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  1. SUMMARY

    Pollen tubes (PTs) grow by the targeted secretion of new cell wall material to their expanding tip region. Sec1/Munc18 (SM) proteins promote membrane fusion through regulation of the SNARE complex. We have previously shown that disruption of protein glycosylation in theArabidopsis thaliana hpat1 hpat3double mutant leads to PT growth defects that can be suppressed by reducing secretion. Here, we identified five point mutant alleles of the SM proteinSEC1Aashpat1/3suppressors. The suppressors increased seed set, reduced PT growth defects and reduced the rate of glycoprotein secretion. In the absence of thehpatmutations,sec1areduced pollen germination and PT elongation producing shorter and wider PTs. Consistent with a defect in membrane fusion,sec1aPTs accumulated secretory vesicles. Thoughsec1ahad significantly reduced male transmission, homozygoussec1aplants maintained full seed set, demonstrating thatSEC1Awas ultimately dispensable for pollen fertility. However, when combined with a mutation in anotherSEC1‐likeSMgene,keule, pollen fertility was totally abolished. Mutation insec1b, the final member of the Arabidopsis SEC1 clade, did not enhance thesec1aphenotype. Thus, SEC1A is the major SM protein promoting pollen germination and tube elongation, but in its absence KEULE can partially supply this activity. When we examined the expression of the SM protein family in other species for which pollen expression data were available, we found that at least one Sec1‐like protein was highly expressed in pollen samples, suggesting a conserved role in pollen fertility in other species.

     
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  2. Summary

    HYDROXYPROLINEO‐ARABINOSYLTRANSFERASEs (HPATs) initiate a post‐translational protein modification (Hyp‐Ara) found abundantly on cell wall structural proteins. InArabidopsis thaliana,HPAT1andHPAT3are redundantly required for full pollen fertility. In addition to the lack of Hyp‐Ara inhpat1/3pollen 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 thehpat1/3low seed‐set phenotype, we identified a missense mutation inexo70a2, 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 ofexo70a2also suppressed thehpat1/3fertility phenotype, as did mutants of core exocyst complex membersec15a, indicating that reduced exocyst function bypassed the PT requirement for Hyp‐Ara. In a wild‐type background,exo70a2reduced 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 onEXO70A2and was significantly increased inhpat1/3PTs compared with the wild type, but was reduced in the suppressedexo70a2 hpat1/3tubes.

     
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