Early Responsive To Dehydration Research Articles

A putative NF-Y complex interacting with ERD15 may positively regulate the expression of a peroxidase gene in response to stress in rapeseed (Brassica napus L.)

Drought stress is one of the major constraints on crop productivity, including rapeseed (Brassica napus L.). Nuclear factors Y (NF-Ys) are important transcription factors involved in plant responses to drought and other stresses. However, the underlying molecular mechanisms remain unclear in rapeseed. By silencing BnaNF-YA9 in rapeseed and transforming BnaNF-YA9 into the Arabidopsis mutant Atnf-ya5, we demonstrated that BnaNF-YA9 plays a positive role in drought resistance. To explore its regulatory mechanism, we performed protein-protein interaction analyses using various approaches. Our study revealed complex interactions among BnaNF-YA9, BnaNF-YB2, BnaNF-YC4, and EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15), suggesting that these proteins form a multimember complex. We also showed that BnaNF-YA9 binds to the CCAAT element in the promoter of a BnaPRX gene, which encodes a peroxidase. Interestingly, overexpression of BnaNF-YC4 or BnaERD15 in Arabidopsis increased sensitivity to salt stress, drought, and abscisic acid. Our results support an NF-Y/ERD15/PRX cascade and suggest a complex regulatory network in rapeseed that may be important in maintaining ROS homeostasis during abiotic stress responses. Our findings provide insights into potential targets for improving drought resilience in crops.

A viral small interfering RNA-host plant mRNA pathway modulates virus-induced drought tolerance by enhancing autophagy.

Virus-induced drought tolerance presents a fascinating facet of biotic-abiotic interaction in plants, yet its molecular intricacies remain unclear. Our study shows that cowpea mild mottle virus (CPMMV) infection enhances drought tolerance in common bean (Phaseolus vulgaris) plants through a virus-derived small interfering RNA (vsiRNA)-activated autophagy pathway. Specifically, a 21 nt vsiRNA originating from the CPMMV Triple Gene Block1 (TGB1) gene targeted the 5' untranslated region (UTR) of the host Teosinte branched 1, Cycloidea, Proliferating Cell Factor (TCP) transcription factor gene PvTCP2, independent of the known role of TGB1 as an RNA silencing suppressor. This targeting attenuated the expression of PvTCP2, which encodes a transcriptional repressor, and in turn upregulated the core autophagy-related gene (ATG) PvATG8c, leading to activated autophagy activity surpassing the level induced by drought or CPMMV infection alone. The downstream EARLY RESPONSIVE TO DEHYDRATION (ERD) effector PvERD15 is a homologue of Arabidopsis thaliana AtERD15, which positively regulates stomatal aperture. PvERD15 was degraded in PvATG8c-mediated autophagy. Therefore, we establish a TGB1-PvTCP2-PvATG8c-PvERD15 module as a trans-kingdom fine-tuning mechanism that contributes to virus-induced drought tolerance in plant-drought-virus interactions.

Arabidopsis ERD15 regulated by BBX24 plays a positive role in UV-B signaling

Ultraviolet-B (UV-B, 280-315 nm) is a minor component of solar radiation, but it has a major regulatory impact on plant growth and development. Solar UV-B regulates numerous aspects of plant metabolism, morphology and physiology through altering the expression of hundreds of genes. EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) is a drought-induced rapid response gene, formerly known as a negative regulator of the abscisic acid (ABA) signaling pathway. It is unclear whether ERD15 is involved in UV-B-induced photomorphogenesis. Previously, we reported that the BBX24 transcriptional factor negatively regulated UV-B signaling. In the present study, we identified that ERD15 is involved in UV-B photomorphogenesis as a positive regulator at phenotypic, physiological and molecular levels. Our results indicated that ERD15 expression is suppressed by UV-B, inhibited the elongation of Arabidopsis hypocotyls in a UV-B-dependent manner, promoted the expression of related UV-B signaling genes and increased the total antioxidant capacity of Arabidopsis under UV-B. Genetic hybridization results show that ERD15 acts downstream of BBX24, and BBX24 protein mediated the expression of ERD15 by binding to its promoter. Thus, ERD15 is a novel positive regulator of the UV-B signaling pathway, which is downstream of BBX24 and regulated by BBX24 protein to participate in UV-B photomorphogenesis.

Phosphorylation of Thr-225 and Ser-262 on ERD7 Promotes Age-Dependent and Stress-Induced Leaf Senescence through the Regulation of Hydrogen Peroxide Accumulation in Arabidopsis thaliana.

As the final stage of leaf development, leaf senescence is affected by a variety of internal and external signals including age and environmental stresses. Although significant progress has been made in elucidating the mechanisms of age-dependent leaf senescence, it is not clear how stress conditions induce a similar process. Here, we report the roles of a stress-responsive and senescence-induced gene, ERD7 (EARLY RESPONSIVE TO DEHYDRATION 7), in regulating both age-dependent and stress-induced leaf senescence in Arabidopsis. The results showed that the leaves of erd7 mutant exhibited a significant delay in both age-dependent and stress-induced senescence, while transgenic plants overexpressing the gene exhibited an obvious accelerated leaf senescence. Furthermore, based on the results of LC-MS/MS and PRM quantitative analyses, we selected two phosphorylation sites, Thr-225 and Ser-262, which have a higher abundance during senescence, and demonstrated that they play a key role in the function of ERD7 in regulating senescence. Transgenic plants overexpressing the phospho-mimetic mutant of the activation segment residues ERD7T225D and ERD7T262D exhibited a significantly early senescence, while the inactivation segment ERD7T225A and ERD7T262A displayed a delayed senescence. Moreover, we found that ERD7 regulates ROS accumulation by enhancing the expression of AtrbohD and AtrbohF, which is dependent on the critical residues, i.e., Thr-225 and Ser-262. Our findings suggest that ERD7 is a positive regulator of senescence, which might function as a crosstalk hub between age-dependent and stress-induced leaf senescence.

Analyses of leaf morphological changes and expressed genes related to water shortage of Fagus orientalis populations in a drought gradient

Climate change models predict an increase in mean annual temperature, a decrease in precipitation and consequently a scarcity of water resources during the growing season for Iran, including the northern forests of the country. One of the consequences is increased water shortage stress during the summer season. Fagus orientalis Lipsky is one of the most important commercial broad leaf species in the Hyrcanian forests and its potential of phenotypic and genetic adaptation to the climate change is essentially vital. The present research was carried out along two precipitation gradients from very humid to semi-humid conditions on four beech populations. The results of the flexibility of the leaf morphological traits showed that the populations of dryer regions had smaller and more elongated leaves than other populations. Evaluation of the expression of seven candidate genes in response to water shortage showed that the expression of aldehyde dehydrogenase, ascorbate peroxidase, Dehydrin (DHN), early responsive to dehydration, late embryogenesis abundant and Cys/His-Type Zinc-Finger in the eastern population was higher than that in the western population, while a difference in elevation gradient was only observed in the expression of some genes. Therefore, the natural selection pressure in the past evolutionary periods in the westeast gradient seems to be higher than the elevation gradient and has led to greater resistance to drought. Principal component analysis revealed that aldehyde dehydrogenase genes (ALDH), early responsive to dehydration (ERD), late embryogenesis abundant (LEA) and DHN are the most effective genes responding to water shortage stress for Oriental beech in the Hyrcanian forests. Keywords: drought gradient, Fagus orientalis, gene expression, leaf morphology

Overexpression of the VaERD15 gene increases cold tolerance in transgenic grapevine

In plants, the Early Responsive to Dehydration (ERD) genes are rapidly activated in response to drought and other abiotic stresses. Here, we characterize the VaERD15 gene from the cold-resistant grapevine Vitis amurensis accession 'Shuangyou', and its counterpart VvERD15 from the cold-sensitive V. vinifera cultivar 'Red Globe'. To evaluate a role for VaERD15 in promoting cold tolerance, we expressed the VaERD15 in transgenic V. vinifera 'Red Globe' and evaluated its influence on type and cold stress-related physiological and biochemical indices. We also assessed the response of the ERD15 promoter and its deletion fragments from 'Shuangyou' and 'Red Globe' to low temperature, ABA, SA and PEG. Additionally, several proteins interacting with VaERD15 were identified and the expression profiles for the respective genes were evaluated in response to low temperature. We found that the VaERD15 protein contains a PAM2 domain and localizes in the nucleus and cell membrane. Under low-temperature stress, the expression of ERD15 in 'Shuangyou' was greater than that in 'Red Globe’. Transgenic 35S::VaERD15 plants showed greater cold tolerance and had lower electrical conductivity and malondialdehyde content than non-transgenic controls, while the contents of proline, soluble sugar, soluble protein and the activities of SOD, POD and CAT were higher than those in non-transgenic controls. The ERD15 promoter and its deletions could respond to low temperature, ABA, SA and PEG, but the degree of response to the treatments varied. Proteins interacting with VaERD15 were identified as xyloglucan endotransglucosylase/hydrolase protein 7, glutamine synthetase, 40S ribosomal protein 23 and disulfide-isomerase LQY1, and the corresponding genes responded to cold temperatures to varying degrees.

Zinc-finger protein MdBBX7/MdCOL9, a target of MdMIEL1 E3 ligase, confers drought tolerance in apple.

Water deficit is one of the main challenges for apple (Malus × domestica) growth and productivity. Breeding drought-tolerant cultivars depends on a thorough understanding of the drought responses of apple trees. Here, we identified the zinc-finger protein B-BOX 7/CONSTANS-LIKE 9 (MdBBX7/MdCOL9), which plays a positive role in apple drought tolerance. The overexpression of MdBBX7 enhanced drought tolerance, whereas knocking down MdBBX7 expression reduced it. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis identified one cis-element of MdBBX7, CCTTG, as well as its known binding motif, the T/G box. ChIP-seq and RNA-seq identified 1,197 direct targets of MdBBX7, including ETHYLENE RESPONSE FACTOR (ERF1), EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15), and GOLDEN2-LIKE 1 (GLK1) and these were further verified by ChIP-qPCR and electronic mobility shift assays. Yeast two-hybrid screen identified an interacting protein of MdBBX7, RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1). Further examination revealed that MdMIEL1 could mediate the ubiquitination and degradation of MdBBX7 by the 26S proteasome pathway. Genetic interaction analysis suggested that MdMIEL1 acts as an upstream factor of MdBBX7. In addition, MdMIEL1 was a negative regulator of the apple drought stress response. Taken together, our results illustrate the molecular mechanisms by which the MdMIEL1-MdBBX7 module influences the response of apple to drought stress.

Arabidopsis thaliana EARLY RESPONSIVE TO DEHYDRATION 7 Localizes to Lipid Droplets via Its Senescence Domain.

Lipid droplets (LDs) are neutral-lipid-containing organelles found in all kingdoms of life and are coated with proteins that carry out a vast array of functions. Compared to mammals and yeast, relatively few LD proteins have been identified in plants, particularly those associated with LDs in vegetative (non-seed) cell types. Thus, to better understand the cellular roles of LDs in plants, a more comprehensive inventory and characterization of LD proteins is required. Here, we performed a proteomics analysis of LDs isolated from drought-stressed Arabidopsis leaves and identified EARLY RESPONSIVE TO DEHYDRATION 7 (ERD7) as a putative LD protein. mCherry-tagged ERD7 localized to both LDs and the cytosol when ectopically expressed in plant cells, and the protein’s C-terminal senescence domain (SD) was both necessary and sufficient for LD targeting. Phylogenetic analysis revealed that ERD7 belongs to a six-member family in Arabidopsis that, along with homologs in other plant species, is separated into two distinct subfamilies. Notably, the SDs of proteins from each subfamily conferred targeting to either LDs or mitochondria. Further, the SD from the ERD7 homolog in humans, spartin, localized to LDs in plant cells, similar to its localization in mammals; although, in mammalian cells, spartin also conditionally localizes to other subcellular compartments, including mitochondria. Disruption of ERD7 gene expression in Arabidopsis revealed no obvious changes in LD numbers or morphology under normal growth conditions, although this does not preclude a role for ERD7 in stress-induced LD dynamics. Consistent with this possibility, a yeast two-hybrid screen using ERD7 as bait identified numerous proteins involved in stress responses, including some that have been identified in other LD proteomes. Collectively, these observations provide new insight to ERD7 and the SD-containing family of proteins in plants and suggest that ERD7 may be involved in functional aspects of plant stress response that also include localization to the LD surface.

Investigation of the response to salinity of transgenic potato plants overexpressing the transcription factor StERF94

Salinity is one of the most important constraints threatening the cultivation of potato plants (Solanum tuberosum L.). It affects plant growth and leads to significant yield loss. Consequently, it is important to improve the tolerance of potato plants to salinity. In this context, we investigated the involvement of a potato ethylene responsive factor (StERF94) in plant response to salinity, since our previous genome-wide analysis showed that it may be related to biotic and abiotic stress response. ERF proteins belong to a large family of transcription factors that participate in plant response to abiotic stresses. We have previously identified the StERF94 gene which shows increased expression in potato plants submitted to salt treatment. In this study, transgenic potato plants overexpressing StERF94 were produced and submitted to salt treatment (100 mM NaCl) in vitro and under greenhouse culture conditions. StERF94 transgenic lines showed lower decrease of stem elongation under salt treatment in comparison to non-transgenic wild-type plants. Moreover, these plants showed a low level of H2O2 and Malondialdehyde content, and an increase in catalase and GPX (Gluthation peroxidase) activities compared to non-transgenic plants. In a second step, enhanced expression of some target genes for example CuZn-SOD, DHN25 (Dehydrin) and ERD (Early Responsive to Dehydration) was noted in the StERF94 transgenic plants, submitted to salt treatment. The StERF94 factor was also involved in the activation of osmoprotectant synthesis. Taken together, all these data suggest that overexpression of the StERF94 transcription factor increases the tolerance of potato plants to salinity by improving plant growth, osmoprotectant synthesis and antioxidant activityleading to low oxidative stress damage.

Cloning and Expression Analysis of ZmERD3 Gene From Zea mays.

BackgroundStresses (such as drought, salt, viruses, and others) seriously affect plant productivity. To cope with these threats, plants express a large number of genes, including several members of ERD (early responsive to dehydration) genes to synthesize and assemble adaptive molecules. But, the function of ERD3 gene hasn’t been known so far.ObjectivesThe purpose of the present study was to clone the stress-resistance gene: ZmERD3, and to analyze its expression pattern in the maize plant organs at different stages and under various stress treatments.Materials and MethodsMaizeGDB database search together with the bioinformatics analysis led to the identification of ZmERD3 gene in Zea mays. The cDNA sequence and promoter of ZmERD3 gene were obtained through PCR. Bioinformatics analysis was performed through online tools. The tissue-specific expression profile of the ZmERD3 gene in maize plant was carried out using the quantitative real time PCR (qRT-PCR) technique and its expression pattern in response to stress treatments (such as PEG, NaCl, ABA, and low temperature) was also analyzed through qRT-PCR method.ResultsBased on the homology alignment with AtERD3 (XP_002867953) in MaizeGDB (http://www. maizegdb.org/), the cDNA sequence and promoter region of the ZmERD3 gene were obtained. The bioinformatic analysis showed that ZmERD3 protein has one specific hit of methyltransferase and a high probability of location in the cytoplasm, and there are many cis-regulatory elements responsive to light, heat, cold, dehydration, as well as other stresses in its promoter sequence. Expression analysis revealed that the amount of ZmERD3 mRNA is different in all indicated organs of the maize plant. In addition, the ZmERD3 expression could be induced by abiotic stress treatments. Compared to the control, treatment with NaCl or PEG-6000 could significantly enhance the expression ability of ZmERD3 gene. As well, its expression level was increased about 20 times above the control after exposure to NaCl and PEG-6000 treatments for 3-6 h.ConclusionsOne putative methyltransferase gene, ZmERD3 was cloned. ZmERD3 expression exhibited an obvious tissue-specificity, and its expression could make a significant response to NaCl and PEG-6000 treatments.

VaERD15, a Transcription Factor Gene Associated with Cold-Tolerance in Chinese Wild Vitis amurensis.

Early responsive to dehydration (ERD) genes can be rapidly induced to counteract abiotic stresses, such as drought, low temperatures or high salinities. Here, we report on an ERD gene (VaERD15) related to cold tolerance from Chinese wild Vitis amurensis accession ‘Heilongjiang seedling’. The full-length VaERD15 cDNA is 685 bp, including a 66 bp 5′-untranslated region (UTR), a 196 bp 3′-UTR region and a 423 bp open reading frame encoding 140 amino acids. The VaERD15 protein shares a high amino acid sequence similarity with ERD15 of Arabidopsis thaliana. In our study, VaERD15 was shown to have a nucleic localization function and a transcriptional activation function. Semi-quantitative PCR and Western blot analyses showed that VaERD15 was constitutively expressed in young leaves, stems and roots of V. amurensis accession ‘Heilongjiang seedling’ plants, and expression levels increased after low-temperature treatment. We also generated a transgenic Arabidopsis Col-0 line that over-expressed VaERD15 and carried out a cold-treatment assay. Real-time quantitative PCR (qRT-PCR) and Western blot analyses showed that as the duration of cold treatment increased, the expression of both gene and protein levels increased continuously in the transgenic plants, while almost no expression was detected in the wild type Arabidopsis. Moreover, the plants that over-expressed VaERD15 showed higher cold tolerance and accumulation of proline, soluble sugars, proteins, malondialdehyde and three antioxidases (superoxide dismutase, peroxidase, and catalase). Lower levels of relative ion leakage also occurred under cold stress. Taken together, our results indicate that the transcription factor VaERD15 was induced by cold stress and was able to enhance cold tolerance.

Transcription activation activity of ERD15 protein from Morus indica

Early Responsive to Dehydration (ERD) genes are described as rapid response mediators of dehydration stress. Recently, ERD15 has emerged as a novel stress induced transcription factor which might be involved in mediating distinct stress responses in plants. In order to determine whether mulberry ERD15 can act as functional transcription factor, yeast-based assays were performed. Mulberry ERD15 was found to drive high level reporter gene expression in yeast which suggests it may function as a transcription factor. However, due to lack of an identifiable DNA binding domain, deletion analysis was carried out to determine the putative region of the protein involved in mediating protein-DNA interaction. Our results indicate that the region between 70 and 100 amino acids is critical in conferring transcription activation activity and might harbor the DNA binding region of ERD15.

English

Plant physiology, morphology and biochemistry are genetically controlled by a large set of genes. Expression of these genes is influenced by a number of factors, internal as well as external, including stage of plant development, diseases, drought, salinity, cold, and many others (Yoshioka et al., 2003; Zhang et al., 2001). Some genes are activated or suppressed by a specific stress factor while, many genes respond to more than one stimulus (Kariola et al., 2006). These genes can be classified, on the basis of their expression patterns and/or their responsiveness to environmental stressors, in different groups viz., responsive to dehydration (RD), cold regulated (COR), cold inducible (KIN), lowtemperature induced (LTI), early responsive to dehydration (ERD), salt overly sensitive (SOS), dehydration-responsive element-binding proteins (DREBs) and many more (Shinozaki and Yamaguchi-Shinozaki, 2007; Mahajan and Tuteja, 2005). Among these genes, ERDs have emerged as a new class or group of genes, identified first time by Kiyosue et al. (1994) in Arabidopsis plants dehydrated for one hour. Some members of the ERD group have been studied such as ERD1 (that encodes a Clp protease regulatory subunit; Nakashima et al., 1997), ERD5 (proline dehydrogenase; Kiyosue et al., 1996), ERD8 (hsp8-1; Takahashi et al., 1992), ERD10 (Group II LEA protein = lti29/lti45; Welin et al., 1995) and ERD15 (Kariola et al., 2006; Alves et al., 2011; Ziaf et al., 2011). Among these ERD genes, ERD15 has been used as a stress responsive gene in various stress experiments (Dunaeva and Adamska, 2001; Park et al., 2009; Li et al., 2010) in Arabidopsis and wheat. Nevertheless, its induction and function has been contradictory (Kariola et al., 2006; Ziaf et al., 2011), which can be due to difference in sequence among the species and/or due to some mutations in the sequence from specific crop. Transcript of several genes is accumulated at a specific stage of plant growth or in specific organs, varying to some extent with their function. Some are under the control of endogenous circadian system and thus affect the physiology and metabolism (Yakir et al., 2007). About 6% genes in Arabidopsis, involved in various functions such as photosynthesis, responses to stresses, sugar metabolism and flowering in response to day-length, fluctuate daily and are controlled by circadian clock (Harmer et al., 2000). Wang and Grumet (2004) reported diurnal pattern for transcript level of Pak. J. Agri. Sci., Vol. 53(1), 27-33; 2016 ISSN (Print) 0552-9034, ISSN (Online) 2076-0906 DOI: 10.21162/PAKJAS/16.3695 http://www.pakjas.com.pk

Intra-specific variations in expression of stress-related genes in beech progenies are stronger than drought-induced responses

Rapidly decreasing water availability as a consequence of climate change is likely to endanger the range of long-lived tree species. A pressing question is, therefore, whether adaptation to drought exists in important temperate tree species like European beech (Fagus sylvatica L.), a wide-spread, dominant forest tree in Central Europe. Here, five beech stands were selected along a precipitation gradient from moist to dry conditions. Neutral genetic markers revealed strong variation within and little differentiation between the populations. Natural regeneration from these stands was transferred to a common garden and used to investigate the expression of genes for abscisic acid (ABA)-related drought signaling [9-cis-epoxy-dioxygenase (NCED), protein phosphatase 2C (PP2C), early responsive to dehydration (ERD)] and stress protection [ascorbate peroxidase (APX), superoxide dismutase (SOD), aldehyde dehydrogenase (ALDH), glutamine amidotransferase (GAT)] that are involved in drought acclimation. We hypothesized that progenies from dry sites exhibit constitutively higher expression levels of ABA- and stress-related genes and are less drought responsive than progenies from moist sites. Transcript levels and stress responses (leaf area loss, membrane integrity) of well-irrigated and drought-stressed plants were measured during the early, mid- and late growing season. Principal component (PC) analysis ordered the beech progenies according to the mean annual precipitation at tree origin by the transcript levels of SOD, ALDH, GAT and ERD as major loadings along PC1. PC2 separated moist and drought treatments with PP2C levels as important loading. These results suggest that phosphatase-mediated signaling is flexibly acclimated to the current requirements, whereas stress compensatory measures exhibited genotypic variation, apparently underlying climate selection. In contrast to expectation, the drought responses were less pronounced than the progeny-related differences and the transcript levels were constitutively lower in beeches from dry than from moist sites. These results imply that beeches from dry origins may have evolved mechanisms to avoid oxidative stress.

Membrane Topology and Predicted RNA-Binding Function of the ‘Early Responsive to Dehydration (ERD4)’ Plant Protein

Functional annotation of uncharacterized genes is the main focus of computational methods in the post genomic era. These tools search for similarity between proteins on the premise that those sharing sequence or structural motifs usually perform related functions, and are thus particularly useful for membrane proteins. Early responsive to dehydration (ERD) genes are rapidly induced in response to dehydration stress in a variety of plant species. In the present work we characterized function of Brassica juncea ERD4 gene using computational approaches. The ERD4 protein of unknown function possesses ubiquitous DUF221 domain (residues 312–634) and is conserved in all plant species. We suggest that the protein is localized in chloroplast membrane with at least nine transmembrane helices. We detected a globular domain of 165 amino acid residues (183–347) in plant ERD4 proteins and expect this to be posited inside the chloroplast. The structural-functional annotation of the globular domain was arrived at using fold recognition methods, which suggested in its sequence presence of two tandem RNA-recognition motif (RRM) domains each folded into βαββαβ topology. The structure based sequence alignment with the known RNA-binding proteins revealed conservation of two non-canonical ribonucleoprotein sub-motifs in both the putative RNA-recognition domains of the ERD4 protein. The function of highly conserved ERD4 protein may thus be associated with its RNA-binding ability during the stress response. This is the first functional annotation of ERD4 family of proteins that can be useful in designing experiments to unravel crucial aspects of stress tolerance mechanism.

A gene expression analysis of syncytia laser microdissected from the roots of the Glycine max (soybean) genotype PI 548402 (Peking) undergoing a resistant reaction after infection by Heterodera glycines (soybean cyst nematode)

The syncytium is a nurse cell formed within the roots of Glycine max by the plant parasitic nematode Heterodera glycines. Its development and maintenance are essential for nematode survival. The syncytium appears to undergo two developmental phases during its maturation into a functional nurse cell. The first phase is a parasitism phase where the nematode establishes the molecular circuitry that during the second phase ensures a compatible interaction with the plant cell. The cytological features of syncytia undergoing susceptible or resistant reactions appear the same during the parasitism phase. Depending on the outcome of any defense response, the second phase is a period of syncytium maintenance (susceptible reaction) or failure (resistant reaction). In the analyses presented here, the localized gene expression occurring at the syncytium during the resistant reaction was studied. This was accomplished by isolating syncytial cells from Glycine max genotype Peking (PI 548402) by laser capture microdissection. Microarray analyses using the Affymetrix soybean GeneChip directly compared Peking syncytia undergoing a resistant reaction to those undergoing a susceptible reaction during the parasitism phase of the resistant reaction. Those analyses revealed lipoxygenase-9 and lipoxygenase-4 as the most highly induced genes in the resistant reaction. The analysis also identified induced levels of components of the phenylpropanoid pathway. These genes included phenylalanine ammonia lyase, chalcone isomerase, isoflavone reductase, cinnamoyl-CoA reductase and caffeic acid O-methyltransferase. The presence of induced levels of these genes implies the importance of jasmonic acid and phenylpropanoid signaling pathways locally at the site of the syncytium during the resistance phase of the resistant reaction. The analysis also identified highly induced levels of four S-adenosylmethionine synthetase genes, the EARLY-RESPONSIVE TO DEHYDRATION 2 gene and the 14-3-3 gene known as GENERAL REGULATORY FACTOR 2. Subsequent analyses studied microdissected syncytial cells at 3, 6 and 9 days post infection (dpi) during the course of the resistant reaction, resulting in the identification of signature gene expression profiles at each time point in a single G. max genotype, Peking.

A Maize Early Responsive to Dehydration Gene, ZmERD4, Provides Enhanced Drought and Salt Tolerance in Arabidopsis

Early responsive to dehydration (ERD) genes could be rapidly induced to respond to dehydration and to various other abiotic stresses. Here, we report on an ERD gene (ZmERD4) from maize cloned by rapid amplification of complementary DNA (cDNA) ends. The ZmERD4 cDNA had a total length of 2,536 bp with an open reading frame of 2,196 bp, 5′-untranslated region (UTR) of 48 bp, and 3′-UTR of 292 bp. The gene encoded a predicted polypeptide of 732 amino acids. The ZmERD4 protein shared a high amino acid sequence similarity with ERD4 of Oryza sativa and Arabidopsis thaliana. A reverse-transcription polymerase chain reaction analysis revealed that ZmERD4 was constitutively expressed in different tissues. RNA gel blot showed that ZmERD4 could be induced by both drought and salt stress and also responded to abscisic acid treatment, but it was not induced by low temperature (4°C). Transgenic Arabidopsis plants constitutively expressing the ZmERD4 gene under the control of the 35S cauliflower mosaic virus 35S promoter exhibited slightly smaller-sized leaves under normal growing conditions. Moreover, 35S::ZmERD4 transgenic plants displayed enhanced tolerance to water deficit and high salinity when compared to wild-type plants. Altogether, these findings suggested that ZmERD4 played an important role in early stages of plant adaptation to stress conditions and might be useful in improving plant tolerance to abiotic stress.

EARLY RESPONSIVE TO DEHYDRATION 15, a negative regulator of abscisic acid responses in Arabidopsis.

EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) is rapidly induced in response to various abiotic and biotic stress stimuli in Arabidopsis (Arabidopsis thaliana). Modulation of ERD15 levels by overexpression or RNAi silencing altered the responsiveness of the transgenic plants to the phytohormone abscisic acid (ABA). Overexpression of ERD15 reduced the ABA sensitivity of Arabidopsis manifested in decreased drought tolerance and in impaired ability of the plants to increase their freezing tolerance in response to this hormone. In contrast, RNAi silencing of ERD15 resulted in plants that were hypersensitive to ABA and showed improved tolerance to both drought and freezing, as well as impaired seed germination in the presence of ABA. The modulation of ERD15 levels not only affected abiotic stress tolerance but also disease resistance: ERD15 overexpression plants showed improved resistance to the bacterial necrotroph Erwinia carotovora subsp. carotovora accompanied with enhanced induction of marker genes for systemic acquired resistance. We propose that ERD15 is a novel mediator of stress-related ABA signaling in Arabidopsis.

Mapping of 25 drought-inducible genes, RD and ERD, in Arabidopsis thaliana.

We mapped 25 Arabidopsis thaliana drought-inducible genes. Responsive to Dehydration (RD) and Early Responsive to Dehydration (ERD), to the Arabidopsis genome and compared map positions with those of mutants that show environmental stress response. We hybridized CIC yeast artificial chromosome (YAC) library filters with the cDNAs and determined the map positions of 18 corresponding genes. We screened the P1 library with 7 other clones and analyzed segregation of their restriction fragment length polymorphisms (RFLP) in recombinant inbred (RI) lines. One cDNA could be mapped because it had been sequenced by the Arabidopsis genome sequencing program.

Cloning of cDNAs for genes that are early-responsive to dehydration stress (ERDs) inArabidopsis thaliana L.: identification of three ERDs as HSP cognate genes

In Arabidopsis thaliana L., accumulation of abscisic acid (ABA) began to increase 2 h after plants had been subjected to dehydration stress and reached maximum levels after 10 h. Differential hybridization was used to isolate 26 Arabidopsis cDNAs with gene expression induced by a 1 h dehydration treatment. The cDNA clones were classified into 16 groups based on Southern blot hybridization, and named ERD (early-responsive to dehydration) clones. Partial sequencing of the cDNA clones revealed that three ERDs were identical to those of HSP cognates (Athsp70-1, Athsp81-2, and ubiquitin extension protein). Dehydration stress strongly induced the expression of genes for the three ERDs, while application of ABA, which is known to act as a signal transmitter in dehydration-stressed plants, did not significantly affect the ERD gene expression. This result suggests that these HSP cognates are preferentially responsive to dehydration stress in A. thaliana, and that signaling pathways for the expression of these genes under conditions of dehydration stress are not mainly mediated by ABA. We also discuss the possible functions of these three ERD gene products against dehydration stress.