Annexin5 Is Essential for Pollen Development in Arabidopsis Zhu Jingen , Yuan Shunjie , Wei Guo , Qian Dong , Wu Xiaorong , Jia Honglei , Gui Mengyuan , Liu Wenzhe , An Lizhe , Xiang Yun   Molecular Plant  Volume 7, Issue 4, Pages 751-754 (April 2014) DOI: 10.1093/mp/sst171 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Down-Regulation of Ann5 in the RNAi Lines Aborts Pollen Development. (A) Expression pattern of Ann5 in various tissues and organs of Arabidopsis as determined by RT–PCR. C, 3-day-old cotyledon; R, root; S, stem; L, leaf; OF, open flower; EB, early bud. Total RNA was extracted from individual tissues of 6-week-old plants. EF4A was used as an internal positive control. (B, C) Histochemical staining for GUS activity in transgenic plants carrying the GUS gene driven by the Ann5 promoter sequence in Columbia (Col) wild-type inflorescence (B), mature pollen, and pollen tube (C). GUS activity was detected in pollen grains and pollen tubes. The black arrows indicate early buds. Bars = 1mm in (B) and 20 μm in (C). (D) Temporal expression profile of Ann5 in transgenic Arabidopsis Ann5Pro–YFP-NLS lines at the early microspore, late microspore, bicellular, and tricellular stages of pollen development. Pollen grains from homozygous transgenic plants were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Yellow fluorescent protein (YFP), DAPI fluorescence, and bright-field signals are displayed in the top, middle, and bottom panels, respectively. Vegetative nuclei are indicated by arrowheads and generative nuclei by arrows. The YFP signal accumulated beginning in the bicellular pollen grain stage in transgenic lines. Bars = 5 μm. (E) The observations of the pollen lethal phenotype in wild-type and pCAMBIA1300–Lat52–UTRRNAi-1 (designated as Lat52–UTRi) plants. Bright-field, Alexander’s viability staining, and scanning electron microscopy (SEM) images are shown in the left, middle, and right panels, respectively. Arrowheads indicate the representative collapsed pollen grains. Bars = 20 μm. (F) Comparison of the progression of mitotic division between wild-type and Ann5Pro–UTRi-1 plants. The uninucleate microspores, bicellular, and tricellular pollen released from anthers in wild-type and Ann5Pro–UTRi-1 plants were identified by nuclear DAPI staining. We visualized pollen under a light microscope with or without a UV fluorescent filter. Representative pollen from anthers at different developmental stages is shown. Vegetative nuclei are indicated by arrowheads and generative nuclei by arrows. Sterile pollen grains (red asterisks) were observed in Ann5Pro–UTRi-1 anthers in both the bicellular and tricellular stages. Bars = 20 μm. (G) TEM micrographs of pollen cross-sections from wild-type and Ann5Pro–UTRi-1 anthers during the bicellular stage. (a) TEM cross-sections of pollen from wild-type. (b–e) Progression of pollen abortion in Ann5Pro–UTRi-1 lines. Arrowheads indicate plasmolysis and red asterisks point to breaks in the plasma membrane. VN, vegetative nucleus; GN, generative nucleus; V, vesicle; Ex, exine; In, intine. Bars = 2 μm. Molecular Plant 2014 7, 751-754DOI: (10.1093/mp/sst171) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions