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.

Drought is one of the main abiotic stresses limiting the quality and yield of citrus. Cuticular waxes play an important role in regulating plant drought tolerance and water use efficiency (WUE). However, the contribution of cuticular waxes to drought tolerance, WUE and the underlying molecular mechanism is still largely unknown in citrus. ‘Longhuihong’ (MT) is a bud mutant of ‘Newhall’ navel orange with curly and bright leaves. In this study, significant increases in the amounts of total waxes and aliphatic wax compounds, including n-alkanes, n-primary alcohols and n-aldehydes, were overserved in MT leaves, which led to the decrease in cuticular permeability and finally resulted in the improvements in drought tolerance and WUE. Compared to WT leaves, MT leaves possessed much lower contents of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), significantly higher levels of proline and soluble sugar, and enhanced superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities under drought stress, which might reduce reactive oxygen species (ROS) damage, improve osmotic regulation and cell membrane stability, and finally, enhance MT tolerance to drought stress. Transcriptome sequencing results showed that seven structural genes were involved in wax biosynthesis and export, MAPK cascade, and ROS scavenging, and seven genes encoding transcription factors might play an important role in promoting cuticular wax accumulation, improving drought tolerance and WUE in MT plants. Our results not only confirmed the important role of cuticular waxes in regulating citrus drought resistance and WUE but also provided various candidate genes for improving citrus drought tolerance and WUE.

The U-box E3 ubiquitin ligase (PUB) gene family plays an important role in regulating plant responses to abiotic stress. Poncirus trifoliata (trifoliate orange), a citrus rootstock with notable cold, drought, and salt tolerance, serves as an excellent model for studying stress-responsive genes. In this study, a total of 47 PUB genes (PtrPUBs) were identified in the trifoliate orange genome. Chromosomal distribution analysis indicated that PtrPUB genes were unevenly distributed across nine trifoliate orange chromosomes. Phylogenetic tree analysis indicated that 170 PUB proteins from trifoliate orange, Arabidopsis thaliana, and tomato were clustered into five subfamilies. Gene structure, conserved domain, and motif analyses revealed diverse exon–intron and motif organizations of PtrPUB genes, suggesting potential functional differentiation among PtrPUBs. Cis-acting analysis indicated that the promoters of PtrPUB genes harbor elements related to hormone signaling (ABA, MeJA), drought stress, and low-temperature responses. Transcriptomic data and qRT-PCR results suggested that PtrPUB genes are responsive to ABA and dehydration treatments. This study provides a foundation for understanding the functional roles of PUB genes in trifoliate orange and offers insights for improving stress tolerance in citrus breeding programs.