Carbon usage in yellow-fleshed Manihot esculenta storage roots shifts from starch biosynthesis to cell wall and raffinose biosynthesis via the myo-inositol pathway.

Cassava is a crucial staple crop for smallholder farmers in tropical Asia and Sub-Saharan Africa. Although high yield remains the top priority for farmers, the significance of nutritional values has increased in cassava breeding programs. A notable negative correlation between provitamin A and starch accumulation poses a significant challenge for breeding efforts. The negative correlation between starch and carotenoid levels in conventional and genetically modified cassava plants implies the absence of a direct genomic connection between the two traits. The competition among various carbon pathways seems to account for this relationship. In this study, we conducted a thorough analysis of 49 African cassava genotypes with varying levels of starch and provitamin A. Our goal was to identify factors contributing to differential starch accumulation. Considering carotenoid levels as a confounding factor in starch production, we found that yellow- and white-fleshed storage roots did not differ significantly in most measured components of starch or de novo fatty acid biosynthesis. However, genes and metabolites associated with myo-inositol synthesis and cell wall polymer production were substantially enriched in high provitamin A genotypes. These results indicate that yellow-fleshed cultivars, in comparison to their white-fleshed counterparts, direct more carbon toward the synthesis of raffinose and cell wall components. This finding is underlined by a significant rise in cell wall components measured within the 20 most contrasting genotypes for carotenoid levels. Our findings enhance the comprehension of the biosynthesis of starch and carotenoids in the storage roots of cassava.

The relationship between cell wall and postharvest physiological deterioration of fresh produce.

Postharvest physiological deterioration (PPD) reduces the availability and economic value of fresh produces, resulting in the waste of agricultural products and becoming a worldwide problem. Therefore, many studies have been carried out at the anatomical structural, physiological and biochemical levels and molecular levels of PPD of fresh produces to seek ways to manage the postharvest quality of fresh produce. The cell wall is the outermost structure of a plant cell and as such represents the first barrier to prevent external microorganisms and other injuries. Many studies on postharvest quality of crop storage organs relate to changes in plant cell wall-related components. Indeed, these studies evidence the non-negligible role of the plant cell wall in postharvest storage ability. However, the relationship between cell wall metabolism and postharvest deterioration of fresh produces has not been well summarized. In this review, we summarize the structural changes of cell walls in different types of PPD, metabolic changes, and the possible molecular mechanism regulating cell wall metabolism in PPD of fresh produce. This review provides a basis for further research on delaying the occurrence of PPD of fresh produce.

PYRIDOX(AM)INE 5'-PHOSPHATE OXIDASE3 of Arabidopsis thaliana maintains carbon/nitrogen balance in distinct environmental conditions.

The identification of factors that regulate C/N utilization in plants can make a substantial contribution to optimization of plant health. Here, we explored the contribution of pyridox(am)ine 5'-phosphate oxidase3 (PDX3), which regulates vitamin B6 homeostasis, in Arabidopsis (Arabidopsis thaliana). Firstly, N fertilization regimes showed that ammonium application rescues the leaf morphological phenotype of pdx3 mutant lines but masks the metabolite perturbance resulting from impairment in utilizing soil nitrate as a source of N. Without fertilization, pdx3 lines suffered a C/N imbalance and accumulated nitrogenous compounds. Surprisingly, exploration of photorespiration as a source of endogenous N driving this metabolic imbalance, by incubation under high CO2, further exacerbated the pdx3 growth phenotype. Interestingly, the amino acid serine, critical for growth and N management, alleviated the growth phenotype of pdx3 plants under high CO2, likely due to the requirement of pyridoxal 5'-phosphate for the phosphorylated pathway of serine biosynthesis under this condition. Triggering of thermomorphogenesis by growth of plants at 28 °C (instead of 22 °C) did not appear to require PDX3 function, and we observed that the consequent drive toward C metabolism counters the C/N imbalance in pdx3. Further, pdx3 lines suffered a salicylic acid-induced defense response, probing of which unraveled that it is a protective strategy mediated by nonexpressor of pathogenesis related1 (NPR1) and improves fitness. Overall, the study demonstrates the importance of vitamin B6 homeostasis as managed by the salvage pathway enzyme PDX3 to growth in diverse environments with varying nutrient availability and insight into how plants reprogram their metabolism under such conditions.

Efficient sugar utilization and transition from oxidative to substrate-level phosphorylation in high starch storage roots of African cassava genotypes.

Cassava's storage roots represent one of the most important sources of nutritional carbohydrates worldwide. Particularly, smallholder farmers in sub-Saharan Africa depend on this crop plant, where resilient and yield-improved varieties are of vital importance to support steadily increasing populations. Aided by a growing understanding of the plant's metabolism and physiology, targeted improvement concepts already led to visible gains in recent years. To expand our knowledge and to contribute to these successes, we investigated storage roots of eight cassava genotypes with differential dry matter content from three successive field trials for their proteomic and metabolic profiles. At large, the metabolic focus in storage roots transitioned from cellular growth processes toward carbohydrate and nitrogen storage with increasing dry matter content. This is reflected in higher abundance of proteins related to nucleotide synthesis, protein turnover, and vacuolar energization in low starch genotypes, while proteins involved in sugar conversion and glycolysis were more prevalent in high dry matter genotypes. This shift in metabolic orientation was underlined by a clear transition from oxidative- to substrate-level phosphorylation in high dry matter genotypes. Our analyses highlight metabolic patterns that are consistently and quantitatively associated with high dry matter accumulation in cassava storage roots, providing fundamental understanding of cassava's metabolism as well as a data resource for targeted genetic improvement.

Understanding source-sink interactions: Progress in model plants and translational research to crops.

Agriculture is facing a massive increase in demand per hectare as a result of an ever-expanding population and environmental deterioration. While we have learned much about how environmental conditions and diseases impact crop yield, until recently considerably less was known concerning endogenous factors, including within-plant nutrient allocation. In this review, we discuss studies of source-sink interactions covering both fundamental research in model systems under controlled growth conditions and how the findings are being translated to crop plants in the field. In this respect we detail efforts aimed at improving and/or combining C3, C4, and CAM modes of photosynthesis, altering the chloroplastic electron transport chain, modulating photorespiration, adopting bacterial/algal carbon-concentrating mechanisms, and enhancing nitrogen- and water-use efficiencies. Moreover, we discuss how modulating TCA cycle activities and primary metabolism can result in increased rates of photosynthesis and outline the opportunities that evaluating natural variation in photosynthesis may afford. Although source, transport, and sink functions are all covered in this review, we focus on discussing source functions because the majority of research has been conducted in this field. Nevertheless, considerable recent evidence, alongside the evidence from classical studies, demonstrates that both transport and sink functions are also incredibly important determinants of yield. We thus describe recent evidence supporting this notion and suggest that future strategies for yield improvement should focus on combining improvements in each of these steps to approach yield optimization.

The Arabidopsis electron-transfer flavoprotein:ubiquinone oxidoreductase is required during normal seed development and germination.

The importance of the alternative donation of electrons to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex has been demonstrated. However, the functional significance of this pathway during seed development and germination remains to be elucidated. To assess the function of this pathway, we performed a detailed metabolic and transcriptomic analysis of Arabidopsis mutants to test the molecular consequences of a dysfunctional ETF/ETFQO pathway. We demonstrate that the disruption of this pathway compromises seed germination in the absence of an external carbon source and also impacts seed size and yield. Total protein and storage protein content is reduced in dry seeds, whilst sucrose levels remain invariant. Seeds of ETFQO and related mutants were also characterized by an altered fatty acid composition. During seed development, lower levels of fatty acids and proteins accumulated in the etfqo-1 mutant as well as in mutants in the alternative electron donors isovaleryl-CoA dehydrogenase (ivdh-1) and d-2-hydroxyglutarate dehydrogenase (d2hgdh1-2). Furthermore, the content of several amino acids was increased in etfqo-1 mutants during seed development, indicating that these mutants are not using such amino acids as alternative energy source for respiration. Transcriptome analysis revealed alterations in the expression levels of several genes involved in energy and hormonal metabolism. Our findings demonstrated that the alternative pathway of respiration mediated by the ETF/ETFQO complex affects seed germination and development by directly adjusting carbon storage during seed filling. These results indicate a role for the pathway in the normal plant life cycle to complement its previously defined roles in the response to abiotic stress.

Metabolic shifts during fruit development in pungent and non-pungent peppers.

Fruit pungency is caused by the accumulation of capsaicinoids, secondary metabolites whose relation to primary metabolism remains unclear. We have selected ten geographically diverse accessions of Capsicum chinense Jacq with different pungency levels. A detailed metabolic profile was conducted in the fruit placenta and pericarp at 20, 45, and 60 days after anthesis aiming at increasing our understanding of the metabolic changes in these tissues across fruit development and their potential connection to capsaicin metabolism. Overall, despite the variation in fruit pungency among the ten accessions, the composition and metabolite levels in both placenta and pericarp were uniformly stable across accessions. Most of the metabolite variability occurred between the fruit developmental stages rather than among the accessions. Interestingly, different metabolite adjustments in the placenta were observed among pungent and non-pungent accessions, which seem to be related to differences in the genetic background. Furthermore, we observed high coordination between metabolites and capsaicin production in C. chinense fruits, suggesting that pungency in placenta is adjusted with primary metabolism.

Ascorbate and Thiamin: Metabolic Modulators in Plant Acclimation Responses.

Cell compartmentalization allows incompatible chemical reactions and localised responses to occur simultaneously, however, it also requires a complex system of communication between compartments in order to maintain the functionality of vital processes. It is clear that multiple such signals must exist, yet little is known about the identity of the key players orchestrating these interactions or about the role in the coordination of other processes. Mitochondria and chloroplasts have a considerable number of metabolites in common and are interdependent at multiple levels. Therefore, metabolites represent strong candidates as communicators between these organelles. In this context, vitamins and similar small molecules emerge as possible linkers to mediate metabolic crosstalk between compartments. This review focuses on two vitamins as potential metabolic signals within the plant cell, vitamin C (L-ascorbate) and vitamin B1 (thiamin). These two vitamins demonstrate the importance of metabolites in shaping cellular processes working as metabolic signals during acclimation processes. Inferences based on the combined studies of environment, genotype, and metabolite, in order to unravel signaling functions, are also highlighted.

Metabolic profiles of six African cultivars of cassava (Manihot esculenta Crantz) highlight bottlenecks of root yield.

Cassava is an important staple crop in sub-Saharan Africa, due to its high productivity even on nutrient poor soils. The metabolic characteristics underlying this high productivity are poorly understood including the mode of photosynthesis, reasons for the high rate of photosynthesis, the extent of source/sink limitation, the impact of environment, and the extent of variation between cultivars. Six commercial African cassava cultivars were grown in a greenhouse in Erlangen, Germany, and in the field in Ibadan, Nigeria. Source leaves, sink leaves, stems and storage roots were harvested during storage root bulking and analyzed for sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, protein, activities of enzymes in central metabolism and yield traits. High ratios of RuBisCO:phosphoenolpyruvate carboxylase activity support a C3 mode of photosynthesis. The high rate of photosynthesis is likely to be attributed to high activities of enzymes in the Calvin-Benson cycle and pathways for sucrose and starch synthesis. Nevertheless, source limitation is indicated because root yield traits correlated with metabolic traits in leaves rather than in the stem or storage roots. This situation was especially so in greenhouse-grown plants, where irradiance will have been low. In the field, plants produced more storage roots. This was associated with higher AGPase activity and lower sucrose in the roots, indicating that feedforward loops enhanced sink capacity in the high light and low nitrogen environment in the field. Overall, these results indicated that carbon assimilation rate, the K battery, root starch synthesis, trehalose, and chlorogenic acid accumulation are potential target traits for genetic improvement.

Cassava Metabolomics and Starch Quality.

Cassava plays an important role as a staple food for more than 800 million people in the world due to its ability to maintain relatively high productivity even in nutrient-depleted soils. Even though cassava has been the focus of several breeding programs and has become a strong focus of research in the last few years, relatively little is currently known about its metabolism and metabolic composition in different tissues. In this article, the absolute content of sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, carotenoids, chlorophylls, tocopherols, and total protein as well as starch quality is described based on multiple analytical techniques, with protocols specifically adjusted for material from different cassava tissues. Moreover, quantification of secondary metabolites relative to internal standards is presented using both non-targeted and targeted metabolomics approaches. The protocols have also been adjusted to apply to freeze-dried material in order to allow processing of field harvest samples that typically will require long-distance transport. © 2019 The Authors. Basic Protocol 1: Metabolic profiling by gas chromatography-mass spectrometry (GC-MS) Support Protocol 1: Preparation of freeze-dried cassava material Support Protocol 2: Preparation of standard compound mixtures for absolute quantification of metabolites by GC-MS Support Protocol 3: Preparation of retention-time standard mixture Basic Protocol 2: Determination of organic acids and phosphorylated intermediates by ion chromatography-mass spectrometry (IC-MS) Support Protocol 4: Preparation of standards and recovery experimental procedure Basic Protocol 3: Determination of soluble sugars, starch, and free amino acids Alternate Protocol: Determination of soluble sugars and starch Basic Protocol 4: Determination of anions Basic Protocol 5: Determination of elements Basic Protocol 6: Determination of total protein Basic Protocol 7: Determination of non-targeted and targeted secondary metabolites Basic Protocol 8: Determination of carotenoids, chlorophylls, and tocopherol Basic Protocol 9: Determination of starch quality.

Appropriate Thiamin Pyrophosphate Levels Are Required for Acclimation to Changes in Photoperiod.

Thiamin pyrophosphate (TPP) is the active form of vitamin B1 and works as an essential cofactor for enzymes in key metabolic pathways, such as the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. Although its action as a coenzyme has been well documented, the roles of TPP in plant metabolism are still not fully understood. Here, we investigated the functions of TPP in the regulation of the metabolic networks during photoperiod transition using previously described Arabidopsis (Arabidopsis thaliana) riboswitch mutant plants, which accumulate thiamin vitamers. The results show that photosynthetic and metabolic phenotypes of TPP riboswitch mutants are photoperiod dependent. Additionally, the mutants are more distinct from control plants when plants are transferred from a short-day to a long-day photoperiod, suggesting that TPP also plays a role in metabolic acclimation to the photoperiod. Control plants showed changes in the amplitude of diurnal oscillation in the levels of metabolites, including glycine, maltose, and fumarate, following the photoperiod transition. Interestingly, many of these changes are not present in TPP riboswitch mutant plants, demonstrating their lack of metabolic flexibility. Our results also indicate a close relationship between photorespiration and the TCA cycle, as TPP riboswitch mutants accumulate less photorespiratory intermediates. This study shows the potential role of vitamin B1 in the diurnal regulation of central carbon metabolism in plants and the importance of maintaining appropriate cellular levels of thiamin vitamers for the plant's metabolic flexibility and ability to acclimate to an altered photoperiod.

The genetic architecture of photosynthesis and plant growth-related traits in tomato.

To identify genomic regions involved in the regulation of fundamental physiological processes such as photosynthesis and respiration, a population of Solanum pennellii introgression lines was analyzed. We determined phenotypes for physiological, metabolic, and growth related traits, including gas exchange and chlorophyll fluorescence parameters. Data analysis allowed the identification of 208 physiological and metabolic quantitative trait loci with 33 of these being associated to smaller intervals of the genomic regions, termed BINs. Eight BINs were identified that were associated with higher assimilation rates than the recurrent parent M82. Two and 10 genomic regions were related to shoot and root dry matter accumulation, respectively. Nine genomic regions were associated with starch levels, whereas 12 BINs were associated with the levels of other metabolites. Additionally, a comprehensive and detailed annotation of the genomic regions spanning these quantitative trait loci allowed us to identify 87 candidate genes that putatively control the investigated traits. We confirmed 8 of these at the level of variance in gene expression. Taken together, our results allowed the identification of candidate genes that most likely regulate photosynthesis, primary metabolism, and plant growth and as such provide new avenues for crop improvement.

Coupling Radiotracer Experiments with Chemical Fractionation for the Estimation of Respiratory Fluxes.

Carbohydrates catabolized via respiratory processes are not only used for energy production but also for biosynthesis of cellular components including soluble molecules (sugars, amino acids, organic acids, and their derivatives) and insoluble macromolecules (proteins, starch, and cell wall). Radiotracer experiments using 14C-labeled glucose provide a global picture of the fate of respired carbon in the metabolic network. This method is based on a chemical fractionation of biomolecules in 14C-glucose fed plant materials and the subsequent determination of radioactivity in each fraction. Metabolic flux into each fraction can be estimated from the specific activity of the hexose phosphate pool. Here, we describe the procedure for glucose metabolism in potato tuber but similar protocols can be adopted for various plant organs and substrates.

Regulation of ascorbate biosynthesis in green algae has evolved to enable rapid stress-induced response via the VTC2 gene encoding GDP-l-galactose phosphorylase.

Ascorbate (vitamin C) plays essential roles in stress resistance, development, signaling, hormone biosynthesis and regulation of gene expression; however, little is known about its biosynthesis in algae. In order to provide experimental proof for the operation of the Smirnoff-Wheeler pathway described for higher plants and to gain more information on the regulation of ascorbate biosynthesis in Chlamydomonas reinhardtii, we targeted the VTC2 gene encoding GDP-l-galactose phosphorylase using artificial microRNAs. Ascorbate concentrations in VTC2 amiRNA lines were reduced to 10% showing that GDP-l-galactose phosphorylase plays a pivotal role in ascorbate biosynthesis. The VTC2 amiRNA lines also grow more slowly, have lower chlorophyll content, and are more susceptible to stress than the control strains. We also demonstrate that: expression of the VTC2 gene is rapidly induced by H2 O2 and 1 O2 resulting in a manifold increase in ascorbate content; in contrast to plants, there is no circadian regulation of ascorbate biosynthesis; photosynthesis is not required per se for ascorbate biosynthesis; and Chlamydomonas VTC2 lacks negative feedback regulation by ascorbate in the physiological concentration range. Our work demonstrates that ascorbate biosynthesis is also highly regulated in Chlamydomonas albeit via mechanisms distinct from those previously described in land plants.

Exploring natural variation of photosynthetic, primary metabolism and growth parameters in a large panel of Capsicum chinense accessions.

MAIN CONCLUSION: Collectively, the results presented improve upon the utility of an important genetic resource and attest to a complex genetic basis for differences in both leaf metabolism and fruit morphology between natural populations. Diversity of accessions within the same species provides an alternative method to identify physiological and metabolic traits that have large effects on growth regulation, biomass and fruit production. Here, we investigated physiological and metabolic traits as well as parameters related to plant growth and fruit production of 49 phenotypically diverse pepper accessions of Capsicum chinense grown ex situ under controlled conditions. Although single-trait analysis identified up to seven distinct groups of accessions, working with the whole data set by multivariate analyses allowed the separation of the 49 accessions in three clusters. Using all 23 measured parameters and data from the geographic origin for these accessions, positive correlations between the combined phenotypes and geographic origin were observed, supporting a robust pattern of isolation-by-distance. In addition, we found that fruit set was positively correlated with photosynthesis-related parameters, which, however, do not explain alone the differences in accession susceptibility to fruit abortion. Our results demonstrated that, although the accessions belong to the same species, they exhibit considerable natural intraspecific variation with respect to physiological and metabolic parameters, presenting diverse adaptation mechanisms and being a highly interesting source of information for plant breeders. This study also represents the first study combining photosynthetic, primary metabolism and growth parameters for Capsicum to date.

Iron excess affects rice photosynthesis through stomatal and non-stomatal limitations.

Iron toxicity is the most important stressor of rice in many lowland environments worldwide. Rice cultivars differ widely in their ability to tolerate excess iron. A physiological evaluation of iron toxicity in rice was performed using non-invasive photosynthesis, photorespiration and chlorophyll a fluorescence imaging measurements and chlorophyll content determination by SPAD. Four rice cultivars (BR IRGA 409; BR IRGA 412; BRA 041171 and BRA 041152) from the Brazilian breeding programs were used. Fe(2+) was supplied in the nutrient solution as Fe-EDTA (0.019, 4, 7 and 9 mM). Increases in shoot iron content due to Fe(2+) treatments led to changes in most of the non-invasive physiological variables assessed. The reduction in rice photosynthesis can be attributed to stomatal limitations at moderate Fe(2+) doses (4mM) and both stomatal and non-stomatal limitations at higher doses. Photorespiration was an important sink for electrons in rice cultivars under iron excess. A decreased chlorophyll content and limited photochemical ability to cope with light excess were characteristic of the more sensitive and iron accumulator cultivars (BRA 041171 and BRA 041152). Chlorophyll fluorescence imaging revealed a spatial heterogeneity of photosynthesis under excessive iron concentrations. The results showed the usefulness of non-invasive physiological measurements to assess differences among cultivars. The contributions toward understanding the rice photosynthetic response to toxic levels of iron in the nutrient solution are also discussed.