These authors contributed equally to this work. SUMMARY WRINKLED1 (WRI1) is a key transcription factor governing plant oil biosynthesis. We characterized three intrinsically disordered regions (IDRs) in Arabidopsis WRI1, and found that one C-terminal IDR of AtWRI1 (IDR3) affects the stability of AtWRI1. Analysis by bimolecular fluorescence complementation and yeasttwo-hybrid assays indicated that the IDR3 domain does not determine WRI1 stability by interacting with BTB/POZ-MATH proteins connecting AtWRI1 with CULLIN3-based E3 ligases. Analysis of the WRI1 sequence revealed that a putative PEST motif (proteolytic signal) is located at the C-terminal region of AtWRI1 IDR3 . We also show that a 91 amino acid domain at the C-terminus of AtWRI1 without the PEST motif is sufficient for transactivation. We found that removal of the PEST motif or mutations in putative phosphorylation sites increased the stability of AtWRI1, and led to increased oil biosynthesis when these constructs were transiently expressed in tobacco leaves. Oil content was also increased in the seeds of stable transgenic wri1-1 plants expressing AtWRI1 with mutations in the IDR3-PEST motif. Taken together, our data suggest that intrinsic disorder of AtWRI1 IDR3 may facilitate exposure of the PEST motif to protein kinases. Thus, phosphorylation of the PEST motif in the AtWRI1 IDR3 domain may affect AtWRI1-mediated plant oil biosynthesis. The results obtained here suggest a means to increase accumulation of oils in plant tissues through WRI1 engineering.

Summary Plant 14‐3‐3 proteins are phosphopeptide‐binding proteins, belonging to a large family of proteins involved in numerous physiological processes including primary metabolism, although knowledge about the function of 14‐3‐3s in plant lipid metabolism is sparse. WRINKLED1 (WRI1) is a key transcription factor that governs plant oil biosynthesis. At present, AtWRI1‐interacting partners remain largely unknown. Here, we show that 14‐3‐3 proteins are able to interact with AtWRI1, both in yeast and plant cells. Transient co‐expression of 14‐3‐3‐ and AtWRI1‐encoding cDNAs led to increased oil biosynthesis in Nicotiana benthamiana leaves. Stable transgenic plants overproducing a 14‐3‐3 protein also displayed increased seed oil content. Co‐production of a 14‐3‐3 protein with AtWRI1 enhanced the transcriptional activity of AtWRI1. The 14‐3‐3 protein was found to increase the stability of AtWRI1. A possible 14‐3‐3 binding motif was identified in one of the two AP2 domains of AtWRI1, which was also found to be critical for the interaction of AtWRI1 with an E3 ligase linker protein. Thus, we hypothesize a regulatory mechanism by which the binding of 14‐3‐3 to AtWRI1 interferes with the interaction of AtWRI1 and the E3 ligase, thereby protecting AtWRI1 from degradation. Taken together, our studies identified AtWRI1 as a client of 14‐3‐3 proteins and provide insights into a role of 14‐3‐3 in mediating plant oil biosynthesis.

WRINKLED1 (WRI1) has targets other than fatty acid biosynthesis genes that mediate the growth of roots.

Characterization of the TCP4-WRI1 complex regulating Arabidopsis seed oil biosynthesis expands our understanding of WRI1-interacting factors and describes a further role for TCP transcription factors.