We plan to use SharePoint 2010 application pages and wondered about our current setup, which has the default master page (v4.master) with no changes but a heavily customized page layout for the Publishing Pages. A question: Can an application page access the page layout that the Publishing Pages use or it can only access the master page?
Are you certain you need an application page? You can provision publishing pages via code in a document library set for Publishing Pages and also specify the layout. In publishing scenarios this is the normal pattern.
Application Pages are typically for more generic feature support not tied to any particular site collection which is why you cannot point to a specified site collection resource; in this case the Page Layout.
My understanding would be that an application page wouldn't have the publishing page content type, so publishing page layouts could not be applied to it. You may be able to work around some of it via custom code, but out of the box just creating one, I don't believe you'd have that option.
The application page will use the master, but not a publishing page layout.