miRNAs are post transcriptional regulators that cause down regulation of target genes. Therefore, if a target gene is down-regulated by a miRNA, a negative correlation between miRNA expression and the target mRNA expression is expected. No statistically significant differentially expressed miRNAs were observed in our fruit small RNA seq data, so we instead predicted miRNAs that are potentially targeting DEGs found in fruit tissues. This approach was taken due to the complex regulatory networks that are known to exist in plants and other higher organisms. One miRNA may regulate many genes as its targets, while one gene may be targeted by many miRNAs. Both of these scenarios were observed in citrus roots in response to dehydration and salt stress. To evaluate these potential relationships in this study, the psRNA Target program was utilized, which accepts a list of known plant miRNAs in citrus and the coding sequence of the DEGs reported here to predict miRNAs according to the criteria described by Meyers et al.. Over 15,000 miRNA-mRNA interactions were predicted using psRNA Target. The RNAseq data was then utilized with an in-house R-script in order to select potential interacting pairs with an expected negative correlation in gene expression. After removal of genes that did not have any functional annotation, there were 366 combinations of miRNA-mRNA pairs that showed reciprocal expression patterns. Comparing these genes with the enriched GO terms and KEGG pathways led to several candidate miRNA-mRNA interactions that could be causing changes in fruit traits when differentially expressed between root stocks.
These genes included transcriptional regulators, hormone signal transduction genes, transporters,nft growing system and sugar metabolism genes. Based on the interacting pairs predicted and their relevance to fruit quality, 10 pairs of miRNAs and target mRNAs were selected for validation via qRT-PCR analysis . Samples collected at time points two and three were chosen for validation due to the larger differences in expression levels of genes in fruit grown on trifoliate orange compared to rough lemon root stocks at those times. For qRT-PCR, two biological replicates and three technical replicates were analyzed to quantify expression of each gene. Three miRNAs were up-regulated at both time points, while their target genes were down-regulated, two miRNAs were down-regulated at both time points, while their target genes were up-regulated, and the remaining five miRNAs validated were down-regulated at one time point and up-regulated at the other . The correlation between the relative expression level detected by qRT-PCR and by RNA-sequencing was calculated. Pearson correlation values were highly significant with r = 0.94, which strongly supported the sequencing data . However, certain miRNA-mRNA pairs did not have the expected fold changes from one time point to the next. For example, Csi-miR171a shows an increased fold change from September to November. This should correlate with a decreased fold change from September to November in the target gene , but instead, we see an increase in the target mRNA expression from September to November. Only this pair and Csi-miR1863 – ATEXP1 show this inconsistency. The results for the remaining eight pairs were consistent with their expected expression levels.
Figure 1.12 shows that seven of the miRNAs had increased expression levels in November compared with September, while three miRNAs decreased in expression from the during fruit development. The objective of this study was to correlate changes in gene expression of grafted citrus trees to effects in fruit quality due to varying root stocks. In this study, four root stocks were chosen from a root stock trial with Washington navel orange scion in Riverside, CA to assess for various fruit quality traits; Argentina sweet orange, Schaub rough lemon, Carrizo citrange, and Rich 16-6 trifoliate orange. Fruit quality data was collected from fruit grown on each of the four root stocks at the end of the growing season when fruit were ripe. In the present study, weight, height, width, rind color, rind texture, peel thickness, internal texture, juice weight, percent juice, total soluble solid and titratable acid levels were measured. The total yield and average fruit weights were markedly higher in navel orange fruit from trees grafted onto rough lemon compared to sweet orange, Carrizo citrange, or trifoliate orange root stocks. The rind thickness was also greatest on rough lemon root stocks compared with the other root stock-scion combinations. The most substantial differences could be seen in total soluble solids and acid levels. The highest levels of sugars and acids were found in fruit grown on Carrizo citrange. Trifoliate orange and sweet orange root stocks produced fruit with only slightly lower sugar and acid levels, while rough lemon produced fruit with significantly lower levels . This is consistent with the previously mentioned reports of root stock effects on fruit quality3,7-14. Presently, there is very little understanding of how root stocks influence citrus fruit quality, especially at the level of gene regulation. In this study, an integrated mRNA and miRNA high throughput sequencing analysis in fruit grafted onto genetically diverse root stocks was performed to help resolve potential mechanisms of root stock-scion effects on fruit quality. In the present study, RNA-seq was used to investigate transcriptome differences in the fruit of ‘Washington’ navel sweet orange grafted onto different root stocks and explore genes that may influence fruit quality traits. Juice vesicles from fruit grafted onto four genetically diverse root stocks at four different fruit development periods were sequenced. The RNA-seq approach detected a similar number of genes in all samples .
A large number of these genes were identified as differentially expressed over the course of fruit development, which is consistent with previous studies of transcriptome changes during fruit ripening in sweet orange . However, most genes showed similar temporal expression patterns among all root stock genotypes. Furthermore, only ~15% of the genes were genotype-specific . Therefore, the remainder of this study focused on DEGs identified between these root stock genotypes during fruit development.The majority of the differentially expressed genes are observed in comparisons involving rough lemon root stocks, especially compared to trifoliate orange. This is consistent with the observed differences in fruit quality traits, as fruit of trees grafted on rough lemon root stock showed consistent significant differences from fruit of trees grafted on the other three root stocks in many of the traits measured . These results suggest that rough lemon and trifoliate root stocks show the greatest effects on the scion and are good candidates to identify graft-related genes playing a role in fruit quality. The largest and most significant changes in gene expression between root stocks were observed at time points two and three . Among the DEGs were several genes with functions involved in fruit quality traits, such as those relating to starch and sucrose metabolism, fructose metabolism,nft hydroponic system and hormone signaling related genes. KEGG pathway analysis displayed plant hormone signal transduction, carotenoid biosynthesis, and fructose and mannose metabolism pathways to be significantly enriched. Several genes involved in various hormone-signaling pathways were DE, mainly genes in the abscisic acid and auxin-response pathways. Several genes involved in these pathways were chosen to validate the RNA-seq data by qRT-PCR due to their potential biological significance regarding root stock effects on fruit quality. ABA has been known to be a regulator of fruit ripening and response to abiotic stress in non-climacteric fruit. AHG1, a homolog of Arabidopsis PP2C family protein, was DE in this study. This gene was slightly up-regulated when comparing fruit of trees grafted on trifoliate to fruit of trees grafted on rough lemon root stock at time two and significantly down-regulated at time three . Upregulation of AHG1 is in accordance with previous studies showing this gene being induced by water stress, which may have occurred in September.
The down regulation of this gene later in the season could be correlated with increased fruit maturation in fruit grown on trifoliate root stocks. This is in agreement with a study in tomato where suppression of PP2C expression led to increased ABA accumulation and higher levels of ABA-signaling genes that increase the expression of ABA-mediated ripening-related genes. Auxin signal transduction is mediated by Aux/IAA and ARF genes. Aux/IAA proteins are negative regulators of the auxin signal transduction pathway. In this study, a gene encoding an Aux/IAA protein, IAA16, was up-regulated in fruit grown on trifoliate compared to rough lemon root stocks at time two and three . A previous study revealed that a gain-of-function mutation in IAA16 displayed reduced response to auxin and ABA, which led to reduced plant growth89. Silencing of related Aux/IAA genes increased fruit size in tomato due to auxin control of cell expansion and elongation. In addition to Aux/IAA, another early auxin-response gene, SAUR78, was DE in this study. This gene was down-regulated in fruit grown on trees grafted onto trifoliate compared to rough lemon root stocks at time two and three . Small Auxin Up RNA genes are a group of auxin-inducible proteins. SAUR78 over expression lines in Arabidopsis increased plant growth through interaction with ethylene receptor. Other SAUR genes have also been shown to promote cell expansion. Furthermore, a MYB77 gene encoding a transcription factor was DE in this study, displaying a slight increase in expression in fruit grown on trifoliate root stock at time two, but a large decrease in expression at time three . This gene was previously described as a regulator of the auxin signal transduction pathway. This protein was shown to interact with ARFs to promote plant growth. Interestingly, the effects of MYB77 in Arabidopsis were found to be increased by endogenous exposure to ABA and further promote plant growth. While these two studies were performed in roots, this transcription factor was shown to be involved in citrus fruit ripening, where it was highly correlated with ABA and suggested to have a similar function in response to the hormone28 . Although there were not statistically significantly differences seen in other genes in the auxin- and ABA-signaling pathways, trends could be observed during hierarchical clustering of these genes. Many of the genes within a family shared common expression levels and generally follow the predicted regulatory patterns in their respective pathways . Taken together, the changes in ABA- and auxin responsive genes suggest a potential mechanism for induced ripening by trifoliate root stock and larger fruit produced when rough lemon is used as a root stock. The expansion phase of citrus fruit development involves cell enlargement and water accumulation. Given the changes in hormone-signaling pathways that likely lead to changes in fruit size, other genes related to fruit growth, such as transporters and genes related to cell wall metabolism were investigated. This led to the identification of two DEGs that could be influencing fruit size. The first, a Plasma membrane Intrinsic Protein 2gene encoding an aquaporin was down-regulated in fruit grown on trifoliate root stock . Water import in plants is mediated by aquaporins and essential for cell expansion. These genes were highly expressed in expanding green grapes and one was identified as a candidate gene under the QTL for berry weight. PIP genes were also associated with an increase in volume of fruit in apple and strawberry. The second DEG, an expansin , was also down-regulated in fruit grown on trifoliate root stock . Expansins play various roles in fruit development, including cell elongation and cell wall softening. A homolog of EXP1 in tomato was expressed during green fruit cell division and expansion with maximum accumulation of EXP1 during the late phase of green fruit expansion and early maturation. The increase in expression of these two genes in fruit grown on rough lemon root stock could contribute to the larger fruit size observed. In addition to cell division and cell expansion, during fruit development, fruit softening is also an important feature that relies on cell wall metabolism. The Trichome Birefringence-Likegene, which encodes a protein required for cellulose biosynthesis, was identified in our study as DE. Mutations in this gene caused a reduction in the amount of pectins and an increase in pectin methylesterase activity. PME catalyses the demethylesterification of pectin, which may undergo depolymerisation by glycosidases. TBL23 was up-regulated in fruit grown on trifoliate root stock compared to rough lemon , suggesting a potential role in fruit softening during citrus ripening. Transcription factors also play an important role in plant development and fruit ripening.