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Strigolactones (SLs) represent a class of plant hormones that regulate developmental processes and play a role in the response of plants to various biotic and abiotic stresses. Both in planta hormonal roles and ex planta signalling effects of SLs are potentially interesting agricultural targets. In this review, we explore various aspects of SL function and highlight distinct areas of agriculture that may benefit from the use of synthetic SL analogues, and we identify possible bottlenecks. Our objective is to identify where the contributions of science and stakeholders are still needed to achieve harnessing the benefits of SLs for a sustainable agriculture of the near future.

Pollen tubes are one of the fastest growing eukaryotic cells. Rapid anisotropic growth is supported by highly active exocytosis and endocytosis at the plasma membrane, but the subcellular localization of these sites is unknown. To understand molecular processes involved in pollen tube growth, it is crucial to identify the sites of vesicle localization and trafficking. This report presents novel strategies to identify exocytic and endocytic vesicles and to visualize vesicle trafficking dynamics, using pulse-chase labelling with styryl FM dyes and refraction-free high-resolution time-lapse differential interference contrast microscopy. These experiments reveal that the apex is the site of endocytosis and membrane retrieval, while exocytosis occurs in the zone adjacent to the apical dome. Larger vesicles are internalized along the distal pollen tube. Discretely sized vesicles that differentially incorporate FM dyes accumulate in the apical, subapical, and distal regions. Previous work established that pollen tube growth is strongly correlated with hydrodynamic flux and cell volume status. In this report, it is shown that hydrodynamic flux can selectively increase exocytosis or endocytosis. Hypotonic treatment and cell swelling stimulated exocytosis and attenuated endocytosis, while hypertonic treatment and cell shrinking stimulated endocytosis and inhibited exocytosis. Manipulation of pollen tube apical volume and membrane remodelling enabled fine-mapping of plasma membrane dynamics and defined the boundary of the growth zone, which results from the orchestrated action of endocytosis at the apex and along the distal tube and exocytosis in the subapical region. This report provides crucial spatial and temporal details of vesicle trafficking and anisotropic growth.

A micropore-filtration method was used to reduce the proportion of plant DNA in microbial DNA samples isolated from roots prior to sequencing. We tested the impact of this pre-sequencing filtration methodology and used it to characterize the root microbiome of maize grown on two soils with different fertility levels. The micropore filtration reduced plant DNA contamination and unveiled potential in the N-poor soil for N fixation in roots and phosphate uptake by roots in the phosphate-poor soil. Our methodology and findings allude to the potential capability of plants to initiate plant-microbe interactions under sub-optimal soil fertility.

• Pollen tubes require a tightly regulated pectin secretion machinery to sustain the cell wall plasticity required for polar tip growth. Involved in this regulation at the apical plasma membrane are proteins and signaling molecules, including phosphoinositides and phosphatidic acid (PA). However, the contribution of diacylglycerol kinases (DGKs) is not clear. • We transiently expressed tobacco DGKs in pollen tubes to identify a plasma membrane (PM)-localized isoform, and then to study its effect on pollen tube growth, pectin secretion and lipid signaling. In order to potentially downregulate DGK5 function, we overexpressed an inactive variant. • Only one of eight DGKs displayed a confined localization at the apical PM. We could demonstrate its enzymatic activity and that a kinase-dead variant was inactive. Overexpression of either variant led to differential perturbations including misregulation of pectin secretion. One mode of regulation could be that DGK5-formed PA regulates phosphatidylinositol 4-phosphate 5-kinases, as overexpression of the inactive DGK5 variant not only led to a reduction of PA but also of phosphatidylinositol 4,5-bisphosphate levels and suppressed related growth phenotypes. • We conclude that DGK5 is an additional player of polar tip growth that regulates pectin secretion probably in a common pathway with PI4P 5-kinases.

Phospholipids are emerging as novel second messengers in plant cells. They are rapidly formed in response to a variety of stimuli via the activation of lipid kinases or phospholipases. These lipid signals can activate enzymes or recruit proteins to membranes via distinct lipid-binding domains, where the local increase in concentration promotes interactions and downstream signaling. Here, the latest developments in phospholipid-based signaling are discussed, including the lipid kinases and phospholipases that are activated, the signals they produce, the domains that bind them, the downstream targets that contain them and the processes they control.

The activation of phosphoinositide-specific phospholipase C (PI-PLC) is one of the earliest responses triggered by the recognition of several microbe-associated molecular patterns (MAMPs) in plants. The Arabidopsis (Arabidopsis thaliana) PI-PLC gene family is composed of nine members. Previous studies suggested a role for PLC2 in MAMP-triggered immunity, as it is rapidly phosphorylated in vivo upon treatment with the bacterial MAMP flg22. Here, we analyzed the role of PLC2 in plant immunity using an artificial microRNA to silence PLC2 expression in Arabidopsis. We found that PLC2-silenced plants are more susceptible to the type III secretion system-deficient bacterial strain Pseudomonas syringae pv tomato (Pst) DC3000 hrcC⁻ and to the nonadapted pea (Pisum sativum) powdery mildew Erysiphe pisi. However, PLC2-silenced plants display normal susceptibility to virulent (Pst DC3000) and avirulent (Pst DC3000 AvrRPM1) P. syringae strains, conserving typical hypersensitive response features. In response to flg22, PLC2-silenced plants maintain wild-type mitogen-activated protein kinase activation and PHI1, WRKY33, and FRK1 immune marker gene expression but have reduced reactive oxygen species (ROS)-dependent responses such as callose deposition and stomatal closure. Accordingly, the generation of ROS upon flg22 treatment is compromised in the PLC2-defficient plants, suggesting an effect of PLC2 in a branch of MAMP-triggered immunity and nonhost resistance that involves early ROS-regulated processes. Consistently, PLC2 associates with the NADPH oxidase RBOHD, suggesting its potential regulation by PLC2.

Phosphatidylinositol 3-P (PI3P) is a signaling molecule that controls a variety of processes in endosomal, autophagic, and vacuolar/lysosomal trafficking in yeasts and mammals. Vacuolar protein sorting 34 (Vps34) is a conserved PI3K present in multiple complexes with specific functions and regulation. In yeast, the PI3K complex II consists of Vps34p, Vps15p, Vps30p/Atg6p, and Vps38p, and is essential for vacuolar protein sorting. Here, we describe the Arabidopsis (Arabidopsis thaliana) homolog of yeast Vps38p and human UV radiation resistance-associated gene protein. Arabidopsis VPS38 interacts with VPS30/ATG6 both in yeast and in planta. Although the level of PI3P in Arabidopsis vps38 mutants is similar to that in wild type, vps38 cells contain enlarged multivesicular endosomes and fewer organelles enriched in PI3P than the wild type. The vps38 mutants are defective in the trafficking of vacuolar cargo and its receptor VACUOLAR SORTING RECEPTOR2;1. The mutants also exhibit abnormal cytoplasmic distributions of endocytic cargo, such as auxin efflux carriers PINFORMED1 (PIN1) and PIN2. Constitutive autophagy is normal in the mutants but starvation-induced autophagy was slightly inhibited. We conclude that Arabidopsis VPS38 is dispensable for autophagy but essential for efficient targeting of biosynthetic and endocytic cargo to the vacuole.

Phosphatidic acid (PtdOH) is emerging as an important signaling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using (32)P-labeled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid (32)P-PtdOH increase, peaking within 2 and 5 min, respectively. In principle, PtdOH can be generated through three different pathways, i.e., (1) via de novo phospholipid biosynthesis (through acylation of lyso-PtdOH), (2) via phospholipase D hydrolysis of structural phospholipids, or (3) via phosphorylation of diacylglycerol (DAG) by DAG kinase (DGK). Using a differential (32)P-labeling protocol and a PLD-transphosphatidylation assay, evidence is provided that the rapid (32)P-PtdOH response was primarily generated through DGK. A simultaneous decrease in the levels of (32)P-PtdInsP, correlating in time, temperature dependency, and magnitude with the increase in (32)P-PtdOH, suggested that a PtdInsP-hydrolyzing PLC generated the DAG in this reaction. Testing T-DNA insertion lines available for the seven DGK genes, revealed no clear changes in (32)P-PtdOH responses, suggesting functional redundancy. Similarly, known cold-stress mutants were analyzed to investigate whether the PtdOH response acted downstream of the respective gene products. The hos1, los1, and fry1 mutants were found to exhibit normal PtdOH responses. Slight changes were found for ice1, snow1, and the overexpression line Super-ICE1, however, this was not cold-specific and likely due to pleiotropic effects. A tentative model illustrating direct cold effects on phospholipid metabolism is presented.

Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth and resistance to certain plant pathogens. Despite its potential use in agriculture, little is known on the molecular basis of this beneficial plant-fungal interaction. In a genetic screen for plants, which do not show a P. indica- induced growth response, we isolated an Arabidopsis mutant in the OXI1 (Oxidative Signal Inducible1) gene. OXI1 has been characterized as a protein kinase which plays a role in pathogen response and is regulated by H₂O₂ and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1). A genetic analysis showed that double mutants of the two closely related PDK1.1 and PDK1.2 genes are defective in the growth response to P. indica. While OXI1 and PDK1 gene expression is upregulated in P. indica-colonized roots, defense genes are downregulated, indicating that the fungus suppresses plant defense reactions. PDK1 is activated by phosphatidic acid (PA) and P. indica triggers PA synthesis in Arabidopsis plants. Under beneficial co-cultivation conditions, H₂O₂ formation is even reduced by the fungus. Importantly, phospholipase D (PLD)α1 or PLDδ mutants, which are impaired in PA synthesis do not show growth promotion in response to fungal infection. These data establish that the P. indica-stimulated growth response is mediated by a pathway consisting of the PLD-PDK1-OXI1 cascade.

Polyamines, such as putrescine (Put), spermidine (Spd), and spermine (Spm), are low-molecular-weight polycationic molecules found in all living organisms. Despite the fact that they have been implicated in various important developmental and adaptative processes, their mode of action is still largely unclear. Here, we report that Put, Spd, and Spm trigger a rapid increase in the signaling lipid, phosphatidic acid (PA) in Arabidopsis seedlings but also mature leaves. Using time-course and dose-response experiments, Spm was found to be the most effective; promoting PA responses at physiological (low μM) concentrations. In seedlings, the increase of PA occurred mainly in the root and partly involved the plasma membrane polyamine-uptake transporter (PUT), RMV1. Using a differential P -labeling strategy combined with transphosphatidylation assays and T-DNA insertion mutants, we found that phospholipase D (PLD), and in particular was the main contributor of the increase in PA. Measuring non-invasive ion fluxes (MIFE) across the root plasma membrane of wild type and mutant seedlings, revealed that the formation of PA is linked to a gradual- and transient efflux of K . Potential mechanisms of how and the increase of PA are involved in polyamine function is discussed.