Two Arabidopsis late pollen transcripts are detected in cytoplasmic granules

Abstract

Many of mRNAs synthesized during pollen development are translated after germination, and we hypothesize that they are stored in cytoplasmic granules. We analyzed the cellular localization of the SKS14 and AT59 Arabidopsis mRNAs, which are orthologues of the tobacco NTP303 and tomato LAT59 pollen mRNAs, respectively, by artificially labeling the transcripts with a MS2-GFP chimera. A MATLAB-automated image analysis helped to identify the presence of cytoplasmic SKS14 and AT59 mRNA granules in mature pollen grains. These mRNA granules partially colocalized with VCS and DCP1, two processing body (PB) proteins. Finally, we found a temporal correlation between SKS14 protein accumulation and the disappearance of SKS14 mRNA granules during pollen germination. These results contribute to unveil a mechanism for translational regulation in Arabidopsis thaliana pollen.

Keywords: MATLAB; MS2; pollen; processing body; translational regulation.

Figure 1

mRNA detection by the MS2 system. The two depicted constructs were inserted in the same vector: GFP fused to the MS2 coat protein (MCP) with a nuclear localization signal (NLS), and the SKS14 or AT59 transcripts fused to MCP‐binding site (MCP‐bs). A control construct termed MS2 encodes the GFP‐MCP chimera and no target mRNA. The three constructs SKS14‐MS2,AT59‐MS2, and MS2 were under the control of the pollen‐specific promoter LAT52. LB and RB, left and right borders of the translocation cassette, respectively

Figure 2

Arabidopsis SKS14 and AT59 mRNAs form cytoplasmic granules in mature pollen grains. (a) Confocal images of a representative pollen grain from the control MS2 line. A mask was applied to eliminate vegetative nucleus and facilitate cytoplasm visualization. (b‐e) Representative images of two independent SKS14‐MS2 (b and c) and two AT59‐MS2 lines (d and e). In the left panels, white arrowheads show examples of cytoplasmic granules identified by the MATLAB script while empty arrowheads show cytoplasmic aggregates that were not detected by the MATLAB script. Right panels, DIC images. Size bar, 5 μm. (f) Quantification of cytoplasmic granules. Each point corresponds to the mean value of an independent sample that included 20 pollen grains. The media and standard error for each transgenic line are shown. Statistical significance (Mann–Whitney test) relative to the control line MS2 3‐1 is indicated (***p < .001 and **p < .01)

Figure 3

The PB proteins VCS and DCP1 form cytoplasmic foci in mature pollen. Representative images of mature pollen grains in the absence (‐) or presence (+) of puromycin 50 μg/ml. DIC images are shown in the right panels. Size bar, 5 μm

Figure 4

Colocalization of SKS14 mRNA with RFP‐VCS. (a) Confocal image of a representative mature pollen grain showing high colocalization between SKS14 mRNA and a RFP‐VCS body. (b) Confocal image of a representative mature pollen grain showing a SKS14 mRNA cytoplasmic granule contiguous to a RFP‐VCS body. (c) Confocal image of a representative cytoplasmic granule with no relationship with any RFP‐VCS body. In the left panels, white arrowheads show examples of cytoplasmic granules identified by MATLAB while empty arrowheads show cytoplasmic aggregates not detected by MATLAB. The insets in the merged (“Overlay”) column are enlarged on the 10X panels. In the “Colocalization” column, the blue point and black circle indicate the localization and size of the VCS body, respectively, and the red point and circle correspond to the SKS14 mRNA granule. DIC images are shown. Size bar, 5 μm

Figure 5

SKS14‐RFP protein is expressed from early stages of germination and localizes at the margins of pollen tubes. Representative images of S2 line pollen grains expressing SKS14‐RFP protein (left panels). Right panels, DIC images. Size bars, 5 μm for early and late stages (a and b) and 15 μm for the pollen tube (c). (d) A 5X magnification shows localization at the tip (Size bar, 5 μm). (e) Quantification of SKS14 mRNA cytoplasmic granules per pollen grain compared to a MS2 control (MS2 3‐1). ES and LS, early and late stages of pollen germination, respectively. Each point represents the mean value of independent samples including 9‐10 pollen grains. Lines link data from paired samples (same experiment). Statistically different values are indicated (***p < .001 and **p < .01). The p‐value for the ES‐MS2/LS‐MS2 pair was 0.99, and for the LS‐MS2/LS‐S13 pair, 0.48 (two‐way ANOVA randomized block, Bonferroni post‐test)