Formate-tetrahydrofolate ligase: supplying the cytosolic one-carbon network in roots with one-carbon units originating from glycolate.

The metabolism of tetrahydrofolate (H4PteGlun)-bound one-carbon (C1) units (C1 metabolism) is multifaceted and required for plant growth, but it is unclear what of many possible synthesis pathways provide C1 units in specific organelles and tissues. One possible source of C1 units is via formate-tetrahydrofolate ligase, which catalyzes the reversible ATP-driven production of 10-formyltetrahydrofolate (10-formyl-H4PteGlun) from formate and tetrahydrofolate (H4PteGlun). Here, we report biochemical and functional characterization of the enzyme from Arabidopsis thaliana (AtFTHFL). We show that the recombinant AtFTHFL has lower Km and kcat values with pentaglutamyl tetrahydrofolate (H4PteGlu5) as compared to monoglutamyl tetrahydrofolate (H4PteGlu1), resulting in virtually identical catalytic efficiencies for the two substrates. Stable transformation of Arabidopsis plants with the EGFP-tagged AtFTHFL, followed with fluorescence microscopy, demonstrated cytosolic signal. Two independent T-DNA insertion lines with impaired AtFTHFL function had shorter roots compared to the wild type plants, demonstrating the importance of this enzyme for root growth. Overexpressing AtFTHFL led to the accumulation of H4PteGlun + 5,10-methylene-H4PteGlun and serine, accompanied with the depletion of formate and glycolate, in roots of the transgenic Arabidopsis plants. This metabolic adjustment supports the hypothesis that AtFTHFL feeds the cytosolic C1 network in roots with C1 units originating from glycolate, and that these units are then used mainly for biosynthesis of serine, and not as much for the biosynthesis of 5-methyl-H4PteGlun, methionine, and S-adenosylmethionine. This finding has implications for any future attempts to engineer one-carbon unit-requiring products through manipulation of the one-carbon metabolic network in non-photosynthetic organs.

Identification of triacylglycerol remodeling mechanism to synthesize unusual fatty acid containing oils.

Typical plant membranes and storage lipids are comprised of five common fatty acids yet over 450 unusual fatty acids accumulate in seed oils of various plant species. Plant oils are important human and animal nutrients, while some unusual fatty acids such as hydroxylated fatty acids (HFA) are used in the chemical industry (lubricants, paints, polymers, cosmetics, etc.). Most unusual fatty acids are extracted from non-agronomic crops leading to high production costs. Attempts to engineer HFA into crops are unsuccessful due to bottlenecks in the overlapping pathways of oil and membrane lipid synthesis where HFA are not compatible. Physaria fendleri naturally overcomes these bottlenecks through a triacylglycerol (TAG) remodeling mechanism where HFA are incorporated into TAG after initial synthesis. TAG remodeling involves a unique TAG lipase and two diacylglycerol acyltransferases (DGAT) that are selective for different stereochemical and acyl-containing species of diacylglycerol within a synthesis, partial degradation, and resynthesis cycle. The TAG lipase interacts with DGAT1, localizes to the endoplasmic reticulum (with the DGATs) and to puncta around the lipid droplet, likely forming a TAG remodeling metabolon near the lipid droplet-ER junction. Each characterized DGAT and TAG lipase can increase HFA accumulation in engineered seed oils.

The microtubule-nucleating factor MACERATOR tethers AUGMIN7 to microtubules and governs phragmoplast architecture.

The plant cytokinetic microtubule array, called the phragmoplast, exhibits higher microtubule dynamics in its center (midzone) than at the periphery (distal zone). This behavior is known as the axial asymmetry. Despite being a major characteristic of the phragmoplast, little is known about regulators of this phenomenon. Here we address the role of microtubule nucleation in axial asymmetry by characterizing MACERATOR (MACET) proteins in Arabidopsis thaliana and Nicotiana benthamiana with a combination of genetic, biochemical, and live-cell imaging assays, using photo-convertible microtubule probes, and modeling. MACET paralogs accumulate at the shrinking microtubule ends and decrease the tubulin OFF rate. Loss of MACET4 and MACET5 function abrogates axial asymmetry by suppressing microtubule dynamicity in the midzone. MACET4 also narrows the microtubule nucleation angle at the phragmoplast leading edge and functions as a microtubule tethering factor for AUGMIN COMPLEX SUBUNIT 7 (AUG7). The macet4 macet5 double mutant shows diminished clustering of AUG7 in the phragmoplast distal zone. Knockout of AUG7 does not affect MACET4 localization, axial asymmetry, or microtubule nucleation angle, but increases phragmoplast length and slows down phragmoplast expansion. The mce4-1 mce5 aug7-1 triple knockout is not viable. Experimental data and modeling demonstrate that microtubule nucleation factors regulate phragmoplast architecture and axial asymmetry directly by generating new microtubules and indirectly by modulating the abundance of free tubulin.

A plant virus protein, NIa-pro, interacts with Indole-3-acetic acid-amido synthetase, whose levels positively correlate with disease severity.

Potato virus Y (PVY) is an economically important plant pathogen that reduces the productivity of several host plants. To develop PVY-resistant cultivars, it is essential to identify the plant-PVY interactome and decipher the biological significance of those molecular interactions. We performed a yeast two-hybrid (Y2H) screen of Nicotiana benthamiana cDNA library using PVY-encoded NIa-pro as the bait. The N. benthamiana Indole-3-acetic acid-amido synthetase (IAAS) was identified as an interactor of NIa-pro protein. The interaction was confirmed via targeted Y2H and bimolecular fluorescence complementation (BiFC) assays. NIa-pro interacts with IAAS protein and consequently increasing the stability of IAAS protein. Also, the subcellular localization of both NIa-pro and IAAS protein in the nucleus and cytosol was demonstrated. By converting free IAA (active form) to conjugated IAA (inactive form), IAAS plays a crucial regulatory role in auxin signaling. Transient silencing of IAAS in N. benthamiana plants reduced the PVY-mediated symptom induction and virus accumulation. Conversely, overexpression of IAAS enhanced symptom induction and virus accumulation in infected plants. In addition, the expression of auxin-responsive genes was found to be downregulated during PVY infection. Our findings demonstrate that PVY NIa-pro protein potentially promotes disease development via modulating auxin homeostasis.

Organellomic gradients in the fourth dimension.

Organelles function as hubs of cellular metabolism and elements of cellular architecture. In addition to 3 spatial dimensions that describe the morphology and localization of each organelle, the time dimension describes complexity of the organelle life cycle, comprising formation, maturation, functioning, decay, and degradation. Thus, structurally identical organelles could be biochemically different. All organelles present in a biological system at a given moment of time constitute the organellome. The homeostasis of the organellome is maintained by complex feedback and feedforward interactions between cellular chemical reactions and by the energy demands. Synchronized changes of organelle structure, activity, and abundance in response to environmental cues generate the fourth dimension of plant polarity. Temporal variability of the organellome highlights the importance of organellomic parameters for understanding plant phenotypic plasticity and environmental resiliency. Organellomics involves experimental approaches for characterizing structural diversity and quantifying the abundance of organelles in individual cells, tissues, or organs. Expanding the arsenal of appropriate organellomics tools and determining parameters of the organellome complexity would complement existing -omics approaches in comprehending the phenomenon of plant polarity. To highlight the importance of the fourth dimension, this review provides examples of organellome plasticity during different developmental or environmental situations.

Quantification of Microtubule-Bundling Activity of MAPs Using TIRF Microscopy.

Cross-linking of microtubules by microtubule-associated proteins (MAPs) results in the formation of microtubule bundles. It has been shown that a majority of microtubules in interphase plant cells are bundled. Bundling can contribute to maintaining structural stability and sustaining spatial organization of microtubule arrays. While bundling can be readily detected by an electron or fluorescent microscope, quantifying this activity remains technically challenging. Here we describe a method for quantifying microtubule-bundling in vitro using green and red stable microtubules. Furthermore, this method distinguishes between different types of microtubule-microtubule interactions: bundling, annealing, and branching. Our technique can be used to compare bundling activity of different MAPs and generate parameters for modeling their contribution to organization and dynamics of microtubule arrays.

Heat and drought induced transcriptomic changes in barley varieties with contrasting stress response phenotypes.

Drought and heat stress substantially impact plant growth and productivity. When subjected to drought or heat stress, plants exhibit reduction in growth resulting in yield losses. The occurrence of these two stresses together intensifies their negative effects. Unraveling the molecular changes in response to combined abiotic stress is essential to breed climate-resilient crops. In this study, transcriptome profiles were compared between stress-tolerant (Otis), and stress-sensitive (Golden Promise) barley genotypes subjected to drought, heat, and combined heat and drought stress for five days during heading stage. The major differences that emerged from the transcriptome analysis were the overall number of differentially expressed genes was relatively higher in Golden Promise (GP) compared to Otis. The differential expression of more than 900 transcription factors in GP and Otis may aid this transcriptional reprogramming in response to abiotic stress. Secondly, combined heat and water deficit stress results in a unique and massive transcriptomic response that cannot be predicted from individual stress responses. Enrichment analyses of gene ontology terms revealed unique and stress type-specific adjustments of gene expression. Weighted Gene Co-expression Network Analysis identified genes associated with RNA metabolism and Hsp70 chaperone components as hub genes that can be useful for engineering tolerance to multiple abiotic stresses. Comparison of the transcriptomes of unstressed Otis and GP plants identified several genes associated with biosynthesis of antioxidants and osmolytes were higher in the former that maybe providing innate tolerance capabilities to effectively combat hostile conditions. Lines with different repertoire of innate tolerance mechanisms can be effectively leveraged in breeding programs for developing climate-resilient barley varieties with superior end-use traits.

Drought Tolerance Strategies and Autophagy in Resilient Wheat Genotypes.

Drought resiliency strategies combine developmental, physiological, cellular, and molecular mechanisms. Here, we compare drought responses in two resilient spring wheat (Triticum aestivum) genotypes: a well-studied drought-resilient Drysdale and a resilient genotype from the US Pacific North-West Hollis. While both genotypes utilize higher water use efficiency through the reduction of stomatal conductance, other mechanisms differ. First, Hollis deploys the drought escape mechanism to a greater extent than Drysdale by accelerating the flowering time and reducing root growth. Second, Drysdale uses physiological mechanisms such as non-photochemical quenching (NPQ) to dissipate the excess of harvested light energy and sustain higher Fv/Fm and ϕPSII, whereas Hollis maintains constant NPQ but lower Fv/Fm and ϕPSII values. Furthermore, more electron donors of the electron transport chain are in the oxidized state in Hollis than in Drysdale. Third, many ROS homeostasis parameters, including peroxisome abundance, transcription of peroxisome biogenesis genes PEX11 and CAT, catalase protein level, and enzymatic activity, are higher in Hollis than in Drysdale. Fourth, transcription of autophagy flux marker ATG8.4 is upregulated to a greater degree in Hollis than in Drysdale under drought, whereas relative ATG8 protein abundance under drought stress is lower in Hollis than in Drysdale. These data demonstrate the activation of autophagy in both genotypes and a greater autophagic flux in Hollis. In conclusion, wheat varieties utilize different drought tolerance mechanisms. Combining these mechanisms within one genotype offers a promising strategy to advance crop resiliency.

Response of Plant-Associated Microbiome to Plant Root Colonization by Exogenous Bacterial Endophyte in Perennial Crops.

The application of bacterial inoculums for improving plant growth and production is an important component of sustainable agriculture. However, the efficiency of perennial crop inoculums depends on the ability of the introduced endophytes to exert an impact on the host-plant over an extended period of time. This impact might be evaluated by the response of plant-associated microbiome to the inoculation. In this study, we monitored the effect of a single bacterial strain inoculation on the diversity, structure, and cooperation in plant-associated microbiome over 1-year period. An endophyte (RF67) isolated from Vaccinium angustifolium (wild blueberry) roots and annotated as Rhizobium was used for the inoculation of 1-year-old Lonicera caerulea (Haskap) plants. A significant level of bacterial community perturbation was detected in plant roots after 3 months post-inoculation. About 23% of root-associated community variation was correlated with an application of the inoculant, which was accompanied by increased cooperation between taxa belonging to Proteobacteria and Actinobacteriota phyla and decreased cooperation between Firmicutes in plant roots. Additionally, a decrease in bacterial Shannon diversity and an increase in the relative abundances of Rhizobiaceae and Enterobacteriaceae were detected in the roots of inoculated plants relative to the non-inoculated control. A strong effect of the inoculation on the bacterial cooperation was also detected after 1 year of plant field growth, whereas no differences in bacterial community composition and also alpha and beta diversities were detected between bacterial communities from inoculated and non-inoculated roots. These findings suggest that while exogenous endophytes might have a short-term effect on the root microbiome structure and composition, they can boost cooperation between plant-growth-promoting endophytes, which can exist for the extended period of time providing the host-plant with long-lasting beneficial effects.

Impact of Wheat Streak Mosaic Virus on Peroxisome Proliferation, Redox Reactions, and Resistance Responses in Wheat.

Although peroxisomes play an essential role in viral pathogenesis, and viruses are known to change peroxisome morphology, the role of genotype in the peroxisomal response to viruses remains poorly understood. Here, we analyzed the impact of wheat streak mosaic virus (WSMV) on the peroxisome proliferation in the context of pathogen response, redox homeostasis, and yield in two wheat cultivars, Patras and Pamir, in the field trials. We observed greater virus content and yield losses in Pamir than in Patras. Leaf chlorophyll and protein content measured at the beginning of flowering were also more sensitive to WSMV infection in Pamir. Patras responded to the WSMV infection by transcriptional up-regulation of the peroxisome fission genes PEROXIN 11C (PEX11C), DYNAMIN RELATED PROTEIN 5B (DRP5B), and FISSION1A (FIS1A), greater peroxisome abundance, and activation of pathogenesis-related proteins chitinase, and ß-1,3-glucanase. Oppositely, in Pamir, WMSV infection suppressed transcription of peroxisome biogenesis genes and activity of chitinase and ß-1,3-glucanase, and did not affect peroxisome abundance. Activity of ROS scavenging enzymes was higher in Patras than in Pamir. Thus, the impact of WMSV on peroxisome proliferation is genotype-specific and peroxisome abundance can be used as a proxy for the magnitude of plant immune response.

Apoptosis is not conserved in plants as revealed by critical examination of a model for plant apoptosis-like cell death.

BACKGROUND: Animals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant "apoptosis-like programmed cell death" (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis, and in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies. RESULTS: Here, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40-55 °C and 85 °C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca2+ and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85 °C, the only striking morphological difference between cell deaths induced by 40-55 °C or 85 °C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents. CONCLUSIONS: We conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways.

Analysis of formin functions during cytokinesis using specific inhibitor SMIFH2.

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

The Arabidopsis R-SNARE VAMP714 is essential for polarisation of PIN proteins and auxin responses.

The plant hormone auxin and its directional intercellular transport play a major role in diverse aspects of plant growth and development. The establishment of auxin gradients requires the asymmetric distribution of members of the auxin efflux carrier PIN-FORMED (PIN) protein family to the plasma membrane. An endocytic pathway regulates the recycling of PIN proteins between the plasma membrane and endosomes, providing a mechanism for dynamic localisation. N-Ethylmaleimide-sensitive factor adaptor protein receptors (SNAP receptors, SNAREs) mediate fusion between vesicles and target membranes and are classed as Q- or R-SNAREs based on their sequence. We analysed gain- and loss-of-function mutants, dominant-negative transgenics and localisation of the Arabidopsis R-SNARE VAMP714 protein to understand its function. We demonstrate that VAMP714 is essential for the insertion of PINs into the plasma membrane, for polar auxin transport, root gravitropism and morphogenesis. VAMP714 gene expression is upregulated by auxin, and the VAMP714 protein co-localises with endoplasmic reticulum and Golgi vesicles and with PIN proteins at the plasma membrane. It is proposed that VAMP714 mediates the delivery of PIN-carrying vesicles to the plasma membrane, and that this forms part of a positive regulatory loop in which auxin activates a VAMP714-dependent PIN/auxin transport system to control development.

A guide to plant TPX2-like and WAVE-DAMPENED2-like proteins.

TPX2 proteins were first identified in vertebrates as a key mitotic spindle assembly factor. Subsequent studies demonstrated that TPX2 is an intricate protein, with functionally and structurally distinct domains and motifs including Aurora kinase-binding, importin-binding, central microtubule-binding, and C-terminal TPX2 conserved domain, among others. The first plant TPX2-like protein, WAVE-DAMPENED2, was identified in Arabidopsis as a dominant mutation responsible for reducing the waviness of roots grown on slanted agar plates. Each plant genome encodes at least one 'canonical' protein with all TPX2 domains and a family of proteins (20 in Arabidopsis) that diversified to contain only some of the domains. Although all plant TPX2-family proteins to date bind microtubules, they function in distinct processes such as cell division, regulation of hypocotyl cell elongation by hormones and light signals, vascular development, or abiotic stress tolerance. Consequently, their expression patterns, regulation, and functions have diverged considerably. Here we summarize the current body of knowledge surrounding plant TPX2-family proteins.

Brachypodium distachyon MAP20 functions in metaxylem pit development and contributes to drought recovery.

Pits are regions in the cell walls of plant tracheary elements that lack secondary walls. Each pit consists of a space within the secondary wall called a pit chamber, and a modified primary wall called the pit membrane. The pit membrane facilitates transport of solutions between vessel cells and restricts embolisms during drought. Here we analyzed the role of an angiosperm-specific TPX2-like microtubule protein MAP20 in pit formation using Brachypodium distachyon as a model system. Live cell imaging was used to analyze the interaction of MAP20 with microtubules and the impact of MAP20 on microtubule dynamics. MAP20-specific antibody was used to study expression and localization of MAP20 in different cell types during vascular bundle development. We used an artificial microRNAs (amiRNA) knockdown approach to determine the function of MAP20. MAP20 is expressed during the late stages of vascular bundle development and localizes around forming pits and under secondary cell wall thickenings in metaxylem cells. MAP20 suppresses microtubule depolymerization; however, unlike the animal TPX2 counterpart, MAP20 does not cooperate with the γ-tubulin ring complex in microtubule nucleation. Knockdown of MAP20 causes bigger pits, thinner pit membranes, perturbed vasculature development, lower reproductive potential and higher drought susceptibility. We conclude that MAP20 may contribute to drought adaptation by modulating pit size and pit membrane thickness in metaxylem.

Identification and characterization of the land-plant-specific microtubule nucleation factor MACET4.

Here, we show that the embryophyte (land-plant)-specific protein MACERATOR4 (MACET4) binds microtubules in vitro and in vivo, promotes microtubule polymerization at sub-critical tubulin concentrations, decreases the lag phase in microtubule bulk polymerization assays, and colocalizes with microtubule nucleation sites. Furthermore, we find that MACET4 forms oligomers that induce aster formation in vitro in a manner that is similar to aster formation mediated by centrosomes and TPX2. MACET4 is expressed during cell division and accumulates at the microtubule nucleation regions of the plant-specific cytokinetic microtubule array, the phragmoplast. We found that MACET4 localizes to the preprophase band and the cortical division zone, but not the spindle. MACET4 appears as cytoplasmic foci in vivo and forms octamers in vitro Transient expression in tobacco leaf pavement cells results in labeling of shrinking plus- and minus-ends. MACET4 facilitates microtubule depolymerization by increasing the frequency of catastrophes in vivo and by suppressing rescues in vitro Microtubules formed in the presence of MACET4 in vitro are shorter, most likely due to the depletion of the free tubulin pool. Accordingly, MACET4 knockdown results in longer phragmoplasts. We conclude that the direct activity of MACET4 is in promoting microtubule nucleation.This article has an associated First Person interview with the first author of the paper.

Impact of heat and drought stress on peroxisome proliferation in quinoa.

Although peroxisomes play a key role in plant metabolism under both normal and stressful growth conditions, the impact of drought and heat stress on the peroxisomes remains unknown. Quinoa represents an informative system for dissecting the impact of abiotic stress on peroxisome proliferation because it is adapted to marginal environments. Here we determined the correlation of peroxisome abundance with physiological responses and yield under heat, drought and heat plus drought stresses in eight genotypes of quinoa. We found that all stresses caused a reduction in stomatal conductance and yield. Furthermore, H2 O2 content increased under drought and heat plus drought. Principal component analysis demonstrated that peroxisome abundance correlated positively with H2 O2 content in leaves and correlated negatively with yield. Pearson correlation coefficient for yield and peroxisome abundance (r = -0.59) was higher than for commonly used photosynthetic efficiency (r = 0.23), but comparable to those for classical stress indicators such as soil moisture content (r = 0.51) or stomatal conductance (r = 0.62). Our work established peroxisome abundance as a cellular sensor for measuring responses to heat and drought stress in the genetically diverse populations. As heat waves threaten agricultural productivity in arid climates, our findings will facilitate identification of genetic markers for improving yield of crops under extreme weather patterns.

Differential Dynamic Changes of Reduced Trait Model for Analyzing the Plastic Response to Drought Phases: A Case Study in Spring Wheat.

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 STATEMENT: This 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.