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.

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.

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.

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.

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.

Transcriptional profiling of canker-resistant transgenic sweet orange (Citrus sinensis Osbeck) constitutively overexpressing a spermidine synthase gene.

Citrus canker disease caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases affecting the citrus industry worldwide. In our previous study, the canker-resistant transgenic sweet orange (Citrus sinensis Osbeck) plants were produced via constitutively overexpressing a spermidine synthase. To unravel the molecular mechanisms underlying Xcc resistance of the transgenic plants, in the present study global transcriptional profiling was compared between untransformed line (WT) and the transgenic line (TG9) by hybridizing with Affymetrix Citrus GeneChip. In total, 666 differentially expressed genes (DEGs) were identified, 448 upregulated, and 218 downregulated. The DEGs were classified into 33 categories after Gene ontology (GO) annotation, in which 68 genes are in response to stimulus and involved in immune system process, 12 genes are related to cell wall, and 13 genes belong to transcription factors. These genes and those related to starch and sucrose metabolism, glutathione metabolism, biosynthesis of phenylpropanoids, and plant hormones were hypothesized to play major roles in the canker resistance of TG9. Semiquantitative RT-PCR analysis showed that the transcript levels of several candidate genes in TG9 were significantly higher than in WT both before and after Xcc inoculation, indicating their potential association with canker disease.

Different transcriptional response to Xanthomonas citri subsp. citri between kumquat and sweet orange with contrasting canker tolerance.

Citrus canker disease caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating biotic stresses affecting the citrus industry. Meiwa kumquat (Fortunella crassifolia) is canker-resistant, while Newhall navel orange (Citrus sinensis Osbeck) is canker-sensitive. To understand the molecular mechanisms underlying the differences in responses to Xcc, transcriptomic profiles of these two genotypes following Xcc attack were compared by using the Affymetrix citrus genome GeneChip. A total of 794 and 1324 differentially expressed genes (DEGs) were identified as canker-responsive genes in Meiwa and Newhall, respectively. Of these, 230 genes were expressed in common between both genotypes, while 564 and 1094 genes were only significantly expressed in either Meiwa or Newhall. Gene ontology (GO) annotation and Singular Enrichment Analysis (SEA) of the DEGs showed that genes related to the cell wall and polysaccharide metabolism were induced for basic defense in both Meiwa and Newhall, such as chitinase, glucanase and thaumatin-like protein. Moreover, apart from inducing basic defense, Meiwa showed specially upregulated expression of several genes involved in the response to biotic stimulus, defense response, and cation binding as comparing with Newhall. And in Newhall, abundant photosynthesis-related genes were significantly down-regulated, which may be in order to ensure the basic defense. This study revealed different molecular responses to canker disease in Meiwa and Newhall, affording insight into the response to canker and providing valuable information for the identification of potential genes for engineering canker tolerance in the future.

Cloning and molecular characterization of a mitogen-activated protein kinase gene from Poncirus trifoliata whose ectopic expression confers dehydration/drought tolerance in transgenic tobacco.

The mitogen-activated protein kinase (MAPK) cascade plays pivotal roles in diverse signalling pathways related to plant development and stress responses. In this study, the cloning and functional characterization of a group-I MAPK gene, PtrMAPK, in Poncirus trifoliata (L.) Raf are reported. PtrMAPK contains 11 highly conserved kinase domains and a phosphorylation motif (TEY), and is localized in the nucleus of transformed onion epidermal cells. The PtrMAPK transcript level was increased by dehydration and cold, but was unaffected by salt. Transgenic overexpression of PtrMAPK in tobacco confers dehydration and drought tolerance. The transgenic plants exhibited better water status, less reactive oxygen species (ROS) generation, and higher levels of antioxidant enzyme activity and metabolites than the wild type. Interestingly, the stress tolerance capacity of the transgenic plants was compromised by inhibitors of antioxidant enzymes. In addition, overexpression of PtrMAPK enhanced the expression of ROS-related and stress-responsive genes under normal or drought conditions. Taken together, these data demonstrate that PtrMAPK acts as a positive regulator in dehydration/drought stress responses by either regulating ROS homeostasis through activation of the cellular antioxidant systems or modulating transcriptional levels of a variety of stress-associated genes.

Ectopic expression of MdSPDS1 in sweet orange (Citrus sinensis Osbeck) reduces canker susceptibility: involvement of H2O2 production and transcriptional alteration.

BACKGROUND: Enormous work has shown that polyamines are involved in a variety of physiological processes, but information is scarce on the potential of modifying disease response through genetic transformation of a polyamine biosynthetic gene. RESULTS: In the present work, an apple spermidine synthase gene (MdSPDS1) was introduced into sweet orange (Citrus sinensis Osbeck 'Anliucheng') via Agrobacterium-mediated transformation of embryogenic calluses. Two transgenic lines (TG4 and TG9) varied in the transgene expression and cellular endogenous polyamine contents. Pinprick inoculation demonstrated that the transgenic lines were less susceptible to Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker, than the wild type plants (WT). In addition, our data showed that upon Xac attack TG9 had significantly higher free spermine (Spm) and polyamine oxidase (PAO) activity when compared with the WT, concurrent with an apparent hypersensitive response and the accumulation of more H2O2. Pretreatment of TG9 leaves with guazatine acetate, an inhibitor of PAO, repressed PAO activity and reduced H2O2 accumulation, leading to more conspicuous disease symptoms than the controls when both were challenged with Xac. Moreover, mRNA levels of most of the defense-related genes involved in synthesis of pathogenesis-related protein and jasmonic acid were upregulated in TG9 than in the WT regardless of Xac infection. CONCLUSION: Our results demonstrated that overexpression of the MdSPDS1 gene prominently lowered the sensitivity of the transgenic plants to canker. This may be, at least partially, correlated with the generation of more H2O2 due to increased production of polyamines and enhanced PAO-mediated catabolism, triggering hypersensitive response or activation of defense-related genes.

An arginine decarboxylase gene PtADC from Poncirus trifoliata confers abiotic stress tolerance and promotes primary root growth in Arabidopsis.

Arginine decarboxylase (ADC) is an important enzyme responsible for polyamine synthesis under stress conditions. In this study, the gene encoding ADC in Poncirus trifoliata (PtADC) was isolated and it existed as a single-copy member. Transcript levels of PtADC were up-regulated by low temperature and dehydration. Overexpression of PtADC in an Arabidopsis thaliana ADC mutant adc1-1 promoted putrescine synthesis in the transgenic line and the stomatal density was reverted to that in the wild type. The transgenic line showed enhanced resistance to high osmoticum, dehydration, long-term drought, and cold stress compared with the wild type and the mutant. The accumulation of reactive oxygen species (ROS) in the transgenic line was appreciably decreased under the stresses, but ROS scavenging capacity was compromised when the transgenic plants were treated with the ADC inhibitor D-arginine prior to stress treatment. In addition, the transgenic line had longer roots than the wild type and the mutant under both normal and stressful conditions, consistent with larger cell number and length of the root meristematic zone. Taken together, these results demonstrated that PtADC is involved in tolerance to multiple stresses, and its function may be due, at least partly, to efficient ROS elimination and to its influence on root growth conducive to drought tolerance.

Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response.

Polyamines, small aliphatic polycations, have been suggested to play key roles in a number of biological processes. In this paper, attempts were made to investigate the possibility of improving dehydration tolerance of citrus in vitro plants by exogenous application of spermine (Spm). 'Red Tangerine' (Citrus reticulata Blanco) in vitro plants pretreated with 1 mM Spm exhibited less wilted phenotype and lower water loss and electrolyte leakage than the control under dehydration. Spm-pretreated plants contained higher endogenous polyamine content during the course of the experiment relative to the control, particularly at the end of dehydration, coupled with higher expression levels of ADC and SPMS. Histochemical staining showed that the Spm-pretreated leaves were stained to a lower extent than those without Spm pretreatment, implying generation of less reactive oxygen species (ROS). On the contrary, activities of peroxidase (POD) and superoxide dismutase (SOD) in the Spm-pretreated samples were higher than the control at a given time point or during the whole experiment, suggesting that Spm exerted a positive effect on antioxidant systems. In addition, significantly smaller stomatal aperture size was observed in Spm-pretreated epidermal peels, which showed that stomatal closure was promoted by polyamines. All of these data suggest that Spm pretreatment causes accumulation of higher endogenous polyamines and accordingly leads to more effective ROS scavenging (less tissue damage) and stimulated stomatal closure (lower water loss) upon dehydration, which may function collectively to enhance dehydration tolerance.