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ABSTRACT Of the 61 kinesins annotated inArabidopsis thaliana, many are still without assigned function. Here, we have screened an insertional mutant library of Arabidopsis pollen‐expressed kinesins for fertility defects. Insertional mutants for three kinesins showed a significant reduction in seed set. Among them, we focused on the sole kinesin‐4 expressed in pollen (kinesin‐4C, here Pollen‐Expressed Kinesin 14, PEK14). We show a seed‐set defect in the three independent allelespek14‐1, pek14‐2, and pek14‐3. This defect is male‐derived and is equally distributed throughout the silique. Maturepek14‐1anthers contain about 10% inviable pollen grains.pek14‐1pollen tubes grow 20% more slowly and show reduced pollen tube bending. Analysis of the male germ unit (MGU), as it travels through the pollen tube, demonstrates an aberrant organization of thepek14‐1MGU in 30% of pollen tubes and an increase in the distance of the MGU to the tip by 24%. Expression of GFP‐tagged PEK14 successfully complemented the observed seed set defect, as well as the growth rate, bending, and MGU organization defects observed inpek14‐1. In pollen, PEK14‐GFP is located diffusely at the pollen tube tip. PEK14‐GFP is also expressed in the root meristematic zone and is located at the mid‐zone of the phragmoplast, but no apparent root growth phenotype was observed, likely due to redundancy in this organ.
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Exocyst mutants suppress pollen tube growth and cell wall structural defects of hydroxyproline O‐arabinosyltransferase mutants
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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- Award ID(s):
- 1755482
- PAR ID:
- 10450010
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 103
- Issue:
- 4
- ISSN:
- 0960-7412
- Page Range / eLocation ID:
- p. 1399-1419
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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