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Pepino (Solanum muricatum) is an herbaceous crop phylogenetically related to tomato and potato. Pepino fruit vary in color, size and shape, and are eaten fresh. In this study, we use pepino as a fruit model to understand the transcriptional regulatory mechanisms controlling fruit quality. To identify the key genes involved in anthocyanin biosynthesis in pepino, two genotypes were studied that contrasted in foliar and fruit pigmentation. Anthocyanin profiles were analyzed, as well as the expression of genes that encode enzymes for anthocyanin biosynthesis and transcriptional regulators using both RNA-seq and quantitative PCR. The differential expression of the transcription factor genes R2R3 MYB SmuMYB113 and R3MYB SmuATV suggested their association with purple skin and foliage phenotype. Functional analysis of these genes in both tobacco and pepino showed that SmuMYB113 activates anthocyanins, while SmuATV suppresses anthocyanin accumulation. However, despite elevated expression in all tissues, SmuMYB113 does not significantly elevate flesh pigmentation, suggesting a strong repressive background in fruit flesh tissue. These results will aid understanding of the differential regulation controlling fruit quality aspects between skin and flesh in other fruiting species.

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Certain viruses dramatically affect yield and quality of potatoes and have proved difficult to eradicate with current approaches. Here, we describe a reliable and efficient virus eradication method that is high throughput and more efficacious at producing virus-free potato plants than current reported methods. Thermotherapy, chemotherapy, and cryotherapy treatments were tested alone and in combination for ability to eradicate single and mixed Potato virus S (PVS), Potato virus A (PVA), and Potato virus M (PVM) infections from three potato cultivars. Chemotherapy treatments were undertaken on in vitro shoot segments for four weeks in culture medium supplemented with 100 mg L-1 ribavirin. Thermotherapy on in vitro shoot segments was applied for two weeks at 40°C (day) and 28°C (night) with a 16 h photoperiod. Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture followed by exposure to PVS2 either without or with liquid nitrogen (LN, cryotherapy) treatment. The virus status of control and recovered plants following therapies was assessed in post-regeneration culture after 3 months and then retested in plants after they had been growing in a greenhouse for a further 3 months. Microtuber production was investigated using in vitro virus-free and virus-infected segments. We found that thermotherapy and cryotherapy (60 min PVS2 + LN) used alone were not effective in virus eradication, while chemotherapy was better but with variable efficacy (20-100%). The most effective result (70-100% virus eradication) was obtained by combining chemotherapy with cryotherapy, or by consecutive chemotherapy, combined chemotherapy and thermotherapy, then cryotherapy treatments irrespective of cultivar. Regrowth following the two best virus eradication treatments was similar ranging from 8.6 to 29% across the three cultivars. The importance of virus removal on yield was reflected in "Dunluce" free of PVS having higher numbers of microtubers and in "V500' free of PVS and PVA having a greater proportion of microtubers > 5 mm. Our improved procedure has potential for producing virus-free planting material for the potato industry. It could also underpin the global exchange of virus-free germplasm for conservation and breeding programs.

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OBJECTIVE: To investigate whether muscle energy technique (MET) to the thoracic spine decreases the pain and disability associated with shoulder impingement syndrome (SIS). DESIGN: Single-center, 3-arm, randomized controlled trial, single-blind, placebo control with concealed allocation and a 12-month follow-up. SETTING: Private osteopathic practice. PARTICIPANTS: Three groups of 25 participants (N=75) 40 years or older with SIS received allocated intervention once a week for 15 minutes, 4 consecutive weeks. INTERVENTIONS: Participants were randomly allocated to MET to the thoracic spine (MET-only), MET plus soft tissue massage (MET+STM), or placebo. MAIN OUTCOME MEASURES: Primary outcome measure: Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire. SECONDARY OUTCOME MEASURES: Shoulder Pain and Disability Index (SPADI) questionnaire; visual analog scale (VAS) (mm/100): current, 7-day average, and 4-week average; Patient-Specific Functional Scale (PSFS); and Global Rating of Change (GROC). Measures recorded at baseline, discharge, 4-week follow-up, 6 months, and 12 months. Also baseline and discharge thoracic posture and range of motion (ROM) measured using an inclinometer. Statistical analysis included mixed-effects linear regression model for DASH, SPADI, VAS, PSFS, GROC, and thoracic posture and ROM. RESULTS: MET-only group demonstrated significantly greater improvement in pain and disability (DASH, SPADI, VAS 7-day average) than placebo at discharge (mean difference, DASH=-8.4; 95% CI, -14.0 to -2.8; SPADI=-14.7; 95% CI, -23.0 to -6.3; VAS=-15.5; 95% CI, -24.5 to -6.5), 6 months (-11.1; 95% CI, -18.6 to -3.7; -14.9; 95% CI, -26.3 to -3.5; -14.1; 95% CI, -26.0 to -2.2), and 12 months (-13.4; 95% CI, -23.9 to-2.9; -19.0; 95% CI, -32.4 to -5.7; -17.3; 95% CI, -30.9 to -3.8). MET+STM group also demonstrated greater improvement in disability but not pain compared with placebo at discharge (DASH=-8.2; 95% CI, -14.0 to -2.3; SPADI=-13.5; 95% CI, -22.3 to -4.8) and 6 months (-9.0; 95% CI, -16.9 to -1.2; -12.4; 95% CI, -24.3 to -0.5). For the PSFS, MET-only group improved compared with placebo at discharge (1.3; 95% CI, 0.1-2.5) and 12 months (1.8; 95% CI, 0.5-3.2); MET+STM at 12 months (1.7; 95% CI, 0.3-3.0). GROC: MET-only group improved compared with placebo at discharge (1.5; 95% CI, 0.9-2.2) and 4 weeks (1.0; 95% CI, 0.1-1.9); MET+STM at discharge (1.2; 95% CI, 0.5-1.9) and 6 months (1.2; 95% CI, 0.1-1.3). There were no differences between MET-only group and MET+STM, and no between-group differences in thoracic posture or ROM. CONCLUSIONS: MET of the thoracic spine with or without STM improved the pain and disability in individuals 40 years or older with SIS and may be recommended as a treatment approach for SIS.

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BACKGROUND: Shoulder impingement syndrome (SIS) is the most common form of shoulder pain. Conservative and surgical treatments for SIS are often not effective. One such surgical intervention is subacromial decompression, aimed at widening the subacromial space (SAS). A better understanding of the changes in the SAS may help explain the relative ineffectiveness of current interventions. OBJECTIVE: To measure the acromiohumeral distance (AHD) and supraspinatus tendon thickness (STT) in people with SIS using a case control study. METHODS: The AHD and STT of 39 participants with SIS ≥3 months and 39 age, gender and dominant arm matched controls were measured using ultrasound imaging. Between-group differences for AHD and STT were compared using t-tests. A linear regression was used to determine if there was a relationship between AHD and STT measures, with group as a covariate. RESULTS: Compared to controls (mean age 55.7 years, SD 10.6), individuals with SIS (mean age 57.1 years, SD 11.1) had a significantly larger AHD (mean difference 2.14 mm, 95% CI 1.21, 3.07, p < 0.001) and STT (mean difference 1.25 mm, 95% CI 0.60, 1.90, p < 0.001). The linear regression model indicated an association between AHD and STT (ß = 0.59, 95% CI 0.29, 0.89, p < 0.01, R2 = 0.35, n = 78), suggesting that as STT increases in size, so does the AHD. CONCLUSION: Individuals with SIS had a larger AHD and greater STT than controls. These results suggest the SAS is already wider in people with SIS and that the symptoms associated with SIS may be more related to an increased STT than a smaller SAS.

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Tomato fruit stored below 12°C lose quality and can develop chilling injury upon subsequent transfer to a shelf temperature of 20°C. The more severe symptoms of altered fruit softening, uneven ripening and susceptibility to rots can cause postharvest losses. We compared the effects of exposure to mild (10°C) and severe chilling (4°C) on the fruit quality and transcriptome of 'Angelle', a cherry-type tomato, harvested at the red ripe stage. Storage at 4°C (but not at 10°C) for 27 days plus an additional 6 days at 20°C caused accelerated softening and the development of mealiness, both of which are commonly related to cell wall metabolism. Transcriptome analysis using RNA-Seq identified a range of transcripts encoding enzymes putatively involved in cell wall disassembly whose expression was strongly down-regulated at both 10 and 4°C, suggesting that accelerated softening at 4°C was due to factors unrelated to cell wall disassembly, such as reductions in turgor. In fruit exposed to severe chilling, the reduced transcript abundances of genes related to cell wall modification were predominantly irreversible and only partially restored upon rewarming of the fruit. Within 1 day of exposure to 4°C, large increases occurred in the expression of alternative oxidase, superoxide dismutase and several glutathione S-transferases, enzymes that protect cell contents from oxidative damage. Numerous heat shock proteins and chaperonins also showed large increases in expression, with genes showing peak transcript accumulation after different times of chilling exposure. These changes in transcript abundance were not induced at 10°C, and were reversible upon transfer of the fruit from 4 to 20°C. The data show that genes involved in cell wall modification and cellular protection have differential sensitivity to chilling temperatures, and exhibit different capacities for recovery upon rewarming of the fruit.

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Flower sepals are critical for flower development and vary greatly in life span depending on their function post-pollination. Very little is known about what controls sepal longevity. Using a sepal senescence mutant screen, we identified two Arabidopsis mutants with delayed senescence directly connecting strigolactones with senescence regulation in a novel floral context that hitherto has not been explored. The mutations were in the strigolactone biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) and in the strigolactone receptor gene DWARF14 (AtD14). The mutation in AtD14 changed the catalytic Ser97 to Phe in the enzyme active site, which is the first mutation of its kind in planta. The lesion in MAX1 was in the haem-iron ligand signature of the cytochrome P450 protein, converting the highly conserved Gly469 to Arg, which was shown in a transient expression assay to substantially inhibit the activity of MAX1. The two mutations highlighted the importance of strigolactone activity for driving to completion senescence initiated both developmentally and in response to carbon-limiting stress, as has been found for the more well-known senescence-associated regulators ethylene and abscisic acid. Analysis of transcript abundance in excised inflorescences during an extended night suggested an intricate relationship among sugar starvation, senescence, and strigolactone biosynthesis and signalling.

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BACKGROUND: Shoulder impingement syndrome (SIS) is the most common form of shoulder pain and a persistent musculoskeletal problem. Conservative and invasive treatments, aimed at the shoulder joint, have had limited success. Research suggests shoulder function is related to thoracic posture, but it is unknown whether thoracic posture is associated with SIS. OBJECTIVE: The objective of this study was to investigate whether there is a relationship between SIS and thoracic posture. DESIGN: This was a case control study. METHODS: Thoracic posture of 39 participants with SIS and 39 age-, gender-, and dominant arm-matched controls was measured using the modified Cobb angle from a standing lateral radiograph. Thoracic range of motion (ROM) was also measured using an inclinometer. Between-group differences were compared using t tests. The relationship between thoracic posture and thoracic ROM was determined with linear regression. RESULTS: Twenty women and 19 men with SIS (mean age = 57.1 years, SD = 11.1) and 39 age-matched, gender-matched, and dominant arm-matched controls (mean age = 55.7years, SD = 10.6) participated. Individuals with SIS had greater thoracic kyphosis (mean difference = 6.2o, 95% CI 2.0-10.4) and less active thoracic extension (7.8o, 95% CI = 2.2-13.4). Greater thoracic kyphosis was associated with less extension ROM (ie, more flexion when attempting full extension: ß = 0.71, 95% CI = 0.45-0.97). LIMITATIONS: These cross-sectional data can only demonstrate association and not causation. Both radiographic measurements and inclinometer measurements were not blinded. CONCLUSION: Individuals with SIS had a greater thoracic kyphosis and less extension ROM than age- and gender-matched healthy controls. These results suggest that clinicians could consider addressing the thoracic spine in patients with SIS.

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Selenium (Se) is an essential micronutrient for human health, entering the diet mainly through the consumption of plant material. Members of the Brassicaceae are Se-accumulators that can accumulate up to 1g Se kg-1 dry weight (DW) from the environment without apparent ill effect. The Brassicaceae also produce glucosinolates (GSLs), sulfur (S)-rich compounds that benefit human health. Radish (Raphanus sativus) has a unique GSL profile and is a Se-accumulating species that is part of the human diet as sprouts, greens and roots. In this report we describe the effects of Se-fertilisation on GSL production in radish during five stages of early development (from seed to mature salad greens) and on the transcript abundance of eight genes encoding enzymes involved in GSL metabolism. We tentatively identified (by tandem mass spectrometry) the selenium-containing glucosinolate, 4-(methylseleno)but-3-enyl glucosinolate, with the double bond geometry not resolved. Two related isothiocyanates were tentatively identified by Gas Chromatography-Mass Spectrometry as (E/Z?) isomers of 4-(methylseleno)but-3-enyl isothiocyanate. Se fertilisation of mature radish led to the presence of selenoglucosinolates in the seed. While GSL concentration generally reduced during radish development, GSL content was generally not affected by Se fertilisation, aside from the indole GSL, indol-3-ylmethyl glucosinolate, which increased on Se treatment, and the Se-GSLs, which significantly increased during development. The transcript abundance of genes involved in aliphatic GSL biosynthesis declined with Se treatment while that of genes involved in indole GSL biosynthesis tended to increase. APS kinase transcript abundance increased significantly in three of the four developmental stages following Se treatment. The remaining genes investigated were not significantly changed following Se treatment. We hypothesise that increased APS kinase expression in response to Se treatment is part of a general protection mechanism controlling the uptake of S and the production of S-containing compounds such as GSLs. The upregulation of genes encoding enzymes involved in indole GSL biosynthesis and a decrease in those involved in aliphatic GSL biosynthesis may be part of a similar mechanism protecting the plant's GSL complement whilst limiting the amount of Se-GSLs produced.

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KEY MESSAGE: Viral-induced gene silencing of selected biosynthetic genes decreased overall carotenoid accumulation in California poppy. Regulation of carotenogenesis was linked with pigment sequestration, not changes in biosynthetic gene expression. Genes of carotenogenesis are well described, but understanding how they affect carotenoid accumulation has proven difficult because of plant lethality when the pigments are lacking. Here, we used a Tobacco Rattle Virus-based virus-induced-gene-silencing (VIGS) approach in California poppy (Eschscholzia californica) to investigate how silencing of the carotenoid biosynthetic pathway genes affects carotenoid metabolite accumulation and RNA transcript abundance of the carotenoid biosynthetic pathway genes. VIGS of upstream (PDS and ZDS) and downstream (ßOH and ZEP) genes reduced transcript abundance of the targeted genes in the poppy petals while having no effect on abundance of the other carotenogenesis genes. Silencing of PDS, ZDS, ßOH and ZEP genes reduced total pigment concentration by 75-90% and altered petal colour. HPLC and LC-MS measurements suggested that petal colour changes were caused by substantially altered pigment profiles and quantity. Carotenoid metabolites were different to those normally detected in wild-type petals accumulated but overall carotenoid concentration was less, suggesting the chemical form of carotenoid was important for whether it could be stored at high amounts. In poppy petals, eschscholtzxanthin and retro-carotene-triol were the predominant carotenoids, present mainly as esters. Specific esterification enzymes for specific carotenoids and/or fatty acids appear key for enabling petal carotenoids to accumulate to high amounts. Our findings argue against a direct role for carotenoid metabolites regulating carotenogenesis genes in the petals of California poppy as transcript abundance of carotenogenesis genes studied was unchanged, while the petal carotenoid metabolite profile changed substantially.

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BACKGROUND: Radiographs are used to monitor thoracic kyphosis in individuals with certain pathologies (e.g. osteoporosis), exposing patients to potentially harmful radiation. Thus, other measures for monitoring the progression of thoracic kyphosis are desirable. The gravity-dependent inclinometer has been shown to be reliable but its validity as a measure of thoracic kyphosis has not been investigated. OBJECTIVES: To determine the validity of the gravity-dependent (analogue) inclinometer for measuring thoracic kyphosis. DESIGN: Cross-sectional study. METHOD: Participants (n=78) were recruited as part of a larger study of shoulder impingement syndrome. Healthy participants (n=39) were age and gender matched to the shoulder impingement syndrome group (n=39). Measurements of thoracic kyphosis using a gravity-dependent inclinometer were compared with modified Cobb angle results obtained from a sagittal view of lateral radiographs. A Bland-Altman plot assessed agreement. The Pearson correlation coefficient and linear regression was used to determine the association between modified Cobb angles and inclinometer measurements. RESULTS: The Bland-Altman plot demonstrated good agreement. The Pearson correlation coefficient, r=0.62 (p<0.001), and linear regression model established a strong association between the thoracic kyphosis angle from the inclinometer readings and the modified Cobb angle measured from the radiographs (ß=0.47, 95% CI 0.29, 0.65, p<0.001, R2=0.52, n=78). Age as a confounder was included in the model (ß=0.35, 95% CI 0.19, 0.51, p<0.001). CONCLUSIONS: The gravity-dependent (analogue) inclinometer produces angles that are comparable to the modified Cobb angle obtained from radiographs, establishing its criterion validity as a safe clinical tool for measuring thoracic kyphosis.

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Postharvest deterioration of fruits and vegetables can be accelerated by biological, environmental, and physiological stresses. Fully understanding tissue response to harvest will provide new opportunities for limiting postharvest losses during handling and storage. The model plant Arabidopsis thaliana (Arabidopsis) has many attributes that make it excellent for studying the underlying control of postharvest responses. It is also one of the best resourced plants with numerous web-based bioinformatic programs and large numbers of mutant collections. Here we introduce a novel assay system called AIDA (the Arabidopsis Inflorescence Degreening Assay) that we developed for understanding postharvest response of immature tissues. We also demonstrate how the high-throughput screening capability of AIDA can be used with mapping technologies (high-resolution melting [HRM] and needle in the k-stack [NIKS]) to identify regulators of postharvest senescence in ethyl methanesulfonate (EMS) mutagenized plant populations. Whether it is best to use HRM or NIKS or both technologies will depend on your laboratory facilities and computing capabilities.

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Broccoli (Brassica oleracea L. var. italica) sprouts contain glucosinolates (GLs) that when hydrolysed yield health promoting isothiocyanates such as sulforaphane (SF). SF content can be increased by salt (NaCl) stress, although high salt concentrations negatively impact plant growth. Salicylic acid (SA) treatments can attenuate the negative effects of salt on growth. To test whether sprout isothiocyanate content could be elevated without sprout growth being compromised, broccoli seed were germinated and grown for seven days in salt (0, 80 and 160 mM) alone and in combination with 100 µM SA. Increasing concentrations of salt lowered transcript accumulation of GL biosynthetic genes which was reflected in lowered content of Gluconapin, 4-methoxyglucobrassicin and neoglucobrassicin glucosinolates. Other glucosinolates such as glucoraphanin did not alter significantly. Salt (160 mM) increased transcript abundance of the GL hydrolytic gene MYROSINASE (BoMYO) and its cofactor EPITHIOSPECIFIER MODIFIER1 (BoESM1) whose encoded product directs MYROSINASE to produce isothiocyanate rather than nitrile forms. SF content was increased 6-fold by the 160 mM salt treatment, but the salt treatment reduced percentage seed germination, slowed seed germination, and reduced sprout hypocotyl elongation. This growth inhibition was prevented if 100 µM SA was included with the salt treatment. These findings suggest that the increase in SF production by salt occurs in part because of increased transcript abundance of genes in the hydrolytic pathway, which occurs independently of the negative impact of salt on sprout growth.

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Galactose (Gal) is incorporated into cell wall polysaccharides as flowers open, but then is lost because of ß-galactosidase activity as flowers mature and wilt. The significance of this for flower physiology resides in the role of galactan-containing polysaccharides in the cell wall, which is still largely unresolved. To investigate this, transcript accumulation of six cell wall-associated ß-galactosidases was simultaneously knocked down in 'Mitchell' petunia (Petunia axillaris x (P. axillaris x P. hybrida)) flower petals. The multi-PhBGAL RNAi construct targeted three bud- and three senescence-associated ß-galactosidase genes. The petals of the most down-regulated line (GA19) were significantly disrupted in galactose turnover during flower opening, and at the onset of senescence had retained 86% of their galactose compared with 20% in the controls. The Gal content of Na2CO3-soluble cell wall extracts and the highly insoluble polysaccharides associated with cellulose were particularly affected. Immunodetection with the antibody LM5 showed that much of the cell wall Gal in GA19 was retained as galactan, presumably the side-chains of rhamnogalacturonan-I. The flowers of GA19, despite having retained substantially more galactan, were no different from controls in their internal cell arrangement, dimensions, weight or timing of opening and senescence. However, the GA19 petals had less petal integrity (as judged by force required to cause petal fracture) after opening and showed a greater decline in this integrity with time than controls, raising the possibility that galactan loss is a mechanism for helping to maintain petal tissue cohesion after flower opening.

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The signal that initiates the age-regulated senescence program in flowers is still unknown. Here we propose for the ephemeral Arabidopsis thaliana flower that it dies because of continued expression of the MADS-box transcription factor AGAMOUS (AG). AG is necessary for specifying the reproductive structures of the flower. Flowers of ag-1, which lack AG, exhibited delayed sepal senescence and abscission. The flowers also had reduced jasmonic acid (JA) content. Other anther-defective sterile mutants deficient in JA, defective in anther dehiscence 1 (dad1) and delayed dehiscence 2 (dde2), exhibited delayed sepal senescence and abscission as well. Manually pollinated dad1 flowers produced siliques but still had delayed senescence, demonstrating that absence of pollination does not cause delayed senescence. When ag-1, dad1 and dde2 flowers were sprayed with 100 µM methyl jasmonate, the sepal senescence and abscission phenotypes were rescued, suggesting that JA has a role in these processes. Our study uncovers a novel role for AG in determining the timing of death of the flower it helps develop and highlights a role for JA in sepal senescence.

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Flowers are complex systems whose vegetative and sexual structures initiate and die in a synchronous manner. The rapidity of this process varies widely in flowers, with some lasting for months while others such as Hibiscus rosa-sinensis survive for only a day. The genetic regulation underlying these differences is unclear. To identify key genes and pathways that coordinate floral organ senescence of ephemeral flowers, we identified transcripts in H. rosa-sinensis floral organs by 454 sequencing. During development, 2053 transcripts increased and 2135 decreased significantly in abundance. The senescence of the flower was associated with increased abundance of many hydrolytic genes, including aspartic and cysteine proteases, vacuolar processing enzymes, and nucleases. Pathway analysis suggested that transcripts altering significantly in abundance were enriched in functions related to cell wall-, aquaporin-, light/circadian clock-, autophagy-, and calcium-related genes. Finding enrichment in light/circadian clock-related genes fits well with the observation that hibiscus floral development is highly synchronized with light and the hypothesis that ageing/senescence of the flower is orchestrated by a molecular clock. Further study of these genes will provide novel insight into how the molecular clock is able to regulate the timing of programmed cell death in tissues.

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Stresses such as energy deprivation, wounding and water-supply disruption often contribute to rapid deterioration of harvested tissues. To uncover the genetic regulation behind such stresses, a simple assessment system was used to detect senescence mutants in conjunction with two rapid mapping techniques to identify the causal mutations. To demonstrate the power of this approach, immature inflorescences of Arabidopsis plants that contained ethyl methanesulfonate-induced lesions were detached and screened for altered timing of dark-induced senescence. Numerous mutant lines displaying accelerated or delayed timing of senescence relative to wild type were discovered. The underlying mutations in three of these were identified using High Resolution Melting analysis to map to a chromosomal arm followed by a whole-genome sequencing-based mapping method, termed 'Needle in the K-Stack', to identify the causal lesions. All three mutations were single base pair changes and occurred in the same gene, NON-YELLOW COLORING1 (NYC1), a chlorophyll b reductase of the short-chain dehydrogenase/reductase (SDR) superfamily. This was consistent with the mutants preferentially retaining chlorophyll b, although substantial amounts of chlorophyll b were still lost. The single base pair mutations disrupted NYC1 function by three distinct mechanisms, one by producing a termination codon, the second by interfering with correct intron splicing and the third by replacing a highly conserved proline with a non-equivalent serine residue. This non-synonymous amino acid change, which occurred in the NADPH binding domain of NYC1, is the first example of such a mutation in an SDR protein inhibiting a physiological response in plants.

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BACKGROUND: With the explosive numbers of sequences generated by next generation sequencing, the demand for high throughput screening to understand gene function has grown. Plant viral vectors have been widely used as tools in down-regulating plant gene expression. However, plant viral vectors can also express proteins in a very efficient manner and, therefore, can also serve as a valuable tool for characterizing proteins and their functions in metabolic pathways in planta. RESULTS: In this study, we have developed a Gateway®-based high throughput viral vector cloning system from Narcissus Mosaic Virus (NMV). Using the reporter genes of GFP and GUS, and the plant genes PAP1 (an R2R3 MYB which activates the anthocyanin pathway) and selenium-binding protein 1 (SeBP), we show that NMV vectors and the model plant Nicotiana benthamiana can be used for efficient protein expression, protein subcellular localization and secondary metabolite production. CONCLUSIONS: Our results suggest that not only can the plant viral vector system be employed for protein work but also can potentially be amenable to producing valuable secondary metabolites on a large scale, as the system does not require plant regeneration from seed or calli, which are stages where certain secondary metabolites can interfere with development.

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Leaf senescence is a genetically controlled dismantling programme that enables plants to efficiently remobilise nutrients to new growing sinks. It involves substantial metabolic reprogramming whose timing is affected by developmental and environmental signals. Plant hormones have long been known to affect the timing of leaf senescence, but they also affect plant development and stress responses. It has therefore been difficult to tease apart how the different hormones regulate the onset and progression of leaf senescence, i.e., whether they directly affect leaf senescence or affect it indirectly by altering the developmental programme or by altering plants' response to stress. Here we review research on hormonal regulation of leaf senescence and propose that hormones affect senescence through differential responses to developmental and environmental signals. We suggest that leaf senescence strictly depends on developmental changes, after which senescence can be induced, depending on the type of hormonal and environmental cues.

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O-acetylserine (thiol) lyases (OASTLs) are evolutionarily conserved proteins among many prokaryotes and eukaryotes that perform sulfur acquisition and synthesis of cysteine. A mutation in the cytosolic OASTL-A1 protein ONSET OF LEAF DEATH3 (OLD3) was previously shown to reduce the OASTL activity of the old3-1 protein in vitro and cause auto-necrosis in specific Arabidopsis accessions. Here we investigated why a mutation in this protein causes auto-necrosis in some but not other accessions. The auto-necrosis was found to depend on Recognition of Peronospora Parasitica 1 (RPP1)-like disease resistance R gene(s) from an evolutionarily divergent R gene cluster that is present in Ler-0 but not the reference accession Col-0. RPP1-like gene(s) show a negative epistatic interaction with the old3-1 mutation that is not linked to reduced cysteine biosynthesis. Metabolic profiling and transcriptional analysis further indicate that an effector triggered-like immune response and metabolic disorder are associated with auto-necrosis in old3-1 mutants, probably activated by an RPP1-like gene. However, the old3-1 protein in itself results in largely neutral changes in primary plant metabolism, stress defence and immune responses. Finally, we showed that lack of a functional OASTL-A1 results in enhanced disease susceptibility against infection with virulent and non-virulent Pseudomonas syringae pv. tomato DC3000 strains. These results reveal an interaction between the cytosolic OASTL and components of plant immunity.

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