A Novel bHLH Transcription Factor PtrbHLH66 from Trifoliate Orange Positively Regulates Plant Drought Tolerance by Mediating Root Growth and ROS Scavenging

Drought limits citrus yield and fruit quality worldwide. The basic helix-loop-helix (bHLH) transcription factors (TFs) are involved in plant response to drought stress. However, few bHLH TFs related to drought response have been functionally characterized in citrus. In this study, a bHLH family gene, named PtrbHLH66, was cloned from trifoliate orange. PtrbHLH66 contained a highly conserved bHLH domain and was clustered closely with bHLH66 homologs from other plant species. PtrbHLH66 was localized to the nucleus and had transcriptional activation activity. The expression of PtrbHLH66 was significantly induced by polyethylene glycol 6000 (PEG6000) and abscisic acid (ABA) treatments. Ectopic expression of PtrbHLH66 promoted the seed germination and root growth, increased the proline and ABA contents and the activities of antioxidant enzymes, but reduced the accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS) under drought stress, resulting in enhanced drought tolerance of transgenic Arabidopsis. In contrast, silencing the PtrbHLH66 homolog in lemon plants showed the opposite effects. Furthermore, under drought stress, the transcript levels of 15 genes involved in ABA biosynthesis, proline biosynthesis, ROS scavenging and drought response were obviously upregulated in PtrbHLH66 ectopic-expressing Arabidopsis but downregulated in PtrbHLH66 homolog silencing lemon. Thus, our results suggested that PtrbHLH66 acted as a positive regulator of plant drought resistance by regulating root growth and ROS scavenging.