Chickpea (Cicer arietinum L.), a rainfed crop in semi-arid regions, experiences frequent intermittent drought stress, hindering growth. This study explored morpho-physiological and molecular responses of six chickpea genotypes (ICC 4958, BG 4005, Pusa 362, BGM 10218, RSG 888, ICC 1882) under terminal drought to enhance yield and adaptability. Drought stress was imposed during early podding in a controlled glasshouse experiment. Various physiological, root growth, fertility, and yield parameters were measured. Proteomics analysis was conducted on leaf, pod, and root tissue samples, collected at similar to 0.3 fraction of transpirable soil water (FTSW). Significant differences occurred for various traits, including phenologies, water relations, pollen viability and germination, flower and pod numbers, abortion rates, photosynthetic apparatus and pigments, osmotic adjustments, canopy temperatures, root length, and densities, and overall yield and biomass. FTSW dynamics can aid genotype screening during early drought. Characterizing stress-responsive proteins can provide insights into cellular processes and adaptations. Terminal drought affected leaf central shoot metabolism, reducing photosynthetic capacity, while roots and pods reconfigured enzyme abundances to promote root growth and pod development. The extensive genotypic variation in proteome data, morpho-physiological traits, and water-use patterns presents opportunities for developing drought-adapted cultivars.

Analysis of salinity tolerance processes in wheat has focused on salt exclusion from shoots while root phenotypes have received limited attention. Here, we consider the varying phenotypic response of four bread wheat varieties that differ in their type and degree of salt tolerance and assess their molecular responses to salinity and changes in root cell wall lignification. These varieties were Westonia introgressed with Nax1 and Nax2 root sodium transporters (HKT1;4-A and HKT1;5-A) that reduce Na+ accumulation in leaves, as well as the 'tissue tolerant' Portuguese landrace Mocho de Espiga Branca that has a mutation in the homologous gene HKT1;5-D and has high Na+ concentration in leaves. These three varieties were compared with the relatively more salt-sensitive cultivar Gladius. Through the use of root histochemical analysis, ion concentrations, as well as differential proteomics and targeted metabolomics, we provide an integrated view of the wheat root response to salinity. We show different metabolic re-arrangements in energy conversion, primary metabolic machinery and phenylpropanoid pathway leading to monolignol production in a genotype and genotype by treatment-dependent manner that alters the extent and localisation of root lignification which correlated with an improved capacity of wheat roots to cope better under salinity stress.

Root secreted acid phosphatases and organic anions are widely perceived as major players of plant phosphorus (P) mobilisation from the rhizosphere under P limiting growth conditions. Previous research indicated that other mechanisms play a role, especially in species with fine roots, such as wheat. In this study we characterised the plant-derived extracellular proteome of wheat roots by profiling root tip mucilage, soluble root secreted and root tip proteomes. Extracellular acid phosphatases and enzymes of the central carbon metabolism were targeted using selected reaction monitoring. More than 140 proteins with extracellular localisation prediction were identified in mucilage. P starvation induced proteins predicted to be localised to the apoplast which are related to cell wall modification and defence in both, root tip and soluble root-secreted proteomes. Glycolytic enzymes were strongly increased in abundance by P limitation in root tips, as were PEPC and plastidial MDH. Soluble acid phosphatases were not identified in extracellular protein samples. Our results indicate that root tip mucilage contains proteins with the functional potential to actively shape their immediate environment by modification of plant structural components and biotic interactions. Wheat acid phosphatases appear to play a minor role in P mobilisation beyond the immediate root surface. SIGNIFICANCE: Phosphorus (P) is a plant growth limiting nutrient in many agricultural situations and the development of phosphorus efficient crops is of paramount importance for future agricultural management practices. As P is relatively immobile in soils, processes occurring at the root-soil interface, the rhizosphere, are suspected to play a key role in plant-induced P mobilisation. According to the current view, the secretion of extracellular acid phosphatases and organic anions enhances P mobilisation within several millimetres beyond the root surface, either directly or indirectly through the selection and appropriate soil microbes. However, the mechanisms of P mobilisation in species with fine roots, such as wheat, and the role of other secreted root proteins are poorly understood. Here, we carried out the profiling of wheat root tip mucilage, soluble root secreted and root tip proteomes. We analysed proteome changes in response to P starvation. We found that proteins with a predicted localisation to the apoplast made up a major proportion of stress-responsive proteins. Acid phosphatases were not identified within extracellular protein samples, which were enriched in proteins with predicted extracellular localisation. The absence of extracellular APases was further validated by multiple reaction monitoring. Our data indicates that wheat acid phosphatases play a minor role in P mobilisation beyond the immediate root surface and provides a resource for breeding strategies and further investigations of the functional roles of root tip-released proteins in the rhizosphere under P limitation.

The impact of salinity on wheat plants is often studied by analysis of shoot responses, even though the main mechanism of tolerance is shoot Na+ exclusion. Wheat roots directly experience rising NaCl concentrations and show more physiological responses in root tips than in mature roots and altered responses with time; but the molecular reason for these differential responses is unclear. We have found that there is a distinct difference between the proteome responses of wheat root tip and mature root tissues to salinity. Translation and protein synthesis related proteins showed a significant decrease in abundance, most of the glycolytic enzymes and selected TCA cycle enzymes and ATP synthase subunits were significantly decreased in abundance under salt stress in root tips only. The root tip response in wheat indicates the protein synthesis capacity and energy production were impaired under salt stress which correlated with the anatomical response of root growth decrease and its respiratory rate. Wheat root responses are direct and rapid effects of the soil salinity in this species, therefore shoot responses such as reduction in shoot growth and photosynthetic capacity need to be considered in light of these effects on root metabolism. Significance: Salinity is a critical environmental factor limiting crop production throughout the world. Wheat (Triticum aestivum) is the most significant cereal crop for human nutrition and both its growth and yield is negatively impacted by salinity. Salinity stress is known to impose osmotic stress in plants during the initial phase of exposure and ion toxicity in the later stages of development. Roots are the first plant organ to perceive the salt. However, intensive breading approaches to develop salt tolerant crops have mainly focussed on exclusion of salt from above ground tissues, and only achieved limited success to date. Wheat roots physiologically respond to salinity by overall reduction in the length of seminal mots. The stunting of the wheat root system is considered to be a result of higher sensitivity of root tips to salinity. However, the metabolic changes that underpin selective root tip sensitivity is largely unknown. Here, we carried out non-targeted profiling of mature root versus root tip proteomes under control and salt stress conditions. We found distinct changes in abundance of proteins involved in carbon and energy metabolism and protein metabolism in mature roots and root tips in response to salt stress. We further investigated the impact of these changes on metabolic machinery in the wheat root proteome using a targeted MS approach. We found evidence that protein synthesis and energy production machinery becomes limiting in mot tips, while the same processes in mature root remains less affected by salt stress. Our proteomic data explain the impairment of root growth and physiological characteristics as well as improve the understanding of wheat root responses under salinity which is an essential first step for further investigation of molecular traits underpinning root characteristics to improve salt tolerance of wheat.

Summary The impact of salinity on wheat plants is often studied by analysis of shoot responses, even though the main mechanism of tolerance is shoot Na + exclusion. There is a need to understand the molecular responses of root tissues that directly experience rising NaCl concentrations. We have combined analysis of root growth, ion content and respiration with proteome responses in wheat root tip and mature root tissues under saline conditions. We find significant changes in translation and protein synthesis, energy metabolism and amino acid metabolism in a root tissue specific manner. Translation and protein synthesis related proteins showed significant decreases in abundance only in root tips, as did most of the glycolytic enzymes and selected TCA cycle enzymes and ATP synthase subunits. This selective root tip proteome response indicates protein synthesis capacity and energy production were impaired under salt stress, correlating with the anatomical response of roots and reduced root tip respiration rate. Wheat roots respond directly to soil salinity, therefore shoot responses such as reduction in shoot growth and photosynthetic capacity need to be considered in light of these effects.