Insights into genomic variations in rice Hsp100 genes across diverse rice accessions.

MAIN CONCLUSION: The Hsp101 gene is present across all sequenced rice genomes. However, as against Japonica rice, Hsp101 protein of most indica and aus rice contain insertion of glutamic acid at 907th position. The understanding of the heat stress response of rice plants is important for worldwide food security. We examined the presence/absence variations (PAVs) of heat shock proteins (Hsps)/heat shock transcription factor (Hsf) genes in cultivated rice accessions. While 53 Hsps/Hsfs genes showed variable extent of PAVs, 194 genes were the core genes present in all the rice accessions. ClpB1/Hsp101 gene, which is critically important for thermotolerance in plants, showed 100% distribution across the rice types. Within the ClpB1 gene sequence, 40 variation sites consisting of nucleotide polymorphisms (SNPs) and short insertion/deletions (InDels) were discerned. An InDel in ClpB1 leading to an in-frame insertion of 3 nucleotides (TCC) thereby an additional amino acid (glutamic acid) at 907th amino acid position was noted in most of the indica and aus as against japonica rice types. Three rice types namely Moroberekan (japonica), IR64 (indica) and N22 (aus) were further analyzed to address the question of ClpB1 genomic variations and its protein levels with the heat tolerance phenotype. The growth profiling analysis in the post heat stress (HS) period showed that N22 seedlings were most tolerant, IR64 moderately tolerant and Moroberekan highly sensitive. Importantly, the ClpB1 protein sequences of these three rice types showed distinct differences in terms of SNPs. As the ClpB1 protein levels accumulated post HS were generally higher in Moroberekan than N22 seedlings in our study, it is proposed that some additional gene loci in conjunction with ClpB1 regulate the overall rice heat stress response.

Meta-QTL and haplo-pheno analysis reveal superior haplotype combinations associated with low grain chalkiness under high temperature in rice.

Chalk, an undesirable grain quality trait in rice, is primarily formed due to high temperatures during the grain-filling process. Owing to the disordered starch granule structure, air spaces and low amylose content, chalky grains are easily breakable during milling thereby lowering head rice recovery and its market price. Availability of multiple QTLs associated with grain chalkiness and associated attributes, provided us an opportunity to perform a meta-analysis and identify candidate genes and their alleles contributing to enhanced grain quality. From the 403 previously reported QTLs, 64 Meta-QTLs encompassing 5262 non-redundant genes were identified. MQTL analysis reduced the genetic and physical intervals and nearly 73% meta-QTLs were narrower than 5cM and 2Mb, revealing the hotspot genomic regions. By investigating expression profiles of 5262 genes in previously published datasets, 49 candidate genes were shortlisted on the basis of their differential regulation in at least two of the datasets. We identified non-synonymous allelic variations and haplotypes in 39 candidate genes across the 3K rice genome panel. Further, we phenotyped a subset panel of 60 rice accessions by exposing them to high temperature stress under natural field conditions over two Rabi cropping seasons. Haplo-pheno analysis uncovered haplotype combinations of two starch synthesis genes, GBSSI and SSIIa, significantly contributing towards the formation of grain chalk in rice. We, therefore, report not only markers and pre-breeding material, but also propose superior haplotype combinations which can be introduced using either marker-assisted breeding or CRISPR-Cas based prime editing to generate elite rice varieties with low grain chalkiness and high HRY traits.

Arabidopsis plants overexpressing additional copies of heat shock protein Hsp101 showed high heat tolerance and endo-gene silencing.

Hsp101 chaperone is vital for survival of plants under heat stress. We generated transgenic Arabidopsis thaliana (Arabidopsis) lines with extra copies of Hsp101 gene using diverse approaches. Arabidopsis plants transformed with rice Hsp101 cDNA driven by Arabidopsis Hsp101 promoter (IN lines) showed high heat tolerance while the plants transformed with rice Hsp101 cDNA driven by CaMV35S promoter (C lines) were like wild type plants in heat stress response. Transformation of Col-0 plants with 4633 bp Hsp101 genomic fragment (GF lines) from A. thaliana containing both its coding and the regulatory sequence resulted in mostly over-expressor (OX) lines and a few under-expressor (UX) lines of Hsp101. OX lines showed enhanced heat tolerance while the UX lines were overly heat sensitive. In UX lines, silencing of not only Hsp101 endo-gene was noted but also transcript of choline kinase (CK2) was silenced. Previous work established that in Arabidopsis, CK2 and Hsp101 are convergent gene pairs sharing a bidirectional promoter. The elevated AtHsp101 protein amount in most GF and IN lines was accompanied by lowered CK2 transcript levels under HS. We observed increased methylation of the promoter and gene sequence region in UX lines; however, methylation was lacking in OX lines.

Heat-induced proteomic changes in anthers of contrasting rice genotypes under variable stress regimes.

Heat stress drastically affects anther tissues resulting in poor plant fertility, necessitating an urgent need to determine the key proteome regulation associated with mature anther in response to heat stress. We identified several genotype - specific protein alterations in rice anthers of Moroberekan (Japonica, heat sensitive), IR64 (Indica, moderately heat tolerant), and Nagina22 (Aus, heat tolerant) in the short-term (ST_HS; one cycle of 42°C, 4 hours before anthesis) and long-term (LT_HS; 6 cycles of 38°C, 6 hours before anthesis) heat stress. The proteins upregulated in long-term heat stress in Nagina22 were enriched in biological processes related to unfolded protein binding and carboxylic acid metabolism, including amino acid metabolism. In short-term heat stress, Nagina22 anthers were enriched in proteins associated with vitamin E biosynthesis and GTPase activator activity. In contrast, downregulated proteins were related to ribosomal proteins. The expression of different Hsp20 and DnaJ was genotype specific. Overall, the heat response in Nagina22 was associated with its capacity for adequate metabolic control and cellular homeostasis, which may be critical for its higher reproductive thermotolerance. This study improves our understanding of thermotolerance mechanisms in rice anthers during anthesis and lays a foundation for breeding thermotolerant varieties via molecular breeding.

Hsp70, sHsps and ubiquitin proteins modulate HsfA6a-mediated Hsp101 transcript expression in rice (Oryza sativa L.).

Hsp100 chaperones disaggregate the aggregated proteins and are vital for maintenance of protein homeostasis. The level of Hsp100 synthesised in the cells has a bearing on the survival of plants under heat stress. The Hsp100 transcription machinery is activated within minutes of the onset of heat stress. The heat shock factor HsfA6a plays a major role in the transcriptional regulation of the Hsp101 gene in rice plants. Through yeast-2-hybrid library screening, we identified small heat shock proteins (sHSPs), Hsp70 and ubiquitin as HsfA6a interacting proteins (HIPs). The bimolecular fluorescence complementation assays showed the physical interaction of HsfA6a with Hsp16.9A-CI and Hsp18.0-CII in the cytosolic region and with cHsp70-1 in the nucleus. With the Hsp101 promoter: reporter gene assays, using yeast cells and rice protoplasts, we show that CI-sHsps and CII-sHsps are negative regulators and Hsp70 positive regulator of the HsfA6a activity in modulation of Hsp101 transcription. We also noted that the HsfA6a interactors, Hsp70 and CI-sHsps and CII-sHsps, physically interact with each other. We noted that HsfA6a binds with the CI-sHsp and Hsp70 promoters, implying that HsfA6a has a role in transcriptional regulation of its interacting proteins. Furthermore, we noted that the mutation of the ubiquitin/sumoylation acceptor site lysine 10 to alanine (K10A) of HsfA6a enhanced its DNA binding potential on the Hsp101 promoter, implying that these modifiers are possibly involved in modulation of HsfA6a activity. Our work shows that Hsp70, CI-sHsps and CII-sHsp, and ubiquitin proteins coordinate with HsfA6a in mediating the Hsp101 transcription process in rice.

Tango between Ethylene and HSFA2 Settles Heat Tolerance.

The phytohormone ethylene has roles in senescence, fruit ripening, and biotic and abiotic stress responses. However, the detailed mechanism(s) by which ethylene affects the plant heat stress response (HSR) is not well understood. Two recent studies by Huang et al. and Shekhawat et al. now reveal that ethylene signaling converges on HSFA2 to bring about heat stress (HS) tolerance in plants.

AtHsc70-1 negatively regulates the basal heat tolerance in Arabidopsis thaliana through affecting the activity of HsfAs and Hsp101.

Heat shock protein 70 (Hsp70) chaperones are highly conserved and essential proteins with diverse cellular functions, including plant abiotic stress tolerance. Hsp70 proteins have been linked with basal heat tolerance in plants. Hsp101 likewise is an important chaperone protein that plays a critical role in heat tolerance in plants. We observed that Arabidopsis hsc70-1 mutant seedlings show elevated basal heat tolerance compared with wild-type. Over-expression of Hsc70-1 resulted in increased heat sensitivity. Hsp101 transcript and protein levels were increased during non-heat stress (HS) and post-HS conditions in hsc70-1 mutant seedlings. In contrast, Hsp101 was repressed in Hsc70-1 over-expressing plants after post-HS conditions. Hsc70-1 showed physical interaction with HsfA1d and HsfA1e protein in the cytosol under non-HS conditions. In transient reporter gene analysis, HsfA1d, HsfA1e and HsfA2 showed transcriptional response on the Hsp101 promoter. HsfA1d and HsfA2 transcripts were at higher levels in hsc70-1 mutant compared with wild-type. We provide genetic evidence that Hsc70-1 is a negative regulator affecting HsfA1d/A1e/A2 activators, which in turn regulate Hsp101 expression and basal thermotolerance.

Heat-Induced Oxidation of the Nuclei and Cytosol.

The concept that heat stress (HS) causes a large accumulation of reactive oxygen species (ROS) is widely accepted. However, the intracellular compartmentation of ROS accumulation has been poorly characterized. We therefore used redox-sensitive green fluorescent protein (roGFP2) to provide compartment-specific information on heat-induced redox changes of the nuclei and cytosol of Arabidopsis leaf epidermal and stomatal guard cells. We show that HS causes a large increase in the degree of oxidation of both compartments, causing large shifts in the glutathione redox potentials of the cells. Heat-induced increases in the levels of the marker transcripts, heat shock protein (HSP)101, and ascorbate peroxidase (APX)2 were maximal after 15 min of the onset of the heat treatment. RNAseq analysis of the transcript profiles of the control and heat-treated seedlings revealed large changes in transcripts encoding HSPs, mitochondrial proteins, transcription factors, and other nuclear localized components. We conclude that HS causes extensive oxidation of the nucleus as well as the cytosol. We propose that the heat-induced changes in the nuclear redox state are central to both genetic and epigenetic control of plant responses to HS.

Silencing of class I small heat shock proteins affects seed-related attributes and thermotolerance in rice seedlings.

MAIN CONCLUSION: Silencing of CI-sHsps by RNAi negatively affected the seed germination process and heat stress response of rice seedlings. Seed size of RNAiCI-sHsp was reduced as compared to wild-type plants. Small heat shock proteins (sHsps) are the ATP-independent chaperones ubiquitously expressed in response to diverse environmental and developmental cues. Cytosolic sHsps constitute the major repertoire of sHsp family. Rice cytosolic class I (CI)-sHsps consists of seven members (Hsp16.9A, Hsp16.9B, Hsp16.9C, Hsp17.4, Hsp17.7, Hsp17.9A and Hsp18). Purified OsHsp17.4 and OsHsp17.9A proteins exhibited chaperone activity by preventing formation of large aggregates with model substrate citrate synthase. OsHsp16.9A and OsHsp17.4 showed nucleo-cytoplasmic localization, while the localization of OsHsp17.9A was preferentially in the nucleus. Transgenic tobacco plants expressing OsHsp17.4 and OsHsp17.9A proteins and Arabidopsis plants ectopically expressing OsHsp17.4 protein showed improved thermotolerance to the respective trans-hosts during the post-stress recovery process. Single hairpin construct was designed to generate all CI-sHsp silenced (RNAiCI-sHsp) rice lines. The major vegetative and reproductive attributes of the RNAiCI-sHsp plants were comparable to the wild-type rice plants. Basal and acquired thermotolerance response of RNAiCI-sHsp seedlings of rice was mildly affected. The seed length of RNAiCI-sHsp rice plants was significantly reduced. The seed germination process was delayed and seed thermotolerance of RNAiCI-sHsp was negatively affected than the non-transgenic seeds. We, thus, implicate that sHsp genes are critical in seedling thermotolerance and seed physiology.

Cpn60ß4 protein regulates growth and developmental cycling and has bearing on flowering time in Arabidopsis thaliana plants.

Chloroplastic Cpn60 proteins are type I chaperonins comprising of Cpn60α and Cpn60ß subunits. Arabidopsis genome contains six entries in Cpn60 family, out of which two are for Cpn60α subunit and four for Cpn60ß subunit. We noted that the cpn60ß4 knockout mutant plants (T-DNA insertion salk_064887 line) differed from the wild type Col-0 plants in the developmental programming. cpn60ß4 mutant plants showed early seed germination. Radical emergence, hypocotyl emergence and cotyledons opening were faster in cpn60ß4 mutant plants than WT. Importantly, cpn60ß4 mutant plants showed early-flowering phenotype. The number of flowers and siliques as well as weight of the seeds were higher in cpn60ß4 mutant plants as compared to Col-0 plants. These effects were reverted to wild type like growth and developmental patterns when genomic fragment of Arabidopsis encompassing Cpn60ß4 gene was complemented in the mutant background. The overexpression of Cpn60ß4 gene using CaMV35 promoter in wild type background (OE-Cpn60ß4) delayed the floral transition as against wild type plants. The plastid division were affected in cpn60ß4 mutant plants compared to Col-0. The results of this study suggest that Cpn60ß4 plays important role(s) in chloroplast development and is a key factor in plant growth, development and flowering in Arabidopsis.

In vivo promoter engineering in plants: Are we ready?

Engineering plant promoter sequence for optimal expression of a gene has been a long standing goal for plant scientists. In recent times, Sequence Specific Nucleases (SSNs) like CRISPR/Cas9 are enabling researchers to achieve this goal, in vivo in the genome. It is well known that SSNs have met with unprecedented success in rapid transgene free crop improvement largely by targeting the coding sequence. Here, we discuss the strategies being employed by plant scientists in targeting SSNs to non-coding promoter regions/Cis Regulatory Elements (CRE). We collectively refer all such endeavors as in vivo promoter engineering (IPE). We further classify the IPE efforts into CRE addition, CRE deletion/disruption, promoter swap/insertion and targeted promoter polymorphism. Till date, IPE has proven useful in altering plant architecture in tomato, developing resistance against Xanthomonas sp in rice and citrus, and engineering drought tolerance in maize. However it is quite challenging to achieve predictable changes in gene expression using IPE at this point. In future years, data generated from high throughput techniques to investigate non coding genome may immensely augment the efforts in this direction. As IPE does not involve addition of the transgene for modifying crop traits, it will be relatively more conducive to public acceptance in crop improvement programs.

Mapping of domains of heat stress transcription factor OsHsfA6a responsible for its transactivation activity.

Elevated temperatures affect the growth and reproduction of crop plants and thus have become concern worldwide. Hsp101/ClpB protein is a major molecular chaperone, performing dis-aggregation of protein aggregates formed during heat stress. In rice, OsHsfA6a binds to the promoter of OsHsp101/ClpB-C and regulates its expression. In this study, analysis of C-terminal domains of ClassA OsHsfs revealed the presence of aromatic, hydrophobic, acidic (AHA) and nuclear export signal (NES) motifs in all the members. Using deletion constructs, we show that the activation potential of OsHsfA6a is confined in the C-terminal activation domain comprising of AHA and NES sequences. The results obtained in yeast were complemented with transient expression of reporter in protoplast (TERP) based assay. Detailed analysis of OsHsfA6a splice variants shows the presence of one full version and a DBD truncated smaller version whose existence needs experimental evidences. Phylogeny analysis revealed that OsHsfA6a has diverged from A6a/A6b forms of Arabidopsis and tomato and has no expressologs. OsHsfA6a in-silico network was enriched in MAP kinases along with Hsp70 and Hsp90 proteins. Thus, it appears that regulation of OsClpB-C by HsfA6a is unique in rice and activation potential of OsHsfA6a resides in the single AHA motif located in the C-terminal domain.

Analysis of transactivation potential of rice (Oryza sativa L.) heat shock factors.

MAIN CONCLUSION: Based on yeast one-hybrid assays, we show that the presence of C-terminal AHA motifs is not a prerequisite for transactivation potential in rice heat shock factors. Transcriptional activation or transactivation (TA) of heat stress responsive genes takes place by binding of heat shock factors (Hsfs) to heat shock elements. Analysis of TA potential of thirteen rice (Oryza sativa L.) Hsfs (OsHsfs) carried out in this study by yeast one-hybrid assay showed that OsHsfsA3 possesses strong TA potential while OsHsfs A1a, A2a, A2b, A4a, A4d, A5, A7b, B1, B2a, B2b, B2c and B4d lack TA potential. From a near complete picture of TA potential of the OsHsf family (comprising of 25 members) emerging from this study and an earlier report from our group (Mittal et al. in FEBS J 278(17):3076-3085, 2011), it is concluded that (1) overall, six OsHsfs, namely A3, A6a, A6b, A8, C1a and C1b possess TA potential; (2) four class A OsHsfs, namely A3, A6a, A6b and A8 have TA potential out of which A6a and A6b contain AHA motifs while A3 and A8 lack AHA motifs; (3) nine class A OsHsfs, namely A1a, A2a, A2b, A2e, A4a, A4d, A5, A7a and A7b containing AHA motif(s) lack TA function in the yeast assay system; (4) all class B OsHsfs lack AHA motifs and TA potential (B4a not analyzed) and (5) though all class C OsHsf members lack AHA motifs, two members C1a and C1b possess TA function, while one member C2a lacks TA potential (C2b not analyzed). Thus, the presence or absence of AHA motif is possibly not the only factor determining TA potential of OsHsfs. Our findings will help to identify the transcriptional activators of rice heat shock response.

Constitutive over-expression of rice ClpD1 protein enhances tolerance to salt and desiccation stresses in transgenic Arabidopsis plants.

Caseinolytic proteases (Clps) perform the important role of removing protein aggregates from cells, which can otherwise prove to be highly toxic. ClpD system is a two-component protease complex composed of a regulatory ATPase module ClpD and a proteolytic component ClpP. Under desiccation stress condition, rice ClpD1 (OsClpD1) gene encoding for the regulatory subunit, was represented by four variant transcripts differing mainly in the expanse of their N-terminal amino acids. These transcripts were expressed in a differential manner in response to salt, mannitol and polyethylene glycol stresses in rice. Purified OsClpD1.3 protein exhibited intrinsic chaperone activity, shown using citrate synthase as substrate. Arabidopsis (Col-0) plants over-expressing OsClpD1.3 open reading frame downstream to CaMV35S promoter (ClpD1.3 plants) showed higher tolerance to salt and desiccation stresses as compared to wild type plants. ClpD1.3 seedlings also showed enhanced growth during the early stages of seed germination under unstressed, control conditions. The free proline levels and starch breakdown activities were higher in the ClpD1.3 seedlings as compared to the wild type Arabidopsis seedlings. It thus emerges that increasing the potential of ClpD1 chaperoning activity may be of advantage in protection against abiotic stresses.

Expression analysis of ClpB/Hsp100 gene in faba bean (Vicia faba L.) plants in response to heat stress.

Heat stress adversely affects the growth and yield of faba bean crop. Accumulation of ClpB/Hsp100 class of proteins is a critical parameter in induction of acquired heat stress tolerance in plants. Heat-induced expression of ClpB/Hsp100 genes has been noted in diverse plant species. Using primers complementary to soybean ClpB/Hsp100 gene, we analyzed the transcript expression profile of faba bean ClpB/Hsp100 gene in leaves of seedlings and flowering plants and in pollen grains. ClpB/Hsp100 protein accumulation profile was analyzed in leaves of faba bean seedlings using Arabidopsis thaliana cytoplasmic Hsp101 antibodies. The transcript and protein levels of faba bean ClpB/Hsp100 were significantly induced in response to heat stress.

Characterization of 5'UTR of rice ClpB-C/Hsp100 gene: evidence of its involvement in post-transcriptional regulation.

Rice (Oryza sativa) ClpB-C (OsClpB-C) protein is expressed upon heat stress in vegetative tissues and constitutively in seeds. We produced stably transformed Arabidopsis plants carrying ß-glucuronidase (Gus) reporter gene downstream to 1-kb OsClpB-C promoter (1kbPro plants). In the 1kbPro plants, expression of Gus transcript and protein followed the expression pattern of OsClpB-C gene in rice plants, i.e., heat induced in vegetative tissues and constitutive in seeds. Next, we produced transgenic Arabidopsis plants containing Gus downstream to 862-bp fragment of OsClpB-C promoter [lacking 138 nucleotides from 3' end of the 5'untranslated region (5'UTR); ∆UTR plants). In ∆UTR plants, Gus transcript was expressed in heat-inducible manner, but strikingly, Gus protein levels were negligible after heat treatment. However, Gus protein was expressed in ∆UTR seedlings at levels comparable to 1kbPro seedlings when recovery treatment of 22 °C/2 h was given post heat stress (38 °C/15 min). This suggests that 5'UTR of OsClpB-C gene is involved in its post-transcriptional regulation and is an obligate requirement for protein expression during persistent heat stress. Furthermore, the Gus transcript levels were higher in the polysomal RNA fraction in heat-stressed seedlings of 1kbPro plants as compared to ∆UTR plants, indicating that 5'UTR aids in assembly of ribosomes onto the Gus transcript during heat stress. Unlike the case of seedlings, Gus protein was formed constitutively in ∆UTR seeds at levels comparable to 1kbPro seeds. Hence, the function of 5'UTR of OsClpB-C is dispensable for expression in seeds.

ClpB/Hsp100 proteins and heat stress tolerance in plants.

High-temperature stress can disrupt cellular proteostasis, resulting in the accumulation of insoluble protein aggregates. For survival under stressful conditions, it is important for cells to maintain a pool of native soluble proteins by preventing and/or dissociating these aggregates. Chaperones such as GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE (Hsp70/Hsp40/nucleotide exchange factor) help cells minimize protein aggregation. Protein disaggregation is accomplished by chaperones belonging to the Caseinolytic Protease (Clp) family of proteins. ClpB/Hsp100 proteins are strikingly ubiquitous and are found in bacteria, yeast and multi-cellular plants. The expression of these proteins is regulated by heat stress (HS) and developmental cues. Bacteria and yeast contain one and two forms of ClpB proteins, respectively. Plants possess multiple forms of these proteins that are localized to different cellular compartments (i.e. cytoplasm/nucleus, chloroplast or mitochondria). Overwhelming evidence suggests that ClpB/Hsp100 proteins play decisive roles in cell adaptation to HS. Mutant bacteria and yeast cells lacking active ClpB/Hsp100 proteins are critically sensitive to high-temperature stress. Likewise, Arabidopsis, maize and rice mutants lacking cytoplasmic ClpB proteins are very sensitive to heat. In this study, we present the structural and functional attributes of plant ClpB forms.

Morphological and physiological characterization of different genotypes of faba bean under heat stress.

Heat stress (HS) is the major constraint to crop productivity worldwide. The objective of the present experiment was to select the tolerant and sensitive genotype(s) on the basis of morpho-physiological and biochemical characteristics of ten Vicia faba genotypes. These genotypes were as follows: Zafar 1, Zafar 2, Shebam 1, Makamora, Espan, Giza Blanka, Giza 3, C4, C5 and G853. The experimental work was undertaken to study the effects of different levels of temperature (control, mild, and modest) on plant height (PH) plant(-1), fresh weight (FW) and dry weight (DW) plant(-1), area leaf(-1), content of leaf relative water (RWC), proline content (Pro) and total chlorophyll (Total Chl), electrolyte leakage (EL), malondialdehyde level (MDA), hydrogen peroxide (H2O2), and activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) enzymes. HS significantly affected growth performance of all genotypes. However, the magnitude of reduction in genotypes 'C5' was relatively low, possibly due to its better antioxidant activities (CAT, POD and SOD), and accumulation of Pro and Total Chl, and leaf RWC. In the study, 'C5' was noted to be the most HS tolerant and 'Espan' most HS sensitive genotypes. It was concluded that the heat-tolerant genotypes may have better osmotic adjustment and protection from free radicals by increasing the accumulation of Pro content with increased activities of antioxidant enzyme.

Response of different genotypes of faba bean plant to drought stress.

Drought stress is one of the major abiotic stresses that are a threat to crop production worldwide. Drought stress impairs the plants growth and yield. Therefore, the aim of the present experiment was to select the tolerant genotype/s on the basis of moprpho-physiological and biochemical characteristics of 10 Vicia faba genotypes (Zafar 1, Zafar 2, Shebam, Makamora, Espan, Giza Blanka, Giza 3, C4, C5 and G853) under drought stress. We studied the effect of different levels of drought stress i.e., (i) normal irrigation (ii) mild stress (iii) moderate stress, and (iv) severe stress on plant height (PH) plant-1, fresh weight (FW) and dry weight (DW) plant-1, area leaf-1, leaf relative water content (RWC), proline (Pro) content, total chlorophyll (Total Chl) content, electrolyte leakage (EL), malondialdehyde (MDA), hydrogen peroxide (H2O2) content, and activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) of genotypes of faba bean. Drought stress reduced all growth parameters and Total Chl content of all genotypes. However, the deteriorating effect of drought stress on the growth performance of genotypes "C5" and "Zafar 1" were relatively low due to its better antioxidant enzymes activities (CAT, POD and SOD), and accumulation of Pro and Total Chl, and leaf RWC. In the study, genotype "C5" and "Zafar 1" were found to be relatively tolerant to drought stress and genotypes "G853" and "C4" were sensitive to drought stress.