Figure 2.

NSE4 Expression Analysis during Pollen, Ovule, and Seed Development.

(A) Test for functionality of NSE4-VENUS translational fusion line. Wild-type (WT), nse4a-2 (4a-2), and nse4a-2 plants complemented with ProNSE4A:NSE4A:VENUS (4A-VENUS) were germinated and grown on the control and zebularine-containing media for 7 d. Restoration of root growth in 4a-2 NSE4A-VENUS indicates full functionality of the translational fusion protein.

(B) The first two columns show DAPI- and GUS-stained pollen of ProNSE4A:GUS (Pro4A:GUS) reporter line. Stage (Stg) 10 corresponds to the microspore, Stg 11 to bicellular pollen, Stg 12 to tricellular pollen, and Stg 14 to mature pollen from open anthers. The last column shows pollen from the ProNSE4A:NSE4A:VENUS (4A-VENUS) reporter line. Bar = 5 µm.

(C) The ProNSE4B:GUS (Pro4B:GUS) reporter line presented in the same way as in (A). Bar = 5 µm. Stg, stage.

(D) GUS activity of ProNSE4A:GUS (Pro4A:GUS; left) and ProNSE4B:GUS (Pro4B:GUS; right) from ovule primordia to early postfertilization. Stage (Stg) 10, 11, and 12 to 14 show ovule primordia, the nucellus, and developing the embryo sac, respectively. Bars = 50 µm.

(E) ProNSE4A:NSE4A:VENUS (4A-VENUS) signals at the same stages as described in (C). In the ovule primordia of stage (Stg) 11, the megaspore mother cell is almost free of 4A-VENUS signal (arrowheads). However, its expression is greatly increased in the female meiocyte of Stg 11 (arrowhead). Bar = 10 µm.

(F) GUS activity driven by the NSE4A and NSE4B promoters at the indicated hours after pollination (HAP). Reporter lines were pollinated with their own pollen 48 h after emasculation. Bars = 50 µm. e, embryo; ce, chalazal endosperm.

(G) Accumulation of ProNSE4A:NSE4A:VENUS (4A-VENUS) in nuclei of globular-, heart-, torpedo-, and bent cotyledon–stage embryos and syncytial endosperm 72 h after pollination. Left images represent differential interference contrast (DIC), and the right images show the VENUS signal. Bars = 50 µm.