Phenotyping for physiological traits under plant stress: methods, challenges, and future perspectives

BackgroundLentil is an essential cool-season food legume that offers several benefits in human nutrition and cropping systems. Drought stress is the major environmental constraint affecting lentil plants’ growth and productivity by altering various morphological, physiological, and biochemical traits. Our previous research provided physiological and biochemical evidence showing the role of silicon (Si) in alleviating drought stress in lentil plants, while the molecular mechanisms are still unidentified. Understanding the molecular mechanisms of Si-mediated drought stress tolerance can provide fundamental information to enhance our knowledge of essential gene functions and pathways modulated by Si during drought stress in plants. Thus, the present study compared the transcriptomic characteristics of two lentil genotypes (drought tolerant-ILL6002; drought sensitive-ILL7537) under drought stress and investigated the gene expression in response to Si supplementation using high-throughput RNA sequencing.ResultsThis study identified 7164 and 5576 differentially expressed genes (DEGs) from drought-stressed lentil genotypes (ILL 6002 and ILL 7537, respectively), with Si treatment. RNA sequencing results showed that Si supplementation could alter the expression of genes related to photosynthesis, osmoprotection, antioxidant systems and signal transduction in both genotypes under drought stress. Furthermore, these DEGs from both genotypes were found to be associated with the metabolism of carbohydrates, lipids and proteins. The identified DEGs were also linked to cell wall biosynthesis and vasculature development. Results suggested that Si modulated the dynamics of biosynthesis of alkaloids and flavonoids and their metabolism in drought-stressed lentil genotypes. Drought-recovery-related DEGs identified from both genotypes validated the role of Si as a drought stress alleviator. This study identified different possible defense-related responses mediated by Si in response to drought stress in lentil plants including cellular redox homeostasis by reactive oxygen species (ROS), cell wall reinforcement by the deposition of cellulose, lignin, xyloglucan, chitin and xylan, secondary metabolites production, osmotic adjustment and stomatal closure.ConclusionOverall, the results suggested that a coordinated interplay between various metabolic pathways is required for Si to induce drought tolerance. This study identified potential genes and different defence mechanisms involved in Si-induced drought stress tolerance in lentil plants. Si supplementation altered various metabolic functions like photosynthesis, antioxidant defence system, osmotic balance, hormonal biosynthesis, signalling, amino acid biosynthesis and metabolism of carbohydrates and lipids under drought stress. These novel findings validated the role of Si in drought stress mitigation and have also provided an opportunity to enhance our understanding at the genomic level of Si’s role in alleviating drought stress in plants.

Combining biodegradable chelating agents with phytoextraction is an efficient technique to amend metal-contaminated soils, but most studies have addressed remediation efficiency rather than a comprehensive understanding of the interactions among plant stress, metal accumulation, and metal bioavailability. This study aimed to investigate the effects of biodegradable chelating agents on improving the efficiency of phytoextraction for cobalt (Co)-contaminated soil by sweet alyssum (Lobularia maritima (L.)) and to explore the interrelationships among plant stress, Co accumulation, and Co bioavailability. Sweet alyssum (three plants per pot) was grown in pots containing soil with Co added at 0, 40, and 60 mg kg−1, respectively. After 70 days of growth, we added four biodegradable chelating agents (EDDS, NTA, CA, and OA) at various concentrations (0, 2.5, 5.0, and 7.5 mmol kg−1). The plants were harvested after 7 days, and the biomass, reactive oxygen species (ROS) parameters, Co concentrations of the shoot and root, and available Co content in the soil were analyzed. The results demonstrate that chelating agents significantly (p < 0.05) improved the phytoextraction capability of sweet alyssum and influenced plant growth and stress. The capability of EDDS to activate Co was higher than that of other chelating agents at identical concentrations in Co-contaminated soils. Furthermore, we observed that a moderate concentration (40 mg kg−1) of Co could promote plant growth and that high concentrations of Co (60 mg kg−1) and EDDS (7.5 mmol kg−1) cause enhanced stress to plant growth, even resulting in lower shoot Co accumulation than that in the moderate EDDS treatment (5.0 mmol kg−1). The present study demonstrates that the application of EDDS may be a better choice for Co phytoextraction than NTA, CA, and OA; nevertheless, a high concentration of EDDS may enhance the negative effects on plant growth, physiological traits, and Co accumulation.

The impact of biochar and chitosan on barley plants under drought stress conditions was investigated during two field experiments. Our results confirmed that drought stress negatively affected morphological and physiological growth traits of barley plants such as plant height, number of leaves, chlorophyll concentrations, and relative water content. However, electrolyte leakage (EL%), lipid peroxidation (MDA), soluble sugars, sucrose and starch contents significantly increased as a response to drought stress. Additionally, 1000 grain weight, grains yield ha−1 and biological yield significantly decreased in stressed barley plants, also anatomical traits such as upper epidermis, lower epidermis, lamina, and mesophyll tissue thickness as well as vascular bundle diameter of flag leaves significantly decreased compared with control. The use of biochar and chitosan led to significant increases in plant height, number of leaves, and chlorophyll concentrations as well as relative water content; nevertheless these treatments led to significant decreases in electrolyte leakage (EL%) and lipid peroxidation (MDA) in the stressed plants. Moreover, anatomical and yield characters of stressed barley plants were improved with application of biochar and chitosan. The results proved the significance of biochar and chitosan in alleviating the damaging impacts of drought on barley plants.

Symbiotic associations with microbes can contribute to mitigating abiotic environmental stress in plants. In this study, we investigated individual and interactive effects of two root endophytic fungal species on physiological and biochemical mechanisms of the crop Chenopodium quinoa in response to salinity. Fungal endophytes (FE) Talaromyces minioluteus and Penicillium murcianum, isolated from quinoa plants that occur naturally in the Atacama Desert, were used for endophyte inoculation. A greenhouse experiment was developed using four plant groups: (1) plants inoculated with T. minioluteus (E1+), (2) plants inoculated with P. murcianum (E2+), (3) plants inoculated with both fungal species (E1E2+), and (4) non-inoculated plants (E-). Plants from each group were then assigned to either salt (300 mM) or control (no salt) treatments. Differences in morphological traits, photosynthesis, stomatal conductance, transpiration, superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase, (POD), phenylalanine ammonia-lyase (PAL), phenolic content, and lipid peroxidation between plant groups under each treatment were examined. We found that both endophyte species significantly improved morphological and physiological traits, including plant height, number of shoots, photosynthesis, stomatal conductance, and transpiration, in C. quinoa in response to salt, but optimal physiological responses were observed in E1E2+ plants. Under saline conditions, endophyte inoculation improved SOD, APX, and POD activity by over 50%, and phenolic content by approximately 30%, with optimal enzymatic responses again observed in E1E2+ plants. Lipid peroxidation was significantly lower in inoculated plants than in non-inoculated plants. Results demonstrate that both endophyte species enhanced the ability of C. quinoa to cope with salt stress by improving antioxidative enzyme and non-enzyme systems. In general, both FE species interacting in tandem yielded better morphological, physiological, and biochemical responses to salinity in quinoa than inoculation by a single species in isolation. Our study highlights the importance of stress-adapted FE as a biological agent for mitigating abiotic stress in crop plants.

Water deficit is one of the major limitations to food production worldwide and most climate change scenarios predict an aggravation of the situation. To face the expected increase in drought stress in the coming years, breeders are working to elucidate the genetic control of barley growth and productivity traits under water deficit. Barley is known as a relatively drought tolerant crop and genetic variability was observed for drought tolerance traits. The objectives of the present study were the quantification of morphological and physiological responses in a collection of 209 spring barley genotypes to drought stress, and the genetic analysis by genome-wide association study to find quantitative trait loci (QTL) and the allele contributions for each of the investigated traits. In six pot experiments, 209 spring barley genotypes were grown under a well-watered and water-limited regime. Stress phases were initiated individually for each genotype at the beginning of tillering and spiking for the vegetative- and the generative stage experiments, respectively, and terminated when the transpiration rates of stress treatments reached 10% of the well-watered control. After the stress phase, a total of 42 productivity related traits such as the dry matter of plant organs, tiller number, leaf length, leaf area, amount of water soluble carbohydrates in the stems, proline content in leaves and osmotic adjustment of corresponding well-watered and stressed plants were analysed, and QTL analyses were performed to find marker-trait associations. Significant water deficit effects were observed for almost all traits and significant genotype x treatment interactions (GxT) were observed for 37 phenotypic traits. Genome-wide association studies (GWAS) revealed 77 significant loci associated with 16 phenotypic traits during the vegetative stage experiment and a total of 85 significant loci associated with 13 phenotypic traits during the generative stage experiment for traits such as leaf area, number of green leaves, grain yield, harvest index and stem length. For traits with significant GxT interactions, genotypic differences for relative values were analysed using one way ANOVA. More than 110 loci for GxT interaction were found for 17 phenotypic traits explaining in many cases more than 50% of the genetic variance.

Basil is one of the most widespread aromatic and medicinal plants, which is often grown in drought- and salinity-prone regions. Often co-occurrence of drought and salinity stresses in agroecosystems and similarities of symptoms which they cause on plants complicates the differentiation among them. Development of automated phenotyping techniques with integrative and simultaneous quantification of multiple morphological and physiological traits enables early detection and quantification of different stresses on a whole plant basis. In this study, we have used different phenotyping techniques including chlorophyll fluorescence imaging, multispectral imaging, and 3D multispectral scanning, aiming to quantify changes in basil phenotypic traits under early and prolonged drought and salinity stress and to determine traits which could differentiate among drought and salinity stressed basil plants. Ocimum basilicum “Genovese” was grown in a growth chamber under well-watered control [45–50% volumetric water content (VWC)], moderate salinity stress (100 mM NaCl), severe salinity stress (200 mM NaCl), moderate drought stress (25–30% VWC), and severe drought stress (15–20% VWC). Phenotypic traits were measured for 3 weeks in 7-day intervals. Automated phenotyping techniques were able to detect basil responses to early and prolonged salinity and drought stress. In addition, several phenotypic traits were able to differentiate among salinity and drought. At early stages, low anthocyanin index (ARI), chlorophyll index (CHI), and hue (HUE2D), and higher reflectance in red (RRed), reflectance in green (RGreen), and leaf inclination (LINC) indicated drought stress. At later stress stages, maximum fluorescence (Fm), HUE2D, normalized difference vegetation index (NDVI), and LINC contribute the most to the differentiation among drought and non-stressed as well as among drought and salinity stressed plants. ARI and electron transport rate (ETR) were best for differentiation of salinity stressed plants from non-stressed plants both at early and prolonged stress.

Members of the aldo–keto reductase family including aldose reductases are involved in antioxidant defense by metabolizing a wide range of lipid peroxidation-derived cytotoxic compounds. Therefore, we produced transgenic wheat genotypes over-expressing the cDNA of alfalfa aldose reductase gene. These plants consequently exhibit 1.5–4.3 times higher detoxification activity for the aldehyde substrate. Permanent drought stress was generated in the greenhouse by growing wheat plants in soil with 20 % water capacity. The control and stressed plants were monitored by a semi automatic phenotyping platform providing computer-controlled watering, digital and thermal imaging. Calculation of biomass values was based on the correlation (R 2 = 0.7556) between fresh weight and green pixel-based shoot surface area. The green biomass production by plants of the three transgenic lines was 12–26–41 % higher than the non-transgenic plants' grown under water limitation. Thermal imaging of stressed non-transgenic plants indicated an elevation in the leaf temperature. The thermal status of transformants was similar at both normal and suboptimal water regime. In drought, the transgenic plants used more water during the growing season. The described phenotyping platform provided a comprehensive data set demonstrating the improved physiological condition of the drought stressed transgenic wheat plants in the vegetative growth phase. In soil with reduced water capacity two transgenic genotypes showed higher seed weight per plant than the control non-transgenic one. Limitation of greenhouse-based phenotyping in analysis of yield potential is discussed.

Flooding stress declines oxygen availability in roots that induce hypoxia stress in plants, thereby severely decline plant growth. Study was undertaken to clarify how potassium supplementation alleviates flooding stress in cotton plants. We applied potassium (K) fertilizer in pot soils at different levels; K 0 = Control (No K), K 1 = 30 mg kg−1, K 2 = 60 mg kg−1 and K 3 = 80 mg kg−1 with different duration of flooding stress s. D 0 = Normal irrigation (0 day flooding), D 1 = three days flooding, D 2 = six days flooding, D 3 = nine days flooding, D4 = twelve days flooding. The K supplementation (80 mg kg−1) in soil with 3-day flooding stress showed better combination for mitigating flooding stress. The supplementation of K3 dose (80 mg kg−1) alleviated flooding against 3-day initial flooding (D1) of which physiological traits showed better response compared to prolonged flooding stress (12-day flooding stress). The result suggests that K-involving flooding stress mitigation strategy in cotton plants was active for initial flooding stress (3-day flooding stress), while it was not fully active for pronged (upto 12-day flooding stress). Furthermore, we found K significantly improved antioxidant enzymes, photosynthetic performance, yield attributes, and lint quality.

Implementation of an algorithm for automated phenotyping through plant 3D-modeling: A practical application on the early detection of water stress

Current limited water availability due to climate changes results in severe drought stress and desiccation in plants. Phenotyping drought tolerance remains challenging. In particular, our knowledge about the discriminating power of traits for capturing a plastic phenotype in high-throughput settings is scant. The study is designed to investigate the differential performance and broad-sense heritability of a battery set of morphological, physiological, and cellular traits to understand the adaptive phenotypic response to drought in spring wheat during the tillering stage. The potential of peroxisome abundance to predict the adaptive response under severe drought was assessed using a high-throughput technique for peroxisome quantification in plants. The research dissected the dynamic changes of some phenological traits during three successive phases of drought using two contrasting genotypes of adaptability to drought. The research demonstrates 5 main findings: (1) a reduction of the overall dimension of the phenological traits for robust phenotyping of the adaptive performance under drought; (2) the abundance of peroxisomes in response to drought correlate negatively with grain yield; (3) the efficiency of ROS homeostasis through peroxisome proliferation which seems to be genetically programmed; and (4) the dynamics of ROS homeostasis seems to be timing dependent mechanism, the tolerant genotype response is earlier than the susceptible genotype. This work will contribute to the identification of robust plastic phenotypic tools and the understanding of the mechanisms for adaptive behavior under drought conditions.Summary statementThis study presents the estimated broad-sense heritability of 24 phenological traits under drought compared with non-stressed conditions. The results demonstrated a reduced model of the overall dimension of the phenological traits for phenotyping drought tolerant response including a novel trait (peroxisome abundance). Also, it displays that the adaptive mechanism through peroxisomes proliferation that is a genetic-dependent manner and related to the stress phase, since tolerant plants can sense the stress and maintain the cellular balance earlier than the sensitive plants.

Salinity has a huge negative impact on plant growth and development by increasing sodium ions accumulation and potassium ions loss that deeply disturbs the plant cell homeostasis and can lead to plant cell death. The imbalance between Na+ and K+ could be solved by applying potassium silicate (K2SiO3). The glycine betaine (GB) is well-known to play a crucial role against oxidative stress in plants by improving the antioxidant machinery. Thus, this research aimed to apply K2SiO3 (1 mM) and GB (10 mM) alone or in combination against 200 mM NaCl-induced damages in Dalbergia odorifera. The results showed a significant amelioration of negative effects of salt stress on the phenotypic traits, chlorophyll content, net photosynthetic rate, stomatal conductance, transpiration rate, and water use efficiency by applied substances. The contents of saccharides and proline were down-regulated by K2SiO3, GB, and K2SiO3-GB, whereas the proteins content was increased by these treatments. The contents of lipid peroxidation, superoxide anion, hydrogen peroxide were reduced by exogenous substances under stress. The activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) and the accumulation of antioxidants (glutathione and ascorbate) were enhanced by exogenous substances. The K2SiO3-GB combination mostly showed better effects on antioxidant machinery compared to a single treatment.

Summary A phenotypic syndrome refers to complex patterns of integration among functionally related traits in an organism that defines how the organism interacts with its environment and sustains itself. Human‐induced biological invasions have become important sources of environmental modifications. However, the extent to which invasive species affect the phenotypic syndromes of individuals in a native is currently unknown. Such knowledge has important implications for understanding ecological interactions and the management of biological invasions. Here, field monitoring in a natural stream were combined with standardized estimates of behavioral, physiological and morphological traits to address the hypothesis that coexistence with a non‐native invader induces a novel environmental pressure that disrupts the adaptive integration among phenotypic traits of the native species. We compared the strength of integration among key phenotypic traits (i.e. aerobic scope, standard metabolic rate, body growth, activity, and body shape) and ecological niche traits (i.e. spring and summer diet, home range size, daily movements) of an allopatric group of native brown trout (Salmo trutta) with a group of brown trout living in sympatry with non‐native brook trout (Salvelinus fontinalis). We found that the integration of phenotypic traits was substantially reduced in the sympatric brown trout and that allopatric and sympatric brown trout differed in key phenotypic and ecological niche traits. Brown trout living in sympatry with non‐native brook trout consumed more terrestrial prey, had smaller home ranges, and a stouter body shape. Sympatric brown trout also had lower specific growth rate, suggesting a lower fitness. The results are generally in line with our hypothesis suggesting that the reduction in fitness observed in sympatric brown trout is caused by the breakdown of their adaptive phenotypic syndrome. This may be caused by differences in the plasticity of the response of phenotypic traits to the novel selection pressure induced by the non‐native species. Our results may help explaining deleterious effects of non‐native species reported in the absence of direct competition with the native species. A lay summary is available for this article.

The volume deals with other aspect (s) of metabolic activities of plants under abiotic stress. This book discusses techniques for improving yield potential and adaptiveness to unfavorable environmental conditions. This book also addresses how knowledge of the changes in physiological mechanisms can contribute in understanding the yield structure under abiotic stress. • Abiotic Stress and Metabolic Responses in Plants • Abiotic Stress and Secondary Metabolites in Plants • Microbes: Support and Protect Plants against Abiotic Stresses • Abiotic Stress and Physiological Traits in Plants • Abiotic Stress and Heat Shock Proteins (HSPs) in Plants

ABSTRACTRelationships between ribotypic and phenotypic traits of protists across life cycle stages remain largely unknown. Herein, we used single cells of two soil and two marine ciliate species to examine phenotypic and ribotypic traits and their relationships across lag, log, plateau, cystic stages and temperatures. We found that Colpoda inflata and Colpoda steinii demonstrated allometric relationships between 18S ribosomal DNA (rDNA) copy number per cell (CNPC), cell volume (CV), and macronuclear volume across all life cycle stages. Integrating previously reported data of Euplotes vannus and Strombidium sulcatum indicated taxon-dependent rDNA CNPC-CV functions. Ciliate and prokaryote data analysis revealed that the rRNA CNPC followed a unified power-law function only if the rRNA-deficient resting cysts were not considered. Hence, a theoretical framework was proposed to estimate the relative quantity of resting cysts in the protistan populations with total cellular rDNA and rRNA copy numbers. Using rDNA CNPC was a better predictor of growth rate at a given temperature than rRNA CNPC and CV, suggesting replication of redundant rDNA operons as a key factor that slows cell division. Single-cell high-throughput sequencing and analysis after correcting sequencing errors revealed multiple rDNA and rRNA variants per cell. Both encystment and temperature affected the number of rDNA and rRNA variants in several cases. The divergence of rDNA and rRNA sequence in a single cell ranged from 1% to 10% depending on species. These findings have important implications for inferring cell-based biological traits (e.g., species richness, abundance and biomass, activity, and community structure) of protists using molecular approaches.IMPORTANCE Based on phenotypic traits, traditional surveys usually characterize organismal richness, abundance, biomass, and growth potential to describe diversity, organization, and function of protistan populations and communities. The rRNA gene (rDNA) and its transcripts have been widely used as molecular markers in ecological studies of protists. Nevertheless, the manner in which these molecules relate to cellular (organismal) and physiological traits remains poorly understood, which could lead to misinterpretations of protistan diversity and ecology. The current research highlights the dynamic nature of cellular rDNA and rRNA contents, which tightly couple with multiple phenotypic traits in ciliated protists. We demonstrate that quantity of resting cysts and maximum growth rate of a population can be theoretically estimated using ribotypic trait-based models. The intraindividual sequence polymorphisms of rDNA and rRNA can be influenced by encystment and temperature, which should be considered when interpreting species-level diversity and community structure of microbial eukaryotes.

Herbaceous species can modify leaf structure during the growing season in response to drought stress and water loss. Evolution can select combinations of traits in plants for efficient water use in restricted environments. We investigated plant traits that mediate adaptation and acclimation to water stress in two herbaceous drought-tolerant species. Anatomical, morphological and physiological traits related to stems and leaves were examined under optimal watering (OW) and a long period of restricted watering (RW) in 11 accessions from three Solanaceae species (Solanum chilense, S.peruvianum and S.lycopersicum). The relationships between these traits were tested using linear regression and PCA. There were significant differences in anatomical traits between the species under both OW and RW, where leaf area correlated with stem diameter. Proline and total carbohydrates accumulated highly in S.chilense and S.peruvianum, respectively, and these osmolytes were strongly correlated with increased osmotic potential. Stomatal density varied between species but not between acclimation treatments, while stomatal rate was significantly higher in wild tomatoes. There was a strong positive relationship between stem growth rate and a group of traits together expressed as total stomatal number. Total stomata is described by integration of leaf area, stomatal density, height and internode length. It is proposed that constitutive adaptations and modifications through acclimation that mediate RW play an important role in tolerance to drought stress in herbaceous plants. The capacity for growth under drought stress was not associated with any single combination of traits in wild tomatoes, since the two species differed in relative levels of expression of various phenotypic traits.