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  • Autophagy-mediated compartmental cytoplasmic deletion is essential for tobacco pollen germination and male fertility.

Autophagy-mediated compartmental cytoplasmic deletion is essential for tobacco pollen germination and male fertility.

Autophagy (2020-01-28)
Peng Zhao, Xue-Mei Zhou, Lin-Lin Zhao, Alice Y Cheung, Meng-Xiang Sun
ABSTRACT

In plants, macroautophagy/autophagy has mainly been associated with stress-related processes but how it impacts normal physiological and developmental processes remains largely unexplored. Pollen germination is the critical first step toward fertilization in flowering plants. It is metabolically demanding and relies on high levels of cytoplasmic reorganization activities to support a dramatic morphological transformation that underlies the development of a pollen tube as the conduit to deliver sperm for fertilization. The role of autophagy in this process remains unclear. Here we provide evidence that pollen germination is accompanied by elevated autophagic activity and successful pollen tube emergence depends on autophagy-mediated cytoplasmic deletion. Genetic and cytological experiments demonstrate that inhibition of autophagy prevents pollen germination while induces the persistence of a layer of undegraded cytoplasm at the germination aperture. Together, these results unveil a novel compartmentalized autophagy. Furthermore, high-throughput comparative lipidomic analyses show that suppressed autophagy-induced inhibition of pollen germination is accompanied by altered profiles of stored and signaling lipids. Proteomic analyses reveal that autophagy likely exert its role in pollen germination via downstream mitochondria-related pathways. These findings reveal a critical role for autophagy in initiating pollen germination and provide evidences for compartmental cytoplasmic deletion being crucial for male fertility. Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy-related gene; Cer: ceramide; CL: cardiolipin; Con A: concanamycin A; DAG: diradylglycerol; GO: gene ontology; HAG: hour after germination; LC-MS: liquid chromatography-mass spectrometry; MAG: min after germination; MDC: monodansylcadaverine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PLD: phospholipase D; PtdIns3K: phosphatidylinositol 3-kinase; RT-qPCR: quantitative real-time reverse transcription PCR; TAG: triradylglycerol; TEM: transmission electron microscopy; TMT: tandem mass tagging.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Propidium iodide, ≥94% (HPLC)
Sigma-Aldrich
3-Methyladenine, autophagy inhibitor
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Trichloroacetic acid, ACS reagent, ≥99.0%
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Agarose, Ultra-low Gelling Temperature, molecular biology grade
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Rosetta(DE3) Competent Cells - Novagen, Rosetta host strains are BL21 derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli.
Sigma-Aldrich
tert-Butyl methyl ether, HPLC Plus, for HPLC, GC, and residue analysis, 99.9%
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Methanol, suitable for HPLC, gradient grade, ≥99.9%
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Triton X-100, laboratory grade
Sigma-Aldrich
Urea, suitable for electrophoresis
Sigma-Aldrich
Triton X-100, for molecular biology
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Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
Sigma-Aldrich
Iodoacetamide, BioUltra
Sigma-Aldrich
Tetraethylammonium borohydride, technical, ≥95% (T)
Supelco
Triethylammonium bicarbonate buffer, 1 M, suitable for HPLC, LiChropur