Characterization of Extracellular Vesicles from Entamoeba histolytica Identifies Roles in Intercellular Communication That Regulates Parasite Growth and Development.

Extracellular vesicles (EVs) secreted by eukaryotic and prokaryotic cells to transport lipids, proteins, and nucleic acids to the external environment have important roles in cell-cell communication through cargo transfer. We identified and characterized EVs from Entamoeba histolytica, a protozoan parasite and a human pathogen. Conditioned medium from amebic parasites contained particles consistent with the expected size and morphology of EVs. Mass spectrometry was used to characterize the EV proteome and showed that it was enriched in common exosome marker proteins, including proteins associated with vesicle formation, cell signaling, and metabolism, as well as cytoskeletal proteins. Additionally, the EVs were found to selectively package small RNAs (sRNA), which were protected within the vesicles against RNase treatment. Sequencing analysis of the sRNA contained in EVs revealed that the majority were 27 nucleotides (nt) in size and represented a subset of the cellular antisense small RNA population that has previously been characterized in Entamoeba RNA interference (RNAi) pathway proteins, including Argonaute, were also present in amebic EVs. Interestingly, we found that the amebic EVs impacted intercellular communication between parasites and altered encystation efficiency. EVs isolated from encysting parasites promoted encystation in other parasites, whereas EVs from metabolically active trophozoites impeded encystation. Overall, the data reveal that Entamoeba secrete EVs that are similar in size and shape to previously characterized exosomes from other organisms and that these EVs contain a defined protein and small RNA cargo and have roles in intercellular communication among parasites and influence growth kinetics.

Microplastics in the soil-water-food nexus: Inclusive insight into global research findings.

Nuisance imposed by biotic and abiotic stressors on diverse agroecosystems remains an area of focus for the scientific fraternity. However, emerging contaminants such as microplastics (MP) have imposed additional dimension (alone or in combinations with other stressors) in agroecosystems and keep escalating the challenges to achieve sustainability. MP are recognized as persistent anthropogenic contaminants, fetch global attention due to their unique chemical features that keeps themselves unresponsive to the decaying process. This review has been theorized to assess the current research trends (along with possible gap areas), widespread use of MP, enhancement of the harshness of heavy metals (HMs), complex interactions with physico-chemical constituents of arable soil, accumulation in the edible parts of field crops, dairy products, and other sources to penetrate the food web. So far, the available review articles are oriented to a certain aspect of MP and lack a totality when considered from in soil-water-food perspective. In short, a comprehensive perspective of the adverse effects of MP on human health has been assessed. Moreover, an agro-techno-socio-health prospective-oriented critical assessment of policies and remedial measures linked with MP has provided an extra edge over other similar articles in influential future courses of research.

Giant cells: multiple cells unite to survive.

Multinucleated Giant Cells (MGCs) are specialized cells that develop from the fusion of multiple cells, and their presence is commonly observed in human cells during various infections. However, MGC formation is not restricted to infections alone but can also occur through different mechanisms, such as endoreplication and abortive cell cycle. These processes lead to the formation of polyploid cells, eventually resulting in the formation of MGCs. In Entamoeba, a protozoan parasite that causes amoebic dysentery and liver abscesses in humans, the formation of MGCs is a unique phenomenon and not been reported in any other protozoa. This organism is exposed to various hostile environmental conditions, including changes in temperature, pH, and nutrient availability, which can lead to stress and damage to its cells. The formation of MGCs in Entamoeba is thought to be a survival strategy to cope with these adverse conditions. This organism forms MGCs through cell aggregation and fusion in response to osmotic and heat stress. The MGCs in Entamoeba are thought to have increased resistance to various stresses and can survive longer than normal cells under adverse conditions. This increased survival could be due to the presence of multiple nuclei, which could provide redundancy in case of DNA damage or mutations. Additionally, MGCs may play a role in the virulence of Entamoeba as they are found in the inflammatory foci of amoebic liver abscesses and other infections caused by Entamoeba. The presence of MGCs in these infections suggests that they may contribute to the pathogenesis of the disease. Overall, this article offers valuable insights into the intriguing phenomenon of MGC formation in Entamoeba. By unraveling the mechanisms behind this process and examining its implications, researchers can gain a deeper understanding of the complex biology of Entamoeba and potentially identify new targets for therapeutic interventions. The study of MGCs in Entamoeba serves as a gateway to exploring the broader field of cell fusion in various organisms, providing a foundation for future investigations into related cellular processes and their significance in health and disease.

Efficient Synergistic Antibacterial Activity of α-MSH Using Chitosan-Based Versatile Nanoconjugates.

The application of antimicrobial peptides has emerged as an alternative therapeutic tool to encounter against multidrug resistance of different pathogenic organisms. α-Melanocyte stimulating hormone (α-MSH), an endogenous neuropeptide, is found to be efficient in eradicating infection of various kinds of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA). However, the chemical stability and efficient delivery of these biopharmaceuticals (i.e., α-MSH) to bacterial cells with a significant antibacterial effect remains a key challenge. To address this issue, we have developed a chitosan-cholesterol polymer using a single-step, one-pot, and simple chemical conjugation technique, where α-MSH is loaded with a significantly high amount (37.7%), and the final product is obtained as chitosan-cholesterol α-MSH polymer-drug nanoconjugates. A staphylococcal growth inhibition experiment was performed using chitosan-cholesterol α-MSH and individual controls. α-MSH and chitosan-cholesterol both show bacterial growth inhibition by a magnitude of 50 and 79%, respectively. The killing efficiency of polymer-drug nanoconjugates was very drastic, and almost no bacterial colony was observed (∼100% inhibition) after overnight incubation. Phenotypic alternation was observed in the presence of α-MSH causing changes in the cell structure and shape, indicating stress on Staphylococcus aureus. As a further consequence, vigorous cell lysis with concomitant release of the cellular material in the nearby medium was observed after treatment of chitosan-cholesterol α-MSH nanoconjugates. This vigorous lysis of the cell structure is associated with extensive aggregation of the bacterial cells evident in scanning electron microscopy (SEM). The dose-response experiment was performed with various concentrations of chitosan-cholesterol α-MSH nanoconjugates to decipher the degree of the bactericidal effect. The concentration of α-MSH as low as 1 pM also shows significant inhibition of bacterial growth (∼40% growth inhibition) of Staphylococcus aureus. Despite playing an important role in inhibiting bacterial growth, our investigation on hemolytic assay shows that chitosan-cholesterol α-MSH is significantly nontoxic at a wide range of concentrations. In a nutshell, our analysis demonstrated novel antimicrobial activity of nanoparticle-conjugated α-MSH, which could be used as future therapeutics against multidrug-resistant Staphylococcus aureus and other types of bacterial cells.

Development of a CRISPR/Cas9 system in Entamoeba histolytica: proof of concept.

The protozoan parasite Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death on a global scale. The lack of molecular tools for genome editing hinders the study of important biological functions of this parasite. Due to its versatility, the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system has been successfully used to induce site-specific genomic alterations, including in protozoan parasites. In this study, we optimised CRISPR-Cas9 for use as a genetic tool in E. histolytica. We chose a single plasmid approach containing both guide RNA (gRNA) and Cas9 nuclease expression cassettes. The amebic U6 promoter was used to drive the expression of the gRNA and its expression was confirmed by Northern blot analysis. Stable transfectant cell lines were obtained using a destabilising domain of dihydrofolate reductase fused to myc-tagged Cas9 (ddCas9). With this system, we were able to induce ddCas9 expression 16 h following treatment with the small molecule ligand trimethoprim (TMP). Stable cell lines expressing ddCas9 and Luc-gRNA or non-specific (NS)-gRNA were transiently transfected with a plasmid containing a mutated luciferase gene (pDeadLuc) targeted by Luc-gRNA and another plasmid with a truncated luciferase gene (pDonorLuc) to restore luciferase expression and consequent activity. We observed that luminescence signal increased for the cell line expressing Luc-gRNA, suggesting that homologous recombination was facilitated by Cas9 activity. This evidence is supported by the presence of chimeric DNA detected by PCR and confirmed by sequencing of the resulting repaired DNA obtained by homologous recombination. We believe this represents the first report of a CRISPR/Cas9 system use in Entamoeba and provides evidence that this genome editing approach can be useful for genetic studies in this early branching eukaryote.

A novel galacto-glycerolipid from Oxalis corniculata kills Entamoeba histolytica and Giardia lamblia.

Oxalis corniculata is a naturally occurring weed that has been used in traditional medicine for the cure of dysentery and diarrhea in India. One of the common causes of dysentery is due to infection by the protist pathogen Entamoeba histolytica. Bioactivity profiling of extracts from O. corniculata identified several compounds that showed antiamoebic activity in axenic cultures of E. histolytica. These were characterized by nuclear magnetic resonance, infrared, and mass spectrometry as (i) Oc-1, a mixture of saturated fatty acids C24 to C28; (ii) Oc-2, a mixture of long-chain alcohols C18 to C28; and (iii) Oc-3, a single compound that was a galacto-glycerolipid (GGL). Of the different compounds that were obtained, the strongest antiamoebic activity was found in GGL. The addition of GGL to E. histolytica xenic cultures containing other microbial flora from the large intestine did not affect its antiamoebic activity. Amoebicidal concentrations of GGL had no effect on intestinal microbial flora or on the mammalian cell line HEK-293. GGL was also found to be equally effective in killing another protist pathogen, Giardia lamblia, that causes diarrhea in humans. The importance of this study is based on the identification of novel natural products and the possibility of developing these compounds as active agents to treat at least two pathogenic parasitic intestinal infections endemic to tropical regions.

Entamoeba stage conversion: progress and new insights.

Entamoeba histolytica, an anaerobic protozoan, is an important global health problem. This parasite has a biphasic life cycle consisting of a dormant cyst stage which is environmentally resistant and transmits the infection, and the proliferative trophozoite stage which is motile and causes invasive disease. The stage conversion process remains poorly understood despite being central to amoebic biology. In this review, we will highlight recent progress in our understanding of Entamoeba stage conversion including dissecting transcriptome analysis in development, characterization of transcriptional networks, demonstration of epigenetic regulation, and role of small molecules that regulate Entamoeba development.

The NAD+ Responsive Transcription Factor ERM-BP Functions Downstream of Cellular Aggregation and Is an Early Regulator of Development and Heat Shock Response in Entamoeba.

Entamoeba histolytica is a protozoan parasite and a major cause of dysentery and diarrheal disease in developing countries. Disease transmission from one host to another occurs via cysts which can survive in environmental extremes and are transmitted through contaminated food and water. Recent studies in our lab identified a novel transcription factor, Encystation Regulatory Motif- Binding Protein (ERM-BP), which is responsive to NAD+ and has an important role in encystation. The key residues important for ERM-BP function were demonstrated in vitro using recombinant protein. In this study we demonstrate the in vivo functional consequences of mutations in key domains and their impact on Entamoeba encystation. Our results show that mutations in the DNA binding domain (ERM-BP-DBM) and in the nicotinamidase domain (ERM-BP-C198A) lead to protein mis-localization in both trophozoites and cysts and significantly reduce encystation efficiency. Additionally, we showed that silencing of ERM-BP significantly decreased the size and number of multi-nucleated giant cells (MGC) that form during encystation, indicating that ERM-BP functions upstream of the cellular aggregation that precedes stage conversion. Dissection of epistatic interactions between ERM-BP and a second encystation-related transcription factor, NF-Y revealed that ERM-BP is upstream of NF-Y in controlling the developmental cascade and appears to be one of the earliest regulators of development identified to date in Entamoeba. We also demonstrated that ERM-BP is upregulated during heat stress in Entamoeba, another condition which increases intracellular NAD+ levels and that overexpression of ERM-BP makes E. histolytica and E. invadens parasites more resistant to heat stress. Overexpression of ERM-BP in E. histolytica also induced the formation of cyst-like quadrinucleated cells and formation of MGCs. Overall, our work has identified an important role of ERM-BP in Entamoeba stress response and links an NAD+-responsive transcription factor to both development and heat shock response. Characterization of stress and developmental cascades are important avenues to investigate for Entamoeba, an important human parasitic pathogen.

Nuclear Factor Y (NF-Y) Modulates Encystation in Entamoeba via Stage-Specific Expression of the NF-YB and NF-YC Subunits.

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor composed of three subunits, namely, NF-YA, NF-YB, and NF-YC, which are conserved throughout evolution. In higher eukaryotes, NF-Y plays important roles in several cellular processes (development, cell cycle regulation, apoptosis, and response to growth, stress, and DNA damage) by controlling gene expression through binding to a CCAAT promoter motif. We demonstrated that NF-Y subunits in the protist Entamoeba, while significantly divergent from those of higher eukaryotes, have well-conserved domains important for subunit interactions and DNA binding and that NF-YB and NF-YC are developmentally expressed during encystation. Electrophoretic mobility shift assays confirmed that the NF-Y protein(s) from Entamoeba cysts binds to a CCAAT motif. Consistent with a role as a transcription factor, the NF-Y proteins show nuclear localization during development. Additionally, we demonstrated that NF-YC localizes to the chromatoid body (an RNA processing center) during development, indicating that it may have a role in RNA processing. Finally, silencing of the NF-YC subunit resulted in reduced stability of the NF-Y complex and decreased encystation efficiency. We demonstrated that the NF-Y complex functions at a time point subsequent to the NAD+ flux and expression of the transcription factor encystation regulatory motif-binding protein, both of which are early regulators of Entamoeba development. Taken together, our results demonstrate that the NF-Y complex plays an important role in regulating encystation in Entamoeba and add to our understanding of the transcriptional networks and signals that control this essential developmental pathway in an important human pathogen.IMPORTANCE The human parasite Entamoeba histolytica is an important pathogen with significant global impact and is a leading cause of parasitic death in humans. Since only the cyst form can be transmitted, blocking encystation would prevent new infections, making the encystation pathway an attractive target for the development of new drugs. Identification of the genetic signals and transcriptional regulatory networks that control encystation would be an important advance in understanding the developmental cascade. We show that the Entamoeba NF-Y complex plays a crucial role in regulating the encystation process in Entamoeba.

Functional Characterization of Entamoeba histolytica Argonaute Proteins Reveals a Repetitive DR-Rich Motif Region That Controls Nuclear Localization.

The RNA interference (RNAi) pathway regulates gene expression in many eukaryotic organisms. Argonaute (Ago) proteins, together with bound small RNAs (sRNAs), are key effectors that mediate gene silencing function. However, there is limited knowledge of Ago proteins and their functions in nonmodel systems. In the protozoan parasite Entamoeba histolytica, RNAi is a robust means for stable gene silencing mediated via large populations of antisense sRNAs. Here, we report functional characterization of three Ago proteins in E. histolytica (EhAgo2-1, EhAgo2-2, and EhAgo2-3). Our data show that each EhAgo protein has a distinct subcellular localization and binds 27-nucleotide (nt) sRNAs and that the localization of EhAgo proteins is altered in response to stress conditions. Via mutagenesis analyses, we demonstrated that the Ago PAZ (Piwi/Argonaute/Zwille) domain in all three EhAgos is essential for sRNA binding. With mutation of the PAZ domain in EhAgo2-2, there was no effect on the nuclear localization of the protein but a strong phenotype and a growth defect. We further show that EhAgo2-2 contains an unusual repetitive DR-rich (aspartic acid, arginine-rich) motif region which functions as a nuclear localization signal (NLS) and is both necessary and sufficient to mediate nuclear localization. Overall, our data delineate the localization and sRNA binding features of the three E. histolytica Ago proteins and demonstrate that the PAZ domain is necessary for sRNA binding. The repetitive DR-rich motif region in EhAgo2-2 has not previously been defined in other systems, which adds to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems.IMPORTANCE The protozoan parasite Entamoeba histolytica, which causes amebiasis and affects over 50 million people worldwide, contains an important RNAi pathway for gene silencing. Gene silencing via the RNAi pathway is mediated by the Argonaute (Ago) proteins. However, we lack knowledge on Ago function(s) in this nonmodel system. In this paper, we discovered that three E. histolytica Ago proteins (EhAgo2-1, EhAgo2-2, and EhAgo2-3) all bind 27-nt small RNAs and have distinct subcellular localizations, which change in response to stress conditions. The EhAgos bind small RNA populations via their PAZ domains. An unusual repetitive DR-rich motif region is identified in EhAgo2-2 that functions as a nuclear localization signal. Our results show for the first time an active nuclear transport process of the EhAgo2-2 RNA-induced silencing complex (RISC) in this parasite. These data add to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems.

An NAD+-dependent novel transcription factor controls stage conversion in Entamoeba.

Developmental switching between life-cycle stages is a common feature among parasitic pathogens to facilitate disease transmission and pathogenesis. The protozoan parasite Entamoeba switches between invasive trophozoites and dormant cysts, but the encystation process remains poorly understood despite being central to amoebic biology. We identify a transcription factor, Encystation Regulatory Motif-Binding Protein (ERM-BP), that regulates encystation. Down-regulation of ERM-BP decreases encystation efficiency resulting in abnormal cysts with defective cyst walls. We demonstrate that direct binding of NAD+ to ERM-BP affects ERM-BP conformation and facilitates its binding to promoter DNA. Additionally, cellular NAD+ levels increase during encystation and exogenous NAD+ enhances encystation consistent with the role of carbon source depletion in triggering Entamoeba encystation. Furthermore, ERM-BP catalyzes conversion of nicotinamide to nicotinic acid, which might have second messenger effects on stage conversion. Our findings link the metabolic cofactors nicotinamide and NAD+ to transcriptional regulation via ERM-BP and provide the first mechanistic insights into Entamoeba encystation.

Recent advances in Entamoeba biology: RNA interference, drug discovery, and gut microbiome.

In recent years, substantial progress has been made in understanding the molecular and cell biology of the human parasite Entamoeba histolytica, an important pathogen with significant global impact. This review outlines some recent advances in the Entamoeba field in the last five years, focusing on areas that have not recently been discussed in detail: (i) molecular mechanisms regulating parasite gene expression, (ii) new efforts at drug discovery using high-throughput drug screens, and (iii) the effect of gut microbiota on amoebiasis.

High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress.

The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.

Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns.

Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points. In this study RNA-Seq data was utilised to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5' and 3' untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3'-U-Rich Motif (Ei3'-URM) (TTTGTT) in the 5' and 3' flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analysed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic manipulation of E. invadens and allowing further dissection of factors controlling Entamoeba developmental biology.

Novel isoforms of the transport regulator klar.

Klar is a regulator of microtubule-motor dependent transport processes in Drosophila, including nuclear migration, vesicle motility, and lipid-droplet transport. The single klar locus gives rise to multiple isoforms that presumably have unique functions. Up to now, three Klar isoforms (α, ß, γ) were known. Here we describe two novel isoforms, δ and ε, whose expression depends on a previously uncharacterized promoter. Klar δ and/or ε are widely expressed during development, including in the embryonic and larval nervous system as well as in ovaries. When we specifically ablate Klar δ and ε expression genetically, no gross organismal phenotypes are apparent. However, ectopic expression of these isoforms causes nuclear mispositioning in developing photoreceptors and in oocytes, demonstrating their biological activity. Our analysis identifies novel forms of the Klar protein and provides new tools for functionally dissecting the complex klar locus.

Polyunsaturated fatty acids induce polarized submembranous F-actin aggregates and kill Entamoeba histolytica.

We have recently identified a novel galacto-glycerolipid (GGL) from the plant Oxalis corniculata that killed the human pathogen Entamoeba histolytica. In this study, we show that the anti-amoebic activity of GGL was due to the polyunsaturated fatty acid α-linolenic acid (C18:3 ) side chain. Treatment of α-linolenic acid to E. histolytica trophozoites disrupted the cytoskeletal network and led to polarization of F-actin at one end of the cells with prominent filopodial extensions. In addition, clustering of surface receptors and signaling molecules was also observed adjacent to the polarized actin similar to concanavalin-A-(Con-A) induced capping. But, in contrast to Con-A-induced capping, α-linolenic acid induced caps were not shed and showed accumulation of long and numerous filopodia at the cap site. We found that α-linolenic acid disrupts the actin cytoskeletal network, which led to the detachment of plasma membrane from the underlying cytoskeleton. A similar effect was observed with other dietary fatty acids such as linoleic acid (C18:2 ), arachidonic acid (C20:4 ), eicosapentaenoic acid (C20:5 ), and docosahexaenoic acid (C22:6 ). Our findings showed that dietary polyunsaturated fatty acids are powerful anti-amoebic agents that lead to disruption of the actin cytoskeleton.

Long term transcript accumulation during the development of dehydration adaptation in Cicer arietinum.

Cool season crops face intermittent drought. Exposure to drought and other abiotic stresses is known to increase tolerance of the plants against subsequent exposure to such stresses. Storage of environmental signals is also proposed. Preexposure to a dehydration shock improved adaptive response during subsequent dehydration treatment in a cool season crop chickpea (Cicer arietinum). We have identified 101 dehydration-inducible transcripts of chickpea by repetitive rounds of cDNA subtraction; differential DNA-array hybridization followed by northern-blot analysis and analyzed their responses to exogenous application of abscisic acid (ABA). Steady-state expression levels of the dehydration-induced transcripts were monitored during the recovery period between 2 consecutive dehydration stresses. Seven of them maintained more than 3-fold of expression after 24 h and more than 2-fold of expression level even at 72 h after the removal of stress. Noticeably, all of them were inducible by exogenous ABA treatment. When the seedlings were subjected to recover similarly after an exposure to exogenous ABA, the steady-state abundances of 6 of them followed totally different kinetics returning to basal level expression within 24 h. This observation indicated a correlation between the longer period of abundance of those transcripts in the recovery period and improved adaptation of the plants to subsequent dehydration stress and suggested that both ABA-dependent and -independent mechanisms are involved in the maintenance of the messages from the previous stress experience.