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, blueberry container size 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. Garden or English peas are hardy, cool season, vining annuals grown for their fresh immature green seeds and pods. Peas are classified in the Fabaceae family, which consists of approximately seven hundred genera and seventeen thousand species, with cosmopolitan distribution throughout the temperate, subtropical, and tropical zones of the world. Many species in this family are used as food, forage, timber, and dye plants. Peas are thought to have originated on the eastern rim of the Mediterranean into the mideast. Remains of 7,000- year-old carbonized seeds have been found in Switzerland. By the height of the Greek and Roman civilizations, peas were well established garden, field, and green manure crops. Although peas are not heavy yielders , they are well worth the effort in small gardens. A fresh garden pea’s taste is so far superior to its store-bought equivalent that it is in fact a different vegetable—sugar vs. starch, fresh and lively vs. dull and soggy. Along with spinach, growing raspberries in container peas usually herald the first working of the soil and planting in spring. If all goes well, sweetness and succulence await you 50 to 70 days after planting seeds. Because they need to be trellised, peas afford excellent opportunities for intercropping . Once established, peas don’t require much work. They are able to grab onto the trellis and spread themselves out for greater exposed photosynthetic area and better air circulation to reduce the incidence of mildew. They are not very sensitive to weed pressure. In fact, weeding established pea patches can do more harm than good, as peas have numerous surface roots that are sensitive to disturbance.Cultivation. All peas are emphatically cool season crops. Optimally, they are direct sown when the soil temperature averages over 50° F. Sixty to eighty days of temperatures below 80° F are requisite for good production. Soil temperatures of 55°–75° F will yield germinating seedlings in 7–10 days. Overly wet and cold or wet and warm soil increases the percentage of pre-emergent rot. In fact, because pea seeds are large and can imbibe and hold so much water, allowing the soil to dry down significantly between waterings will reduce rot and ensure good germination. Soils. Good drainage is essential for vigorous growth. Early cropping favors sandy soils as they drain and warm more quickly than clays. Peas, as do most legumes, prefer a slightly acid to slightly alkaline soil pH. This higher pH range also provides for the high calcium needs of peas. Peas are intolerant of acid soils. Planting. Peas should be direct seeded or gently transplanted from speedling/plug trays. Seeds can either be drilled in rows or broadcast sown. Drills can be single or double rows 2–4 inches from the trellis to facilitate the tendrils finding the fence. Seeds should be sown heavily , as pea seeds generally have a moderate germination percentage even under ideal conditions. Thin to 8–12 seeds per foot if necessary. The general adage about covering seed two to three times its narrowest diameter applies here; seeds should be planted 1–1 1/2 inches deep, then gently tamped or watered in. Pea seeds can also be thickly broadcast at 2–3 seeds per square inch, and raked in or covered with soil. Twiggy brush or one to two layers of horizontal netting creates the trellis for support. This broadcast method nets a higher yield per area but can increase incidence of powdery mildew due to restricted air circulation. Note that pea seed viability is relatively short under ideal circumstances. In most home garden situations seed will only last 1–3 years. When ordering pea seeds, think in 1/2 and 1 pound increments . Varietal selection is important as to plant height and time to maturation, but most importantly, pay attention to disease resistance. Basically, if a varietal description doesn’t tout or mention disease resistance, be a smart shopper and realize it probably has none.
The more recent the varietal introduction, the more disease resistant it is. Unfortunately, the converse is true as regards heirloom varieties. Nutrients. Because legumes are capable of fixing nitrogen via association with soil bacteria, garden peas are mistakenly thought to need little or no supplemental nitrogen. The truth is that they fix very little nitrogen unless inoculated with the appropriate species of bacterium . They will also use most of the nitrogen they fix and thus don’t particularly enrich the soil for the following crop. Phosphorous is an important nutrient for early root development and to assist with flowering, fruiting and sugar development. Fortunately legumes are efficient at gathering and concentrating phosphorous. Fertilizing the peas prior to planting is optional on enriched or improved soils. A compost of chicken manure, mixed greens, and straw or leaves will boost nitrogen and phosphorous levels. Composts of brassicas and legumes will concentrate phosphorous and calcium. With a single or double row of peas on a trellis in the center of a 48-50-inch-wide raised bed, a crop of quick-maturing plants can be grown along the bed edges for more efficient use of space. These include — Planting Days to Crop method harvest baby spinach direct sown 20-30 days mature spinach direct sown 40-50 days cutting lettuces transplanted 20-30 day mature leaf or transplanted 40-50 days mini romaine lettuce butter lettuce transplanted 50-60 days arugula direct sown 20-30 days direct-seeded radishes direct sown 30-40 days Inoculation. Pea plant vigor and thus production is markedly increased when the seed is inoculated with the appropriate species of Rhizobium bacteria . These bacteria can be purchased in a powdered carrying agent from most seed catalogues and nurseries. To inoculate, simply dampen the seed, add powdered inoculate and mix until the seeds have a blackened, peppered look. Plant as soon as possible as the water activates the bacterial population and desiccation is harmful. Watering. Peas require 1–2 inches of water per week. They are intolerant of water stress ; stress will reduce plant size, decrease yield quality—resulting in tough, starchy peas—and severely shorten the length of cropping. Flowering and early fruit set are key times to ensure an even flow of water. While peas have a tap root that can penetrate up to 3 feet, most of the effective feeding roots range from just under the surface to 12–15 inches deep. Once peas are established, and especially as they begin fruiting, they are subject to a fungal disease called powdery mildew. To help avoid the problem, either water overhead in the morning prior to a sunny stretch of weather so that the plants will dry out, or use drip tape or soaker hose around the base of the plants. Trellis or Fencing. All but the shortest varieties of peas need some sort of support. Although many varieties are advertised as self-supporting , this is not true. Fencing allows closer plant spacing , because the plants can spread out on the trellis or fence. Fencing also increases sunlight interception, minimizes disease, and facilitates easier picking. One age-old tradition for trellising peas is what the British refer to as “twiggy brush.” The branched prunings of last year’s growth from fruit trees inserted into the soil make an excellent, cheap, and somewhat artistic fence.