skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Overexpressing Vitamin C Defective 2 reduces fertility and alters Ca2+ signals in Arabidopsis pollen
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
Award ID(s):
2016143
PAR ID:
10465441
Author(s) / Creator(s):
; ; ; ; ; ; ; ;
Date Published:
Journal Name:
Plant Physiology
Volume:
191
Issue:
4
ISSN:
0032-0889
Page Range / eLocation ID:
2276 to 2287
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; (, Plant Physiology)
    null (Ed.)
    Abstract Generating cellular Ca2+ signals requires coordinated transport activities from both Ca2+ influx and efflux pathways. In Arabidopsis (Arabidopsis thaliana), multiple efflux pathways exist, some of which involve Ca2+-pumps belonging to the Autoinhibited Ca2+-ATPase (ACA) family. Here, we show that ACA1, 2, and 7 localize to the endoplasmic reticulum (ER) and are important for plant growth and pollen fertility. While phenotypes for plants harboring single-gene knockouts (KOs) were weak or undetected, a triple KO of aca1/2/7 displayed a 2.6-fold decrease in pollen transmission efficiency, whereas inheritance through female gametes was normal. The triple KO also resulted in smaller rosettes showing a high frequency of lesions. Both vegetative and reproductive phenotypes were rescued by transgenes encoding either ACA1, 2, or 7, suggesting that all three isoforms are biochemically redundant. Lesions were suppressed by expression of a transgene encoding NahG, an enzyme that degrades salicylic acid (SA). Triple KO mutants showed elevated mRNA expression for two SA-inducible marker genes, Pathogenesis-related1 (PR1) and PR2. The aca1/2/7 lesion phenotype was similar but less severe than SA-dependent lesions associated with a double KO of vacuolar pumps aca4 and 11. Imaging of Ca2+ dynamics triggered by blue light or the pathogen elicitor flg22 revealed that aca1/2/7 mutants display Ca2+ transients with increased magnitudes and durations. Together, these results indicate that ER-localized ACAs play important roles in regulating Ca2+ signals, and that the loss of these pumps results in male fertility and vegetative growth deficiencies. 
    more » « less
  2. ; ; ; ; ; ; ; ; ; (, The Plant Cell)
    Abstract Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here we developed a genetically encoded FRET (Förster resonance energy transfer)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses. 
    more » « less
  3. ; ; ; ; ; (, Journal of Experimental Botany)
    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. 
    more » « less
  4. ; ; ; ; (, Plant Physiology)
    Abstract As a universal second messenger, calcium (Ca2+) transmits specific cellular signals via a spatiotemporal signature generated from its extracellular source and internal stores. Our knowledge of the mechanisms underlying the generation of a Ca2+ signature is hampered by limited tools for simultaneously monitoring dynamic Ca2+ levels in multiple subcellular compartments. To overcome the limitation and to further improve spatiotemporal resolutions, we have assembled a molecular toolset (CamelliA lines) in Arabidopsis (Arabidopsis thaliana) that enables simultaneous and high-resolution monitoring of Ca2+ dynamics in multiple subcellular compartments through imaging different single-colored genetically encoded calcium indicators. We uncovered several Ca2+ signatures in three types of Arabidopsis cells in response to internal and external cues, including rapid oscillations of cytosolic Ca2+ and apical plasma membrane Ca2+ influx in fast-growing Arabidopsis pollen tubes, the spatiotemporal relationship of Ca2+ dynamics in four subcellular compartments of root epidermal cells challenged with salt, and a shockwave-like Ca2+ wave propagating in laser-wounded leaf epidermis. These observations serve as a testimony to the wide applicability of the CamelliA lines for elucidating the subcellular sources contributing to the Ca2+ signatures in plants. 
    more » « less
  5. ; ; ; ; ; ; (, Cytoskeleton)
    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. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email