Rangjin Xie

Anthocyanin biosynthesis in fruit tree crops: Genes and their regulation

The anthocyanin biosynthesis pathway is a little complex with branches responsible for the synthesis of a variety of metabolites. In fruit tree crops, during the past decade, many structural genes encoding enzymes in the anthocyanin biosynthetic pathway and various regulatory genes encoding transcription factors that regulate the expression of structural genes have been cloned and then functionally characterized in detail. In general, the structural genes involved in anthocyanin synthesis were coordinately expressed and their levels of expression were positively related to the degree of anthocyanin concentration; while, the coordinated expression pattern is striking a diverse among fruit crop species. Regulatory genes regulate spatiotemporally the structural genes and then form complicated metabolic network. Anthocyanin biosynthesis can be affected by external and internal factors, such as light, UV-B, low temperature and ABA through changes in expression of structural and regulatory genes. Key words: Anthocyanin, regulatory genes, structural genes, fruit tree crops, factors.

Recent advances in molecular events of fruit abscission

It is widely accepted that fruit abscission is a highly regulated developmental process that is both influenced and activated in response to changing environment and plays crucial roles in the health and reproductive success of plants. Recent evidences showed that numerous genes related to metabolic and signalling pathways were coordinately implicated in regulating fruit abscission. Cross talks within hormones, between saccharides and hormones, as well as between polyamines and ethylene result in synergetic or antagonistic interactions which together play an important role in adjusting fruit abscission. Although hormones are the most studied internal factors related to abscission, the role of saccharides and polyamines during fruit abscission is emerging now. The characterizations of the molecular mechanisms of regulating fruit abscission are essential to develop effective strategies for controlling this process in plants.

Combined analysis of mRNA and miRNA identifies dehydration and salinity responsive key molecular players in citrus roots

Citrus is one of the most economically important fruit crops around world. Drought and salinity stresses adversely affected its productivity and fruit quality. However, the genetic regulatory networks and signaling pathways involved in drought and salinity remain to be elucidated. With RNA-seq and sRNA-seq, an integrative analysis of miRNA and mRNA expression profiling and their regulatory networks were conducted using citrus roots subjected to dehydration and salt treatment. Differentially expressed (DE) mRNA and miRNA profiles were obtained according to fold change analysis and the relationships between miRNAs and target mRNAs were found to be coherent and incoherent in the regulatory networks. GO enrichment analysis revealed that some crucial biological processes related to signal transduction (e.g. ‘MAPK cascade’), hormone-mediated signaling pathways (e.g. abscisic acid- activated signaling pathway’), reactive oxygen species (ROS) metabolic process (e.g. ‘hydrogen peroxide catabolic process’) and transcription factors (e.g., ‘MYB, ZFP and bZIP’) were involved in dehydration and/or salt treatment. The molecular players in response to dehydration and salt treatment were partially overlapping. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-seq and sRNA-seq analysis. This study provides new insights into the molecular mechanisms how citrus roots respond to dehydration and salt treatment.

The ARF, AUX/IAA and GH3 gene families in citrus: genome-wide identification and expression analysis during fruitlet drop from abscission zone A.

Completion of the whole genome sequencing of citrus enabled us to perform genome-wide identification and functional analysis of the gene families involved in agronomic traits and morphological diversity of citrus. In this study, 22 CitARF, 11 CitGH3 and 26 CitAUX/IAA genes were identified in citrus, respectively. Phylogenetic analysis revealed that all the genes of each gene family could be subdivided into three groups and showed strong evolutionary conservation. The GH3 and AUX/IAA gene families shrank and ARF gene family was highly conserved in the citrus genome after speciation from Arabidopsis thaliana. Tissue-specific expression profiles revealed that 54 genes were expressed in at least one tissue while just 5 genes including CitARF07, CitARF20, CitGH3.04, CitAUX/IAA25 and CitAUX/IAA26 with very low expression level in all tissues tested, suggesting that the CitARF, CitGH3 and CitAUX/IAA gene families played important roles in the development of citrus organs. In addition, our data found that the expression of 2 CitARF, 4 CitGH3 and 4 AUX/IAA genes was affected by IAA treatment, and 7 genes including, CitGH3.04, CitGH3.07, CitAUX/IAA03, CitAUX/IAA04, CitAUX/IAA18, CitAUX/IAA19 and CitAUX/IAA23 were related to fruitlet abscission. This study provides a foundation for future studies on elucidating the precise role of citrus ARF, GH3 and AUX/IAA genes in early steps of auxin signal transduction and open up a new opportunity to uncover the molecular mechanism underlying citrus fruitlet abscission.

Comprehensive analysis of SAUR gene family in citrus and its transcriptional correlation with fruitlet drop from abscission zone A.

Small auxin-up RNA (SAUR) gene family is large, and the members of which can be rapidly induced by auxin and encode highly unstable mRNAs. SAUR genes are involved in various developmental and physiological processes, such as leaf senescence, fruitlet abscission, and hypocotyl development. However, their modes of action in citrus remain unknown. Hereby, a systematic analysis of SAUR gene family in citrus was conducted through a genome-wide search. In this study, a total of 70 SAUR genes, referred to as CitSAURs, have been identified in citrus. The evolutionary relationship and the intro-exon organization were analyzed, revealing strong gene conservation and the expansion of particular functional genes during plant evolution. Expression analysis showed that the major of CitSAUR genes were expressed in at least one tissue and showed distinctive expression levels, indicating the SAUR gene family play important roles in the development and growth of citrus organs. However, there were more than 20 CitSAUR genes such as CitSARU36, CitSAUR37, and CitSAUR54 exhibiting very low expression level in all tissue tested. Twenty-three out of 70 CitSAUR genes were responded to indole-3-acetic acid (IAA) treatment, of which just CitSAUR19 was down-regulated. Additionally, 14 CitSAUR genes exhibited distinct changes during fruitlet abscission, however just 5 of them including CitSAUR06, CitSAUR08, CitSAUR44, CitSAUR61, and CitSAUR64 were associated with fruitlet abscission. The current study provides basic information for the citrus SAUR gene family and will pave the way for deciphering the precise role of SAURs in citrus development and growth as well as fruitlet abscission.

Genome-Wide Analysis of Citrus R2R3MYB Genes and Their Spatiotemporal Expression under Stresses and Hormone Treatments

The R2R3MYB proteins represent one of the largest families of transcription factors, which play important roles in plant growth and development. Although genome-wide analysis of this family has been conducted in many species, little is known about R2R3MYB genes in citrus, In this study, 101 R2R3MYB genes has been identified in the citrus (Citrus sinesis and Citrus clementina) genomes, which are almost equal to the number of rice. Phylogenetic analysis revealed that they could be subdivided into 21 subgroups. The evolutionary relationships and the intro-exon organizations were also analyzed, revealing strong gene conservation but also the expansions of particular functional genes during the plant evolution. Tissue-specific expression profiles showed that 95 citrus R2R3MYB genes were expressed in at least one tissue and the other 6 genes showed very low expression in all tissues tested, suggesting that citrus R2R3MYB genes play important roles in the development of all citrus organs. The transcript abundance level analysis during abiotic conditions (NaCl, abscisic acid, jasmonic acid, drought and low temperature) identified a group of R2R3MYB genes that responded to one or multiple treatments, which showed a promising for improving citrus adaptation to stresses. Our results provided an essential foundation for the future selection of the citrus R2R3MYB genes for cloning and functional dissection with an aim of uncovering their roles in citrus growth and development.

Ca(NO 3 ) 2 canopy spraying during physiological fruit drop period has a better influence on the tree character and fruit quality of Newhall navel orange ( Citrus sinensis Osbeck)

Abstract This study aimed to provide a theoretical basis for adopting suitable cultivation measures to tackle calcium (Ca) deficiency in citrus leaves. The Newhall navel orange (Citrus sinensis Osbeck) canopy was sprayed with 20.0 mmol L−1 of Ca(NO3)2 during physiological fruit drop period, fruit expanding period, and fruit maturing period on 30, 90, and 210 days after full bloom (DAFB), respectively, and its effects on leaf gas exchange parameters and leaf mineral nutrition and fruit quality were analyzed. The results showed that: (1) The photosynthetic rate (ACO2) at 9:00 a.m. and 16:00 p.m. of fruit expanding period with 30 and 90 DAFB Ca(NO3)2 treatments slightly or significantly improved mainly by decreasing stomatal limitation and nonstomatal limitation, respectively. (2) Compared with control (CK), the Ca concentration in leaves with 30, 90, and 240 DAFB Ca(NO3)2 treatments increased by 127.16, 97.53, and 33.33%, respectively, and the leaf magnesium concentration also increased by more than 32.26%. However, Ca(NO3)2 canopy spraying on 30 DAFB significantly reduced the leaf potassium concentration, by 22.14% compared with CK. (3) Ca(NO3)2 canopy spraying on 30 DAFB decreased the second fruit drop rate by 30.55% and increased the weight per fruit by 25.04%, thus resulting in a significant increase in citrus yield. (4) Spraying Ca(NO3) on 30 DAFB mainly affected the metabolism of titratable acid (TA) to improve the maturity of citrus fruits. Whilst it improved the external quality and the coloring of citrus fruit significantly. Therefore, Ca(NO3)2 canopy spraying during physiological fruit drop period has a better influence on the tree character and fruit quality of Newhall navel orange (Citrus sinensis Osbeck).

Role of Ca2+ and calmodulin in on-tree oleocellosis tolerance of Newhall navel orange

Previous studies have shown that spraying calcium on the canopy can significantly reduce the incidence of on-tree oleocellosis (OTO). However, the OTO regulation with calcium spraying has not been reported clearly. Therefore, the physiologic mechanism of spraying Ca(NO3)2 and calmodulin (CaM) inhibitors (trifluoperazine-TFP) on the canopy of 8-year-old Newhall navel orange on Lichi16-6 trifoliata (P. trifoliate) including the ratio (RO) and degree of OTO (DO) have been studied under high summer temperature. The results showed that exogenous Ca(NO3)2 treatment significantly decreased DO value by increase in CaM content of leaves, peroxidase (POD) activity in leaves, and fruit peels. However, TFP treatment significantly decreased CaM content in leaves, SOD and CAT activity in fruit peels, while the POD activity in fruit peels significantly increased, and the formation of Ca(NO3)2-induced DO tolerance in citrus fruits was weakened by TFP treatment. Exogenous Ca(NO3)2 treatment increased stomatal conductance (Gs) and transpiration rate (Tr), and decreased the daily range of rind oil release pressure (△RORP) significantly. However, TFP treatment had no significant influence on transpiration rate (Tr) and △RORP. The results were consistent with the RO of different treatments. These results confirm that Ca2+ and CaM regulate DO value, and the RO of OTO was mainly related to the regulation of △RORP through water metabolism.

The molecular events of IAA inhibiting citrus fruitlet abscission revealed by digital gene expression profiling

Citrus fruits possess two abscission zones (AZ), AZ A and AZ C located at the pedicel and calyx, respectively. Early citrus fruitlet abscission (CFA) exclusively occurs at AZ A. Previous data have shown that indole-3-acetic acid (IAA) could inhibit fruitlet abscission. However, its role in CFA remains vague. In this study, we first removed the ovaries of fruitlets in order to exclude their interferences. Then, the calyxes were treated with IAA, gibberellin 3 (GA3) and 6-benzylaminopurine (6-BA), respectively. The results have shown that IAA could prevent CFA from taking place, while either GA3 or 6-BA could not. When IAA concentration decreased to a value between 30 mg/L and 40 mg/L, CFA occurred, showing a concentration-dependent manner. Digital gene expression analysis revealed that 2317 corresponded to IAA treatment, of which 1226 genes were closely related to CFA. The most affected genes included those related to biosynthesis, transport and signaling of phytohormones, primarily ethylene (ET), abscisic acid (ABA) and auxin as well as protein ubiquitination, ROS response, calcium signal transduction, cell wall and transcription factors (TFs). The results obtained in this study suggested that the IAA in AZ A could suppress ethylene biosynthesis and signaling, and then inhibit abscission signaling. To our knowledge, it is the first time to reveal the key role of IAA in CFA, which will contribute to a better understanding for the mechanism underlying CFA.

Genome-wide identification of citrus CAMTA genes and their expression analysis under stress and hormone treatments

ABSTRACT In plants, CAMTA (Calmodulin-binding transcription activator) proteins have been widely analysed, due to their vital roles in developmental regulation, environmental stress response, and hormone crosstalk. However, in citrus, this gene family, to date, has not been systematically characterised. In this study, 9 CitCAMTA genes have been identified from the citrus (Citrus sinensis and Citrus clementina) genomes. Their phylogenetic relationship, protein domain, exon–intron structure, and so forth were investigated in detail, revealing the divergence in their functions. Tissue-specific expression analysis showed that these CitCAMTA genes – with the exception of CitCAMTA4 – could be expressed in at least 1 tissue, indicating their roles in the development of root, stem, leaf, cotyledon, seed, and fruit peel. Under stress and hormone treatments, the expression patterns of each CitCAMTA remarkably varied, which showed a promising for improving citrus adaptation to stresses. Unexpectedly, the expression level of CitCAMTA4 was undetectable under all treatments. Taken together, our work lays a foundation for further analysis on citrus CAMTA genes, and provides a basic understanding of their roles in stress responses.