Project description:To date, little is known about molecular mechanisms by which woody plants engage symbiosis with arbuscular mycorrhizal (AM) fungi. Here we investigated transcriptome changes in the roots of Poncirus trifoliata (the most common citrus rootstock) that are induced during colonization of an AM fungus Glomus versiforme (Gv). A total of 282 Poncirus genes were differentially expressed in response to Gv colonization, of which 138 could identify homologous genes from the model legume Medicago truncatula that also exhibit similar AM-induced transcriptional changes, while the remaining 144 do not. A high proportion of the AM-responsive Poncirus genes are predicted to be involved in transcription regulation, transport process, cellular organization and protein degradation, implicating these processes in the establishment of AM symbiosis. Promoter-GUS analysis of six AM-induced Poncirus genes [encoding an exocyst subunit (PtrEXO70I), two transcription factors (PtNAC1 and PtPALM1), one chitinase (PtrChit2), one plastid movement associated protein (PtrPMI2) and one lipase (PtrLipase3)] showed that all of them exhibit specific expression in arbuscule-containing root cortical cells, suggesting their potential involvement in establishing AM symbiosis. Notably, down-regulation of the ortholog of PtrExo70I in Medicago by RNAi significantly impaired arbuscule development, indicating that Exo70I is an important host component required for arbuscule development in root cortical cells. This study not only helps identify conserved host genes engaged in AM symbiosis but should also guide future mechanistic studies of potentially Poncirus-specific events during its symbiosis with AM fungi.
Project description:Transcription profiling of citrus rootstock Poncirus trifoliata (L.) Raf. Keywords: Abiotic stress (Iron chlorosis) Total RNA from four replicates for each sample category (Poncirus trifoliata (L.) Raf watered for 60 days with 18 uM Fe-EDDHA or without Fe-EDDHA) were generated and compared.
Project description:Salt stress, especially saline-alkali stress, has seriously negative effect on citrus production. Ziyang xiangcheng (Citrus junos Sieb.) (Cj) has been reported as a saline-alkali stress and iron deficiency tolerant citrus rootstock. However, the molecular mechanism of its saline-alkali stress tolerance is still not clear. Two citrus rootstocks and one navel orange scion, Cj, Poncirus trifoliate (Poncirus trifoliata (L.) Raf.) (Pt) and ‘Lane Late’ navel orange (Citrus sinensis (L.) Osb.) (LL), were used in this study. The grafted materials Cj+LL and Pt+LL grown in calcareous soil were used to identify genes and pathways responsive to saline-alkali stress using RNA-seq. The seedlings of Cj and Pt grown in the solutions with different gradient pH value were used to perform a supplement experiment. Comprehensively analyzing the data of RNA-seq, physiology and biochemistry, agronomic traits and mineral elements of Cj+LL, Pt+LL, Cj and Pt, several candidate pathways and genes were identified to be highly regulated under saline-alkali stress. Here, we propose citrate is important for the tolerance to iron deficiency and the jasmonate (JA) biosynthesis and signal transduction pathway may play a crucial role in tolerance to saline-alkali stress in citrus by interacting with other plant hormones, calcium signaling, ROS scavenging system and lignin biosynthesis.
Project description:MicroRNAs (miRNAs) play a critical role in post-transcriptional gene regulation. miRNAs have been shown to control many genes involved in various biological and metabolic processes. Deep sequencing technologies have facilitated identification of species-specific or lowly expressed as well as conserved or highly expressed miRNAs in plants. In this research, we used Solexa sequencing to discover new microRNAs in trifoliate orange (Citrus trifoliata) an important rootstock of citrus. A total of 13,106,753 reads representing 4,876,395 distinct sequences were obtained from a short RNA library generated from small RNA extracted from C. trifoliata flower and fruit tissues, Based on sequence similarity and hairpin structure prediction, we found that 178,102 reads representing 89 sequences from 42 highly conserved miRNA families, have perfect matches to known miRNAs. We also identified 10 novel miRNA candidates, whose precursors were all potentially generated from citrus ESTs. And of them five miRNA* sequences were also sequenced. These sequences had not been described in other plant species and accumulation of these 10 novel miRNAs were confirmed by qRT-PCR analysis. Potential target genes were predicted for most conserved and novel miRNAs. Moreover, four target genes included one encoding IRX12 copper ion binding/ oxidoreductase and three genes encoding NB-LRR disease resistance protein have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in C. trifoliata.
Project description:Alkaline stress has serious negative effects on citrus production. Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka) (Cj) has been reported to be a rootstock that is tolerant to alkaline stress and iron deficiency. Poncirus trifoliata (Poncirus trifoliata (L.) Raf.) (Pt), the most widely used rootstock in China, is sensitive to alkaline stress. To investigate the molecular mechanism underlying the tolerance of Cj to alkaline stress, next-generation sequencing was employed to profile the root transcriptomes and small RNAs of Cj and Pt seedlings which were cultured in nutrient solution with three gradient pH. This two-regulation level data set provides a system-level view of molecular events with precise resolution. The data suggest that the auxin pathway may play a central role in inhibitory effect of alkaline stress on root growth, and the regulation of auxin homeostasis under alkaline stress was important for citrus adapting to alkaline stress. Moreover, the JA pathway shown an opposite response to alkaline stress in Cj and Pt may contributes to the differentials of root system architecture and iron deficiency tolerance between Cj and Pt. The data set provides a wealth of genomic resources and new clues for further studying the mechanisms underlying Cj that resist alkaline stress.
Project description:Alkaline stress has serious negative effects on citrus production. Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka) (Cj) has been reported to be a rootstock that is tolerant to alkaline stress and iron deficiency. Poncirus trifoliata (Poncirus trifoliata (L.) Raf.) (Pt), the most widely used rootstock in China, is sensitive to alkaline stress. To investigate the molecular mechanism underlying the tolerance of Cj to alkaline stress, next-generation sequencing was employed to profile the root transcriptomes and small RNAs of Cj and Pt seedlings which were cultured in nutrient solution with three gradient pH. This two-regulation level data set provides a system-level view of molecular events with precise resolution. The data suggest that the auxin pathway may play a central role in inhibitory effect of alkaline stress on root growth, and the regulation of auxin homeostasis under alkaline stress was important for citrus adapting to alkaline stress. Moreover, the JA pathway shown an opposite response to alkaline stress in Cj and Pt may contributes to the differentials of root system architecture and iron deficiency tolerance between Cj and Pt. The data set provides a wealth of genomic resources and new clues for further studying the mechanisms underlying Cj that resist alkaline stress.
Project description:MicroRNAs (miRNAs) play a critical role in post-transcriptional gene regulation. miRNAs have been shown to control many genes involved in various biological and metabolic processes. Deep sequencing technologies have facilitated identification of species-specific or lowly expressed as well as conserved or highly expressed miRNAs in plants. In this research, we used Solexa sequencing to discover new microRNAs in trifoliate orange (Citrus trifoliata) an important rootstock of citrus. A total of 13,106,753 reads representing 4,876,395 distinct sequences were obtained from a short RNA library generated from small RNA extracted from C. trifoliata flower and fruit tissues, Based on sequence similarity and hairpin structure prediction, we found that 178,102 reads representing 89 sequences from 42 highly conserved miRNA families, have perfect matches to known miRNAs. We also identified 10 novel miRNA candidates, whose precursors were all potentially generated from citrus ESTs. And of them five miRNA* sequences were also sequenced. These sequences had not been described in other plant species and accumulation of these 10 novel miRNAs were confirmed by qRT-PCR analysis. Potential target genes were predicted for most conserved and novel miRNAs. Moreover, four target genes included one encoding IRX12 copper ion binding/ oxidoreductase and three genes encoding NB-LRR disease resistance protein have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in C. trifoliata. Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.
Project description:Citrus and most other fruit crops are commercially propagated via grafting, which ensures trees have consistent fruit traits combined with favorable traits from the rootstock such as soil adaptability, vigor, and resistance to soil pathogens. Breeding new rootstocks requires careful agronomic evaluations, and widespread use of new rootstocks and scions requires graft compatibility with commercially important scions and rootstocks. Graft incompatibility can occur when the scion and rootstock are not able to form a permanent, healthy union. Understanding and preventing graft incompatibility is therefore of paramount importance in the breeding of new fruit cultivars and in the choice of scion and rootstock by growers. The rootstock US-1283 is a citrandarin generated from a cross of ‘Ninkat’ mandarin (Citrus reticulata) and ‘Gotha Road’ #6 trifoliate orange (Poncirus trifoliata). It was released in 2014 after years of field evaluation because of its superior productivity and good fruit quality on ‘Hamlin’ sweet orange (C. sinensis) under Florida’s growing conditions. Subsequently, it was observed that trees of ‘Bearss’ lemon (C. limon) and ‘Valencia’ sweet orange (C. sinensis) grafted onto US-1283 exhibited apparent incompatible and unhealthy growth near the graft union. The incompatibility manifested as stem grooving and necrosis underneath the bark on the rootstock side of the graft. A genetically similar citrandarin rootstock, US-812 (C. reticulata ‘Sunki’ × P. trifoliata ‘Benecke’), is fully graft compatible with the same scions. Transcriptome analysis was performed on the vascular tissues above and below the graft union of compatible US-812 and incompatible US-1283 graft combinations with ‘Bearss’ and ‘Valencia’ to identify expression networks associated with incompatibility and help understand the processes and potential causes of incompatibility in citrus. Transcriptional reprogramming was stronger in the incompatible rootstock than in the grafted scions. Functional analysis of the transcriptional events below the graft unions of US-1283 incompatible combinations revealed differentially expression genes (DEGs) associated with oxidative stress and plant defense, among other pathways, similar to a pathogen-induced immune response localized to the rootstock, although no known pathogens were detected in the assayed plants. These changes were not observed above the graft unions.Differentially expressed genes (DEGs) in US-1283, but not the scions, were associated with oxidative stress and plant defense, among others, similar to a pathogen-induced immune response localized to the rootstock. No pathogen infection was detected. It is hypothesized this response could have been triggered by signaling miscommunications between rootstock and scion either through 1) unknown molecules from the scion that were perceived as danger signals by the rootstock, 2) missing signals from the scion or missing receptors in the rootstock necessary for the formation of a healthy graft union, 3) the overall perception of the scion by the rootstock as non-self, or 4) a combination of the above.