The outline of miRNA accumulation across samples is different from that of sRNA-producing loci. While the expression of sRNA-generating regions allows distinguishing very well between ripened and green berries and also between cultivars , the accumulation of miRNAs shows a clear distinction only between ripened and green berries, and when the berries were green, we observe a further dichotomy separating the two cultivars and the two green developmental stages. The same pattern of miRNA accumulation among green and ripened berries of grapevine was observed when we described the miRNA expression atlas of Vitis vinifera . Comparing the distribution of miRNAs expressed throughout our samples, we found a set of 39 miRNAs ubiquitous or nearly ubiquitous to all the libraries, and very few miRNAs specific of a cultivar, vineyard or developmental stage. All these 39 miRNAs belong to known vvi-miRNA families. With few exceptions, the same set of miRNAs was also found expressed in all the small RNA libraries constructed with different tissues of the grapevine cv. Corvina , where the population of expressed miRNAs appears highly variable apart from a well-defined group of miRNAs, plastic flower pots probably related to the basal metabolism. These findings are also consistent with previous report in grapevine where a small number of known tissue-specific miRNAs was described .

Considering the ripening process as shown in the heat maps , and the correlation dendrogram, it is clear that most miRNAs are modulated during the developmental process. For some miRNA families, we observed the same peculiar patterns of miRNA accumulation, previously described in the grapevine miRNA atlas , e.g., an increase of accumulation toward ripening for miR156 f/g/i, and a decrease for miR166c/e, miR172d, miR319, and miR396a/b, but this is not the main focus of our paper. To establish genotype and environmental influence on miRNA modulation, we performed a statistical analysis that revealed a number of miRNAs differentially expressed. Being aware of the fact that we had only two biological replicates, we applied the exact test as implemented in the EdgeR package. This test has been recently judged a very robust tool that can be used in experiments similar to our, because of its low false positive rate and relative high true positive ratein the presence of a fold change higher than 4 . Considering berries at the same developmental stages, we compared Sangiovese vs. Cabernet Sauvignon in a given vineyard and Montalcino vs. Bolgheri, Montalcino vs. Riccione, and Bolgheri vs. Riccione keeping the cultivar fixed. In total we performed 9 pairwise comparisons for each developmental stage. In general, we observed that berries at 19 ◦Brix and at harvest show a higher number of differentially expressed miRNAs. The most interesting examples are represented by two novel miRNAs, whose predicted targets are related to the biosynthesis and accumulation of secondary metabolites, which are of crucial importance in grapevine berries, since its quality depends mainly on its metabolites .

The candidate grapem1191 is differentially expressed in Sangiovese between Riccione and Bolgheri and was predicted to target the transparent-testa 12 gene that encodes a multidrug secondary transporter-like protein involved in the vacuolar accumulation of the flavonoid proanthocyanidin in different species including grapevine . Also, in grapevine some studies provide evidences that the intracellular transport of acylated anthocyanins is catalyzed by a MATE transporter . The grape-m1355 seems to be involved in four different pathways, all related to secondary metabolites. It is differentially expressed in Montalcino between the two varieties and was predicted to target a cinnamoyl reductase-like protein , which is part of the of the polyphenol biosynthetic pathway ; a cinnamyl alcohol dehydrogenase involved in the lignin biosynthesis ; a phenylacetaldehyde reductase , which catalyzes, in tomato, the last step in the synthesis of the volatile 2-phenylethanol, important for the aroma and flavor of many foods ; and different bifunctional dihydroflavonol 4-reductases . DFR catalyzes the first step in the conversion of dihydroflavonols to anthocyanins and are responsible for the production of colored anthocyanins . The same miRNA candidate was described in the grape miRNA atlas also predicted to target several genes of DFR-like and one CCR. As for known miRNAs, several members of the miR395 family are differentially expressed at 19 ◦Brix and at harvest in Bolgheri and in both Bolgheri and Riccione, respectively, when comparing the two cultivars. Moreover, miR395f is differentially expressed also in CS at harvest between Montalcino and Bolgheri. This miRNA has been shown to target genes involved in Sulphate assimilation and metabolism , and hence it could be connected to flavonoid and stilbene pathways as suggested by Tavares et al. . miR399 family members are also differentially expressed in several comparisons: at 19 ◦Brix between Riccione and Bolgheri in CS and between Riccione and Montalcino in SG, plus in Montalcino between CS and SG.

At harvest, miR399 are differentially expressed in SG in all the three comparisons among vineyards and in Riccione between CS and SG. miR399 is implicated in Phosphate homeostasis being rapidly up-regulated upon Pi starvation . miR399 regulatory network has been shown to be important in flowering time and was identified as a temperature-sensitive miRNA , however its characterization in fruit ripening is lacking, although intriguing. miR396 family members are known to be regulated during organ development, targeting Growth Regulating Factors and also in berry development , and we observed their modulation during berry ripening in our data as well, but more interestingly, they are also differentially expressed between CS and SG in berries sampled in Bolgheri at 19 ◦Brix. Finally, the investigation of the global relationships of different small RNA classes and miRNAs expressed in different grapevine cultivars, collected in different vineyards and developmental stages, suggests that although the vineyard may influence their profile of abundance it probably does in less proportion than developmental stage and cultivar. Somehow, this behavior would be expected because although the epigenetic state is dynamic and responsive to both developmental and environmental signals, small RNAs in general and even more miRNAs are well known to play numerous crucial roles at each major stage of plants development . The results here described are in agreement with those reported in the grapevine miRNA atlas , especially with respect to the clustering of berries according to their developmental stage, sustaining the idea that miRNAs influence organ identity and clearly separate green and ripened berries. Also, in the study of the grapevine transcriptome performed by Dal Santo et al. , they observed that other factors such as year and developmental stage had more influence on the gene expression, rather than the environment.Humans began burning fossil fuels in large quantities around 300 years ago and as a result, the carbon dioxide concentration in the atmosphere has risen from 275 ppm to around 420 ppm – more than a 50% increase . Rising atmospheric CO2 and the associated global warming has profound impacts on plants and agriculture. Extreme temperature and precipitation changes are projected to reduce the yields of many commodity and specialty crops . The atmospheric CO2 concentration itself also directly affects plants but how this will impact crop yields is still controversial. One prediction is that elevated CO2 will stimulate photosynthesis and have a net “fertilization” effect on global vegetation, plastic garden container increasing gross primary productivity . Elevated CO2concentrations also decrease stomatal conductance because relatively less exchange with the atmosphere is required to achieve sufficient carbon fixation. Reduced stomatal conductance may partially counteract water stress caused by climate change related drought and higher temperatures . Flux tower measurements and satellite observations in recent decades have shown increases in forest net ecosystem productivity and leaf area index supporting the CO2-fertilization effect hypotheses. On the other hand, some observational studies have shown any fertilization effects may be short-lived as the relative increase in productivity per unit CO2concentration decreases over time .Nitrogen availability is implicated as a major limiting factor in plant’s response to elevated CO2. In long term CO2enrichment experiments nitrogen limitation has been found to significantly reduce CO2fertilization effects . Moreover, atmospheric CO2concentration and crop nutritional quality are inversely related: protein content declines in C3crops grown under elevated CO2vs. those grown in ambient CO2. This finding further supports a link between nitrogen limitation and elevated CO2and has important implications for the future of human and animal nutrition because C3crops like wheat are an important protein source globally .

Elevated CO2has been shown in multiple studies to inhibit nitrate assimilation in C3 crops which would, at least in part, explain the decline in protein under elevated CO2and the nitrogen dependence of the CO2fertilization effect. Nitrate is the most abundant form of nitrogen in most agricultural soils and in many global ecosystems making plants’ ability to effectively utilize this form of nitrogen especially important. The mechanisms of nitrate assimilation inhibition by CO2are not yet fully understood but one promising hypothesis is that C3plants are able to use some of the energy which was traditionally thought to be dissipated as heat during photo respiration and use it for the reduction of nitrate to nitrite . Reducing nitrate to a usable form during nitrate assimilation is a very energetically expensive process, which is one of the reasons nitrate is so abundant in agricultural soils. By harnessing energy from photo respiration, which has previously been considered wasteful, plants may more easily access a reservoir of soil nitrogen that is untapped by other organisms.Further research is needed to find strategies to mitigate the impact of rising atmospheric CO2 on our food supply, but one possible strategy under investigation is to provide other forms of nitrogen. Ammonium and other reduced forms of nitrogen are much less energetically expensive for plants to assimilate, and the rate of assimilation for these compounds is not inhibited by elevated CO2. Providing C3 crops like wheat with more ammonium could help prevent protein decline under elevated CO2 but ammonium has two major issues that need to be addressed: nitrification and ammonium toxicity. Most fertilizers applied to crops contain ammonia but soil nitrifiers quickly oxidize this form of nitrogen into nitrate. Farmers often apply synthetic nitrification inhibitors alongside fertilizers in an attempt to slow this process. More recently, the root exudates of some important crop species have been found to contain biological nitrification inhibitors . Even if these compounds can effectively inhibit nitrification in agricultural soils, crops would still have to contend with toxicity from ammonium accumulation. The mechanisms behind ammonium toxicity are not yet fully understood and neither are those behind ammonium tolerance . There is great natural variation in plants’ tolerance to ammonium, even among varieties within the same species . Ammonium tolerance can, therefore, be treated as a beneficial crop trait and selected for in breeding programs.Measuring nitrate assimilation in plants can be used to study variation in their responses to elevated CO2 as well as quantify the relative utilization of ammonium vs nitrate. Various methods have been used to quantify nitrate assimilation but one of particular interest is Assimilatory Quotient which refers to the ratio of CO2consumed to O2evolved during photosynthesis . AQ measurements are able to estimate nitrate assimilation because plants have coupled this energetically expensive reaction with photosynthesis. During photosynthesis, reduced ferredoxin generated during the light-dependent reactions can either provide reducing power to the CBB cycle or to nitrite reductase . When plants are assimilating nitrate, some of the reduced ferredoxin generated from the oxygen-evolving step of photosynthesis goes towards nitrate assimilation rather than carbon fixation, thereby lowering the amount of CO2fixed per O2 evolved – in other words, lowering the AQ. AQ measurements have the advantage of allowing for non-destructive real-time estimation of nitrate assimilation in living plants but are not widely used due to the difficulty of measuring very small O2fluxes against the large ambient background concentration of 20.9%. Currently there are no commercially available O2analyzers with a high enough precision for measuring AQ. AQ measurement in previous studies has either been done with room-sized custom-built O2analyzers or with commercial analyzers that have since been discontinued indicating a need for new instrumentation development. The following three chapters describe different tools that were developed for research needs related to investigating the impacts of rising CO2on crop nutrition, but many have additional applications beyond their original purpose. The first chapter describes an image analysis tool that was originally developed to measure the rosette area of Arabidopsis seedlings grown on agar plates as part of a larger study investigating the genetic basis of plant response to nitrogen form and concentration.