Members of the ELMOD protein family specify formation of distinct aperture domains on the Arabidopsis pollen surface

Pollen apertures, the characteristic gaps in pollen wall exine, have emerged as a model for studying the formation of distinct plasma membrane domains. In each species, aperture number, position, and morphology are typically fixed; across species they vary widely. During pollen development, certain plasma membrane domains attract specific proteins and lipids and become protected from exine deposition, developing into apertures. However, how these aperture domains are selected is unknown. Here, we demonstrate that patterns of aperture domains in Arabidopsis are controlled by the members of the ancient ELMOD protein family, which, although important in animals, has not been studied in plants. We show that two members of this family, MACARON (MCR) and ELMOD_A, act upstream of the previously discovered aperture proteins and that their expression levels influence the number of aperture domains that form on the surface of developing pollen grains. We also show that a third ELMOD family member, ELMOD_E, can interfere with MCR and ELMOD_A activities, changing aperture morphology and producing new aperture patterns. Our findings reveal key players controlling early steps in aperture domain formation, identify residues important for their function, and open new avenues for investigating how diversity of aperture patterns in nature is achieved.

Members of the ELMOD protein family specify formation of distinct aperture domains on the Arabidopsis pollen surface

Pollen apertures, the characteristic gaps in pollen wall exine, have emerged as a model for studying the formation of distinct plasma-membrane domains. In each species, aperture number, position, and morphology are typically fixed; across species they vary widely. During pollen development certain plasma-membrane domains attract specific proteins and lipids and become protected from exine deposition, developing into apertures. However, how these aperture domains are selected is unknown. Here, we demonstrate that patterns of aperture domains in Arabidopsis are controlled by the members of the ancient ELMOD protein family, which, although important in animals, has not been studied in plants. We show that two members of this family, MACARON (MCR) and ELMOD_A, act upstream of the previously discovered aperture proteins and that their expression levels influence the number of aperture domains that form on the surface of developing pollen grains. We also show that a third ELMOD family member, ELMOD_E, can interfere with MCR and ELMOD_A activities, changing aperture morphology and producing new aperture patterns. Our findings reveal key players controlling early steps in aperture domain formation, identify residues important for their function, and open new avenues for investigating how diversity of aperture patterns in nature is achieved.

The hexose transporter SWEET5 confers galactose sensitivity to Arabidopsis pollen via the galactokinase GALK

Galactose is an abundant and essential sugar used for the biosynthesis of many macromolecules in different organisms, including plants. Galactose metabolism is tightly and finely controlled since excess galactose and derivatives are inhibitory. In Arabidopsis, root growth and pollen germination were strongly inhibited upon excess galactose. However, the mechanism of galactose induced inhibition during pollen germination remains obscure. In this study, we characterized AtSWEET5 as a glucose and galactose plasma-membrane transporter localized in the pollen. SWEET5 protein level start to accumulate since tricellular stage of pollen development and peaked in mature pollen before rapidly declining after pollen was germinated. SWEET5 levels are responsible for the dosage-dependent sensitivity of galactose and GALK is essential for the inhibitory effects of galactose during pollen germination. The unexpected observation that GALK is required for efficient galactose uptake in pollen may reveal an unknown regulatory mechanism for galactose transporters. Overall, SWEET5 and GALK contribute to the maintenance of galactose metabolic homeostasis during pollen germination, and galactose transport is positively regulated by GALK. The study of SWEET5 upon galactose condition also suggests SWEET5 is a major low-affinity hexose transporter at the early stage of pollen germination.One-sentence summarySWEET5 mediates pollen galactose sensitivity via GALK that is required for efficient galactose uptake in pollen during pollen germination.

Rho GTPase ROP1 Interactome Analysis Reveals Novel ROP1-Associated Pathways for Pollen Tube Polar Growth in Arabidopsis

ROP (Rho-like GTPases from plants) GTPases are polarly localized key regulators of polar growth in pollen tubes and other cells in plants. However, how ROP GTPases are regulated and how they control polar growth remains to be fully understood. To gain new insights into ROP-dependent mechanisms underlying polar cell growth, we characterized the interactome of ROP1 GTPase that controls Arabidopsis pollen tube (PT) tip growth, an extreme form of polar cell growth. We established an efficient method for culturing Arabidopsis pollen tubes in liquid medium, which was used for immunoprecipitation/mass spectrometry-based identification of ROP1-associated proteins. A total of 654 candidates were isolated from the ROP1 interactome in Arabidopsis pollen tubes, and GO (Gene Ontology) classification and pathway analysis revealed multiple uncharacterized ROP1-dependent processes including translation, cell wall modification, post transcriptional modification, and ion homeostasis, in addition to known ROP1-dependent pathways. The ROP1-interactome data was further supported by the co-expression of the candidate interactors in highly mature pollen with PT germination and growth defects being discovered in 25% (8/32) of the candidate mutant genes. Taken together, our work uncovers valuable information for the identification and functional elucidation of ROP-associated proteins in the regulation of polar growth, and provides a reliable reference to identify critical regulators of polar cell growth in the future.

Ultrastructural characterization of microlipophagy induced by the interaction of vacuoles and lipid bodies around generative and sperm cells in Arabidopsis pollen

During pollen maturation, various organelles change their distribution and function during development as male gametophytes. We analyzed the behavior of lipid bodies and vacuoles involved in lipophagy in Arabidopsis pollen using serial section SEM and conventional TEM. At the bicellular pollen stage, lipid bodies in the vegetative cells lined up at the surface of the generative cell. Vacuoles then tightly attached, drew in, and degraded the lipid bodies and eventually occupied the space of the lipid bodies. Degradation of lipid began before transfer of the entire contents of the lipid body. At the tricellular stage, vacuoles instead of lipid bodies surrounded the sperm cells. The degradation of lipid bodies is morphologically considered microautophagy. The atg2-1 Arabidopsis mutant is deficient in one autophagy-related gene (ATG). In this mutant, the assembly of vacuoles around sperm cells was sparser than that in wild-type pollen. The deficiency of ATG2 likely prevents or slows lipid degradation, although it does not prevent contact between organelles. These results demonstrate the involvement of microlipophagy in the pollen development of Arabidopsis.

First Steps in the Successful Fertilization of Rice and Arabidopsis: Pollen Longevity, Adhesion and Hydration

Understanding the behavior of pollen during pollination is important for food security in the future. The elucidation of pollen development and growth regulation largely relies on the study of the dicotyledonous model plant Arabidopsis thaliana. However, rice (Oryza sativa) pollen exhibits different characteristics to that of Arabidopsis. The latter undergoes programmed dehydration and withstands adverse environmental conditions, whereas rice pollen is sensitive to desiccation. Moreover, the short longevity of rice pollen significantly hampers hybrid seed production. Although the “omics” data for mature rice pollen have been accumulated, few genes that control pollination and pollen hydration have been identified. Therefore, to facilitate future studies, it is necessary to summarize the developmental processes involved in pollen production in rice and to consolidate the underlying mechanisms discovered in previous studies. In this review, we describe the pollen developmental processes and introduce gametophytic mutants, which form defective pollen in Arabidopsis and rice. In addition, we discuss the perspectives on the research on pollen longevity, adhesion and hydration.

New opportunities and insights into Papaver self-incompatibility by imaging engineered Arabidopsis pollen

Pollen tube growth is essential for plant reproduction. Their rapid extension using polarized tip growth provides an exciting system for studying this specialized type of growth. Self-incompatibility (SI) is a genetically controlled mechanism to prevent self-fertilization. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy). This utilizes two S-determinants: stigma-expressed PrsS and pollen-expressed PrpS. Interaction of cognate PrpS–PrsS triggers a signalling network, causing rapid growth arrest and programmed cell death (PCD) in incompatible pollen. We previously demonstrated that transgenic Arabidopsis thaliana pollen expressing PrpS–green fluorescent protein (GFP) can respond to Papaver PrsS with remarkably similar responses to those observed in incompatible Papaver pollen. Here we describe recent advances using these transgenic plants combined with genetically encoded fluorescent probes to monitor SI-induced cellular alterations, including cytosolic calcium, pH, the actin cytoskeleton, clathrin-mediated endocytosis (CME), and the vacuole. This approach has allowed us to study the SI response in depth, using multiparameter live-cell imaging approaches that were not possible in Papaver. This lays the foundations for new opportunities to elucidate key mechanisms involved in SI. Here we establish that CME is disrupted in self-incompatible pollen. Moreover, we reveal new detailed information about F-actin remodelling in pollen tubes after SI.