Caleb M. Rounds

Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.

Background Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments. Methodology/Principal Findings In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore. Conclusions/Significance Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actins role in tip growing plant cells.

atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins

The members of the Toc159 family of GTPases act as the primary receptors for the import of nucleus-encoded preproteins into plastids. Toc159, the most abundant member of this family in chloroplasts, is required for chloroplast biogenesis (Bauer, J., K. Chen, A. Hiltbunner, E. Wehrli, M. Eugster, D. Schnell, and F. Kessler. 2000. Nature. 403:203–207) and has been shown to covalently cross-link to bound preproteins at the chloroplast surface (Ma, Y., A. Kouranov, S. LaSala, and D.J. Schnell. 1996. J. Cell Biol. 134:1–13; Perry, S.E., and K. Keegstra. 1994. Plant Cell. 6:93–105). These reports led to the hypothesis that Toc159 functions as a selective import receptor for preproteins that are required for chloroplast development. In this report, we provide evidence that Toc159 is required for the import of several highly expressed photosynthetic preproteins in vivo. Furthermore, we demonstrate that the cytoplasmic and recombinant forms of soluble Toc159 bind directly and selectively to the transit peptides of these representative photosynthetic preproteins, but not representative constitutively expressed plastid preproteins. These data support the function of Toc159 as a selective import receptor for the targeting of a set of preproteins required for chloroplast biogenesis.

Exocytosis Precedes and Predicts the Increase in Growth in Oscillating Pollen Tubes

We examined exocytosis during oscillatory growth in lily (Lilium formosanum and Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes using three markers: (1) changes in cell wall thickness by Nomarski differential interference contrast (DIC), (2) changes in apical cell wall fluorescence in cells stained with propidium iodide (PI), and (3) changes in apical wall fluorescence in cells expressing tobacco pectin methyl esterase fused to green fluorescent protein (PME-GFP). Using PI fluorescence, we quantified oscillatory changes in the amount of wall material from both lily and tobacco pollen tubes. Measurement of wall thickness by DIC was only possible with lily due to limitations of microscope resolution. PME-GFP, a direct marker for exocytosis, only provides information in tobacco because its expression in lily causes growth inhibition and cell death. We show that exocytosis in pollen tubes oscillates and leads the increase in growth rate; the mean phase difference between exocytosis and growth is –98° ± 3° in lily and –124° ± 4° in tobacco. Statistical analyses reveal that the anticipatory increase in wall material predicts, to a high degree, the rate and extent of the subsequent growth surge. Exocytosis emerges as a prime candidate for the initiation and regulation of oscillatory pollen tube growth.

Growth Mechanisms in Tip-Growing Plant Cells

Tip growth is employed throughout the plant kingdom. Our understanding of tip growth has benefited from modern tools in molecular genetics, which have enabled the functional characterization of proteins mediating tip growth. Here we first discuss the evolutionary role of tip growth in land plants and then describe the prominent model tip-growth systems, elaborating on some advantages and disadvantages of each. Next we review the organization of tip-growing cells, the role of the cytoskeleton, and recent developments concerning the physiological basis of tip growth. Finally, we review advances in the understanding of the extracellular signals that are known to guide tip-growing cells.

Control of Cell Wall Extensibility during Pollen Tube Growth

In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expansion rate is controlled by local changes in the viscosity of the apical wall. We focus on several different structures and underlying processes that are thought to be major participants including exocytosis, the organization and activity of the actin cytoskeleton, calcium and proton physiology, and cellular energetics. We think that the actin cytoskeleton, in particular the apical cortical actin fringe, directs the flow of vesicles to the apical domain, where they fuse with the plasma membrane and contribute their contents to the expanding cell wall. While pH gradients, as generated by a proton-ATPase located on the plasma membrane along the side of the clear zone, may regulate rapid actin turnover and new polymerization in the fringe, the tip-focused calcium gradient biases secretion towards the polar axis. The recent data showing that exocytosis of new wall material precedes and predicts the process of cell elongation provide support for the idea that the intussusception of newly secreted pectin contributes to decreases in apical wall viscosity and to cell expansion. Other prime factors will be the localization and activity of the enzyme pectin methyl-esterase, and the chelation of calcium by pectic acids. Finally, we acknowledge a role for reactive oxygen species in the control of wall viscosity.

Calcium entry into pollen tubes

Growing pollen tubes require calcium to maintain a tip-focused cytosolic gradient and as a constituent of the constantly expanding cell wall. Advances in cell and molecular biology as well as electrophysiology implicate several candidate channels and receptors in the flow of calcium into the cell. In this review we discuss the channels that have been identified and consider the role of the growing tip cell wall acting as a sink for calcium thus accounting for differences in oscillatory phase between influx measured on the outside of the cell and changes in tip concentration inside the cell. We also briefly draw attention to uptake mechanisms that restrict and shape the calcium signature in the growing pollen tube.

Propidium iodide competes with Ca2+ to label pectin in pollen tubes and arabidopsis root hairs

We have used propidium iodide (PI) to investigate the dynamic properties of the primary cell wall at the apex of Arabidopsis (Arabidopsis thaliana) root hairs and pollen tubes and in lily (Lilium formosanum) pollen tubes. Our results show that in root hairs, as in pollen tubes, oscillatory peaks in PI fluorescence precede growth rate oscillations. Pectin forms the primary component of the cell wall at the tip of both root hairs and pollen tubes. Given the electronic structure of PI, we investigated whether PI binds to pectins in a manner analogous to Ca2+ binding. We first show that Ca2+ is able to abrogate PI growth inhibition in a dose-dependent manner. PI fluorescence itself also relies directly on the amount of Ca2+ in the growth solution. Exogenous pectin methyl esterase treatment of pollen tubes, which demethoxylates pectins, freeing more Ca2+-binding sites, leads to a dramatic increase in PI fluorescence. Treatment with pectinase leads to a corresponding decrease in fluorescence. These results are consistent with the hypothesis that PI binds to demethoxylated pectins. Unlike other pectin stains, PI at low yet useful concentration is vital and specifically does not alter the tip-focused Ca2+ gradient or growth oscillations. These data suggest that pectin secretion at the apex of tip-growing plant cells plays a critical role in regulating growth, and PI represents an excellent tool for examining the role of pectin and of Ca2+ in tip growth.

The Molecular Basis for Distinct Pathways for Protein Import into Arabidopsis Chloroplasts

This work investigates the structural determinants for receptor and pathway specificity in chloroplast protein targeting. It provides evidence that the A-domains of the Toc159 family of import receptors are major determinants of distinct pathways for protein import into plastids. The translocons at the outer envelope membrane of chloroplasts (TOCs) initiate the import of thousands of nucleus-encoded proteins into the organelle. The identification of structurally and functionally distinct TOC complexes has led to the hypothesis that the translocons constitute different import pathways that are required to coordinate the import of sets of proteins whose expression varies in response to organelle biogenesis and physiological adaptation. To test this hypothesis, we examined the molecular basis for distinct TOC pathways by analyzing the functional diversification among the Toc159 family of TOC receptors. We demonstrate that the N-terminal A-domains of the Toc159 receptors regulate their selectivity for preprotein binding. Furthermore, the in vivo function of the two major Toc159 family members (atToc159 and atToc132) can be largely switched by swapping their A-domains in transgenic Arabidopsis thaliana. On the basis of these results, we propose that the A-domains of the Toc159 receptors are major determinants of distinct pathways for protein import into chloroplasts.

Pollen tube energetics: respiration, fermentation and the race to the ovule

This review covers historical and recent progress in understanding how respiration, fermentation and mitochondria contribute to pollen tube growth. It also summarizes what is known about the energetic requirements of this growth. Molecular mechanisms are viewed in the context of pollen tube physiology, a necessary perspective to understanding pollen tube growth.

The apical actin fringe contributes to localized cell wall deposition and polarized growth in the lily pollen tube

Inhibition of lily pollen tube growth with three different agents, brefeldin A, latrunculin B, and potassium cyanide, provides evidence that the apical actin fringe contributes to localized pectin deposition and polarized cell growth. In lily (Lilium formosanum) pollen tubes, pectin, a major component of the cell wall, is delivered through regulated exocytosis. The targeted transport and secretion of the pectin-containing vesicles may be controlled by the cortical actin fringe at the pollen tube apex. Here, we address the role of the actin fringe using three different inhibitors of growth: brefeldin A, latrunculin B, and potassium cyanide. Brefeldin A blocks membrane trafficking and inhibits exocytosis in pollen tubes; it also leads to the degradation of the actin fringe and the formation of an aggregate of filamentous actin at the base of the clear zone. Latrunculin B, which depolymerizes filamentous actin, markedly slows growth but allows focused pectin deposition to continue. Of note, the locus of deposition shifts frequently and correlates with changes in the direction of growth. Finally, potassium cyanide, an electron transport chain inhibitor, briefly stops growth while causing the actin fringe to completely disappear. Pectin deposition continues but lacks focus, instead being delivered in a wide arc across the pollen tube tip. These data support a model in which the actin fringe contributes to the focused secretion of pectin to the apical cell wall and, thus, to the polarized growth of the pollen tube.