Pollen tube NAD(P)H oxidases act as a speed control to dampen growth rate oscillations during polarized cell growth

Abstract

SUMMARYReactive oxygen species (ROS) produced by NAD(P)H oxidases play a central role in plant stress responses and development. To better understand the function of NAD(P)H oxidases in plant development, we characterized the Arabidopsis thaliana NAD(P)H oxidases RBOHH and RBOHJ. Both proteins were specifically expressed in pollen and dynamically targeted to distinct and overlapping plasma membrane domains at the pollen tube tip. Functional loss of RBOHH and RBOHJ in homozygous double mutants resulted in reduced fertility. Analyses of pollen tube growth revealed remarkable differences in growth dynamics between Col-0 and rbohh-1 rbohj-2 pollen tubes. Growth rate oscillations of rbohh-1 rbohj-2 pollen tubes showed strong fluctuations in amplitude and frequency, ultimately leading to pollen tube collapse. Prior to disintegration, rbohh-1 rbohj-2 pollen tubes exhibit high-frequency growth oscillations, with significantly elevated growth rates, suggesting that an increase in the rate of cell-wall exocytosis precedes pollen tube collapse. Time-lapse imaging of exocytic dynamics revealed that NAD(P)H oxidases slow down pollen tube growth to coordinate the rate of cell expansion with the rate of exocytosis, thereby dampening the amplitude of intrinsic growth oscillations. Using the Ca 2+ reporter Yellow Cameleon 3.6, we demonstrate that high-amplitude growth rate oscillations in rbohh-1 rbohj-2 pollen tubes are correlated with growth-dependent Ca 2+ bursts. Electrophysiological experiments involving double mutant pollen tubes and pharmacological treatments also showed that ROS influence K + homeostasis. Our results indicate that, by limiting pollen tube growth, ROS produced by NAD(P)H oxidases modulate the amplitude and frequency of pollen tube growth rate oscillations.

EXO70C2 Is a Key Regulatory Factor for Optimal Tip Growth of Pollen

“…Indeed, too high growth 435 rate has been documented in loss-of-function mutants in NADPH oxidases to precede a pollen 436 tube collapse (Lassig et al, 2014). The documented weakened cell wall at the tube apex 437 during the rapid growth is most likely the primary cause of the exo70C2 phenotype.…”

“…This interpretation is in full agreement 464 with previous experiments on NADPH oxidases in the tip growth. When two NADPH 465 oxidases RBOHH and RBOHJ were knocked-out, the mutant pollen tubes exhibited higher 466 expansion rates than WT causing their burst (Lassig et al, 2014) resulting in optimal balance between the cell wall biogenesis and surface growth. 479…”

Apoplastic ROS production upon pollination by RbohH and RbohJ in Arabidopsis

“…[2][3][4] Apoplastic ROS are suggested to play a role in cell wall loosening in maize coleoptiles, leaves, and roots.…”

“…1 Reactive oxygen species (ROS) accumulate at the tip of growing pollen tubes and 2 NADPH oxidases, respiratory burst oxidase homolog H (RbohH) and RbohJ, specifically localized in the plasma membrane of the growing tip 2 are involved in this accumulation. [2][3][4] The ROS are suggested to stimulate the plasma membrane Ca 2C -permeable channel(s) and induce Ca 2C influx into the cytoplasm. 5 The cytosolic Ca 2C rise in turn activates the ROS-producing activity of Rboh proteins by direct binding to the EF-hand motifs and by phosphorylation through the Ca 2C -dependent protein kinases, generating a positive feedback regulation that maintains the growth site to the tip during elongation (Fig.…”

Pollen tube growth: where does the energy come from?

“…However, most of the data consider only proteins which are localized in mitochondria (NAD(P)H-dehydrogenases), at the plasma membrane (NAD(P)H oxidase) or which are directly involved in NAD C biosynthesis (nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT), nucleotide diphosphate kinase (NDP)). 86,87,91,92 But again, it is necessary to consider the importance of the malate valves as well as of cytosolic and plastidial glycolysis in parallel, because these pathways act as powerful systems for balancing the required NAD(P)H/ NAD(P)…”