Cu levels recorded at planting of this root stock trial were just above optimal levels . It is plausible that trifoliate roots could be better adapted to a potential deficiency of Cu in subsequent years. Further testing of nutrient levels in the plant and soil will need to be performed to confirm these hypotheses. An interesting gene was identified as DE that could potentially contribute to the dwarfing phenotype sometimes seen when trifoliate is used as a root stock. SWEETIE, identified in Arabidopsis, is a sugar transporter that controls sugar flux and modulates many important processes96. A mutation in this gene causes modified carbohydrate metabolism that leads to a dwarfed phenotype.While this dwarfed phenotype was observed in roots and shoots in Arabidopsis, it is conceivable that themolecular mechanism of dwarfing in grafted citrus plants could involve the movement of sugars from the root stock to the scion via the transporter encoded by SWEETIE. This would be consistent with previous studies reporting that the graft union can cause differences in carbohydrate transport, resulting in dwarfing. In the University of California, Riverside root stock trial sampled, trees on rough lemon were 28.7.% larger than trees on Rich 16-6 trifoliate orange, potentially due to differing abilities of the root stocks to transport carbohydrates. Plants, being immobile,bato bucket endure a variety of environmental stresses over the course of their lifetime.

Therefore, they have acquired specific and sensitive ways to sense and react to each type of stress. For example, abiotic stress can trigger the production of ABA, which leads to the activation of the expression of an assortment of stress responsive genes. Cold stress can lead to water-deficit conditions in plant cells, which induces changes in cytosolic calcium levels. Ca2+, acting as a messenger, is able to activate signaling pathways and induce a variety of plant growth, development and stress responses. Annexin is a Ca2+ -binding protein that senses Ca2+ and transmits the signal to downstream signaling components of the ABA and abiotic signaling pathway, regulating stress response in plants106. In Arabidopsis, a mutation in AnnAt4 displayed tolerance to dehydration, while AnnAt4 over expressing transgenic plants were more sensitive to this stress. AnnAt4, was down-regulated in trifoliate roots, suggesting that it may play a role in the ability of this root stock to respond to cold stress induced dehydration .Cell wall architecture is important in plant resistance to abiotic stress. Cell wall-related proteins are thought to play a central role in modulating cell wall elasticity, which facilitates cell enlargement and expansion. These proteins include xyloglucan endo-β- transglucosylases/hydrolases , endo-1,4-β-D-glucanase , and expansins. Most cases of abiotic stress are associated with an increase in XTH proteins, which helps maintain cell wall plasticity in response to stress. The over expression of XET-related genes in Arabidopsis has been associated with improved freezing tolerance. Similar results of increased XET expression in response to cold were also seen in rice and poplar. In the present study, there was increased in XTR4/XTH30 in trifoliate compared to rough lemon roots .

It is conceivable that this gene is having a similar effect in trifoliate roots, enhancing tolerance of this root stock to cold stress.The Arabidopsis Ethylene Response DNA Binding Factor/Related to ABI3/VP1 subfamily is a group of plant-specific B3 transcription factors. In Arabidopsis, expression of EDF2/RAV2 was repressed by ABA and overexpression of this gene negatively regulated plant growth and development. Similar results were displayed while over expressing the soybean homolog GmRAV. GmRAV-OX lines exhibited slower plant growth rate , reduced root elongation, delayed flowering time, and reduced photosynthetic rate. Additionally, RAV2 was found to influence plant defense response in Arabidopsis, specifically it was required by viral suppressors of silencing. Decreased expression of this gene restored the silencing defense mechanism of the plant, allowing the plant to defend itself against plant viruses. EDF2/RAV2 was down regulated in trifoliate roots compared to rough lemon, suggesting the trifoliate root stock has increased plant defense mechanisms, as well as increased growth and development . Trifoliate root stock confers better tolerance to several diseases than rough lemon, including Citrus tristeza virus, Phytophthora root rot, and citrus nematodes. This gene could be a good candidate for further studies of plant defense and the relation to subsequent signaling pathways induced by pathogens. Small RNA-seq reads that did not match to any known plant miRNA were used in prediction tools that combine the expression patterns, Dicer cleavage site, and secondary structure of miRNA precursor molecules to more accurately predict novel miRNAs. Several studies have predicted miRNAs using the Citrus clementina and Citrus sinensis reference genomes for prediction of miRNA precursor molecules. Using recently available SNP variant calls from whole genome sequencing information of Poncirus trifoliata and Citrus jambhiri, we created ‘pseudo’ reference genomes for these varieties.

These were created by building a consensus sequence based off the Citrus clementina reference genome and replacing any of the newly acquired SNP variant data for each respective genotype. By utilizing this method, nine novel miRNAs were discovered using the trifoliate pseudo-reference genome and three were discovered using the rough lemon pseudo-reference genome. The increased number of novel miRNAs in trifoliate is likely due to the fact that it is more divergent from clementine than rough lemon, resulting in more polymorphisms that were only detected in the trifoliate pseudo-reference genome. Typically, an increase in miRNA expression will lead to the down regulation of its mRNA target, while a decrease in miRNA activity will lead to upregulation of its target. In plants, miRNAs generally regulate target gene expression through cleavage, and subsequent degradation of the target mRNA. An integrated analysis of miRNA and mRNA expression profiles can help identify miRNA-mRNA interaction pairs involved in regulating specific biological processes. By using this approach, we identified several important regulatory miRNAs potentially involved in control of root system architecture and response to abiotic and biotic stressors. Interestingly, some of these miRNAs had multiple mRNA targets, suggesting there is a complicated regulatory network in citrus root stocks and some miRNAs may control multiple aspects of root growth.For Carex aquatilis and Salix rotundifolia, the observed NH4 + uptake profiles were consistent with the prevailing hypothesis that fine-root biomass density,dutch bucket hydroponic as functionally absorptive tissues, exerts first-order control on nutrient uptake [De Baets et al., 2007; Vamerali et al., 2003]. For Eriophorum angustifolium, however, the observed uptake profile did not follow the prevailing hypothesis. We showed that this pattern resulted from decreased competition between roots and microbial decomposers in mineral soils. The ECA competition hypothesis as integrated in the N-COM model explicitly represents these competitive interactions and accurately predicted the NH4 + uptake profile . The model results indicate that since Eriophorum angustifolium is a relatively poor competitor for NH4 + , it shifts its uptake profile deeper in the soil, in order to avoid NH4 + competition with microbial decomposers in the organic layer. Root physiology traits suggest that the Eriophorum angustifolium root system is less carbon efficient. In particular, compared with Carex aquatilis, maintenance and growth respiration per gram of root are higher but root longevity is much shorter for Eriophorum angustifolium [Billings et al., 1977; Shaver and Billings, 1975]. Although root morphological traits suggest that Eriophorum angustifolium has higher root length per gram root biomass [Eissenstat et al., 2000], total root density is much lower than Carex aquatilis . Furthermore, the Eriophorum angustifolium NH4 + uptake pattern is also consistent with the idea that microbial activity and N immobilization are highly limited by carbon availability. Compared with mineral soil layers, relatively higher carbon availability in surface organic layer will lead to higher potential of microbial activity and consequently higher microbial N immobilization demand and stronger nitrogen competition between plant and microbe [Booth et al., 2005]. Although both gross nitrogen mineralization and immobilization rates are commonly high in surface soils, net immobilization typically occurs because of strong microbial demand [e.g., Iversen et al., 2011].

Overall, our 15N tracer measurements and modeling analysis at Barrow, Alaska, showed that plant nitrogen uptake patterns emerge from root and soil biotic competition, which could be predicted by essential root traits and appropriate treatment of microbial competitive interaction. Although not studied here, mineral surfaces are also effective competitors for enzymes [Sulman et al., 2014; Tang and Riley, 2015], and further research is required to determine when those processes need to be included in nutrient and carbon cycle models.In this study, we showed that an important complication in predicting arctic tundra vegetation species responses to warming is associated with their different root characteristics, which can affect their ability to compete for elevated nitrogen availability throughout the soil profile. In this sense, explicitly considering key root functional traits is particularly important for studying warming-induced fertilization effects on arctic vegetation. Here we highlight the importance of several essential root traits in controlling nitrogen uptake patterns. First, maximum rooting depth is an important plant functional trait in modeling plant nitrogen uptake and response to arctic warming. More deeply rooted species can access existing and newly thawed deep soil nitrogen [Keuper, 2012]. In addition, roots acclimated to low temperatures in deep soil may have higher nutrient uptake capacity than roots in warmer surface soils [Chapin, 1974]. However, nitrogen in deeper soil is available for plant acquisition for a relatively shorter period than nitrogen in near-surface soil because the active layer thaws and increases in thickness throughout the growing season. Shallow-rooting species access soil nitrogen nearer the surface, and do so in the context of stronger microbial competition, but with more abundant soil nitrogen and over longer periods during the growing season. Therefore, different tundra species may respond dramatically differently to climate warming-induced soil nitrogen availability changes. The trade offs and ecological significance of plant carbon investments to compete for nitrogen in relatively warm shallow soils with high microbial competition, or to access nitrogen in relatively cold deeper soils with less microbial competition warrant further investigation. Second, root nitrogen uptake capacity is also an important trait for nutrient competitiveness. Species with low nitrogen uptake capacity must develop dense or long-lived roots in order to acquire enough soil nitrogen. For example, Carex aquatilis’s fine roots live for multiple years, and the fine root to leaf biomass ratio can be as large as 16 [Iversen et al., 2015b]. In contrast, species with high nitrogen uptake capacity invest less carbon for the growth of relatively short-lived roots [Eissenstat et al., 2000]. Third, tundra species with different carbon allocation strategies may contribute differently to carbon-climate interactions. For example, Carex aquatilis may fix more carbon per unit additional nitrogen uptake than Eriophorum angustifolium, because the former allocate more carbon to grow roots and root C:N ratios are much higher than leaves . Carbon costs of constructing roots are commonly lower than above ground tissues [Poorter, 1994]. In addition, tissue lifespan [Withington et al., 2006], decomposability [Hobbie et al., 2010], maintenance respiration [Segal and Sullivan, 2014], and contribution to soil carbon accumulation [Hu et al., 2016] differ among leaves and roots. Integration of these essential root traits into ESMs will improve understanding of how arctic tundra plants will respond to climate warming, through informing the magnitude of warming-induced increases in nitrogen availability on tundra carbon production.Current ESM land models have rudimentary representations of plant traits because of a lack of mechanistic understanding of how those traits control plant and ecosystem biogeochemical processes and a lack of trait data to structure and parameterize large-scale simulations. We have recommended several key traits, which should improve predictions of root nitrogen uptake and how arctic tundra plants may respond to warming-induced elevated nitrogen availability. Some knowledge of the global spatial distributions of several of the aforementioned root traits is available. For root biomass profiles, the first global database was presented by Jackson et al. [1996]. Zeng [2001] further analyzed those biomass profile data according to Plant Functional Types and derived PFT-based root distribution data needed for large-scale land models. Schenk and Jackson [2002] expanded the Jackson et al. [1996] data set to include 475 root biomass profiles. However, most of those profile data are from temperate regions .