In order to avoid the overhead of establishing a new connection each time a query needs fired against MySQL, there are two options available:
Regarding your question about should the application server wait for a connection, the answer is yes.
MySQL connections are blocking. When you issue a request from MySQL server over a connection, the connection will wait, idle, until a response is received from the server.
There is no way to send two requests on the same connection and see which returns first. You can only send one request at a time.
So, generally, a single thread in a connection pool consists of one client side connection (in your case, the application server is the client) and one server side connection (database).
Your application should wait for an available connection thread from the pool, allowing the pool to grow when it's needed, and to shrink back to your default number of threads, when it's less busy.
Having persistent connections does not imply that all threads use the same connection. It just "says" that you keep the connection open (in contradiction to open a connection each time you need one). Opening a connection is an expensive operation, so - in general - you try to avoid opening connections more often than necessary.
This is the reason why multithreaded applications often use connection pools. The pool takes care of opening and closing connections and every thread that needs a connection requests one from the pool. It is important to take care that the thread returns the connection as soon as possible to the pool, so that another thread can use it.
If your application has only a few long running threads that need connections you can also open a connection for each thread and keep this open.
Using just one connection (as you described it) is equal to a connection pool with the maximum size one. This will be sooner or later your bottleneck as all threads will have to wait for the connection. This could be an option to serialize the database operations (perform them in a certain order), although there are better options to ensure serialisation.