Plant organ growth and final size are determined by coordinated cell proliferation and expansion. The BIGPETALp (BPEp) basic helix-loop-helix (bHLH) transcription factor was shown to limit Arabidopsis thaliana petal growth by influencing cell expansion. We demonstrate here that BPEp interacts with AUXIN RESPONSE FACTOR8 (ARF8) to affect petal growth. This interaction is mediated through the BPEp C-terminal domain (SD BPEp ) and the C-terminal domain of ARF8. Site-directed mutagenesis identified an amino acid consensus motif in SD BPEp that is critical for mediating BPEp-ARF8 interaction. This motif shares sequence similarity with motif III of ARF and AUXIN/INDOLE-3-ACETIC ACID proteins. Petals of arf8 mutants are significantly larger than those of the wild type due to increased cell number and increased cell expansion. bpe arf8 double mutant analyses show that during early petal development stages, ARF8 and BPEp work synergistically to limit mitotic growth. During late stages, ARF8 and BPEp interact to limit cell expansion. The alterations in cell division and cell expansion observed in arf8 and/or bpe mutants are associated with a change in expression of early auxin-responsive genes. The data provide evidence of an interaction between an ARF and a bHLH transcription factor and of its biological significance in regulating petal growth, with local auxin levels likely influencing such a biological function.

SUMMARYIn Arabidopsis, four homeotic gene classes, A, B, C and E, are required for the patterning of floral organs. However, very little is known about how the activity of these master genes is translated into regulatory processes leading to specific growth patterns and the formation of organs with specific shapes and sizes. Previously we showed that the transcript variant BPEp encodes a bHLH transcription factor that is involved in limiting petal size by controlling post-mitotic cell expansion. Here we show that the phytohormone jasmonate is required for control of BPEp expression. Expression of BPEp was negatively regulated in opr3 mutant flowers that are deficient in jasmonate synthesis. Moreover, the expression of BPEp was restored in opr3 flowers following exogenous jasmonate treatments. Expression of the second transcript variant BPEub, which originates from the same gene as BPEp via an alternative splicing event, was not affected, indicating that BPEp accumulation triggered by jasmonate occurs at the post-transcriptional level. Consistent with these data, opr3 exhibited an increase in petal size as a result of increased cell size, as well as a modified vein pattern, phenotypes that are similar to those of the bpe-1 mutant. Furthermore, exogenous treatments with jasmonate rescued petal phenotypes associated with loss of function of OPR3. Our data demonstrate that jasmonate signaling downstream of OPR3 is involved in the control of cell expansion and in limiting petal size, and that BPEp is a downstream target that functions as a component mediating jasmonate signaling during petal growth.

Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform maturation of seed storage protein upon cleavage-dependent autoactivation in the low-pH environment of storage vacuoles. The AEPs have attracted attention for their macrocyclization reactions, and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show that butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide-binding region are the source of its efficient macrocyclization. All considered, it seems that either butelase 1 has not fully specialized or a requirement for autocatalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.