A MADS-Box Gene CiMADS43 Is Involved in Citrus Flowering and Leaf Development through Interaction with CiAGL9.

MADS-box genes are involved in various developmental processes including vegetative development, flower architecture, flowering, pollen formation, seed and fruit development. However, the function of most MADS-box genes and their regulation mechanism are still unclear in woody plants compared with model plants. In this study, a MADS-box gene (CiMADS43) was identified in citrus. Phylogenetic and sequence analysis showed that CiMADS43 is a GOA-like Bsister MADS-box gene. It was localized in the nucleus and as a transcriptional activator. Overexpression of CiMADS43 promoted early flowering and leaves curling in transgenic Arabidopsis. Besides, overexpression or knockout of CiMADS43 also showed leaf curl phenotype in citrus similar to that of CiMADS43 overexpressed in Arabidopsis. Protein-protein interaction found that a SEPALLATA (SEP)-like protein (CiAGL9) interacted with CiMADS43 protein. Interestingly, CiAGL9 also can bind to the CiMADS43 promoter and promote its transcription. Expression analysis also showed that these two genes were closely related to seasonal flowering and the development of the leaf in citrus. Our findings revealed the multifunctional roles of CiMADS43 in the vegetative and reproductive development of citrus. These results will facilitate our understanding of the evolution and molecular mechanisms of MADS-box genes in citrus.

Functional analysis of a PISTILLATA-like gene CcMADS20 involved in floral organs specification in citrus.

PISTILLATA (PI), as a member of MADS-box transcription factor, plays an important role in petal and stamen specification in Arabidopsis. However, little is known about PI-like genes in citrus. To understand the molecular mechanism of PI during the developmental process of citrus flower, a PI-like gene CcMADS20 was isolated from Citrus Clemantina. Sequence alignment and phylogenetic analysis revealed that CcMADS20 had relatively high similarity with PI-like homolog and was classified in the core dicotyledonous group. The temporal and spatial expression analyses showed that CcMADS20 was specifically expressed in petal and stamen of citrus flower, which was consistent with PI expression pattern in Arabidopsis. Protein interaction revealed that CcMADS20 could form heterodimer with AP3-like proteins. Furthermore, ectopic overexpression of CcMADS20 in Arabidopsis resulted in transformation of sepals into petal-like structure, as observed in other plants overexpressing a functional PI-like homolog. Additionally, promoter fragments of CcMADS20 were also cloned in the representative 21 citrus varieties. Interestingly, four types of promoters were discovered in these citrus varieties, resulting from two stable insert/deletion fragments (Locus1 and Locus2). The homo/hetero-zygosity of promoter alleles in each variety was strongly related to the evolutionary origin of citrus. Four promoters activity analysis indicated that Locus1 presence inhibited CcMADS20 transcriptional activity and Locus2 presence promoted its transcriptional activity. These findings suggested that CcMADS20 determines petal and stamen development during the evolutionary process of citrus and four promoters discovered, as effective genetic markers, are valuable for citrus breeding practices.

Identification of Genes Associated with Lemon Floral Transition and Flower Development during Floral Inductive Water Deficits: A Hypothetical Model.

Water deficit is a key factor to induce flowering in many woody plants, but reports on the molecular mechanisms of floral induction and flowering by water deficit are scarce. Here, we analyzed the morphology, cytology, and different hormone levels of lemon buds during floral inductive water deficits. Higher levels of ABA were observed, and the initiation of floral bud differentiation was examined by paraffin sections analysis. A total of 1638 differentially expressed genes (DEGs) were identified by RNA sequencing. DEGs were related to flowering, hormone biosynthesis, or metabolism. The expression of some DEGs was associated with floral induction by real-time PCR analysis. However, some DEGs may not have anything to do with flowering induction/flower development; they may be involved in general stress/drought response. Four genes from the phosphatidylethanolamine-binding protein family were further investigated. Ectopic expression of these genes in Arabidopsis changed the flowering time of transgenic plants. Furthermore, the 5' flanking region of these genes was also isolated and sequence analysis revealed the presence of several putative cis-regulatory elements, including basic elements and hormone regulation elements. The spatial and temporal expression patterns of these promoters were investigated under water deficit treatment. Based on these findings, we propose a model for citrus flowering under water deficit conditions, which will enable us to further understand the molecular mechanism of water deficit-regulated flowering in citrus. HIGHLIGHT: Based on gene activity during floral inductive water deficits identified by RNA sequencing and genes associated with lemon floral transition, a model for citrus flowering under water deficit conditions is proposed.

Genome-wide identification, isolation and expression analysis of auxin response factor (ARF) gene family in sweet orange (Citrus sinensis).

Auxin response factors (ARFs) are an important family of proteins in auxin-mediated response, with key roles in various physiological and biochemical processes. To date, a genome-wide overview of the ARF gene family in citrus was not available. A systematic analysis of this gene family in citrus was begun by carrying out a genome-wide search for the homologs of ARFs. A total of 19 nonredundant ARF genes (CiARF) were found and validated from the sweet orange. A comprehensive overview of the CiARFs was undertaken, including the gene structures, phylogenetic analysis, chromosome locations, conserved motifs of proteins, and cis-elements in promoters of CiARF. Furthermore, expression profiling using real-time PCR revealed many CiARF genes, albeit with different patterns depending on types of tissues and/or developmental stages. Comprehensive expression analysis of these genes was also performed under two hormone treatments using real-time PCR. Indole-3-acetic acid (IAA) and N-1-napthylphthalamic acid (NPA) treatment experiments revealed differential up-regulation and down-regulation, respectively, of the 19 citrus ARF genes in the callus of sweet orange. Our comprehensive analysis of ARF genes further elucidates the roles of CiARF family members during citrus growth and development process.

Genome-wide classification and evolutionary and expression analyses of citrus MYB transcription factor families in sweet orange.

MYB family genes are widely distributed in plants and comprise one of the largest transcription factors involved in various developmental processes and defense responses of plants. To date, few MYB genes and little expression profiling have been reported for citrus. Here, we describe and classify 177 members of the sweet orange MYB gene (CsMYB) family in terms of their genomic gene structures and similarity to their putative Arabidopsis orthologs. According to these analyses, these CsMYBs were categorized into four groups (4R-MYB, 3R-MYB, 2R-MYB and 1R-MYB). Gene structure analysis revealed that 1R-MYB genes possess relatively more introns as compared with 2R-MYB genes. Investigation of their chromosomal localizations revealed that these CsMYBs are distributed across nine chromosomes. Sweet orange includes a relatively small number of MYB genes compared with the 198 members in Arabidopsis, presumably due to a paralog reduction related to repetitive sequence insertion into promoter and non-coding transcribed region of the genes. Comparative studies of CsMYBs and Arabidopsis showed that CsMYBs had fewer gene duplication events. Expression analysis revealed that the MYB gene family has a wide expression profile in sweet orange development and plays important roles in development and stress responses. In addition, 337 new putative microsatellites with flanking sequences sufficient for primer design were also identified from the 177 CsMYBs. These results provide a useful reference for the selection of candidate MYB genes for cloning and further functional analysis forcitrus.

Proteomics-based identification of DEAD-box protein 48 as a novel autoantigen, a prospective serum marker for pancreatic cancer.

Patients with cancer frequently develop autoantibodies, and the identification of panels of tumor autoantigens may have utility in early cancer diagnosis and immunotherapy. This study aims to exploit the autoantibody repertoire in pancreatic cancer and identify the possible serum marker for pancreatic cancer. Sera from 55 newly diagnosed patients with pancreatic cancer and 52 healthy controls were analyzed for antibody-based reactivity against Hep-2, a human larynx epithelioma cancer cell line, with one-dimensional immunoblot assay. From this analysis, we observed a prominent band with a molecular weight of 47 kDa in 63.64% (35/55) patients, while in only 1.9% normal group (1/52). Using immunoblot analysis after two-dimensional electrophoresis combined with liquid chromatography-electrospray ionization tandem mass spectrometry, this target antigen was identified as DEAD-box protein 48 (DDX48). BLAST analysis showed that it was highly similar to eukaryotic initiation factor 4A and might play a role in pre-mRNA processing. An enzyme-linked immunosorbent assay was performed using recombinant, purified DDX48 as an antigen to detect anti-DDX48 autoantibodies in sera. Reactivity was observed in 20 of 60 (33.33%) pancreatic cancer patients, 3 of 30 (10.00%) colorectal cancer patients, 2 of 30 (6.67%) gastric cancer patients, 2 of 30 (6.67%) hepatocellular cancer patients, while none of the 20 chronic pancreatitis patients, 30 lung cancer patients, and 60 normal individuals. Together, these results demonstrate that the detection of autoantibodies to DDX48 may have clinical utility for the improved diagnosis of pancreatic cancer.