Defective pollen tube tip growth induces neo-polyploid infertility.

Genome duplication (generating polyploids) is an engine of novelty in eukaryotic evolution and a promising crop improvement tool. Yet newly formed polyploids often have low fertility. Here we report that a severe fertility-compromising defect in pollen tube tip growth arises in new polyploids of Arabidopsis arenosa. Pollen tubes of newly polyploid A. arenosa grow slowly, have aberrant anatomy and disrupted physiology, often burst prematurely, and have altered gene expression. These phenotypes recover in evolved polyploids. We also show that gametophytic (pollen tube) genotypes of two tip-growth genes under selection in natural tetraploid A. arenosa are strongly associated with pollen tube performance in the tetraploid. Our work establishes pollen tube tip growth as an important fertility challenge for neo-polyploid plants and provides insights into a naturally evolved multigenic solution.

Two Is Company, but Four Is a Party-Challenges of Tetraploidization for Cell Wall Dynamics and Efficient Tip-Growth in Pollen.

Some cells grow by an intricately coordinated process called tip-growth, which allows the formation of long tubular structures by a remarkable increase in cell surface-to-volume ratio and cell expansion across vast distances. On a broad evolutionary scale, tip-growth has been extraordinarily successful, as indicated by its recurrent 're-discovery' throughout evolutionary time in all major land plant taxa which allowed for the functional diversification of tip-growing cell types across gametophytic and sporophytic life-phases. All major land plant lineages have experienced (recurrent) polyploidization events and subsequent re-diploidization that may have positively contributed to plant adaptive evolutionary processes. How individual cells respond to genome-doubling on a shorter evolutionary scale has not been addressed as elaborately. Nevertheless, it is clear that when polyploids first form, they face numerous important challenges that must be overcome for lineages to persist. Evidence in the literature suggests that tip-growth is one of those processes. Here, I discuss the literature to present hypotheses about how polyploidization events may challenge efficient tip-growth and strategies which may overcome them: I first review the complex and multi-layered processes by which tip-growing cells maintain their cell wall integrity and steady growth. I will then discuss how they may be affected by the cellular changes that accompany genome-doubling. Finally, I will depict possible mechanisms polyploid plants may evolve to compensate for the effects caused by genome-doubling to regain diploid-like growth, particularly focusing on cell wall dynamics and the subcellular machinery they are controlled by.

A Comprehensive Toolkit for Quick and Easy Visualization of Marker Proteins, Protein-Protein Interactions and Cell Morphology in Marchantia polymorpha.

Even though stable genomic transformation of sporelings and thalli of Marchantia polymorpha is straightforward and efficient, numerous problems can arise during critical phases of the process such as efficient spore production, poor selection capacity of antibiotics or low transformation efficiency. It is therefore also desirable to establish quick methods not relying on stable transgenics to analyze the localization, interactions and functions of proteins of interest. The introduction of foreign DNA into living cells via biolistic mechanisms has been first reported roughly 30 years ago and has been commonly exploited in established plant model species such as Arabidopsis thaliana or Nicotiana benthamiana. Here, we report the fast and reliable transient biolistic transformation of Marchantia thallus epidermal cells using fluorescent protein fusions. We present a catalog of fluorescent markers which can be readily used for tagging of a variety of subcellular compartments. Moreover, we report the functionality of the bimolecular fluorescence complementation (BiFC) in M. polymorpha with the example of the p-body markers MpDCP1/2. Finally, we provide standard staining procedures for live cell imaging in M. polymorpha, applicable to visualize cell boundaries or cellular structures, to complement or support protein localizations and to understand how results gained by transient transformations can be embedded in cell architecture and dynamics. Taken together, we offer a set of easy and quick tools for experiments that aim at understanding subcellular localization, protein-protein interactions and thus functions of proteins of interest in the emerging early diverging land plant model M. polymorpha.

An Evolutionarily Conserved Receptor-like Kinases Signaling Module Controls Cell Wall Integrity During Tip Growth.

Rooting cells and pollen tubes-key adaptative innovations that evolved during the colonization and subsequent radiation of plants on land-expand by tip growth. Tip growth relies on a tight coordination between the protoplast growth and the synthesis/remodeling of the external cell wall. In root hairs and pollen tubes of the seed plant Arabidopsis thaliana, cell wall integrity (CWI) mechanisms monitor this coordination through the Malectin-like receptor kinases (MLRs), such as AtANXUR1 and AtFERONIA, that act upstream of the AtMARIS PTI1-like kinase. Here, we show that rhizoid growth in the early diverging plant, Marchantia polymorpha, is also controlled by an MLR and PTI1-like signaling module. Rhizoids, root hairs, and pollen tubes respond similarly to disruption of MLR and PTI1-like encoding genes. Thus, the MLR and PTI1-like signaling module that controls CWI during tip growth is conserved between M. polymorpha and A. thaliana, suggesting that it was active in the common ancestor of land plants.

The Protein Phosphatases ATUNIS1 and ATUNIS2 Regulate Cell Wall Integrity in Tip-Growing Cells.

Fast tip-growing plant cells such as pollen tubes (PTs) and root hairs (RHs) require a robust coordination between their internal growth machinery and modifications of their extracellular rigid, yet extensible, cell wall (CW). Part of this essential coordination is governed by members of the Catharanthus roseus receptor-like kinase1-like (CrRLK1L) subfamily of RLKs with FERONIA (FER) and its closest homologs, ANXUR1 (ANX1) and ANX2, controlling CW integrity during RH and PT growth, respectively. Recently, Leucine-Rich Repeat Extensin 8 (LRX8) to LRX11 were also shown to be important for CW integrity in PTs. We previously reported an anx1 anx2 suppressor screen in Arabidopsis thaliana that revealed MARIS (MRI) as a positive regulator of both FER- and ANX1/2-dependent CW integrity pathways. Here, we characterize a suppressor that exhibits a weak rescue of the anx1 anx2 PT bursting phenotype and a short RH phenotype. The corresponding suppressor mutation causes a D94N substitution in a Type One Protein Phosphatase we named ATUNIS1 (AUN1). We show that AUN1 and its closest homolog, AUN2, are nucleocytoplasmic negative regulators of tip growth. Moreover, we demonstrate that AUN1D94N and AUN1H127A harboring mutations in key amino acids of the conserved catalytic site of phosphoprotein phosphatases function as dominant amorphic variants that repress PT growth. Finally, genetic interaction studies using the hypermorph MRIR240C and amorph AUN1D94N dominant variants indicate that LRX8-11 and ANX1/2 function in distinct but converging pathways to fine-tune CW integrity during tip growth.

Plant Malectin-Like Receptor Kinases: From Cell Wall Integrity to Immunity and Beyond.

Plant cells are surrounded by cell walls protecting them from a myriad of environmental challenges. For successful habitat adaptation, extracellular cues are perceived at the cell wall and relayed to downstream signaling constituents to mediate dynamic cell wall remodeling and adapted intracellular responses. Plant malectin-like receptor kinases, also known as Catharanthus roseus receptor-like kinase 1-like proteins (CrRLK1Ls), take part in these perception and relay processes. CrRLK1Ls are involved in many different plant functions. Their ligands, interactors, and downstream signaling partners are being unraveled, and studies about CrRLK1Ls' roles in plant species other than the plant model Arabidopsis thaliana are beginning to flourish. This review focuses on recent CrRLK1L-related advances in cell growth, reproduction, hormone signaling, abiotic stress responses, and, particularly, immunity. We also give an overview of the comparative genomics and evolution of CrRLK1Ls, and present a brief outlook for future research.

Imaging Ca2+ Dynamics in Wild-Type and NADPH Oxidase-Deficient Mutant Pollen Tubes with Yellow Cameleon and Confocal Laser Scanning Microscopy.

While cytosolic calcium (Ca2+) plays a central role in a myriad of signaling pathways as a secondary messenger, how dynamic changes of cytosolic calcium relate to cell growth control remains poorly understood. The engineering and continuous improvements of genetically encoded calcium sensors such as the Yellow Cameleon (YC) sensors combined with advances in microscopy have allowed imaging with great resolution of the spatiotemporal characteristics of cytosolic [Ca2+]cyt in individual cells. An exciting new step consists therefore in cautiously studying calcium dynamics in mutant backgrounds that display disturbed cellular growth behavior to further enhance our understanding on growth-related processes. Here, we describe methods to perform imaging of [Ca2+]cyt dynamics in growing Arabidopsis thaliana wild-type and NADPH-oxidase deficient rbohH rbohJ pollen tubes stably expressing YC3.6 using confocal laser scanning microscopy. We also present different ways to extract meaningful qualitative and quantitative information about calcium dynamics during growth.

Sexual reproduction, sporophyte development and molecular variation in the model moss Physcomitrella patens: introducing the ecotype Reute.

Rich ecotype collections are used for several plant models to unravel the molecular causes of phenotypic differences, and to investigate the effects of environmental adaption and acclimation. For the model moss Physcomitrella patens collections of accessions are available, and have been used for phylogenetic and taxonomic studies, for example, but few have been investigated further for phenotypic differences. Here, we focus on the Reute accession and provide expression profiling and comparative developmental data for several stages of sporophyte development, as well as information on genetic variation via genomic sequencing. We analysed cross-technology and cross-laboratory data to define a confident set of 15 mature sporophyte-specific genes. We find that the standard laboratory strain Gransden produces fewer sporophytes than Reute or Villersexel, although gametangia develop with the same time course and do not show evident morphological differences. Reute exhibits less genetic variation relative to Gransden than Villersexel, yet we found variation between Gransden and Reute in the expression profiles of several genes, as well as variation hot spots and genes that appear to evolve under positive Darwinian selection. We analyzed expression differences between the ecotypes for selected candidate genes in the GRAS transcription factor family, the chalcone synthase family and in genes involved in cell wall modification that are potentially related to phenotypic differences. We confirm that Reute is a P. patens ecotype, and suggest its use for reverse-genetics studies that involve progression through the life cycle and multiple generations.