Discovering new genes for alfalfa (Medicago sativa) growth and biomass resilience in combined salinity and Phoma medicaginis infection through GWAS.

Salinity and Phoma medicaginis infection represent significant challenges for alfalfa cultivation in South Africa, Europe, Australia, and, particularly, Tunisia. These constraints have a severe impact on both yield and quality. The primary aim of this study was to establish the genetic basis of traits associated with biomass and growth of 129 Medicago sativa genotypes through genome-wide association studies (GWAS) under combined salt and P. medicaginis infection stresses. The results of the analysis of variance (ANOVA) indicated that the variation in these traits could be primarily attributed to genotype effects. Among the test genotypes, the length of the main stem, the number of ramifications, the number of chlorotic leaves, and the aerial fresh weight exhibited the most significant variation. The broad-sense heritability (H²) was relatively high for most of the assessed traits, primarily due to genetic factors. Cluster analysis, applied to morpho-physiological traits under the combined stresses, revealed three major groups of accessions. Subsequently, a GWAS analysis was conducted to validate significant associations between 54,866 SNP-filtered single-nucleotide polymorphisms (SNPs) and seven traits. The study identified 27 SNPs that were significantly associated with the following traits: number of healthy leaves (two SNPs), number of chlorotic leaves (five SNPs), number of infected necrotic leaves (three SNPs), aerial fresh weight (six SNPs), aerial dry weight (nine SNPs), number of ramifications (one SNP), and length of the main stem (one SNP). Some of these markers are related to the ionic transporters, cell membrane rigidity (related to salinity tolerance), and the NBS_LRR gene family (associated with disease resistance). These findings underscore the potential for selecting alfalfa genotypes with tolerance to the combined constraints of salinity and P. medicaginis infection.

Improving productivity and soil fertility in Medicago sativa and Hordeum marinum through intercropping under saline conditions.

BACKGROUND AND AIMS: Intercropping is an agriculture system used to enhance the efficiency of resource utilization and maximize crop yield grown under environmental stress such as salinity. Nevertheless, the impact of intercropping forage legumes with annual cereals on soil salinity remains unexplored. This research aimed to propose an intercropping system with alfalfa (Medicago sativa)/sea barley (Hordeum marinum) to explore its potential effects on plant productivity, nutrient uptake, and soil salinity. METHODS: The experiment involved three harvests of alfalfa and Hordeum marinum conducted under three cropping systems (sole, mixed, parallel) and subjected to salinity treatments (0 and 150 mM NaCl). Agronomical traits, nutrient uptake, and soil properties were analyzed. RESULTS: revealed that the variation in the measured traits in both species was influenced by the cultivation mode, treatment, and the interaction between cultivation mode and treatment. The cultivation had the most significant impact. Moreover, the mixed culture (MC) significantly enhanced the H. marinum and M. sativa productivity increasing biomass yield and development growth under salinity compared to other systems, especially at the second harvest. Furthermore, both intercropping systems alleviated the nutrient uptake under salt stress, as noted by the highest levels of K+/Na+ and Ca2+/Mg2+ ratios compared to monoculture. However, the intercropping mode reduced the pH and the electroconductivity (CEC) of the salt soil and increased the percentage of organic matter and the total carbon mostly with the MC system. CONCLUSIONS: Intercropped alfalfa and sea barely could mitigate the soil salinity, improve their yield productivity, and enhance nutrient uptake. Based on these findings, we suggest implementing the mixed-culture system for both target crops in arid and semi-arid regions, which further promotes sustainable agricultural practices.

Variability in Responses to Phoma medicaginis Infection in a Tunisian Collection of Three Annual Medicago Species.

Spring black stem and leaf spot, caused by Phoma medicaginis, is an issue in annual Medicago species. Therefore, in this study, we analyzed the response to P. medicaginis infection in a collection of 46 lines of three annual Medicago species (M. truncatula, M. ciliaris, and M. polymorpha) showing different geographic distribution in Tunisia. The reaction in the host to the disease is explained by the effects based on plant species, lines nested within species, treatment, the interaction of species × treatment, and the interaction of lines nested within species × treatment. Medicago ciliaris was the least affected for aerial growth under infection. Furthermore, the largest variation within species was found for M. truncatula under both conditions. Principal component analysis and hierarchical classification showed that M. ciliaris lines formed a separate group under control treatment and P. medicaginis infection and they are the most vigorous in growth. These results indicate that M. ciliaris is the least susceptible in response to P. medicaginis infection among the three Medicago species investigated here, which can be used as a good candidate in crop rotation to reduce disease pressure in the field and as a source of P. medicaginis resistance for the improvement of forage legumes.

Comparative Proteomics Analysis between Maize and Sorghum Uncovers Important Proteins and Metabolic Pathways Mediating Drought Tolerance.

Drought severely affects crop yield and yield stability. Maize and sorghum are major crops in Africa and globally, and both are negatively impacted by drought. However, sorghum has a better ability to withstand drought than maize. Consequently, this study identifies differences between maize and sorghum grown in water deficit conditions, and identifies proteins associated with drought tolerance in these plant species. Leaf relative water content and proline content were measured, and label-free proteomics analysis was carried out to identify differences in protein expression in the two species in response to water deficit. Water deficit enhanced the proline accumulation in sorghum roots to a higher degree than in maize, and this higher accumulation was associated with enhanced water retention in sorghum. Proteomic analyses identified proteins with differing expression patterns between the two species, revealing key metabolic pathways that explain the better drought tolerance of sorghum than maize. These proteins include phenylalanine/tyrosine ammonia-lyases, indole-3-acetaldehyde oxidase, sucrose synthase and phenol/catechol oxidase. This study highlights the importance of phenylpropanoids, sucrose, melanin-related metabolites and indole acetic acid (auxin) as determinants of the differences in drought stress tolerance between maize and sorghum. The selection of maize and sorghum genotypes with enhanced expression of the genes encoding these differentially expressed proteins, or genetically engineering maize and sorghum to increase the expression of such genes, can be used as strategies for the production of maize and sorghum varieties with improved drought tolerance.

Water Deficit-Induced Changes in Phenolic Acid Content in Maize Leaves Is Associated with Altered Expression of Cinnamate 4-Hydroxylase and p-Coumaric Acid 3-Hydroxylase.

The amino acid phenylalanine is a precursor to phenolic acids that constitute the lignin biosynthetic pathway. Although there is evidence of a role of some phenolic acids in plant responses to pathogens and salinity, characterization of the involvement of phenolic acids in plant responses to drought is limited. Drought reduces water content in plant tissue and can lead to decreased cell viability and increased cell death. We thus subjected maize seedlings to water deficit and evaluated relative water content and cell viability together with p-coumaric acid, caffeic acid and ferulic acid contents in the leaves. Furthermore, we measured the enzymatic activity of cinnamate 4-hydroxylase (EC 1.14.13.11) and p-coumarate 3-hydroxylase (EC 1.14.17.2) and associated these with the expression of genes encoding cinnamate 4-hydroxylase and p-coumarate-3 hydroxylase in response to water deficit. Water deficit reduced relative water content and cell viability in maize leaves. This corresponded with decreased p-coumaric acid but increased caffeic and ferulic acid content in the leaves. Changes in the phenolic acid content of the maize leaves were associated with increased enzymatic activities of cinnamate 4-hydroxylase and p-coumarate hydroxylase. The increased enzymatic activity of p-coumarate 3-hydroxylase was associated with increased expression of a gene encoding p-coumarate 3-hydroxylase. We thus conclude that metabolic pathways involving phenolic acids may contribute to the regulation of drought responses in maize, and we propose that further work to elucidate this regulation may contribute to the development of new maize varieties with improved drought tolerance. This can be achieved by marker-assisted selection to select maize lines with high levels of expression of genes encoding cinnamate 4-hydroxylase and/or p-coumarate 3-hydroxylase for use in breeding programs aimed and improving drought tolerance, or by overexpression of these genes via genetic engineering to confer drought tolerance.

Evaluation of the Morpho-Physiological, Biochemical and Molecular Responses of Contrasting Medicago truncatula Lines under Water Deficit Stress.

Medicago truncatula is a forage crop of choice for farmers, and it is a model species for molecular research. The growth and development and subsequent yields are limited by water availability mainly in arid and semi-arid regions. Our study aims to evaluate the morpho-physiological, biochemical and molecular responses to water deficit stress in four lines (TN6.18, JA17, TN1.11 and A10) of M. truncatula. The results showed that the treatment factor explained the majority of the variation for the measured traits. It appeared that the line A10 was the most sensitive and therefore adversely affected by water deficit stress, which reduced its growth and yield parameters, whereas the tolerant line TN6.18 exhibited the highest root biomass production, a significantly higher increase in its total protein and soluble sugar contents, and lower levels of lipid peroxidation with greater cell membrane integrity. The expression analysis of the DREB1B gene using RT-qPCR revealed a tissue-differential expression in the four lines under osmotic stress, with a higher induction rate in roots of TN6.18 and JA17 than in A10 roots, suggesting a key role for DREB1B in water deficit tolerance in M. truncatula.

Exogenous 3,3'-Diindolylmethane Improves Vanadium Stress Tolerance in Brassica napus Seedling Shoots by Modulating Antioxidant Enzyme Activities.

3,3'-diindolylmethane (DIM) belongs to a family of indole glucosinolate compounds that have been shown to improve Brassica napus growth through the modulation of reactive oxygen species when applied exogenously. The B. napus cultivar AV Garnet was previously identified as a vanadium-sensitive cultivar. Therefore, in this study we investigated whether exogenous DIM could improve the vanadium tolerance of AV Garnet. We performed the following experiments: seed germination assessment, dry weight assessment, cell viability assay, chlorophyll content assay, malondialdehyde (MDA) assay, conjugated diene (CD) content assay, hydrogen peroxide (H2O2) content assay, superoxide (O2-) content determination, methylglyoxal (MG) content determination, hydroxyl radical (·OH) concentration determination, ascorbate peroxidase (APX) activity assay, superoxide dismutase (SOD) activity assay, glyoxalase I (Gly I) activity assay, glutathione S-transferase (GST) activity assay and inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis for vanadium content determination. Under vanadium stress, exogenous DIM increased the seed germination percentage, shoot dry weight, cell viability and chlorophyll content. Exogenous DIM also led to a decrease in MDA, CD, H2O2, O2-, MG and ·OH, under vanadium stress in the shoots. Furthermore, DIM application led to an increase in the enzymatic activities of APX, SOD, Gly I and GST under vanadium stress. Interestingly, under vanadium stress, DIM treatment did not alter vanadium content in B. napus shoots. Our results indicate that exogenous application of DIM can improve B. napus seedling shoot growth and biomass under vanadium stress by priming the antioxidant enzymes via reactive oxygen species (ROS) signaling.

Morpho-Physiological, Biochemical, and Genetic Responses to Salinity in Medicago truncatula.

We used an integrated morpho-physiological, biochemical, and genetic approach to investigate the salt responses of four lines (TN1.11, TN6.18, JA17, and A10) of Medicago truncatula. Results showed that TN1.11 exhibited a high tolerance to salinity, compared with the other lines, recording a salinity induced an increase in soluble sugars and soluble proteins, a slight decrease in malondialdehyde (MDA) accumulation, and less reduction in plant biomass. TN6.18 was the most susceptible to salinity as it showed less plant weight, had elevated levels of MDA, and lower levels of soluble sugars and soluble proteins under salt stress. As transcription factors of the APETALA2/ethylene responsive factor (AP2/ERF) family play important roles in plant growth, development, and responses to biotic and abiotic stresses, we performed a functional characterization of MtERF1 gene. Real-time PCR analysis revealed that MtERF1 is mainly expressed in roots and is inducible by NaCl and low temperature. Additionally, under salt stress, a greater increase in the expression of MtERF1 was found in TN1.11 plants than that in TN6.18. Therefore, the MtERF1 pattern of expression may provide a useful marker for discriminating among lines of M. truncatula and can be used as a tool in breeding programs aiming at obtaining Medicago lines with improved salt tolerance.

Decoding Heavy Metal Stress Signalling in Plants: Towards Improved Food Security and Safety.

The mining of heavy metals from the environment leads to an increase in soil pollution, leading to the uptake of heavy metals into plant tissue. The build-up of toxic metals in plant cells often leads to cellular damage and senescence. Therefore, it is of utmost importance to produce plants with improved tolerance to heavy metals for food security, as well as to limit heavy metal uptake for improved food safety purposes. To achieve this goal, our understanding of the signaling mechanisms which regulate toxic heavy metal uptake and tolerance in plants requires extensive improvement. In this review, we summarize recent literature and data on heavy metal toxicity (oral reference doses) and the impact of the metals on food safety and food security. Furthermore, we discuss some of the key events (reception, transduction, and response) in the heavy metal signaling cascades in the cell wall, plasma membrane, and cytoplasm. Our future perspectives provide an outlook of the exciting advances that will shape the plant heavy metal signaling field in the near future.

Genetic Diversity, Population Structure and Marker-Trait Association for 100-Seed Weight in International Safflower Panel Using SilicoDArT Marker Information.

Safflower is an important oilseed crop mainly grown in the arid and semi-arid regions of the world. The aim of this study was to explore phenotypic and genetic diversity, population structure, and marker-trait association for 100-seed weight in 94 safflower accessions originating from 26 countries using silicoDArT markers. Analysis of variance revealed statistically significant genotypic effects (p < 0.01), while Turkey samples resulted in higher 100-seed weight compared to Pakistan samples. A Constellation plot divided the studied germplasm into two populations on the basis of their 100-seed weight. Various mean genetic diversity parameters including observed number of alleles (1.99), effective number of alleles (1.54), Shannon's information index (0.48), expected heterozygosity (0.32), and unbiased expected heterozygosity (0.32) for the entire population exhibited sufficient genetic diversity using 12232 silicoDArT markers. Analysis of molecular variance (AMOVA) revealed that most of the variations (91%) in world safflower panel are due to differences within country groups. A model-based structure grouped the 94 safflower accessions into populations A, B, C and an admixture population upon membership coefficient. Neighbor joining analysis grouped the safflower accessions into two populations (A and B). Principal coordinate analysis (PCoA) also clustered the safflower accessions on the basis of geographical origin. Three accessions; Egypt-5, Egypt-2, and India-2 revealed the highest genetic distance and hence might be recommended as candidate parental lines for safflower breeding programs. The mixed linear model i.e., the Q + K model, demonstrated that two DArTseq markers (DArT-45483051 and DArT-15672391) had significant association (p < 0.01) for 100-seed weight. We envisage that identified DArTseq markers associated with 100-seed weight will be helpful to develop high-yielding cultivars of safflower through marker-assisted breeding in the near future.

Modelling predicts that soybean is poised to dominate crop production across Africa.

The superior agronomic and human nutritional properties of grain legumes (pulses) make them an ideal foundation for future sustainable agriculture. Legume-based farming is particularly important in Africa, where small-scale agricultural systems dominate the food production landscape. Legumes provide an inexpensive source of protein and nutrients to African households as well as natural fertilization for the soil. Although the consumption of traditionally grown legumes has started to decline, the production of soybeans (Glycine max Merr.) is spreading fast, especially across southern Africa. Predictions of future land-use allocation and production show that the soybean is poised to dominate future production across Africa. Land use models project an expansion of harvest area, whereas crop models project possible yield increases. Moreover, a seed change in farming strategy is underway. This is being driven largely by the combined cash crop value of products such as oils and the high nutritional benefits of soybean as an animal feed. Intensification of soybean production has the potential to reduce the dependence of Africa on soybean imports. However, a successful "soybean bonanza" across Africa necessitates an intensive research, development, extension, and policy agenda to ensure that soybean genetic improvements and production technology meet future demands for sustainable production.

Inhibition of NOS- like activity in maize alters the expression of genes involved in H2O2 scavenging and glycine betaine biosynthesis.

Nitric oxide synthase-like activity contributes to the production of nitric oxide in plants, which controls plant responses to stress. This study investigates if changes in ascorbate peroxidase enzymatic activity and glycine betaine content in response to inhibition of nitric oxide synthase-like activity are associated with transcriptional regulation by analyzing transcript levels of genes (betaine aldehyde dehydrogenase) involved in glycine betaine biosynthesis and those encoding antioxidant enzymes (ascorbate peroxidase and catalase) in leaves of maize seedlings treated with an inhibitor of nitric oxide synthase-like activity. In seedlings treated with a nitric oxide synthase inhibitor, transcript levels of betaine aldehyde dehydrogenase were decreased. In plants treated with the nitric oxide synthase inhibitor, the transcript levels of ascorbate peroxidase-encoding genes were down-regulated. We thus conclude that inhibition of nitric oxide synthase-like activity suppresses the expression of ascorbate peroxidase and betaine aldehyde dehydrogenase genes in maize leaves. Furthermore, catalase activity was suppressed in leaves of plants treated with nitric oxide synthase inhibitor; and this corresponded with the suppression of the expression of catalase genes. We further conclude that inhibition of nitric oxide synthase-like activity, which suppresses ascorbate peroxidase and catalase enzymatic activities, results in increased H2O2 content.

Drought and exogenous abscisic acid alter hydrogen peroxide accumulation and differentially regulate the expression of two maize RD22-like genes.

Increased biosynthesis of abscisic acid (ABA) occurs in plants in response to water deficit, which is mediated by changes in the levels of reactive oxygen species such as H2O2. Water deficit and ABA induce expression of some RD22-like proteins. This study aimed to evaluate the effect of water deficit and exogenous ABA (50 µM ABA applied every 24 hours for a total of 72 hours) on H2O2 content in Zea mays (maize) and to characterise genes encoding two putative maize RD22-like proteins (designated ZmRD22A and ZmRD22B). The expression profiles of the two putative maize RD22-like genes in response to water deficit and treatment with ABA were examined in leaves. In silico analyses showed that the maize RD22-like proteins share domain organisation with previously characterized RD22-like proteins. Both water deficit and exogenous ABA resulted in increased H2O2 content in leaves but the increase was more pronounced in response to water deficit than to exogenous ABA. Lignin content was not affected by exogenous ABA, whereas it was decreased by water deficit. Expression of both RD22-like genes was up-regulated by drought but the ZmRD22A gene was not influenced by exogenous ABA, whereas ZmRD22B was highly responsive to exogenous ABA.

Modification of cadaverine content by NO in salt-stressed maize.

NO has an important role in the control of plant development, growth, and the response to abiotic stress. In our recent paper it was demonstrated that NO affected the salt-induced changes in free amino acid levels in maize. (1) Since polyamines are synthesized from lysine and arginine, it was supposed that their concentrations are also influenced by NO. Cadaverine levels were increased by a NO donor and decreased by an inhibitor of NO synthesis in salt-stressed maize. These findings indicate that NO participates in the mediation of the effect of salt on cadaverine content. The coordinated changes in the NO and cadaverine levels may be involved in regulating of the response to salt stress in maize.

Measurement of nitric oxide in plant tissue using difluorofluorescein and oxyhemoglobin.

Nitric oxide (NO) is now well established as a signalling molecule in plants, regulating various physiological processes ranging from development to responses to pathogens and changes in the physical environment. Various methods for the detection of NO in plant tissue have been described, and all of these methods have serious limitations that impact their utility for accurate detection of NO in plant tissues. Despite such limitations, both difluorofluorescein diacetate and oxyhemoglobin present convenient and relatively easy approaches for measuring NO in plant tissue and their utility can be enhanced by including appropriate controls to address some of the limitations that these two methods have. This chapter provides methods for measuring or detecting NO production in plant tissue using either difluorofluorescein diacetate or oxyhemoglobin.

Nitric oxide affects salt-induced changes in free amino acid levels in maize.

It was assumed that salt-induced redox changes affect amino acid metabolism in maize (Zea mays L.), and this influence may be modified by NO. The applied NaCl treatment reduced the fresh weight of shoots and roots. This decrease was smaller after the combined application of NaCl and an NO-donor ((Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, DETA/NO) in the shoots, while it was greater after simultaneous treatment with NaCl and nitro-L-arginine (L-NNA, inhibitor of NO synthesis) in the roots. The quantum yield efficiency of photosystem II was not influenced by the treatments. NaCl had a significant effect on the redox environment in the leaves as it was shown by the increase in the amount of glutathione disulphide and in the redox potential of the glutathione/glutathione disulphide redox pair. This influence of NaCl was modified by DETA/NO and L-NNA. Pharmacological modification of NO levels affected salt-induced changes in both the total free amino acid content and in the free amino acid composition. NaCl alone increased the concentration of almost all amino acids which effect was strengthened by DETA/NO in the case of Pro. L-NNA treatment resulted in a significant increase in the Ala, Val, Gly and Tyr contents. The Ile, Lys and Val concentrations rose considerably after the combined application of NaCl and DETA/NO compared to NaCl treatment alone in the recovery phase. NaCl also increased the expression of several genes related to the amino acid and antioxidant metabolism, and this effect was modified by DETA/NO. In conclusion, modification of NO levels affected salt-induced, glutathione-dependent redox changes and simultaneously the free amino acid composition and the level of several free amino acids. The observed much higher Pro content in plants treated with both NaCl and DETA/NO during recovery may contribute to the protective effect of NO against salt stress.

Caspase-like enzymatic activity and the ascorbate-glutathione cycle participate in salt stress tolerance of maize conferred by exogenously applied nitric oxide.

Salinity stress causes ionic stress (mainly from high Na⁺ and Cl⁻ levels) and osmotic stress (as a result of inhibition of water uptake by roots and amplified water loss from plant tissue), resulting in cell death and inhibition of growth and ultimately adversely reducing crop productivity. In this report, changes in root nitric oxide content, shoot and root biomass, root H2O2 content, root lipid peroxidation, root cell death, root caspase-like enzymatic activity, root antioxidant enzymatic activity and root ascorbate and glutathione contents/redox states were investigated in maize (Zea mays L. cv Silverking) after long-term (21 d) salt stress (150 mM NaCl) with or without exogenously applied nitric oxide generated from the nitric oxide donor 2,2'-(Hydroxynitrosohydrazano)bis-ethane. In addition to reduced shoot and root biomass, salt stress increased the nitric oxide and H2O2 contents in the maize roots and resulted in elevated lipid peroxidation, caspase-like activity and cell death in the roots. Altered antioxidant enzymatic activities, along with changes in ascorbate and glutathione contents/redox status were observed in the roots in response to salt stress. The detrimental effects of salt stress in the roots were reversed by exogenously applied nitric oxide. These results demonstrate that exogenously applied nitric oxide confers salt stress tolerance in maize by reducing salt stress-induced oxidative stress and caspase-like activity through a process that limits accumulation of reactive oxygen species via enhanced antioxidant enzymatic activity.

Identification of a novel Arabidopsis thaliana nitric oxide-binding molecule with guanylate cyclase activity in vitro.

While there is evidence of nitric oxide (NO)-dependent signalling via the second messenger cyclic guanosine 3',5'-monophosphate (cGMP) in plants, guanylate cyclases (GCs), enzymes that catalyse the formation of cGMP from guanosine 5'-triphosphate (GTP) have until recently remained elusive and none of the candidates identified to-date are NO-dependent. Using both a GC and heme-binding domain specific (H-NOX) search motif, we have identified an Arabidopsis flavin monooxygenase (At1g62580) and shown electrochemically that it binds NO, has a higher affinity for NO than for O(2) and that this molecule can generate cGMP from GTP in vitro in an NO-dependent manner.

Nitric oxide increases the enzymatic activity of three ascorbate peroxidase isoforms in soybean root nodules.

Ascorbate peroxidase is one of the major enzymes regulating the levels of H2O2 in plants and plays a crucial role in maintaining root nodule redox status. We used fully developed and mature nitrogen fixing root nodules from soybean plants to analyze the effect of exogenously applied nitric oxide, generated from the nitric oxide donor 2,2'-(hydroxynitrosohydrazono)bis-ethanimine, on the enzymatic activity of soybean root nodule ascorbate peroxidase. Nitric oxide caused an increase in the total enzymatic activity of ascorbate peroxidase. The nitric oxide-induced changes in ascorbate peroxidase enzymatic activity were coupled to altered nodule H2O2 content. Further analysis of ascorbate peroxidase enzymatic activity identified three ascorbate peroxidase isoforms for which augmented enzymatic activity occurred in response to nitric oxide. Our results demonstrate that nitric oxide regulates soybean root nodule ascorbate peroxidase activity. We propose a role of nitric oxide in regulating ascorbate-dependent redox status in soybean root nodule tissue.