skip to main content

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

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.

more » « less
Award ID(s):
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  
Publisher / Repository:
Date Published:
Journal Name:
The Plant Journal
Page Range / eLocation ID:
p. 1399-1419
Medium: X
Sponsoring Org:
National Science Foundation
More Like this

    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.

    more » « less
  2. Abstract A potential strategy to mitigate oxidative damage in plants is to increase the abundance of antioxidants, such as ascorbate (i.e. vitamin C). In Arabidopsis (A. thaliana), a rate-limiting step in ascorbate biosynthesis is a phosphorylase encoded by Vitamin C Defective 2 (VTC2). To specifically overexpress VTC2 (VTC2 OE) in pollen, the coding region was expressed using a promoter from a gene with ∼150-fold higher expression in pollen, leading to pollen grains with an eight-fold increased VTC2 mRNA. VTC2 OE resulted in a near-sterile phenotype with a 50-fold decrease in pollen transmission efficiency and a five-fold reduction in the number of seeds per silique. In vitro assays revealed pollen grains were more prone to bursting (greater than two-fold) or produced shorter, morphologically abnormal pollen tubes. The inclusion of a genetically encoded Ca2+ reporter, mCherry-GCaMP6fast (CGf), revealed pollen tubes with altered tip-focused Ca2+ dynamics and increased bursting frequency during periods of oscillatory and arrested growth. Despite these phenotypes, VTC2 OE pollen failed to show expected increases in ascorbate or reductions in reactive oxygen species, as measured using a redox-sensitive dye or a roGFP2. However, mRNA expression analyses revealed greater than two-fold reductions in mRNA encoding two enzymes critical to biosynthetic pathways related to cell walls or glyco-modifications of lipids and proteins: GDP-d-mannose pyrophosphorylase (GMP) and GDP-d-mannose 3′,5′ epimerase (GME). These results support a model in which the near-sterile defects resulting from VTC2 OE in pollen are associated with feedback mechanisms that can alter one or more signaling or metabolic pathways critical to pollen tube growth and fertility. 
    more » « less
  3. Summary

    We investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth inArabidopsis thaliana(Col‐0).

    Patch‐clamp whole‐cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca2+]cyt). We investigated the pollen‐expressed proteinsAtSLAH3,AtALMT12,AtTMEM16 andAtCCCas the putative anion transporters responsible for these currents.

    AtCCCGFPwas observed at the shank andAtSLAH3‐GFPat the tip and shank of thePTplasma membrane. Both are likely to carry the majority of anion current at negative potentials, as extracellular anionic fluxes measured at the tip ofPTs with an anion vibrating probe were significantly lower inslah3−/−andccc−/−mutants, but unaffected inalmt12−/−andtmem16−/−. We further characterised the effect ofpHandGABAby patch clamp. Strong regulation by extracellularpHwas observed in the wild‐type, but not intmem16−/−. Our results are compatible withAtTMEM16 functioning as an anion/H+cotransporter and therefore, as a putativepHsensor.GABApresence: (1) inhibited the overall currents, an effect that is abrogated in thealmt12−/−and (2) reduced the current inAtALMT12 transfectedCOS‐7 cells, strongly suggesting the direct interaction ofGABAwithAtALMT12.

    Our data show thatAtSLAH3 andAtCCCactivity is sufficient to explain the major component of extracellular anion fluxes, and unveils a possible regulatory system linkingPTgrowth modulation bypH,GABA, and [Ca2+]cytthrough anionic transporters.

    more » « less
  4. null (Ed.)
    A wide range of proteins with diverse functions in development, defense, and stress responses are O -arabinosylated at hydroxyprolines (Hyps) within distinct amino acid motifs of continuous stretches of Hyps, as found in the structural cell wall extensins, or at non-continuous Hyps as, for example, found in small peptide hormones and a variety of plasma membrane proteins involved in signaling. Plant O -glycosylation relies on hydroxylation of Prolines to Hyps in the protein backbone, mediated by prolyl-4-hydroxylase (P4H) which is followed by O -glycosylation of the Hyp C 4 -OH group by either galactosyltransferases (GalTs) or arabinofuranosyltranferases (Ara f Ts) yielding either Hyp-galactosylation or Hyp-arabinosylation. A subset of the P4H enzymes with putative preference to hydroxylation of continuous prolines and presumably all Ara f T enzymes needed for synthesis of the substituted arabinose chains of one to four arabinose units, have been identified and functionally characterized. Truncated root-hair phenotype is one common denominator of mutants of Hyp formation and Hyp-arabinosylation glycogenes, which act on diverse groups of O -glycosylated proteins, e.g., the small peptide hormones and cell wall extensins. Dissection of different substrate derived effects may not be regularly feasible and thus complicate translation from genotype to phenotype. Recently, lack of proper arabinosylation on arabinosylated proteins has been shown to influence their transport/fate in the secretory pathway, hinting to an additional layer of functionality of O -arabinosylation. Here, we provide an update on the prevalence and types of O -arabinosylated proteins and the enzymatic machinery responsible for their modifications. 
    more » « less
  5. Introduction

    VPS45 belongs to the Sec1/Munc18 family of proteins, which interact with and regulate Qa-SNARE function during membrane fusion. We have shown previously thatArabidopsis thalianaVPS45 interacts with the SYP61/SYP41/VTI12 SNARE complex, which locates on thetrans-Golgi network (TGN). It is required for SYP41 stability, and it functions in cargo trafficking to the vacuole and in cell expansion. It is also required for correct auxin distribution during gravitropism and lateral root growth.


    Asvps45knockout mutation is lethal in Arabidopsis, we identified a mutant,vps45-3, with a point mutation in theVPS45gene causing a serine 284-to-phenylalanine substitution. The VPS45-3 protein is stable and maintains interaction with SYP61 and SYP41. However,vps45-3plants display severe growth defects with significantly reduced organ and cell size, similar tovps45RNAi transgenic lines that have reduced VPS45 protein levels. Root hair and pollen tube elongation, both processes of tip growth, are highly compromised invps45-3. Mutant root hairs are shorter and thicker than those of wild-type plants, and are wavy. These root hairs have vacuolar defects, containing many small vacuoles, compared with WT root hairs with a single large vacuole occupying much of the cell volume. Pollen tubes were also significantly shorter invps45-3compared to WT.


    We thus show that VPS45 is essential for proper tip growth and propose that the observed vacuolar defects lead to loss of the turgor pressure needed for tip growth.

    more » « less