HYDROXYPROLINE
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 the
- NSF-PAR ID:
- 10446537
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 110
- Issue:
- 5
- ISSN:
- 0960-7412
- Page Range / eLocation ID:
- p. 1353-1369
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary O ‐ARABINOSYLTRANSFERASEs (HPATs) initiate a post‐translational protein modification (Hyp‐Ara) found abundantly on cell wall structural proteins. InArabidopsis thaliana ,HPAT1 andHPAT3 are redundantly required for full pollen fertility. In addition to the lack of Hyp‐Ara inhpat1/3 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 thehpat1/3 low 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 ofexo70a2 also suppressed thehpat1/3 fertility 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,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 onEXO70A2 and was significantly increased inhpat1/3 PTs compared with the wild type, but was reduced in the suppressedexo70a2 hpat1/3 tubes. -
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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 that
Arabidopsis thaliana VPS45 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.Results As
vps45 knockout mutation is lethal in Arabidopsis, we identified a mutant,vps45-3 , with a point mutation in theVPS45 gene causing a serine 284-to-phenylalanine substitution. The VPS45-3 protein is stable and maintains interaction with SYP61 and SYP41. However,vps45-3 plants display severe growth defects with significantly reduced organ and cell size, similar tovps45 RNAi 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 invps45-3 compared to WT.Discussion 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.
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Abstract The sorting of eukaryotic proteins to various organellar destinations requires receptors that recognize cargo protein targeting signals and facilitate transport into the organelle. One such receptor is the peroxin
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Abstract Brassinosteroids (
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Abstract Cells employ multiple systems to maintain cellular integrity, including mechanosensitive ion channels and the cell wall integrity (CWI) pathway. Here, we use pollen as a model system to ask how these different mechanisms are interconnected at the cellular level. MscS-Like 8 (MSL8) is a mechanosensitive channel required to protect Arabidopsis thaliana pollen from osmotic challenges during in vitro rehydration, germination, and tube growth. New CRISPR/Cas9 and artificial miRNA-generated msl8 alleles produced unexpected pollen phenotypes, including the ability to germinate a tube after bursting, dramatic defects in cell wall structure, and disorganized callose deposition at the germination site. We document complex genetic interactions between MSL8 and two previously established components of the CWI pathway, MARIS and ANXUR1/2. Overexpression of MARISR240C-FP suppressed the bursting, germination, and callose deposition phenotypes of msl8 mutant pollen. Null msl8 alleles suppressed the internalized callose structures observed in MARISR240C-FP lines. Similarly, MSL8-YFP overexpression suppressed bursting in the anxur1/2 mutant background, while anxur1/2 alleles reduced the strong rings of callose around ungerminated pollen grains in MSL8-YFP overexpressors. These data show that mechanosensitive ion channels modulate callose deposition in pollen and provide evidence that cell wall and membrane surveillance systems coordinate in a complex manner to maintain cell integrity.
