| dc.description.abstract | Leaves are initiated as primordia at the flanks of the shoot apical meristem. Primordia in the simple-leaved species such as Arabidopsis produce single lamina, whereas those in compound-leaved species such as tomato form secondary outgrowths at the margin, which leads to the formation of dissected lamina with multiple leaflets at maturity. It has been shown that the underlying molecular mechanism of leaflet formation in compound leaves is an extended expression of KNOX-I genes, which are otherwise restricted to the meristem zone in simple-leaved species, in early leaf primordia. Compound leaves such as those of tomato can be converted to simple form by reducing the KNOX-I level, or to a super-compound structure by increasing the KNOX-I expression. However, overexpression of KNOX-I genes fails to convert the simple Arabidopsis leaves to compound structure, primarily due to the presence of strong KNOX-I suppressors such as JAW-TCP and KNOX-II proteins in the leaf primordia. Simultaneous down-regulation of multiple JAW-TCP and KNOX-II genes converts simple Arabidopsis leaves to super-compound form that continues to make higher-order leaflets for ever, with concomitant ect= opic expression of meristem genes such as KNAT2, KNAT6 (both belonging to KNOX-I class) and CUC2. This suggests that the JAW-TCP proteins, along with KNOX-II members, suppress KNOX-I genes in the leaf primordia. However, the JAW-TCP proteins have been shown to act as transcriptional activators and are not known for their transcriptional repression activity. It is possible that the CIN-TCPs interact with the chromatic modifiers and recruit them to their target genes to suppress their transcription. I have investigated how TCP/KNOX-II factors establish stable repression of meristem genes via chromatin modifications and identify downstream effectors that enforce simple leaf architecture.
Part I: TCP-Mediated Epigenetic Repression of KNOX-I Genes
To test the hypothesis that CIN-TCP proteins recruit epigenetic silencers to KNOX-I loci, we generated a loss-of-function mutant (named jk-M) by combining down-regulation of five CIN-TCPs and three KNOX-II genes, which converts simple leaves into indeterminately branching “super-compound” forms. We performed targeted ChIP-qPCR to profile the repressive mark H3K27me3 and the activation mark H3K4me3 at KNAT2 and KNAT6 upstream regulatory regions (URRs) at 12 and 30 days after stratification (DAS). In jk-M leaves, H3K27me3 were reduced compare to wild type, whereas a 24-hour dexamethasone-induced pulse of TCP4 in a dexamethasone-inducible line (jk-D) restored H3K27me3 deposition to levels exceeding those of Col-0. RT-qPCR analysis also indicates towards these chromatin changes.
To elucidate how TCP4 engages the Polycomb Repressive Complex 2 (PRC2), we conducted a yeast two-hybrid screen against 35 candidate chromatin remodelers and histone modifiers. TCP4 interacted strongly with SWI3C, an ATP-dependent remodeler, and with LEC1, a histone-modifying factor, however no physical interaction was found with CLF, PRC2 component. EMSA assays confirmed weak direct binding of TCP4 to KNAT2 URR fragments, suggesting that TCP4 may serve as a targeting module for chromatin modifiers rather than as a classical DNA-binding repressor. ChIP-qPCR using FLAG-tagged TCP4 transgenics detected enrichment at KNAT2 and KNAT6 URRs in vivo, indicating locus-specific recruitment.
Finally, genetic interactions with the PRC2 catalytic mutant curly leaf (clf-28) revealed that TCP4-mediated repression of meristem genes and the associated H3K27me3 deposition are dependent on functional PRC2. When we mutated clf-28 in the jk-M background, the double mutants exhibited the wild type flowering which was not alter after Dex treatment. This indicates that, PRC2 activity is essential for the rescue of late flowering in TCP-KNOX–deficient plants.
Part II. Genome-Wide H3K27me3 Profiling Identifies Target Genes of TCP/KNOX-II Activity
To capture the global landscape of H3K27me3 in relation to TCP/KNOX-II activity, we performed ChIP-seq on the 5th and 6th leaves of Col-0, jk-M (reduced TCP/KNOX-II activity), and jk-D (induced TCP-only activity) lines. Peak calling identified 9,056 H3K27me3-marked genes in Col-0, which increased to 11,196 in jk-M but decreased to 10,232 in jk-D, illustrating that loss of TCP/KNOX-II expands the repressive landscape while TCP4 induction refines it.
To pinpoint genes whose repression in wild type depends on TCP/KNOX-II, we applied a stringent filter This yielded 391 candidates, of which 100 high-confidence genes were functionally categorized into developmental regulators, hormone signaling components, stress-response factors, DNA-repair enzymes, and metabolic genes. Among these, one AINTEGUMENTA-LIKE (AIL/PLT) clade members, AIL7, and the flavonoid biosynthesis enzyme TT7, emerged as top candidates. ChIP-qPCR validation in independent biological replicates confirmed their enrichment. Motif analysis highlighted enrichment of TCP-like binding motifs in proximal promoters, implicating direct or indirect TCP/KNOX-II targeting. Integrating transcriptomic data revealed 27 high-confidence targets that exhibited both H3K27me3 loss and concomitant transcript upregulation in jk-M. These data establish a genome-wide role for TCP/KNOX-II in shaping the H3K27me3 epigenome to reinforce simple leaf identity.
Part III. Functional Validation of AIL-7 and Genetic Interactions with TCP/KNOX Networks
Focusing on AIL7 or PLT7 as a candidate effector of leaf determinacy, we first conducted a phylogenetic analysis of the AIL family and generated pPLT7:GUS reporter lines. To visualize PLT7 expression dynamics, we generated pPLT7:GUS reporter lines in three genetic contexts. In Col-0; pPLT7:GUS leaves, no GUS activity was detected, indicating tight repression under wild-type conditions. In the jk-M; pPLT7:GUS background (mock-treated), we observed strong, margin-localized GUS staining precisely at lobe junctions, revealing ectopic PLT7 activation in the absence of CIN-TCP and KNOX-II function. Upon dexamethasone induction (jk-D; pPLT7:GUS), GUS staining was abolished, demonstrating that reporter activation depends entirely on the jk-M trans-activation system.
Genetic interaction studies were performed to dissect the contributions of TCP, KNOX, and PLT genes to leaf form. First, loss-of-function in TCP–KNOX pathways was established using a pRPS5A:MIR319a line (knocking down CIN-TCPs) and a knat3,4,5 triple mutant. To determine whether PLT factors act downstream or in parallel, plt3,5,7 triple mutants were crossed into each TCP–KNOX loss-of-function line. Through multi-generational crosses, we obtained homozygous single, double, and higher-order mutants that were rigorously genotyped and phenotyped. Quantitative comparison of leaflet number across Col-0, single mutants, double mutants, and higher-order combinations showed that removal of PLT activity significantly suppressed the compound leaf phenotype of pRPS5A:MIR319a;knat3,4,5 mutant suggesting PLTs act as key effectors by which TCP/KNOX-II factors maintain simple leaf architecture.
Collectively, our multidisciplinary approach—from locus-specific histone profiling and protein interaction assays to genome-wide epigenomic mapping and genetic validation—reveals that CIN-TCP proteins like TCP4 recruit PRC2 component and by histone methylation (H3K27me3), keep meristem genes (KNOX-I and PLT) turned off in leaf primordia. This epigenetic control ensures leaves develop a simple form. When PLT genes escape this repression, they trigger extra leaflet growth, which turn a simple Arabidopsis leaf into a compound one. Together, our findings suggest the moonlight activity of an activating transcription factor as a suppressor of meristematic gene expression. | en_US |
