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Contemporary ecology and agronomy point to the many benefits of agroforestry crop systems for the provision of ecosystem services by regenerating native ecologies, and in many contexts, socio-economic benefits for coffee farmers, especially the contribution of nitrogen-fixing trees’ litter to soil nutrition and water retention. However, the implementation of agroforestry in coffee cultivation is thus far incomplete and uptake has been uneven. In this paper, we examine the adoption and non-adoption of agroforestry coffee growing techniques as a reflection of the historical, social and cultural embeddedness of smallholder coffee cultivation. It is structured as a narrative literature review contextualized with the results of surveys of smallholder coffee farmers in Colombia, Malawi and Uganda regarding their perceptions of agroforestry coffee in their respective contexts. Findings suggest that coffee farmers’ perceptions of agroforestry and the decision to implement or remove it are influenced by factors included in the notion of embeddedness, involving social relations, historical memory and formal and informal institutions, as well as practical capabilities. Intention and action are sometimes discordant due to the complex interactions of these institutional factors, and they often conflict with outside interveners’ expectations based on epistemological differences. The study illuminates some of the main sources, manifestations and dimensions of the social embeddedness of agricultural practices which mediate the perception of current practices, the sacrifice implied by potential changes, the credibility of theories linking action with outcome and the desirability of expected outcomes.

Background & Aims:\ud The current global pandemic of Coronavirus (COVID-19), and measures adopted to reduce its spread, threaten the nutritional status of populations in Low- and middle-income countries (LMICs). Documenting how the COVID-19 affects diets, nutrition and food security can help generating evidence-informed recommendations for mitigating interventions and policies.\ud Methods:\ud We carried out a systematic literature review. A structure search strategy was applied in MEDLINE (Pubmed®), EMBASE®, Scopus® and Web of Science®. Grey literature was retrieved by screening a pre-set list of institutions involved in monitoring the impact of COVID-19 pandemic on nutrition and food security. The first search was done on 20th August 2020, and updated in mid-November 2020 and mid-January 2021. All research steps were described as recommended in the PRISMA statement.\ud \ud Results:\ud Out of the 2085 references identified, thirty-five primary studies were included. In spite of their heterogeneity, studies converge to demonstrate a detrimental effect of COVID-19 pandemic and associated containment measures on diet quality and food insecurity. One of the major direct effects of COVID-19 on food and nutrition outcomes has been through its impact on employment, income generating activities and associated purchasing power. Other channels of impact, such as physical access, availability and affordability of food provided a heterogeneous picture and were assessed via binary and often simplistic questions. The impacts of COVID-19 manifested with various intensity degrees, duration and in different forms. Factors contributing to these variations between and within countries were: 1) timing, duration and stringency of national COVID-19 restriction measures and policies to mitigate their adverse impacts; 2) context specific food value chain responses to domestic and international containment measures; 3) differentiated impacts of restriction measures on different groups, along lines of gender, age, socio-economic status and employment conditions. Dietary changes and food insecurity manifested various intensity degrees, duration and in different forms between and within countries. Shorter value chains and traditional smallholder farms were somewhat more resilient in the face of COVID-19 pandemic. Additionally, the impact of the pandemic has been particularly adverse on women, individuals with a low socio-economic status, informal workers and young adults that relied on daily wages. Finally, there were heterogeneous government responses to curb the virus and to mitigate the damaging effects of the pandemic. It has been demonstrated that existing and well-functioning social protection programmes and public distribution of food can buffer the adverse effects on food insecurity. But social safety nets cannot be effective on their own and there is a need for broader food systems interventions and investments to support sustainable and inclusive food systems to holistic achieve food and nutrition security.\ud \ud Conclusion:\ud In conclusion, the current economic and heath crisis impact diet quality and food security, and this raises concerns about long term impacts on access to and affordability of nutrient-rich, healthy diets and their health implications. Women and individuals with a low socio-economic are the most at risk of food insecurity. Social safety nets can be effective to protect them and must be urgently implemented. We advocate for improved data collection to identify vulnerable groups and measure how interventions are successful in protecting them.

The Inquiry-Based approach to Science Education (IBSE) is presented in scienti#c literature (Hake, 1998; Sharma et al., 2010) and in research projects reports (e.g. ESTABLISH, 2010; SAILS, 2014) as a credible solution to the reported lack of ef#cacy of more ‘traditional’ educative approaches, especially when focused on Research and Innovation (RRI) (Sutcliffe, 2006; IRRESISTIBLE, 2013; Ark of Inquiry, 2013). IBSE is an inductive approach to science teaching that considers direct experience at the center of learning. IBSE activities and strategies actively involve students in the identi#cation of relevant evidence, in critical and logical reasoning on the evidence collected and in re+ection on their interpretation. Students learn to conduct investigations, but are also led by the teachers to understand the typical processes scientists use to build knowledge. IBSE strategies are credited to improve student understanding in many conceptual #elds, due to their strongly contextualized nature, that focuses on the interdependence of situation and cognition, and on the ways they facilitate learning processes (Fazio, 2020) info:eu-repo/semantics/publishedVersion

PTDC/ASP-AGR/31257/2017 UIDB/00239/2020 UIDP/04035/2020 UID/AGR/04129/2020 UIDB/04551/2020 CRA-RED-00053-16 As atmospheric [CO2] continues to rise to unprecedented levels, understanding its impact on plants is imperative to improve crop performance and sustainability under future climate conditions. In this context, transcriptional changes promoted by elevated CO2 (eCO2) were studied in genotypes from the two major traded coffee species: the allopolyploid Coffea arabica (Icatu) and its diploid parent, C. canephora (CL153). While Icatu expressed more genes than CL153, a higher number of differentially expressed genes were found in CL153 as a response to eCO2. Although many genes were found to be commonly expressed by the two genotypes under eCO2, unique genes and pathways differed between them, with CL153 showing more enriched GO terms and metabolic pathways than Icatu. Divergent functional categories and significantly enriched pathways were found in these genotypes, which altogether supports contrasting responses to eCO2. A considerable number of genes linked to coffee physiological and biochemical responses were found to be affected by eCO2 with the significant upregulation of photosynthetic, antioxidant, and lipidic genes. This supports the absence of photosynthesis down-regulation and, therefore, the maintenance of increased photosynthetic potential promoted by eCO2 in these coffee genotypes. publishersversion published

Purpose This paper aims to understand if a project work methodology proposed to students, based on an inquiry perspective and dealing with different dimensions of sustainable development, contributed to creating an interdisciplinary solution for a problem on sustainability challenged by food production and consumption, and also to understand if this methodological approach is perceived as important to their learning as professionals and citizens. Data were collected by direct observation, a questionnaire applied to the students and students’ individual reflections. Design/methodology/approach This paper aims to present the research results of the impact of a pedagogical approach on students, implemented as a part of the Doctoral Programme in Sustainability Science, which was designed following an innovative model at the University of Lisbon, Portugal. The main drivers include building a new body of interdisciplinary knowledge leading to the application of science to address real problems towards transdisciplinary education. Findings The results suggest great potential for an inquiry perspective in trying to solve a real problem. Students’ proposals were realistic, viable and complementary enough to collectively contribute in response to the global problem. The use of approaches acquired from different areas of knowledge was clear, and the project methodology was well understood. Students considered the experience very rewarding in terms of learning and contributing positively to their personal and professional development. Originality/value This Doctoral programme is anchored in a progressive continuum encompassing holistic debates with a multidisciplinary team of professors in environments that promote interdisciplinary attitudes and new knowledge, and also project work aimed at guiding students to transdisciplinary learning, which constitutes an innovative form of dealing with the complex challenges created by the science of sustainability.

The use of food dietary supplements, commonly called “multimisturas”, has been adopted, in developing countries, as a strategy to combat micronutrient deficiency in the population.. The goal of this work was to study the composition and nutritional potential of a “multimistura” that was developed and used in a vulnerable community of Nampula, Mozambique to fight micronutrient deficiency in pregnant or lactating women and in children up to 8 years old. The micronutrient composition of the “multimistura” developed with locally available products did not show statistically significant differences when compared to commercial food supplements used in industrialized countries. Taking in account the composition results and the nutritional requirements of each target group, it was observed that the currently recommended daily intake should be, at least, doubled to fulfil the nutritional needs of each of the groups. The exception regards Children in the 1-3 year old group, would be an exception, as the doubling of ingestion would be detrimental since it would exceed the tolerable daily intake of magnesium. This work allowed the suggestion of some procedures aimed at balancing the supplement nutritional composition to leverage adequate levels of household nutritional security in the target community.

The impact on cell wall (CW) of the deficiency in nitrogen (–N), phosphorus (–P) or sulphur (–S), known to impair essential metabolic pathways, was investigated in the economically important fruit species Vitis vinifera L. Using cuttings as an experimental model a reduction in total internode number and altered xylem shape was observed. Under –N an increased internode length was also seen. CW composition, visualised after staining with calcofluor white, Toluidine blue and ruthenium red, showed decreased cellulose in all stresses and increased pectin content in recently formed internodes under –N compared to the control. Using CW-epitope specific monoclonal antibodies (mAbs), lower amounts of extensins incorporated in the wall were also observed under –N and –P conditions. Conversely, increased pectins with a low degree of methyl-esterification and richer in long linear 1,5-arabinan rhamnogalacturonan-I (RG-I) side chains were observed under –N and –P in mature internodes which, in the former condition, were able to form dimeric association through calcium ions. –N was the only condition in which 1,5-arabinan branched RG- content was not altered, as –P and –S older internodes showed, respectively, lower and higher amounts of this polymer. Higher xyloglucan content in older internodes was also observed under –N. The results suggest that impairments of specific CW components led to changes in the deposition of other polymers to promote stiffening of the CW. The unchanged extensin amount observed under –S may contribute to attenuating the effects on the CW integrity caused by this stress. Our work showed that, in organized V. vinifera tissues, modifications in a given CW component can be compensated by synthesis of different polymers and/or alternative linking between polymers. The results also pinpoint different strategies at the CW level to overcome mineral stress depending on how essential they are to cell growth and plant development.

Mozambican forests are exposed to risks that contribute to the loss of biodiversity and associated ecosystem services. Thus, ex situ conservation represents a key strategy to reduce genetic erosion. In this study, we evaluated the ecological status of the ex situ conservation plantation in Michafutene, Maputo province, one of the most important repositories of forest genetic resources in the country. Thirty plots were established in which all trees, shrubs and grass species were identified. A total of 2092 individuals spanning 39 species were scored. Afzelia quanzensis was the most important species (Importance Value Index – IVI = 203), but with a low silvicultural performance. Other important trees were Albizia adianthifolia (IVI = 32), Albizia versicolor (IVI = 16) and Pterocarpus angolensis (IVI = 12). A complementary genotyping analysis of A. quanzensis was conducted by intersimple sequence repeats, indicating that the germplasm collection has different provenances and represents a wide genetic pool. Thus, despite the poor management, there is a considerable potential for the conservation of A. quanzensis provided immediate and appropriate management activities are implemented to improve its ecological performance. Resume Mozambican forests are exposed to risks that contribute to the loss of biodiversity and associated ecosystem services. Thus, ex situ conservation represents a key strategy to reduce genetic erosion. In this study, we evaluated the ecological status of the ex situ conservation plantation in Michafutene, Maputo province, one of the most important repositories of forest genetic resources in the country. Thirty plots were established in which all trees, shrubs and grass species were identified. A total of 2092 individuals spanning 39 species were scored. Afzelia quanzensis was the most important species (Importance Value Index – IVI = 203), but with a low silvicultural performance. Other important trees were Albizia adianthifolia (IVI = 32), Albizia versicolor (IVI = 16) and Pterocarpus angolensis (IVI = 12). A complementary genotyping analysis of A. quanzensis was conducted by Inter-Simple Sequence Repeats, indicating that the germplasm collection has different provenances and represents a wide genetic pool. Thus, despite the poor management there is a considerable potential for the conservation of A. quanzensis provided immediate and appropriate management activities are implemented to improve its ecological performance. Les forets mozambicaines sont exposees a des risques qui peuvent contribuer a la perte de leur biodiversite et des services ecosystemiques qui y sont associes. La conservation ex situ constitue donc une strategie cle pour reduire cette erosion genetique. Dans cette etude, nous avons evalue le statut ecologique de la plantation ex situ de Michafutene, dans la province de Maputo, qui compte parmi les plus importants depositaires des ressources genetiques forestieres du pays. Trente parcelles ont ete etablies, ou toutes les especes d'arbres, arbustes et herbes ont ete identifiees. Au total, 2092 individus appartenant a 39 especes ont ete denombres. L'espece la plus importante etait Afzelia quanzensis (Indice de valeur d'importance – IVI = 32), mais avec une faible performance sylvicole. Parmi les autres arbres importants, citons Albizia adianthifolia (IVI = 32), Albizia versicolor (IVI = 16) et Pterocarpus angolensis (IVI = 12). Une analyse genotypique complementaire d’A. quanzensis a ete realisee au niveau des microsatellites (Inter-Simple Sequence Repeats – ISSR), qui indique que la collection de germoplasmes vient de differentes provenances et represente un large pool genetique. Donc, malgre une gestion mediocre, la conservation d’A. quanzensis garde un potentiel considerable pour autant que des activites de gestion appropriees soient realisees immediatement pour en ameliorer les performances ecologiques. .

In seedless table grapes, the excessive natural fruit set leads to compact bunches, small berries with poor colour, low uniformity of maturation and higher incidence of diseases, requiring flower and fruit thinning to attain a profitable production. Gibberellic acid spraying (GAc) and imposition of shading nets, cutting incident irradiance by 90, 98 or 100% at flowering, were tested in consecutive years aiming to examine their influence on flower drop and consequent quality of ‘Sugraone’, ‘Thompson Seedless’ and ‘Crimson Seedless’ grapes. Shade treatments reduced leaf gas exchange and leaf growth rates while no differences were found in GAc-treated vines. Effects on leaf gas exchange showed to be reversible after shading net removal. Flower drop percentages increased in ‘Sugraone’ in response to 90% light reduction imposed at 50% bloom, and in ‘Thompson Seedless’ in response to GAc spraying and shade imposition, when compared to untreated vines. Bunches were significantly less compact by reducing 90% and 100% of the incident light at 50% bloom, during 15 and 24 days, on ‘Sugraone’ and ‘Crimson Seedless’, respectively, when compared to untreated vines. In ‘Thompson Seedless’ vines, bunches were less compact after shade imposed both at 50 and 100% bloom, during 14 and 10 days, respectively. In general, shade-promoted thinning induced a decrease on berry weight and diameter compared to untreated vines. GAc treatment resulted in lower bunch compactness and improved berry quality in ‘Thompson Seedless’, while the effects on berry quality observed in ‘Sugraone’ and ‘Crimson Seedless’ were not consistent across the trials conducted in different years. ‘Thompson Seedless’ was the more sensitive cultivar to GAc and shade thinning methods.

Flower-to-fruit transition depends of nutrient availability and regulation at the molecular level by sugarand hormone signalling crosstalk. However, in most species, the identities of fruit initiation regulatorsand their targets are largely unknown. To ascertain the main pathways involved in stenospermocarpictable grape fruit set, comprehensive transcriptional and metabolomic analyses were conducted specifi-cally targeting the early phase of this developmental stage in ‘Thompson Seedless’. The high-throughputanalyses performed disclosed the involvement of 496 differentially expressed genes and 28 differentlyaccumulated metabolites in the sampled inflorescences. Our data show broad transcriptome reprogram-ming of molecule transporters, globally down-regulating gene expression, and suggest that regulation ofsugar- and hormone-mediated pathways determines the downstream activation of berry development.The most affected gene was the SWEET14 sugar transporter. Hormone-related transcription changeswere observed associated with increased indole-3-acetic acid, stimulation of ethylene and gibberellinmetabolisms and cytokinin degradation, and regulation of MADS-box and AP2-like ethylene-responsivetranscription factor expression. Secondary metabolism, the most representative biological process attranscriptome level, was predominantly repressed. The results add to the knowledge of molecular eventsoccurring in grapevine inflorescence fruit set and provide a list of candidates, paving the way for geneticmanipulation aimed at model research and plant breeding info:eu-repo/semantics/publishedVersion

The tropical coffee crop has been predicted to be threatened by future climate changes and global warming. However, the real biological effects of such changes remain unknown. Therefore, this work aims to link the physiological and biochemical responses of photosynthesis to elevated air [CO2 ] and temperature in cultivated genotypes of Coffea arabica L. (cv. Icatu and IPR108) and Coffea canephora cv. Conilon CL153. Plants were grown for ca. 10 months at 25/20°C (day/night) and 380 or 700 μl CO2 l(-1) and then subjected to temperature increase (0.5°C day(-1) ) to 42/34°C. Leaf impacts related to stomatal traits, gas exchanges, C isotope composition, fluorescence parameters, thylakoid electron transport and enzyme activities were assessed at 25/20, 31/25, 37/30 and 42/34°C. The results showed that (1) both species were remarkably heat tolerant up to 37/30°C, but at 42/34°C a threshold for irreversible nonstomatal deleterious effects was reached. Impairments were greater in C. arabica (especially in Icatu) and under normal [CO2 ]. Photosystems and thylakoid electron transport were shown to be quite heat tolerant, contrasting to the enzymes related to energy metabolism, including RuBisCO, which were the most sensitive components. (2) Significant stomatal trait modifications were promoted almost exclusively by temperature and were species dependent. Elevated [CO2 ], (3) strongly mitigated the impact of temperature on both species, particularly at 42/34°C, modifying the response to supra-optimal temperatures, (4) promoted higher water-use efficiency under moderately higher temperature (31/25°C) and (5) did not provoke photosynthetic downregulation. Instead, enhancements in [CO2 ] strengthened photosynthetic photochemical efficiency, energy use and biochemical functioning at all temperatures. Our novel findings demonstrate a relevant heat resilience of coffee species and that elevated [CO2 ] remarkably mitigated the impact of heat on coffee physiology, therefore playing a key role in this crop sustainability under future climate change scenarios.

Cuticles are plant structures, composed mostly by lipidic layers, synthesized by nonwoody aerial plant organs and deposited on the surface of outer epidermal cell walls. Although its significance has been often disregarded, cuticle deposition modifies organ chemistry, influences mechanical properties, and plays a central role in sensing and interacting with the surrounding environment. Even though some research has been undertaken addressing cuticle biosynthesis and composition in vegetative plant tissues, comparatively less information is available regarding cuticle composition in the epidermis of fruits. However, recent work points to a role for cuticles in the modulation of fruit quality and postharvest performance, indicating that current models for the investigation of fruit development, metabolism, and quality need to integrate a comprehensive knowledge of the cuticle layer. This paper provides an overview of recent findings and observations regarding cuticle biosynthesis and composition in fruits from species of agronomic and economic relevance. Important, but often neglected differences in cuticle composition and biosynthesis patterns among diverse fruit species are described herein to generate an atlas of what is currently known about fruit cuticles and to highlight what remains to be explored. Emphasis is placed on the need to investigate each genetic background considering its own specificities, to permit correlations with the particular physiology of each species considered. Both specific composition and changes during maturation and ripening are reviewed. B.B. is the recipient of a FI-DGR grant from AGAUR (Generalitat de Catalunya). Financial support through the AGL2010-14801/ALI project, granted by the Ministerio de Ciencia e Innovación (MICINN) of Spain, is gratefully acknowledged.

The noteworthy fine remodeling that plant cell walls (CWs) undergo to adapt to developmental, physiological and environmental cues and the observation that its composition and dynamics differ between species represents an opportunity to couple crop species agronomic studies with research on CW modifications. Vitis vinifera is one of the most important crops from an economic point-of-view due to the high value of the fruit, predominantly for winemaking. The availability of some information related to this species’ CWs allows researching its responses to imposed conditions that affect the plant’s development. Mineral deficiency, in particular nitrogen, phosphorus, potassium and sulfur, strongly affects plant metabolism, reducing both growth and crop yield. Despite the importance of mineral nutrition in development, its influence on CW synthesis and modifications is still insufficiently documented. Addressing this knowledge gap, V. vinifera experimental models were used to study CW responses to imposed mineral depletion in unorganized (callus) and organized (shoots) tissues. The discussion of the obtained results is the main focus of this review. Callus and shoots submitted to mineral restriction are impaired in specific CW components, predominantly cellulose. Reorganization on structure and deposition of several other polymers, in particular the degree and pattern of pectin methyl-esterification and the amount of xyloglucan (XyG), arabinan and extensin, is also observed. In view of recently proposed CW models that consider biomechanical hotspots and direct linkages between pectins and XyG/cellulose, the outcome of these modifications in explaining maintenance of CW integrity through compensatory stiffening can be debated. Nutrient stresses do not affect evenly all tissues with undifferentiated callus tissues showing more pronounced responses, followed by shoot mature internodes, and then newly formed internodes. The impact of nitrogen depletion leads to more noticeable responses, supporting this nutrient’s primary role in plant development and metabolism. The consequential compensatory mechanisms highlight the pivotal role of CW in rearranging under environmental stresses info:eu-repo/semantics/publishedVersion

Ripening-related firmness loss shortens considerably the storage potential of sweet cherry (Prunus avium L.), thus limiting postharvest handling, transportation, and commercialisation. The biochemical mechanisms underlying this process in cherries are not fully understood, and the mechanisms operating in a given fruit may be not extrapolated to a different species. Cell wall materials obtained from untreated and calcium-treated 'Celeste' sweet cherries were fractionated and analysed after cold storage, and related enzyme activities and gene expression were assessed. Calcium-treated fruit were firmer, with lower weight loss and decay incidence than the controls. The accumulation of PaβGal and PaEXP1 transcripts was strongly inhibited in cold-stored fruit, although expression levels recovered largely after three days of shelf life. Data suggest that pectin methylesterase, β-galactosidase and expansin activities may control the access of additional proteins to their substrates. A possible role for the apoplastic redox status in the modulation of the process is also discussed B. Belge was the recipient of a FI-DGR grant from AGAUR (Generalitat de Catalunya). This work was supported through the AGL2010-14801/ALI project, funded by the Ministerio de Ciencia e Innovación (MICINN) of Spain.

Rice is the world's most important staple food and the basis of the diet of the majority of the population. In small farm agriculture, the yields obtained in cereal production are usually low and losses, both in the field and during storage, are dramatically high, particularly in developing countries. In Mozambique, these aspects, together with an increased frequency of floods, are hindering advances in rice production. Aimed at contributing to the reduction of losses in stored rice, trials were carried out to compare the effectiveness of traditional raffia bags and of hermetic storage using single and double plastic bags concerning quantitative losses and seed quality, including germination potential, after three and six months of storage. Pest identification, insect populations estimates, percentage of weight loss, germination power and seedling vigor were evaluated. The results showed that, in descending order of density, Angoumois grain moth (Sitotroga cerealella Olivier), lesser grain borer (Rhyzopertha dominica F.), rice/maize weevil (Sitophilus zeamais Mostch. and Sitophilus oryzae L.) and red flour beetle [Tribolium castaneum (Herbst)] were the main insects infesting the rice. When compared to hermetic storage with both single and duplicate airtight bags, traditional storage presented statistically significant higher mean infestation density (30.63–53.94 individuals/kg in traditional and 0.71–3.50 individuals/kg in hermetic storage) and percentage of weight loss (3.03–3.44% in traditional contrasting with 0.27–0.47% in hermetic conditions). In traditional storage a significant 38.25% drop in the germination potential was also observed, attaining values below the established minimum tolerated in Mozambique (80%), while under hermetic storage, that reduction remained within the acceptable values of 13.9–17.5%. The distinct storage methods did not produce significant differences on the moisture content of the grain. These results demonstrate that the use of hermetic storage has resulted in a safe, pesticide-free, and sustainable storage method, suitable for rice seeds, with advantages over traditional bagging. The results presented here lead to propose hermetic storage to be adopted by paddy small farmers, in order to lever food security and income generation in the country.

Temperature and mineral nutrition are major environmental factors regulating plant growth and development. Yet, cold impact on mineral contents and the ability of the plants to perform changes in specific elements as a part of the acclimation process received little attention. Using five Coffea genotypes previously characterized concerning their cold sensitivity, a mineral analysis was performed considering macro (N, P, K, Ca, Mg, and S) and micro (Na, Fe, Mn, Zn, Cu, and B) nutrients in order to predict their importance in cold tolerance. The results showed a cold-induced dynamics of mineral nutrients in recently mature leaves. The less cold sensitive Icatu, and partially Catuai, accumulated N, Ca, Mn, Cu, and Zn with potential implications in the maintenance of photosynthetic performance, the reinforcement of the antioxidative defense system, lipid metabolism, and the expression of cold regulated genes, thus constituting interesting traits to evaluate the cold acclimation ability. After a principal component analysis (PCA), N, Fe, Mn, and Cu were further confirmed as strong candidates for an early cold tolerance evaluation due to their dynamics and to specific roles in the activities of Cu/Zn-SOD (Cu), APX (Fe), and PSII (Mn).

In spite of the large number of reports on the aerobic respiratory chain of Escherichia coli, from gene transcription regulation to enzyme kinetics and structural studies, an integrative perspective of this pathway is yet to be produced. Here, a multi-level analysis of the aerobic respiratory chain of E. coli was performed to find correlations between gene transcription, enzyme activity, growth dynamics, and supercomplex formation and composition. The transcription level of all genes encoding the aerobic respiratory chain of E. coli varied significantly in response to bacterial growth. Coordinated expression patterns were observed between the genes encoding NADH : quinone oxidoreductase and complex I (NDH-1), alternative NADH : quinone oxidoreductase (NDH-2) and cytochrome bdI, and also between sdhA and appC, encoding succinate dehydrogenase and cytochrome bdII, respectively. In general, the rates of the respiratory chain activities increased from mid-exponential to late-stationary phase, with no significant further variation occurring until the mid-stationary phase. Multi-level correlations between gene transcription, enzyme activity and growth dynamics were also found in this study. The previously reported NADH dehydrogenase and formate : oxygen oxidoreductase supercomplexes of E. coli were already assembled at mid-exponential phase and remained throughout growth. A new succinate oxidase supercomplex composed of succinate dehydrogenase and cytochrome bdII was identified, in agreement with the suggestion provided by the coordinated transcription of sdhA and appC.

Selection and validation of appropriate reference genes should be the first step to consider in experiments based on quantitative real-time polymerase chain reaction (qRT-PCR). In this study, ten candidate genes were investigated for their stability as suitable reference genes in qRT-PCR data normalization using a diverse set of 12 Coffea cDNAs from plants from three different species/genotypes exposed to single or multiple abiotic stresses (drought and chilling, alone or in combination). Primer amplification efficiencies were calculated for all of the selected genes and varied according to each individual genotype. The expression of each gene was measured by qRT-PCR to evaluate its stability. A multiple analytical approach was followed, based on consensus merged data from four different complementary statistics, namely geNorm, BestKeeper, NormFinder, and coefficient of variation, which produced comparable but not identical results. According to this approach, the most suitable sets of reference genes for data normalization in the five experimental datasets are (1) total assay: GAPHD, Cycl, and UBQ10; (2) genotype: GAPDH, UBQ10, Ap47, and EF-1A; (3) cold stress: UBQ10, GAPDH, ACT, and EF-1A; (4) drought stress: GAPDH, ACT, EF1A, and Apt; and (5) multiple stress: UBQ10, GAPDH, ACT, and elf-4A. Normalization of gene expression using these selected genes was validated by examination of the expression of the photosynthetic-related ApoA2 gene in samples from non-stressed and stressed plants. Our results are useful to assist studies on Coffea physiology with the aim of breeding for increased tolerance to abiotic stress conditions.

Textural changes that lead to softening of fruits are accompanied by loss of neutral sugars, solubilisation and depolymerisation of the polysaccharides of the cell wall, and rearrangements of their associations, as the result of the combined action of several cell wall-modifying enzymes, acting in both pectic and hemicellulosic fractions. Recent studies on the structure of the plant cell wall have disclosed a large number and type of biochemical linkages between the components. Such linkages are potential targets for enzymatic action and draw attention to the putative involvement of several members of enzymes able to act and modify its structure in a developmental and coordinated way. Extensive work on fruit ripening has been done using tomato (Solanum lycopersicum [Lycopersicon esculentum Mill.]) as a plant model and the information concerning fruits other than model species is fragmented and incomplete. However, recent data from the literature had disclosed that differences exist between fruits, and even between cultivars of the same fruit species. These differences exist in the type and extent of the modification of the polysaccharides of the cell wall and in the expression and regulation of cell wall-modifying enzymes. In addition, genetic manipulation of cell wall-modifying genes re-opened the discussion about the real effect of these enzymes in the cell wall and their role in fruit softening. Moreover, the function of each enzyme has been proposed based on its homology with other annotated sequences, but, in most cases, confirmation of activity in planta and substrate specificity remains to be investigated. This aspect and recognized limitations of the in vitro enzymatic activity assays also need to be considered when discussing their role. This paper provides a critical review on the current knowledge concerning these differences and emphasises the need of using other species and more accurate methodologies to investigate general mechanisms and fruit specificities of softening among different fleshy fruits.

To gain a better insight on the molecular regulation of apple ( Malus × domestica Borkh. cv ‘Mondial Gala’) fruit ripening, the mRNA Differential Display technique coupled with silver-staining was used with success as a method to identify and isolate cDNAs representing transcripts differentially expressed during this developmental process. Using 25 primer combinations and three populations of mRNA (samples from unripe but fully expanded fruits, from fruits at harvest and from overripe fruits), a total of 927 bands were obtained, of which 14 non-redundant cDNAs were confirmed to be up-regulated during apple ripening, after elimination of false-positives by RT-PCR (reverse transcription polymerase chain reaction) expression studies. DNA sequence analyses of the clones identified revealed significant homology to known plant genes and proteins reported in public databases, namely a 14-3-3 protein, a zinc-finger protein, a lipoxygenase, a flavonol synthase, a WD-40 repeat protein, a protein disulphide isomerase, an l -allo-threonine aldolase, an anthranilate N -hydroxycinnamoyl/benzoyltransferase, an homologue to a phototropic-response protein, a known apple allergen (Mal d1), two CTP synthase clones, an IAA-amidohydrolase and a calcium-binding EF hand protein. These proteins can be associated to biological processes like lipid metabolism, secondary metabolism, signal transduction, gene transcription and RNA metabolism, stress responses or defense. Still, for some of the clones, this is the first report in which they are associated with ripening. Even so, the homology found with previously described proteins allows disclosing possible biochemical roles for the proteins putatively encoded by these gene families and their putative significance in apple ripening is discussed. The results provide a contribution to better characterise the changes in gene expression that accompanies the ripening process in apple; a specie which lacks information about the molecular regulation of ripening.

Background Abscission is a highly coordinated developmental process by which plants control vegetative and reproductive organs load. Aiming at get new insights on flower abscission regulation, changes in the global transcriptome, metabolome and physiology were analyzed in ‘Thompson Seedless’ grapevine (Vitis vinifera L.) inflorescences, using gibberellic acid (GAc) spraying and shading as abscission stimuli, applied at bloom. Results Natural flower drop rates increased from 63.1 % in non-treated vines to 83 % and 99 % in response to GAc and shade treatments, respectively. Both treatments had a broad effect on inflorescences metabolism. Specific impacts from shade included photosynthesis inhibition, associated nutritional stress, carbon/nitrogen imbalance and cell division repression, whereas GAc spraying induced energetic metabolism simultaneously with induction of nucleotide biosynthesis and carbon metabolism, therefore, disclosing alternative mechanisms to regulate abscission. Regarding secondary metabolism, changes in flavonoid metabolism were the most represented metabolic pathways in the samples collected following GAc treatment while phenylpropanoid and stilbenoid related pathways were predominantly affected in the inflorescences by the shade treatment. However, both GAc and shade treated inflorescences revealed also shared pathways, that involved the regulation of putrescine catabolism, the repression of gibberellin biosynthesis, the induction of auxin biosynthesis and the activation of ethylene signaling pathways and antioxidant mechanisms, although often the quantitative changes occurred on specific transcripts and metabolites of the pathways. Conclusions Globally, the results suggest that chemical and environmental cues induced contrasting effects on inflorescence metabolism, triggering flower abscission by different mechanisms and pinpointing the participation of novel abscission regulators. Grapevine showed to be considered a valid model to study molecular pathways of flower abscission competence acquisition, noticeably responding to independent stimuli. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0722-7) contains supplementary material, which is available to authorized users.

Cell wall (CW) is a dynamic structure that determines the plant form, growth and response to environmental conditions. Vitis vinifera callus grown under nitrogen (-N), phosphorous (-P) and sulfur (-S) deficiency were used as a model system to address the influence of mineral stress in CW remodeling. Callus cells morphology was altered, mostly under -N, resulting in changes in cell length and width compared with the control. CW composition ascertained with specific staining and immuno-detection showed a decrease in cellulose and altered pattern of pectin methylesterification. Under mineral stress genes expression from candidate families disclosed mainly a downregulation of a glycosyl hydrolase family 9C (GH9C), xyloglucan transglycosylase/hydrolases (XTHs) with predicted hydrolytic activity and pectin methylesterases (PMEs). Conversely, upregulation of PMEs inhibitors (PMEIs) was observed. While methylesterification patterns can be associated to PME/PMEI gene expression, the lower cellulose content cannot be attributed to altered cellulose synthase (CesA) gene expression suggesting the involvement of other gene families. Salt extracts from -N and -P callus tissues increased plastic deformation in cucumber hypocotyls while no effect was observed with -S extracts. The lower endo-acting glycosyl hydrolase activity of -N callus extracts pinpoints a more expressive impact of -N on CW-remodeling.

Understanding abscission is both a biological and an agronomic challenge. Flower abscission induced independently by shade and gibberellic acid (GAc) sprays was monitored in grapevine (Vitis vinifera L.) growing under a soilless greenhouse system during two seasonal growing conditions, in an early and late production cycle. Physiological and metabolic changes triggered by each of the two distinct stimuli were determined. Environmental conditions exerted a significant effect on fruit set as showed by the higher natural drop rate recorded in the late production cycle with respect to the early cycle. Shade and GAc treatments increased the percentage of flower drop compared to the control, and at a similar degree, during the late production cycle. The reduction of leaf gas exchanges under shade conditions was not observed in GAc treated vines. The metabolic profile assessed in samples collected during the late cycle differently affected primary and secondary metabolisms and showed that most of the treatment-resulting variations occurred in opposite trends in inflorescences unbalanced in either hormonal or energy deficit abscission-inducing signals. Particularly concerning carbohydrates metabolism, sucrose, glucose, tricarboxylic acid (TCA) metabolites and intermediates of the raffinose family oligosaccharides pathway were lower in shaded and higher in GAc samples. Altered oxidative stress remediation mechanisms and indolacetic acid (IAA) concentration were identified as abscission signatures common to both stimuli. According to the global analysis performed, we report that grape flower abscission mechanisms triggered by GAc application and C-starvation are not based on the same metabolic pathways.

The cell wall (CW) is a dynamic structure that responds to stress. Water shortage (WS) impacts grapevine berry composition and its sensorial quality. In the present work, berry texture, skin CW composition, and expression of remodeling genes were investigated in two V. vinifera varieties, Touriga Nacional (TN) and Trincadeira (TR), under two water regimes, Full Irrigation (FI) and No Irrigation (NI). The global results allowed an evident separation between both varieties and the water treatments. WS resulted in increased anthocyanin contents in both varieties, reduced amounts in cellulose and lignin at maturation, but an increase in arabinose-containing polysaccharides more tightly bound to the CW in TR. In response to WS, the majority of the CW related genes were down-regulated in a variety dependent pattern. The results support the assumption that WS affects grape berries by stiffening the CW through alteration in pectin structure, supporting its involvement in responses to environmental conditions.

Textural changes that lead to softening of apples are accompanied by enzymatic degradation and loss of neutral sugars of the pectic polysaccharides of the cell-wall. Recently, it has been suggested that the process is the result of the combined action of several cell-wall degrading enzymes. This work aims to establish a temporal sequence of the action of cell-wall degrading enzymes during apple growth and ripening. A cDNA enriched with mRNA isolated from over-ripe fruits was constructed and screened to clone: an endo-1,4-β-glucanase (EGase), two xyloglucan endotransglycosylase/hydrolases (XTH), an expansin, an α-L-arabinofuranosidase (AFase), a pectin methylesterase (PME), and a pectate lyase (PL) full-length cDNAs encoding late isoforms. These clones were characterized and their accumulation during fruit growth and ripening was examined by semi-quantitative RT-PCR analyses. Expression of β-galactosidase (βGal) and polygalacturonase (PG) mRNAs previously reported was also included in the analyses. Transcripts of all enzymes, except PME, could be detected in fruits at harvest. However, transcripts of EGase were more abundant at fruit set and both expansin and pectate lyase had high expression before ripening. The strongest expression in over-ripe fruits was observed for βGal and PG clones. RT-PCR analyses were also conducted in flowers, peduncles, young and expanded leaves and senescent petioles, and the results showed that the cloned cDNAs are not fruit-specific. The activity for each enzyme was also monitored in fruits at the same stages as for mRNA accumulation. Comparison of the patterns of enzyme activity with mRNA expression suggests the presence of isoforms acting in fruit growth and ripening-specific isoforms or post-transcriptional regulation. Furthermore, the results indicate that the pattern of softening of apple fruits different from the model plant tomato.

In this work the results previously reported from random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLPTM) and inter-simple sequence repeated (ISSR) techniques were used to compare their capacity to discriminate cultivars and to evaluate the genetic diversity among 24 cultivars of Pyrus communis. The three marker systems studied did not show relevant differences in polymorphism detection (informativeness) nor in genetic similarity estimates among cultivars. However, they presented considerable divergences in the number of markers generated per primer (Multiplex Index), which influenced the differences observed in efficiency of the marker systems. AFLP obtained the highest Marker Index value (11.73), followed by ISSR (8.81), while RAPD presented the lowest (2.06). Nevertheless ISSR markers were technically simpler, faster and cheaper than AFLP and they appeared to be the best technique for large-scale DNA fingerprinting analysis, for genetic diversity studies and germplasm evaluation. In spite of low correlation values of genetic similarities calculated between marker systems, the ISSR and AFLP dendrograms showed similar cultivars cluster. A more consistent and robust dendrogram was constructed, combining the results from the three marker systems, and could confirm or reinforce the results previously reported with each marker alone.

The composition and structure of the surface tissues have a noticeable influence on the postharvest storage potential of fruits, inasmuch as they behave as a barrier against drying, chemical attacks, mechanical injuries and microbial infections. Cuticle is made of cutin - a biological insoluble polyester - embedded in an impermeable wax complex, and its inner side interacts intimately with the underlying epidermal cell walls. The cuticle plays a decisive role in plant development, being its first communication system with the surrounding biotic and abiotic environment. Published reports on composition and biosynthesis of fruit cuticles are comparatively scarce, and many knowledge gaps exist as to what part they play in quality determination and postharvest performance. This review aims at collecting available information in relation to the role of fruit cuticle as a determinant factor of some important traits related to postharvest quality of produce, including water loss, susceptibility to several physical and biological stresses, and decreased fruit firmness. To our best knowledge, this is the first published work focusing on fruit cuticle as a major modulator of postharvest quality, and interlinking pre-existing disperse literature on this topic. A deeper comprehension of cuticle structure and functions will be of help in understanding postharvest biology and in designing new technological solutions. B. Belge is the recipient of a FI-DGR grant from AGAUR (Generalitat de Catalunya). Financial support through the AGL2010-14801/ALI project, granted by the Ministerio de Ciencia eInnovación (MICINN) of Spain, is gratefully acknowledged.

Molecular markers which sample multiple loci simultaneously, like amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSR), and selective amplification of microsatellite polymorphic loci (SAMPL), produce highly informative fingerprints due to their high effective multiplex ratio and expected heterozygosity. Moreover, these markers can be generated for DNA of any organism without initial investment in primer/probe development or in sequence analyses. The fragments produced can be visualized either by agarose or polyacrylamide gel electrophoresis followed by autoradiography or silver staining or via separation and detection on automatic DNA sequencers. Here, we describe detailed protocols based on the original methods aimed to obtain these markers optimized to be resolved on polyacrylamide gel electrophoresis and detected by silver staining which provides a fast, sensitive, and cost-effective method.

Patterns of phenotypic and phenetic variability in sixPortuguese cultivars of chestnut (Castanea sativa Mill.) are evaluated. Morphological characterisationwas based on the quantification of seventeen traits. Varianceanalysis showed significant differences among cultivars, and cultivar× year for all the traits studied, and trees within cultivarsshowed also some significant differences for some of themorphological variables. A significant correlation was obtainedbetween length of the leaf blade and the percentage of unisexual andandrogynic inflorescence with the effective thermal index,accumulated rainfall from April to October and from July to October,or the accumulated temperature below seven during the dormant period.Principal Component and cluster analysis were performed to group thecultivars, according to their similarity coefficients. For molecularcharacterisation, 125 RAPD and 157 ISSR polymorphic markers wereamplified using 28 and 7 primers respectively. High level ofcongruence among the two marker systems (r =90.5%) was obtained from comparison of pheneticsimilarities based on the percentage of shared fragments. ISSRmarkers revealed important advantages over RAPDs, due to a higheffective multiplex ratio (12.5 for ISSR compared with 2.2 forRAPD analysis) and reproducibility. Although morphological andmolecular results are comparable, slight differences are showed incluster analysis UPGMA dendrograms. Molecular analysis explainedhomonym situations among 'Martainha' and'Longal' cultivars in Portugal.

In this study, two microsatellite-based methodologies (SSR and ISSR) were evaluated for potential use in fingerprinting and determination of the similarity degree between 41 commercial cultivars of apple previously characterised using RAPD and AFLP markers. A total of 13 SSR primer sets was used and 84 polymorphic alleles were amplified. Seven ISSR primers yielded a total of 252 bands, of which 176 (89.1%) were polymorphic. Except for cultivars obtained from somatic mutations, all cultivars were easily distinguishable employing both methods. The similarity coefficient between cultivars ranged from 0.20 to 0.87 for SSR analysis and from 0.71 to 0.92 using the ISSR methodology. Dendrograms constructed using UPGMA cluster analysis revealed a phenetic classification that emphasises the existence of a narrow genetic base among the cultivars used, with the Portuguese cultivars revealing higher diversity. This study indicates that the results obtained based on the RAPD, AFLP, SSR and ISSR techniques are significantly correlated. The marker index, based on the effective multiplex ratio and expected heterozygosity, was calculated for both analyses (MI = 1.7 for SSR and MI = 8.4 for ISSR assays) and the results obtained were directly compared with previous RAPD and AFLP data from the same material. The SSR and ISSR markers were found to be useful for cultivar identification and assessment of phenetic relationships, revealing advantages, due to higher reproducibility, over other commonly employed PCR-based methods, namely RAPD and AFLP.

Forty-one apple (Malus × domestica Borkh.) cultivars were screened for RAPD (Random Amplified Polymorphic DNA) and AFLP(Amplified Fragment Length Polymorphism) markers. RAPD analysis was performed with 35 arbitrary 10-mer primers, selected from 60 primers tested (kits A, C and E, Operon Technologies, Inc.). Of a total of 362bands observed, 208 (57.5%) were polymorphic. Three-hundred-and-eighty-one AFLP fragments were obtained with 8primer combinations, of which 218 (57.2%) were polymorphic. Cultivars differentiated through mutation were included in this study and showed identical patterns when analysed with both RAPD and AFLP analysis. The estimated genetic relationships were correlated (r = 73.7%) between the analysis with the two different markers. UPGMA analysis was performed and dendrograms were constructed using either the data apart from each(RAPD and AFLP) method or combined in a single joint matrix. The relationships among the forty-one studied cultivars were basically consistent with the known lineage and geographic origins of the cultivars. The four Portuguese cultivars included in this study clustered together and diverged from the other cultivars. Apparently they constitute an independent genetic pool, which could be of interest for apple plant breeders.

Molecular characterisation and phenetic similarities between several cultivars of P. communis and P. pyrifolia, and genotypes of P. cordata, P. bourgaeana and P. pyraster were investigated through RAPD markers. Sixty decamer primers were screened, generating polymorphic patterns of Occidental and Oriental pear genotypes. Twenty-two selected primers originated clear and reproducible patterns, produced a total of 358 bands, 327 of them polymorphic. For 10 of the 12 genotypes analysed it was possible to find genotype-specific RAPDs and fragment patterns which could be used for cultivar identification. The patterns distinguished between genotypes and their analysis established a first approach to phenetic classification within the Pyrus genus based on DNA markers, clustering the genotypes according to their geographic origin. RAPD analysis of in vitro and in vivo material of seven cultivars was also performed, resulting in identical patterns for each genotype.

Molecular markers which sample multiple loci simultaneously, like amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSR), and selective amplification of microsatellite polymorphic loci (SAMPL), produce highly informative fingerprints due to their high effective multiplex ratio and expected heterozygosity. Moreover, these markers can be generated for DNA of any organism without initial investment in primer/probe development or in sequence analyses. The fragments produced can be visualized either by agarose or polyacrylamide gel electrophoresis followed by autoradiography or silver staining or via separation and detection on automatic DNA sequencers. Here, we describe detailed protocols based on the original methods aimed to obtain these markers optimized to be resolved on polyacrylamide gel electrophoresis and detected by silver staining which provides a fast, sensitive, and cost-effective method.

Versão provisória aceite p. publicação na Plant Science (ISSN 0168-9452)em 19-01-2013 Grapevine (Vitis vinifera L.) is one of the most economically important fruit crops in the world. Deficit in nitrogen, phosphorus and sulfur nutrition impairs essential metabolic pathways. The influence of mineral stress in the composition of the plant cell wall (CW) has received residual attention. Using grapevine callus as a model system, 6-weeks deficiency of those elements caused a significant decrease in growth. Callus CWs were analyzed by Fourier transform infrared spectroscopy (FT-IR), by quantification of CW components and by immunolocalization of CW epitopes with monoclonal antibodies. PCA analysis of FT-IR data suggested changes in the main components of the CW in response to individual mineral stress. Decreased cellulose, modifications in pectin methyl esterification and increase of structural proteins were among the events disclosed by FT-IR analysis. Chemical analyses supported some of the assumptions and further disclosed an increase in lignin content under nitrogen deficiency, suggesting a compensation of cellulose by lignin. Moreover, polysaccharides of callus under mineral deficiency showed to be more tightly bonded to the CW, probably due to a more extensive cross-linking of the cellulose-hemicellulose network. Our work showed that mineral stress impacts the CW at different extents according to the withdrawn mineral element, and that the modifications in a given CW component are compensated by the synthesis and/or alternative linking between polymers. The overall results here described for the first time pinpoint the CW of Vitis callus different strategies to overcome mineral stress, depending on how essential they are to cell growth and plant development.

International audience; Grapes (Vitis spp.) are economically significant fruit species. Many scientific advances have been achieved in understanding physiological, biochemical, and molecular aspects of grape berry maturation. Some of these advances have led to the improvement of wine quality through better grape growing practices; but this avenue of research is still wide open due to the complexity of the biochemical mechanisms involved in the development and ripening of grapes and their response to environment. The Biochemistry of the Grape Berry is a pioneer offering for readers - giving a focused and integrated coverage of the key biochemical and molecular aspects of grape berry development and ripening. Readers will benefit from a comprehensive and updated coverage on key steps, mechanisms and regulation in grapevine biochemistry and molecular biology, provided by an international team of leading scientists. It provides useful references on this research area and original data from laboratories worldwide. The comprehensive range of topics covered in this e-book is valuable to a multidisciplinary readership including plant physiologists, enologists, microbiologists, wine makers, biochemists and students. Thus The Biochemistry of the Grape Berry represents a step forward in the comprehension of grape berry biochemistry, wine quality and health benefits.

Textural characteristics of fruits and vegetables are important in determining consumer acceptance, and even minor deviations from the expected texture can result in produce rejection. The textural properties of a food are the group of physical characteristics that arise from its structural elements, are sensed by touch, and are related to the deformations, fracture, disintegration, and ow under force; these physical properties are measured objectively and expressed as functions of mass, time, and distance (Bourne, 2002). Although the term is used widely and loosely, texture is not a single, well-dened attribute. It is a multi-trait attribute encompassing individual characteristics described by terms like rm, stiff, breakdown, crisp, granular, hard, juicy, spongy, melty, oury, or gritty (Harker et al., 1997ab; King et al., 2000). Each of the mentioned attributes is likely to reect particular facets of cell wall structure, especially cell wall strength and cell-to-cell adhesion. Hence, texture should be dened as a collective term that encompasses the structural and mechanical properties of a plant organ and their sensory perception by the consumer (Figure 4.1). Depending on the specic fruit or vegetable, one or a few textural attributes are appropriate to dene its texture for the purpose of quality control throughout the supply chain.

Fruit softening is accompanied by modifications of the cell wall pectic and hemicellulosic fractions, as the result of the combined action of several cell wall-modifying enzymes. The objective of this work was to clone specific cDNAs that encode isoforms of cell wall-modifying enzymes, which are expressed during the final stages of apple softening, and to establish a temporal sequence of their accumulation. A cDNA library enriched with mRNA isolated from over-ripe fruit was constructed and screened. A pectin methylesterase (MdPME1), a pectate lyase (MdPL1), an α- l -arabinofuranosidase (MdAF1) an endo-1,4-β-glucanase (MdEG1), two xyloglucan endotransglucosylase/hydrolases (Md-XTH1 and Md-XTH2), and an alpha-expansin (MdEXPA3) specific cDNAs were identified by homology-based cloning, and their mRNA accumulation was examined during fruit growth and ripening. The expression of an apple β-galactosidase (β-Gal; pABG1) and a polygalacturonase (PG; pGDPG-1) mRNA previously reported was also investigated using the same biological material. Transcripts of all enzymes, except MdPME1, could be unambiguously detected by semi-quantitative RT-PCR in fruit during ripening. However, transcripts of MdEG1 were more abundant at fruit set and MdPL1 exhibited higher expression before commercial maturity. The strongest RT-PCR signals in over-ripe fruit were observed for PG, β-Gal and Md-XTH1 clones. Two XTHs were detected in over-ripe fruit. While Md-XTH1 acts constitutively during fruit development, Md-XTH2 showed a ripening-related pattern. The Md-XTH2-encoded protein was heterologously expressed in Saccharomyces cerevisiae and showed both transglycosylase and hydrolase activities. Expression analyses conducted in flowers, peduncles, young and expanded leaves, and petioles of senescent leaves revealed that none of the cloned cDNAs is fruit specific.

Fruit softening is thought to result from extensive cell wall modifications that occur during ripening. These modifications are the result, at least in part, of the activity of members of cell wall-modifying enzymes from the same families involved in the cell wall loosening which promote tissue extension and growth. In this work, the activities of a set of pectolytic and non-pectolytic cell wall-modifying enzymes, namely polygalacturonase (PG; endo-and exo-acting), pectin methylesterase (PME), pectate lyase (PL),-galactosidase (-Gal),-l-arabinofuranosidase (AFase), endo-1,4--glucanase (EGase), xyloglucan endotransglycosylase (XET) and expansin, were monitored during growth and ripening of ‘Mondial Gala’ apple (Malus × domestica Borkh.) fruit. After optimisation of extraction protocols and standard activity assays, activity could be detected in all the assays, except for endo-PG. The overall results suggest that fruit growth and ripening are possibly coordinated by members of the same families of cell wall-modifying enzymes, although different isoforms may be involved in distinct developmental processes. Based on the trend of total activity measured in vitro using equal amounts of protein per developmental stage, the role of EGase seems to be more prominent during growth than during ripening, and XET activity is most important only after the fruit stopped growing and is maintained throughout ripening. -Gal and AFase activities increased after harvest as the fruit became over-ripe. On the other hand, exo-PG, PL and expansin activities increase from that in unripe fruit to fruit at harvest but are maintained at similar levels thereafter, throughout the over-ripe stages. The patterns of activity observed are discussed in relation to published information about ripening of apples and to results reported using other species. 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Sweet potato (Ipomoea batatas (L.) Lam) is one of the most important root crops in Mozambique, ranking in the 3rd position, after cassava and maize. Within the scope of the national and regional strategies/initiatives, we have used a multi-analysis approach to characterize the national sweet potato germplasm collection at two different levels: i) genetic, morphological and agronomic diversity; and ii) agronomic potential (storage root yield, vine weight, biomass, harvest index and dry matter content) toward drought tolerance. This collection, composed by 44 accessions, comprises 28 genotypes cultivated in three different provinces of Mozambique (Gaza, Inhambane and Zambezia), nine from other African countries (Kenya, South Africa, Uganda and Zimbabwe), one from the United States of America, and six from CGIAR research centers (IITA and CIP). According to our results, the Mozambican germplasm bank presents a high level of diversity, comparable to those from the collections of the primary centers of origin and South Africa, therefore constituting of a good source of agronomic traits for breeding. Regarding drought tolerance, six Mozambican genotypes (Admarc, Chingova, Nhacoongo-1, Xihetamakote, Nwanatuyo, and Chissicuana-2), one from Uganda (NASPOT-5), one from Zimbabwe (Moz_white), one from Kenya (SPK 004), and one from the USA (Resisto) seem to have the highest potential to be used in regions with frequent drought seasons and in future breeding programs. The results showed that such integrated analysis can be used to successfully characterize the genetic material in terms of suitability to drought-prone regions, therefore helping sweet potato crop management, with economic and food security impacts.

Inter-simple sequence repeat (ISSR) markers were used for cultivar identification and for determination of the phenetic relationships among 24 pear cultivars (Pyrus communis L.). The ability of several molecular marker systems including randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP), inter-simple sequence repeats (ISSR), simple sequence repeats (SSR), and selective amplification of microsatellite polymorphic loci (SAMPL) to detect variation among clones of the most significant Portuguese cultivar, Rocha, was also investigated. Each of the eight ISSR primers tested was able to distinguish the 24 pear cultivars. The ISSR primers generated 337 markers, 79.5% of which were polymorphic. The cultivar dendrogram obtained with the ISSR marker data was very similar to that obtained with previous RAPD+AFLP analysis, confirming the genetic divergence of `Pérola', `Carvalhal' and `Lawson' from the other cultivars. Eight out of 15 apple [Malus sylvestris (L.) Mill. var domestica (Borkh.) Mansf.] SSR primers tested also amplified microsatellites in pear. None of the five molecular marker systems analyzed (with a total of 1082 markers) detected reproducible polymorphisms among the nine `Rocha' clones, in spite of the presence of clear phenotypic differences.

Fruit cuticles influence greatly postharvest quality of produce. Yet, few published studies have addressed changes in cuticle composition and related gene expression from a postharvest perspective, particularly for fruit species within the Rosaceae family. Because physical treatments have proved effective in improving postharvest potential in some commodities, we were interested in assessing whether such benefits could arise, at some extent, from treatment-related alterations in cuticular components. In this work, commercially ripe 'October Sun' peaches were submitted to heat or CO2 shocks, and then stored at 0 °C for two weeks followed by five days at 20 °C. Wax and cutin composition was analyzed from cuticle samples isolated enzymatically from fruit skin, and expression of selected genes PpCER1, PpLACS1 and PpLipase, putatively involved in cuticle deposition, was quantified by quantitative real-time PCR. The amount (g m−2) of the main cuticular wax families identified was higher in treated fruit in comparison with the controls, particularly after the shelf life period. Treatment influence on cutin composition was less clear. Gene expression was generally impaired under cold storage, to recover to a variable extent upon transfer to 20 °C. The differential gene expression shown in response to treatments may relate to the observed compositional changes in the cuticle. This work was supported through the AGL2010-14801/ALI project, funded by the Ministerio de Ciencia e Innovación (MICINN) of Spain. B. Belge was the recipient of a FI-DGR grant from AGAUR (Generalitat de Catalunya).

Variability of commercial plum (Prunus L. sp.) cultivars is unknown since breeding often involves intercrossing hybrids with several species but has been based on a low number of parents. Molecular markers like amplified fragment length polymorphisms (AFLP) and inter-simple sequence repeats (ISSR), which sample multiple loci simultaneously, have become increasingly popular, and were used to characterize 24 diploid and four hexaploid cultivars of plum. Seven AFLP and six ISSR primers were used, and resulted in amplification of 379 and 270 products, respectively. Unweighted pair-group method with arithmetic averages (UPGMA) dendrograms, based on similarity coefficients, reflected a clear separation between diploid and hexaploid plums. Among diploid plums, two pairs of cultivars were relatively distinct from the rest, namely `Golden Japan' and `Methley' and `Ozark Premier' and `Songold'. Furthermore, several cultivars were grouped together both with AFLP and ISSR analysis: 1) `Ambra', `Red Beaut', and `Black Beaut', 2) `Black Diamond' and `Royal Diamond', 3) `June Rose', `Santa Rosa', and `Royal Red', and iv) `Freedom', `Larry Ann', and `Queen Rosa'. Although the phenetic classification obtained by the two methods were similar (r = 0.73, for the diploid group), ISSR had a higher reproducibility and percentage of polymorphisms (87.4% vs. 62.8%) than AFLP. Methodological aspects of both markers systems are discussed. Results obtained suggest that the AFLP and ISSR approaches are valuable tools for identification of specific genotypes and analysis of phenetic relationships in plum.

only loosely associated as proved by Blue-native PAGE/Immuno blot studies [1]. Interestingly, there is evidence that the specific proteasome inhibitor lactacystin elevates Tom20 protein level [2]. Furthermore, this level might also depend on physiological demands [3]. To investigate possible variations in Tom20/TOM assembly on the single cell level, we searched for a method compatible with measurements in living cells. We employed fluorescence resonance energy transfer in combination with fluorescence lifetime imaging microscopy (FRET-FLIM) to investigate protein–protein interaction. Tom22 was fused to eGFPm as FRET donor and Tom20 to DsRedm or DsRedm-Halo/TMR as FRET acceptor. Donor and acceptor-tagged Tom22 and Tom20 were coexpressed in mammalian HeLa cells. The average fluorescence lifetime tamp was measured in presence and absence of acceptor. A mean FRET (E=(1− tDA/tA)×100) of 3.49±0.43 SD could be measured between Tom20-eGFPm and Tom22-DsRedm. By using the double acceptor construct, the determined average FRET efficiency was significantly increased (E=6.57±0.82 SD). This is an almost twofold enhancement compared to using DsRedm as the only acceptor. Introduction of a 20 amino acid linker between Tom20 and the fluorescent protein tags decreased the FRET efficiency, indicating an unfavourable constitution for the acceptor function. For mitochondria treated with lactacystin, a specific inhibitor of the proteasome, we detected a significant rise in average FRET efficiency. We show that interaction between two candidate proteins of the TOM complex, Tom22 and Tom20, fused to eGFPm and DsRedm-Halo, respectively, can be monitored in living cells using FRET-FLIM. Moreover, we found that specific inhibition of the proteasome apparently enhances the assembly of Tom20 into the TOM complex, resulting in increased FRET.