Figure 2.

NSPHAN Expression Analysis.

Expression patterns for NSPHAN were examined in situ with digoxygenin-labeled probes ([A] to [C]), by RT-PCR (D), and in RNA gel blots (E). Bars in (A), (B), and (C) = 60, 80, and 100 μm, respectively.

(A) In situ hybridization with an antisense RNA probe in a juvenile shoot apex shows NSPHAN mRNA is undetectable in the central zone of the meristem (cz) but is present throughout P1 and P2 leaf primordia.

(B) In stages P4 to P5, NSPHAN is expressed throughout emerging leaf blades (lb) and in leaf midveins (mv).

(C) In the expanding lamina, NSPHAN is localized to middle mesophyll (mm) and lateral veins (lv). mv, midveins.

(D) RT-PCR amplification of NSPHAN and NTH23 as control from total RNA samples shows expression in leaves up through the 10-cm stage and in the stems.

(E) RNA gel blot analysis with an NSPHAN-specific probe (pNAM5.2.3) shows similar mRNA levels at the tip and base of juvenile wild-type leaves (NSWT) and undetectable levels in three antisense transgenics (AF6J.4, AF34.1, and AF18B.1) relative to a ubiquitin control (pUBI). Expression levels of the antisense transgene were analyzed with the pRH81 probe.