Optimus and Cheetor fighting over the possibility of inacting his plan to obliterate all the mechanical life on Cybertron. It was pretty hard watching two of the characters that got along best during the Beast Wars fighting with each other.
Optimus (and the audiance) learning what really happened to Rhinox.
The real Savage/Noble's death while trying to help Nightscream.
The death of Optimus Primal.
Rattrap's feelings of uselessness early on, which all come to a head in The Weak Component when he botches up a mission; gets chewed out; and even hears his teammates talking smack about him behind his back.
Rattrap: Still think you gotta cover for me?! ...You still wanna CUT ME LOOSE?! ...You never believed in me. Ever.