete loss of growth anisotropy, suggesting a genetic interaction of actin and microtubules. Green fluorescent protein labeling of F-actin or microtubules in tortifolia2 distorted1 double mutants indicated that F-actin enhances microtubule dynamics and enables reorientation. Together, our results suggest actin-dependent and -independent functions of cortical microtubules in trichome differentiation.     

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

Myrosin cells, which accumulate myrosinase to produce toxic compounds when they are ruptured by herbivores, form specifically along leaf veins in Arabidopsis thaliana. However, the mechanism underlying this pattern formation is unknown. Here, we show that myrosin cell development requires the endocytosis-mediated polar localization of the auxin-efflux carrier PIN1 in leaf primordia. Defects in the endocytic/vacuolar SNAREs (syp22 and syp22 vti11) enhanced myrosin cell development. The syp22 phenotype was rescued by expressing SYP22 under the control of the PIN1 promoter. Additionally, myrosin cell development was enhanced either by lacking the activator of endocytic/vacuolar RAB5 GTPase (VPS9A) or by PIN1 promoter-driven expression of a dominant-negative form of RAB5 GTPase (ARA7). By contrast, myrosin cell development was not affected by deficiencies of vacuolar trafficking factors, including the vacuolar sorting receptor VSR1 and the retromer components VPS29 and VPS35, suggesting that endocytic pathway rather than vacuolar trafficking pathway is important for myrosin cell development. The phosphomimic PIN1 variant (PIN1-Asp), which is unable to be polarized, caused myrosin cells to form not only along leaf vein but also in the intervein leaf area. We propose that Brassicales plants might arrange myrosin cells near vascular cells in order to protect the flux of nutrients and water via polar PIN1 localization.     

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.     

Photosynthetic Control of Arabidopsis Leaf Cytoplasmic Translation Initiation by Protein Phosphorylation

Photosynthetic CO₂ assimilation is the carbon source for plant anabolism, including amino acid production and protein synthesis. The biosynthesis of leaf proteins is known for decades to correlate with photosynthetic activity but the mechanisms controlling this effect are not documented. The cornerstone of the regulation of protein synthesis is believed to be translation initiation, which involves multiple phosphorylation events in Eukaryotes. We took advantage of phosphoproteomic methods applied to Arabidopsis thaliana rosettes harvested under controlled photosynthetic gas-exchange conditions to characterize the phosphorylation pattern of ribosomal proteins (RPs) and eukaryotic initiation factors (eIFs). The analyses detected 14 and 11 new RP and eIF phosphorylation sites, respectively, revealed significant CO₂-dependent and/or light/dark phosphorylation patterns and showed concerted changes in 13 eIF phosphorylation sites and 9 ribosomal phosphorylation sites. In addition to the well-recognized role of the ribosomal small subunit protein RPS6, our data indicate the involvement of eIF3, eIF4A, eIF4B, eIF4G and eIF5 phosphorylation in controlling translation initiation when photosynthesis varies. The response of protein biosynthesis to the photosynthetic input thus appears to be the result of a complex regulation network involving both stimulating (e.g. RPS6, eIF4B phosphorylation) and inhibiting (e.g. eIF4G phosphorylation) molecular events.     

Maize Leaf Rolling Initiation

Maize plants of CPB2 and CPB8 hybrids were kept under water deficit for 22 d. In the CPB8 hybrid, leaf rolling initiated at the 9^th d of water deficit period, while in CPB2 hybrid it was at the 15^th d. Both hybrids showed leaf rolling initiation at the same leaf water potential, Ψ_W of -0.480±0.095 MPa. At leaf rolling initiation, the leaf osmotic potential, Ψ_S was -0.730±0.085 MPa in CPB8 and 0.630±0.110 MPa in CPB2. The leaf temperature and stomatal conductance were higher in CPB8 than in CPB2. Values of leaf Ψ_W, ribulose-1,5-bisphosphate carboxylase activity, chlorophyll content, and specific leaf area were similar in both hybrids. Phosphoenolpyruvate carboxylase activity and protein content were lower in the CPB2 hybrid than in CPB8. In both hybrids leaf rolling initiation was associated with: (1) higher leaf temperature, with leaf rolling effect related to leaf temperature reduction, and (2) lower leaf Ψ_S, related to osmotic adjustment as an additional component of drought-tolerance strategy. This revised version was published online in June 2006 with corrections to the Cover Date.     

Maternal Control of PIN1 Is Required for Female Gametophyte Development in Arabidopsis

Land plants are characterised by haplo-diploid life cycles, and developing ovules are the organs in which the haploid and diploid generations coexist. Recently it has been shown that hormones such as auxin and cytokinins play important roles in ovule development and patterning. The establishment and regulation of auxin levels in cells is predominantly determined by the activity of the auxin efflux carrier proteins PIN-FORMED (PIN). To study the roles of PIN1 and PIN3 during ovule development we have used mutant alleles of both genes and also perturbed PIN1 and PIN3 expression using micro-RNAs controlled by the ovule specific DEFH9 (DEFIFICENS Homologue 9) promoter. PIN1 down-regulation and pin1-5 mutation severely affect female gametophyte development since embryo sacs arrest at the mono- and/or bi-nuclear stages (FG1 and FG3 stage). PIN3 function is not required for ovule development in wild-type or PIN1-silenced plants. We show that sporophytically expressed PIN1 is required for megagametogenesis, suggesting that sporophytic auxin flux might control the early stages of female gametophyte development, although auxin response is not visible in developing embryo sacs.     

Cell polarity and PIN protein positioning in Arabidopsis require STEROL METHYLTRANSFERASE1 function

Plants have many polarized cell types, but relatively little is known about the mechanisms that establish polarity. The orc mutant was identified originally by defects in root patterning, and positional cloning revealed that the affected gene encodes STEROL METHYLTRANSFERASE1, which is required for the appropriate synthesis and composition of major membrane sterols. smt1(orc) mutants displayed several conspicuous cell polarity defects. Columella root cap cells revealed perturbed polar positioning of different organelles, and in the smt1(orc) root epidermis, polar initiation of root hairs was more randomized. Polar auxin transport and expression of the auxin reporter DR5-beta-glucuronidase were aberrant in smt1(orc). Patterning defects in smt1(orc) resembled those observed in mutants of the PIN gene family of putative auxin efflux transporters. Consistently, the membrane localization of the PIN1 and PIN3 proteins was disturbed in smt1(orc), whereas polar positioning of the influx carrier AUX1 appeared normal. Our results suggest that balanced sterol composition is a major requirement for cell polarity and auxin efflux in Arabidopsis.     

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.     

Inflorescence Initiation and Leaf Size in Some Gramineae

The morphology of successive leaves on the flowering shoot wasstudied in species of Glyceria, Lolium, and Triticum. The bladesof successive leaves were progressively longer, eventually reachinga maximum, after which the blades of the last few leaves producedbefore heading were shorter. When the longest leaf blade waselongating, dissection of the shoot apices showed that inflorescenceinitiation was taking place. Epidermal cell measurements inTriticum indicate that differences in blade length are due todifferences in the amount of cell extension. It appears that a correlated change occurs in blade morphologyassociated with the onset of the reproductive state of the shootapex, brought about via changes in the amount of cell extension. A study of the effect of different amounts of low-temperatureand different day-lengths on the relation between inflorescenceinitiation and the production of the longest leaf blade showedthat, under some conditions, this relation can be disturbed.     

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.     

Clathrin-mediated constitutive endocytosis of PIN auxin efflux carriers in Arabidopsis

Endocytosis is an essential process by which eukaryotic cells internalize exogenous material or regulate signaling at the cell surface [1]. Different endocytic pathways are well established in yeast and animals; prominent among them is clathrin-dependent endocytosis [2, 3]. In plants, endocytosis is poorly defined, and no molecular mechanism for cargo internalization has been demonstrated so far [4, 5], although the internalization of receptor-ligand complexes at the plant plasma membrane has recently been shown [6]. Here we demonstrate by means of a green-to-red photoconvertible fluorescent reporter, EosFP [7], the constitutive endocytosis of PIN auxin efflux carriers [8] and their recycling to the plasma membrane. Using a plant clathrin-specific antibody, we show the presence of clathrin at different stages of coated-vesicle formation at the plasma membrane in Arabidopsis. Genetic interference with clathrin function inhibits PIN internalization and endocytosis in general. Furthermore, pharmacological interference with cargo recruitment into the clathrin pathway blocks internalization of PINs and other plasma-membrane proteins. Our data demonstrate that clathrin-dependent endocytosis is operational in plants and constitutes the predominant pathway for the internalization of numerous plasma-membrane-resident proteins including PIN auxin efflux carriers.     

Initiation of axillary and floral meristems in Arabidopsis

Shoot development is reiterative: shoot apical meristems (SAMs) give rise to branches made of repeating leaf and stem units with new SAMs in turn formed in the axils of the leaves. Thus, new axes of growth are established on preexisting axes. Here we describe the formation of axillary meristems and floral meristems in Arabidopsis by monitoring the expression of the SHOOT MERISTEMLESS and AINTEGUMENTA genes. Expression of these genes is associated with SAMs and organ primordia, respectively. Four stages of axillary meristem development and previously undefined substages of floral meristem development are described. We find parallels between the development of axillary meristems and the development of floral meristems. Although Arabidopsis flowers develop in the apparent absence of a subtending leaf, the expression patterns of AINTEGUMENTA and SHOOT MERISTEMLESS RNAs during flower development suggest the presence of a highly reduced, "cryptic" leaf subtending the flower in Arabidopsis. We hypothesize that the STM-negative region that develops on the flanks of the inflorescence meristem is a bract primordium and that the floral meristem proper develops in the "axil" of this bract primordium. The bract primordium, although initially specified, becomes repressed in its growth.     

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.