Figure 4.

Centromere Pairing Is Defective during Prophase I of Meiosis in arp6-1 Megasporocytes.

(A) to (D) and (I) to (L) Epifluorescence microscopy images of megasporocyte spreads subjected to FISH (red signal).

(E) to (H) and (M) to (P) Merged images of centromere (red) and DAPI-stained chromosomes (blue) of megasporocyte spreads.

(A) and (E) Wild-type megasporocyte in leptotene showing 10 unpaired and dispersed centromeres.

(B) and (F) Wild-type megasporocyte in pachytene with five pairs of centromeres.

(C) and (G) Wild-type megasporocyte in diakinesis with five pairs of centromeres of the five bivalents.

(D) and (H) Wild-type megasporocyte in metaphase I containing five pairs of condensed chromosomes with separated centromeres in each pair pointing to opposite poles.

(I) and (M) arp6-1 megasporocyte in leptotene showing 10 centromeres, many of which are abnormally diffused.

(J) and (N) arp6-1 megasporocyte in pachytene with at least one abnormally diffused centromere (white arrow in [N]).

(K) and (O) arp6-1 megasporocyte in diakinesis with a mixture of univalents (white arrows) and bivalents.

(L) and (P) arp6-1 megasporocyte in metaphase I containing abnormally diffused centromeres with chromosome bridges (white arrows).

(Q) and (R) Immunoblotting of ARP6 (Q) or PEPC protein (R) in wild-type, arp6-1, and arp6-2 ovaries containing stage 2-III/IV ovules undergoing meiosis. Blots were first probed with ARP6 monoclonal antibody (mAbARP6a; α-ARP6), or the immunoblot was stripped and reprobed with anti-PEPC polyclonal antibody (α-PEPC). Protein sizes in kilodaltons; *, 50 kD; ⁺, 100 kD.

(S) to (U) In situ localization of ARP6 mRNA in Arabidopsis whole-mount ovules.

(S) In situ hybridization of ARP6 antisense RNA to wild-type ovules (89% of stained ovules [n = 106] showed this pattern).

(T) In situ hybridization of ARP6 antisense RNA to arp6-1 ovules (100% of stained ovules [n = 65] showed this pattern).

(U) Control hybridization of wild-type ovules using ARP6 sense RNA did not give any signal (100% of stained ovules [n = 51] showed this pattern).

Bar in (A) = 10 µm for (A) to (P), and bars in (S) to (U) = 10 μm.