Physalis, a berry, is encapsulated by a novel morphological feature of the post-floral, accrescent calyx that is referred to as a Chinese lantern. The evolutionary developmental genetics of the Chinese lantern have been investigated in the last decade; however, the selective values of the morphological novelty remain elusive. Here, we measured the photosynthetic parameters of the fruiting calyces, monitored microclimatic variation within the Chinese lanterns during fruit development, performed floral-calyx-removal experiments, and recorded the fitness-related traits in Physalis floridana. Ultimately, we show that the green-fruiting calyx of Physalis has photosynthetic capabilities, thus serving as an energy source for fruit development. Moreover, the developing Chinese lantern provides a microclimate that benefits the development and maturation of berry and seed, and it improves plant fitness in terms of fruit/seed weight and number, and fruit maturation under low-temperature environments. Furthermore, the lantern structure facilitates the dispersal of fruits and seeds by water and wind. Our results suggest that the Chinese lantern morphology of Physalis is an evolutionary adaptive trait and improves plant fitness, thus providing new insight into the origin of morphological novelties.The origin of morphological novelties is an important but neglected issue of evolutionary biology. The fruit of the genus  Flowers are a characteristic of angiosperms, and the evolution of the flower form is largely related to facilitating pollination to increase the rate of reproduction6. The perianth  is generally considered to be a protective structure for the central reproductive organs and an attractant for pollinators7. In some plants the perianth persists and continues to grow in order to enhance the attraction to pollinators, such as the sepals of the genus Helleborus, which are enlarged and as brightly colored as petals8. Nonetheless, persistent sepals , e.g. in Solanaceae, are not necessarily modified for this purpose. The post-floral calyx morphology, including size and shape, at the fruiting stages in Solanaceae is extremely diversified10. In Physalis, the post-floral calyx becomes inflated and resembles a Chinese lantern. This structure is also called inflated calyx syndrome (ICS) and this morphological novelty distinguishes Physalis from most other genera within the Solanaceae11.Morphological variations and morphological novelties are assumed to be a result of adaptive evolution. The developmental mechanisms for evolution are summarized for morphological diversificationPhysalis floridana MADS-box gene 2 (MPF2) in floral organs11. Compared to its ortholog in Solanum tuberosum, the heterotopic expression of MPF2 in the floral organs of Physalis may result from variation in the CArG-boxes in its promoter11. The phenotypic variation of MPF2 knockdowns supports the role of MPF2 in male fertility and the post-fruiting growth of the calyx in P. floridana11, which was further justified by overexpression of this gene synthesized ICS-like trait in Solanum12. The Chinese lantern is derived from the floral calyx after pollination without changing organ identity because downregulation of another P. floridana MADS-box gene 3 (MPF3), which is an euAP1 gene, generates a leaf-like floral calyx, a small lantern, and poor male fertility13. Moreover, MPF3 interacts with MPF2 to bind the variants of the CArG-boxes in the MPF2 promoter13, thus governing the floral expression of MPF2, which is the key to the origin of the Chinese lantern in Physalis.How did the novelty arise? We previously found that the origin of the Chinese lantern is associated with the heterotopic expression of the Margaranthus, Przewalskia, and Withania10, which evidence for its phylogenetic polyphyly15; however, the expression of MPF2-like genes in floral organs is plesiomorphic within the family Solanaceae10. Moreover, MPF2-like variations  define the evolutionary pattern of the ICS in Solanaceae17. Furthermore, fossils of lantern-like fruits from the Eocene (52.2-Ma) suggest the structure has an ancient history and that the origin of ICS occurred in a lakeside rainforest paleoenvironment18. Therefore, Solanaceae is a logical family with which to study the genetics, biosynthesis, and evolution of ICS-like traits18. The selective value of the Chinese lantern remains unknown; its potential adaptive functions have been discussed previously19, and include facilitating wind-mediated fruit dispersal9, maintaining an adaptive microclimate16, originating as a byproduct of fertility evolution20, and being a source of photosynthesis and protection of inner organs12. This accessory structure may benefit berry development and seed dispersal, but empirical evidence is lacking. Thus, this study aimed to assess the potential selective values of the Chinese lantern in Physalis by evaluating its biological and ecological roles.The Chinese lantern-like structure is found in a few additional genera of the Solanaceae, such as Physalis floridana started to inflate and surround the developing berry, as documented by images of 5 days after fertilization (5DF), 20 days after fertilization (20DF), and mature calyces , seed number per berry, and 100-seed weight, were recorded. When calyx and carpopodium turned yellow . Besides the obvious differences between the fruit-character values of naked and bagged Physalis berries as demonstrated above, the survival rate of mature fruits showed a similar reduction pattern in these cases compared to the intact one .To reveal the benefits of the calyx to the berry, we conducted calyx-removal experiments at the blooming stage. Calyces were completely removed, and the remaining floral organs were left untouched and covered with small waterproof and non-transparent paper bags. These were designated the bagged low Fig. , the fruits Fig. . Howeverits Fig. . Seed nuits Fig. , and 100its Fig. . The extone Fig. . In addiier Fig. . These o(Ti-Te)] of three periods all appeared around 13:00, the hottest time point in a day, and the internal temperature of 5 DF lantern was lower than the external one, which is opposite to that of Chinese lantern stages of MFr and 20 DF  within the calyx lantern under various conditions. Under normal growth conditions, diurnal variation of temperature and RH was monitored. Temperature within the lantern (Ti) of three developmental stages  varied differently as external temperature (Te) changed Fig. . Peak of DF Fig. . Besides DF Fig. . However DF Fig. , indicatWe also observed that both Ti and RHi were higher than external ones in 20 DF lanterns as the Te dropped from 28 °C to 10 °C, and as the RHe changed from 55% to 30% Fig. . FurtherPhysalis berries that had the calyx removed became shriveled and wrinkled after one month, and had significantly lower weight  plants that were placed in 16 °C incubator after flowering Fig. . MaturatPhysalis and tomato; however, the Chinese lanterns protect berries from low-temperature environments and thus improve plant fitness.These results suggest low temperature is deleterious to fruit development in Physalis and tomato fruits in water and found that Physalis fruits floated for a long period of time until water entered the lantern; in contrast, when the calyx was removed, the naked Physalis berries immediately sank (as the tomato berries did)  Fig. , indicatets Fig. . Tomato iod Fig. . If wateint Fig. . No signies Fig. . TherefoPhysalis berries and the intact Physalis fruits were placed into three circles  and naked Physalis berries (21.7%) had moved <50 cm  might have the primary function of green leaves in producing photosynthate23. Calyx removal has an effect on the delivery of sucrose into sink organs, and leads to reduction in fruit weight in Diospyros kaki25. In tomato, the green calyx contributes to 2% of fruit weight22. In Physalis, through calyx removal and physiological tests, we found that the inflated green calyx also serves a photosynthetic source for fruit and seed development. The wounding effect on fruit development was not excluded; nonetheless, calyx removal at the blooming stage led to significant reductions in fruit and seed weight in the naked Physalis berries, indicating that photosynthesis in the inflated calyx of Physalis very likely contributes to fruit and seed development. The photosynthetic role of the green calyx is not surprising because this activity is a fundamental attribute of green plant tissues27. However, bagging the naked Physalis berry after calyx removal rescued around 10% of the weights of berries and seeds, implying that the observed weight reduction resulted from more than the photosynthetic capability of the green lanterns.The Chinese lantern structure is derived from an accrescent calyx, collective of sepalsHelleborus foetidus (Ranunculaceae), which has large, persistent green sepals that contribute to fitness (enhanced seed number and size)8. Our data also suggest that the inflated calyx could improve plant fitness in Physalis. However, unlike the enlarged and flat sepals in H. foetidus8, the fruiting calyx of Physalis develops into a lantern with a small opening at its tips, thus forming a relatively closed microclimate or chamber. The differences we observed in fruit and seed weights between naked and bagged Physalis berries in these calyx-removal experiments implies that the Chinese lantern structure provides a microclimate beneficial to fruit and seed development. Moreover, the amount of chlorophyll was extremely small and the rate of photosynthesis of the Chinese lanterns was quite low, and they had a tendency to decline as the lanterns developed. Calyx removal at later stages (after pollination) had less of an effect on berry development12; however, the photosynthetic output of the lanterns, which revealed by removing the calyx at the blooming stage, seemed to be significant. The underlying mechanisms for this striking contrast are unclear, but it might be partially associated with microclimate effects of the Chinese lanterns (see below).Our findings are consistent with previous observations in 28, and thus plant fitness. Calyx-removal from floral buds suggested that the floral calyx is important to fruit development in Physalis12. In this study, seed number per berry and seed size were reduced; additionally, fruit setting, survival rate, and fruit weight were reduced among calyx-removed treatments at the blooming stage following artificial pollination. We thus predicted that the Chinese lantern structure is beneficial to fitness. We also observed that the lantern can buffer the inside temperature and humidity as the external environment changes. Under 16 °C conditions, the internal temperature and relative humidity were higher inside the Chinese lanterns, providing relatively optimal microenvironments. The crucial contributing role of Chinese lantern to fruit development and fitness, such as by controlling microclimate, was further demonstrated under the low-temperature conditions. Moderate low temperature (approximately 19 °C) disrupts pollen development and causes severe reductions in rice grain yields via reducing gibberellin29. To avoid the cool temperature damage at floral stages, Physalis seedlings at the blooming stage were transferred into 16 °C constant incubators, and artificial pollination was carried out. Under the low temperature, intact Physalis fruit development was significantly retarded and fruits needed twice the amount of time to mature compared with naturally developed fruits; moreover, fruit setting rate , and the weights of berries and seeds were dramatically reduced. After removing the floral calyx under low temperature, although fertilization could be done, the naked Physalis berries hardly ripened, and the seeds in these berries never became mature. However, the bagged berries could counter the negative effects that resulted from calyx removal under the low-temperature conditions. Furthermore, Chinese lanterns, as a natural barrier, might also protect the developing berry from animals and improve the survival of mature berries30  or humidity (<5%) between the inside and outside of the Chinese lantern could make such a tremendous effect on berry development and fitness. However, the reproductive phase in flowering plants is often highly sensitive to external environmental alterations; for example, pollen development and fertilization may often be the most sensitive reproductive stages33. We, therefore, surmise that the microclimate provided by the Chinese lantern structure formed just after fertilization might mainly exert its role to improve fitness during the short time surrounding fertilization.Variation in the external environment affects seed number and sizes30 Fig. . Therefo36, whereas wind-dispersed seeds depend on high seed production, aerodynamic seed morphology, and small seed size41. Fruits may develop certain novel structures, like wings, thorns, or an explosive apparatus to facilitate seed dispersal45. Solanaceous species present remarkable fruit morphological diversity46, which contributes to a wide dispersal of fruits and seeds. Most berries, like tomato and eggplants, are dispersed by animals (including humans), while dispersal of a few could be mediated by non-organism forces9. The Chinese lantern, as the accessory organ of fruit, can facilitate fruit and seed dispersal. In water, tomatoes basically sink to the bottom immediately, whereas intact Physalis fruits usually float for several days. Moreover, intact Physalis fruits moved farther than the tomato berries as water flowed. Lantern removal made the naked Physalis berries disperse more like tomatoes in water. These observations suggested potential adaptive functions of the Chinese lantern in fruit dispersal in water. Interestingly, fossil discovery of the inflated calyces suggest that Physalis originated in ancient, humid, and riparian environments, such as the lakeside rainforest paleoenvironment18, thus suggesting the possible primary role of the Chinese lantern in floatation dispersal.A ripe fruit can be dispersed away from its mother plant by multiple mechanisms. Seed dispersal of sweet, fleshy fruits, such as berries, is performed by frugivorous animals, such as bats and birdsPhysalis fruits is efficiently mediated by wind; however, lantern removal disabled the capability of long distance dispersal. The mobility of tomato berries in wind was severely limited, as was the naked Physalis berries. Consistent with this, wind plays a role in fruit dispersal of Przewalskia tangutica9 which has a lantern-like structure, the homolog of the Chinese lantern in Physalis.Nonetheless, the nature of the lantern- or balloon-like structure permits other strategies to fulfill long distance dispersal. Long distance dispersal of the intact Physalis fruits in these abiotic conditions is attributed to the nature of the Chinese lantern structure. However, animal dispersal (human and birds) of Physalis fruits and seeds does exist48. Unlike P. floridana, mature fruits of Physalis alkekengi are not easy to separate from mother plants , suggesting that the novel structure, although immediately maladaptive to the plant in decreasing reproductive fitness, might play a role in maintaining a local ecological system. The origin of the lantern trait is still in debate, but the Chinese lantern, as a masterpiece of nature, clearly influences both plant fitness and ecological relationships.The persistence and accrescence of the calyx may be an advantageous trait in plants. In Physalis floridana P10611 and Solanum pimpinellifolium (LA1589) were grown in an experimental field at the Institute of Botany, Chinese Academy of Sciences, under natural conditions from May to September in the years 2014, 2015, and 2016. The P106 and Micro-Tom plants for all controlled experiments were grown in plant-growth incubators either under 16 °C or 28 °C.The unfolded floral and fruiting calyces were imaged, and then calyx areas were quantified using the software, NIS-Elmenys3.1. Twenty calyces at each defined developmental stage were measured.57.The leaf near to the fruit and calyces of 5 days post fertilization (DF), 20 DF and mature fruit (MFr) were collected and crushed in absolute alcohol. The mixture was thoroughly shaken and then kept in the dark for 16 h. Absorbance of the supernatant was recorded at 663 and 645 nm on a UV-visible spectrophotometer . The chlorophyll content was estimated according to Arnon (1949)−2·s−1) was determined under the following eight photon flux density gradients: PS1450, PS918, PS606, PS325, PS167, PS89, PS31, and PS0. Further light response curve fitting was done using a non-rectangular hyperbola model to confirm photosynthetic characteristics58. Apparent quantum efficiency , the light compensation point  and light saturation point  difference, and the max net photosynthetic rate  were used to reflect capacity of light utilization and photosynthetic productivity efficiency of green tissues, while dark respiration rate  quantifies the material consumption of respiration.Photon flux density response curves were measured by GFS-3000 and DUAL-PAM-100 measuring systems . The net photosynthetic rate  were simultaneously measured as the temperature was dropped (in 3 °C decrements). We also measured these parameters at a constant temperature of 16 °C. Ten lanterns for each developmental stage from three plants were treated in each experiment. Temperature and relative humidity were recorded using a dew point measuring instrument .For the diurnal variation, both temperature and RH of inner calyx and external environment were recorded every 1 h on Aug. 15, 23, and 27 of the three cultivated years in the experimental field. Five lanterns for each developmental stage  from three plants were measured for three days in each experiment per year. For temperature analyses, the P. floridana at the blooming stage, and then flowers were artificially pollinated; the resultant fruits were designated as the naked Physalis berries. In parallel, some naked berries were covered using waterproof and non-transparent paper bag (7.5 cm × 10.5 cm) . These berries as well as the intact fruits from artificial pollination were labeled simultaneously. Fifty samples were included in one treatment, and each treatment was replicated at least three times. The fruit setting or survival rate, wet and dry fruit weight, seed weight and seed number per berry were measured. The fruit maturation time was defined as the time from pollination to maturation. The experiments were performed in an experimental field and in 16 °C incubators.Calyces were completely removed from Physalis (P106) and tomato (Micro-Tom) seedlings at the blooming stage were transferred into 16 °C constant incubators. For Physalis, the experiment was done as in the section “Calyx-removal analysis”. Pollen activity was detected using the iodine-potassium iodide (I2–KI) staining. For tomato, 40 blooming flowers were bagged with waterproof paper (7.5 cm × 10.5 cm), and 40 flowers were kept naked. We recorded the number of growing days from flowers to mature fruits, the rate of fruit set, fruit weight, and number of seeds per berry. Tomato plants growing in normal conditions were used as control.Physalis were placed as two groups on an experimental bench at 27 °C for one month. Fruit weight was measured weekly. Thirty fruit samples were measured in each group. One typical fruit image for each group was photographed.Naked berries and intact fruits of Physalis were placed into water at one vertical plane, and both the inlets and the outlets were opened simultaneously. The flow distances of the subjected berries/fruits were measured after 1 min. Ten fruits or berries were used in each experiment, and the experiment was repeated 20 times. To test dispersal ability in wind, we chose three consecutive days during which there was a northwest wind of 20–38 km/h. The naked berries and intact fruits of Physalis were placed in a circle with 19-cm radius  with two inlets and three outlets was made as a water flow device .The recorded data were analyzed in EXCEL, and the significance between treatments was evaluated on the two-tailed Student’s Electronic supplementary material


Rhizobium leguminosarum bv. phaseoli LCS0306, to assess its performance in the field, and to determine the genomic features contributing to the high effectiveness of its symbiosis with common bean. Plants inoculated with the autochthonous strain LCS0306 fixed significantly more nitrogen than those with the allochthonous strains R. phaseoli ATCC 14482T and R. etli CFN42T, and had grain yield similar to the nitrogen-fertilized controls. Inoculation with LCS0306 was particularly efficacious when formulated with a carrier based upon a mixture of perlite and biochar. Whole genome comparisons revealed no differences in the classical symbiotic genes of strain LCS0306 within the symbiovar phaseoli. However, its symbiotic superior performance might be due to its genomic versatility, as it harbors a large assortment of genes contributing to fitness and competitiveness. It is concluded that inoculation with elite rhizobia formulated with perlite-biochar carriers might constitute a step-change in the sustainable cultivation of common bean in Spanish soils.Common bean is a poor symbiotic N-fixer, with a low response to inoculation owing to its promiscuous nodulation with competitive but inefficient resident rhizobia. Consequently, farmers prefer to fertilize them rather than rely on their capacity for Biological Nitrogen Fixation (BNF). However, when rhizobial inoculants are based on autochthonous strains, they often have superior BNF performance in the field due to their genetic adaptations to the local environment. Nevertheless, there is scant information at the genomic level explaining their superiority or on how their genomes may influence the inoculant performance. This information is especially important in technologically advanced agri-systems like Europe, where environmental concerns and increasingly stringent fertilizer regulations are encouraging a return to the use of rhizobial inoculants, but based upon strains that have been thoroughly characterized in terms of their symbiotic performance and their genetics. The aim of this study was to design an inoculant formulation based on a superior autochthonous strain,  Phaseolus vulgaris L.) is an outstanding pulse crop with more than 35 million ha cultivated per year worldwide  and Pararhizobium (Bradyrhizobium (nod) and nitrogen fixation , are plasmid-borne in Rhizobium  is indigenous to the Protected Geographic Indication (PGI) “Alubia de La Bañeza-León,” which is the region with the most ancient tradition of common bean cultivation and has the largest common bean-cropped area in Spain. Isolated from a root nodule of common bean, it was selected for its high N-fixation effectiveness under hydroponic conditions , belongs to the symbiovar phaseoli and carries the nodC γ-allele present in R. etli Viking 1 . Therefore, the general aim of this study was to design a successful inoculant for common bean based on an elite autochthonous strain with an adequate formulation which can result in grain yields that are at least equal to those obtained through current fertilization practices. The study involved first the design of the formulation based on the elite local strain R. leguminosarum bv. phaseoli LCS0306 (Rlp LCS0306) and bio-based carriers. The agronomic performance of these innovative inoculants was tested in two field trials to appraise the superiority of the inoculant containing the autochthonous Rlp LCS0306 compared to the inoculants based on the type strains of Rhizobium etli (Re CFN42T) and Rhizobium phaseoli (Rp ATCC 14482T), which are allochthonous. As the formulation based on the elite local strain Rlp LCS0306 performed better than the type strains, we then attempted to explain its superiority from a genomic perspective.Given the global importance of common bean as a crop, the improvement of its BNF capacity would be advantageous both to the environment and the economy. Currently, in the PGI “Alubia de La Bañeza-León” the BNF ability of common bean is under-used as farmers prefer to fertilize it with ammonium nitrate, which constitutes a significant financial cost (>100 Euros haR. leguminosarum bv. viciae (Rlv UPM791) (R. phaseoli ATCC 14482T (Rp ATCC 14482T) and (4) R. etli CFN42T (Re CFN42T). Rlv UPM791 was included because it showed the highest similarity with Rlp LCS0306 in a genome BLAST comparison, and the other two strains were included as allochthonous controls, because they both belong to sv. phaseoli, a symbiovar that only nodulates legumes in the genus Phaseolus (and it is not currently possible to find strains recommended for common bean inoculation in Spain).Four strains were used in this study: (1) the autochthonous strain Rlp LCS0306 isolated from Sueros de Cepeda located in the PGI “Alubia de La Bañeza-León,” as described by  UPM791) , (3) R. The common bean cultivar used was “Riñón” also known as “Riñón de León,” the most important cultivar in cropping area in the PGI “Alubia de La Bañeza – León.”9 cfu ml–1 of the corresponding strain. Five additional plants with no rhizobial suspension added were grown as uninoculated controls. The plants were grown in a growth chamber under controlled conditions (16 h light at 24°C and 8 h darkness at 18°C) for 4 weeks.Nodulation tests were assessed in a hydroponic experiment under axenic conditions. Five plants were used per strain in independent 1 L pots, filled with sterile washed vermiculite and irrigated from a bottom reservoir with sterile N-free solution . Each plRhizobium strains. The liquid inoculum was produced in a pilot fermenter  at 28°C and with 10% dissolved oxygen for 5 days to achieve a concentration > 1 × 109 cfu ml–1. Following centrifugation at 8,000 g, the cfu ml–1 concentration was increased by one order of magnitude.The growth medium was Yeast Mannitol Agar (YMA) or broth (YMB)  for the The individual components for the carriers were perlite, compost and biochar from pyrolysis. The compost was derived from de-alcoholized grape pomace together with vinasses of lees and lignocellulosic plant material . The bio9 cfu for Pe and 3 × 109 cfu for the other formulations. After preparation, the inoculants with an available carbon source (Co and CC) were incubated for 15 days at 28°C, and then stored at 4–6°C until the sampling time, whereas those of mineral origin (Pe) or with short-time unavailable carbon sources (PB) were immediately transferred to 4–6°C.To prepare the inoculum, all the carrier materials were ground, passed through an 80 μm sieve, and autoclaved in pots at 120°C for 20 min, except for the carriers with compost, which were autoclaved for 40 min. The inoculum obtained as indicated above was combined with a cell protector, consisting of 1% locust bean plus 1% trehalose (weight:volume) (unpublished data). The cellular suspension was uniformly and aseptically mixed with the carrier, according to the moisture retention characteristic curves of each carrier (data not shown). The final moisture was selected to allow a maximum volume of bacterial culture in the inoculant but providing an adequate consistency in the final mix as follows: 50% for Pe and 33% for Co, CC and PB. Therefore, the theoretical concentration of viable cells per g of inoculant after inoculation was 5 × 10post hoc means comparisons.The survival of the strain Rlp LCS0306 was assessed for each formulation at different time intervals . The obtained information served for a pre-selection of carriers, allowing to reject those which did not have adequate compatibility with the strain. At each sampling date, three samples for each formulation were used for the inoculum survival analysis. Viable bacteria were estimated by plating 10-fold serial dilutions on YMA plates supplemented with Congo red in duplicate for each sample. The mean values of the viable number of rhizobia per g of inoculant were then calculated for the different times and plotted on a logarithmic scale. One-way ANOVA was used to analyze the effect of the carrier in the bacterial survival at each sampling date, and Tukey test was used for Two field experiments within the demarcation of the PGI “Alubia de la Bañeza-León,” were conducted one in 2017 and one in 2018, in two different plots in order to preserve the principles of the existing crop rotation. The plots were more than 45 km away from the place where the strain Rlp LCS0306 was isolated. In the statistical analysis, the field experiment 2017 and 2018 respectively, were considered as the environment. The coordinates of each field, as well as the Edapho-climatic conditions and count of nodulating rhizobia based in the Most Probable Number , are sho2 (7 × 7) plot, with rows 0.5 m apart and a space between plants of 0.15 m. Experimental units were spaced 2 m apart to prevent spread of rhizobia in the soil solution. The six treatments were the following six inoculants: Rlp LCS0306 formulated with the carriers Pe, P-B, Co and CC; Re CFN42T formulated with Pe, and Rp ATCC14482T formulated with Pe. Two uninoculated controls were also included, one fertilized with mineral nitrogen (N) and one without. Prior to sowing, seeds were dried in the shade and the appropriate quantity of seeds was then mixed with 2% by weight of the inoculant, plus 1% (weight:volume) of gum arabic solution (40% weight of gum arabic in water) as binder.The experimental design followed a statistical pattern of randomized complete blocks with three replications. The experimental unit was a 49 m–1. Hence the P rates, expressed as kg P ha–1 were 21 kg ha–1 for 2017 and 24 kg ha–1 for 2018. All applications were as triple superphosphate . With regard to K, the plots received 126 kg ha–1 in 2017 and 131 kg ha–1 in 2018 as KCl . The N-fertilized control plot received 170 kg N ha–1 which corresponds to the expected total N extraction  and potassium (K), taking into account the soil texture, the soil content of P and K, respectively, and for P, the soil pH, and in accordance with traction . NitrogeAt the phenological stage of early pod set, R3 (one pod at maximum length), five central plants from the third row of each treatment and replication were randomly collected for nodulation assessment to appraise the number of nodules per plant and the dry nodule biomass per plant (g). In order to check the presence of the inoculated strain in each of the five plants, one random nodule was surface-sterilized, crushed in sterile distilled water, plated onto YMA and incubated at 28°C for 72 h. Following this, five isolated colonies with the morphology of strain Rlp LCS0306 were selected for DNA extraction and RAPD profiling with the M13 primer , which i–1. A sub-sample consisting on a proportional basis of the above ground biomass components was ground at 0.85 mm for 15N isotopic analysis. Non-legume weed species  from within the plots were collected, processed in the same way, and used as reference plants as a proxy for the 15N natural abundance of plant-available soil mineral N. Isotopic analysis was performed at SIDI (Universidad Autónoma de Madrid). The isotopic composition of plant samples was expressed as δ15NAIR (‰). Raw data from δ15NAIR (‰) are in 2 (% Ndfa) by the common bean plants was calculated from the 15N abundance of the legume species and that of the non-fixing reference plant as indicated by B value was determined as proposed by At the phenological stage of physiological maturity, R7, eight central plants from the fifth row of each replicate plot were randomly collected and oven-dried at 70°C for 48 h. The aerial dry biomass of the common bean plants was expressed as kg ha–1) = aerial dry biomass (kg ha–1) × N content in the aerial biomass (%); the last was determined using the Kjeldahl method. The amount of N-fixed was calculated as: N-fixed (kg N ha–1) = %Ndfa × Aerial biomass N (kg N ha–1) (–1) was calculated as the difference between aerial biomass N (kg N ha–1) and N-fixed.The N content in the common bean aerial biomass was calculated as: Aerial biomass N (kg N ha N ha–1) . The soi2 plot, and finally expressed as kg ha–1. The following yield components were also recorded for each plant: (i) number of pods per plant; (ii) number of seeds per pod; and (iii) 100-seeds dry weight in g. Finally, the harvest index (HI) was calculated on the basis of dry matter.Sampling at harvesting was carried out in the rows that remained complete after the intermediate samplings described above, leaving at least one untouched row at each edge as a border. The six central meters of the 7th to 13th rows were hand-harvested at the harvest maturity stage. The yield was recorded as weight of air-dried beans which corresponds to the commercial grain, and then corrected to absolute dry weight after drying the seeds at 80°C to a constant weight. The dry matter of air-dried beans was 88.43%. The yield was calculated from each corresponding 21 mT and Rp ATCC 14482T, formulated with perlite, plus the two uninoculated controls. The year of the experiment was considered the environment. The dependent variables were the parameters about nodulation and N fixation  appropriate to a randomized complete block design for all the dependent variables, considering the year of the experiment as the environment. For those parameters in which the ANOVA detected significant differences, the mean values were compared using the LSD test. A pair-wise correlation analysis between the dependent variables was carried out using the Pearson coefficient.The treatment considered for this analysis was the formulation of the strain LCS0306, with four levels, corresponding to the four different carriers, Pe, Co, CC, and PB. The data were subjected to ANOVA and the means comparison was performed with the Dunnet test, using the formulation Pe as reference for comparison. All the statistical analyses were carried out with IBM-SPSS v.24.de novo assembly was performed using SPAdes 3.7 (Genome sequencing was performed by MicrobesNG (Birmingham) by Illumina NGS with a coverage of 30%. The reads were trimmed using Trimmomatic  and de nAdes 3.7 . AnnotatAdes 3.7 .T , Rp ATCC 14482T (accessions RJJV01000001–RJJV01000081) and Rlv UPM791 (accessions CP025505.1–CP025510.1) using a custom BLAST database on Geneious 10.0.9 (Biomatters). Clusters of orthologous groups (COGs) of proteins were predicted using the WebMGA server (Average Nucleotide Identity (ANI) using MUMmer  as the aA server  and KAASA server . BLAST RA server .The draft of this whole-genome shotgun project has been deposited in GenBank under the provisional accession no WNKD00000000.–1 of the inoculant was slightly but significantly higher in Pe and CC than in Co. PB showed the significantly lowest load compared to the rest of the carriers, with 0.22 logarithmic units less than Pe which had the highest value, due to the preparation process (see Inoculum Production and Design of the Inoculant Formulations)  . All theT produced an average of 31.6 nodules per plant, significantly lower than the average number of nodules produced by Rlp LCS0306 (65.6) and Rp ATCC 14482T (78.6). The latter two strains did not significantly differ in this respect . These strains were then evaluated under field conditions to compare their symbiotic performance according to various parameters.The initial nodulation assay under hydroponic conditions showed that the strain Re CFN4215N natural abundance method. The combined ANOVA for all the parameters  is shown in p ≤ 0.01 or p ≤ 0.001, depending on parameter), except for the number of nodules per plant. There was no significant interaction between the treatment and the environment.In the field, the parameters evaluated were the average number of nodules, dry nodule biomass per plant and symbiotic nitrogen fixation, evaluated by the –1), Ndfa (50%) (–1)  and the soil N uptake (48.7 kg ha–1), compared to the other treatments (T (43.3%)   and N-fi0%) (–1) , althougch other . As expeeatments . The pos (43.3%) .T showed a high nodule occupancy both in 2017 and 2018. Rlp LCS0306 had a recovery rate from nodules of 84% in 2017 and 72% in 2018, whereas Rp ATCC 14482T had a recovery of 80% in 2017 and 72% in 2018. However, for Re CFN42T recovery was lower at 60% in 2017 and 44% in 2018. The bacteria isolated from the root nodules which were not the inoculated strain could be either rhizobia which actively induced nodule formation, or other endophytic bacteria which entered the nodule. In order to elucidate the identity of the other strains isolated, it would be necessary to sequence of specific genes of the unknown isolates, which is out of the scope of the present work.The nodule occupancy was tested in 25 independent nodules per treatment each year. Rlp LCS0306 and Rp ATCC 14482p ≤ 0.001), but not in the 100-seeds weight. The interaction between the inoculation treatment and the year was significantly higher for the yield and all the yield components (p ≤ 0.001 or p ≤ 0.01) (–1), compared to the inoculation with Re CFN42T  or Rp ATCC 14482T , which did not differ from each other (–1) and was more than 1200 kg ha–1 greater than the uninoculated negative control plants  . Such inch other . Moreovel plants . These sl plants .R value 0.7, p ≤ 0.001) and the N-fixed , and weakly correlated with the aerial biomass  and the grain yield   and the grain yield, although in this case, only in 2017 . N-fixed showed a significant correlation with the soil N uptake , the aerial biomass  and the grain yield .The parameters to estimate nodulation  did not show any significant correlation between them ; althoug ≤ 0.05) . The cor ≤ 0.05)  showed tR. etli, R. phaseoli and the closely related strain, Rlv UPM791. The highest ANI scores were obtained against Rlv UPM791  cycle, required for aerobic respiration and energy production; the pentose phosphate pathway, required for the oxidation of glucose and the synthesis of nucleotides, and the Entner–Doudoroff pathway, for the catabolism of glucose to pyruvate. These similarities were reflected in the growth pattern with different carbon sources  analysis reflected a large number of protein families involved in metabolism . The met sources . A noticThe ability to persist in the soil and outcompete local rhizobia populations is based on many different parameters. At the genomic level, different traits have been described as having a role in competitiveness, such as motility and chemotaxis, exopolysaccharide (EPS) production, ABC transporters or secretion systems among others. Some of these traits have been analyzed in Rlp LCS0306 to give an overview of its genomic potential in terms of competition  and symbT, Rp ATCC 14482T and Rlv UPM791. For instance, Rlp LCS0306 contains the T1SSd proteins orthologous to the PrsD and PrsE proteins required for biofilm formation. The Rlp LCS0306 genome also harbors a putative T4SS-pili (virB1–virB11) system homologous to the cluster in pRlvA, involved in colonization of surfaces in Gram-negative bacteria. Strains Rlp ATCC14482T and Rlp LCS0306 harbor syntenic imp (tss) and hcp clusters encoding a putative T6SS. As with Re CFN42T, Rlp LCS0306 contains a homologous T3SS cluster that might also play a role in symbiosis, and the T4SS traGDCAFBHMR genes involved in conjugal transfer, followed by the nocQMT nopaline transporter, a signal involved in DNA transfer.Given the proposed role of secretion systems in rhizosphere colonization ability, the secretion systems of the strains were also analyzed in order to explain their competitiveness . Strain Rhizobium, the symbiotic genes, i.e., the genes involved in nodulation and nitrogen fixation are plasmid-borne. When the Rlp LCS0306 genome was aligned against the genome of Rlv UPM791 (T (T (p42d), indicating that Rlp LCS0306 contained a putative symbiotic plasmid belonging to the symbiovar phaseoli. As expected from its high efficiency in N-fixation, Rlp LCS0306 harbors all the nodulation and nitrogen fixation genes required to establish a successful symbiotic relationship with Phaseolus , grain yield, pods per plant and 100-seeds weight (p < 0.01). There was a significant interaction between the formulation and the year for 100-seeds weight (p < 0.01) due to the fact that better results were achieved in 2017 for CC formulation and in 2018 for PB formulation.In consideration of previous inoculant formulation designs and the symbiotic performance parameters of Rlp LCS0306, the formulation of the LCS0306-containing inoculant was tested under agronomically realistic conditions in the field. The combined ANOVA for all the parameters combined  is shown in p < 0.01 in the Dunnet test). Moreover, CC formulation produced significantly higher number of pods per plant and 100-seeds weight (p < 0.05). The best performance of the Rlp LCS0306 inoculant was obtained with the PB formulation, with a 15% higher yield compared to the perlite control.The formulations with biochar produced significantly higher values for the following parameters, compared to the control (Pe) : PB formDespite the advantages of BNF and other microbial processes in agriculture, the use of bacterial inoculants to provide nutrients to crops or to promote their nutrient use efficiency, tolerance to abiotic stress, or crop quality, is increasing but still not a common practice . EspeciaRegarding the strain, one of the main challenges is the selection of superior rhizobial strains by inferring a high performance only based on their genetic features . It is n–1 above uninoculated plants. As symbiotic N-fixation abilities are uncoupled from colonization and nodulation abilities  and hcp clusters encoding a T6SS  secrete nodulation outer proteins (Nops) in the presence of flavonoids, inducing the transcription of nodulation genes  and rhizobial strains has been proposed to play a role in their rhizosphere colonization ability . The T3Sg a T6SS . Impaireexudates . Rlp LCSon genes .R. leguminosarum bv. viciae, Che1, present in Rlp LCS0306, has shown to be essential for competitive nodulation  cv. Tendergreen   and Rp CFN42T  . The quooduction  and presp CFN42T  is also –1 for 2017 and 2018. Thus, in our field trials the increase in the gross income due to the inoculation would have been 1,245 eur in 2017 and 1,352 eur in 2018. Therefore, the present study has indicated that rhizobial inoculation with elite strains like Rlp LCS0306, if applied, could constitute a step-change in the sustainable cultivation of common bean in Spanish soils.Although all of the inoculated strains produced higher yields than the native ones, the particularly high N-fixing ability of Rlp LCS0306 in the field has been demonstrated, i.e., Ndfa of 50%, compared to 40% for native rhizobia, and an almost doubling of total N-fixed. Moreover, the inoculation with the strain Rlp LCS0306 produced the same aerial biomass and grain yield as the N-fertilized control. Thus, this autochthonous strain produced considerably higher aerial biomass and yield than the resident strains, and the yield was even higher than in the treatments inoculated with the other strains, hence confirming the agronomic potential of Rlp LCS0306. According to the Observatory of prices of agriculture and livestock products of Castille and León (Spain), the medium sale price of dry beans in the PGI “Alubia de La Bañeza-León” was 100.54 eur 100 kgP. vulgaris, a high inorganic N uptake combined with relatively low values of %Ndfa maximizes N use efficiency (NUE) in soils with relatively high N-levels (a legacy owing to applications of fertilizer to previous seasons non-legume crops), which in turn reduces the risk of nitrogen leaching from the soil.However, the improvement of the grain yield or the aerial biomass produced by the crop as a consequence of inoculation, was only partially explained on the basis of the Ndfa (%) or the Nodule biomass, i.e., the correlation between aerial biomass or the grain yield with the Ndfa or the Nodule biomass was, at the most, weakly significant and not in all the cases there was statistical significance. The aforementioned results indicate that even if the functional link between plant growth and symbiotic functioning proposed by other authors  has beenPhaseolus. Apart from characterizing the N-fixing genetic machinery of Rlp LCS0306, the genomic analysis has also revealed other data of interest that taken together help to explain its superiority compared to strains Re CFN42T and Rp ATCC 14482T. Despite having a genomic backbone homologous to the biovar viciae, Rlp LCS0306 contains the symbiotic repertoire of Re CFN42T. Rhizobial genomes are extremely variable  biosynthesis, a carbon polymer that seems to play a role during root infection and invasion . Interestingly, this was not accompanied with an increase either in the Ndfa (%), N-fixed in kg ha–1, nodule number, or nodule biomass compared to the other formulations used in the experiment. Thus, the improvement in field performance of the PB formulation suggests that it is related to the plant growth promoting effect assigned to biochar  and also a significantly higher grain yield  and nodule biomass. However, there may be other factors contributing to the superiority of LCS0306 in field trials, potentially derived from its gene assortment, as the strain harbors a R. leguminosarum scaffold with a symbiotic plasmid characteristic of strains nodulating Phaseolus, containing the genes required for an efficient symbiosis. Rlp LCS0306 has evolved in the local conditions of northern central plateau in Spain, and it has gathered several genomic characteristics as enumerated above, putatively involved in its adaptation to such local conditions. The ensemble of its diverse genomic characteristics, rather than a specific characteristic itself, seems to contribute to the superior performance of Rlp LCS0306 in the field. To date, cultivation of common bean with the available commercial inoculants has resulted in a suboptimal nodulation and BNF, as this crop is not native to Europe. This study constitutes the first evidence of a native inoculant enhancing BNF and grain yield in common bean in Spain, stressing its economic value for the future sustainable cultivation of this important crop.The results obtained in this study explained the success under field conditions of an outstanding inoculant for common bean based on the autochthonous strain Rlp LCS0306 when it has been appropriately formulated, in terms of its symbiotic performance, genomic features and agronomic traits. Overall, the superior performance of strain Rlp LCS0306 appears to be due to the combination of different modes of action, which together produced a significantly higher grain yield and a high rate of recovery from nodules, compared to the resident rhizobia and to other common bean-nodulating strains like Re CFN42PRJNA552714.The datasets generated for this study can be found in the NCBI BioProject no. RP-B executed the field trial, collected the data and wrote the manuscript. CS-C analyzed the genomic data and wrote the manuscript. EJ worked on the general structure and the integration of the different parts. FG-A designed the field trial, analyzed the data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This suggests an inhibitory effect of leptin signal on the transcription of these orexigenic genes during short-term fasting and refeeding in functional zebrafish. Most pronounced effects were observed in the group of anorexigenic genes, where the impairment of leptin signalling resulted in reduced gene expression in several genes, including cart family, crhb, gnrh2, mc4r, pomc and spx, in the control group. This suggests a stimulatory effect of leptin signal on the transcription of these anorexigenic genes under normal feeding condition. In addition, we found multiple gain and loss in expression correlations between the appetite-regulating genes, in zebrafish with impaired leptin signal, suggesting the presence of gene regulatory networks downstream of leptin signal in zebrafish brain. The results provide the first evidence for the effects of leptin signal on the transcription of various appetite-regulating genes in zebrafish brain, under different feeding conditions. Altogether, these transcriptional changes suggest an anorexigenic role for leptin signal, which is likely to be mediated through distinct set of appetite-regulating genes under different feeding conditions.The hormone leptin is a key regulator of body weight, food intake and metabolism. In mammals, leptin acts as an anorexigen and inhibits food intake centrally by affecting the appetite centres in the hypothalamus. In teleost fish, the regulatory connections between leptin and other appetite-regulating genes are largely unknown. In the present study, we used a zebrafish mutant with a loss of function leptin receptor to investigate brain expression patterns of 12 orexigenic and 24 anorexigenic genes under different feeding conditions . Expression patterns were compared to wild-type zebrafish, in order to identify leptin-dependent differentially expressed genes under different feeding conditions. We provide evidence that the transcription of certain orexigenic and anorexigenic genes is influenced by leptin signalling in the zebrafish brain. We found that the expression of orexigenic genes was not affected by impaired leptin signalling under normal feeding conditions; however, several orexigenic genes showed increased transcription during fasting and refeeding, including  The central regulation of feeding behaviour in the brain is under the influence of endocrine signals, originating from the brain itself, as well as peripheral organs, after exposure to different metabolic and nutritional conditions. The regulation of food intake, in both mammals and fish, takes place in the hypothalamus in the central nervous system. In mammals, appetite regulation through hormones is mediated by their receptors, located on a group of key neurons called arcuate nucleus neurons2. In fish, similar neurons exist in the periventricular and lateral parts of the hypothalamus3. Nevertheless, it appears that neurons generating neuroendocrine signals display a wider anatomical distribution in the fish brain than in mammals, as it has been observed for appetite-regulating genes expressed in different parts of the fish brain4. The neurons mediating the appetite-regulating effects can be divided into two main types: orexigenic, stimulating food intake and/or related locomotor activity; and anorexigenic, reducing food intake and/or related locomotor activity5. At the core of these processes are the key molecular players, the so-called appetite-regulation genes, which encode a range of neuropeptides and their cognate receptors. The appetite-regulation genes can also be classified in two categories of orexigenic and anorexigenic genes based on their functions7. Due to the large diversity of teleost fish in diet, feeding habits, physiology and adaptation to various environmental factors influencing their feeding behaviour, the number of studies addressing molecular mechanisms underlying appetite regulation in fish has been growing in recent years8. However, transcriptional activity and regulatory connections between the appetite-regulating genes, under different feeding conditions, are still not fully explored in fish.Feeding behaviour involves complex processes of foraging controlled by appetitive behaviours (hunger-driven activities) and food ingestion10. Zebrafish has two leptin hormone genes, lepa and lepb, which are differently expressed in several organs11. Both leptin isoforms mediate their signal through a single leptin receptor gene in zebrafish (lepr), which is expressed in several brain regions, including the hypothalamic lateral tuberal nucleus12. Most studies of leptin functions in fish are conducted on Cypriniformes (mainly goldfish and zebrafish) and Salmoniformes8. Several studies in goldfish showed that excessive level of leptin can reduce both feeding behavior and locomotor activity (anorexigenic effects)16. The regulatory effects of leptin signal on appetite can be exerted through stimulation of anorexigenic genes, while inhibiting orexigenic genes in the brain18. However, the leptin expression in the brain and other organs, in response to different feeding conditions, seems to be highly variable across fish species19. For instance, post-prandial increase of leptin expression has been observed in goldfish and zebrafish in different organs20, whereas such an increase was absent in rainbow trout21. On the other hand, fasting did not affect the hepatic expression of leptin in goldfish, common carp, pacu and Nile tilapia, while on the contrary, its hepatic expression was affected during fasting in most Perciformes and Salmoniformes examined22. Furthermore, in zebrafish, inhibiting lepa leads to a decrease in metabolic rate23, whereas leptin treatment can increase energy expenditure24. After feeding the expression of leptin increases in most fishes including goldfish and zebrafish20. Despite the fact that the number of molecular studies on leptin-dependent regulation of feeding in fish is consistently growing, little is known about regulatory connections between leptin signal and other appetite-regulating genes under different feeding conditions.One of the signals, transduced by the leptin hormone and its receptor, is an appetite-regulating molecular mechanism, among its other regulatory roles, which include controlling body weight, reproduction, development and stress responses in vertebratesi.e. 12 orexigenic and 24 anorexigenic genes (Table lepr mutant). We first identified a stably expressed gene in zebrafish brain under different feeding conditions and used it as normalization factor for the analysis of gene expression by qPCR. Next, we aimed to profile the expression dynamics of the appetite-regulating genes during different feeding conditions: normal feeding (control group), fasting and two refeeding stages (2 and 6 hours after refeeding) in wild-type zebrafish and compared the results to the expression patterns observed in the lepr mutant. This allowed us to identify genes for which transcriptional changes, in different feeding conditions, may be under the influence of functional leptin signalling. Finally, we predicted loss and gain of potential regulatory connections between the appetite regulating genes in defective leptin signalling,through pairwise expression correlation analysis. Our findings provide comprehensive information about the expression dynamics of known appetite-regulating genes in relation to different feeding conditions, as well as non-functional leptin signalling in the brain of adult zebrafish.To further explore the role of leptin in appetite regulation, we compared in this study the expression levels of 36 genes, which have already been shown in other studies of Cypriniformes to have appetite-regulating functions, 27. We quantified the expression levels of 8 candidate reference genes in the brains of wild-type and lepr mutant, exposed to different feeding conditions. The expression levels of the candidate reference genes varied considerably in the brain of zebrafish with highest expression (lowest Cq) for actb2, around 19 average Cq value, to tmem50a with lowest expression (highest Cq), around 28 average Cq value   in every specific experimental conditions is a prerequisite for the analysis of relative gene expression levels by qPCRlepr mutant brain during the fasting-refeeding experiment, and found expression changes in a few of the genes for each genotype , aplnra, galr1a and galr2a. Among these DE genes, none of them displayed changes in both genotypes, and even their expression dynamics were not following similar patterns between the genotypes . This shows more appetite stimulation by orexigenic genes, during changes in feeding condition, in defective leptin signalling. Furthermore, we only found one of the genes, apln, with increased brain expression in both refeeding groups of lepr mutant. Altogether, these findings demonstrate noticeable differences in transcriptional regulation of orexigenic genes in the brain between wild-type and lepr mutant strains, under different feeding conditions.Next, we compared the expression of the orexigenic genes, between wild-type and nes Fig. . Intereslepr mutant during the fasting-refeeding experiment, and found 15 genes showing expression changes in at least one of the genotypes . Interestingly, we found all cart genes displaying similar changes within the wild-type group while no difference was identified in the mutant. Spx was the only gene showing similar differential expression pattern in both genotypes; however, it seems that in zebrafish with impaired leptin signal, its induction appeared earlier during refeeding. These findings demonstrate weakened transcriptional regulation for certain anorexigenic genes in functional or defective leptin signalling during the fasting-refeeding experiment . These findings, during fasting and refeeding conditions, are similar to the orexigenic genes, where only increased expression of the genes was found in the mutant compared to wild-type zebrafish. Therefore, the results demonstrate that functional leptin signalling is required for transcriptional suppression of certain anorexigenic and orexigenic genes, in changes of feeding condition.We directly compared the expression of the anorexigenic genes between the genotypes, and observed expression changes opposite to the orexigenic genes. For instance, while no difference was observed in the control group for orexigenic genes, for the anorexigenic genes, most differences were found in the control group Fig. . All of 28. Most of the identified correlations appeared to be positive in both genotypes; however, not all of the positive correlations were similar between the genotypes, indicating gain or loss of certain regulatory connections between the genes in defective leptin signal , we observed strong connections between cart1,2,3,4/crhb/gnrh2/mc4r/pomc, cnr1/kiss1, nucb2/pacap, cnr1/pacap, and agrp/spx. We found only one negative correlation, trh/mchr1, which showed similarity between the genotypes  demonstrates potential molecular interactions between products of cart3, kiss1r, nmu, trh, mc4r, pomca, agrp and mchr1a in zebrafish and/or other vertebrates , and its cognate receptor, aplnra, to be differentially expressed in wild-type and lepr mutant, respectively. However, the changes seemed to differ in pattern between the two genotypes. Apln encodes a peptide that functions as an endogenous ligand for the G-protein coupled apelin receptor (aplnra), which in turn activates different tissue specific signalling pathways with mainly metabolic effects29. The observed difference was due to the increase in expression of apln during fasting in wild-type and expression induction of aplnra immediately after refeeding in the brain of lepr mutant  exhibit up-regulation in Apln expression35. We did not find expression difference between the two genotypes in the control group; however, the difference emerged after fasting and in the refeeding groups. The induction of apln by fasting in the wild-type group indicates its potential orexigenic effect in zebrafish, which seems to be weakened by the impaired leptin signal. On the other hand, we found apln to have higher expression in the lepr mutant than the wild-type during refeeding  in the nervous system, and is essential for suppression of cart3 expression during fasting45. We did not find expression correlation between cnr1 and cart3 in both genotypes, but we found a gain of negative expression correlation between cnr1 and pomc in the lepr mutant 51. Among teleost fish, in both Salmoniformes and Cypriniformes, leptin increases pomc expression in the brain53, and in medaka, knockout of lepr decreases pomc expression in the brain as well41. These findings are consistent with our results of pomc expression, which shows expression reduction in the lepr mutant at the normal feeding condition. Furthermore, the expression suppression of cnr1 by leptin signal during 2hrs refeeding indicates potential anorexigenic role of leptin signal at early refeeding in zebrafish.We found that two receptors of galanin, ant Fig. . Interestrh, encodes thyrotropin-releasing hormone and only in goldfish it has been shown that trh can increase feeding and locomotor behaviours54. In addition to higher expression of trh in the lepr mutant at 6 hrs refeeding . Perhaps the most striking findings about anorexigenic genes was the consistent and similar expression differences for all of the cart genes (Cocaine and amphetamine regulated transcripts) between the two genotypes. The analysis of expression dynamics for cart1,2,3,4 genes revealed their reduced expression in the wild-type zebrafish, only in the treatment groups 65.Among the anorexigenic genes tested, we found distinct expression dynamics between the genotypes for almost all of the DE genes  and its receptors . The ligand encoding gene, mch, showed reduced expression in the treatment groups than the control group but only in the wild-type, whereas its receptors showed increased expression in the refeeding groups compared to the control group in the mutant 79, the rest of these genes are shown to have anorexigenic role in at least one Cypriniforme species, mainly goldfish86. Except for nucb2, for which the anorexignenic role already is described in zebrafish84, the transcriptional changes of the rest of the genes suggest their potential appetite-regulating role in zebrafish as well. In mice, leptin increases the brain expression of Mc4r87. Consistently, the zebrafish lepr mutant in our study shows reduced mc4r expression under normal feeding condition, indicating a conserved positive regulatory connection between leptin signal and mc4r. The potential regulatory connections between these genes and leptin singalling has not been studied in fish, but our results indicate the existence of such connections in zebrafish brain. In goldfish, nmu and pacap88, and in zebrafish, nmu89, act upstream of crh in the brain, and in this study, we also found positive expression correlation between nmu and crh in both genotypes, and between pacap and crh in the wild-type , are useful to evaluate the level of impairment of leptin signal in the lepr mutant used in this study.The study provides evidence that both orexigenic and anorexigenic genes in the zebrafish brain are influenced by leptin signalling. We observed the most pronounced effect in the group of anorexigenic genes, where impaired leptin signalling resulted in reduced gene expression in several genes of the control group. This suggests a stimulatory effect of leptin on transcription of these anorexigenic genes in wild-type zebrafish under normal feeding conditions. We also observed effects of the impaired leptin signal on expression correlations between appetite regulating genes in zebrafish brain, implicating the existence of complex gene regulatory networks, which are under the influence of leptin signal. The study further indicates the potential appetite-regulating role of several of the investigated genes; however, additional studies are required to confirm the role of these genes in appetite regulation in zebrafish. Taken together, based on the expression changes of the appetite regulating genes, in the impaired leptin receptor signal transduction, we propose an anorexigenic role for leptin signalling in zebrafish. It should be emphasized, however, that all the observed transcriptional changes might not necessarily be translated into changes at the protein level. Furthermore, functional investigations, such as leptin-dependent phosphorylation of sa12593, obtained from European Zebrafish Resource Centre. The mutation was created by the Sanger Institute for the Zebrafish Mutation Project by replacing a cytosine with an adenine on chromosome 6, leading to the formation of a premature codon stop, causing an incomplete translation. For this study, we kept the lepr mutants and their sibling wild-type zebrafish in 3-liter recirculating tanks at 28.4 °C at the SciLife lab zebrafish facility at Uppsala University. The water parameters were checked regularly and maintained by facility staff members. We set up the dark-light conditions to 10 and 14 hours, respectively. Before the experiment, we fed the fishes with dry pellets once in the morning and with Artemia twice a day (middays and evenings). We divided a total number of 40 zebrafish of similar age, into four categories: fish fed by the facility as control group and fish fasted for a week; fish fasted for a week and sampled 2 hours after the refeeding or sampled 6 hours after the refeeding. Each group was subdivided into two categories: homozygote fish for the mutation on the leptin receptor and wild type fish, with 5 fish of each genotype per treatment group. It should be noted that within each group and its counterpart in the other genotype, we tried to include similar female/male ratios  , at 4 °C for one day and at −20 °C the next day until RNA isolation step.In this study, we used zebrafish strain leptin receptor later (Ambion) and were put into a new 1.5 ml Eppendorf tube containing 200 μl of Trizol. Then, we homogenized the samples using a syringe needle (25 G Terumo needle and BD Plastipak 1 mL syringe). A quantity of 40 μl of chloroform (Sigma-Aldrich) was added, followed by room temperature incubation for 5 minutes. After incubation, we centrifuged the samples at 12 000 g/min for 20 minutes at 4 °C. The aqueous upper phase was carefully transferred into new RNAse-free microfuge tubes, and 1 μl of glycoblue (Ambion) and 100 μl of ice cold (−20 °C) isopropyl alcohol (Sigma-Aldrich) were added to the tubes. Again, we centrifuged the samples at 13 000 g/min for 20 minutes at 4 °C. The upper phase was removed and the RNA pellet was washed by adding 200 μl of ice cold (−20 °C) ethanol (VWR) at 75% mixed with DMSO solution, followed by centrifugation at 9 000 g/min for 5 minutes at 4 °C. Subsequently, a washing step was performed with 75% ethanol and repeated 3 times. The samples were then dried by opening the tube under a fume hood and solubilized by adding 10 μl of Nuclease-free water (Ambion). We performed a DNAse treatment on each sample to remove genomic DNA by Turbo DNA-free kit (Ambion) according to the manufacturer’s instructions. The quantity and quality of RNA was measured with a NanoDrop 1000 v3.7  and 1000 ng of RNA input was used for the reverse transcription (RT). For the RT step, we used Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen). In brief, the RNA samples were mixed with 0.5 μl of random primers (50 ng/μl) and 0.5 dNTP (10 nM), incubated at 65 °C for 5 minutes and then cooled down on ice for a minute. A mix composed of 2 μl of 5X First-Strand Buffer, 0.5 μl of DTT (0.1 M), 0.5 μl of RNase OUT (40 U/μl) and 0.5 μl of Superscript III RT (200 U/μl) was made and added to each sample. An incubation time of 5 minutes at 25 °C was set up, followed by an enzyme reaction time of 50 minutes at 50 °C and an enzyme inhibition time of 15 minutes at 70 °C. The final volume of 10 μl of cDNA was stored at −20 °C until the qPCR step.We isolated the RNA of sampled brains using Trizol (Ambion). The dissected brains were removed from RNA95. As target genes, we selected 36 candidates involved in appetite regulation in Cypriniformes (mainly studied on goldfish and zebrafish) with the exception of kiss1 and kiss1r which are only studied in sea bass (Perciformes)  and specified the exon/exon boundaries using annotated Danio rerio genome in the Ensembl database, http://www.ensembl.org 97. The primers with short amplicon sizes (<200 bp) were designed by Primer Express 3.0  and their dimerization and secondary structure formation were evaluated using OligoAnalyzer 3.1 (Integrated DNA Technology) , we selected 8 candidate reference genes based on studies of zebrafish, which had investigated suitable references genes across different experimental conditions, developmental stages and tissuesfree water, for a total volume of 15 μl per tube. The qPCR instrument used was MxPro-3000 PCR machine  together with MxPro software (Stratagene) for data mining. We conducted each biological replicate in two technical replicates for each gene and we followed a sample maximization method98 to have an optimal experimental set-up in each run. The reaction program was as follow: 50 °C for 2 minutes (1 cycle), 95 °C for 2 minutes (1 cycle), 95 °C for 15 second and 62 °C for 1 minute (40 cycles). A dissociation step (60–95 °C) was performed at the end of the amplification step. We estimated threshold cycles, number of copies and efficiencies by the software. To calculate primer efficiencies , we used generated standard curves using serial dilution steps of pooled cDNA samples. For each gene, we ran standard curves in triplicates, and calculated the results using the following formula: E = 10[−1/slope]. R2 values were higher than 0.990 and efficiencies were ranging between 94.1–108.7% for all assays  of each sample was mixed with 7.5 μl of qPCR Master mix called PowerUp SYBR Green Master Mix (Thermo Fischer Scientific), 0.3 μl of forward and reverse primers (10 uM) and 6.2 μl of RNA-99, NormFinder100 and geNorm101. BestKeeper uses two algorithms for ranking of candidate reference genes; first one is based on the standard deviation (SD) of Cq values across all the samples and the second one considers expression correlations or BestKeeper index (r)99. NormFinder determines the most stable genes, through calculating expression stability values, which are based on analysis of inter- and intra-group variation in expression100. Finally, geNorm calculates mean pairwise variations in a stepwise manner between each gene and the other candidates 101. The ranking outcomes, from each of these alone, cannot be reliable unless consistency of the results are observed between the software for the top ranked genes103. In this study, we used the Cq value of the most stable reference gene across both genotypes and different feeding conditions, Cqreference, to normalize Cq values of target genes for each sample (ΔCqtarget = Cqtarget − Cqreference). To calculate ΔΔCq values, we chose a biological replicate with lowest expression  across all the samples for each gene , and then subtracted the ΔCq from the calibrator ΔCq value . We calculated the relative expression quantities  through 2−ΔΔCq, and their logarithmic values (or fold changes) were used for statistical analysis104. We used student t-tests for the direct comparison gene expression levels of a target gene between wild-type and lepr mutant in each feeding condition. To analyse the expression dynamic of a target gene for each genotype, we implemented analysis of variance (ANOVA) test across the different feeding conditions followed by Tukey’s honest significant difference (HSD) post hoc tests. We used Benjamini-Hochberg procedure to correct for the false positive rate in the multiple comparisons105. Moreover, to identify the similarities in expression patterns between the target genes, we used pairwise Pearson correlation coefficients across the feeding conditions in each genotype. We implemented a dendrogram hierarchical clustering of expression values of the target genes to identify overall similarities between the feeding conditions and the genotypes. R (http://www.r-project.org) was used for all statistical analyses106. We explored the knowledge based interactions between the gene products by STRING v10 (http://string-db.org/), using zebrafish database for protein interactome107.To identify the most suitable reference gene(s), we used three different software for calculating the expression stabilities; BestKeeperThe fish handling procedures were approved by the Swedish Ethical Committee on Animal Research in Uppsala (permit C10/16). All methods were carried out in accordance with the guidelines and regulations of the Swedish Ethical Committee on Animal Research in Uppsala.Supplementary Information.Supplementary Information2.


After 7 days of storage, HiOxMAP samples exhibited inferior consumer acceptability for tenderness, flavor, overall liking, quality and re-purchase intention as well as higher shear force and hardness, relative to VAC samples . Loins stored in HiOxMAP had higher lightness (L*), redness (a*) and yellowness (b*) values at 3 and 7 days, but lower ratio of oxymoglobin to metmyoglobin (oxy/met) values in the meat surface at 14 days of display, relative to VAC samples . The oxy/met ratio declined from 2.3 to 1.7 between days 3 and 14 of display in HiOxMAP samples (p < 0.05), whereas the ratio was similar and stayed relatively high for VAC samples. VAC samples produced consistently higher colour values  when left to bloom 30 min after removal from packaging (p < 0.05). Lipid oxidation values, measured using thiobarbituric acid reactive substances, in HiOxMAP pork loins, were higher at all time points compared to VAC during the 14 day storage period (p < 0.05). The use of vacuum packing for retail shelves, should be considered as the preferred option, over HiOxMAP.Current trends in meat packaging have seen a shift from conventional overwrap to vacuum packing (VAC) and modified atmosphere packaging (MAP). The aim of this experiment was to investigate the effects of high oxygen MAP (HiOxMAP) of pork loins compared with vacuum packed (VAC) on eating quality and colour, after storage in simulated illuminated retail display conditions. Pork loins ( Pork is the one of the most consumed meats and is the most common protein source in the human diet in the world. Meeting the consumers’ demand for high quality pork is a main focus for the pork industry  and it hPorks’ presentation is a fundamental gateway between consumers and producers. It has been shown that the consumers’ decision to purchase fresh meat is initially based on the visual qualities, such as colour, marbling and purge loss , while sThe surface colour of meat is predominantly dependent on the pigment myoglobin (Mb), an iron and oxygen binding protein. Mb incorporates an oxygen atom into its structure when exposed to an oxygen-rich environment. Oxygen-bound Mb (oxymyoglobin) is primarily responsible for the bright cherry-red colour in meat, particularly in the surface of meat in HiOxMAP . In an oAs discussed above, although the effects of HiOxMAP and vacuum packaging (VAC) on colour stability and lipid oxidation have been extensively researched for beef ,20,21, lSensory procedures used in this study were approved by the Human Research Ethics Committee of the University of Melbourne. The experiment was split into two replicates, being two collection days which were two weeks apart. Forty loins from the left and right side of 20 pig carcasses were collected at 1–2 days post-slaughter, from carcasses weighing about 65 kg. Loin samples were placed in a refrigerated trailer at 4 °C and transferred to the meat laboratory in the University of Melbourne.Before pork loins arrived at the sensory lab, all the tools and equipment were sterilised with 80% ethanol. Sequentially, two paired loins from the one carcass were transferred from the chiller, 2–3 cm was removed from the posterior end of each loin and discarded. Then, from the anterior end and middle of both loins, three × 12.5 cm sections and five × 4 cm sections were removed and allocated to treatments as shown in 2 (24 h) and water transmission less than 5 g/m2/24 h. HiOxMAP packaging was conducted on a Multivac T200 . Packing trays were clear Cryovac , a cello soaker pad  was used in all MAP trays and a Biaxially Oriented PolyAmide/Polyethylene/Ethylene vinyl alcohol based film with less than 10 cc/m2/24 h and less than 3 g/m2/24 h was also used. The gas composition in the MAP was 80% O2 and 20% CO2. The gas ratio of two packaging packs after ageing treatments were checked by a gas analyser and was 80% O2, 20% CO2 ± 0.1%. Thiobarbituric acid reactive substances (TBARS) are formed as a by-product of lipid peroxidation (as degradation products of fats), which can be detected by the TBARS assay using thiobarbituric acid as a reagent. The TBARS assay measures malondialdehyde (MDA) present in the sample, as well as malondialdehyde generated from lipid hydroperoxides by the hydrolytic conditions of the reaction [All the chops (2.5 cm and 4 cm) were first weighed, then packed according to treatment, shown in Control samples for sensory assessment and for objective and chemical measurement were trimmed and packed in vacuum bags on day 0 and stored in a −20 °C freezer. Vacuum packed and HiOxMAP samples were displayed in simulated retail display in a double flat glass door upright refrigerator with LED light  at 2 ± 1 °C for 3, 7 or 14 days. The samples were rotated between shelves and position daily to minimise the effects of any variation in illumination between shelves. After display, samples for sensory assessment were packed in vacuum bags and stored at −20 °C. Samples for objective measurements were trimmed, then packed in vacuum bags for storage at −20 °C.Instrumental colour analysis on the surface of meat samples was conducted using a Hunterlab Miniscan EZ  that had been calibrated against white and black reference tiles. The settings used for colour measurement were D65 illuminant and 10° observer angle. CIE L* (lightness), a* (redness) and b* (yellowness) values  were obtFor purge loss, samples from 3 and 7 days in VAC or HiOxMAP were weighed before and after packaging. The purge loss was calculated as the initial weight minus the final weight, divided by the initial weight, multiplied by 100 to obtain percentage as described previously .Cooking of pork samples was performed according to procedures outlined in , with mo2 × 4–5 cm rectangular strips were cut parallel to the direction of muscle fibres, and the WBSF was measured using a Warner-Bratzler shear force blade (V-shaped) adapted to a Lloyd machine  with a 500 N load cell, and the shearing speed was set at 300 mm/min. The peak of the shear force was recorded and the mean was calculated from the 6 sub-samples.Objective texture measurements were conducted, using the Warner-Bratzler shear force (WBSF) method of  with modHardness: the force work required for the initial penetration,Cohesiveness : the reduced force work required for second penetration from work needed for the initial penetration,Chewiness: the force required to achieve hardness and cohesiveness.A modified compression method was used to analyse the effects of connective tissue on tenderness of meat using methods of  with modTBARS analysis was conducted using a modified extraction method of Witte, et al. . 20 g duThe design of this sensory assessment was in accordance with previous sensory studies on pork  but withThe allocation of samples within one sensory session was randomized based on 10 consumers consuming pieces of meat from 10 carcasses, and was designed by using Latin–square design. Every consumer tasted the three treatments twice, enabling a high degree of comparison between treatments and between carcasses. Serving orders can be a factor in influencing sensory results and therefore random serving orders were used in the sensory evaluation. The randomisation of samples presented to consumers was also designed based on Latin-square design. Allocation of frozen samples into sensory sessions was achieved by sorting samples in the sensory lab three days before sensory sessions. Samples were taken out from freezer and laid out on tables. Using an allocation sheet, samples were sorted into different sessions with 30 steaks in each session, then placed back in the freezer. Before thawing, carton numbers and samples in cartons were checked to avoid errors and also samples were placed in a single layer to ensure consistent rates of sample thawing. Samples were thawed in a refrigerator at 2 ± 1 °C for 24 h before cooking. Prior to cooking, the temperature of steaks were recorded and ranged between 5 and 8 °C. After thawing, samples were taken out from vacuum bags ready for cooking.Cooking was conducted on two Silex grillers . Five steaks were cooked at a time on each Silex griller, thus 10 samples were served simultaneously to 10 consumers. As outlined by Channon, Taverner, D‘Souza and Warner , pork saIn each session, consumers were seated in individual sensory booths. Before evaluation, consumers were given instruction and explanations and were asked to fill out a questionnaire to record the demographic information including name, age group, and pork consumption habit. Each consumer was asked to assess each sample for aroma (dislike extremely to like extremely), tenderness (not tender to very tender), juiciness (not juicy to very juicy), flavour (dislike extremely to like extremely), overall liking (dislike extremely to like extremely) and quality grade  and purchase intention  on a 100 mm line scale with 0 representing the minimum and 100 representing the maximum. In the case of quality grade and purchase intention, they were anchored with words at either end of the scale, as for the other traits, but also had the words ‘average’, and ‘might buy it’ in the middle of the scale. The 10 tastings for each sample were averaged to give the final eating quality score for each. The methodology for sample collection, allocation to treatments, allocation to consumer sessions, cooking, running of consumer sessions, analysis of the line scales, and averaging were first described in Watson, Gee, Polkinghorne and Porter , and havThe GENSTAT program  was used to do an analysis of variance to determine the effects of packaging and ageing on the texture and the sensory attributes of the pork loin samples. Block factors of carcass and replicate were used in ANOVA for purge loss, cooking loss, WBSF, hardness, chewiness, cohesiveness, TBARS and colour measurements. The analysis of variance for sensory attributes included blocking factors of carcass, replicate and session in the ANOVA. p < 0.05 for all except flavour, p < 0.10). In addition, the 7 day VAC samples had higher scores for re-purchase intention, relative to the 7 day HiOxMAP samples (p < 0.05). In terms of objectively measured tenderness, vacuum packaging of pork resulted in an average WBSF value of 54.1N, which were approximately 5 units higher than the control and HiOxMAP treated samples (see below for further results for WBSF). Generally, meat must have a PSF of <40 N (4.1 kg) in order to ensure high levels of consumer acceptability [The impact of packaging and ageing on sensory attributes of pork are shown in tability . Packaging and ageing treatments did not result in a change in sensory scores for aroma of pork in the current study. This is in agreement with a study by Lund, Lametsch, Hviid, Jensen and Skibsted  who examp < 0.01), as presented in Both packaging and ageing influenced the tenderness of pork (p < 0.001) as presented in p < 0.05) such that for HiOxMAP, cohesiveness increased between days 3 and 7 whereas there was no change for VAC samples. The influence of packaging and ageing on chewiness is also shown in p < 0.05).There were effects of packaging type and ageing period on the hardness of pork loins  and tensile force. These mechanical forces are defined as objective assessments in estimating the tenderness of meat . Data prp < 0.001 for both). Also, there was a trend for an interaction between packaging and ageing (p < 0.10). Over the ageing period from 3 to 7 days ageing, purge loss of VAC pork increased from 6.8% to 10.5%, while purge loss of pork in HiOxMAP only slightly increased from 5.1% to 7.0%. Purge loss is associated with fluid loss from meat during storage [The influence of packaging and ageing on purge loss is presented in  storage , and is  storage  who show storage . The hig storage . Howeverng p < 0.. Over thp < 0.05). Cooking loss for control and pork loin steaks aged in HiOxMAP for 3 days were the highest, HiOxMAP and VAC samples stored for 7 days were intermediate and 3 day VAC samples had the lowest cook loss. Cooking loss is defined as the loss of fluid, along with soluble substances, from meat during the cooking process, and it has been established that it could affect tenderness and juiciness [Differences were observed in cooking loss among packaging × ageing treatments as displayed in uiciness . In thisuiciness . Lagerstuiciness  found thp > 0.05). After prolonged storage, HiOxMAP packaging produced the palest colour samples and this was significantly different after 7 and 14 days of storage (p < 0.05). An increase in lightness is attributed to relative contents of chemical forms of myoglobin and increased light scattering due to protein denaturation [The effect of time and packaging on L*a*b* values and oxy/met ratio is given in turation . The incturation .p < 0.001), but gradually fell as storage time progressed. The a* values of HiOxMAP and VACb after 14 days of storage were not significantly different (p > 0.05). However, HiOxMAP loins still produced higher redness compared to VACnb after 14 days of storage (p < 0.001). Redness level is an important factor in producing the desired pink colour in pork [The a* values of pork loins were highest at 3 days of storage when packed in HiOxMAP , but gradually decreased as storage time progresses. The b* values of VACb and HiOxMAP at day 14 were not different (p > 0.05) but VACnb and HiOxMAP were different (p < 0.001). O’Sullivan, Byrne, Martens, Gidskehaug, Andersen and Martens [The b* values of pork loins followed a similar trend to a* values, where HiOxMAP loins produced the highest b* values after 3 days of storage ( Martens  and Chiz Martens  indicatep < 0.05) but declined after this time period. At 7 days storage, the oxy/met ratio of HiOxMAP meat was lower than VACb, and was lower than both VACb and VACnb after 14 days of storage (p < 0.001). The oxy/met ratio is measured from reflectance and is a measurement which closely relates to what the human eye and brain can see [For the oxy/met ratio, HiOxMAP produced the highest value after 3 days of storage ( can see . This va can see . The Aus can see . AccordiBrewer and McKeith  suggestep < 0.001), suggesting the beneficial effects of VAC and 30 min blooming on minimising metmyoglobin formation and subsequently general appearance of pork loins in long term storage. The results have indicated that HiOxMAP packaging caused an increased rate of decline in the oxy/met ratio on the surface of pork loins, indicating that metmyoglobin formation is accelerated in a high oxygen environment which produced the undesirable pale, brown colour. Blooming of VAC loins seemed to have a significant effect in increasing oxy/met ratio compared to no blooming  pork —which is described as L* > 57 . This gip < 0.001 for all). HiOxMAP had a significant impact on lipid oxidation development, with TBARS values being higher at 3, 7 and 14 days of storage (p < 0.001) relative to VAC. VAC samples did not show an overall increase in TBARS values—with day 0 samples and day 14 samples being similar (p > 0.05) and were consistent with previous studies [ studies ,52,53. TLipid oxidation is closely correlated to pigment oxidation due to production of free radicals and ROS , and theRancidity is described as a flavour, which develops due to various factors that create undesirable and unacceptable characteristics in meat. The connection of lipid oxidation and rancidity has not been fully established. Campo, et al.  attempteThe values of TBARS found in this study have indicated that malonaldehyde production in HiOxMAP and VAC meat did not reach unacceptable levels of lipid oxidation—with the largest TBARS value being 0.37 MDA mg/kg. However, the elevated rate of lipid oxidation found in HiOxMAP product provides further explanation of the deterioration of colour properties, as well as indicating that shelf life qualities were negatively impacted compared to VAC packaging. Pork loin samples subjected to HiOxMAP exhibited inferior consumer acceptability for tenderness, flavour, overall liking, quality and re-purchase intention relative to samples vacuum packed for 7 days. Samples vacuum packed for 7 days also had lower shear force and hardness values compared to samples undergoing HiOxMAP for 7 days. In addition, HiOxMAP maintained a high a* value in samples during 3 days of storage and higher L* value at the end of storage. Blooming of vacuum packaged meat after long term storage resulted in greater colour stability, as shown by the same a* values, lower L* values and less browning (higher oxy/met) compared to HiOxMAP packaging. HiOxMAP packaging resulted in higher rates of lipid oxidation compared to VAC, which indicated a reduction in shelf life quality. This research, based on a small number of samples, has shown that pork loins have lower ‘colour and oxidation’ shelf life when packed in HiOxMAP compared to vacuum packed samples. The results have also shown that pork loins stored in HiOxMAP have elevated lipid oxidation, reduced contrast in colour and increased browning. The findings suggest that pork loins are suited for packaging in HiOxMAP for less than 7 days, possibly only 3 days, and VAC packaging should be considered for storage for any time period longer than 3–7 days. Hence, the use of vacuum packing of pork for retail shelves should be considered as the preferred option, over HiOxMAP.Using similar packaging and consumer sensory methodology, it has been shown that beef and sheep meat packed in HiOxMAP have reduced consumer sensory scores of 10–12 for beef  and 10–1