The advantages of a PVC drain and associated storage tank are that potentially pathogenic liquid is contained, can be recycled to maintain optimal pile moisture levels under drier conditions and, if sanitized, the nutrient content of the leachate can be recycled . In contrast, a soil floor costs less, but it is important to consider, and monitor for, the potential leaching of pathogens, nutrients that can causes algalblooms, and trace metals that could contaminate drinking water when using a permeable floor . Future studies should further explore the quantity, composition, and timing of pile leaching, and assess the efficacy of soil as a filter to avoid contamination of groundwater alongside lowering GHG emissions. Though use of a permeable soil floor and/or PVC overflow drain showed potential to reduce EcoSan composting GHG emissions, the effects of turning the pile e even once e were even greater. Emissions of CH4 dropped two orders of magnitude, approaching zero, within one day after turning and stayed comparably low through the third day. Piles in the EcoSan second stage are turned every 7e10 days , therefore it is likely that CH4 emissions remain low throughout this entire phase, as originally evidenced by the >3-month time points in the initial measurements at Compost CH and Compost PAP. From these results, it may appear to be beneficial from a climate forcing perspective to reduce the time spent in the first static phase, however this must be balanced by the need to safely manage the pathogen burden at this early treatment stage, especially if piles are turned using manual labor. Turning must only begin when pathogen abundance in the material has been reduced to a safe level, thus safeguarding the health of employees and local environment . Furthermore, though not observed in this study,black plastic planting pots past work has also shown that pile turning can increase N losses. Significant spikes in ammonia and N2O emissions follow mechanical turning of composting manure .

It is therefore possible that within EcoSan composting there may be a trade-off between N2O and CH4 emissions between the initial static and later turned stages, similar to our observations across different sanitation pathways. Our gridded sampling scheme also allowed us to test the hypothesis that aeration drives CH4 emissions within piles. The results confirmed the utility of our model-based sampling design, with mean CH4 emissions four to five times higher from pile centers than pile corners or edges, regardless of the general drainage characteristics of the pile. An alternative to early turning may be the use of additional engineering to further aerate the middle of large piles where, even under well-drained pile conditions, we observed steep increases in CH4 emissions. One solution may be use of perforated PVC pipes for passive aeration of the pile at relatively low cost.Understanding how management can best support aerobic conditions provides a win-win opportunity to increase the operational efficiency composting for treating waste while reducing the associated GHG emissions . The preliminary comparisons in this study captured significant effects of pile lining permeability and pile turning on GHG emissions during thermophilic composting, and helped us interpret the longer-term dynamics of GHG emissions during composting. Although our targeted measurements identify two of the management controls of GHG differences , robust estimates of emission factors for EcoSan composting requires a more comprehensive assessment of GHG dynamics, considering different management options, and with more extensive sampling throughout the composting operational stages. In sum these results support the potential for EcoSan composting to further reduce CH4 and overall GHG emissions associated with waste containment and treatment if piles are carefully designed and effectively managed to support aerobic metabolism.Te California poppy , also known as the golden poppy, is native to the West Coast of the United States. Te flowers are brilliant golden-orange, instantly recognizable, and widely drawn and photographed. Native Americans valued the golden poppy as a food source. First catalogued from a Russian seafaring expedition to the San Francisco Bay in the early1800s, the golden poppy was designated the state flower of California in 1903. Te golden poppy has since been inextricably linked to California pop culture, even eulogized by the novelist John Steinbeck. Te golden-orange color results from carotenoid pigments. Te carotenoid biosynthetic pathway in plants has been well characterized.

Te first committed step is the condensation of two geranylgeranyl diphosphate molecules to phytoene , catalyzed by phytoene synthase . Subsequent enzymatic steps that include desaturation, isomerization, cyclization, hydroxylation and epoxidation sequentially generate carotenoids that appear red , orange , and yellow , and combinations of these pigments create the observed palette. Notably, California poppy petals also contain abundant retro-carotenoids , generated from antheraxanthin and violaxanthin by as yet unknown enzymes. Additional proteins have been reported to modulate carotenoid biosynthesis or degradation. Carotenoids serve not only as chromoplast pigments to attract pollinators and horticulturalists, but also as chloroplast accessory pigments and antioxidants crucial for photosynthesis. For those living in or visiting California, it is not uncommon to spot the occasional white-petal California poppy in a field of orange poppies. Indeed, white-petal varieties were described from English garden hybrids as early as the 1880s, and in scientific literature from the 1930s. Biochemical and genetic studies ensued, defining the white-petal trait to be recessive and based on near absence of carotenoid pigment. By crossing different white-petal variants, including 8 originating from natural populations and 7 from commercial sources, Barrell et al. reported lack of complementation indicative of a single genetic locus. However, the gene and mutation underlying white-petal variants have yet to be discovered.To investigate the genetic basis of white-petal poppy variants, we carried out transcriptome sequencing of developing flower buds from four different commercial white poppy varieties displaying varied shades of white: Ivory Castle, White Linen, Alba, and White . Tree orange-petal poppy varieties served as controls. RNA was isolated from developing flower buds, where pigment production was presumed ongoing. Since no poppy reference genome was available, RNAseq reads were assembled de novo into transcript contigs, which were then annotated by homology to an orthologous reference transcriptome,black plastic pots for plants for which we selected another eudicot clade flowering plant, the garden tomato .Since white poppy petals are deficient in carotenoid pigments, we focused on genes of the carotenoid biosynthetic pathway. Comparing expression of carotenoid biosynthesis genes between white and orange poppy varieties, only phytoene synthase showed significantly altered expression, with an average 2.5-fold reduced transcript levels in white varieties .

While this finding focused attention on PSY, the modest reduction in white varieties was unlikely to account for a near absence of carotenoid pigment. Comparing the aligned PSY transcript reads between white and orange petal poppy varieties, all four white varieties exhibited an apparent 5bp gap within the PSY transcript . Te gap occurred within the coding region, at the site of an inferred exon-exon junction . To define the alteration at the genome level, we designed PCR primers to amplify and sequence across the exon-exon junction from genomic DNA . In the white petal varieties, the resultant PCR product was consistently smaller . Sequence alignment revealed a 76bp deletion within the PSY intron, which extended through the 3′ splice acceptor site and 5 bp of the downstream exon . By comparing the white-petal PSY genome sequence and assembled transcript contig, loss of the splice acceptor site led to usage of a cryptic splice acceptor site within the intron, resulting in a coding frame shif with early translational termination and predicted C-terminal truncation of the PSY protein . Early translational termination is associated with nonsense-mediated mRNA decay, consistent with our observed reduced PSY transcript levels. Notably, the C-terminal truncation abolishes a highly-conserved putative enzyme active site in PSY. Tat the PSY deletion is present in all four white petal varieties , but in none of the three orange petal varieties , demonstrates strong segregation with the white petal phenotype . Together, the genetic and inferred biochemical data provide strong evidence that the 76bp deletion underlies the white petal trait. Moreover, that all four white petal poppy varieties harbor precisely the same deletion suggests that they were likely all derived from a single white-petal poppy origin. In addition to studying commercial varieties, we also sought to examine white-petal specimens among wild poppy plants. To this end, we undertook expeditions to find and collect white petal specimens among California poppy fields across Santa Clara, San Mateo, and Solano counties. Of three specimens collected, all harbored precisely the same PSY mutation , suggesting that they likely represent commercial seed contaminants among orange petal varieties that were seeded rather than wild plants. Interestingly, in one field we noted orange and white poppies together with an uncommon yellow-orange petal variant. PCR analysis revealed that the yellow-orange poppy carried both the wild type and deletion PSY allele, suggesting an F1 hybrid between previously seeded orange and white petal varieties . Carotenoids are flower petal pigments, but they also provide essential roles as accessory pigments and antioxidants in chloroplasts for photosynthesis. Thus, given the PSY null mutation identified from flower buds, the existence of other PSY encoding genes seemed likely. To investigate that possibility, we carried out RNAseq from green leaf material from orange and white petal poppy varieties. Aligning the reads, only a small fraction of the PSY reads from the white-petal leaf specimen exhibited the deletion.

A distinct set of single nucleotide polymorphisms present only in the leaf RNA segregated with the non-deletion reads, allowing us to design haplotype-specifc PCR primers to amplify across the exon-exon junction. Notably, PCR of genomic DNA using the non-deletion haplotype-specific primers revealed two larger PCR products , where sequencing disclosed two different intron sequences . This finding indicates the presence of two additional PSY genes , expressed in poppy leaves. Based on the relative frequencies of SNPs in the PSY transcripts from petal and leaf tissue , we can infer that PSY1A is the only PSY gene expressed in California poppy petals, while PSY1B and PSY1C are expressed only in leaves. Nonetheless, PSY1A is also expressed in leaves where indeed it is more abundantly expressed compared to PSY1B/1C. California poppy PSY1A exhibits high homology to PSY1B/1C at the nucleotide sequence level, and 100% identity at the amino acid sequence level, suggesting relatedness by recent gene duplication. However, we note limitations of our analysis, including variable read coverages , and the challenges of phasing short reads and assigning SNPs to individual genes. A definitive analysis will require cloning the individual PSY cDNAs and genome loci. Te finding of multiple PSY gene paralogs in plants, first detailed in the tomato, is now common. Like for the California poppy, some such PSY paralogs are expressed primarily in green tissues, while others drive carotenoid accumulation in flowers, fruits, or roots. For example, in the tomato , PSY1 is predominantly expressed in the petals and ripening fruit, while PSY2 is predominant in leaves. In the loquat , PSY1 is expressed in the fruit peel, PSY2A in the ripening fruit flesh, and PSY2B in leaves. And in the carrot , PSY1 and PSY2 are expressed in the root, while PSY1 is also expressed in leaves. A comparative analysis of PSY protein sequences among eudicots reveals California poppy PSY to be most closely related to PSY from the recently sequenced opium poppy genome, and overall more closely related to the so-called Eudicot PSY1 clade. In summary, by transcriptome sequencing of California poppy flower buds, we have identified a frameshifing deletion in phytoene synthase that is common to multiple commercial white petal varieties. All have distinct white hues , but nonetheless appear to have been bred from the same common originator. Importantly, the white-petal trait in 15 different natural and commercial California poppy variants was previously shown to map to a single genetic locus. Tat study included the Alba and Ivory Castle varieties also analyzed here. Thus, we can infer that PSY1A mutations underlie all previously studied white-petal California poppy variants. Whether other variants share the same 76bp frame shifting deletion mutation remains to be determined. PSY variants/mutations have previously been associated with agriculturally important traits, e.g., color variation in tomatoes, peppers, cassavas, and loquats.