Fig. 5

SPL9 directly promotes CBF2 expression in Arabidopsis. A RT-qPCR analysis of miR172B and CBF2 expression, respectively, after SPL9 activation in the presence of DEX. Three biological replicates were performed. Asterisks indicate significant difference from mock using Student’s t-test (P < 0.01). B ChIP-qPCR analysis of SPL9 binding sites in the promoter of CBF2. Chromatins from 9-day-old pSPL9:3 × FLAG-rSPL9 and rSPL9 (as negative control) seedlings grown in long day condition were immunoprecipitated with a polyclonal antibody to FLAG. Values are the means of three biological replicates, each having three technical replicates. Site1 and Site2 denote different PCR-amplified regions in the promoter region of CBF2. ACT2 was a negative control. Asterisks indicate significant difference from the value of ACT2 using Student’s t-test (P < 0.01). C Activation of CBF2 transcription by direct binding of SPL9 to the cis-regulatory sequence in the promoter of CBF2. GUS expression was quantitated using RT-qPCR in leaves of N. benthamiana infiltrated with Agrobacterium with different combinations of constructs, including Vec + pCBF2:GUS, Vec + pΔCBF2:GUS, rSPL9 + pCBF2:GUS, and rSPL9 + pΔCBF2:GUS. pSY06 (Vec): the control vector. Values were normalized to that of Vec + pCBF2:GUS and are the mean of three biological replicates; Asterisks indicate significant difference from Vec + pCBF2:GUS using Student’s t-test (** P < 0.01)