Nitrate at this depth is neither available for trees, since the majority of active fibrous roots of orange trees are in the top 15–30 cm depth , nor can it be easily transformed because of the limited microbial population and available carbon at this depth . This can potentially lead to eventual leaching losses to receiving water bodies. Hence over-irrigation not only led to a profound water loss, but also brought about a reduction in plant nitrogen uptake and an increase in potential danger of appreciable NO3-N leaching losses in all fertigation scenarios. Therefore, the combination of inadequate management of irrigation and nitrogen fertilizers in commercial agriculture may lead to considerable nitrate losses out of the root zone, and may increase the risk of nitrate contamination of ground water aquifers.Petal senescence is the irreversible and final stage of floral differentiation and development, associated with dynamic alterations once a flower has been successfully pollinated. However, it is not clear how the process is regulated genetically. Homeostasis or alterations of plant hormones is involved in the onset of floral senescence. In ethylene -sensitive flowers, the first sign of visible senescence is accompanied by a transient and sudden rise of ET production. Other hormones such as cytokinin, abscisic acid , auxin, gibberellic acid,hydroponic nft system and jasmonic acid are also involved in ET-sensitive petal senescence. ABA accelerates petal senescence.
Treatment with ABA promotes the large increase in ET production and hastens petal wilting in carnation flowers. Pretreatments with silver thiosulfate , a chemical that inhibits the perception of ET by the ET receptor, completely prevents the increase in ABA levels. A negative relationship was observed between the level of CKs and petal senescence in petunia and carnation. In rose, the increase of CK content antagonized petal senescence prompted by ET. Applying CKs delayed petunia petal senescence. Auxin also plays a role in ET-sensitive petal senescence. Application of auxin prompted ET production and petal wilting in cut carnation flowers. In addition, 2,4-dichlorophenoxyacetic acid , a synthetic auxin, induced the expression of 1-aminocyclopropane-1-carboxylic acid synthase genes in petals. In most research, these hormones are used as exogenous regulators to observe ET sensitivity and floral longevity in ET-sensitive species. Although the enrichment of ‘response to 1-aminocyclopropase-1- carboxylic acid and auxin stimulus’ was observed 12 h after pollination in the petals of petunia, the differential expression patterns of genes related to these hormones in petal senescence is unclear. Petal senescence is regulated by transcription factors . On one hand, ethylene-insensitve-like and ethylene response factors are correlated with the ET response signaling pathway. EIL3, a homolog of ETinsensitive 3 in carnation, is a pivotal switch of ET induced gene expression. DAFSAG9, which is homologous to ERF2, was significantly upregulated in senescing daffodil petals. On the other hand, a large group of other TFs, such as B-box zinc finger, bHLH DNA-binding, homeodomain-like , MADS-box, MYB, and NAC, display differential expression when ET-insensitivity is induced in the etr1-1 transgenic petunia. More than 20 members from the ERF, NAC, bZIP, HD-Zip, and WRKY TF families showed differential expression in petals at the early stage of pollination-induced senescence in petunia.
In addition, NAC, Aux/IAA, MYB, bZIP, and MADS-box are differentially expressed during carnation petal senescence. These studies indicate that these TFs play regulating roles in ET-dependent petal senescence. However, the biological functions of these TFs are largely unknown. High-throughput gene expression analysis using messenger RNA sequencing represents the most powerful tool to elucidate the underlying regulatory mechanism of corolla senescence. Recently, pollination- and ET-induced corolla senescence in petunia has been studied through RNA-Seq analysis16,20, however, the regulatory mechanisms that govern the onset of natural corolla senescence from opening to wilting in petunia is unclear. Therefore, identifying the dynamic processes and regulatory factors in transcription is a crucial step in determining the master switches in corolla senescence. We employed RNA-seq technology to investigate the global and chronological sequence of transcriptional events during the initial corolla senescence in petunia. Furthermore, virus-induced gene silencing system was used to dissect biological functions of potential regulatory genes such as TFs. Our data suggest that hormonal interactions between auxin and ET may play a critical role in the regulation of onset of corolla senescence in petunia.One microgram of total RNA was reverse-transcribed using PrimeScript RT reagent with gDNA Eraser Kit , according to the manufacturer’s instructions. Specific primers were designed by the Primer 3 program and listed in Supplementary Table S1. Amplifications were performed in an Applied Bio-systems 7300 system . Melting curve analysis was performed and the absence of non-specific products and primer dimers were verified. For data analysis, average threshold cycle values were calculated for each gene of interest, on the basis of three independent biological samples and were normalized and used to calculate relative transcript levels as described elsewhere. 26S ribosomal RNA was used as an internal standard for normalization.ET emission was monitored using a laser-based ET detector and a gas handling system as described previously. Briefly, flowers collected at D0 were placed into 70 ml sealed glass vials.
The air was passed through a platinum-based catalyzer before entering the cuvettes in order to remove external ET and other hydrocarbons. A scrubber with KOH and CaCl2 was used to reduce the CO2 and the water content in the gas flow. ET emission was monitored and recorded in real time. Three biological replicates of every flowering stage were performed. Each experiment was repeated three times.To measure longevity of intact flowers, white flowers from pTRV/CHS-TFs inoculated petunia ‘Primetime Blue’ plants were tagged at D0. The time when the corollas wilted and the edges collapsed was recorded. At least 20 flowers of three plants from each of the three independent biological replicates were monitored. Purple flowers from water-inoculated wild-type and white flowers from pTRV/CHS-silenced plants were used as controls. Statistical analyses were performed using the SPSS package . One-way analysis of variance was performed for experiments with one independent variable. Duncan’s test was used as the post hoc test if significant differences were found.Flowers that were fully opened but anthers not yet dehisced were marked as D0. The corollas continued to expand for 2 days. Visible senescence symptoms, such as curving of the corolla edges,nft channels were observed at an average of 4 days. Corolla wilting was found at about 7–8 days . We measured ET production using a real-time ET detection system, EDT-300. An increase and decrease of ET emission was detected during D2–D7 stage. The level spiked around D4, reaching the maximum level at 5.5 days, and then decreasing sharply .In order to determine the alteration in gene expression during corolla senescence, we generated cDNA libraries composed of the samples collected from four developmental stages with two biological replicates. RNA sequencing of these libraries produced 49,421,030, 52,985,600, 47,813,446, and 56,552,704 clean reads at D0, D2, D4, and D7, respectively . The sequences were mapped to the P. axillaris reference genome for annotation of all unigenes. The mapping rate was over 93% for samples of each stage . Differential expression analysis was conducted by comparing four different developmental stages. Analysis on all four stages generated 5167 unigenes that were significantly differentially expressed across these stages. The number of DEGs was decreased from 4626 between D0 and D2, to 1116 between D2 and D4, and to 327 between D4 and D7 . DEGs were clustered to generate expression patterns based on time series using the STEM software. Cluster analysis of the data from four time points generated 26 clusters, including down regulated genes in clusters 0 through 12 and upregulated genes in clusters 13 through 25 . A few clusters displayed a more complex pattern. For instance, clusters 2, 5, 7, 8, and 11 showed an initial decrease followed by upregulation. However, clusters 14 and 17 exhibited an initial increase followed by a decline . In addition, the down regulated clusters 3 and 4 and the upregulated clusters 15, 16, 21, 24, and 25 were statistically significant .In order to identify up and down regulated GO at each selected time point, seven gene clusters exhibiting either significantly decreased or increased expression were further analyzed using Cytoscape software with its GO enrichment tool BiNGO. At the transition from D0 to D2, the metabolic processes of major macro-nutrients including ‘carbohydrates, lipids, aromatic amino acids, and nitrogen compounds’ were down regulated . In addition, ‘cell wall organization and bio-genesis’, ‘Sadenosylmethionine biosynthesis’, and ‘negative regulation of transcription, DNA-dependent’ and ‘RNA metabolism’ were down regulated . However, ‘CK pathway’, ‘RNA modification’, ‘macromolecule methylation’, ‘DNA metabolism’, ‘ATP activity’, and ‘S-adenosylmethionine-dependent methyltransferase activity’ were upregulated . At the transition from D2 to D4, ‘monosaccharide metabolism ’, ‘polysaccharide metabolism ’, ‘lipid catabolism’, ‘amino acid metabolism ’, ‘Sadenosylmethionine biosynthesis’, and ‘L-phenylalanine biosynthesis’ were significantly upregulated . ‘Response to auxin stimulus’ was also significantly upregulated .
Down regulated GO terms were mainly ‘nicotianamine metabolism and biosynthesis’ .At the transition from D4 to D7, ‘iron ion binding’ was significantly upregulated. The only down regulated biological process was the auxinmediated signaling pathway . The over representation of ‘ribosome and cytosolic small ribosomal subunit’ was also enriched in the down regulated GO group .ET is a key flower senescence promoting hormone in ET-sensitive species. In this study, transcriptional dynamics at four distinct developmental stages of corollain petunia were monitored. The ‘S-adenosylmethionine biosynthesis’ GO term was significantly upregulated at the D2 and D4 transition . Expression of ACS and ACO genes was upregulated through D2 to D4 and D4 to D7 transitions. The increase of ET emission was initially detected at the D2 to D4 transition, while the spike of ET emission occurred at the D4 to D7 transition . These data suggest that early onset of corolla senescence may occur in the transition from D2 to D4, and execution of senescence takes place in the transition from D4 to D7.Notably, large alterations in abundances of auxinrelated transcripts occurred throughout the four developmental stages, especially through the transition from D2 to D4. Although, at present, the role of auxin in plant senescence remain poorly defined, and contrasting observations have been obtained from different species. Several studies have reported an involvement of auxin in the process of senescence, especially in petal senescence. For example, in cut carnation flowers, exogenous application of IAA hastened the rise in ET production and flower wilting. 2,4-dichlorophenoxyacetic acid , a synthetic auxin, induced the expression of ACC synthase genes in the styles, ovaries, and petals. It was reported that in the corollas of pollinated petunias, ‘response to auxin stimulus’ and ‘response to ACC’ were significantly enriched at 12 hap. Interaction between auxin and ET occurred at the early stage of pollination. Furthermore, the interaction between ET and auxin was also reported in ET-induced corolla senescence in petunia. Interestingly, during pear ripening, the auxin-associated transcripts are significantly upregulated in the S2 to S3 transition before pear ripening and down regulated in the S3 to S4 transition. In addition, auxin level declined prior to ripening in tomato, grape, and strawberry fruit. Moreover, the largest number of DEGs related to auxin were observed in the abscission process of rose petal. Down regulation of RhIAA16 by VIGS in rose promoted petal abscission. In our transcriptome data, DEGs in the auxin pathway, including auxin-responsive genes , auxin-induced genes , and auxin efflux carrier were all induced at the D2 to D4 transition, where ET production was increased. However, those auxin-related genes were down regulated in the D4 to D7 transition , while expression of ACO and ACS genes was upregulated and ET production reached a peak at 5.5 days . Taken together, we postulate that auxin might play common and vital positive roles in activating ET production and regulating developmental process that lead to subsequent attainment of ripening, senescence, and abscission capacity.Ecolabels are part of a new wave of environmental policy that emphasizes information disclosure as a tool to induce environmentally conscious behavior by both firms and consumers. The goal of ecolabels is to provide easily understood information and thereby elicit increased demand for products perceived as environmentally friendly. An important concern among consumers is that ecolabeled products might entail a trade-off between product quality and environmental impact. In other words, in order to achieve low environmental impact, green products would have to be of lower quality. In this study, we use the case of ecocertification in the wine industry to test the link between environmentally friendly production and product quality.