Systematic analysis of the basic/helix-loop-helix (bHLH) transcription factor family in pummelo (Citrus grandis) and identification of the key members involved in the response to iron deficiency.

BACKGROUND: Iron (Fe) deficiency is a common problem in citrus production. As the second largest superfamily of transcription factors (TFs), the basic/helix-loop-helix (bHLH) proteins have been shown to participate in the regulation of Fe homeostasis and a series of other biological and developmental processes in plants. However, this family of members in citrus and their functions in citrus Fe deficiency are still largely unknown. RESULTS: In this study, we identified a total of 128 CgbHLHs from pummelo (Citrus grandis) genome that were classified into 18 subfamilies by phylogenetic comparison with Arabidopsis thaliana bHLH proteins. All of these CgbHLHs were randomly distributed on nine known (125 genes) and one unknown (3 genes) chromosomes, and 12 and 47 of them were identified to be tandem and segmental duplicated genes, respectively. Sequence analysis showed detailed characteristics of their intron-exon structures, bHLH domain and conserved motifs. Gene ontology (GO) analysis suggested that most of CgbHLHs were annotated to the nucleus, DNA-binding transcription factor activity, response to abiotic stimulus, reproduction, post-embryonic development, flower development and photosynthesis. In addition, 27 CgbHLH proteins were predicted to have direct or indirect protein-protein interactions. Based on GO annotation, RNA sequencing data in public database and qRT-PCR results, several of CgbHLHs were identified as the key candidates that respond to iron deficiency. CONCLUSIONS: In total, 128 CgbHLH proteins were identified from pummelo, and their detailed sequence and structure characteristics and putative functions were analyzed. This study provides comprehensive information for further functional elucidation of CgbHLH genes in citrus.

Comprehensive Analysis of Autophagy-Related Genes in Sweet Orange (Citrus sinensis) Highlights Their Roles in Response to Abiotic Stresses.

Autophagy is a highly conserved intracellular degradation pathway that breaks down damaged macromolecules and/or organelles. It is involved in plant development and senescence, as well as in biotic and abiotic stresses. However, the autophagy process and related genes are largely unknown in citrus. In this study, we identified 35 autophagy-related genes (CsATGs-autophagy-related genes (ATGs) of Citrus sinensis, Cs) in a genome-wide manner from sweet orange (Citrus sinensis). Bioinformatic analysis showed that these CsATGs were highly similar to Arabidopsis ATGs in both sequence and phylogeny. All the CsATGs were randomly distributed on nine known (28 genes) and one unknown (7 genes) chromosomes. Ten CsATGs were predicted to be segmental duplications. Expression patterns suggested that most of the CsATG were significantly up- or down-regulated in response to drought; cold; heat; salt; mannitol; and excess manganese, copper, and cadmium stresses. In addition, two ATG18 members, CsATG18a and CsATG18b, were cloned from sweet orange and ectopically expressed in Arabidopsis. The CsATG18a and CsATG18b transgenic plants showed enhanced tolerance to osmotic stress, salt, as well as drought (CsATG18a) or cold (CsATG18b), compared to wild-type plants. These results highlight the essential roles of CsATG genes in abiotic stresses.

Whole-transcriptome RNA sequencing reveals the global molecular responses and ceRNA regulatory network of mRNAs, lncRNAs, miRNAs and circRNAs in response to copper toxicity in Ziyang Xiangcheng (Citrus junos Sieb. Ex Tanaka).

BACKGROUND: Copper (Cu) toxicity has become a potential threat for citrus production, but little is known about related mechanisms. This study aims to uncover the global landscape of mRNAs, long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) in response to Cu toxicity so as to construct a regulatory network of competing endogenous RNAs (ceRNAs) and to provide valuable knowledge pertinent to Cu response in citrus. RESULTS: Tolerance of four commonly used rootstocks to Cu toxicity was evaluated, and 'Ziyang Xiangcheng' (Citrus junos) was found to be the most tolerant genotype. Then the roots and leaves sampled from 'Ziyang Xiangcheng' with or without Cu treatment were used for whole-transcriptome sequencing. In total, 5734 and 222 mRNAs, 164 and 5 lncRNAs, 45 and 17 circRNAs, and 147 and 130 miRNAs were identified to be differentially expressed (DE) in Cu-treated roots and leaves, respectively, in comparison with the control. Gene ontology enrichment analysis showed that most of the DEmRNAs and targets of DElncRNAs and DEmiRNAs were annotated to the categories of 'oxidation-reduction', 'phosphorylation', 'membrane', and 'ion binding'. The ceRNA network was then constructed with the predicted pairs of DEmRNAs-DEmiRNAs and DElncRNAs-DEmiRNAs, which further revealed regulatory roles of these DERNAs in Cu toxicity. CONCLUSIONS: A large number of mRNAs, lncRNAs, circRNAs, and miRNAs in 'Ziyang Xiangcheng' were altered in response to Cu toxicity, which may play crucial roles in mitigation of Cu toxicity through the ceRNA regulatory network in this Cu-tolerant rootstock.

Exogenous spermine pretreatment confers tolerance to combined high-temperature and drought stress in vitro in trifoliate orange seedlings via modulation of antioxidative capacity and expression of stress-related genes.

Spermine (Spm) is thought to play an important role in drought or high-temperature (HT) tolerance. However, it is not clear whether Spm confers similar resistance in the presence of both drought and HT, which often occur simultaneously. In the present study, the trifoliate orange (Poncirus trifoliata (L.) Raf.) seedlings were pretreated with 1 mmol L-1 Spm to evaluate their tolerance to combined drought and HT (45 ºC) stress. Spm-pretreated seedlings showed less leaf wilting, less water loss and less electrolyte leakage than control leaves not treated with Spm within 180 min of treatment. Histochemical staining with diaminobenzidine and nitro blue tetrazolium showed that Spm-pretreated seedlings accumulated less hydrogen peroxide and superoxide than those of control plants 60, 120 and 180 min after treatment when exposed to both drought and HT (45 ºC). However, superoxide dismutase, peroxidase and catalase were significantly more active in Spm-pretreated seedlings than in control seedlings. In addition, Spm-pretreated seedlings showed significantly higher expression of heat shock proteins, abscisic acid (ABA)-responsive element binding factor and 9-cis-epoxycarotenoid dioxygenase 3 than controls either before (0 min) or after (60, 120 and 180 min) combined drought and HT treatment. All of these data suggest that exogenous Spm pretreatment confers tolerance to simultaneously occurring drought and HT stresses. Spm may influence this by activating antioxidant enzymes, increasing the effectiveness of scavenging of reactive oxygen species. It may also increase the expression levels of stress-related genes that protect trifoliate orange seedlings from stress damage.

Genome-wide identification of sweet orange (Citrus sinensis) metal tolerance proteins and analysis of their expression patterns under zinc, manganese, copper, and cadmium toxicity.

Plant metal tolerance proteins (MTPs) play important roles in heavy metal homeostasis; however, related information in citrus plants is limited. Citrus genome sequencing and assembly have enabled us to perform a systematic analysis of the MTP gene family. We identified 12 MTP genes in sweet orange, which we have named as CitMTP1 and CitMTP3 to CitMTP12 based on their sequence similarity to Arabidopsis thaliana MTPs. The CitMTPs were predicted to encode proteins of 864 to 2556 amino acids in length that included 4 to 6 putative transmembrane domains (TMDs). Furthermore, all the CitMTPs contained a highly conserved signature sequence encompassing the TMD-II and the start of the TMD-III. Phylogenetic analysis further classified the CitMTPs into Fe/Zn-MTP, Mn-MTP, and Zn-MTP subgroups, which coincided with the MTPs of A. thaliana and rice. The closely clustered CitMTPs shared a similar gene structure. Expression analysis indicated that most CitMTP transcripts were upregulated to various extents under heavy metal stress. Among these, CitMTP5 in the roots and CitMTP11 in the leaves during Zn stress, CitMTP8 in the roots and CitMTP8.1 in the leaves during Mn stress, CitMTP12 in the roots and CitMTP1 in the leaves during Cu stress, and CitMTP11 in the roots and CitMTP1 in the leaves during Cd stress showed the highest extent of upregulation. These findings are suggestive of their individual roles in heavy metal detoxification.

Genome-Wide Identification, Cloning and Functional Analysis of the Zinc/Iron-Regulated Transporter-Like Protein (ZIP) Gene Family in Trifoliate Orange (Poncirus trifoliata L. Raf.).

Zinc (Zn) and iron (Fe) deficiency are widespread among citrus plants, but the molecular mechanisms regarding uptake and transport of these two essential metal ions in citrus are still unclear. In the present study, 12 members of the Zn/Fe-regulated transporter (ZRT/IRT)-related protein (ZIP) gene family were identified and isolated from a widely used citrus rootstock, trifoliate orange (Poncirus trifoliata L. Raf.), and the genes were correspondingly named as PtZIPs according to the sequence and functional similarity to Arabidopsis thaliana ZIPs. The 12 PtZIP genes were predicted to encode proteins of 334-419 amino acids, harboring 6-9 putative transmembrane (TM) domains. All of the PtZIP proteins contained the highly conserved ZIP signature sequences in TM-IV, and nine of them showed a variable region rich in histidine residues between TM-III and TM-IV. Phylogenetic analysis subdivided the PtZIPs into four groups, similar as found for the ZIP family of A. thaliana, with clustered PtZIPs sharing a similar gene structure. Expression analysis showed that the PtZIP genes were very differently induced in roots and leaves under conditions of Zn, Fe and Mn deficiency. Yeast complementation tests indicated that PtIRT1, PtZIP1, PtZIP2, PtZIP3, and PtZIP12 were able to complement the zrt1zrt2 mutant, which was deficient in Zn uptake; PtIRT1 and PtZIP7 were able to complement the fet3fet4 mutant, which was deficient in Fe uptake, and PtIRT1 was able to complement the smf1 mutant, which was deficient in Mn uptake, suggesting their respective functions in Zn, Fe, and Mn transport. The present study broadens our understanding of metal ion uptake and transport and functional divergence of the various PtZIP genes in citrus plants.

[Citrus boron nutrient level and its impact factors in the Three Gorges Reservoir region of Chongqing, China].

To investigate the level of boron nutrient in citrus and its impact factors, a total of 954 citrus leaf samples and 302 soil samples were collected from representative orchards in the 12 main citrus production counties in the Three Gorges Reservoir region of Chongqing to determine the boron content in citrus leaves, as well as the relationships between leaf boron content with soil available boron content, soil pH value, cultivar, rootstock and the age of tree. Results indicated that the leaf samples from 41.6% orchards (< 35 mg x kg(-1)) and the soil samples from 89.4% orchards (< 0.5 mg x kg(-1)) were boron insufficient. The correlation of leaf boron content and soil available boron content was not significant. The soil pH, cultivar, rootstock and the age of tree did affect the leaf boron content. The leaves from the orchards with soil pH of 4.5-6.4 demonstrated significantly higher boron contents than with the soil pH of 6.5-8.5. The leaf boron contents in the different cultivars was ranged as Satsuma mandarin > pomelo > valencia orange > sweet orange > tangor > navel orange. The citrus on trifoliate orange and sour pomelo rootstocks had significantly higher leaf boron contents than on Carrizo citrange and red tangerine rootstocks. Compared with the adult citrus trees (above 8 year-old), 6.6% more of leaf samples of younger trees (3 to 8 year-old) contained boron contents in the optimum range (35-100 mg x kg(-1)).