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1.  Accumulation of dehydrin-like proteins in the mitochondria of cereals in response to cold, freezing, drought and ABA treatment 
BMC Plant Biology  2002;2:5.
Background
Dehydrins are known as Group II late embryogenesis abundant proteins. Their high hydrophilicity and thermostability suggest that they may be structure stabilizers with detergent and chaperone-like properties. They are localised in the nucleus, cytoplasm, and plasma membrane. We have recently found putative dehydrins in the mitochondria of some cereals in response to cold. It is not known whether dehydrin-like proteins accumulate in plant mitochondria in response to stimuli other than cold stress.
Results
We have found five putative dehydrins in the mitochondria of winter wheat, rye and maize seedlings. Two of these polypeptides had the same molecular masses in all three species (63 and 52 kD) and were thermostable. Drought, freezing, cold, and exogenous ABA treatment led to higher accumulation of dehydrin-like protein (dlp) 63 kD in the rye and wheat mitochondria. Protein 52 kD was induced by cold adaptation and ABA. Some accumulation of these proteins in the maize mitochondria was found after cold exposition only. The other three proteins appeared to be heat-sensitive and were either slightly induced or not induced at all by all treatments used.
Conclusions
We have found that, not only cold, but also drought, freezing and exogenous ABA treatment result in accumulation of the thermostable dehydrins in plant mitochondria. Most cryotolerant species such as wheat and rye accumulate more heat-stable dehydrins than cryosensitive species such as maize. It has been supposed that their function is to stabilize proteins in the membrane or in the matrix. Heat-sensitive putative dehydrins probably are not involved in the stress reaction and adaptation of plants.
doi:10.1186/1471-2229-2-5
PMCID: PMC116594  PMID: 12057012
2.  Plant dehydrins and stress tolerance 
Plant Signaling & Behavior  2011;6(10):1503-1509.
Dehydrins (DHNs), or group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response and adaptation to abiotic stresses. They accumulate typically in maturing seeds or are induced in vegetative tissues following salinity, dehydration, cold and freezing stress. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y, S and K segments. The K segment representing a highly conserved 15 amino acid motif forming amphiphilic a-helix is especially important since it has been found in all dehydrins. Since more than 20 y, they are thought to play an important protective role during cellular dehydration but their precise function remains unclear. This review outlines the current status of the progress made toward the structural, physico-chemical and functional characterization of plant dehydrins and how these features could be exploited in improving stress tolerance in plants.
doi:10.4161/psb.6.10.17088
PMCID: PMC3256378  PMID: 21897131
abiotic stress; dehydration stress; drought; cold acclimation; freezing tolerance; LEA proteins; dehydrins
3.  Involvement of Multiple Types of Dehydrins in the Freezing Response in Loquat (Eriobotrya japonica) 
PLoS ONE  2014;9(1):e87575.
Dehydrins (DHNs) are a family of plant proteins typically induced in response to stress conditions that cause cellular dehydration, such as low temperatures, high salinity, and drought. Loquat (Eriobotrya japonica) is a perennial fruit crop that blossoms during winter. Loquat fruitlets are frequently injured by freezing. To evaluate the role of the EjDHNs in freezing resistance in loquat fruitlets, two cultivars of loquat, the freezing-sensitive ‘Ninghaibai’ (FS-NHB) and the freezing-tolerant ‘Jiajiao’ (FT-JJ), were analyzed under induced freezing stress. Freezing stress led to obvious accumulation of reactive oxygen species and considerable lipid peroxidation in membranes during the treatment period. Both these phenomena were more pronounced in ‘FS-NHB’ than in ‘FS-JJ.’ Immunogold labeling of dehydrin protein was performed. DHN proteins were found to be concentrated mainly in the vicinity of the plasma membrane, and the density of the immunogold labeling was significantly higher after freezing treatment, especially in the more freezing-tolerant cultivar ‘FT-JJ.’ Seven DHNs, showing four different structure types, were obtained from loquat fruitlets and used to study the characteristics of different EjDHN proteins. These DHN proteins are all highly hydrophilic, but they differ significantly in size, ranging from 188 to 475 amino acids, and in biochemical properties, such as theoretical pI, aliphatic index, and instability index. Freezing treatment resulted in up-regulation of the expression levels of all seven EjDHNs, regardless of structure type. The accumulation of the transcripts of these EjDHN genes was much more pronounced in ‘FT-JJ’ than in ‘FS-NHB.’ Altogether, this study provides evidence that EjDHNs are involved in the cryoprotection of the plasma membrane during freeze-induced dehydration in loquat fruitlets.
doi:10.1371/journal.pone.0087575
PMCID: PMC3909202  PMID: 24498141
4.  Transcriptional responses of winter barley to cold indicate nucleosome remodelling as a specific feature of crown tissues 
Functional & Integrative Genomics  2011;11(2):307-325.
We report a series of microarray-based comparisons of gene expression in the leaf and crown of the winter barley cultivar Luxor, following the exposure of young plants to various periods of low (above and below zero) temperatures. A transcriptomic analysis identified genes which were either expressed in both the leaf and crown, or specifically in one or the other. Among the former were genes responsible for calcium and abscisic acid signalling, polyamine synthesis, late embryogenesis abundant proteins and dehydrins. In the crown, the key organ for cereal overwintering, cold treatment induced transient changes in the transcription of nucleosome assembly genes, and especially H2A and HTA11, which have been implicated in cold sensing in Arabidopsis thaliana. In the leaf, various heat-shock proteins were induced. Differences in expression pattern between the crown and leaf were frequent for genes involved in certain pathways responsible for osmolyte production (sucrose and starch, raffinose, γ-aminobutyric acid metabolism), sugar signalling (trehalose metabolism) and secondary metabolism (lignin synthesis). The action of proteins with antifreeze activity, which were markedly induced during hardening, was demonstrated by a depression in the ice nucleation temperature.
Electronic supplementary material
The online version of this article (doi:10.1007/s10142-011-0213-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s10142-011-0213-8
PMCID: PMC3098344  PMID: 21360135
Barley; Differentially expressed genes; Cold acclimation; Crown; Leaf; Metabolic pathways
5.  A KS-type dehydrin and its related domains reduce Cu-promoted radical generation and the histidine residues contribute to the radical-reducing activities 
Journal of Experimental Botany  2013;64(6):1615-1624.
Dehydrin is a plant disordered protein whose functions are not yet totally understood. Here it is reported that a KS-type dehydrin can reduce the formation of reactive oxygen species (ROS) from Cu. AtHIRD11, which is the Arabidopsis KS-type dehydrin, inhibited generation of hydrogen peroxide and hydroxyl radicals in the Cu–ascorbate system. The radical-reducing activity of AtHIRD11 was stronger than those of radical-silencing peptides such as glutathione and serum albumin. The addition of Cu2+ reduced the disordered state, decreased the trypsin susceptibility, and promoted the self-association of AtHIRD11. Domain analyses indicated that the five domains containing histidine showed ROS-reducing activities. Histidine/alanine substitutions indicated that histidine is a crucial residue for reducing ROS generation. Using the 27 peptides which are related to the KnS-type dehydrins of 14 plant species, it was found that the strengths of ROS-reducing activities can be determined by two factors, namely the histidine contents and the length of the peptides. The degree of ROS-reducing activities of a dehydrin can be predicted using these indices.
doi:10.1093/jxb/ert016
PMCID: PMC3617826  PMID: 23382551
Circular dichroism; dehydrin; disordered protein; heavy metal; histidine; reactive oxygen species.
6.  Dehydrin-like Proteins in the Necrotrophic Fungus Alternaria brassicicola Have a Role in Plant Pathogenesis and Stress Response 
PLoS ONE  2013;8(10):e75143.
In this study, the roles of fungal dehydrin-like proteins in pathogenicity and protection against environmental stresses were investigated in the necrotrophic seed-borne fungus Alternaria brassicicola. Three proteins (called AbDhn1, AbDhn2 and AbDhn3), harbouring the asparagine-proline-arginine (DPR) signature pattern and sharing the characteristic features of fungal dehydrin-like proteins, were identified in the A. brassicicola genome. The expression of these genes was induced in response to various stresses and found to be regulated by the AbHog1 mitogen-activated protein kinase (MAPK) pathway. A knock-out approach showed that dehydrin-like proteins have an impact mainly on oxidative stress tolerance and on conidial survival upon exposure to high and freezing temperatures. The subcellular localization revealed that AbDhn1 and AbDhn2 were associated with peroxisomes, which is consistent with a possible perturbation of protective mechanisms to counteract oxidative stress and maintain the redox balance in AbDhn mutants. Finally, we show that the double deletion mutant ΔΔabdhn1-abdhn2 was highly compromised in its pathogenicity. By comparison to the wild-type, this mutant exhibited lower aggressiveness on B. oleracea leaves and a reduced capacity to be transmitted to Arabidopsis seeds via siliques. The double mutant was also affected with respect to conidiation, another crucial step in the epidemiology of the disease.
doi:10.1371/journal.pone.0075143
PMCID: PMC3788798  PMID: 24098369
7.  Identification of Cis-Acting Promoter Elements in Cold- and Dehydration-Induced Transcriptional Pathways in Arabidopsis, Rice, and Soybean 
The genomes of three plants, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and soybean (Glycine max), have been sequenced, and their many genes and promoters have been predicted. In Arabidopsis, cis-acting promoter elements involved in cold- and dehydration-responsive gene expression have been extensively analysed; however, the characteristics of such cis-acting promoter sequences in cold- and dehydration-inducible genes of rice and soybean remain to be clarified. In this study, we performed microarray analyses using the three species, and compared characteristics of identified cold- and dehydration-inducible genes. Transcription profiles of the cold- and dehydration-responsive genes were similar among these three species, showing representative upregulated (dehydrin/LEA) and downregulated (photosynthesis-related) genes. All (46 = 4096) hexamer sequences in the promoters of the three species were investigated, revealing the frequency of conserved sequences in cold- and dehydration-inducible promoters. A core sequence of the abscisic acid-responsive element (ABRE) was the most conserved in dehydration-inducible promoters of all three species, suggesting that transcriptional regulation for dehydration-inducible genes is similar among these three species, with the ABRE-dependent transcriptional pathway. In contrast, for cold-inducible promoters, the conserved hexamer sequences were diversified among these three species, suggesting the existence of diverse transcriptional regulatory pathways for cold-inducible genes among the species.
doi:10.1093/dnares/dsr040
PMCID: PMC3276264  PMID: 22184637
plant genome; cis-acting promoter elements; cold; dehydration; microarray
8.  A novel family of dehydrin-like proteins is involved in stress response in the human fungal pathogen Aspergillus fumigatus 
Molecular Biology of the Cell  2011;22(11):1896-1906.
This article identifies novel stress-protective proteins that belong to the family of intrinsically unstructured proteins, DprA and DprB, which are associated with the cytosol and the peroxisomes in Aspergillus fumigatus.
 During a search for genes controlling conidial dormancy in Aspergillus fumigatus, two dehydrin-like genes, DprA and DprB, were identified. The deduced proteins had repeated stretches of 23 amino acids that contained a conserved dehydrin-like protein (DPR) motif. Disrupted DprAΔ mutants were hypersensitive to oxidative stress and to phagocytic killing, whereas DprBΔ mutants were impaired in osmotic and pH stress responses. However, no effect was observed on their pathogenicity in our experimental models of invasive aspergillosis. Molecular dissection of the signaling pathways acting upstream showed that expression of DprA was dependent on the stress-activated kinase SakA and the cyclic AMP-protein kinase A (cAMP-PKA) pathways, which activate the bZIP transcription factor AtfA, while expression of DprB was dependent on the SakA mitogen-activated protein kinase (MAPK) pathway, and the zinc finger transcription factor PacC. Fluorescent protein fusions showed that both proteins were associated with peroxisomes and the cytosol. Accordingly, DprA and DprB were important for peroxisome function. Our findings reveal a novel family of stress-protective proteins in A. fumigatus and, potentially, in filamentous ascomycetes.
doi:10.1091/mbc.E10-11-0914
PMCID: PMC3103405  PMID: 21490150
9.  Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress 
BMC Plant Biology  2012;12:140.
Background
Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew.
Results
Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles.
Conclusions
The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.
doi:10.1186/1471-2229-12-140
PMCID: PMC3460772  PMID: 22882870
Grapevine; Dehydrin; Stress-induced expression; Powdery mildew; Promoter
10.  Semi-viviparous embryo development and dehydrin expression in the mangrove Rhizophora mucronata Lam. 
Sexual Plant Reproduction  2010;23(2):95-103.
Rhizophora mucronata Lam. is a tropical mangrove with semi-viviparous (cotyledon body protrusion before shedding), non-quiescent and non-desiccating (recalcitrant) seeds. As recalcitrance has been thought to relate to the absence of desiccation-related proteins such as dehydrins, we for the first time systematically described and classified embryogenesis in R. mucronata and assessed the presence of dehydrin-like proteins. Embryogenesis largely follows the classic pattern till stage eight, the torpedo stage, with the formation of a cotyledonary body. Ovule and embryo express radical adaptations to semi-vivipary in the saline environment: (1) A large, highly vacuolated and persistent endosperm without noticeable food reserves that envelopes the developing embryo. (2) Absence of vascular tissue connections between embryo and maternal tissue, but, instead, transfer layers in between endosperm and integument and endosperm and embryo. Dehydrin-like proteins (55–65 kDa) were detected by the Western analysis, in the ovules till stage 10 when the integuments are dehisced. An additional 50 kDa band was detected at stages 6–8. Together these results suggest a continuous flow of water with nutrients from the integument via the endosperm to the embryo, circumventing the vascular route and probably suppressing the initially induced dehydrin expression.
doi:10.1007/s00497-009-0127-y
PMCID: PMC2874033  PMID: 20084524
Dehydrins; Embryo development; Mangroves; Rhizophora mucronata; Semi-vivipary
11.  Isolation and Characterization of cDNA Encoding Three Dehydrins Expressed During Coffea canephora (Robusta) Grain Development 
Annals of Botany  2006;97(5):755-765.
• Background and Aims Dehydrins, or group 2 late embryogenic abundant proteins (LEA), are hydrophilic Gly-rich proteins that are induced in vegetative tissues in response to dehydration, elevated salt, and low temperature, in addition to being expressed during the late stages of seed maturation. With the aim of characterizing and studying genes involved in osmotic stress tolerance in coffee, several full-length cDNA-encoding dehydrins (CcDH1, CcDH2 and CcDH3) and an LEA protein (CcLEA1) from Coffea canephora (robusta) were isolated and characterized.
• Methods The protein sequences deduced from the full-length cDNA were analysed to classify each dehydrin/LEA gene product and RT–PCR was used to determine the expression pattern of all four genes during pericarp and grain development, and in several other tissues of C. arabica and C. canephora. Primer-assisted genome walking was used to isolate the promoter region of the grain specific dehydrin gene (CcDH2).
• Key Results The CcDH1 and CcDH2 genes encode Y3SK2 dehydrins and the CcDH3 gene encodes an SK3 dehydrin. CcDH1 and CcDH2 are expressed during the final stages of arabica and robusta grain development, but only the CcDH1 transcripts are clearly detected in other tissues such as pericarp, leaves and flowers. CcDH3 transcripts are also found in developing arabica and robusta grain, in addition to being detected in pericarp, stem, leaves and flowers. CcLEA1 transcripts were only detected during a brief period of grain development. Finally, over 1 kb of genomic sequence potentially encoding the entire grain-specific promoter region of the CcDH2 gene was isolated and characterized.
• Conclusions cDNA sequences for three dehydrins and one LEA protein have been obtained and the expression of the associated genes has been determined in various tissues of arabica and robusta coffees. Because induction of dehydrin gene expression is associated with osmotic stress in other plants, the dehydrin sequences presented here will facilitate future studies on the induction and control of the osmotic stress response in coffee. The unique expression pattern observed for CcLEA1, and the expression of a related gene in other plants, suggests that this gene may play an important role in the development of grain endosperm tissue. Genomic DNA containing the grain-specific CcDH2 promoter region has been cloned. Sequence analysis indicates that this promoter contains several putative regulatory sites implicated in the control of both seed- and osmotic stress-specific gene expression. Thus, the CcDH2 promoter is likely to be a useful tool for basic studies on the control of gene expression during both grain maturation and osmotic stress in coffee.
doi:10.1093/aob/mcl032
PMCID: PMC2803416  PMID: 16504969
Dehydrins; late embryogenic abundant protein (LEA); seed development; Coffea; C. canephora; C. arabica; Rubiaceae
12.  Lipid and protein accumulation in developing seeds of three lupine species: Lupinus luteus L., Lupinus albus L., and Lupinus mutabilis Sweet 
Journal of Experimental Botany  2009;60(12):3453-3466.
A comparative study was carried out on the dynamics of lipid accumulation in developing seeds of three lupine species. Lupine seeds differ in lipid content; yellow lupine (Lupinus luteus L.) seeds contain about 6%, white lupine (Lupinus albus L.) 7–14%, and Andean lupine (Lupinus mutabilis Sweet) about 20% of lipids by dry mass. Cotyledons from developing seeds were isolated and cultured in vitro for 96 h on Heller medium with 60 mM sucrose (+S) or without sucrose (–S). Each medium was additionally enriched with 35 mM asparagine or 35 mM NaNO3. Asparagine caused an increase in protein accumulation and simultaneously decreased the lipid content, but nitrate increased accumulation of both protein and lipid. Experiments with [1-14C]acetate and [2-14C]acetate showed that the decrease in lipid accumulation in developing lupine seeds resulted from exhaustion of lipid precursors rather than from degradation or modification of the enzymatic apparatus. The carbon atom from the C-1 position of acetate was liberated mainly as CO2, whereas the carbon atom from the C-2 position was preferentially used in anabolic pathways. The dominant phospholipid in the investigated lupine seed storage organs was phosphatidylcholine. The main fatty acid in yellow lupine cotyledons was linoleic acid, in white lupine it was oleic acid, and in Andean lupine it was both linoleic and oleic acids. The relationship between stimulation of lipid and protein accumulation by nitrate in developing lupine cotyledons and enhanced carbon flux through glycolysis caused by the inorganic nitrogen form is discussed.
doi:10.1093/jxb/erp186
PMCID: PMC2724698  PMID: 19635747
Acetate; asparagine; carbon partitioning; fatty acids; nitrate; phospholipids; seed development; storage lipids; storage proteins; sucrose
13.  Sequence Evolution and Expression Regulation of Stress-Responsive Genes in Natural Populations of Wild Tomato 
PLoS ONE  2013;8(10):e78182.
The wild tomato species Solanum chilense and S. peruvianum are a valuable non-model system for studying plant adaptation since they grow in diverse environments facing many abiotic constraints. Here we investigate the sequence evolution of regulatory regions of drought and cold responsive genes and their expression regulation. The coding regions of these genes were previously shown to exhibit signatures of positive selection. Expression profiles and sequence evolution of regulatory regions of members of the Asr (ABA/water stress/ripening induced) gene family and the dehydrin gene pLC30-15 were analyzed in wild tomato populations from contrasting environments. For S. chilense, we found that Asr4 and pLC30-15 appear to respond much faster to drought conditions in accessions from very dry environments than accessions from more mesic locations. Sequence analysis suggests that the promoter of Asr2 and the downstream region of pLC30-15 are under positive selection in some local populations of S. chilense. By investigating gene expression differences at the population level we provide further support of our previous conclusions that Asr2, Asr4, and pLC30-15 are promising candidates for functional studies of adaptation. Our analysis also demonstrates the power of the candidate gene approach in evolutionary biology research and highlights the importance of wild Solanum species as a genetic resource for their cultivated relatives.
doi:10.1371/journal.pone.0078182
PMCID: PMC3799731  PMID: 24205149
14.  A dehydration-inducible gene in the truffle Tuber borchii identifies a novel group of dehydrins 
BMC Genomics  2006;7:39.
Background
The expressed sequence tag M6G10 was originally isolated from a screening for differentially expressed transcripts during the reproductive stage of the white truffle Tuber borchii. mRNA levels for M6G10 increased dramatically during fruiting body maturation compared to the vegetative mycelial stage.
Results
Bioinformatics tools, phylogenetic analysis and expression studies were used to support the hypothesis that this sequence, named TbDHN1, is the first dehydrin (DHN)-like coding gene isolated in fungi. Homologs of this gene, all defined as "coding for hypothetical proteins" in public databases, were exclusively found in ascomycetous fungi and in plants. Although complete (or almost complete) fungal genomes and EST collections of some Basidiomycota and Glomeromycota are already available, DHN-like proteins appear to be represented only in Ascomycota. A new and previously uncharacterized conserved signature pattern was identified and proposed to Uniprot database as the main distinguishing feature of this new group of DHNs. Expression studies provide experimental evidence of a transcript induction of TbDHN1 during cellular dehydration.
Conclusion
Expression pattern and sequence similarities to known plant DHNs indicate that TbDHN1 is the first characterized DHN-like protein in fungi. The high similarity of TbDHN1 with homolog coding sequences implies the existence of a novel fungal/plant group of LEA Class II proteins characterized by a previously undescribed signature pattern.
doi:10.1186/1471-2164-7-39
PMCID: PMC1550403  PMID: 16512918
15.  LEA polypeptide profiling of recalcitrant and orthodox legume seeds reveals ABI3-regulated LEA protein abundance linked to desiccation tolerance 
Journal of Experimental Botany  2013;64(14):4559-4573.
In contrast to orthodox seeds that acquire desiccation tolerance during maturation, recalcitrant seeds are unable to survive drying. These desiccation-sensitive seeds constitute an interesting model for comparative analysis with phylogenetically close species that are desiccation tolerant. Considering the importance of LEA (late embryogenesis abundant) proteins as protective molecules both in drought and in desiccation tolerance, the heat-stable proteome was characterized in cotyledons of the legume Castanospermum australe and it was compared with that of the orthodox model legume Medicago truncatula. RNA sequencing identified transcripts of 16 homologues out of 17 LEA genes for which polypeptides are detected in M. truncatula seeds. It is shown that for 12 LEA genes, polypeptides were either absent or strongly reduced in C. australe cotyledons compared with M. truncatula seeds. Instead, osmotically responsive, non-seed-specific dehydrins accumulated to high levels in the recalcitrant cotyledons compared with orthodox seeds. Next, M. truncatula mutants of the ABSCISIC ACID INSENSITIVE3 (ABI3) gene were characterized. Mature Mtabi3 seeds were found to be desiccation sensitive when dried below a critical water content of 0.4g H2O g DW–1. Characterization of the LEA proteome of the Mtabi3 seeds revealed a subset of LEA proteins with severely reduced abundance that were also found to be reduced or absent in C. australe cotyledons. Transcripts of these genes were indeed shown to be ABI3 responsive. The results highlight those LEA proteins that are critical to desiccation tolerance and suggest that comparable regulatory pathways responsible for their accumulation are missing in both desiccation-sensitive genotypes, revealing new insights into the mechanistic basis of the recalcitrant trait in seeds.
doi:10.1093/jxb/ert274
PMCID: PMC3808335  PMID: 24043848
abi3; Castanospermum australe; desiccation tolerance; late embryogenesis abundant proteins; Medicago truncatula; proteomics; recalcitrant seed; RNAseq.
16.  Interactions between cauliflower and Rhizoctonia anastomosis groups with different levels of aggressiveness 
BMC Plant Biology  2009;9:95.
Background
The soil borne fungus Rhizoctonia is one of the most important plant pathogenic fungi, with a wide host range and worldwide distribution. In cauliflower (Brassica oleracea var. botrytis), several anastomosis groups (AGs) including both multinucleate R. solani and binucleate Rhizoctonia species have been identified showing different levels of aggressiveness. The infection and colonization process of Rhizoctonia during pathogenic interactions is well described. In contrast, insights into processes during interactions with weak aggressive or non-pathogenic isolates are limited. In this study the interaction of cauliflower with seven R. solani AGs and one binucleate Rhizoctonia AG differing in aggressiveness, was compared. Using microscopic and histopathological techniques, the early steps of the infection process, the colonization process and several host responses were studied.
Results
For aggressive Rhizoctonia AGs (R. solani AG 1-1B, AG 1-1C, AG 2-1, AG 2-2 IIIb and AG 4 HGII), a higher developmental rate was detected for several steps of the infection process, including directed growth along anticlinal cell walls and formation of T-shaped branches, infection cushion formation and stomatal penetration. Weak or non-aggressive AGs (R. solani AG 5, AG 3 and binucleate Rhizoctonia AG K) required more time, notwithstanding all AGs were able to penetrate cauliflower hypocotyls. Histopathological observations indicated that Rhizoctonia AGs provoked differential host responses and pectin degradation. We demonstrated the pronounced deposition of phenolic compounds and callose against weak and non-aggressive AGs which resulted in a delay or complete block of the host colonization. Degradation of pectic compounds was observed for all pathogenic AGs, except for AG 2-2 IIIb. Ranking the AGs based on infection rate, level of induced host responses and pectin degradation revealed a strong correlation with the disease severity caused by the AGs.
Conclusion
The differences in aggressiveness towards cauliflower observed among Rhizoctonia AGs correlated with the infection rate, induction of host defence responses and pectin breakdown. All Rhizoctonia AGs studied penetrated the plant tissue, indicating all constitutive barriers of cauliflower were defeated and differences in aggressiveness were caused by inducible defence responses, including cell wall fortifications with phenolic compounds and callose.
doi:10.1186/1471-2229-9-95
PMCID: PMC2719643  PMID: 19622152
17.  The Physiology and Proteomics of Drought Tolerance in Maize: Early Stomatal Closure as a Cause of Lower Tolerance to Short-Term Dehydration? 
PLoS ONE  2012;7(6):e38017.
Understanding the response of a crop to drought is the first step in the breeding of tolerant genotypes. In our study, two maize (Zea mays L.) genotypes with contrasting sensitivity to dehydration were subjected to moderate drought conditions. The subsequent analysis of their physiological parameters revealed a decreased stomatal conductance accompanied by a slighter decrease in the relative water content in the sensitive genotype. In contrast, the tolerant genotype maintained open stomata and active photosynthesis, even under dehydration conditions. Drought-induced changes in the leaf proteome were analyzed by two independent approaches, 2D gel electrophoresis and iTRAQ analysis, which provided compatible but only partially overlapping results. Drought caused the up-regulation of protective and stress-related proteins (mainly chaperones and dehydrins) in both genotypes. The differences in the levels of various detoxification proteins corresponded well with the observed changes in the activities of antioxidant enzymes. The number and levels of up-regulated protective proteins were generally lower in the sensitive genotype, implying a reduced level of proteosynthesis, which was also indicated by specific changes in the components of the translation machinery. Based on these results, we propose that the hypersensitive early stomatal closure in the sensitive genotype leads to the inhibition of photosynthesis and, subsequently, to a less efficient synthesis of the protective/detoxification proteins that are associated with drought tolerance.
doi:10.1371/journal.pone.0038017
PMCID: PMC3374823  PMID: 22719860
18.  A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence. 
Nucleic Acids Research  1997;25(24):4970-4976.
A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.
PMCID: PMC147132  PMID: 9396804
19.  Effect of packaging on storage life and quality of cauliflower stored at low temperature 
Medium sized cauliflower (Brassica oleracea var. botrytis) curds after removing outer leaves, sorting and without washing were packed in different packaging films and stored at 0 ± 1°C, 90–95% RH. Changes in curd colour, texture, physiological loss in weight, spoilage and sensory quality were evaluated at weekly interval upto 28 days. Cauliflower curds individual packed in high density polyethylene bags (20 μm) with perforation (6 holes/bag) can be stored up to 21 days at 0 ± 1°C and 90–95% RH with maximum retention of white colour of curd, minimum spoilage, weight and firmness loss and good sensory quality attributes. The use of cling wrap films should be avoided as this leads to accumulation of excessive moisture resulting in huge spoilage loss.
doi:10.1007/s13197-010-0009-1
PMCID: PMC3550995  PMID: 23572616
Cauliflower; Low temperature storage; Packaging; Quality
20.  Low temperatures induce rapid changes in chromatin state and transcript levels of the cereal VERNALIZATION1 gene 
Journal of Experimental Botany  2013;64(8):2413-2422.
Transcriptional activation of the VERNALIZATION1 gene mediates the acceleration of flowering by prolonged cold (vernalization) in temperate cereals. This study examined the earliest stages of the transcriptional response of VRN1 to low temperatures. Time-course analyses, using a sensitive quantitative PCR assay, showed that in sprouting barley seedlings VRN1 transcripts begin to accumulate within 24 hours of the onset of cold. The kinetics of the initial transcriptional response of VRN1 to cold was similar to the cold-induced genes DEHYDRIN5 (DHN5) and COLD REGULATED 14B (COR14B), but occurred at lower levels compared to cold acclimation genes or the response to longer cold treatments. Temperatures between 15 and –2 °C induced expression of VRN1 within 24 hours, with a maximal response observed between 2 and –2 °C. Transcriptional induction was also observed in undifferentiated callus cells. There were significant increases in histone acetylation levels at the VRN1 locus in response to 24-hour cold treatment. Sodium butyrate, a histone deacetylation inhibitor, triggered an increase in histone acetylation at VRN1 chromatin and elevated VRN1 transcript levels. The transcriptional response of VRN1 to short-term cold treatment was examined in near-isogenic lines that have different VRN1 genotypes, showing that an allele of the barley VRN1 gene with an insertion in the first intron and high basal expression levels has a reduced transcriptional response to short term cold treatment. This study suggests that low-temperature induction of VRN1 is a cellular response to cold triggered by the same mechanisms that mediate low-temperature induction of cold acclimation genes.
doi:10.1093/jxb/ert095
PMCID: PMC3654426  PMID: 23580755
Chromatin; cereal; cold acclimation; MADS box; vernalization; VRN1.
21.  Complex Regulation by Apetala2 Domain-Containing Transcription Factors Revealed through Analysis of the Stress-Responsive TdCor410b Promoter from Durum Wheat 
PLoS ONE  2013;8(3):e58713.
Expression of the wheat dehydrin gene Cor410b is induced several fold above its non-stressed levels upon exposure to stresses such as cold, drought and wounding. Deletion analysis of the TdCor410b promoter revealed a single functional C-repeat (CRT) element. Seven transcription factors (TFs) were shown to bind to this CRT element using yeast one-hybrid screens of wheat and barley cDNA libraries, of which only one belonged to the DREB class of TFs. The remaining six encoded ethylene response factors (ERFs) belong to three separate subfamilies. Analysis of binding selectivity of these TFs indicated that all seven could bind to the CRT element (GCCGAC), and that three of the six ERFs could bind both to the CRT element and the ethylene-responsive GCC-box (GCCGCC). The TaERF4 subfamily members specifically bound the CRT element, and did not bind either the GCC-box or DRE element (ACCGAC). Molecular modeling and site-directed mutagenesis identified a single residue Pro42 in the Apetala2 (AP2) domain of TaERF4-like proteins that is conserved in monocotyledonous plants and is responsible for the recognition selectivity of this subfamily. We suggest that both DREB and ERF proteins regulate expression of the Cor410b gene through a single, critical CRT element. Members of the TaERF4 subfamily are specific, positive regulators of Cor410b gene expression.
doi:10.1371/journal.pone.0058713
PMCID: PMC3602543  PMID: 23527011
22.  A novel method for efficient and abundant production of Phytophthora brassicae zoospores on Brussels sprout leaf discs 
BMC Plant Biology  2009;9:111.
Background
Phytophthora species are notorious oomycete pathogens that cause diseases on a wide range of plants. Our understanding how these pathogens are able to infect their host plants will benefit greatly from information obtained from model systems representative for plant-Phytophthora interactions. One attractive model system is the interaction between Arabidopsis and Phytophthora brassicae. Under laboratory conditions, Arabidopsis can be easily infected with mycelial plugs as inoculum. In the disease cycle, however, sporangia or zoospores are the infectious propagules. Since the current P. brassicae zoospore isolation methods are generally regarded as inefficient, we aimed at developing an alternative method for obtaining high concentrations of P. brassicae zoospores.
Results
P. brassicae isolates were tested for pathogenicity on Brussels sprout plants (Brassica oleracea var. gemmifera). Microscopic examination of leaves, stems and roots infected with a GFP-tagged transformant of P. brassicae clearly demonstrated the susceptibility of the various tissues. Leaf discs were cut from infected Brussels sprout leaves, transferred to microwell plates and submerged in small amounts of water. In the leaf discs the hyphae proliferated and abundant formation of zoosporangia was observed. Upon maturation the zoosporangia released zoospores in high amounts and zoospore production continued during a period of at least four weeks. The zoospores were shown to be infectious on Brussels sprouts and Arabidopsis.
Conclusion
The in vitro leaf disc method established from P. brassicae infected Brussels sprout leaves facilitates convenient and high-throughput production of infectious zoospores and is thus suitable to drive small and large scale inoculation experiments. The system has the advantage that zoospores are produced continuously over a period of at least one month.
doi:10.1186/1471-2229-9-111
PMCID: PMC2752460  PMID: 19698127
23.  Molecular and Functional Characterization of Broccoli EMBRYONIC FLOWER 2 Genes 
Plant and Cell Physiology  2012;53(7):1217-1231.
Polycomb group (PcG) proteins regulate major developmental processes in Arabidopsis. EMBRYONIC FLOWER 2 (EMF2), the VEFS domain-containing PcG gene, regulates diverse genetic pathways and is required for vegetative development and plant survival. Despite widespread EMF2-like sequences in plants, little is known about their function other than in Arabidopsis and rice. To study the role of EMF2 in broccoli (Brassica oleracea var. italica cv. Elegance) development, we identified two broccoli EMF2 (BoEMF2) genes with sequence homology to and a similar gene expression pattern to that in Arabidopsis (AtEMF2). Reducing their expression in broccoli resulted in aberrant phenotypes and gene expression patterns. BoEMF2 regulates genes involved in diverse developmental and stress programs similar to AtEMF2 in Arabidopsis. However, BoEMF2 differs from AtEMF2 in the regulation of flower organ identity, cell proliferation and elongation, and death-related genes, which may explain the distinct phenotypes. The expression of BoEMF2.1 in the Arabidopsis emf2 mutant (Rescued emf2) partially rescued the mutant phenotype and restored the gene expression pattern to that of the wild type. Many EMF2-mediated molecular and developmental functions are conserved in broccoli and Arabidopsis. Furthermore, the restored gene expression pattern in Rescued emf2 provides insights into the molecular basis of PcG-mediated growth and development.
doi:10.1093/pcp/pcs063
PMCID: PMC3391658  PMID: 22537758
Brassica oleracea var. italica; Broccoli; EMBRYONIC FLOWER 2; Flowering; Polycomb Group Protein
24.  Genotypic variation in genome-wide transcription profiles induced by insect feeding: Brassica oleracea – Pieris rapae interactions 
BMC Genomics  2007;8:239.
Background
Transcriptional profiling after herbivore attack reveals, at the molecular level, how plants respond to this type of biotic stress. Comparing herbivore-induced transcriptional responses of plants with different phenotypes provides insight into plant defense mechanisms. Here, we compare the global gene expression patterns induced by Pieris rapae caterpillar attack in two white cabbage (Brassica oleracea var. capitata) cultivars. The two cultivars are shown to differ in their level of direct defense against caterpillar feeding. Because Brassica full genome microarrays are not yet available, 70-mer oligonucleotide microarrays based on the Arabidopsis thaliana genome were used for this non-model plant.
Results
The transcriptional responses of the two cultivars differed in timing as characterized by changes in their expression pattern after 24, 48 and 72 hours of caterpillar feeding. In addition, they also differed qualitatively. Surprisingly, of all genes induced at any time point, only one third was induced in both cultivars. Analyses of transcriptional responses after jasmonate treatment revealed that the difference in timing did not hold for the response to this phytohormone. Additionally, comparisons between Pieris rapae- and jasmonate-induced transcriptional responses showed that Pieris rapae induced more jasmonate-independent than jasmonate-dependent genes.
Conclusion
The present study clearly shows that global transcriptional responses in two cultivars of the same plant species in response to insect feeding can differ dramatically. Several of these differences involve genes that are known to have an impact on Pieris rapae performance and probably underlie different mechanisms of direct defense, present in the cultivars.
doi:10.1186/1471-2164-8-239
PMCID: PMC1940009  PMID: 17640338
25.  Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways 
BMC Genomics  2007;8:125.
Background
The heat shock response of Arabidopsis thaliana is dependent upon a complex regulatory network involving twenty-one known transcription factors and four heat shock protein families. It is known that heat shock proteins (Hsps) and transcription factors (Hsfs) are involved in cellular response to various forms of stress besides heat. However, the role of Hsps and Hsfs under cold and non-thermal stress conditions is not well understood, and it is unclear which types of stress interact least and most strongly with Hsp and Hsf response pathways. To address this issue, we have analyzed transcriptional response profiles of Arabidopsis Hsfs and Hsps to a range of abiotic and biotic stress treatments (heat, cold, osmotic stress, salt, drought, genotoxic stress, ultraviolet light, oxidative stress, wounding, and pathogen infection) in both above and below-ground plant tissues.
Results
All stress treatments interact with Hsf and Hsp response pathways to varying extents, suggesting considerable cross-talk between heat and non-heat stress regulatory networks. In general, Hsf and Hsp expression was strongly induced by heat, cold, salt, and osmotic stress, while other types of stress exhibited family or tissue-specific response patterns. With respect to the Hsp20 protein family, for instance, large expression responses occurred under all types of stress, with striking similarity among expression response profiles. Several genes belonging to the Hsp20, Hsp70 and Hsp100 families were specifically upregulated twelve hours after wounding in root tissue, and exhibited a parallel expression response pattern during recovery from heat stress. Among all Hsf and Hsp families, large expression responses occurred under ultraviolet-B light stress in aerial tissue (shoots) but not subterranean tissue (roots).
Conclusion
Our findings show that Hsf and Hsp family member genes represent an interaction point between multiple stress response pathways, and therefore warrant functional analysis under conditions apart from heat shock treatment. In addition, our analysis revealed several family and tissue-specific heat shock gene expression patterns that have not been previously described. These results have implications regarding the molecular basis of cross-tolerance in plant species, and raise new questions to be pursued in future experimental studies of the Arabidopsis heat shock response network.
doi:10.1186/1471-2164-8-125
PMCID: PMC1887538  PMID: 17519032

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