Plant biomarkers as early detection tools in stress management in food crops: a review.

MAIN CONCLUSION: Plant Biomarkers are objective indicators of a plant's cellular state in response to abiotic and biotic stress factors. They can be explored in crop breeding and engineering to produce stress-tolerant crop species. Global food production safely and sustainably remains a top priority to feed the ever-growing human population, expected to reach 10 billion by 2050. However, abiotic and biotic stress factors negatively impact food production systems, causing between 70 and 100% reduction in crop yield. Understanding the plant stress responses is critical for developing novel crops that can adapt better to various adverse environmental conditions. Using plant biomarkers as measurable indicators of a plant's cellular response to external stimuli could serve as early warning signals to detect stresses before severe damage occurs. Plant biomarkers have received considerable attention in the last decade as pre-stress indicators for various economically important food crops. This review discusses some biomarkers associated with abiotic and biotic stress conditions and highlights their importance in developing stress-resilient crops. In addition, we highlighted some factors influencing the expression of biomarkers in crop plants under stress. The information presented in this review would educate plant researchers, breeders, and agronomists on the significance of plant biomarkers in stress biology research, which is essential for improving plant growth and yield toward sustainable food production.

The MsDHN1-MsPIP2;1-MsmMYB module orchestrates the trade-off between growth and survival of alfalfa in response to drought stress.

Dehydrins and aquaporins play crucial roles in plant growth and stress responses by acting as protector and controlling water transport across membranes, respectively. MsDHN1 (dehydrin) and MsPIP2;1 (aquaporin) were demonstrated to interact with a membrane-anchored MYB protein, MsmMYB (as mMYB) in plasma membrane under normal condition. MsDHN1, MsPIP2;1 and MsDHN1-MsPIP2;1 positively regulated alfalfa tolerance to water deficiency. Water deficiency caused phosphorylation of MsPIP2;1 at Ser 272, which led to release C terminus of mMYB (mMYBΔ83) from plasma membrane and translocate to nucleus, where C terminus of MsDHN1 interacted with mMYBΔ83, and promoted mMYBΔ83 transcriptional activity in response to water deficiency. Overexpression of mMYB and mMYBΔ83 down-regulated the expression of MsCESA3, but up-regulated MsCESA7 expression by directly binding to their promoters, and resulted in high drought tolerance in transgenic hairy roots. These results indicate that the MsDHN1-MsPIP2;1-MsMYB module serves as a key regulator in alfalfa against drought stress.

Citrus FRIGIDA cooperates with its interaction partner dehydrin to regulate drought tolerance.

Drought is a major environmental stress that severely affects plant growth and crop productivity. FRIGIDA (FRI) is a key regulator of flowering time and drought tolerance in model plants. However, little is known regarding its functions in woody plants, including citrus. Thus, we explored the functional role of the citrus FRI ortholog (CiFRI) under drought. Drought treatment induced CiFRI expression. CiFRI overexpression enhanced drought tolerance in transgenic Arabidopsis and citrus, while CiFRI suppression increased drought susceptibility in citrus. Moreover, transcriptomic profiling under drought conditions suggested that CiFRI overexpression altered the expression of numerous genes involved in the stress response, hormone biosynthesis, and signal transduction. Mechanistic studies revealed that citrus dehydrin likely protects CiFRI from stress-induced degradation, thereby enhancing plant drought tolerance. In addition, a citrus brassinazole-resistant (BZR) transcription factor family member (CiBZR1) directly binds to the CiFRI promoter to activate its expression under drought conditions. CiBZR1 also enhanced drought tolerance in transgenic Arabidopsis and citrus. These findings further our understanding of the molecular mechanisms underlying the CiFRI-mediated drought stress response in citrus.

The positive impact of the NtTAS14-like1 gene on osmotic stress response in Nicotiana tabacum.

KEY MESSAGE: NtTAS14-like1 enhances osmotic tolerance through coordinately activating the expression of osmotic- and ABA-related genes. Osmotic stress is one of the most important limiting factors for tobacco (Nicotiana tabacum) growth and development. Dehydrin proteins are widely involved in plant adaptation to osmotic stress, but few of these proteins have been functionally characterized in tobacco. Here, to identify genes required for osmotic stress response in tobacco, an encoding dehydrin protein gene NtTAS14-like1 was isolated based on RNA sequence data. The expression of NtTAS14-like1 was obviously induced by mannitol and abscisic acid (ABA) treatments. Knock down of NtTAS14-like1 expression reduced osmotic tolerance, while overexpression of NtTAS14-like1 conferred tolerance to osmotic stress in transgenic tobacco plants, as determined by physiological analysis of the relative electrolyte leakage and malonaldehyde accumulation. Further expression analysis by quantitative real-time PCR indicated that NtTAS14-like1 participates in osmotic stress response possibly through coordinately activating osmotic- and ABA-related genes expression, such as late embryogenesis abundant (NtLEA5), early responsive to dehydration 10C (NtERD10C), calcium-dependent protein kinase 2 (NtCDPK2), ABA-responsive element-binding protein (NtAREB), ABA-responsive element-binding factor 1 (NtABF1), dehydration-responsive element-binding genes (NtDREB2A), xanthoxin dehydrogenase/reductase (NtABA2), ABA-aldehyde oxidase 3 (NtAAO3), 9-cis-epoxycarotenoid dioxygenase (NtNCED3). Together, this study will facilitate to improve our understandings of molecular and functional properties of plant TAS14 proteins and to improve genetic evidence on the involvement of the NtTAS14-like1 in osmotic stress response of tobacco.

Label-Free Quantitative Proteomics Reveal the Mechanisms of Young Wheat (Triticum aestivum L.) Ears' Response to Spring Freezing.

Late spring frost is an important meteorological factor threatening the safe production of winter wheat in China. The young ear is the most vulnerable organ of the wheat plant to spring frost. To gain an insight into the mechanisms underpinning young wheat ears' tolerance to freezing, we performed a comparative proteome analysis of wheat varieties Xumai33 (XM33, freezing-sensitive) and Jimai22 (JM22, freezing-tolerant) under normal and freezing conditions using label-free quantitative proteomic techniques during the anther connective tissue formation phase (ACFP). Under freezing stress, 392 and 103 differently expressed proteins (DEPs) were identified in the young ears of XM33 and JM22, respectively, and among these, 30 proteins were common in both varieties. A functional characterization analysis revealed that these DEPs were associated with antioxidant capacity, cell wall modification, protein folding, dehydration response, and plant-pathogen interactions. The young ears of JM22 showed significantly higher expression levels of antioxidant enzymes, heat shock proteins, and dehydrin under normal conditions compared to those of XM33, which might help to prepare the young ears of JM22 for freezing stress. Our results lead to new insights into understanding the mechanisms in young wheat ears' response to freezing stress and provide pivotal potential candidate proteins required for improving young wheat ears' tolerance to spring frost.

Heterologous expression of bacterial dehydrin gene in Arabidopsis thaliana promotes abiotic stress tolerance.

Salinity, low temperature, and drought are major environmental factors in agriculture leading to reduced crop yield. Dehydrins (DHNs) are induced transcriptionally during cellular dehydration and accumulate in different tissues during abiotic stresses. Here we isolated and characterized a bacterial gene BG757 in Arabidopsis, encoding a putative dehydrin type protein. ABA induces the expression of various dehydrins in plants, therefore, to elucidate the potential role, ABA sensitivity was examined in Arabidopsis transgenic lines expressing BG757. Interestingly, BG757-expressing plants showed hypersensitivity towards NaCl and ABA during seed germination. In addition to germination, BG757-expressing plants also showed root growth retardation in the presence of ABA and NaCl when compared with wild type (WT), suggesting that BG757 positively regulate salt stress and ABA response. Furthermore, BG757-expressing plants showed significant drought tolerance compared with WT. Consistent with drought tolerance, expression levels of stress inducible genes (DREB2A, RD22, RD26, LEA7 and SOS1) were strongly upregulated in transgenic plants compared with WT. All together these results suggest that heterologous expression of bacterial gene, BG757 in plants promotes resistance to environmental stresses. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01358-w.

Multiple copies of a novel amphipathic α-helix forming segment in Physcomitrella patens dehydrin play a key role in abiotic stress mitigation.

Plants use a diverse set of proteins to mitigate various abiotic stresses. The intrinsically disordered protein dehydrin is an important member of this repertoire of proteins, characterized by a canonical amphipathic K-segment. It can also contain other stress-mitigating noncanonical segments-a likely reflection of the extremely diverse nature of abiotic stress encountered by plants. Among plants, the poikilohydric mosses have no inbuilt mechanism to prevent desiccation and therefore are likely to contain unique noncanonical stress-responsive motifs in their dehydrins. Here we report the recurring occurrence of a novel amphipathic helix-forming segment (D-segment: EGφφD(R/K)AKDAφ, where φ represents a hydrophobic residue) in Physcomitrella patens dehydrin (PpDHNA), a poikilohydric moss. NMR and CD spectroscopic experiments demonstrated the helix-forming tendency of the D-segment, with the shuffled D-segment as control. PpDHNA activity was shown to be size as well as D-segment dependent from in vitro, in vivo, and in planta studies using PpDHNA and various deletion mutants. Bimolecular fluorescence complementation studies showed that D-segment-mediated PpDHNA self-association is a requirement for stress abatement. The D-segment was also found to occur in two rehydrin proteins from Syntrichia ruralis, another poikilohydric plant like P. patens. Multiple occurrences of the D-segment in poikilohydric plant dehydrins/rehydrins, along with the experimental demonstration of the role of D-segment in stress abatement, implies that the D-segment mediates unique resurrection strategies, which may be employed by plant dehydrins that are capable of mitigating extreme stress.

Dehydrin MsDHN1 improves aluminum tolerance of alfalfa (Medicago sativa L.) by affecting oxalate exudation from root tips.

A SK3 -type dehydrin MsDHN1 was cloned from alfalfa (Medicago sativa L.). Its function and gene regulatory pathways were studied via overexpression and suppression of MsDHN1 in alfalfa seedlings or hairy roots. The results showed that MsDHN1 is a typical intrinsically disordered protein that exists in the form of monomers and homodimers in alfalfa. The plant growth rates increased as a result of MsDHN1 overexpression (MsDHN1-OE) and decreased upon MsDHN1 suppression (MsDHN1-RNAi) in seedlings or hairy roots of alfalfa compared with the wild-type or the vector line under Al stress. MsDHN1 interacting with aquaporin (AQP) MsPIP2;1 and MsTIP1;1 positively affected oxalate secretion from root tips and Al accumulation in root tips. MsABF2 was proven to be an upstream transcription factor of MsDHN1 and activated MsDHN1 expression by binding to the ABRE element of the MsDHN1 promoter. The transcriptional regulation of MsABF2 on MsDHN1 was dependent on the abscisic acid signaling pathway. These results indicate that MsDHN1 can increase alfalfa tolerance to Al stress via increasing oxalate secretion from root tips, which may involve in the interaction of MsDHN1 with two AQP.

Linking genome wide RNA sequencing with physio-biochemical and cytological responses to catalogue key genes and metabolic pathways for alkalinity stress tolerance in lentil (Lens culinaris Medikus).

BACKGROUND: Alkaline soils cause low productivity in crop plants including lentil. Alkalinity adaptation strategies in lentil were revealed when morpho-anatomical and physio-biochemical observations were correlated with transcriptomics analysis in tolerant (PDL-1) and sensitive (L-4076) cultivars at seedling stage. RESULTS: PDL-1 had lesser salt injury and performed better as compared to L-4076. Latter showed severe wilting symptoms and higher accumulation of Na+ and lower K+ in roots and shoots. PDL-1 performed better under high alkalinity stress which can be attributed to its higher mitotic index, more accumulation of K+ in roots and shoots and less aberrantly dividing cells. Also, antioxidant enzyme activities, osmolytes' accumulation, relative water content, membrane stability index and abscisic acid were higher in this cultivar. Differentially expressed genes (DEGs) related to these parameters were upregulated in tolerant genotypes compared to the sensitive one. Significantly up-regulated DEGs were found to be involved in abscisic acid (ABA) signalling and secondary metabolites synthesis. ABA responsive genes viz. dehydrin 1, 9-cis-epoxycarotenoid dioxygenase, ABA-responsive protein 18 and BEL1-like homeodomain protein 1 had log2fold change above 4.0. A total of 12,836 simple sequence repeats and 4,438 single nucleotide polymorphisms were identified which can be utilized in molecular studies. CONCLUSIONS: Phyto-hormones biosynthesis-predominantly through ABA signalling, and secondary metabolism are the most potent pathways for alkalinity stress tolerance in lentil. Cultivar PDL-1 exhibited high tolerance towards alkalinity stress and can be used in breeding programmes for improving lentil production under alkalinity stress conditions.

Overexpression of ZmDHN15 Enhances Cold Tolerance in Yeast and Arabidopsis.

Maize (Zea mays L.) originates from the subtropical region and is a warm-loving crop affected by low-temperature stress. Dehydrin (DHN) protein, a member of the Group 2 LEA (late embryogenesis abundant proteins) family, plays an important role in plant abiotic stress. In this study, five maize DHN genes were screened based on the previous transcriptome sequencing data in our laboratory, and we performed sequence analysis and promoter analysis on these five DHN genes. The results showed that the promoter region has many cis-acting elements related to cold stress. The significantly upregulated ZmDHN15 gene has been further screened by expression pattern analysis. The subcellular localization results show that ZmDHN15 fusion protein is localized in the cytoplasm. To verify the role of ZmDHN15 in cold stress, we overexpressed ZmDHN15 in yeast and Arabidopsis. We found that the expression of ZmDHN15 can significantly improve the cold resistance of yeast. Under cold stress, ZmDHN15-overexpressing Arabidopsis showed lower MDA content, lower relative electrolyte leakage, and less ROS (reactive oxygen species) when compared to wild-type plants, as well as higher seed germination rate, seedling survival rate, and chlorophyll content. Furthermore, analysis of the expression patterns of ROS-associated marker genes and cold-response-related genes indicated that ZmDHN15 genes play an important role in the expression of these genes. In conclusion, the overexpression of the ZmDHN15 gene can effectively improve the tolerance to cold stress in yeast and Arabidopsis. This study is important for maize germplasm innovation and the genetic improvement of crops.

Distinct Tomato Cultivars Are Characterized by a Differential Pattern of Biochemical Responses to Drought Stress.

Future climate scenarios suggest that crop plants will experience environmental changes capable of affecting their productivity. Among the most harmful environmental stresses is drought, defined as a total or partial lack of water availability. It is essential to study and understand both the damage caused by drought on crop plants and the mechanisms implemented to tolerate the stress. In this study, we focused on four cultivars of tomato, an economically important crop in the Mediterranean basin. We investigated the biochemical mechanisms of plant defense against drought by focusing on proteins specifically involved in this stress, such as osmotin, dehydrin, and aquaporin, and on proteins involved in the general stress response, such as HSP70 and cyclophilins. Since sugars are also known to act as osmoprotectants in plant cells, proteins involved in sugar metabolism (such as RuBisCO and sucrose synthase) were also analyzed. The results show crucial differences in biochemical behavior among the selected cultivars and highlight that the most tolerant tomato cultivars adopt quite specific biochemical strategies such as different accumulations of aquaporins and osmotins. The data set also suggests that RuBisCO isoforms and aquaporins can be used as markers of tolerance/susceptibility to drought stress and be used to select tomato cultivars within breeding programs.

A YSK-Type Dehydrin from Nicotiana tabacum Enhanced Copper Tolerance in Escherichia coli.

Copper is an essential micronutrient for the maintenance of normal cell function but is toxic in excess. Dehydrins are group two late embryogenesis abundant proteins, which facilitate plant survival in harsh environmental conditions. Here, a YSK-type dehydrin, NtDhn17, was cloned from Nicotiana tabacum under copper toxicity and characterized using a heterologous expression system and in vitro or in vivo experiments and exhibited characteristics of intrinsic disorder during in vitro analyses. Heterologous expression of NtDHN17 enhanced the tolerance of E. coli to various metals, osmotic, and oxidative stress. NtDHN17 showed no Cu2+-binding properties in vivo or in vitro, indicating that metal ion binding is not universal among dehydrins. In vitro and in vivo experiments suggested that NtDHN17 behaved as a potent anti-aggregation agent providing strong protection to aggregated proteins induced by excess copper ions, an effect dependent on the K-segment but not on the Y- or S-segments. In summary, the protective role of NtDHN17 towards E. coli under conditions of copper toxicity may be related to anti-aggregation ability rather than its acting as an ion scavenger, which might be a valuable target for the genetic improvement of resistance to heavy metal stresses in plants.

Proteomics Evidence of a Systemic Response to Desiccation in the Resurrection Plant Haberlea rhodopensis.

Global warming and drought stress are expected to have a negative impact on agricultural productivity. Desiccation-tolerant species, which are able to tolerate the almost complete desiccation of their vegetative tissues, are appropriate models to study extreme drought tolerance and identify novel approaches to improve the resistance of crops to drought stress. In the present study, to better understand what makes resurrection plants extremely tolerant to drought, we performed transmission electron microscopy and integrative large-scale proteomics, including organellar and phosphorylation proteomics, and combined these investigations with previously published transcriptomic and metabolomics data from the resurrection plant Haberlea rhodopensis. The results revealed new evidence about organelle and cell preservation, posttranscriptional and posttranslational regulation, photosynthesis, primary metabolism, autophagy, and cell death in response to desiccation in H. rhodopensis. Different protective intrinsically disordered proteins, such as late embryogenesis abundant (LEA) proteins, thaumatin-like proteins (TLPs), and heat shock proteins (HSPs), were detected. We also found a constitutively abundant dehydrin in H. rhodopensis whose phosphorylation levels increased under stress in the chloroplast fraction. This integrative multi-omics analysis revealed a systemic response to desiccation in H. rhodopensis and certain targets for further genomic and evolutionary studies on DT mechanisms and genetic engineering towards the improvement of drought tolerance in crops.

Investigating the Functional Role of the Cysteine Residue in Dehydrin from the Arctic Mouse-Ear Chickweed Cerastium arcticum.

The stress-responsive, SK5 subclass, dehydrin gene, CaDHN, has been identified from the Arctic mouse-ear chickweed Cerastium arcticum. CaDHN contains an unusual single cysteine residue (Cys143), which can form intermolecular disulfide bonds. Mutational analysis and a redox experiment confirmed that the dimerization of CaDHN was the result of an intermolecular disulfide bond between the cysteine residues. The biochemical and physiological functions of the mutant C143A were also investigated by in vitro and in vivo assays using yeast cells, where it enhanced the scavenging of reactive oxygen species (ROS) by neutralizing hydrogen peroxide. Our results show that the cysteine residue in CaDHN helps to enhance C. arcticum tolerance to abiotic stress by regulating the dimerization of the intrinsically disordered CaDHN protein, which acts as a defense mechanism against extreme polar environments.

Overexpression of ZmDHN11 could enhance transgenic yeast and tobacco tolerance to osmotic stress.

KEY MESSAGE: Maize group II LEA protein ZmDHN11 could protect protein activity and confer resistance to osmotic stress on transgenic yeast and tobacco. Late embryogenesis abundant (LEA) proteins are widely assumed to play crucial roles in environmental stress tolerance, but their function has remained obscure. Dehydrins are group II LEA proteins, which are highly hydrophilic plant stress proteins. In the present study, a novel group II LEA protein, ZmDHN11, was cloned and identified from maize. The expression of ZmDHN11 was induced by high osmotic stress, low temperature, salinity, and ABA (abscisic acid). The ZmDHN11 protein specifically accumulated in the nuclei and cytosol. Further study indicated that ZmDHN11 is phosphorylated by the casein kinase CKII. ZmDHN11 protected the activity of LDH under water-deficit stress. The overexpression of ZmDHN11 endows transgenic yeast and tobacco with tolerance to osmotic stress.

Functional Characteristics Analysis of Dehydrins in Larix kaempferi under Osmotic Stress.

Dehydrins (DHN) belong to the late embryogenesis abundant II family and have been found to enhance plant tolerance to abiotic stress. In the present study, we reported four DHNs in Larix kaempferi (LkDHN) which were identified from the published transcriptome. Alignment analysis showed that these four LkDHNs shared close relationships and belonged to SK3-type DHNs. The electrophoretic mobility shift assay indicated that these four LkDHNs all possess sequence-independent binding capacity for double-strands DNAs. The subcellular localizations of the four LkDHNs were in both the nucleus and cytoplasm, indicating that these LkDHNs enter the nucleus to exert the ability to bind DNA. The preparation of tobacco protoplasts with different concentrations of mannitol showed that LkDHNs enhanced the tolerance of plant cells under osmotic stress. The overexpression of LkDHNs in yeasts enhanced their tolerance to osmotic stress and helped the yeasts to survive severe stress. In addition, LkDHNs in the nucleus of salt treated tobacco increased. All of these results indicated that the four LkDHNs help plants survive from heavy stress by participating in DNA protection. These four LKDHNs played similar roles in the response to osmotic stress and assisted in the adaptation of L. kaempferi to the arid and cold winter of northern China.

Plant Dehydrins: Expression, Regulatory Networks, and Protective Roles in Plants Challenged by Abiotic Stress.

Dehydrins, also known as Group II late embryogenesis abundant (LEA) proteins, are classic intrinsically disordered proteins, which have high hydrophilicity. A wide range of hostile environmental conditions including low temperature, drought, and high salinity stimulate dehydrin expression. Numerous studies have furnished evidence for the protective role played by dehydrins in plants exposed to abiotic stress. Furthermore, dehydrins play important roles in seed maturation and plant stress tolerance. Hence, dehydrins might also protect plasma membranes and proteins and stabilize DNA conformations. In the present review, we discuss the regulatory networks of dehydrin gene expression including the abscisic acid (ABA), mitogen-activated protein (MAP) kinase cascade, and Ca2+ signaling pathways. Crosstalk among these molecules and pathways may form a complex, diverse regulatory network, which may be implicated in regulating the same dehydrin.

Cellular Chaperone Function of Intrinsically Disordered Dehydrin ERD14.

Disordered plant chaperones play key roles in helping plants survive in harsh conditions, and they are indispensable for seeds to remain viable. Aside from well-known and thoroughly characterized globular chaperone proteins, there are a number of intrinsically disordered proteins (IDPs) that can also serve as highly effective protecting agents in the cells. One of the largest groups of disordered chaperones is the group of dehydrins, proteins that are expressed at high levels under different abiotic stress conditions, such as drought, high temperature, or osmotic stress. Dehydrins are characterized by the presence of different conserved sequence motifs that also serve as the basis for their categorization. Despite their accepted importance, the exact role and relevance of the conserved regions have not yet been formally addressed. Here, we explored the involvement of each conserved segment in the protective function of the intrinsically disordered stress protein (IDSP) A. thaliana's Early Response to Dehydration (ERD14). We show that segments that are directly involved in partner binding, and others that are not, are equally necessary for proper function and that cellular protection emerges from the balanced interplay of different regions of ERD14.

Transcription factor TabHLH49 positively regulates dehydrin WZY2 gene expression and enhances drought stress tolerance in wheat.

BACKGROUND: As functional proteins, dehydrins are found in many maturing seeds and vegetable tissues under adverse environmental conditions. However, the regulation of dehydrin expression remains unclear. RESULTS: In this study, a novel drought stress-related bHLH transcription factor, TabHLH49, was isolated from a wheat cDNA library treated with the drought and cold stress by using yeast one-hybrid system. TabHLH49 protein possesses a typical conserved bHLH domain and is a Myc-type bHLH transcription factor. TabHLH49 was detected in the nucleus of tobacco epidermal cells, and the amino acid sequences at the C-terminus (amino acids 323-362) is necessary for its transactivation activity. Real-time PCR analyses revealed the tissue-specific expression and drought stress-responsive expression of TabHLH49 in wheat. In addition, the verification in Y1H and electrophoretic mobility shift assays illustrated that TabHLH49 protein can bind and interact with the promoter of the wheat WZY2 dehydrin. Furthermore, the dual-luciferase assays showed that TabHLH49 can positively regulate the expression of WZY2 dehydrin. The transient expression and BSMV-mediated gene silencing of TabHLH49 also showed that TabHLH49 positively regulates the expression of WZY2 dehydrin and improves drought stress resistance in wheat. CONCLUSIONS: These results provide direct evidences that TabHLH49 positively regulates expression level of dehydrin WZY2 gene and improves drought tolerance of wheat.

Cryoprotective activity of Arabidopsis KS-type dehydrin depends on the hydrophobic amino acids of two active segments.

Dehydrins are intrinsically disordered proteins which are related to cold tolerance in plants. Dehydrins show potent cryoprotective activities for freeze-sensitive enzymes such as lactate dehydrogenase (LDH). Previous studies demonstrated that K-segments conserved in dehydrins had cryoprotective activities and that K-segment activities depended on the hydrophobic amino acids in the segment. However, the cryoprotective roles of hydrophobic amino acids in dehydrin itself have not been reported. Here, we demonstrated that hydrophobic amino acids were required for the cryoprotective activity of Arabidopsis dehydrin AtHIRD11. Cryoprotective activities were compared between AtHIRD11 and the corresponding mutant in which all hydrophobic residues were changed to T (AtHIRD11Φ/T) by using LDH. The change strikingly reduced AtHIRD11 activity. A segmentation analysis indicated that the conserved K-segment (Kseg) and a previously unidentified segment (non-K-segment 1, NK1) showed cryoprotective activities. Circular dichroism indicated that the secondary structures of all peptides showed disorder, but only cryoprotective peptides changed to the ordered forms by sodium dodecyl sulfate. Ultracentrifuge analysis indicated that AtHIRD11 and AtHIRD11Φ/T had similar molecular sizes in solution. These results suggest that not only structural disorder but also hydrophobic amino acids contributed to the cryoprotective activity of AtHIRD11. A possible mechanism based on an extended molecular shield model is proposed.