Fig 8. Proposed Model for Class II CCE.

We previously reported a working model for Class II CCE [25], showing that decreased cell numbers in cotyledons were exclusively attributed to decreased TAG-derived Suc, and that CCE might be mediated by IBA-derived IAA related mechanism. Here, we confirmed that IBA-derived IAA is essential for enhancing post-mitotic cell expansion. Based on our previous findings and this research, the scenario for Class II CCE in fugu5 can be summarized as follows. First, upon seed imbibition, excess cytosolic PPi in fugu5–1 leads to inhibition of Suc synthesis de novo from TAG, by inhibiting the gluconeogenic cytosolic enzyme UDP-glucose pyrophosphorylase (UGPase; [27]). Second, during seedling establishment, reduced Suc contents somehow promote the IBA-to-IAA conversion and lead to increased endogenous IAA concentration at 8–10 DAS, which is apparently a crucial transition point to drive CCE in fugu5 cotyledons (Fig 5). Third, one can assume that high endogenous IAA triggers the TIR/AFB-dependent auxin signaling pathway through ARF7 and ARF19, and subsequently activates the vacuolar type V-ATPase leading to an increase in turgor pressure. This ultimately triggers cell size increase and CCE. This scenario is also valid for other mutants, namely icl-2, mls-2, pck1–2, and ibr10–1, all of which exhibit a typical Class II CCE, not because of excess PPi, but due to compromised gluconeogenesis from TAG [25]. Finally, this IAA-mediated CCE is not valid for Class I [22]. Nonetheless, our findings that V-ATPase activity is critically important for CCE in Class II and Class III [20, 21], may suggest that all three CCE classes may converge at this checkpoint and use the V-ATPase complex activity as the final driving force to inflate cell size. Although the above scenario is plausible, its robustness still needs to be challenged experimentally in the future. Succ, succinate. DAS, days after sowing.