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. 2017 Jan;173(1):206-218.
doi: 10.1104/pp.16.00988. Epub 2016 Oct 31.

MONENSIN SENSITIVITY1 (MON1)/CALCIUM CAFFEINE ZINC SENSITIVITY1 (CCZ1)-Mediated Rab7 Activation Regulates Tapetal Programmed Cell Death and Pollen Development

Yong Cui  1   2   3   4 , Qiong Zhao  1   2   3   4 , Hong-Tao Xie  1   2   3   4 , Wing Shing Wong  1   2   3   4 , Xiangfeng Wang  1   2   3   4 , Caiji Gao  1   2   3   4 , Yu Ding  1   2   3   4 , Yuqi Tan  1   2   3   4 , Takashi Ueda  1   2   3   4 , Yan Zhang  1   2   3   4 , Liwen Jiang  5   6   7   8
Affiliations

MONENSIN SENSITIVITY1 (MON1)/CALCIUM CAFFEINE ZINC SENSITIVITY1 (CCZ1)-Mediated Rab7 Activation Regulates Tapetal Programmed Cell Death and Pollen Development

Yong Cui et al. Plant Physiol. 2017 Jan.

Abstract

Programmed cell death (PCD)-triggered degradation of plant tapetum is essential for microspore development and pollen coat formation; however, little is known about the cellular mechanism regulating tapetal PCD Here, we demonstrate that Rab7-mediated vacuolar transport of tapetum degradation-related cysteine proteases is crucial for tapetal PCD and pollen development in Arabidopsis (Arabidopsis thaliana), with the following evidence: (1) The monensin sensitivity1 (mon1) mutants, which are defective in Rab7 activation, showed impaired male fertility due to a combined defect in both tapetum and male gametophyte development. (2) In anthers, MON1 showed preferential high level expression in tapetal cell layers and pollen. (3) The mon1 mutants exhibited delayed tapetum degeneration and tapetal PCD, resulting in abnormal pollen coat formation and decreased male fertility. (4) MON1/CALCIUM CAFFEINE ZINC SENSITIVITY1 (CCZ1)-mediated Rab7 activation was indispensable for vacuolar trafficking of tapetum degradation-related cysteine proteases, supporting that PCD-triggered tapetum degeneration requires Rab7-mediated vacuolar trafficking of these cysteine proteases. (5) MON1 mutations also resulted in defective pollen germination and tube growth. Taken together, tapetal PCD and pollen development require successful MON1/CCZ1-mediated vacuolar transport in Arabidopsis.

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Figures

Figure 1.
Figure 1.
The mon1 mutants exhibit defects in pollen coat formation, causing decreased male fertility. A to C, Impaired fertility in mon1 was indicated by nonelongated siliques of representative primary inflorescences. Nonelongated siliques were found in mon1-2 mutants (B). Bars = 1 cm. D to G, Nonelongated siliques from mon1-2 mutants did not contain any mature embryos. Bars = 1 mm. H, Significant decrease of mon1 fertility indicated by quantitative analysis of silique developmental phenotype. I to P, SEM and TEM images showed mon1-2−/− mutants (K and O) had abnormal pollen coat compared with the wild type (I and M), mon1-2+/− (J and N), and GFP-MON1/mon1-2−/− (L and P). The arrows in K and O indicate abnormal coat formation in mon1-2−/− mutants. Bars = 10 μm in I to L and 2 μm in M to P. WT, Wild type.
Figure 2.
Figure 2.
Defective pollen coats of mon1 result in decreased pollen adhesion to the stigma and a slower pollen hydration process. A to D, Fewer pollen grains were released and attached to the stigma in the mon1 mutant as shown by light and SEM imaging. Bars = 1 mm in A and B and 20 μm in C and D. E to H, Compared with the wild type (E), pollen (indicated by arrows) from the mon1 mutant (F) showed a slower hydration process when pollinated on the wild-type stigmatic papilla under low-humidity conditions (<40%). High humidity (>80%) partially rescued this defect of mon1 (G). Wild-type pollen showed a normal hydration process when pollinated on the mon1 stigmatic papilla under low-humidity conditions (H). Bars = 10 μm. WT, Wild type.
Figure 3.
Figure 3.
Preferential expression of MON1 in tapetum and pollen. High expression of MON1 in tapetum and pollen was detected by RNA in situ hybridization in anthers at different development stages as indicated using MON1 antisense (A–E) and MON1 sense (F–J) probes. All sections are transverse. The pink-purple color indicates the hybridization signals. The strong signals were detected in tapetum and pollen grains from stage 5 to 11 (A–E). Control hybridizations with sense probes for MON1 do not show any signals above background (F–J). Bars = 10 μm.
Figure 4.
Figure 4.
MON1 mutation results in delayed tapetum degeneration. Semithin sections showed delayed tapetum degeneration in mon1-2 compared with the wild type (WT). Transverse semithin sections of wild-type (A–F) and mon1-2 (G–L) anthers at indicated stages were represented. The tapetal cells were completely degenerated at stage 12 in the wild type (F); however, residue from degenerating tapetal cells remained at stage 12 in mon1-2 (L). The arrows in D, E, and J to L indicate tapetal cells. Bars = 20 μm.
Figure 5.
Figure 5.
The mon1-2 shows delayed tapetum degeneration in TEM analysis. High-pressure frozen/freeze-substituted anthers at stages 11 and 12 were examined. TEM images of 70-nm-thick sections showed close-up views of tapetal cells and pollen coat structures in the wild type (WT; A and C) and mon1-2 (B and D). The arrowheads in A indicate the degradation of tapetal cells in the wild type. The arrow in C shows the fully packed pollen coat in the wild type. Bars = 2 μm.
Figure 6.
Figure 6.
MON1 loss of function results in delayed tapetal PCD. Delayed PCD was detected in mon1 compared with the wild type (WT) using the TUNEL assay. A to L, Fluorescence microscopy of DNA fragmentation detected using the TUNEL assays in sections of wild-type and mon1-2 anthers at indicated stages. Tapetal PCD did not occur until stage 11 (J) in mon1 as indicated by a weak TUNEL signal (indicated by arrowheads), while in the wild type the tapetal PCD occurred from late stage 10 (G). The arrows indicate examples of the TUNEL signals showing up in wild-type tapetal cell layers at late stage 10. Bars = 50 μm.
Figure 7.
Figure 7.
The mon1 mutant has impaired vacuolar processing of Cys protease RD21 with accumulated proform. A, Immunoblot and quantitative analysis of total anther protein extracts from the wild type (WT) and mon1-2 at indicated stages with the antibody against Cys protease RD21 showed that the proform of RD21 accumulated in mon1-2. RD21 was detected as three forms: proform (pRD21), intermediate isoform (iRD21), and mature RD21 (mRD21). B and C, Immunogold electron microscopy images show that at stage 9, Cys protease RD21 accumulated in enlarged PVCs (indicated by stars) in mon1-2 (C), while in wild type RD21 mainly reached the vacuole for processing (B). The arrows indicate the labeled gold particles using anti-RD21. V, Vacuole. Bars = 500 nm. D to G, TEM images showed PVC morphology in wild-type (D and E) and mon1-2 (F and G) tapetum. PVCs were labeled by anti-VSR (E and G). The stars indicate the PVCs. The arrows indicate the labeled gold particles using anti-VSR. Bars = 500 nm.
Figure 8.
Figure 8.
Dominant negative Rab7 mutant (RABG3fT22N) inhibits the vacuolar trafficking of tapetum degradation-related Cys proteases as well as soluble vacuolar marker. Vacuolar transport of soluble vacuolar marker (A–C) and Cys proteases (D–R) as indicated was inhibited as they were accumulated inside the enlarged PVCs when coexpressed with RABG3fT22N in protoplasts. Images were collected using a confocal microscope from cells at 12 to 18 h after transfection. Bars = 10 μm.
Figure 9.
Figure 9.
In mon1-2 mutant leaf protoplasts, vacuolar trafficking of soluble vacuolar marker and RD21-YFP was inhibited. Aleu-GFP (A) and RD21-YFP (B) showed vacuolar pattern in wild-type (WT) leaf protoplasts. However, in mon1-2 mutant protoplasts, Aleu-GFP (C) and RD21-YFP (D) showed inhibited vacuolar transport and were accumulated in the enlarged PVCs as indicated by the PVC marker mRFP-RHA1. Images were collected using a confocal microscope from cells at 12 to 18 h after transfection. Bars = 10 μm. DIC, Differential interference contrast.
Figure 10.
Figure 10.
Working model for the essential role of MON1/CCZ1-mediated Rab7 activation in regulating tapetal PCD. Tapetal PCD requires Rab7-mediated vacuolar transport of tapetum degradation-related Cys proteases. Vacuole rupture releases mature Cys proteases, which triggers the PCD in wild-type (WT) tapetum. The cell contents, including the tapetosomes and elaioplasts, then contribute to the formation of pollen coats (A). However, MON1 mutation inhibits vacuolar transport of Cys proteases and their maturation, resulting in delayed tapetal PCD and formation of abnormal pollen coats (B). Ts, Tapetosome; El, elaioplast.
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