Yanyan Ma

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.

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.

Genome-wide identification and analysis of CKX genes in Poncirus trifoliata

ABSTRACT Cytokinin oxidase/dehydrogenases (CKXs) in plants are coded by a small multigene family and play important roles in maintaining cytokinin homeostasis. In this study, four CKX genes (i.e. PsCKX1, PsCKX2, PsCKX5, and PsCKX7) were cloned from Poncirus trifoliata. All PsCKXs contained a highly conserved flavin adenine dinucleotide (FAD) binding domain and a cytokinin dehydrogenase 1, FAD/cytokinin binding domain. PsCKX1 and PsCKX2 shared 66.2% and 65.4% identity with AtCKX6 and AtCKX1, respectively, while PsCKX5 and PsCKX7 exhibited less than 45% identity with AtCKXs. The expression analysis under abiotic conditions (NaCl, ABA, 6-BA and drought) revealed that the four PsCKX genes could respond to at least one treatment, and the expression patterns were diverse in root and leaf. Overexpressing four PsCKX genes in tobacco led to diverse phenotypic variations in transgenic plant, including leaf shape, root architecture, and plant height. In addition, the data showed that PsCKX2 and PsCKX5 hold promise to obtain citrus dwarf rootstock with a stronger root system, since the overexpression of them resulted in dwarf plants with more lateral roots. Taken together, the work lays the basis for applications of PsCKX genes in future.