The regulation of flowering time has crucial implications for plant fitness. MicroRNA156 (miR156) represses the floral transition in Arabidopsis thaliana, but the mechanisms regulating its transcription remain unclear. Here, we show that two AGAMOUS-like proteins, AGL15 and AGL18, act as positive regulators of the expression of MIR156. Small RNA northern blot analysis revealed a significant decrease in the levels of mature miR156 in agl15 agl18 double mutants, but not in the single mutants, suggesting that AGL15 and AGL18 co-regulate miR156 expression. Histochemical analysis further indicated that the double mutants showed a reduction in MIR156 promoter strength. The double mutants also showed reduced abundance of pri-miR156a and pri-miR156c, two of the primary transcripts from MIR156 genes. Electrophoretic mobility shift assays demonstrated that AGL15 directly associated with the CArG motifs in the MIR156a/c promoters. AGL18 did not show binding affinity to the CArG motifs, but pull-down and yeast two-hybrid assays showed that AGL18 forms a heterodimer with AGL15. GFP reporter assays and bimolecular fluorescence complementation (BiFC) showed that AGL15 and AGL18 co-localize in the nucleus and confirmed their in vivo interaction. Overexpression of miR156 did not affect the levels of AGL15 and AGL18 transcripts. Taking these data together, we present a model for the transcriptional regulation of MIR156. In this model, AGL15 and AGL18 may form a complex along with other proteins, and bind to the CArG motifs of the promoters of MIR156 to activate the MIR156 expression.

Parasitic plants are globally prevalent pathogens that withdraw nutrients from their host plants using an organ known as the haustorium. The external environment including nutrient availability affects the extent of parasitism and to understand this phenomenon, we investigated the role of nutrients and found that nitrogen is sufficient to repress haustoria formation in the root parasite Phtheirospermum japonicum. Nitrogen increases levels of abscisic acid (ABA) in P. japonicum and prevents the activation of hundreds of genes including cell cycle and xylem development genes. Blocking ABA signaling overcomes nitrogen’s inhibitory effects indicating that nitrogen represses haustoria formation by increasing ABA. The effect of nitrogen appears more widespread since nitrogen also inhibits haustoria in the obligate root parasite Striga hermonthica. Together, our data show that nitrogen acts as a haustoria repressing factor and suggests a mechanism whereby parasitic plants use nitrogen availability in the external environment to regulate the extent of parasitism.

Cellular regeneration in response to wounding is fundamental to maintain tissue integrity. Various internal factors including hormones and transcription factors mediate healing but little is known about the role of external factors. To understand how the environment affects regeneration, we investigated the effects of temperature upon the horticulturally relevant process of plant grafting. We found that elevated temperatures accelerated vascular regeneration in Arabidopsis thaliana and tomato grafts. Leaves were critical for this effect since blocking auxin transport or mutating PHYTOCHROME INTERACTING FACTOR4 (PIF4) or YUCCA2/5/8/9 in the cotyledons abolished the temperature enhancement. However, these perturbations did not affect grafting at ambient temperatures and temperature enhancement of callus formation and tissue adhesion did not require PIF4, suggesting leaf-derived auxin specifically enhanced vascular regeneration in response to elevated temperatures. We also found that elevated temperatures accelerated the formation of inter-plant vascular connections between the parasitic plant Phtheirospermum japonicum and host Arabidopsis, and this effect required shoot-derived auxin from the parasite. Taken together, our results identify a pathway whereby local temperature perception mediates long distance auxin signaling to modify regeneration, grafting and parasitism.

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Arabidopsis microRNA169 (miR169) is an ambient temperature-responsive microRNA that plays an important role in stress responses and the floral transition. However, the transcription factors that regulate the expression of MIR169 have remained unknown. In this study, we show that Elongated Hypocotyl 5-Homolog (HYH) directly binds to the promoter of MIR169a and negatively regulates its expression. Absolute quantification identified MIR169a as the major locus producing miR169. GUS reporter assays revealed that the deletion of a 498-bp fragment (–1,505 to –1,007, relative to the major transcriptional start site) of MIR169a abolished its ambient temperature-responsive expression. DNA-affinity chromatography followed by liquid chromatography-mass spectrometry analysis identified transcription factor HYH as a trans-acting factor that binds to the 498-bp promoter fragment of pri-miR169a. Electrophoretic mobility shift assays and chromatin immunoprecipitation–quantitative PCR demonstrated that the HYH.2 protein, a predominant isoform of HYH, directly associated with a G-box-like motif in the 498-bp fragment of pri-miR169a. Higher enrichment of HYH.2 protein on the promoter region of MIR169a was seen at 23°C, consistent with the presence of more HYH.2 protein in the cell at the temperature. Transcript levels of pri-miR169a increased in hyh mutants and decreased in transgenic plants overexpressing HYH. Consistent with the negative regulation of MIR169a by HYH, the diurnal levels of HYH mRNA and pri-miR169a showed opposite patterns. Taken together, our results suggest that HYH is a transcription factor that binds to a G-box-like motif in the MIR169a promoter and negatively regulates ambient temperature-responsive expression of MIR169a at higher temperatures in Arabidopsis.