The ascorbate-glutathione cycle coming of age.

Concepts regarding the operation of the ascorbate-glutathione cycle and the associated water/water cycle in the processing of metabolically generated hydrogen peroxide and other forms of reactive oxygen species (ROS) are well established in the literature. However, our knowledge of the functions of these cycles and their component enzymes continues to grow and evolve. Recent insights include participation in the intrinsic environmental and developmental signalling pathways that regulate plant growth, development, and defence. In addition to ROS processing, the enzymes of the two cycles not only support the functions of ascorbate and glutathione, they also have 'moonlighting' functions. They are subject to post-translational modifications and have an extensive interactome, particularly with other signalling proteins. In this assessment of current knowledge, we highlight the central position of the ascorbate-glutathione cycle in the network of cellular redox systems that underpin the energy-sensitive communication within the different cellular compartments and integrate plant signalling pathways.

Papain-like cysteine proteases are required for the regulation of photosynthetic gene expression and acclimation to high light stress.

Chloroplasts are considered to be devoid of cysteine proteases. Using transgenic Arabidopsis lines expressing the rice cystatin, oryzacystatin I (OC-I), in the chloroplasts (PC lines) or cytosol (CYS lines), we explored the hypothesis that cysteine proteases regulate photosynthesis. The CYS and PC lines flowered later than the wild type (WT) and accumulated more biomass after flowering. In contrast to the PC rosettes, which accumulated more leaf chlorophyll and carotenoid pigments than the WT, the CYS lines had lower amounts of leaf pigments. High-light-dependent decreases in photosynthetic carbon assimilation and the abundance of the Rubisco large subunit protein, the D1 protein, and the phosphorylated form of D1 proteins were attenuated in the CYS lines and reversed in the PC lines relative to the WT. However, the transgenic lines had higher amounts of LHC, rbcs, pasbA, and pasbD transcripts than the WT, and also showed modified chloroplast to nucleus signalling. We conclude that cysteine proteases accelerate the reconfiguration of the chloroplast proteome after flowering and in response to high-light stress. Inhibition of cysteine proteases, such as AtCEP1, slows chloroplast protein degradation and stimulates photosynthetic gene expression and chloroplast to nucleus signalling, enhancing stress tolerance traits.

EMS Derived Wheat Mutant BIG8-1 (Triticum aestivum L.)-A New Drought Tolerant Mutant Wheat Line.

Drought response in wheat is considered a highly complex process, since it is a multigenic trait; nevertheless, breeding programs are continuously searching for new wheat varieties with characteristics for drought tolerance. In a previous study, we demonstrated the effectiveness of a mutant known as RYNO3936 that could survive 14 days without water. In this study, we reveal another mutant known as BIG8-1 that can endure severe water deficit stress (21 days without water) with superior drought response characteristics. Phenotypically, the mutant plants had broader leaves, including a densely packed fibrous root architecture that was not visible in the WT parent plants. During mild (day 7) drought stress, the mutant could maintain its relative water content, chlorophyll content, maximum quantum yield of PSII (Fv/Fm) and stomatal conductance, with no phenotypic symptoms such as wilting or senescence despite a decrease in soil moisture content. It was only during moderate (day 14) and severe (day 21) water deficit stress that a decline in those variables was evident. Furthermore, the mutant plants also displayed a unique preservation of metabolic activity, which was confirmed by assessing the accumulation of free amino acids and increase of antioxidative enzymes (peroxidases and glutathione S-transferase). Proteome reshuffling was also observed, allowing slow degradation of essential proteins such as RuBisCO during water deficit stress. The LC-MS/MS data revealed a high abundance of proteins involved in energy and photosynthesis under well-watered conditions, particularly Serpin-Z2A and Z2B, SGT1 and Calnexin-like protein. However, after 21 days of water stress, the mutants expressed ABC transporter permeases and xylanase inhibitor protein, which are involved in the transport of amino acids and protecting cells, respectively. This study characterizes a new mutant BIG8-1 with drought-tolerant characteristics suited for breeding programs.

Factors facilitating sustainable scientific partnerships between developed and developing countries.

International scientific partnerships are key to the success of strategic investments in plant science research and the farm-level adoption of new varieties and technologies, as well as the coherence of agricultural policies across borders to address global challenges. Such partnerships result not only in a greater impact of published research enhancing the career development of early and later stage researchers, but they also ensure that advances in plant science and crop breeding technologies make a meaningful contribution to society by brokering acceptance of emerging solutions to the world problems. We discuss the evidence showing that despite a lack of funding, scientists in some African countries make a significant contribution to global science output. We consider the criteria for success in establishing long-term scientific partnerships between scientists in developing countries in Southern Africa ("the South") and developed countries such as the UK ("the North"). We provide our own personal perspectives on the key attributes that lead to successful institutional collaborations and the establishment of sustainable networks of successful "North-South" scientific partnerships. In addition, we highlight some of the stumbling blocks which tend to hinder the sustainability of long-term "North-South" scientific networks. We use this personal knowledge and experiences to provide guidelines on how to establish and maintain successful long-term "North-South" scientific partnerships.

Strigolactones positively regulate chilling tolerance in pea and in Arabidopsis.

Strigolactones (SL) fulfil important roles in plant development and stress tolerance. Here, we characterized the role of SL in the dark chilling tolerance of pea and Arabidopsis by analysis of mutants that are defective in either SL synthesis or signalling. Pea mutants (rms3, rms4, and rms5) had significantly greater shoot branching with higher leaf chlorophyll a/b ratios and carotenoid contents than the wild type. Exposure to dark chilling significantly decreased shoot fresh weights but increased leaf numbers in all lines. Moreover, dark chilling treatments decreased biomass (dry weight) accumulation only in rms3 and rms5 shoots. Unlike the wild type plants, chilling-induced inhibition of photosynthetic carbon assimilation was observed in the rms lines and also in the Arabidopsis max3-9, max4-1, and max2-1 mutants that are defective in SL synthesis or signalling. When grown on agar plates, the max mutant rosettes accumulated less biomass than the wild type. The synthetic SL, GR24, decreased leaf area in the wild type, max3-9, and max4-1 mutants but not in max2-1 in the absence of stress. In addition, a chilling-induced decrease in leaf area was observed in all the lines in the presence of GR24. We conclude that SL plays an important role in the control of dark chilling tolerance.

Cysteine proteases and wheat (Triticum aestivum L) under drought: A still greatly unexplored association.

Bread wheat (Triticum aestivum L.) provides about 19% of global dietary energy. Environmental stress, such as drought, affects wheat growth causing premature plant senescence and ultimately plant death. A plant response to drought is an increase in protease-mediated proteolysis with rapid degradation of proteins required for metabolic processes. Among the plant proteases that are increased in their activity following stress, cysteine proteases are the best characterized. Very little is known about particular wheat cysteine protease sequences, their expression and also localization. The current knowledge on wheat cysteine proteases belonging to the five clans (CA, CD, CE, CF and CP) is outlined, in particular their expression and possible function under drought. The first successes in establishing an annotated wheat genome database are further highlighted which has allowed more detailed mining of cysteine proteases. We also share our thoughts on future research directions considering the growing availability of genomic resources of this very important food crop. Finally, we also outline future application of developed knowledge in transgenic wheat plants for environmental stress protection and also as senescence markers to monitor wheat growth under environmental stress conditions.

Unlocking the potential of orphan legumes.

Orphan, or underutilized, legumes are domesticated legumes with useful properties, but with less importance than major world crops due to use and supply constraints. However, they play a significant role in many developing countries, providing food security and nutrition to consumers, as well as income to resource-poor farmers. They have been largely neglected by both researchers and industry due to their limited economic importance in the global market. Orphan legumes are better adapted than the major legume crops to extreme soil and climatic conditions, with high tolerance to abiotic environmental stresses such as drought. As a stress response they can also produce compounds with pharmaceutical value. Orphan legumes are therefore a likely source of important traits for introduction into major crops to aid in combating the stresses associated with global climate change. Modern large-scale genomics techniques are now being applied to many of these previously understudied crops, with the first successes reported in the genomics area. However, greater investment of resources and manpower are necessary if the potential of orphan legumes is to be unlocked and applied in the future.

Enhancing faba bean (Vicia faba L.) genome resources.

Grain legume improvement is currently impeded by a lack of genomic resources. The paucity of genome information for faba bean can be attributed to the intrinsic difficulties of assembling/annotating its giant (~13 Gb) genome. In order to address this challenge, RNA-sequencing analysis was performed on faba bean (cv. Wizard) leaves. Read alignment to the faba bean reference transcriptome identified 16 300 high quality unigenes. In addition, Illumina paired-end sequencing was used to establish a baseline for genomic information assembly. Genomic reads were assembled de novo into contigs with a size range of 50-5000 bp. Over 85% of sequences did not align to known genes, of which ~10% could be aligned to known repetitive genetic elements. Over 26 000 of the reference transcriptome unigenes could be aligned to DNA-sequencing (DNA-seq) reads with high confidence. Moreover, this comparison identified 56 668 potential splice points in all identified unigenes. Sequence length data were extended at 461 putative loci through alignment of DNA-seq contigs to full-length, publicly available linkage marker sequences. Reads also yielded coverages of 3466× and 650× for the chloroplast and mitochondrial genomes, respectively. Inter- and intraspecies organelle genome comparisons established core legume organelle gene sets, and revealed polymorphic regions of faba bean organelle genomes.

Potential use of phytocystatins in crop improvement, with a particular focus on legumes.

Phytocystatins are a well-characterized class of naturally occurring protease inhibitors that function by preventing the catalysis of papain-like cysteine proteases. The action of cystatins in biotic stress resistance has been studied intensively, but relatively little is known about their functions in plant growth and defence responses to abiotic stresses, such as drought. Extreme weather events, such as drought and flooding, will have negative impacts on the yields of crop plants, particularly grain legumes. The concepts that changes in cellular protein content and composition are required for acclimation to different abiotic stresses, and that these adjustments are achieved through regulation of proteolysis, are widely accepted. However, the nature and regulation of the protein turnover machinery that underpins essential stress-induced cellular restructuring remain poorly characterized. Cysteine proteases are intrinsic to the genetic programmes that underpin plant development and senescence, but their functions in stress-induced senescence are not well defined. Transgenic plants including soybean that have been engineered to constitutively express phytocystatins show enhanced tolerance to a range of different abiotic stresses including drought, suggesting that manipulation of cysteine protease activities by altered phytocystatin expression in crop plants might be used to improve resilience and quality in the face of climate change.

From lateral root density to nodule number, the strigolactone analogue GR24 shapes the root architecture of Medicago truncatula.

In the rhizosphere, strigolactones not only act as crucial signalling molecules in the communication of plants with parasitic weeds and arbuscular mycorrhiza, but they also play a key role in regulating different aspects of the root system. Here we investigated how strigolactones influence the root architecture of Medicago truncatula. We provide evidence that addition of the synthetic strigolactone analogue GR24 has an inhibitory effect on the lateral root density. Moreover, treatment with GR24 of Sinorhizobium meliloti-inoculated M. truncatula plants affects the nodule number both positively and negatively, depending on the concentration. Plants treated with 0.1 µM GR24 had a slightly increased number of nodules, whereas concentrations of 2 and 5 µM strongly reduced it. This effect was independent of the autoregulation of nodulation mechanism that is controlled by SUPER NUMERIC NODULE. Furthermore, we demonstrate that GR24 controls the nodule number through crosstalk with SICKLE-dependent ethylene signalling. Additionally, because the expression of the nodulation marker EARLY NODULATION11 was strongly reduced in GR24-treated plants, we concluded that strigolactones influence nodulation at a very early stage of the symbiotic interaction.

Redox markers for drought-induced nodule senescence, a process occurring after drought-induced senescence of the lowest leaves in soybean (Glycine max).

BACKGROUND AND AIMS: Water is an increasingly scarce resource that limits crop productivity in many parts of the world, and the frequency and severity of drought are predicted to increase as a result of climate change. Improving tolerance to drought stress is therefore important for maximizing future crop yields. The aim of this study was to compare the effects of drought on soybean (Glycine max) leaves and nodules in order to define phenotypic markers and changes in cellular redox state that characterize the stress response in different organs, and to characterize the relationships between leaf and nodule senescence during drought. METHODS: Leaf and crown nodule metabolite pools were measured together with leaf and soil water contents, and leaf chlorophyll, total protein contents and chlorophyll a fluorescence quenching parameters in nodulated soybeans that were grown under either well-watered conditions or deprived of water for up to 21 d. KEY RESULTS: Ureides, ascorbate, protein, chlorophyll and the ratios of variable chlorophyll a fluorescence (Fv') to maximal chlorophyll a fluorescence (Fm') fell to levels below detection in the oldest leaves after 21 d of drought. While these drought-induced responses were not observed in the youngest leaf ranks, the Fv'/Fm' ratios, pyridine nucleotide levels and the reduction state of the ascorbate pool were lower in all leaf ranks after 21 d of drought. In contrast to leaves, total nodule protein, pyridine nucleotides, ureides, ascorbate and glutathione contents increased as a result of the drought treatment. However, the nodule ascorbate pool was significantly less reduced as a result of drought. Higher levels of transcripts encoding two peroxiredoxins were detected in nodules exposed to drought stress but senescence-associated transcripts and other mRNAs encoding redox-related proteins were similar under both conditions. CONCLUSIONS: While the physiological impact of the drought was perceived throughout the shoot, stress-induced senescence occurred only in the oldest leaf ranks. At this stage, a number of drought-induced changes in nodule metabolites were observed but no metabolite or transcript markers of senescence could be detected. It is concluded that stress-induced senescence in the lowest leaf ranks precedes nodule senescence, suggesting that leaves of low photosynthetic capacity are sacrificed in favour of nodule nitrogen metabolism.

Cysteine protease and cystatin expression and activity during soybean nodule development and senescence.

BACKGROUND: Nodules play an important role in fixing atmospheric nitrogen for soybean growth. Premature senescence of nodules can negatively impact on nitrogen availability for plant growth and, as such, we need a better understanding of nodule development and senescence. Cysteine proteases are known to play a role in nodule senescence, but knowledge is still fragmented regarding the function their inhibitors (cystatins) during the development and senescence of soybean nodules. This study provides the first data with regard to cystatin expression during nodule development combined with biochemical characterization of their inhibition strength. RESULTS: Seventy nine non-redundant cysteine protease gene sequences with homology to papain, belonging to different subfamilies, and several legumain-like cysteine proteases (vacuole processing enzymes) were identified from the soybean genome assembly with eighteen of these cysteine proteases actively transcribed during nodule development and senescence. In addition, nineteen non-redundant cystatins similar to oryzacystatin-I and belonging to cystatin subgroups A and C were identified from the soybean genome assembly with seven actively transcribed in nodules. Most cystatins had preferential affinity to cathepsin L-like cysteine proteases. Transcription of cystatins Glyma05g28250, Glyma15g12211, Glyma15g36180 particularly increased during onset of senescence, possibly regulating proteolysis when nodules senesce and undergo programmed cell death. Both actively transcribed and non-actively transcribed nodule cystatins inhibited cathepsin-L- and B-like activities in different age nodules and they also inhibited papain and cathepsin-L activity when expressed and purified from bacterial cells. CONCLUSIONS: Overlap in activities and specificities of actively and non-actively transcribed cystatins raises the question if non-transcribed cystatins provide a reservoir for response to particular environments. This data might be applicable to the development of strategies to extend the active life span of nodules or prevent environmentally induced senescence.

Ectopic phytocystatin expression leads to enhanced drought stress tolerance in soybean (Glycine max) and Arabidopsis thaliana through effects on strigolactone pathways and can also result in improved seed traits.

Ectopic cystatin expression has long been used in plant pest management, but the cysteine protease, targets of these inhibitors, might also have important functions in the control of plant lifespan and stress tolerance that remain poorly characterized. We therefore characterized the effects of expression of the rice cystatin, oryzacystatin-I (OCI), on the growth, development and stress tolerance of crop (soybean) and model (Arabidopsis thaliana) plants. Ectopic OCI expression in soybean enhanced shoot branching and leaf chlorophyll accumulation at later stages of vegetative development and enhanced seed protein contents and decreased the abundance of mRNAs encoding strigolactone synthesis enzymes. The OCI-expressing A. thaliana showed a slow-growth phenotype, with increased leaf numbers and enhanced shoot branching at flowering. The OCI-dependent inhibition of cysteine proteases enhanced drought tolerance in soybean and A. thaliana, photosynthetic CO2 assimilation being much less sensitive to drought-induced inhibition in the OCI-expressing soybean lines. Ectopic OCI expression or treatment with the cysteine protease inhibitor E64 increased lateral root densities in A. thaliana. E64 treatment also increased lateral root densities in the max2-1 mutants that are defective in strigolactone signalling, but not in the max3-9 mutants that are defective in strigolactone synthesis. Taken together, these data provide evidence that OCI-inhibited cysteine proteases participate in the control of growth and stress tolerance through effects on strigolactones. We conclude that cysteine proteases are important targets for manipulation of plant growth, development and stress tolerance, and also seed quality traits.

Improving the antinutritional profiles of common beans (Phaseolus vulgaris L.) moderately impacts carotenoid bioaccessibility but not mineral solubility.

Common beans are a common staple food with valuable nutritional qualities, but their high contents in antinutritional factors (ANFs) can decrease the bioavailability of (i) fat-soluble micronutrients including carotenoids and (ii) minerals. Our objective was to select ANF-poor bean lines that would not interfere with carotenoid and mineral bioavailability. To achieve this objective, seeds of commercial and experimental Phaseolus vulgaris L. bean lines were produced for 2 years and the bean's content in ANFs (saponins, phytates, tannins, total polyphenols) was assessed. We then measured carotenoid bioaccessibility and mineral solubility (i.e. the fraction of carotenoid and mineral that transfer into the aqueous phase of the digesta and is therefore absorbable) from prepared beans using in vitro digestion. All beans contained at least 200 mg/100 g of saponins and 2.44 mg/100 g tannins. The low phytic acid (lpa) lines, lpa1 and lpa12 exhibited lower phytate levels (≈ - 80%, p = 0.007 and p = 0.02) than their control BAT-93. However, this decrease had no significant impact on mineral solubility. HP5/1 (lpa + phaseolin and lectin PHA-E free) bean line, induced an improvement in carotenoid bioaccessibility (i.e., + 38%, p = 0.02, and + 32%, p = 0.005, for phytofluene bioaccessibility in 2021 and 2022, respectively). We conclude that decrease in the phytate bean content should thus likely be associated to decreases in other ANFs such as tannins or polyphenols to lead to significant improvement of micronutrient bioaccessibility.

Legumes and common beans in sustainable diets: nutritional quality, environmental benefits, spread and use in food preparations.

In recent decades, scarcity of available resources, population growth and the widening in the consumption of processed foods and of animal origin have made the current food system unsustainable. High-income countries have shifted towards food consumption patterns which is causing an increasingly process of environmental degradation and depletion of natural resources, with the increased incidence of malnutrition due to excess (obesity and non-communicable disease) and due to chronic food deprivation. An urgent challenge is, therefore, to move towards more healthy and sustainable eating choices and reorientating food production and distribution to obtain a human and planetary health benefit. In this regard, legumes represent a less expensive source of nutrients for low-income countries, and a sustainable healthier option than animal-based proteins in developed countries. Although legumes are the basis of many traditional dishes worldwide, and in recent years they have also been used in the formulation of new food products, their consumption is still scarce. Common beans, which are among the most consumed pulses worldwide, have been the focus of many studies to boost their nutritional properties, to find strategies to facilitate cultivation under biotic/abiotic stress, to increase yield, reduce antinutrients contents and rise the micronutrient level. The versatility of beans could be the key for the increase of their consumption, as it allows to include them in a vast range of food preparations, to create new formulations and to reinvent traditional legume-based recipes with optimal nutritional healthy characteristics.

In order to lower the antinutritional activity of serine protease inhibitors, we need to understand their role in seed development.

Proteases, including serine proteases, are involved in the entire life cycle of plants. Proteases are controlled by protease inhibitors (PI) to limit any uncontrolled or harmful protease activity. The role of PIs in biotic and abiotic stress tolerance is well documented, however their role in various other plant processes has not been fully elucidated. Seed development is one such area that lack detailed work on the function of PIs despite the fact that this is a key process in the life cycle of the plant. Serine protease inhibitors (SPI) such as the Bowman-Birk inhibitors and Kunitz-type inhibitors, are abundant in legume seeds and act as antinutrients in humans and animals. Their role in seed development is not fully understood and present an interesting research target. Whether lowering the levels and activity of PIs, in order to lower the anti-nutrient levels in seed will affect the development of viable seed, remains an important question. Studies on the function of SPI in seed development are therefore required. In this Perspective paper, we provide an overview on the current knowledge of seed storage proteins, their degradation as well as on the serine protease-SPI system in seeds and what is known about the consequences when this system is modified. We discuss areas that require investigation. This includes the identification of seed specific SPIs; screening of germplasms, to identify plants with low seed inhibitor content, establishing serine protease-SPI ratios and lastly a focus on molecular techniques that can be used to modify seed SPI activity.

Loop replacement design: a new way to improve potency of plant cystatins.

Plant cystatins function as competitive inhibitors of cysteine proteases. Similar to other defence proteins, cystatins include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Protein engineering approaches, such as point mutations, at these functionally relevant amino acid sites have already been found to be a powerful tool in improving the inhibitory properties of cystatins. Such engineered cystatins not only better protect against digestive proteases of herbivorous arthropods but also against cysteine proteases of several other plant pests as well as against cysteine proteases produced in plant during stress-induced senescence. Despite previous engineering successes, an urgent need still exists to further improve both plant cystatin potency and specificity. Tremblay and colleagues propose in this issue a new cystatin engineering strategy to substitute the function-related structural elements (SEs) of a cystatin by the corresponding elements of an alternative cystatin. This strategy, possibly combined with direct cystatin gene editing in a target plant, might provide an innovative way to control cysteine protease activity. Comment on https://doi.org/10.1111/febs.16288.

Antinutritional factors, nutritional improvement, and future food use of common beans: A perspective.

Common bean seeds are an excellent source of protein as well as of carbohydrates, minerals, vitamins, and bioactive compounds reducing, when in the diet, the risks of diseases. The presence of bioactive compounds with antinutritional properties (e.g., phytic acid, lectins, raffinosaccharides, protease inhibitors) limits, however, the bean's nutritional value and its wider use in food preparations. In the last decades, concerted efforts have been, therefore, made to develop new common bean genotypes with reduced antinutritional compounds by exploiting the natural genetic variability of common bean and also applying induced mutagenesis. However, possible negative, or positive, pleiotropic effects due to these modifications, in terms of plant performance in response to stresses or in the resulting technological properties of the developed mutant genotypes, have yet not been thoroughly investigated. The purpose of the perspective paper is to first highlight the current advances, which have been already made in mutant bean characterization. A view will be further provided on future research directions to specifically explore further advantages and disadvantages of these bean mutants, their potential use in innovative foods and representing a valuable genetic reservoir of combinations to assess the true functional role of specific seed bioactive components directly in the food matrix.

Acclimation to high CO2 in maize is related to water status and dependent on leaf rank.

The responses of C(3) plants to rising atmospheric CO(2) levels are considered to be largely dependent on effects exerted through altered photosynthesis. In contrast, the nature of the responses of C(4) plants to high CO(2) remains controversial because of the absence of CO(2) -dependent effects on photosynthesis. In this study, the effects of atmospheric CO(2) availability on the transcriptome, proteome and metabolome profiles of two ranks of source leaves in maize (Zea mays L.) were studied in plants grown under ambient CO(2) conditions (350 +/- 20 µL L(-1) CO(2) ) or with CO(2) enrichment (700 +/- 20 µL L(-1) CO(2) ). Growth at high CO(2) had no effect on photosynthesis, photorespiration, leaf C/N ratios or anthocyanin contents. However, leaf transpiration rates, carbohydrate metabolism and protein carbonyl accumulation were altered at high CO(2) in a leaf-rank specific manner. Although no significant CO(2) -dependent changes in the leaf transcriptome were observed, qPCR analysis revealed that the abundance of transcripts encoding a Bowman-Birk protease inhibitor and a serpin were changed by the growth CO(2) level in a leaf rank specific manner. Moreover, CO(2) -dependent changes in the leaf proteome were most evident in the oldest source leaves. Small changes in water status may be responsible for the observed responses to high CO(2,) particularly in the older leaf ranks.