Kinase Partner Protein Plays a Key Role in Controlling the Speed and Shape of Pollen Tube Growth in Tomato.

The rapid and responsive growth of a pollen tube requires delicate coordination of membrane receptor signaling, Rho-of-Plants (ROP) GTPase activity switching, and actin cytoskeleton assembly. The tomato (Solanum lycopersicum) kinase partner protein (KPP), is a ROP guanine nucleotide exchange factor (GEF) that activates ROP GTPases and interacts with the tomato pollen receptor kinases LePRK1 and LePRK2. It remains unclear how KPP relays signals from plasma membrane-localized LePRKs to ROP switches and other cellular machineries to modulate pollen tube growth. Here, we biochemically verified KPP's activity on ROP4 and showed that KPP RNA interference transgenic pollen tubes grew slower while KPP-overexpressing pollen tubes grew faster, suggesting that KPP functions as a rheostat for speed control in LePRK2-mediated pollen tube growth. The N terminus of KPP is required for self-inhibition of its ROPGEF activity, and expression of truncated KPP lacking the N terminus caused pollen tube tip enlargement. The C-terminus of KPP is required for its interaction with LePRK1 and LePRK2, and the expression of a truncated KPP lacking the C-terminus triggered pollen tube bifurcation. Furthermore, coexpression assays showed that self-associated KPP recruited actin-nucleating Actin-Related Protein2/3 (ARP2/3) complexes to the tip membrane. Interfering with ARP2/3 activity reduced the pollen tube abnormalities caused by overexpressing KPP fragments. In conclusion, KPP plays a key role in pollen tube speed and shape control by recruiting the branched actin nucleator ARP2/3 complex and an actin bundler to the membrane-localized receptors LePRK1 and LePRK2.

Tomato Pistil Factor STIG1 Promotes in Vivo Pollen Tube Growth by Binding to Phosphatidylinositol 3-Phosphate and the Extracellular Domain of the Pollen Receptor Kinase LePRK2.

The speed of pollen tube growth is a major determinant of reproductive success in flowering plants. Tomato (Solanum lycopersicum) STIGMA-SPECIFIC PROTEIN1 (STIG1), a small Cys-rich protein from the pistil, was previously identified as a binding partner of the pollen receptor kinase LePRK2 and shown to promote pollen tube growth in vitro. However, the in vivo function of STIG1 and the underlying mechanism of its promotive effect were unknown. Here, we show that a 7-kD processed peptide of STIG1 is abundant in the stigmatic exudate and accumulates at the pollen tube surface, where it can bind LePRK2. Antisense LePRK2 pollen was less responsive than wild-type pollen to exogenous STIG1 in an in vitro pollen germination assay. Silencing of STIG1 reduced both the in vivo pollen tube elongation rate and seed production. Using partial deletion and point mutation analyses, two regions underlying the promotive activity of the STIG1 processed peptide were identified: amino acids 80 to 83, which interact with LePRK2; and amino acids 88 to 115, which bind specifically to phosphatidylinositol 3-phosphate [PI(3)P]. Furthermore, exogenous STIG1 elevated the overall redox potential of pollen tubes in both PI(3)P-dependent and LePRK2-dependent manners. Our results demonstrate that STIG1 conveys growth-promoting signals acting through the pollen receptor kinase LePRK2, a process that relies on the external phosphoinositide PI(3)P.

Overexpression of the tomato pollen receptor kinase LePRK1 rewires pollen tube growth to a blebbing mode.

The tubular growth of a pollen tube cell is crucial for the sexual reproduction of flowering plants. LePRK1 is a pollen-specific and plasma membrane-localized receptor-like kinase from tomato (Solanum lycopersicum). LePRK1 interacts with another receptor, LePRK2, and with KINASE PARTNER PROTEIN (KPP), a Rop guanine nucleotide exchange factor. Here, we show that pollen tubes overexpressing LePRK1 or a truncated LePRK1 lacking its extracellular domain (LePRK1ΔECD) have enlarged tips but also extend their leading edges by producing "blebs." Coexpression of LePRK1 and tomato PLIM2a, an actin bundling protein that interacts with KPP in a Ca(2+)-responsive manner, suppressed these LePRK1 overexpression phenotypes, whereas pollen tubes coexpressing KPP, LePRK1, and PLIM2a resumed the blebbing growth mode. We conclude that overexpression of LePRK1 or LePRK1ΔECD rewires pollen tube growth to a blebbing mode, through KPP- and PLIM2a-mediated bundling of actin filaments from tip plasma membranes. Arabidopsis thaliana pollen tubes expressing LePRK1ΔECD also grew by blebbing. Our results exposed a hidden capability of the pollen tube cell: upon overexpression of a single membrane-localized molecule, LePRK1 or LePRK1ΔECD, it can switch to an alternative mechanism for extension of the leading edge that is analogous to the blebbing growth mode reported for Dictyostelium and for Drosophila melanogaster stem cells.

An image skeletonization-based tool for pollen tube morphology analysis and phenotyping.

The mechanism underlying pollen tube growth involves diverse genes and molecular pathways. Alterations in the regulatory genes or pathways cause phenotypic changes reflected by cellular morphology, which can be captured using fluorescence microscopy. Determining and classifying pollen tube morphological phenotypes in such microscopic images is key to our understanding the involvement of genes and pathways. In this context, we propose a computational method to extract quantitative morphological features, and demonstrate that these features reflect morphological differences relevant to distinguish different defects of pollen tube growth. The corresponding software tool furthermore includes a novel semi-automated image segmentation approach, allowing to highly accurately identify the boundary of a pollen tube in a microscopic image.

[Fluorescence study on the interaction of narrow distribution low polymerization degree chitooligosaccharides and human serum albumin].

Chitosan (CTS), a linear binary copolymer of (1-->8)-linked 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc unit) and 2-amino-2-deoxy-beta-D-glucopyranose (GlcNH2 unit), is derived from chitin by alkaline deacetylation. In the present work, the narrow molecular weight distribution chitooligosaccharides were prepared by degraded CTS with a microwave-assisted-cleavage method of metal-coordinating template-absorption catalytic oxidation. Under physiological pH conditions, the interaction of CTS and the narrow distribution chitooligosaccharides with Human serum albumin (HSA) was preliminarily explored by fluorescence spectra. Low molecular weight CTS in different concentration was added into HSA solution respectively, the absorptivity of the HSA solution decreased considerably. This phenomenon indicated that there was an interaction between these six different low molecular weight CTS with HSA, and the smaller the DP of the narrow distribution chitooligosaccharides, the stronger the interaction with HSA. However, the interaction gradually failed in as the DP was less than eight. The interaction almost disappeared when using glucosamine, the final product of degraded CTS, which revealed that there was a scale effect between chain-like CTS molecule and protein biomacromolecule. The results suggest that the strongest interaction binding occurs in CTS with DP approximately =8. Whether the DP increases or decreases, the interaction will weaken.