Gametophytic epigenetic regulators, MEDEA and DEMETER, synergistically suppress ectopic shoot formation in Arabidopsis.

KEY MESSAGE: The gametophytic epigenetic regulators, MEA and DME, extend their synergistic role to the sporophytic development by regulating the meristematic activity via restricting the gene expression in the shoot apex. The gametophyte-to-sporophyte transition facilitates the alternation of generations in a plant life cycle. The epigenetic regulators DEMETER (DME) and MEDEA (MEA) synergistically control central cell proliferation and differentiation, ensuring proper gametophyte-to-sporophyte transition in Arabidopsis. Mutant alleles of DME and MEA are female gametophyte lethal, eluding the recovery of recessive homozygotes to examine their role in the sporophyte. Here, we exploited the paternal transmission of these mutant alleles coupled with CENH3-haploid inducer to generate mea-1;dme-2 sporophytes. Strikingly, the simultaneous loss of function of MEA and DME leads to the emergence of ectopic shoot meristems at the apical pole of the plant body axis. DME and MEA are expressed in the developing shoot apex and regulate the expression of various shoot-promoting factors. Chromatin immunoprecipitation (ChIP), DNA methylation, and gene expression analysis revealed several shoot regulators as potential targets of MEA and DME. RNA interference-mediated transcriptional downregulation of shoot-promoting factors STM, CUC2, and PLT5 rescued the twin-plant phenotype to WT in 9-23% of mea-1-/-;dme-2-/- plants. Our findings reveal a previously unrecognized synergistic role of MEA and DME in restricting the meristematic activity at the shoot apex during sporophytic development.

The Polycomb-Group Repressor MEDEA Attenuates Pathogen Defense.

Plants recruit positive and negative regulators for fine tuning the balance between growth and development. Negative regulators of pathogen defense generally modulate defense hormone biosynthesis and signaling. Here, we report a mechanism for attenuation of the defense response in Arabidopsis (Arabidopsis thaliana), which is mediated by the polycomb-group repressor MEDEA (MEA). Our results showed that pathogen inoculation or exogenous application of salicylic acid, methyl jasmonate, or the bacterial 22-amino acid domain of flagellin peptide induces the expression of MEAMEA expression was higher when plants were inoculated with the avirulent strain of Pseudomonas syringae pv. tomato (Pst) carrying the AvrRpt2 effector (Pst-AvrRpt2) compared to the virulent Pst strain. MEA remains suppressed during the vegetative phase via DNA and histone (H3K27) methylation, and only the maternal copy is expressed in the female gametophyte and endosperm via histone and DNA demethylation. In contrast, Pst-AvrRpt2 induces high levels of MEA expression via hyper-accumulation of H3K4me3 at the MEA locus. MEA-overexpressing transgenic plants are susceptible to the fungal pathogen Botrytis cinerea and bacterial pathogens Pst and Pst-AvrRpt2, whereas mea mutant plants are more resistant to bacterial pathogens. AvrRpt2-mediated immunity requires the function of RESISTANCE TO P. SYRINGAE2 (RPS2) in Arabidopsis. Using transcriptional analysis and chromatin immunoprecipitation, we established that MEA directly targets RPS2 and suppresses its transcription. We screened an Arabidopsis cDNA library using MEA as the bait in a yeast two-hybrid assay and identified DROUGHT-INDUCED19, a transcription factor that interacts with MEA and recruits it at the RPS2 promoter. The results identified a previously unknown mechanism of defense response attenuation in plants.

Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) - a class-I KNOX gene in potato.

Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato.

MicroRNA156: a potential graft-transmissible microRNA that modulates plant architecture and tuberization in Solanum tuberosum ssp. andigena.

MicroRNA156 (miR156) functions in maintaining the juvenile phase in plants. However, the mobility of this microRNA has not been demonstrated. So far, only three microRNAs, miR399, miR395, and miR172, have been shown to be mobile. We demonstrate here that miR156 is a potential graft-transmissible signal that affects plant architecture and tuberization in potato (Solanum tuberosum). Under tuber-noninductive (long-day) conditions, miR156 shows higher abundance in leaves and stems, whereas an increase in abundance of miR156 has been observed in stolons under tuber-inductive (short-day) conditions, indicative of a photoperiodic control. Detection of miR156 in phloem cells of wild-type plants and mobility assays in heterografts suggest that miR156 is a graft-transmissible signal. This movement was correlated with changes in leaf morphology and longer trichomes in leaves. Overexpression of miR156 in potato caused a drastic phenotype resulting in altered plant architecture and reduced tuber yield. miR156 overexpression plants also exhibited altered levels of cytokinin and strigolactone along with increased levels of LONELY GUY1 and StCyclin D3.1 transcripts as compared with wild-type plants. RNA ligase-mediated rapid amplification of complementary DNA ends analysis validated SQUAMOSA PROMOTER BINDING-LIKE3 (StSPL3), StSPL6, StSPL9, StSPL13, and StLIGULELESS1 as targets of miR156. Gel-shift assays indicate the regulation of miR172 by miR156 through StSPL9. miR156-resistant SPL9 overexpression lines exhibited increased miR172 levels under a short-day photoperiod, supporting miR172 regulation via the miR156-SPL9 module. Overall, our results strongly suggest that miR156 is a phloem-mobile signal regulating potato development.