ALTERED MERISTEM PROGRAM1 sustains cellular differentiation by limiting HD-ZIP III transcription factor gene expression.

Plants show remarkable developmental and regenerative plasticity through the sustained activity of stem cells in meristems. Under certain conditions, pluripotency can even be re-established in cells that have already entered differentiation. Mutation of the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis (Arabidopsis thaliana) causes a set of hypertrophic phenotypes, indicating a defect in the suppression of pluripotency. A role of AMP1 in the miRNA-mediated inhibition of translation has previously been reported, however, how this activity is related to its developmental functions is unclear. Here we examined the functional interaction between AMP1 and the Class III homeodomain-leucine zipper (HD-ZIP III) transcription factors, which are miRNA-controlled determinants of shoot meristem specification. We found that the HD-ZIP III transcriptional output is enhanced in the amp1 mutant and that plant lines with increased HD-ZIP III activity not only developed amp1 mutant-like phenotypes but also showed a synergistic genetic interaction with the mutant. Conversely, the reduction of HD-ZIP III function suppressed the shoot hypertrophy defects of the amp1 mutant. We further provide evidence that the expression domains of HD-ZIP III family members are expanded in the amp1 mutant and that this misexpression occurs at the transcriptional level and does not involve the function of miRNA165/166. Finally, amp1 mutant-specific phenotypes cannot be mimicked by a general inhibition of miRNA function in the AMP1 expression domain. These findings lead us to a model in which AMP1 restricts cellular pluripotency upstream of HD-ZIP III proteins and this control appears to be not directly mediated by the canonical miRNA pathway.

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

Higher plants can continuously form new organs by the sustained activity of pluripotent stem cells. These stem cells are embedded in meristems, where they produce descendants, which undergo cell proliferation and differentiation programs in a spatiotemporally-controlled manner. Under certain conditions, pluripotency can be reestablished in descending cells and this reversion in cell fate appears to be actively suppressed by the existing stem cell pool. Mutation of the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis causes defects in the suppression of pluripotency in cells normally programmed for differentiation, giving rise to unique hypertrophic phenotypes during embryogenesis as well as in the shoot apical meristem. A role of AMP1 in the miRNA-dependent control of translation has recently been established, however, how this activity is connected to its developmental functions is not resolved. Here we identify members of the cytochrome P450 clade CYP78A to act in parallel with AMP1 to control cell fate in Arabidopsis. Mutation of CYP78A5 and its close homolog CYP78A7 in a cyp78a5,7 double mutant caused suspensor-to-embryo conversion and ectopic stem cell pool formation in the shoot meristem, phenotypes characteristic for amp1. The tissues affected in the mutants showed pronounced expression levels of AMP1 and CYP78A5 in wild type. A comparison of mutant transcriptomic responses revealed an intriguing degree of overlap and highlighted alterations in protein lipidation processes. Moreover, we also found elevated protein levels of selected miRNA targets in cyp78a5,7. Based on comprehensive genetic interaction studies we propose a model in which both enzyme classes act on a common downstream process to sustain cell fate decisions in the early embryo and the shoot apical meristem.

ALTERED MERISTEM PROGRAM1 Restricts Shoot Meristem Proliferation and Regeneration by Limiting HD-ZIP III-Mediated Expression of RAP2.6L.

Plants show an indeterminate mode of growth by the activity of organ forming stem cell niches in apically positioned meristems. The correct formation and activity of these meristems are a prerequisite for their adaptive development and also allow the maintenance of organogenesis under adverse circumstances such as wounding. Mutation of the putative Arabidopsis (Arabidopsis thaliana) Glu carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) results in Arabidopsis plants with enlarged shoot apical meristems, supernumerary stem cell pools, and higher leaf formation rate. AMP1 deficiency also causes exaggerated de novo formation of shoot meristems. The activity of AMP1 has been implicated in the control of microRNA (miRNA)-dependent translation; however, it is not known how this function contributes to the shoot meristem defects. Here, we show that the transcription factor RAP2.6L is upregulated in the Arabidopsis amp1 mutant. Overexpression of RAP2.6L in the wild type causes amp1 mutant-related phenotypic and molecular defects, including enhanced shoot regeneration in tissue culture. Conversely, inhibition of RAP2.6L in the amp1 mutant suppresses stem cell hypertrophy and the regenerative capacity. We further provide evidence that RAP2.6L is under direct transcriptional control of miRNA-regulated class III homeodomain-Leu zipper (HD-ZIP III) proteins, key regulators of shoot meristem development, which overaccumulate in the amp1 mutant. Our results reveal a transcription factor module acting downstream of AMP1 in the control of shoot stem cell niche patterning. By positioning the HD-ZIP III/RAP2.6L module downstream of AMP1 function, we provide a mechanistic link between the role of AMP1 in miRNA-mediated translational repression and shoot stem cell specification.

The Small Molecule Hyperphyllin Enhances Leaf Formation Rate and Mimics Shoot Meristem Integrity Defects Associated with AMP1 Deficiency.

ALTERED MERISTEM PROGRAM1 (AMP1) 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.

ALTERED MERISTEM PROGRAM1 suppresses ectopic stem cell niche formation in the shoot apical meristem in a largely cytokinin-independent manner.

Plants are able to reiteratively form new organs in an environmentally adaptive manner during postembryonic development. Organ formation in plants is dependent on stem cell niches (SCNs), which are located in the so-called meristems. Meristems show a functional zonation along the apical-basal axis and the radial axis. Shoot apical meristems of higher plants are dome-like structures, which contain a central SCN that consists of an apical stem cell pool and an underlying organizing center. Organ primordia are formed in the circular peripheral zone (PZ) from stem cell descendants in which differentiation programs are activated. One mechanism to keep this radial symmetry integrated is that the existing SCN actively suppresses stem cell identity in the PZ. However, how this lateral inhibition system works at the molecular level is far from understood. Here, we show that a defect in the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) causes the formation of extra SCNs in the presence of an intact primary shoot apical meristem, which at least partially contributes to the enhanced shoot meristem size and leaf initiation rate found in the mutant. This defect appears to be neither a specific consequence of the altered cytokinin levels in amp1 nor directly mediated by the WUSCHEL/CLAVATA feedback loop. De novo formation of supernumerary stem cell pools was further enhanced in plants mutated in both AMP1 and its paralog LIKE AMP1, indicating that they exhibit partially overlapping roles to suppress SCN respecification in the PZ.