The MADS-box family gene PtrANR1 encodes a transcription activator promoting root growth and enhancing plant tolerance to drought stress.

KEY MESSAGE: PtrANR1 positively regulates plant drought tolerance by increasing proline level and reducing ROS accumulation. PtrANR1 directly activates PtrAUX1 expression to promote root growth and improve plant drought tolerance. Citrus quality and yield are severely declined under drought stress. To date, the effects of MADS-box family transcription factors (TFs) on plant drought resistance have made some progress. However, whether MADS-box family TFs are associated with citrus drought response has remained unclear. The current paper identified a MADS-box family gene PtrANR1 encoding anthocyanidin reductase from trifoliate orange. PtrANR1 exhibits high identities with ANR1 proteins found in various plants. PtrANR1 possesses two conserved domains known as MADS and kertanin-like domains. PtrANR1 is a nuclear protein which has transactivation activity. A significant induction of PtrANR1 transcript was detected in leaves and roots of trifoliate orange treated with PEG6000 and ABA. Under drought stress, Arabidopsis ectopic overexpressing PtrANR1 exhibited obviously elevated contents of proline, ABA and IAA, better developed root, enhanced antioxidant enzyme activities, as well as notably reduced accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS) compared with WT plants. However, opposite change trends of these physiological indices were detected in PtrANR1 homolog silencing lemon. Furthermore, transgenic Arabidopsis displayed significantly increased expression levels in genes associated with ABA, IAA and proline production, IAA polar transport, ROS elimination and drought response. However, these genes exhibited noticeably decreased transcript levels in PtrANR1 homolog silencing lemon. Moreover, PtrANR1 could increase IAA content and promote root growth by binding to GArG-box in the promoter of PtrAUX1 to activate its transcript. These findings indicated that PtrANR1 had a beneficial impact on plant drought resistance through promoting root development, increasing proline accumulation and scavenging of ROS.

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.

Ectopic Overexpression of CsECR From Navel Orange Increases Cuticular Wax Accumulation in Tomato and Enhances Its Tolerance to Drought Stress.

Drought stress often occurred in citrus to limit its growth, distribution, and fruit quality. Cuticular waxes play an important role in regulating plant tolerance to drought stress. Plant enoyl-CoA reductase (ECR) is involved in the biosynthesis of cuticular waxes and catalyzes the last step of very long-chain fatty acids (VLCFAs) elongation. In this study, a putative ECR gene, named CsECR, was cloned from "Newhall" navel orange. CsECR protein has high identities with other plant ECR proteins and contained a conserved NADP/NAD-binding motif and three conserved functional sites. The highest expression of CsECR was observed in leaves, followed by stems, flavedos, ovaries, juice sacs, stigmas, stamens, albedos, and petals. Besides, the expression of CsECR was significantly induced by PEG6000 and ABA treatments. Ectopic overexpression of CsECR increased the contents of total waxes and aliphatic wax fractions (n-fatty acids, unsaturated fatty acids, n-alkanes, alkenes, iso-, and anteiso-alkanes) in the leaves and fruits of the transgenic tomato. Furthermore, ectopic overexpression of CsECR reduced the cuticle permeability in the leaves and fruits of the transgenic tomato and increased its tolerance to drought stress. Taken together, our results revealed that CsECR plays an important role in plant response to drought stresses by regulating cuticular wax biosynthesis.

Transcriptome and Physiological Analyses of a Navel Orange Mutant with Improved Drought Tolerance and Water Use Efficiency Caused by Increases of Cuticular Wax Accumulation and ROS Scavenging Capacity.

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.