Several transcription factors were differentially expressed in this study. GO enrichment showed the molecular function GO term ‘DNA-binding transcription factor activity’ was significantly enriched. In addition to the MYB77 transcription factor gene described earlier, a GRAS transcription factor gene, HAM3, was DE in this study. GRAS transcription factors were previously found to play a role in berry development and ripening in grapes, tomato, and citrus. This transcription factor showed increased expression later in the season when fruit were grown on trifoliate root stock, suggesting the root stock influences its role in improved citrus fruit quality. The largest phenotypic differences seen in mature fruit grown on trifoliate compared to rough lemon root stock were in the levels of total soluble sugar and titratable acid in ripe fruit. The levels of sugars and acids and their ratio in fleshy fruits is one of the most important determinants of sensory traits such as taste and flavor. Two genes were identified as differentially expressed that could play a role in the accumulation of these compounds. Firstly, a P-type ATPase was DE in fruit growing on trees grafted onto trifoliate versus rough lemon.
This gene was down-regulated at time two,hydroponic nft system but upregulated at time three . Studies have proposed a number of ATPases as proton pumps that are responsible for organic acid accumulation in citrus fruit. The reduced expression of this ATPase gene later in the season in fruit grown on rough lemon root stocks could contribute to the lower accumulation of titratable acid levels in these fruits. This ATPase gene identified in this study was not identified in the previous citrus studies, but the regulation of acid accumulation is a complex, as can be seen in other fruits, such as papaya and apple. It is possible this is a graftinduced effect observed with these specific root stocks, which were not examined in the previous studies. Secondly, a homolog of Arabidopsis BETAFRUCT4 was down-regulated in fruit of trees grown on trifoliate root stock compared to rough lemon at time three . This gene encodes a vacuolar invertase. Decreased expression of vacuolar invertases has been associated with increased sucrose content and accelerated ripening. Interestingly, by using an antisense acid invertase gene in transgenictomato to reduce acid invertase activity, fruit displayed higher levels of sucrose, as well as smaller fruit. We see similar trends in sugar accumulation and alterations in fruit size in this study. Klann et al. suggested that the water influx that drives fruit expansion is closely related to the concentration of osmotically active soluble sugars and therefore, all genotypes accumulate water until they reach a similar threshold of soluble sugar concentration. This could also contribute to the increased size of fruit grown on rough lemon fruit compared to trifoliate root stocks.
This study did not identify any statistically significant differentially expressed miRNAs from our fruit small RNA seq data. Therefore, potential miRNAs that target DEGs were predicted. An in-house R-script was used to select for miRNA-mRNA interaction pairs with an expected negative correlation in gene expression. These pairs were identified for the ten genes described above. All ten miRNA genes and their target mRNAs were detected by qRT-PCR. Pearson correlation coefficient value between the relative expression level detected by qRT-PCR and by RNA-sequencing was highly significant with r = 0.94. Of the ten interaction pairs, eight followed expected fold changes between time points . Therefore, it is likely that these eight target mRNAs are being regulated to some extent by their respective miRNA. Only two pairs do not follow the expected inverse relationship between time points, suggesting those mRNAs are not being regulated post transcriptionally by their miRNAs. This has been observed in previous integrated miRNA-mRNA studies. There have also been reports of target genes having a negative or positive feedback regulation on their respective miRNA, which could be another explanation for the inconsistent correlations seen in this study.Citrus is now grown in more than 140 countries in tropical, subtropical and Mediterranean regions. It is one of the most economically important crops in the world.This reduces the juvenile phase, allowing for the trees to produce fruit many years earlier than would trees grown from seed. Due to the large variation in growing conditions and climate in the regions where citrus is grown, different citrus root stocks are required to improve yield and fruit quality in numerous diverse climates, as well as resist various pests and diseases.
Root stocks impart certain traits to the scion and the effects of root stocks can be large. The most significant impacts are on growth, vigor and yield, tree nutrition, stress resistance, and fruit quality. The root stock effects on various aspects of tree growth and fruit development are well documented, but the molecular mechanisms underlying most of these differences are unknown. Previous studies have shown changes in the transcriptome of various root stock genotypes, especially in response to biotic and abiotic stressors. These types of changes have been seen in Arabidopsis, corn, mulberry, tomato, and poplar. In citrus, gene expression profiling has been used to understand root stock effects and responses to biotic and abiotic factors. In another study, expression studies of leaves from mandarin grafted onto various root stocks were analyzed in order to explain root stock effects on the growth of scions. There is extremely limited tissue-specific transcriptome knowledge in citrus, especially for root tissue. A small number of studies have evaluated trifoliate, trifoliate hybrid, and mandarin root transcriptomes in response to citrus diseases, but these studies each assessed only one genotype. Only recently has an RNA-seq based approach been used to establish a reference transcriptome for citrus and of the 28 samples used in the study,nft channel only two were obtained from roots. The root samples collected for this study were sour orange and trifoliate genotypes, but samples were grouped by organ to perform differential expression and subsequent analyses. To our knowledge, there are no comparative studies of citrus root transcriptomes between genotypes. In plants, the root system is critical for plant growth and development. It serves the functions of anchorage, nutrient and water uptake, and is the main boundary between the plant and its soil environment. Root growth relies on a specific set of signals that involves hormone signaling, availability of nutrients and carbon supply. There is a large degree of genotypic variation in crop plant root systems that can influence the plants growth and production including root length, root density, root angle, lateral root number, and root:shoot ratio. These parameters can impact the plant’s size, tolerance to biotic and abiotic stressors, and ability to uptake water and nutrients. For this reason, grafting, a process which connects the roots of one plant to the scion of another, has been widely using in plant breeding programs in order to improve vigor, alter plant architecture, enhance tolerance to disease and abiotic stress, and contribute to the quality of crops. In citrus, root stocks are bred for a variety of traits that are imparted to scions, such as tree size, yield, tolerance to salt, cold, and drought, tolerance to various pests and diseases, and improved fruit quality. Many studies in citrus have been conducted to assess the impacts of genetically differing root stocks on these traits. However, the study of molecular mechanisms behind root attributes lags far behind above ground tissues in plants, especially in perennial crops. Understanding the genetics of how root systems develop, and the regulatory controls of these processes will help optimize the improvement of yield and quality in citrus. Root system length, growth, and architecture control the ability of plants to respond to various stress conditions. The development of the root system and its architecture is determined by genetic factors interacting with numerous environmental factors. Plants must adapt to their environment by controlling their physiological reactions and morphogenesis. This can create complex root system architectures. For example, different root types can produce lateral roots that significantly extend the elaborate root system and allow the plant to search the soil for water and nutrient-rich areas. The increase in lateral root formation allows plants to more easily uptake these essential molecules in order to survive in unfavorable conditions.
Root stock genotypes exhibiting higher abilities to adapt to stress and create more extensive root systems improve nutritional status and water uptake, which can increase marketable yield. A clear example of this was seen in grafted mini-watermelon and tomato plants. Studies in Arabidopsis, rice, and corn have identified several genes that influence root development and root system architecture. In citrus, transcriptomic studies have been performed to understand the effect of root stocks on growth and in response to cold, nutrient deficiency, and fungal inoculation. Besides these studies, little effort has been invested into studying the effects of citrus root stocks at the molecular level, especially genotype-specific effects and their relation to fruit quality. Plants have evolved to cope with a constantly changing environment, modifying the root system architecture in response to nutrient availability and soil microorganisms. This flexibility requires fine tuning of gene expression. Among the molecules that control root development, small RNAs play a vital role in regulating genes at the post transcriptional level in plants. The most well-studied class of sRNAs are microRNAs , which are approximately 21 nucleotides in length and are produced from noncoding transcripts. Mature miRNAs have been shown to negatively regulate gene expression at the post-transcriptional level by specific binding and subsequent cleavage of their target mRNAs, or by the repression of target translation. Increasing evidence demonstrates that plant miRNAs play critical roles in almost all biological and metabolic processes. A review of miRNAs in roots indicated that they participate in root development, the modulation of root architecture, and root biotic interactions. This review focuses on numerous studies using Arabidopsis and legume plants as models. In citrus, miRNAs profiling has revealed their involvement in adaptation to nutrient deficiency, drought and salinity stress, and pathogen infection. However, no information, to date, is available about the role of miRNAs underlying differences in fruit quality observed between citrus root stocks. In the present study, trees grafted onto four root stocks were chosen from a root stock trial at the University of California, Riverside to assess for various fruit quality traits; Argentina sweet orange, Schaub rough lemon, Carrizo citrange, and Rich 16-6 trifoliate orange. Generally speaking, rough lemon root stocks produce the highest yield and fruit size, but this fruit is often of lower quality . Trifoliate orange root stock, when well adapted, produces high quality fruit, with high yield on smaller trees. Carrizo citrange root stocks produce intermediate yield with good fruit quality. Sweet orange root stocks produce good quality fruit but are very susceptible to various citrus diseases, and therefore are rarely used as root stocks for commercial trees. An RNA-seq approach was used to assess differences in gene expression between root stocks that produce fruit with varying quality traits with the aim of identifying genes that could potentially play a role in improvement of fruit quality. Moreover, miRNA expression profiles were obtained for each of the root stocks to identify potential regulatory mechanisms associated with their target genes. The grafted trees were part of a root stock trial conducted at the Citrus Research Center and Agricultural Experiment Station at the University of California, Riverside that included 28 root stocks. Trees were planted in 2011 in a randomized block design with ten replications. Trees were planted on berms, irrigated with mini sprinklers according to soil moisture sensors, and treated with fertilizers and pesticides according to standard commercial practices. Plastic mulch was used to cover the berms to suppress weeds and retain soil moisture. Trees were grown in a fine sandy loam and surface soil pH was 7.3 at the time of planting. It is important to note that no trees with Huanglongbing have been identified at UCR. For sequencing, roots from two biological replicate trees were harvested at four time points throughout the 2014-2015 growing season . Young, newly growing fibrous root tissue was collected from the first 6 inches of soil along the outer edge of the canopy.