Exocyst mutants suppress pollen tube growth and cell wall structural defects of hydroxyproline O‐arabinosyltransferase mutants

Abstract

Summary HYDROXYPROLINE O ‐ARABINOSYLTRANSFERASEs (HPATs) initiate a post‐translational protein modification (Hyp‐Ara) found abundantly on cell wall structural proteins. In Arabidopsis thaliana , HPAT1 and HPAT3 are redundantly required for full pollen fertility. In addition to the lack of Hyp‐Ara in hpat1/3 pollen tubes (PTs), we also found broadly disrupted cell wall polymer distributions, particularly the conversion of the tip cell wall to a more shaft‐like state. Mutant PTs were slow growing and prone to rupture and morphological irregularities. In a forward mutagenesis screen for suppressors of the hpat1/3 low seed‐set phenotype, we identified a missense mutation in exo70a2 , a predicted member of the vesicle‐tethering exocyst complex. The suppressed pollen had increased fertility, fewer morphological defects and partially rescued cell wall organization. A transcriptional null allele of exo70a2 also suppressed the hpat1/3 fertility phenotype, as did mutants of core exocyst complex member sec15a , indicating that reduced exocyst function bypassed the PT requirement for Hyp‐Ara. In a wild‐type background, exo70a2 reduced male transmission efficiency, lowered pollen germination frequency and slowed PT elongation. EXO70A2 also localized to the PT tip plasma membrane, consistent with a role in exocyst‐mediated secretion. To monitor the trafficking of Hyp‐Ara modified proteins, we generated an HPAT‐targeted fluorescent secretion reporter. Reporter secretion was partially dependent on EXO70A2 and was significantly increased in hpat1/3 PTs compared with the wild type, but was reduced in the suppressed exo70a2 hpat1/3 tubes.

Plant Protein O-Arabinosylation

“…A recent genetic screen for suppressors of the hpat pollen phenotype identified mutants in the late stages of the secretory pathway, specifically in the vesicle-tethering exocyst complex. Beuder et al (2020) found that hpat pollen tubes had increased rates of HPATmodified protein secretion compared to WT pollen tubes and the rate of secretion was reduced in the suppressed line to near WT levels. This observation is consistent with a "toxic" effect of increased secretion of un-arabinosylated proteins.…”

“…Somewhat surprisingly, the total loss of Hyp-arabinoslyation through mutations of all three Arabidopsis HPAT genes is not lethal and does not result in a severe vegetative phenotype ( MacAlister et al, 2016 ). The major phenotype of the triple mutants is a reduction in pollen fertility leading to reduced seed set ( Beuder et al, 2020 ). Similarly, HPAT activity is not essential in the moss Physcomitrella patens where knockout of both HPATs resulted in increased biomass due to enhanced elongation of tip-growing vegetative filament cells ( MacAlister et al, 2016 ).…”

“…(B) Chemical structure of the pulcherosine cross-link. The blue frame in (A) indicates the smallest extensin motif with experimentally validated arabinosylation ( Beuder et al, 2020 ).…”

“…Despite the differences in their cell wall organization ( Chebli et al, 2012 ), pollen tubes are also dependent on Hyp-arabinosylation ( Ogawa-Ohnishi et al, 2013 ; MacAlister et al, 2016 ). Double mutants of Arabidopsis hpat1 and hpat3 (Hyp-arabinosyltransferases, see below) display severe male fertility defects due to compromised pollen tube cell wall polarity ( Beuder et al, 2020 ). A recent genetic screen for suppressors of the hpat pollen phenotype identified mutants in the late stages of the secretory pathway, specifically in the vesicle-tethering exocyst complex.…”

Regulation of Exocyst Function in Pollen Tube Growth by Phosphorylation of Exocyst Subunit EXO70C2

“…The two EXO70A isoforms, EXO70A1 ( Synek et al, 2006 ) and EXO70A2 ( Marković et al, 2020 ), seem to have a general housekeeping function during exocytosis in the sporophyte and the male gametophyte, respectively. Partially resembling the phenotypes of LOF mutants of core exocyst subunits, exo70A2 pollen grains germinate poorly and produce short, thick, slowly growing pollen tubes ( Beuder et al, 2020 ; Marković et al, 2020 ). In sharp contrast, LOF of Arabidopsis EXO70C2 results in stop-and-go growth dynamics where phases with abnormally high growth rates are interrupted by pollen tube tip bursts and recovery, ultimately resulting in pollen tube rupture ( Synek et al, 2017 ).…”