Pollen-Expressed Leucine-Rich Repeat Extensins Are Essential for Pollen Germination and Growth

Abstract

During pollen tube growth, the walls of the tube provide the mechanical strength resisting turgor pressure to protect two sperm cells. Cell wall proteins may play an important role in this process. Pollen tube cell wall proteins known as leucine-rich repeat extensins (LRXs) harbor a leucine-rich repeat domain and an extensin domain. In this study, the functions of four pollen-expressed LRXs, LRX8, LRX9, LRX10, and LRX11 (LRX8-11), were characterized in Arabidopsis (Arabidopsis thaliana). LRX8-11 displayed a consistent expression pattern in mature pollen grains and pollen tubes. In a phenotypic analysis of four single mutants, six double mutants, four triple mutants, and a quadruple mutant, the triple and quadruple mutant plants displayed markedly reduced seed set and decreased male transmission efficiency accompanied by compromised pollen germination and pollen tube growth. GFP-fused LRX8, LRX10, and LRX11 were found to be localized to pollen tube cell walls. An immunohistochemical analysis of pollen tube cell wall polysaccharides showed an increase in the amount of rhamnogalacturonan I in the subapical walls of pollen tubes of the lrx9 lrx10 lrx11 and lrx8 lrx9 lrx11 mutants and a decrease in the content of fucosylated xyloglucans in lrx8 lrx9 lrx11 compared with wild-type plants. Moreover, the callose content in the apical walls of pollen tubes increased in the lrx8 lrx9 lrx11 mutant. In conclusion, we propose that LRX8-11 function synergistically to maintain pollen tube cell wall integrity; thus, they play critical roles in pollen germination and pollen tube growth.

Figure 1.

LRX8–11 are highly expressed in mature pollen grains and growing pollen tubes. The detection of GUS activity in 5-week-old wild-type plants expressing Pro_LRX8:GUS, Pro_LRX9:GUS, Pro_LRX10:GUS, or Pro_LRX11:GUS is shown. Developing male gametophytes were stained with DAPI and GUS. UMP, Uninuclear microspore; BCP, bicellular pollen; TCP, early tricellular pollen; MP, mature pollen grain; PT, pollen tube. Bars = 10 μm.

Figure 2.

LRX8–11 disruption caused a significant reduction in fertility. A, Schematic diagrams of the genomic loci and positions of the T-DNA insertions in LRX8–11. Exons are represented by black boxes, introns by a line, and untranslated regions by gray boxes. The location of each T-DNA insertion is indicated by an inverted triangle. B, DNA level identification of lrx8, lrx9, lrx10, and lrx11. The primers in the odd lanes are LP and RP, and those in the even lanes are LBb1.3 and RP. C, RT-PCR showed no LRX8–11 transcripts in open flowers from lrx8, lrx9, lrx10, and lrx11 plants, respectively. ACTIN7 served as a loading control. Amplification was performed for 25 cycles for ACTIN7 and 35 cycles for LRX8–11. gDNA, Genomic DNA. D, The mutants exhibited reduced seed set. Bar = 1 mm. E, Statistical analysis of silique seed set in wild-type (WT) and triple and quadruple mutant plants. All values are based on three biological replicates (n = 103, 43, 29, 45, 63, and 89). Error bars show se. Asterisks indicate values that differed significantly from the wild type (***, P < 0.001, calculated using Student’s t test).

Figure 3.

Reciprocal cross-pollination of lrx8 lrx9 lrx10 lrx11 and wild-type (WT) plants. A, Siliques were harvested at ∼15 d after hand pollination and dissected for statistical analysis. Bars = 1 mm. B, Statistical analysis of silique seed set. Values are based on three biological replicates (n = 52, 50, 51, and 41). Error bars represent se (***, P < 0.001; ns, not significant, calculated using Student’s t test).

Figure 4.

The mutation of LRX8–11 caused defects in pollen germination and pollen tube growth in vitro. A to F, Pollen from wild-type (WT) and mutant plants was germinated in vitro for 5 h. Bar = 20 μm. G to L, Images of representative pollen grains and pollen tubes. G, Ungerminated pollen. H, Pollen that burst instantly upon germination. I, Swollen tube. J, Branched tube. K, Burst tube. L, Normal pollen tube. Bar = 20 μm. M, Statistical analysis of the pollen germination rates in wild-type and mutant plants. Data were collected from three independent experiments, and the number of pollen grains collected each time was greater than 300 (n > 1,000).

Figure 5.

Pollen tube growth in the transmitting tract was compromised in the mutant plants. A, Growth of wild-type (WT) and mutant pollen tubes in the wild-type pistils at 12 hap as visualized by Aniline Blue staining. Yellow asterisks indicate the beginning of the pollen tubes, and red asterisks show the locations of the fastest pollen tubes. Bar = 200 μm. B, Statistical analysis of the absolute length of the pollen tube in the transmitting tract at 6, 12, and 24 hap. The data were collected from three independent experiments. n at 6 hap = 52, 43, 52, 52, 52, 52; n at 12 hap = 52, 48, 52, 45, 52, 52; and n at 24 hap = 52, 52, 52, 47, 52, 47. Error bars represent se (*, P < 0.05 and ***, P < 0.001; ns, not significant, calculated using two-way ANOVA).

Figure 6.

GFP-fused LRX8, LRX10, and LRX11 were located in the cytoplasm and walls of pollen tubes. Subcellular localization analysis of Pro_LRX8:LRX8-GFP(C), Pro_LRX10:LRX10-GFP(C), and Pro_LRX11:LRX11-GFP(C) in Arabidopsis is shown. A to C, Fluorescence images showing the GFP signals from LRX8-GFP, LRX10-GFP, and LRX11-GFP in pollen grains and pollen tubes cultured in vitro. pg, Pollen grains; pt, pollen tubes. Bars = 20 μm. D to F, Images of representative pollen tubes. Red asterisks mark the collar region, and red triangles indicate the pollen tube tip. Bars = 10 μm. G, J, and M, LRX8-GFP, LRX10-GFP, and LRX11-GFP localization in germinated pollen grains. Red arrows indicate the tip of the germination site. Bars = 10 μm. H, K, and N, LRX8-GFP, LRX10-GFP, and LRX11-GFP localization in growing pollen tubes. Red triangles indicate the tip of the pollen tube. cy, Cytoplasm. Bars = 5 μm. I, L, and O, A plasmolysis assay shows the locations of LRX8, LRX10, and LRX11 in the cytoplasm and walls of pollen tube cells. Red triangles indicate the tips of the pollen tubes. cw, Cell wall. Bars = 5 μm.

Figure 7.

The abundance of cell wall polysaccharides in the pollen tube was changed in lrx9 lrx10 lrx11 (lrx9,10,11) and lrx8 lrx9 lrx11 (lrx8,9,11). A, Immunolabeling of pollen tube wall polysaccharides in wild-type (WT), lrx9,10,11, and lrx8,9,11 mutant plants using JIM7, LM6, LM15, CCRC-M1, and CBM3a. JIM7 labeled HG in the pollen tubes of wild-type, lrx9,10,11, and lrx8,9,11 plants. LM6 recognized RGI in the pollen tubes of wild-type, lrx9,10,11, and lrx8,9,11 plants. LM15 recognized nongalactosylated (XXXG motif) XyG in the pollen tubes of wild-type, lrx9,10,11, and lrx8,9,11 plants. CCRC-M1 recognized α-l-fucosylated XyG in the pollen tubes of wild-type, lrx9,10,11, and lrx8,9,11 plants. The labeling of crystalline cellulose was performed with CBM3a in the pollen tubes of wild-type, lrx9,10,11, and lrx8,9,11 plants. Callose was labeled using Aniline Blue in pollen tubes from wild-type, lrx9,10,11, and lrx8,9,11 plants. Bars = 5 μm. B, Quantitative analysis of the fluorescence intensity at the apical and subapical cell walls of the pollen tubes, and the ratio of the fluorescence intensity at the apex versus the subapex in wild-type, lrx9,10,11, and lrx8,9,11 plants. A single scan along the median of the pollen tube was used for quantification. The images in the top row show the apical and subapical regions of the pollen tube cell walls used for fluorescence intensity measurement. Apex, 0 to 3 μm away from the tip (marked in yellow); subapex, 3 to 8 μm away from the tip (marked in red). The fluorescence intensity of each region was quantified by measuring the mean gray value using ImageJ2x software. AU, Arbitrary fluorescence intensity units. Pollen grains were germinated in vitro for 3 h. Three biological repeats were performed, and 20 to 30 pollen tubes were used for each repeat. Error bars show se. Asterisks indicate values that differed significantly from the wild type (*, P < 0.05; ns, not significant, calculated using a two-way ANOVA and Student’s t test).