The small molecule hyperphyllin enhances leaf formation rate and mimics shoot meristem integrity defects associated with AMP1 deficiency

Abstract

) is a member of the M28 family of carboxypeptidases with a pivotal role in plant development and stress adaptation. Its most prominent mutant defect is a unique hypertrophic shoot phenotype combining a strongly increased organ formation rate with enhanced meristem size and the formation of ectopic meristem poles. However, so far the role of AMP1 in shoot development could not be assigned to a specific molecular pathway nor is its biochemical function resolved. In this work we evaluated the level of functional conservation between AMP1 and its human homolog HsGCPII, a tumor marker of medical interest. We show that HsGCPII cannot substitute AMP1 in planta and that an HsGCPII-specific inhibitor does not evoke amp1-specific phenotypes. We used a chemical genetic approach to identify the drug hyperphyllin (HP), which specifically mimics the shoot defects of amp1, including plastochron reduction and enlargement and multiplication of the shoot meristem. We assessed the structural requirements of HP activity and excluded that it is a cytokinin analog. HP-treated wild-type plants showed amp1-related tissue-specific changes of various marker genes and a significant transcriptomic overlap with the mutant. HP was ineffective in amp1 and elevated the protein levels of PHAVOLUTA, consistent with the postulated role of AMP1 in miRNA-controlled translation, further supporting an AMP1-related mode of action. Our work suggests that plant and animal members of the M28 family of proteases adopted unrelated functions. With HP we provide a tool to characterize the plant-specific functions of this important class of proteins.Arabidopsis ALTERED MERISTEM PROGRAM1 (AMP1, At3G54720, MEROPS ID: M28.007) belongs to the Zn 2+

AMP1 and CYP78A5/7 act through a common pathway to govern cell fate maintenance in Arabidopsis thaliana

“…If one enzyme class is compromised the other, to a certain extent, is still able to independently maintain the required downstream function. This model is also consistent with the strong up-regulation of CYP78A5 expression in amp1 (S1 Table; [9,16]) and the partial rescue of the amp1 shoot phenotype by CYP78A5 overexpression ( Fig 9). In contrast, AMP1 overexpression did not result in a comparable phenotype to that of CYP78A5 overexpression and did not ameliorate the cyp78a5,7 phenotype (S3 Fig; Fig 10).…”

“…AMP1/LAMP1 deficiency results in protein over-accumulation of various tested miRNA targets, without affecting their transcription levels [17]. To test whether cyp78a5,7 also shares this specific molecular defect with amp/lamp1, we assessed the protein levels of the miRNA165/166 targeted HD-ZIP III transcription factor PHV using an YFP-tagged version driven by the 35S promoter, which has been shown to over-accumulate in amp1 [16]. Using three independent biological repeats we found significant higher PHV-GFP protein levels in cyp78a5,7 compared to wild type (S5A and S5C Fig).…”

“…However, its precise physiological function during normal development and tumor formation is not well understood [14]. HsGCPII and AMP1 share the same domain structure and the zinc-binding catalytic center, however the overall sequence similarity is only 28% and important residues required for GCPII substrate recognition are not conserved in AMP1 [15,16]. Consistent with a functional divergence between the animal and plant proteins, heterologous HsGCPII expression did not rescue any of the amp1 phenotypes [16].…”

Plant Chemical Genetics: From Phenotype-Based Screens to Synthetic Biology

“…Examples of the latter are the discoveries of the compounds hyperphyllin and bubblin (Poretska et al, 2016;Sakai et al, 2017). Application of hyperphyllin mimicked the phenotype of the altered meristem program1 (amp1) mutant, whereas bubblin simulated stomatal clustering mutants.…”

“…By far, the most used library in plant chemical biology is the Chembridge DIVERSet (Armstrong et al, 2004;Zouhar et al, 2004;Surpin et al, 2005;DeBolt et al, 2007;Rojas-Pierce et al, 2007;Christian et al, 2008;Gendron et al, 2008;Savaldi-Goldstein et al, 2008;De Rybel et al, 2009, 2012Lin et al, 2010;Tsuchiya et al, 2010;Kim et al, 2011;Kerchev et al, 2014;Hu et al, 2016;Van de Wouwer et al, 2016). Chembridge also provides focused or targeted small-molecule libraries (Poretska et al, 2016). Additional libraries include the Maybridge Hitfinder (Nishimura et al, 2012(Nishimura et al, , 2014, the Life Chemicals, Inc., collection (Zhao, 2012;Ye et al, 2016), the Korean Chemical Bank (Kim et al, 2010), and the RIKEN Natural Products Depository (Noutoshi et al, 2012a;Ito et al, 2015).…”