Methylations on nucleosomal histones play fundamental roles in regulating eukaryotic transcription. Jumonji C domain–containing histone demethylases (JMJs) dynamically control the level of histone methylations. However, how JMJ activity is generally regulated is unknown. We found that the tricarboxylic acid cycle–associated enzyme α-ketoglutarate (α-KG) dehydrogenase (KGDH) entered the nucleus, where it interacted with various JMJs to regulate α-KG–dependent histone demethylations by JMJs, and thus controlled genome-wide gene expression in plants. We show that nuclear targeting is regulated by environmental signals and that KGDH is enriched at thousands of loci in Arabidopsis thaliana . Chromatin-bound KGDH catalyzes α-KG decarboxylation and thus may limit its local availability to KGDH-coupled JMJs, inhibiting histone demethylation. Thus, our results uncover a regulatory mechanism for histone demethylations by JMJs.

Light-dependent seed germination is a vital process for many seed plants. A decisive event in light-induced germination is degradation of the central repressor PHYTOCHROME INTERACTING FACTOR 1 (PIF1). The balance between gibberellic acid (GA) and abscisic acid (ABA) helps to control germination. However, the cellular mechanisms linking PIF1 turnover to hormonal balancing remain elusive. Here, employing far-red light-induced Arabidopsis thaliana seed germination as the experimental system, we identified PLANTACYANIN (PCY) as an inhibitor of germination. It is a blue copper protein associated with the vacuole that is both highly expressed in mature seeds and rapidly silenced during germination. Molecular analyses showed that PIF1 binds to the miR408 promoter and represses miR408 accumulation. This in turn posttranscriptionally modulates PCY abundance, forming the PIF1-miR408-PCY repression cascade for translating PIF1 turnover to PCY turnover during early germination. Genetic analysis, RNA-sequencing, and hormone quantification revealed that PCY is necessary and sufficient to maintain the PIF1-mediated seed transcriptome and the low-GA-high-ABA state. Furthermore, we found that PCY domain organization and regulation by miR408 are conserved features in seed plants. These results revealed a cellular mechanism whereby PIF1-relayed external light signals are converted through PCY turnover to internal hormonal profiles for controlling seed germination.

Plants possess unique primary cell walls made of complex polysaccharides that play critical roles in determining intrinsic cell and organ size. How genes responsible for synthesizing and modifying the polysaccharides in the cell wall are regulated by microRNAs (miRNAs) to control plant size remains largely unexplored. Here we identified 23 putative cell wall-related miRNAs, termed as CW-miRNAs, in Arabidopsis thaliana and characterized miR775 as an example. We showed that miR775 post-transcriptionally silences GALT9, which encodes an endomembrane-located galactosyltransferase belonging to the glycosyltransferase 31 family. Over-expression of miR775 and deletion of GALT9 led to significantly enlarged leaf-related organs, primarily due to increased cell size. Monosaccharide quantification, confocal Raman imaging, and immunolabeling combined with atomic force microscopy revealed that the MIR775A-GALT9 circuit modulates pectin levels and the elastic modulus of the cell wall. We also showed that MIR775A is directly repressed by the transcription factor ELONGATED HYPOCOTYL5 (HY5). Genetic analysis confirmed that HY5 is a negative regulator of leaf size that acts through the HY5-MIR775A-GALT9 repression cascade to control pectin levels. These findings demonstrate that miR775-regulated cell wall remodeling is an integral determinant of intrinsic leaf size in A. thaliana. Studying other CW-miRNAs would provide more insights into cell wall biology.

Abscisic acid (ABA) plays key roles in plant development and responses to abiotic stresses. A wide number of transcriptional and posttranslational regulatory mechanisms of ABA signaling are known; however, less is known about the regulatory roles of alternative splicing. In this work, we found that SKIP, a splicing factor, positively regulates ABA signaling. SKIP binds to the pre-mRNA of ABA signaling-related genes, such as PYL7, PYL8, ABI1, HAB1 and ABI5, to regulate their splicing. The precursor mRNA alternative splicing of several PYL receptors, PP2C phosphatases and ABF transcriptional factors is disrupted by the skip-1 mutation. The abnormal alternative splicing in skip-1 represses the expression of ABA-positive regulators, including PYLs and ABFs, and activates the expression of ABA-negative regulators, such as PP2Cs, which confers ABA hyposensitive phenotype of skip-1. We also found that ABA-mediated genome-wide alternative splicing and differential gene expression are changed by the skip-1 mutation. The number of the differential splicing events is increased by skip-1; however, the number of differential expressed genes in response to ABA is reduced by skip-1. Our results reveal a principle on how a splicing factor regulates ABA signaling and ABA-mediated genome-wide alternative splicing.

MicroRNAs (miRNAs) are trans-acting small regulatory RNAs that work coordinately with transcription factors (TFs) to shape the repertoire of cellular mRNAs available for translation. Despite our growing knowledge of individual plant miRNAs, their global roles in gene regulatory networks remain mostly unassessed. Based on interactions obtained from public databases and curated from the literature, we reconstructed an integrated miRNA network in Arabidopsis that includes 66 core TFs, 318 miRNAs, and 1712 downstream genes. We found that miRNAs occupy distinct niches and enrich miRNA-containing feed-forward loops (FFLs), particularly those with miRNAs as intermediate nodes. Further analyses revealed that miRNA-containing FFLs coordinate TFs located in different hierarchical layers and that intertwined miRNA-containing FFLs are associated with party and date miRNA hubs. Using the date hub MIR858A as an example, we performed detailed molecular and genetic analyses of three interconnected miRNA-containing FFLs. These analyses revealed individual functions of the selected miRNA-containing FFLs and elucidated how the date hub miRNA fulfills multiple regulatory roles. Collectively, our findings highlight the prevalence and importance of miRNA-containing FFLs, and provide new insights into the design principles and control logics of miRNA regulatory networks governing gene expression programs in plants.