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Promoter regions of the ExtA extensin gene from Brassica napus control activation in response to wounding and tensile stress
Abstract
To identify controlling cis acting promoter regions in the B. napus extA extensin gene, expression in transgenic tobacco of 5' - 159, -433, -664, -789 and -940 bp promoter truncations linked to the uidA (B-glucuronidase) reporter coding sequence were analysed. The - 159 and -433 bp truncations directed non specific expression in all cell types within the plant. An activator region which increased expression levels 10 fold in all cell types was located between - 159 to -433 bp. A repressor region was found between -664 to -789 bp; removal of this region resulted in a 15 fold increase in expression. Histochemical analysis showed that transgenics containing the -664, -789 and -940 bp truncations directed expression of the fusion gene only in the phloem. A negative regulatory region located between -433 to -664 bp repressed expression in non-phloem cell types. In areas of the plant subject to tensile stress, the repression exerted by the negative regulatory region was overcome, allowing expression in all cell types. The quantitative repressor and activator regions which controlled absolute expression levels in all cell types were separate from the negative regulatory region which controlled cell type specific expression in response to tensile stress. A wound responsive region was found to be located between -940 to -3500 bp. Thus, the extA gene is under complex control, being regulated by 4 sets of positively and negatively acting cis regions, which control wound inducibility, activation in response to tensile stress, and quantitative expression levels.
... We have previously shown that genes from Brassica napus and Arabidopsis that code for the structural cell wall hydroxyproline-rich glycoprotein, extensin, are expressed in regions of the plant that are subject to mechanical stresses (Shirsat et al . 1996a;Elliott & Shirsat 1998;Merkouropoulos, Barnett & Shirsat 1999). In particular, promoter-reporter fusions of the B. napus extA gene fused to GUS, demonstrated that expression of the reporter was seen in regions of the plant such as the nodes which experience load-bearing stresses due to the weight of the developing axillary branch (Shirsat et al . ...
... 1996a). Further, we were able to show that this expression pattern appeared to be controlled by a pro-moter region that we (Elliott & Shirsat 1998) termed the negative regulatory region (NRR). The hypothesis was put forward that the NRR acted as a negative regulator repressing expression, and that this repression was overcome at nodal stress bearing regions, resulting in extensin gene activation. ...
... Construction of the truncated extA promoter-GUS reporter gene fusions has already been described (Elliott & Shirsat 1998). Briefly, the plasmid pAS44 containing 940 bp of extA 5 ¢ flanking sequence linked to the coding sequence of the gus reporter gene in the pBI101.2 ...
In the present study the hypothesis that the −433 to −664 bp negative regulatory region (NRR) of the Brassica napus extA extensin promoter controls extA activation in response to externally applied weight loads was tested. When weight loads were applied to the nodal regions of transgenic tobacco plants containing extA promoter deletions fused to GUS, repression controlled by the NRR was overcome and GUS expression was induced only in the transgenics carrying the NRR. This proves that extensin expression in nodal regions is not developmentally controlled, but is induced in response to mechanical stresses, and is controlled by the NRR. It was also shown that the activation of the extA promoter during the development of lateral roots is a stress-related response that is also under the control of the NRR but that the constitutive expression of extensin mRNA in the phloem of roots is not due to the mechanical forces the root experiences as it forces it way through the soil. Electrophoretic mobility shift assays using a 25 bp oligonucleotide have been used to show that an 8 bp consensus sequence from the NRR binds nuclear proteins. Wound-induced signals regulating extensin gene expression are shown to travel bi-directionally through the plant, from root to leaf and vice versa.
... Wound inducibility was also seen in transgenic tobacco carrying the extA gene under the control of its own (3.5 kb) promoter (Elliot and Shirsat 1998). Deletion analysis of the extA promoter has localised the wound-responsive elements to within the −940 to −3500 bp region (Elliott and Shirsat 1998). ...
... It is possible that there is a sequence(s) in the promoter that is acting as a T-DNA border and is prone to endonuclease attack. However, this seems unlikely in view of extensive previous work with the −940 extA promoter which has failed to report such an occurrence (Elliott and Shirsat 1998). ...
... Southern data revealed that 9 other clones also lacked the full-length promoter but showed much lower levels of expression which suggests that differential deletions may have taken place in different clones. Certainly previous histochemical data from tobacco (Elliott and Shirsat 1998) shows that markedly different patterns of gene expression are seen in stem and petiole sections when sequential deletions from −940 to −159 are made. Clearly clone 9 deserves further detailed molecular study and may yield valuable insights into the role of specific regulatory elements involved in controlling gene expression in apple. ...
The Brassica napus extensin A gene is highly expressed in root tissue of oilseed rape. In an attempt to identify an effective root-specific promoter for biotechnological applications, we have examined the ability of the –940 extA promoter to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill cv. Greensleeves). Transgenic apple lines were produced by Agrobacterium tumefaciens-mediated transformation and GUS activity was analysed both quantitatively and qualitatively. The extA promoter was active in all tissues of young plants in all 15 clones examined. However Southern blot data suggested that only a proportion of the population contained the entire promoter and that others had suffered deletions of unknown length. This may have contributed to the variation seen in the quantitative and qualitative expression of GUS. Specific GUS activity was highest in the stems where it approached, and in some clones, exceeded that using the constitutive CaMV35S promoter. Histochemical analysis confirmed that GUS was localised to tissues involved in structural support of the stem. Staining was particularly intense at nodal junctions where high tensile stress is exerted on the tissues. Maturing phloem tissues showed localisation of expression to the phloem parenchyma cells and phloem fibres. Transverse sections of the root revealed staining of primary procambial tissues including the young endodermis but no staining was seen in the cortex. Although the –940 extA promoter is clearly not root-specific in apple, it is likely to have useful biotechnological applications in tree species.
... In addition, LRRs have been shown to potentially mediate pathogen recognition and defense [12]. Extensin proteins are a class of cell wall proteins [13,14] that strengthen the structure of the cell wall by cross-linking with other components of the cell wall to affect its properties [14,15]; these proteins have also been shown to respond to wounding and tensile stress [16] and have a protective function [17]. Extensin proteins A total of 11 AtLRXs were identified based on previous research [20] and a search in the UniProt database (https://www.uniprot.org, ...
... Phylogenetic analyses revealed that BrLRXs had different conserved motifs ( Figure 3A, C and Table 2), but all contained Motifs 1-5 and Motifs 7-8. Ten genes (BrLRX 1, 2,4,5,6,7,8,11,13,17) contained Motif 9, and seven of these BrLRXs contained Motif 7 and Motif 9; BrLRX13 contained Motif 8 in addition to Motif 9. A total of 11 BrLRXs (BrLRX 1, 4,5,7,8,12,13,14,15,16,17) contained Motif 6; and Motif 10 was present in BrLRX 1,4,5,7,8,12,13,14,15,17. ...
Leucine-rich repeat extensin (LRX) is involved in the regulation of crucial cellular processes, such as cell wall growth and development, as well as signaling. However, the presence of the LRX gene family in Brassica rapa (B. rapa) has not been previously reported. This study identified 17 BrLRXs within the Brassica rapa genome by bioinformatic analysis, and these genes were distributed on seven chromosomes. Phylogenetic and covariance analyses indicate that BrLRXs can be categorized into two distinct branches: the trophic branch and the reproductive branch, with a close relationship observed between BrLRXs and AtLRXs. According to cis-acting element analysis, this gene family is rich in hormone-responsive and stress-responsive elements such as drought-inducibility, abscisic acid, methyl jasmonate, and gibberellic acid responsive elements, suggesting a potential role in abiotic stress response. Transcriptomic, proteomic, and RT-qPCR analyses demonstrated significant up-regulation of BrLRX2 and BrLRX6 under salt stress, while BrLRX3, BrLRX6, and BrLRX8 were significantly down-regulated under osmotic stress. Our analysis of the protein tertiary structure predicts a strong association between LRX proteins and RALF. Protein–protein interaction prediction revealed that LRX interacts with the RALF protein and the receptor FER, which have been previously reported to jointly regulate plant stress responses. We propose that BrLRX6 and BrLRX8 are implicated in osmotic stress, while BrLRX2 and BrLRX6 are involved in the modulation of salt stress.
... The last column shows the functional categories enriched among the genes with the shared cis motifs in their promoter regions of these motifs was detected in potato and the second in tobacco (Table 2), both distributed between − 1000 and − 500 bp from the TSS. The extA gene has been shown to be expressed in leaves under wounding and tensile stress (Elliott and Shirsat 1998). The first column indicates the information retrieved in the PLACE database (motif accession codes are shown in bold; the plant species in which they were first described in parentheses; and keywords and the consensus in italics). ...
... Common elements in the promoters of genes up-regulated by drought in both WT and pfld potato plants are summarized in Supplementary Table S5. They are the ABRE2 element originally found in the promoter of the barley ABA-responsive gene HVA22 (Shen and Ho 1995), the ATRICHPSPETE element reported in an enhancer of Pisum sativum (Sandhu et al. 1998), the 'quantitative activator region' ACA CGT T and the 'wounding activating region' WARBNEXTA, both first reported in the promoter of the extA gene from B. napus (Elliott and Shirsat 1998). A common element found in the promoters of genes down-regulated by drought in both potato genotypes was the Box C element (Ngai et al. 1997). ...
Key message
Transcriptomes of solanaceous plants expressing a plastid-targeted antioxidant protein were analysed to identify chloroplast redox networks modulating the expression of nuclear genes associated with stress acclimation.
Abstract
Plastid functions depend on the coordinated expression of nuclear genes, many of them associated to developmental and stress response pathways. Plastid-generated signals mediate this coordination via retrograde signaling, which includes sensing of chloroplast redox state and levels of reactive oxygen species (ROS), although it remains a poorly understood process. Chloroplast redox poise and ROS build-up can be modified by recombinant expression of a plastid-targeted antioxidant protein, i.e., cyanobacterial flavodoxin, with the resulting plants displaying increased tolerance to multiple environmental challenges. Here we analysed the transcriptomes of these flavodoxin-expressing plants to study the coordinated transcriptional responses of the nucleus to the chloroplast redox status and ROS levels during normal growth and stress responses (drought or biotic stress) in tobacco and potato, members of the economically important Solanaceae family. We compared their transcriptomes against those from stressed and mutant plants accumulating ROS in different subcellular compartments and found distinct ROS-related imprints modulated by flavodoxin expression and/or stress. By introducing our datasets in a large-scale interaction network, we identified transcriptional factors related to ROS and stress responses potentially involved in flavodoxin-associated signaling. Finally, we discovered identical cis elements in the promoters of many genes that respond to flavodoxin in the same direction as in wild-type plants under stress, suggesting a priming effect of flavodoxin before stress manifestation. The results provide a genome-wide picture illustrating the relevance of chloroplast redox status on biotic and abiotic stress responses and suggest new cis and trans targets to generate stress-tolerant solanaceous crops.
... "AAC GTG T" motif is found in 14 lncRNAs. This motif controls activation in response to wounding and tensile stress (Elliott and Shirsat 1998). "CAA ACA CC" motif is found in eight lncRNAs. ...
... This motif is necessary for seed-specific expression and regulation of abscisic acid-induced transcription (Ezcurra et al. 1999). Five lncRNAs contain "TAG TGG AT" motif, which controls activation of extensin gene in response to wounding and tensile stress (Elliott and Shirsat 1998). To understand the origin of the predicted lncRNAs, analysis of repetitive sequences is very important as these repetitive sequences, especially the TRs, play important roles in diversification of lncRNAs (Nelson et al. 2016;Zhang et al. 2018). ...
Main conclusion
We have predicted miRNAs, their targets and lncRNAs from the genome of Brassica oleracea along with their functional annotation. Selected miRNAs and their targets are experimentally validated. Roles of these non-coding RNAs in post-transcriptional gene regulation are also deciphered.
Abstract
Cauliflower (Brassica oleracea var. Botrytis) is an important vegetable crop for its dietary and medicinal values with rich source of vitamins, dietary fibers, flavonoids and antioxidants. MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs), which regulate gene expression by inhibiting translation or by degrading messenger RNAs (mRNAs). On the other hand, long non-coding RNAs (lncRNAs) are responsible for the up regulation and the down regulation of transcription. Although the genome of cauliflower is reported, yet the roles of these ncRNAs in post-transcriptional gene regulation (PTGR) remain elusive. In this study, we have computationally predicted 355 miRNAs, of which 280 miRNAs are novel compared to miRBase 22.1. All the predicted miRNAs belong to 121 different families. We have also identified 934 targets of 125 miRNAs along with their functional annotation. These targets are further classified into biological processes, molecular functions and cellular components. Moreover, we have predicted 634 lncRNAs, of which 61 are targeted by 30 novel miRNAs. Randomly chosen 10 miRNAs and 10 lncRNAs are experimentally validated. Five miRNA targets including squamosa promoter-binding-like protein 9, homeobox-leucine zipper protein HDG12-like, NAC domain-containing protein 100, CUP-SHAPED COTYLEDON 1 and kinesin-like protein NACK2 of four miRNAs including bol-miR156a, bol-miR162a, bol-miR164d and bol-miR2673 are also experimentally validated. We have built network models of interactions between miRNAs and their target mRNAs, as well as between miRNAs and lncRNAs. Our findings enhance the knowledge of non-coding genome of cauliflower and their roles in PTGR, and might play important roles in improving agronomic traits of this economically important crop.
... Similar studies on the promoter of a maize ⎯⎯⎯⎯ HRGP gene showed that intronic sequences located at the 3'untranslated region affected gene expression in a tissueand a promoter-dependent manner (Menossi et al. 2003). Elliott and Shirsat (1998) identified cis-elements in the promoter of the extA extensin gene in Brassica napus that positively and negatively regulated responsiveness to wounding and tensile stress. ...
... Total RNA (10 μg) was separated on a 1.3 % formaldehyde-agarose gel and transferred by capillary blotting to a Hybond N nylon membrane as described by Sambrook et al. (1989). The Arabidopsis atExt1 coding sequence (Merkouropoulos et al. 1999), was radio-labelled with [ 32 P]dCTP by random primer labelling and used as a probe in northern hybridisations as described previously (Elliott and Shirsat 1998). Blots were washed to a high stringency with the final wash being at 65 °C in 0.1× SSC with 0.1 % SDS to remove background and non-specific hybridisation signals. ...
The Arabidopsis thaliana (L.) Heynh. atExt1 extensin gene is expressed in a cell and tissue-specific manner, in response to developmental cues, and is inducible by a wide range of biotic and abiotic stresses. Over-expression of this gene has been shown to alter stem morphology and to limit the invasiveness of virulent bacterial pathogens, indicating that this cell wall protein gene plays an important role in plant development and defense. A detailed sequence analysis of 3.2 kb of the atExt1 gene promoter region has identified a large number of putative 5′cis-acting elements. Based on the location of clusters of putative promoter control elements, seven atExt1 5′ promoter truncations were constructed, fused upstream of the β-glucuronidase (GUS) reporter gene, and transformed into A. thaliana. Transgenic plants carrying the various promoter constructs were challenged by wounding and pathogen attack and analysed for GUS expression - this analysis revealed a complex pattern of regulation, involving positive and negative control regions. Northern analysis using wounded tissues from transgenic Arabidopsis plants carrying the 3.2 kb-promoter::GUS construct confirmed the transcriptional activation of the transgene.
... As the extensin gene promoter::GUS fusion genes transformed into tobacco plants (in both homologous and heterologous systems) display similar expression patterns in stem tissues, it is reasonable to assume that these promoter regions contain the necessary cis-acting elements that interact with the tobacco transcription machinery. Elliott and Shirsat (1998) identified the negative regulator region (NRR) in the extA oilseed rape extensin gene promoter as the region where nuclear proteins bind resulting in repression of the transgene. It was suggested that disassociation of this transcription factor results in widespread expression of the transgene in all tissues. ...
... It was suggested that disassociation of this transcription factor results in widespread expression of the transgene in all tissues. Since widespread expression was observed in the nodal regions, which are under tensile stress, it was hypothesized that repression was overcome by tensile stress (Elliott and Shirsat, 1998). If this is the case, then cis-acting elements present in the extA gene promoter NRR would have been expected to be present in the atExt1 gene promoter because the expression of the atExt1::GUS fusion gene in transgenic tobacco is almost identical to that of extA::GUS in transgenic tobacco. ...
Histochemical analysis of transgenic tobacco plants harboring the promoter of the Arabidopsis extensin gene atExt1 fused to the β-glucuronidase gene coding sequence demonstrated expression of the transgene in stem tissues. The transgene was expressed in cells of the internal and external phloem at the nodal and internodal regions of the older parts of mature stems. In younger parts of the same stem, expression of the transgene was slightly modified: expression was detected in the internal phloem in the internodal areas. In the nodal areas, the phloem tissue and the surrounding parenchyma were stained, demonstrating transgene expression. Expression was also seen in the phloem of the inflorescence; it was non-specific at the junctions of the flowers to the inflorescence. β-glucuronidase (reporter gene) staining was also observed in the pollen grains and at the base of the corolla.
... The extA transcript is highly expressed in roots, with lower levels in stems and petioles, and very little, if any, expression in leaves (Shirsat et al., 1996). Extensin genes from a number of plant species are induced by wounding treatments (Elliott and Shirsat, 1998). Deletion analysis of the extA promoter localized the wound responsive elements to within the −940 to −3500 bp region (−940 extA promoter) (Elliott and Shirsat, 1998). ...
... Extensin genes from a number of plant species are induced by wounding treatments (Elliott and Shirsat, 1998). Deletion analysis of the extA promoter localized the wound responsive elements to within the −940 to −3500 bp region (−940 extA promoter) (Elliott and Shirsat, 1998). The ability of the −940 extA promoter to drive expression of the gus reporter gene was determined in the apple genotype Greensleeves (Gittins et al., 2001). ...
... The extA transcript is highly expressed in roots, with lower levels in stems and petioles, and very little, if any, expression in leaves (Shirsat et al., 1996). Extensin genes from a number of plant species are induced by wounding treatments (Elliott and Shirsat, 1998). Deletion analysis of the extA promoter localized the wound responsive elements to within the −940 to −3500 bp region (−940 extA promoter) (Elliott and Shirsat, 1998). ...
... Extensin genes from a number of plant species are induced by wounding treatments (Elliott and Shirsat, 1998). Deletion analysis of the extA promoter localized the wound responsive elements to within the −940 to −3500 bp region (−940 extA promoter) (Elliott and Shirsat, 1998). The ability of the −940 extA promoter to drive expression of the gus reporter gene was determined in the apple genotype Greensleeves (Gittins et al., 2001). ...
... Other sequences identical to the consensus motif of the wounding-responsive element (AACGTGT) described within the promoter region of the extA extensin gene from Brassica napus (Elliott and Shirsat, 1998) were also identi®ed within the strawberry Fxaltp gene promoter region at nucleotide positions 414 (±1223) and 748 (±889). ...
... Although several studies report regulation by biotic and abiotic external stimuli of ltp gene expression, little or no information on the effects of several important environmental factors like wounding, cold stress, etc. on ltp gene expression has so far been reported (Sohal et al., 1999). Two wounding responsive-like sequences (Elliott and Shirsat, 1998) have been predicted and are located within the Fxaltp promoter region. Therefore, the Fxaltp gene expression has been studied under wounding treatment. ...
cDNA and genomic clones encoding a strawberry (Fragaria×ananassa cv. Chandler) non‐specific lipid transfer protein (Fxaltp gene) were isolated and characterized. The spatio‐temporal expression pattern and structural features of this gene were studied
for the first time in strawberry, a non‐climacteric fruit of agricultural importance. The architecture and the encoded amino
acid sequence of this non‐climacteric fruit ltp gene were similar to those of other plant LTPs previously reported, and presents
the eight cysteine residues and other features characteristic of plant LTPs. In addition, the deduced protein posseses an
N‐terminal signal peptide and lacks the K/HDEL retention signal, indicating that the strawberry LTP protein would enter the
secretory pathway. In situ studies have shown that the Fxaltp gene is expressed in the epidermal cell layer of the strawberry fruit receptacle and achenes, flowers, and within the cell
layer surrounding the endosperm. These results suggest that this Fxaltp gene promoter could be used as an endogenous promoter for biotechnological purposes in strawberry. Computer analysis using
the PLACE database predicted the presence of several putative cis‐regulatory sequences in response to abscisic acid and cold or wounding stresses within the Fxaltp 5′‐flanking region. Accordingly, the strawberry gene responds to ABA and SA, but not to salt and heat stresses. It is also
reported that ltp gene expression in strawberry is stimulated by wounding and repressed by cold stresses.
... For instance, WRKY71OS is a binding site of WRKY71 transcription factor, and was reported to be involved various fungal infection and wounding response mediated through SA and JA (Liu et al., 2007). The WBOXN-TERF3 and QARBNEXTA cis-elements were previously reported to be involved in wounding-response (Elliott and Shirsat, 1998;Nishiuchi et al., 2004). WBOXATNPR1 in Arabidopsis NPR1 promoter is responsible for SA-induced defence gene expression to impart disease resistance (Yu et al., 2001). ...
The rice Hybrid Proline Rich Protein (HyPRP) encoding gene, OsHyPRP16 expression exhibit early upregulation in response to Magnaporthe oryzae inoculation. Here, we functionally characterized the OsHyPRP16 promoter through deletion analysis in transgenic Arabidopsis using GUS (b-glucuronidase) reporter assay. The promoter fragments, sequentially deleted from the 5′ end could induce differential GUS activity in response to stress conditions induced by different hormones and abiotic stress conditions. In addition, a strong GUS induction was observed in M. oryzae inoculated transgenic Arabidopsis. Based on the insilico and stress-inducibility of D1 promoter fragment against various phytohormones and rice blast fungus, and with no basal activity under control conditions, we rationally selected D1 promoter fragment to drive the expression of a major rice blast resistance gene; Pi54 in the genetic background of blast susceptible TP309 rice line. The D1 promoter fragment was able to induce the expression of Pi54 at immediate-early stages of M. oryzae infection in transgenic rice. The transgenic plants with Pi54 under the control of D1 promoter fragment displayed complete resistance against M. oryzae infection as compared to control plants. The present study suggests that the D1 fragment of OsHyPRP16 promoter is a valuable tool for breeding and development of rice lines with early-inducible and pathogen-responsive enhanced disease resistance.
... A total of 51 cis-acting elements were found (Fig. 9), and of them, 46 cis-acting elements were found in the promoter regions of the APX and APX-like genes, while only 40 cis-acting elements were found in the promoter regions of GPX family genes. To be specific, MYBGAHV (S000181) (Gubler et al. 1995), NRR (S000242) (Elliott and Shirsat 1998), ARF binding site (S000270) (Ulmasov et al. 1999), DRE2COREZMRAB17 (S000402) (Kizis and Pages 2002), and GAREAT (S000439) (Ogawa et al. 2003) were only found in the promoter regions of APX genes, and DRE1 (S000401) (Kizis and Pages 2002), SEBF biding site (S000391) (Seo et al. 2003) and other 9 cis-acting elements only existed in the prediction of promoter regions GPX family genes in A. nanus. ...
Ascorbate peroxidase (APX) and glutathione peroxidase (GPX) are two families of essential peroxidases that maintain redox balance in cells by catalyzing the reduction of hydrogen peroxide. Ammopiptanthus nanus is a rare broad-leaved evergreen shrub that lives in the temperate desert areas of Central Asia and exhibits strong resistance to low temperature and water stress. GPX and APX family members might contribute to the stress response of A. nanus by participating in reactive oxygen species scavenging. In the present study, APX and GPX family members in A. nanus were identified and their structure, evolution, and expression patterns under stress conditions were investigated. A total of 8 GPX genes, 6 APX genes, and 1 APX-like gene were identified in A. nanus, and these genes were unevenly distributed on 7 chromosomes. These APXs and GPXs showed conservation in amino acid sequence, three-dimensional structure, and intron–exon structure. The GPX gene family in A. nanus expanded in gene number, and the expansions were mainly driven by segmental duplication caused by large-scale duplication events in the evolution of Tribe Sophoreae and might play important roles in the freezing and drought tolerance in A. nanus. Expression profiling based on RNA-seq datasets and qRT-PCR analysis showed that most of the APX and GPX members were differentially expressed under osmotic and cold stress, which is in line with the high copies of stress and hormone response-related cis-acting elements predicted from the promoters of the APX and GPX family genes. The study provided new insight into the evolution of APX and GPX family and promoted the understanding of the molecular mechanism underlying the stress tolerance of A. nanus.
... The authors declare that they have no conflict of interest. Abe et al. (2003), Boter et al. (2004), Buchel et al. (1999), Carrillo-Perdomo et al. (2016), Chakraborty (2008), Chakravarthy et al. (2003), Despres et al. (2003), Elliott and Shirsat (1998), Eulgem et al. (1999), Ezcurra et al. (1999), Fehlberg et al. (2005), Filichkin et al. (2004), Finkelstein and Lynch (2000), Fujiwara and Beachy (1994), Götz et al. (2011), Gowik et al. (2004, Gubler et al. (1995), Haralampidis et al. (2002), Hartmann et al. (2005), Huang et al. (1990), Hwang et al. (1998), Jarmoskaite and Russell (2014), Kagaya et al. (1999), Kaplan et al. (2006), Kapoor and Sugiura (1999), Kim et al. (2002), Lelievre et al. (1992), Lessard et al. (1991), Lu et al. (2002), Luo et al. (2005) ...
In plants, RNA helicases play significant roles in growth, development and stress response. In a previous study, a three-fold upregulation of a DEAD-box RNA helicase in a tomato cultivar tolerant to Tomato leaf curl New Delhi virus (ToLCNDV) as compared to susceptible cultivar during virus infection was shown. Given this, a comprehensive study was performed to identify the members of RNA helicase family in tomato and analyze their functional properties in response to abiotic stresses, hormone treatments and ToLCNDV infection. A total of 131 genes were identified and classified into DEAD- (42), DEAH- (38), and DExD/H-box (51) RNA helicases. Expression profiling of candidate genes in response to abiotic stresses and ToLCNDV infection in contrasting tomato cultivars suggested the putative roles of SlDEAD23 and SlDEAD35 in biotic and abiotic stresses. Heterologous overexpression of these genes in yeast enhanced the tolerance of transgenic cells to salt and cold stresses. Further, virus-induced silencing of SlDEAD35 in ToLCNDV tolerant cultivar resulted in susceptibility to virus infection, thus suggesting its involvement in tolerance mechanism. Altogether, this study provides novel insights into the structure, organization and involvement of DEAD-box RNA helicase genes in biotic and abiotic stress responses in tomato.
... Mechanical stimulation imposed on young internodes of plants reduces their elongation and increases their radial expansion, and experimental evidence obtained in natural conditions such as wind sways reveals that the shaking or bending results in a reduction in elongation, but in some species also leads to a stimulation of radial growth. Applying weights to leaf petioles is also performed to exert tensile stress on axial cells and demonstrated transcriptional regulation of expression of cell wallstrengthening proteins in the stressed cells (Shirsat et al. 1996, Elliott andShirsat 1998). To obtain more insight into the underlying mechanisms, many experimental studies in more controllable conditions have been conducted, using different kinds of mechanical loading systems. ...
Plants are exquisitely sensitive to the physical factors in nature, where exposure to precipitation, wind or touch results in shortened plants which are able to better tolerate stress conditions, and are capable of responding in terms of thigmomorphogenesis, alterations in the growth of plants to cope and compensate for the mechanical variables. An understanding of stress physiology in plant growth is important to many plant scientists, since physical stress has a major impact on biological diversity and agricultural productivity, as well as other environmental and ecological problems. In this chapter, a restricted definition of physical stress is presented as a complicated stress factor whose properties can be separated into several physical aspects, which would be induced by the exterior or internal physical environment and eliciting certain specific or non-specific responses in plant growth. Four interrelated phases that could qualify as primary cues for the differentiated courses induced by stress are restated here, and we are confirmed that there exists undoubtedly a bidirectional function of physical stress. In particular, the elegant experimental methodology designed to investigate the relationship between artificially delivered force and the resultant morphological, physiological, and genetic changes is reviewed. The experimental findings reveal that application of mechanical forces can elicit the response of plant in various aspects: growth and development of plant organism, biophysical characteristics (thermodynamic properties), biochemical metabolism, energy metabolism, signal transduction, resistance formation, and some genetic processes.
... The expression of extensin genes is not only regulated developmentally but can also be induced by various stresses. 1,[3][4][5][6][7][8] We are investigating at the molecular level how plants respond to water stress. By using a differential screening method, we have isolated some A. thaliana cDNAs whose expression was induced or suppressed by water stress. ...
From Arabidopsis thaliana we isolated four different cDNAs that encode extensins, a family of cell-wall hydroxyproline-rich glycoproteins (HRGPs).
Putative proteins (AtExt2–5) contained one open reading frame and characteristic Ser-(Pro)4 sequences organized in a high-order repetitive motif. AtExt2–5 genes were strongly expressed during rehydration after dehydration. They were also expressed after treatment with various
amino acids. In particular, AtExt3 and five mRNAs were abundantly accumulated after treatment with l-Ser, Hyp, and l-Pro,
which are major components ofextensin proteins. The AtExt transcripts were strongly expressed in root tissues of both unbolted and bolted plants. The transcripts of AtExt2, 3, and 5 were also detected in the lower stem and flower buds, and that of AtExt4 was detected in bolted flowers. Therefore, we suggest that these four AtExt genes are novel extensin genes in A. thaliana, because the expression of atExt1, which has already been isolated from A. thaliana, was different from these.
... Similarly, PR2GCNT and WBOXNTCHN48 are reported to be present in stress-and developmentally-regulated pathogenesis-related acidic tobacco β-1,3-glucanase gene [83] and an ethylene inducible tobacco class1 chitinase48 gene [84] respectively. Three motifs each similar to AGCBOXNPGLB and QARBNEXTA were also found, which were earlier reported in tobacco β-1,3-glucanase gene enhancer element similar to GCC element required for ethylene regulated transcription of extA gene in B. napa in response to wounding and tensile stress [85,86]. Many motifs similar to ABRE elements were found like ABREDISTBBNNAPA, ABREMOTIFIIIOSRAB16B, ABREMOTIFIOSRAB16B, ABRECE1HVA22, ABRE3HVA22 and ABRETAEM earlier identified in B. napa, rice, barley and wheat in relation to abscissic acid regulated transcription [64,65,68,87,88]. ...
The nucleosome positioning regulates the gene expression and many other DNA-related processes in eukaryotes. Genome-wide mapping of nucleosome positions and correlation of genome-wide nucleosomal remodeling with the changes in the gene expression can help us understanding gene regulation on genome level.
In the present study, we correlate the gene expression and the genomic nucleosomal remodeling in response to salicylic acid (SA) treatment in A. thaliana. We have mapped genome-wide nucleosomes by performing tiling microarray using 146 bp mononucleosomal template DNA. The average nucleosomal coverage is approximately 346 bp per nucleosome both under the control and the SA-treated conditions. The nucleosomal coverage is more in the coding region than in the 5′ regulatory regions. We observe approximately 50% nucleosomal remodeling on SA treatment where significant nucleosomal depletion and nucleosomal enrichment around the transcription start site (TSS) occur in SA induced genes and SA repressed genes respectively in response to SA treatment. Especially in the case of the SA-induced group, the nucleosomal remodeling over the minimal promoter in response to SA is especially significant in the Non-expresser of PR1 (NPR1)-dependent genes. A detailed investigation of npr1-1 mutant confirms a distinct role of NPR1 in the nucleosome remodeling over the core promoter. We have also identified several motifs for various hormonal responses; including ABRE elements in the remodeled nucleosomal regions around the promoter region in the SA regulated genes. We have further identified that the W-box and TGACG/C motif, reported to play an important role in SA-mediated induction, are enriched in nucleosome free regions (NFRs) of the promoter region of the SA induced genes.
This is the first study reporting genome-wide effects of SA treatment on the chromatin architecture of A. thaliana. It also reports significant role of NPR1 in genome-wide nucleosomal remodeling in response to SA.
... Jasmonic acid and elicitor-responsive elements were only found in MdERF4 and 6, and a wound activating region is present exclusively in MdERF4. To prove the function of the wound activating region (Elliott and Shirsat, 1998), EMSA analysis would be needed. Based on Genevestigator, only AtERF56 of subfamily 1 was induced 8.9-fold by methyl jasmonate, but other AtERFs of subfamily 1 were induced less than 2-fold or suppressed by wounding, ethylene, or methyl jasmonate. ...
... Quantitative activator elements are also distributed along the Ext 1.4 promoter region and appear to be tissue-specific. Such a quantitative activator region (QAR) has been localized in the (± 433/± 159) bp region of the extA promoter (Elliott and Shirsat, 1998 [13] ). In the same way, the distal regions of the promoters of bean HRGP4.1 (Wycoff et al., 1995 [51] ) and of maize HRGP (Tagu et al., 1992 [44] ) extensin genes were shown to contain activator elements. ...
The regulation of the Ext 1.4 gene encoding a tobacco (Nicotiana tabacum L.) extensin was studied in response to mechanical constraints. Transgenic plants carrying chimeric Ext 1.4 promoter/GUS (β-glucuronidase)/nos terminator or Ext 1.4 3′-end constructs were obtained. Expression of gene fusions was found in tissues where mechanical stresses occur, e.g., during germination, as well as in root and stem tissues. Chimeric genes were successively and transiently expressed in different tissues during germination, i.e., at the tip of the root and then in the hypocotyl, during their growth through the seed coat. Moreover, they were expressed in cortical cells surrounding the emergence of adventitious and lateral roots and developmentally-regulated in nodes. The expression of Ext 1.4 could be induced by imposing mechanical constraints due to curving of either the stems or roots. Expression then occurred in cells where it does not normally occur, i.e., in cortical cells of internodes and in the distal piliferous zone of roots. Accumulation of RNAs occurs several days after the start of the constraint. Promoter regions involved in regulation of expression of Ext 1.4 in stems, roots, and in seedlings upon mechanical constraint could be localized. Moreover, the 3′ non-coding region was shown to modulate expression in roots. These results suggest that the regulation of Ext 1.4 following mechanical stress is dependent on both tissue-specific and mechanical-responsive elements.
... The extensins are a group of structural cell wall proteins involved in overall plant development and can contribute to plant protection against several forms of stress such as pathogens, wounding, changes in mechanical pressure by the different tissues, and physiological changes during development (Carpita and Gibeaut 1993;Showalter 1993;Tiré et al. 1994;Ahn et al. 1996;Shirsat et al. 1996;Elliott and Shirsat 1998). Studies on classical extensins revealed a few basic characteristics at the amino acid level such as (1) the presence of a signal peptide for secretion into the plant cell wall, (2) a hydroxyproline-rich amino-acid composition with many repetitions of a Ser-Hyp 4 motif, (3) a high degree of post-translational modifications, (4) a highly basic character, and (5) a high content of tyrosine residues that by intermolecular crosslinks mediate cell wall rigidification. ...
The progamic phase, the period of pollen tube growth through the pistil, is a period of specific interactions between the
male gametophyte and the pistil. Understanding of pollen germination and pollen tube growth are relevant for the study of
pollen-pistil interactions and for understanding the function of components specifically accumulated in the transmitting tissue
cell walls and intercellular matrix that may interact with pollen tubes.
... A TATA (TATATAAA) boxes are located 98 nt upstream of the translation start site (Fig. 1B). As shown in Table 1, the 5V-flanking region contains various potential motifs, such as dehydration-responsive elements (ACGTG, AAACCA and CATGTG) [15,16], a wounding and tensile stress-responsive element (ATCCACTA) [17], several elements related to root-specific expression (ATATT) [18], a G-box (CACGTG) [19], an ABA-responsive element (CTAATGT) [20], a low temperature-responsive element (GTCGG) [21], two W-boxes (GTCAAT) [22] and an ethylene-response element (ERE)-like sequence (ATTTCAAT) [23,24] (Fig. 3). ...
The phenylpropanoid pathway plays an important role when plants are exposed to environmental stresses, such as wounding or
pathogen attack. Its activity leads to the production of lignin, flavonoids, and phytoalexins. Cinnamate 4-hydroxylase (C4H)
is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of cinnamic acid to p-coumaric acid. We isolatedC4H cDNA fromCamptotheca acuminata and investigated the expression pattern of the C.acuminata C4H (CaC4H) gene following stress treatments. A search against the BLOCKS database of conserved protein motifs indicated that
CaC4H shares common features with C4Hs from other species. C4H transcripts accumulated in the leaves in response to mechanical
wounding or the application of molecules involved in the stress response, i.e., ethylene, methyl jasmonate, and hydrogen peroxide.
Interestingly, the application of aminoethoxyvinylglycine, salicylic acid, or diphenylene iodonium, which are biosynthetic
inhibitors of ethylene, methyl jasmonate, and hydrogen peroxide, respectively, did not inhibit this wound-induced expression.
Based on these results, we suggest that C4H functions in response to various stresses in Gacuminata leaves.
... Increased expression of extensin transcripts can be triggered by various biotic and abiotic stresses including wounding and pathogen attack (q.v. ethylene signaling), where it has been suggested to function as part of a plants' primary defense, impeding pathogen ingress until secondary defense genes can be mobilized [71][72][73]. Likewise, strengthening of the wall may provide a barrier to insect stylets, although this has yet to be demonstrated to effectively reduce herbivory. ...
The molecular mechanisms underlying plant defense to sap-feeding insects are slowly being uncovered. In large part, past research has focused on interactions between phloem-feeding insects and their annual host plants with little emphasis on xylem-feeders or woody perennials—especially fruit trees. Using nylon filter cDNA arrays, we analyzed the transcriptional changes of 1731 non-redundant citrus transcripts that resulted from herbivory by a xylem-feeding leafhopper, the glassy-winged sharpshooter (GWSS), Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). In addition, herbivory-elicited changes were compared to those of mechanical damage to better identify GWSS-specific responses. GWSS feeding led to a significant expression change in 50 transcripts. Of these, 14 were also changed by mechanical damage; however, the magnitude was in many cases reduced, suggesting transcriptional modification by GWSS-derived elicitors. Sequence similarity searches with the public database GenBank indicated that the responsive transcripts broadly function in direct defense, defense signaling, ROS scavenging, transport, cell wall modification, photosynthesis and abiotic stress. In particular, GWSS feeding resulted in a transcript profile that resembled wounding, likely through jasmonic acid-independent pathways, as well as an association with dehydration stress. In contrast to similar studies with aphids, salicylic acid-dependent pathogenesis-elated genes were weakly induced. Interestingly, six of the GWSS-responsive transcripts failed to significantly match any public protein sequence signifying their potential as novel genes functioning in plant defense, wound response or abiotic stress.
... More work will be needed to fully elucidate their regulatory role in promoter function. The existence of positive and negative cis-regulatory elements localized in different regions has been reported for a number of plant promoters414243. The results described above showed that the SVBV FLt promoter has a negative and a positive cis-regulatory element, which affects the promoter activity quantitatively in a protoplast transient expression assay. ...
A full-length transcript (FLt) promoter was isolated from a genomic clone of Strawberry vein banding virus (SVBV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family, and its activity was analyzed both in protoplasts and transgenic plants. The 5′–3′-boundaries required for maximal promoter activity were determined by 5′- and 3′-end deletion analysis of the SVBV promoter fused to a β-glucuronidase (GUS) reporter gene. A 371-bp promoter fragment (−352 to +19 from the transcription start site; TSS) was found sufficient for maximal promoter activity in a transient protoplast expression assay, and this was chosen for further analysis. Finer deletion analysis of a 90-bp sequence (coordinates −392 to −302 from TSS) of the SVBV FLt promoter revealed the presence of a negative and a positive regulatory element in this region. In gain-of-function experiment, the fusion of the putative positive regulatory elements with minimal promoter showed very little increment in promoter activity, suggesting that a combinatorial action of various cis-sequences is involved in promoter function. The transcription start site of the full-length transcript promoter was mapped to an A-residue that is located 25-bp downstream of the TATA-box. In protoplast transient expression analysis, the SVBV FLt promoter showed about six-fold higher activity in tobacco compared to maize. A quantitative GUS activity assay showed that in transgenic tobacco plants the average promoter activity was about three-fold higher in roots than in leaves, and this higher activity was due to the accumulation of more GUS specific mRNA in roots. Real-time qRT-PCR analysis and quantitative GUS activity assay showed that the relative strength of the SVBV FLt promoter was greater than the CaMV35S promoter in transgenic tobacco plants. The SVBV FLt promoter is a strong, constitutive promoter and has great application potential in expression of foreign genes in plants.
... The cold-inducible soybean promoters had the most (ACACGT in ABRE; 2.6-fold more abundant than in standardized promoters) and second most (ACGCGT in CE3) conserved sequences, as well as other highly conserved sequences (ACGTGG in ABRE; CGCGTT in CE3; ATGTCG in DRE; and CACGTT, CCGTGT and AACACG sequences) (Fig. 5E; Supplementary Table S35). CACGTT is part of pABRE3, T/G box, QAR, WAR, and OsBP-5 binding site, all of which have been identified as cis-acting elements that regulate gene expression in response to ABA62 or jasmonates.63–65 The dehydration-inducible soybean promoters had the most (ACGTGG in ABRE; 3.5-fold more abundant than in standardized promoters) and second most (CACGTG in G box) conserved sequences, and there were other highly conserved parts of ABRE (Fig. 5F; Supplementary Table S36). ...
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.
... Jasmonic acid and elicitor-responsive elements were only found in MdERF4 and 6, and a wound activating region is present exclusively in MdERF4. To prove the function of the wound activating region (Elliott and Shirsat, 1998), EMSA analysis would be needed. Based on Genevestigator, only AtERF56 of subfamily 1 was induced 8.9-fold by methyl jasmonate, but other AtERFs of subfamily 1 were induced less than 2-fold or suppressed by wounding, ethylene, or methyl jasmonate. ...
Graduation date: 2009 Several plant hormones, including ethylene and jasmonates, are known to mediate responses against biotic and abiotic stress. The first and second objectives of this thesis were (i) to elucidate the role of ethylene in defense responses against Botrytis cinerea and specifically (ii) to determine the potential contribution of ethylene response factors (ERFs) to induced resistance. Besides being involved in plant protection, the gaseous plant hormone ethylene is an important developmental signal, controlling fruit ripening and other events. With respect to plant-pathogen interactions ethylene can accelerate or inhibit infection depending on the plant species, organ, or age. Ethylene is known to contribute to foliar resistance against B. cinerea. This pathogen causes gray mold and other diseases. It attacks more than 200 plant species, including apple (Malus domestica) and pear (Pyrus communis). However, effects of ethylene on B. cinerea infection of fruits are not well understood. I specifically examined the role of ethylene in defenses of pome fruits against B. cinerea. Ethylene was manipulated exogenously and endogenously. Inhibition of ethylene action and biosynthesis was found to stimulate infections of pear and apple fruits by B. cinerea, respectively. This implicates ethylene in protecting fruits against B. cinerea. ERFs are known to specifically bind to 'GCC'-boxes of defense-related target gene and to induce foliar resistance against necrotrophic pathogens. To understand the defensive functions of this class of transcription factors in fruits, I characterized members of the ERF gene family in apple. Four MdERF genes, which are expressed in apple fruits, were identified. To determine dependence of gene expression on ethylene, untransformed and ethylene-silenced apples [Dandekar et al. (2004) Transgenic Res. 13: 373-384] were compared. Only one of them, MdERF3, was induced by wounding and B. cinerea infection in ethylene-silenced apple fruits. To test the function of MdERF3, the gene was transiently expressed in tobacco leaves. Elevated expression of MdERF3 was correlated with increased expression of the GCC-box-containing gene Chitinase 48. Thus, MdERF3 appears to be part of the ethylene/pathogenesis-related defense response against B. cinerea in apple fruits. The third objective of my thesis was to investigate interactions between jasmonate and oligogalacturonic acid (OGA) signaling during defense responses against B. cinerea. The Cervone hypothesis [Cervone et al. (1989) Plant Physiol. 90: 542-548] states that OGAs generated from in vitro interactions between fungal polygalacturonases (PGs) and PG-inhibiting proteins (PGIPs) activate phytoalexin biosynthesis and other plant defense responses. I tested the in vivo significance of this hypothesis using genetics. The tomato mutant coi1 with a defect in the jasmonate signaling was crossed to a transgenic line constitutively expressing PGIP from pear. The results suggest that both jasmonates and PGIP independently alter resistance to this fungal pathogen, thus refuting the hypothesis that OGAs activate the jasmonic acid pathway.
... A GUS-visualization and ⁄ or MUG-quantification in the resulting transgenic plants were used to further elucidate the ability of the promoter truncations to induce the reporter gene upon stress treatments. Based on the characteristics Yamaguchi-Shinozaki & Shinozaki, 2005), a quantitative activating region (QAR) element (Elliott & Shirsat, 1998), a motif IIb element (Lescot et al., 2002) and a GCC-box (Brown et al., 2003). ...
*Previously, it was shown that the Arabidopsis thaliana plant defensins AtPDF1.1 (At1g75830) and AtPDF1.2a (At5g44420) exert in vitro antimicrobial properties and that their corresponding genes are expressed in seeds and induced in leaves upon pathogen attack, respectively. *In this study, the expression profile of both AtPDF1.1 and AtPDF1.2a is analysed in wild-type plants upon different stress-related treatments and the effect of modulation of their expression in transgenic plants is examined in both host and nonhost resistance. *AtPDF1.1, which was originally considered to be seed-specific, is demonstrated to be locally induced in leaves upon fungal attack and exhibits an expression profile distinct from that of AtPDF1.2a, a gene frequently used as marker for the ethylene/jasmonate-mediated signaling pathway. Transgenic plants with modulated AtPDF1.1 or AtPDF1.2a gene expression show no altered phenotype upon Botrytis cinerea inoculation. However, constitutive overexpression of AtPDF1.1 in A. thaliana leads to a reduction in symptoms caused by the nonhost Cercospora beticola causing non-spreading spots on A. thaliana leaves. *These results indicate that AtPDF1.1 and AtPDF1.2a clearly differ regarding their expression profile and functionality in planta. It emphasizes the additional level of complexity and fine-tuning within the highly redundant plant defensin genes in A. thaliana.
... In monocotyledons, the best-characterized CREs associated with seed-specific expression are the GCN4-like (GLM) motif and the prolaminbox, which are bound by TFs of the bZIP and DOF families respectively (Vicente- Carbajosa and Carbonero, 2005). Promoters and CREs driving expression in other tissues and organs have also been identified, including green tissues, vascular tissues, roots, root nodules, pollen and flowers (Elliott and Shirsat, 1998;Eyal, et al., 1995;Huang, et al., 1995;Jensen, et al., 1988;Keller and Heierli, 1994;Klinedinst, et al., 2000;Mizukami, et al., 1996). ...
The regulation of gene expression is driven primarily by transcription factors binding to short DNA sequences. Here three studies related to promoter cis-regulatory motif discovery in plant promoters are presented. In the first study, an exact discriminative seeding DNA motif discovery addressing key issues associated with popular DNA motif discovery algorithms is proposed. The Seeder algorithm outperforms popular motif discovery tools on biological benchmark data. In the second study, the algorithm is applied to the identification of cis-regulatory motifs in seed storage protein gene promoters. Known and new motifs are discovered. In the third study, groups of orthologous genes are identified among five dicotyledonous plant species, and DNA motif discovery is carried out in the proximal promoter sequence within each group. The presence of three large clusters of groups of orthologous promoters sharing similar motifs is revealed. L'expression des gènes est régulée, en grande partie, par la liaison des facteurs de transcription à de courtes séquences d'ADN. Trois études sont présentées, portant sur l'identification in silico de motifs régulateurs dans les séquences promotrices de gènes végétaux. Dans la première étude, un algorithme d'initiation discriminative exacte est présenté. L'algorithme surpasse plusieurs algorithmes populaires lorsque appliqué à des données biologiques de référence. Dans la deuxième étude, l'algorithme est utilisé pour l'identification de motifs cis-régulateurs conservés dans les promoteurs de gènes de protéines de réserve des graines chez diverses espèces végétales. Des motifs connus ainsi que de nouveaux motifs sont identifiés. Dans la troisième étude, des groupes de gènes orthologues sont identifiés chez cinq espèces dicotylédones, et une recherche de motifs cis-régulateurs est réalisée dans les séquences promotrices proximales pour chaque groupe. La présence de trois larges grappes de groupes d'orthologues partageant des motifs similaires est mise en évidence.
... Extensin genes of tobacco were found to be induced by wounding, pathogen infection, ethylene, cytokinin stress [Memelink et al., 1993], salicylic acid [Tire et al., 1994], sucrose in combination with wounding [Ahn et al., 1996] and in Arabidopsis also by methyl jasmonate and abscisic acid [Merkouropoulos et al., 1999]. Different members of the extensin family are expressed in different plant organs [Elliott and Shirsat, 1998] or may contain promoter elements induced by different stimuli [Hirsinger et al., 1997]. ...
Halle, Wittenberg, University, Diss., 2002 (Nicht für den Austausch).
Allene oxide synthase (AOS) and hydroperoxide lyase (HPL) are two important members of P450 enzymes metabolizing hydroperoxy fatty acid to produce jasmonates and aldehydes respectively, which function in response to diverse environmental and developmental stimuli. However, their exact roles in soybean have not been clarified. In present study, we identified a lesion‐mimic mutant in soybean named NT302, which exhibits etiolated phenotype together with chlorotic and spontaneous lesions on leaves at R3 podding stage. The underlying gene was identified as GmHPL encoding hydroperoxide lyase by map‐based cloning strategy. Sequence analysis demonstrated that a single nucleotide mutation created a premature termination codon (Gln20‐Ter), which resulted in a truncated GmHPL protein in NT302. GmHPL RNA was significantly reduced in NT302 mutant, while genes in AOS branch of the 13‐LOX pathway were up‐regulated in NT302. The mutant exhibited higher susceptibility to bacterial leaf pustule (BLP) disease, but increased resistance against common cutworm (CCW) pest. GmHPL was significantly induced in response to MeJA, wounding, and CCW in wild type soybean. Virus induced gene silencing (VIGS) of GhHPL genes gave rise to similar lesion‐mimic leaf phenotypes in upland cotton, coupled with upregulation of the expression of JA biosynthesis and JA‐induced genes. Our study provides evidence that competition exist between HPL and AOS branches in 13‐LOX pathway of the oxylipin metabolism in soybean, thereby plays essential roles in modulation of plant development and defense.
The full-length pathogen-inducible plant defensin gene encoding 80 amino acid sequences of pathogen-related gene PR12 (Bjdefensin) was isolated from Brassica juncea. Conserved domain (CD) search reveals its similarity with gamma thionin and knottins families of plant antimicrobial peptides. The protein blast reveals more than 80% of its similarity with defensin-like protein of Brassica rapa and Camelina sativa. Gene expression studies revealed that the transcript levels of Bjdefensin gene increases significantly upon Alternaria infection, jasmonic acid and wounding treatments but was not induced by salicylic acid. To further study the stress-inducible regulation of Bjdefensin gene, its promoter (2.5 kb) was isolated and cloned upstream of GUS gene in pORER2 vector. In silico studies of Bjdefensin promoter showed many important conserved cis-elements, responsive to biotic and abiotic stresses. Histochemical GUS assay showed pathogen-inducible expression of Bjdefensin promoter after fungal infection. Bjdefensin promoter was also induced by Jasmonic acid and wounding.
Plant primary cell walls appear as complex structures composed of two interconnected networks embedded in a pectin matrix. The first network is that of cellulose microfibrils wrapped in and cross-linked with xyloglucans. The second network constitutes structural proteins connected by intermolecular cross-links. Extensins constitute a major class of structural proteins and have been classified according to the content of characteristic amino acid motifs: Ser-(Pro)4 repeats, Tyr-X-Tyr-Lys motifs involved in the formation of intramolecular isodityrosine cross-links, and X-Pro-Val-Tyr-Lys motifs potentially forming intermolecular cross-links. Several extensin genes were cloned, and transgenic plants were used as tools to characterize their patterns of expression and to define tissue-specific or stress-responsive regulatory elements. Major contributions of such studies were to confirm that all extensin genes are regulated in different ways. Moreover, extensin genes are not expressed in all plant cells: they may be expressed in root or stem phloem cells, in cells under mechanical constraints, in response to wounding, and in cells proliferating under hormone control. Promoter deletion studies allowed us to define regulatory domains using both histochemical detection of GUS (β-glucuronidase) activity driven by extensin promoter fragments and fluorometric analyses. Common regulatory elements remain to be determined. Finally, functional approaches were tentatively performed. However, transgenic plants underexpressing two different extensins did not show any clear phenotype confirming that extensins are not required in all plant cell walls and suggesting that other structural proteins may replace extensins where cell-wall reinforcement is essential.
Within a 12,000 mutagenized Sorghum bicolor (L.) Moench plant population we identified a single cellulose deficient and male gametophyte-dysfunctional mutant, namely dwarf1-1 (dwf1-1). Via S. propinquum male/dwf1-1 female F2 population, we mapped dwf1-1 to a frameshift in GA20-oxidase. Assessment of GAs in dwf1-1 revealed ablation of GA. GA ablation was antagonistic to the expression of three specific CESA genes resulting in cellulose deficiency and growth dwarfism, which were complemented by exogenous bioactive GA3 application. Using quantitative polymerase chain reaction, we found that GA was positively regulating the expression of specific CESAs. To cross reference data from our mapped Sorghum allele with another monocotyledonous plant, a series of rice (Oryza sativa) mutants involved in GA biosynthesis and signaling were isolated and these too displayed cellulose deficit. Using chromatin immunoprecipitation we found that a GA conjugated protein could bind to the promoter of CESA proteins, but the mechanistic GA responsive elements remains to be identified. Taken together, data support a model whereby suppressed expansion in "green revolution" GA genes involves regulation of cellulose biosynthesis.
Copyright © 2015, Plant Physiology.
The extreme challenge for the future is to increase the
plant productivity and yield with respect to climate change.
In the current global status, aberrant climate change leads
to the immense impact on the loss of million tons of crop
productivity in agriculture. The consequence is becoming
more pathetic by the current and imminent global changes
in climate, world population increases, industrialisation
toxicity, deterioration of cultivated land and freshwater
insufficiency. All these stresses emphasising the development
of stress-resistant plants are those that have the
ability to adapt and endure the growth and productivity
in stressful and harsh environmental changes. It is significant
to understand plants stress response mechanism
to augment the crop productivity under unfavourable or
stressful environmental conditions.
A full-length transcript (FLt) promoter was isolated from a genomic clone of Strawberry vein banding virus (SVBV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family, and its activity was analyzed both in protoplasts and transgenic plants. The 5′–3′-boundaries required for maximal promoter activity were determined by 5′- and 3′-end deletion analysis of the SVBV promoter fused to a β-glucuronidase (GUS) reporter gene. A 371-bp promoter fragment (−352 to +19 from the transcription start site; TSS) was found sufficient for maximal promoter activity in a transient protoplast expression assay, and this was chosen for further analysis. Finer deletion analysis of a 90-bp sequence (coordinates −392 to −302 from TSS) of the SVBV FLt promoter revealed the presence of a negative and a positive regulatory element in this region. In gain-of-function experiment, the fusion of the putative positive regulatory elements with minimal promoter showed very little increment in promoter activity, suggesting that a combinatorial action of various cis-sequences is involved in promoter function. The transcription start site of the full-length transcript promoter was mapped to an A-residue that is located 25-bp downstream of the TATA-box. In protoplast transient expression analysis, the SVBV FLt promoter showed about six-fold higher activity in tobacco compared to maize. A quantitative GUS activity assay showed that in transgenic tobacco plants the average promoter activity was about three-fold higher in roots than in leaves, and this higher activity was due to the accumulation of more GUS specific mRNA in roots. Real-time qRT-PCR analysis and quantitative GUS activity assay showed that the relative strength of the SVBV FLt promoter was greater than the CaMV35S promoter in transgenic tobacco plants. The SVBV FLt promoter is a strong, constitutive promoter and has great application potential in expression of foreign genes in plants.
In this paper we compare five heterologous promoters fused to β-glucuronidase gene in their influence on localization of GUS
activity in cauliflower (Brassica oleracea var. botrytis) tissues: roots, leaves, petioles and curds. A constitutive promoter CaMV 35S and four tissue specific promoters were used:
extAP from rape, PsMTAP from pea, RBCS3CP from tomato and SRS1P from soybean, and introduced into cauliflower seedling explants using Agrobacterium rhizogenes mediated transformation. Quantitative and histochemical GUS assays confirmed tissue specific gus expression. It was found that extAP promoter was the most active in petioles but also caused a significant gus expression in curds. GUS activity was hardly observed in curd and restricted only to its epidermis when PsMTAP promoter drove the gene. RBCS3CP and SRS1P promoters controlled similar expression of the gus gene throughout the plant except for curd where RBCS3CP was almost inactive.
HcTPS1 and HcTPS2 are floral-scent genes that mediate terpene formation in Hedychium coronarium. Transcription of these genes is induced by various abiotic stresses and hormonal stimuli. In this study, the regulatory mechanisms responsible for HcTPS1 and HcTPS2 expression were investigated by analyzing their promoters. Two genomic fragments, 1,298-bp and 1,928-bp in length, upstream of HcTPS1 and HcTPS2, respectively, were isolated and identified as the promoters ‘PrHcTPS1’ and ‘PrHcTPS2’. Analysis of both promoter sequences indicated cis-elements associated with abiotic stress and jasmonic acid (JA) responses, as well as tissue-specific expression. To identify the functional regions within each of the two promoters, a series of 5′ deletion fragments of different lengths from each promoter were fused to a β-glucuronidase (GUS) reporter gene (pCAMBIA1300-‘prHcTPS’:: gusA) and subsequently used to transform Nicotiana tobacum L. Wisconsin (w38). Functional properties of each promoter segment were determined by GUS staining and quantitative fluorescence analyses of transgenic plants subjected to various abiotic stresses and plant hormones. GUS histochemical staining in transgenic tobacco containing the full fragment of either promoter sequence was only observed in floral organs, where gusA transcripts accumulated as flower development progressed. Additionally, gusA transcripts were also up-regulated by insect feeding, methyl jasmonate (MeJA), and wounding treatments, indicating that ‘PrHcTPS1’ and ‘PrHcTPS2’ promoters were floral-specific, developmentally-regulated, and stress-inducible. Promoter deletion analysis indicated that the promoter fragments p-420 bp of ‘PrHcTPS1’ and p-320 bp of ‘PrHcTPS2’ possessed sufficient, essential cis-elements to drive the expression patterns observed using the whole fragment of either promoter. The identified promoters could be extremely valuable for future use in molecular breeding of ornamental and horticultural plants for floral scent and stress tolerance.
Multiple, highly similar members of the lipid transfer protein (LTP) family have been identified in Nicotiana glauca L. Here we describe four new members of the NgLTP gene family and further characterise one member. Three genes were isolated from a guard cell cDNA library and one (NgLTP2) was isolated from a genomic library. These four NgLTPs, as well as one described previously, NgLTP1, share > 83% amino acid similarity, but the deduced protein sequence of NgLTP2 lacks the last five residues compared with other LTPs. Since the DNA sequences of the five genes are nearly identical, techniques based on nucleic acid hybridisation or PCR amplification were not sufficient to resolve the expression of the individual genes with confidence. Therefore, we characterised the expression pattern of NgLTP2, the only NgLTP gene that was not found in the guard cell cDNA library, using an NgLTP2 promoter-GUS reporter assay. GUS activity driven by the NgLTP2 promoter was assayed in three species of transgenic plants as an indicator of the endogenous pattern of expression of this gene. GUS was strongly induced upon wounding, whereas NgLTP1 was induced by drought stress. Sequence analysis of the NgLTP2 promoter revealed cis-acting motifs associated with induction by wounding. Differential expression of the NgLTP gene family, revealed by the different expression patterns of NgLTP1 and NgLTP2, is further evidence that these genes have multiple functions in N. glauca.
At least two main groups of glycoproteins can be distinguished in plant cell walls: extensins which are insoluble cell wall proteins; and soluble arabinogalactan proteins (AGPs) which have a high carbohydrate content such that protein content constitutes in some cases only 5 % of the glycoprotein weight. These two groups of proteins together with other cell wall proteins more or less glycosylated, such as proline-rich proteins (PRPs), hybrid PRP (HyPRPs) and expansins, are reviewed and compared with similar proteins present in other cell compartments. Different patterns of N- or O-glycosylation are analysed. In some cases, these cell wall proteins or proteins related to them present patterns of glycosylation that act as epitopes recognizable by IgE in allergic responses.
In this paper we show the effect of leaf tissue sampling on estimation of β-glucuronidase activity. Pieces of leaves taken from Agrobacterium-mediated transformed T0 plants of cauliflower were sampled and the GUS activity was measured fluorometrically. Whole leaf tissue and samples of small pieces representing various leaf zones were compared. A great variation of GUS activity within leaf was observed, for which coefficient of variation reached up to 70%. The GUS activity was nearly symmetrical for the left and right side of a leaf blade, with the highest values along the top and middle parts of a leaf blade edge. The relible and repeatable estimation of GUS activity was obtained only if a whole leaf tissue, except the midrib, was used, which allow to reduce the variation to about 10%.
The first report of transformed apple plants in 1989 raised expectations for new apple cultivars that would be better tasting, healthier, and easier to grow. Although, many different traits have now been introduced successfully into apple, no transformed cultivars have yet made it to commercial production. Most early reports on transformed apple described “proof of concept” experiments involving the development of regeneration protocols, and the choice of appropriate promoters and selectable markers. More recently, the focus has moved onto functional testing of traits of potential commercial interest. These traits can be grouped into two categories: horticultural production traits and fruit-focused traits. Production traits of interest include bacterial, fungal and pest resistance, dwarfing, propagation, stress resistance, precocity, storage life, and self-fertility. Examples of fruit-focused traits include novel health properties, flavor, reduced browning, color, and reduced allergenicity. This review will consider reports of characters introduced into apple that are useful to growers and consumers, and looks toward future trends, targets, and challenges.
Genomic sequence of the ATP-dependent phosphoeno/pyruvate carboxykinase (CsPCK) gene has been determined first from cucumber. Several putative clones were isolated in three rounds of genomic library screening
with designated cDNA probes. These clones were analyzed via restriction digests, Southern hybridization, and nucleotide sequencing
to ascertain the structure of theCsPCK gene. Analysis of a selected positive clone (λcscpk-4A) demonstrated that this gene consists of 13 exons and 12 introns,
spanning 9 kb in the cucumber genome. Exon 1 contains only 23 nucleotides of the 5′-noncoding region of cucumberPCK cDNA, whereas Exon 2 comprises 12 nucleotides of the S′-noncoding region with an N-terminal PEPCK coding sequence. All the
exon-intron junction sequences agree with the GT/AG consensus, except for the 5 donor site of Intron 7, where GC replaces
the GT consensus. As with rice (Oryza sativa), cucumber contains only one copy of theCsPCK gene in its haploid genome. The overall number of exons and the structure of this gene are similar to those for bothArabidopsis Chromosome 4 (Atg4)PCK and the rice PCX genes, which contain 13 and 12 exons, respectively. Two additionalArabidopsis PCK genes can be found in the fifth chromosome (Atg5), which contains 9 exons and 8 introns (with 628 and 670 amino acids, respectively)
of the PEPCK peptide. TheCsPCK gene promoter has conserved plant-specific as-acting elements within 2 kb of the 5’ flanking region. Several common cis-acting
elements of the isocitrate lyase (icl) and malate synthase(ms) gene promoters, identified in theCsPCK gene, are responsible for the sugar response during plant development, especially at germination. These conserved elements
are discussed here.
Hydroxyproline-rich glycoproteins (HRGPs) are ubiquitous architectural components of the growing plant cell wall, accounting
for as much as 10-20% of the dry weight. HRGPs are implicated in all aspects of plant growth and development, including responses
to stress. The HRGP superfamily contains three major groups which represent a continuum of peptide periodicity and hydroxyproline-O-glycosylation.
These groups range from the highly periodic and lightly arabinosylated repetitive proline-rich proteins (PRPs), through the
crosslinked extensins which are periodic and highly arabinosylated, to the arabinogalactan-proteins (AGPs) which are the most
highly glycosylated and least periodic. The repetitive units are small, often only four- to six-residue-glycosylated modules
viewed hypothetically as functional motifs, or glycomodules. The Hyp contiguity hypothesis predicts that Hyp arabinosylation
increases with Hyp contiguity and that clustered noncontiguous Hyp residues are sites of arabinogalactan polysaccharide addition
in the AGPs and gums. Recent results involving glycosylation site mapping of endogenous HRGPs and HRGP design using synthetic
genes have corroborated the hypothesis. The uses of synthetic genes in HRGP glycosylation site mapping and structural/functional
analysis are also discussed.
The plant hormone ethylene regulates fruit ripening, other developmental processes, and a subset of defense responses. Here, we show that 1-aminocyclopropane-1-carboxylic acid synthase (ACS)-silenced apple (Malus domestica) fruit that express a sense construct of ACS were more susceptible to Botrytis cinerea than untransformed apple, demonstrating that ethylene strengthens fruit resistance to B. cinerea infection. Because ethylene response factors (ERFs) are known to contribute to resistance against B. cinerea via the ethylene-signaling pathway, we cloned four ERF cDNAs from fruit of M. domestica: MdERF3, -4, -5, and -6. Expression of all four MdERF mRNAs was ethylene dependent and induced by wounding or by B. cinerea infection. B. cinerea infection suppressed rapid induction of wound-related MdERF expression. MdERF3 was the only mRNA induced by wounding and B. cinerea infection in ACS-suppressed apple fruit, although its induction was reduced compared with wild-type apple. Promoter regions of all four MdERF genes were cloned and putative cis-elements were identified in each promoter. Transient expression of MdERF3 in tobacco increased expression of the GCC-box containing gene chitinase 48.
SUMMARY The function of the cell wall protein extensin has been the subject of much speculation since it was first isolated over 40 years ago. In order to investigate the role of extensins in plant defence, we used the gain-of-function strategy to generate transgenic Arabidopsis plants over-expressing the EXT1 extensin gene. These were infected with the virulent bacterial pathogen Pseudomonas syringae DC3000 and symptom development was monitored. Lesions on the transgenics were on average five-fold smaller than those on the wild-type, did not increase in area over the time period of infection, accumulated a small bacterial load and showed very little chlorosis outside the lesion boundary. By contrast, lesions on the wild-type were large, spread to over 50% of the leaf area, continued to increase in size over the time course of the infection, accumulated a bacterial load 100-fold higher than that found in the transgenics, and showed a large chlorotic area outside the lesion boundary. SEM of lesions showed no evidence of bacteria at the lesion boundary in the extensin-over-expressing transgenics, whereas bacteria were always seen at the lesion boundary on the wild-type. Analysis of transgenics carrying an EXT1-GUS promoter-reporter fusion showed expression of GUS in a ring around the boundary of the lesion. Basal defences and signal transduction pathways involved in plant defence were not perturbed in the transgenics, as shown by the analysis of the expression of PR1 and PDF1.2 genes. These results show that extensin over-expression limits pathogen invasiveness.
Mit Hilfe von Fluorescent Differential Display wurden mehrere durch Stress transkriptionell regulierte Gene isoliert, von denen eines, das frühe Gen PcEF1 innerhalb von Minuten transient durch UV-B Strahlung, mechanischen-, Trocken- und Kältestress sowie durch Elicitoren (PEP13, Pmg) transkriptionell induziert wird. PcEF1 bildet mit homologen Proteinen aus anderen Spezies eine neuartige pflanzenspezifische Proteinfamilie. Das PcEF1-Gen kodiert für ein 13,2 kDa Protein, das eine Ca2+-Bindestelle aufweist. Unter Verwendung von rekombinantem PcEF1-Protein wurde gezeigt, daß PcEF1 Ca2+ unter physiologischen Bedingungen binden kann. Mit Hilfe von transgenen Petersilie-Zellkulturlinien, die Luziferase unter der Kontrolle des PcEF1-Promotors exprimieren, wurde gezeigt, daß die differentielle Regulation der PcEF1-Transkriptmengen nur durch den PcEF1-Promotor und nicht über posttranskriptionelle Mechanismen vermittelt wird. Sowohl die UV-B als auch die durch Elicitierung induzierte Expression des PcEF1-Gens verläuft in Abhängigkeit von der extrazellulären Ca2+-Konzentration. UV-B und Elicitierung wirken zudem additiv bei der PcEF1-Expression. Die konstitutive Expression von PcEF1 in transgenen Petersiliezelllinien hatte keinen Einfluß auf die transkriptionelle Regulation der Flavonoidbiosynthesegene CHS und PAL. Auf posttranskriptioneller Ebene war bei allen analysierten Linien eine erhöhte PAL-Enzymaktivität zu beobachten.
In Arabidopsis thaliana wurde eine entwicklungs- und gewebespezifische Expression des PcEF1-homologen Arabidopsis-Gens AtEF1 gezeigt. AtEF1 wird in adulten Pflanzen innerhalb von einer Stunde durch UV-B Bestrahlung induziert.
PcEF1 und seine homologen Proteine bilden wahrscheinlich eine Familie von extrem schnell auf transkriptioneller Ebene regulierter Signalkomponenten, die eine wichtige Funktion bei der UV-B Signaltransduktion in Pflanzen aufweisen und ein Verbindungsglied zu frühen erhöhten Ca2+-Konzentrationen darstellen könnten.
This review of the living cell wall and its protein components is in two parts. The first is anecdotal. A personal account spanning over 40 years research may perhaps be an antidote to one stereotypical view of scientists as detached and humorless. The second part deals with the meaning of function, particularly as it applies to hydroxyproline-rich glycoproteins. Function is a difficult word to define objectively. However, with help from such luminaries as Humpty Dumpty: "A word means what I want it to mean, neither more nor less," and Wittgenstein: "Giving examples of usage ... is the only way to talk about meaning," it is possible to construct a ziggurat representing increasingly complex levels of organization from molecular structure to ecology. Forty years ago I suggested that hydroxyproline-rich structural proteins played a key role in cell wall functioning. But because the bulk of the wall is carbohydrate, there has been an understandable resistance to paradigm change. Expansins, paradoxically, contribute greatly to this resistance because their modus operandi as cell-wall-loosening proteins is based on the idea that they break hydrogen bonds between polysaccharide chains allowing slippage. However, this view is not consistent with the recent discovery [Grobe et al. (1999) Eur. J. Biochem 263: 33-40] that beta-expansins may be proteases, as it implies that the extensin network is not a straightjacket but a substrate for expansin in muro. Such a direct role for extensins in both negative and positive regulation of cell expansion and elongation may constitute a major morphogenetic mechanism operating at all levels of plant growth and development.
We have previously demonstrated that cutting induces the rapid response of genes for ethylene-responsive transcription factors (ERFs) in leaf strips of tobacco, and that the induction was not interfered but enhanced in the presence of the protein synthesis inhibitor cycloheximide (CHX). In this study, we analyzed the expression of genes for ERFs in tobacco plants by injuring leaf tissues with a hemostat. The results verified that mechanical damage is a trigger for rapid and concurrent induction of both the local and the systemic expression of genes for ERFs in tobacco plants. Further studies on systemic response of ERF genes in response to different severity and position of the wound on a leaf suggested that a threshold value might exist for the magnitude of wound signal to induce systemic activation of these genes. Then, we examined expression of genes for ERFs by analysis in transgenic tobacco plants that harbored reporter genes in which the promoter of the gene for NsERF2, NsERF3 or NsERF4 was fused to a gene for beta-glucuronidase. The results suggested that the local and systemic accumulation of ERF mRNAs after wounding was primarily mediated by the rapid activation of transcription of the respective genes. In addition, we found that cycloheximide triggered rapid activation of genes for ERFs which might be mediated via activation of transcription of the genes for ERFs.
Potato (Solanum tuberosum) cultivars differ quantitatively in their responses to mechanical stress including the ability to synthesize melanin pigments in tuber tissues. Investigations into the cellular events induced by mechanical stress on tuber tissues have shown that an early cellular response is a significant and rapid synthesis of superoxide radicals. This burst of radical production distinctively displays a reproducible biphasic pattern over time with peaks of generation at 2 and 5 h. A concomitant consequence of the generation of these free radicals is elevated levels of oxidatively modified tuber proteins. Both radical generation and protein modification vary between cultivars but both are directly proportional to the amount of melanin pigments produced. Cell-free extracts of mechanically stressed tissues, pectic fragments, and scission products generated from cell walls are able to induce superoxide generation in non-stressed tissues, indicating the participation of a biologically active factor that induces a further a phase of radical synthesis.
Hydroxyproline-rich glycoproteins (HRGP) are structural components of the plant cell wall. Hrgp genes from maize and related species have a conserved 500 bp sequence in the 5'-flanking region, and all Hrgp genes from monocots have an intron located in the 3' untranslated region. To study the role of these conserved regions, several deletions of the Hrgp gene were fused to the beta-glucuronidase ( GUS) gene and used to transform maize tissues by particle bombardment. The overall pattern of GUS activity directed by sequential deletions of the Hrgp promoter was different in embryos and young shoots. In embryos, the activity of the full-length Hrgp promoter was in the same range as that of the p35SI promoter construct, based on the strong 35S promoter, whereas in the fast-growing young shoots it was 20 times higher. A putative silencer element specific for young shoots was found in the -1,076/-700 promoter region. Other major cis elements for Hrgp expression are probably located in the regions spanning -699/-510 and -297/-160. Sequences close to the initial ATG and mRNA leader were also important since deletion of the region -52/+16 caused a 75% reduction in promoter activity. The presence of the Hrgp intron in the 3' untranslated region changed the levels of GUS activity directed by the Hrgp and the 35S promoters. This pattern of activity was complex, and was dependent on the promoter and cell type analysed.
Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the β-glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.
The CACGTG G-box motif is a highly conserved DNA sequence that has been identified in the 5' upstream region of plant genes exhibiting regulation by a variety of environmental signals and physiological cues. Gel mobility shift assays using a panel of G-box oligonucleotides differing in their flanking sequences identified two types of binding activity (A and B) in a cauliflower nuclear extract. Competition gel retardation assays demonstrated that the two types of binding activity were distinct. Type A binding activity interacted with oligonucleotides designated as class I elements, whereas type B binding activity interacted strongly with class II elements and weakly with class I elements. A third class of elements, null elements, did not exhibit any detectable binding under our assay conditions. Gel retardation analysis of nonpalindromic hybrid G-box oligonucleotides indicated that hybrid elements of the same class exhibited binding affinity commensurate with the affinity of the weaker element, hybrid class I/II elements exhibited only type B binding, and hybrid class I/null and class II/null elements did not show any detectable binding activity. These binding activities can be explained by the affinity of bZip G-box binding homo- or heterodimer subunits for G-box half sites. These experiments led to a set of classification rules that can predict the binding activity of all reported plant G-box motifs containing the consensus hexameric core. Tissue- and/or development-specific expression of genes containing G-box motifs may be regulated by the affinity of G-box proteins for the different classes of G-box elements.
Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are plant lipid derivatives that resemble mammalian eicosanoids in structure and biosynthesis. These compounds are proposed to play a role in plant wound and pathogen responses. Here we report the quantitative determination of JA/MeJA in planta by a procedure based on the use of [13C,2H3]MeJA as an internal standard. Wounded soybean (Glycine max [L] Merr. cv. Williams) stems rapidly accumulated MeJA and JA. Addition of MeJA to soybean suspension cultures also increased mRNA levels for three wound-responsive genes (chalcone synthase, vegetative storage protein, and proline-rich cell wall protein) suggesting a role for MeJA/JA in the mediation of several changes in gene expression associated with the plants' response to wounding.
We have previously shown that the expression of a rice gene, rab-16A, is responsive to abscisic acid (ABA) and osmotic stress in plant tissues and cultured suspension cells. We demonstrate here that transcriptional elements between -294 and -52 of this gene are sufficient to confer ABA-dependent expression on the chloramphenicol acetyltransferase reporter gene in rice protoplasts. Sequence motifs within this 242-base-pair region of the rab-16A gene are conserved among the 5' upstream regions of other ABA-responsive genes. Gel retardation and DNAse I experiments show nuclear factor(s) binding to these sequences. This correlative data indicate that these motifs are involved in the transcription of the rab genes and suggest that they may be ABA-responsive-elements (ABREs).
A protein factor, identified in nuclear extracts obtained from tomato (Lycopersicon esculentum, Solanaceae) and Arabidopsis thaliana (Brassicaceae) seedlings, specifically binds upstream sequences from the plant light-regulated gene family encoding the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RBCS). RBCS upstream sequences from tomato, pea (Pisum sativum, Leguminosae), and Arabidopsis are recognized by the factor. The factor recognition occurs via a short conserved sequence (G box) whose consensus sequence is 5'-TCTTACACGTGGCAYY-3' (where Y is pyrimidine). This sequence is distinct from the GT motif described previously in RBCS promoters. Two other conserved sequences, showing a lesser degree of evolutionary conservation, are found upstream of the G box but do not bind to the G box binding factor (GBF). Twelve nucleotides within the G box are sufficient for the formation of a stable DNA-GBF complex. GBF is found in both light-grown and dark-adapted tomato leaf extracts, but it is present in greatly reduced amounts in root extracts.
Two recently isolated cDNAs representing genes that are transcriptionally activated in fungus-infected parsley leaves or elicitor-treated, cultured parsley cells are shown to encode a hydroxyproline-rich glycoprotein (HRGP) and an anionic peroxidase. The deduced HRGP protein is rich in tyrosine residues, a feature also found in other pathogen- and wound-induced plant HRGPs. Expression of the peroxidase gene(s) is induced rapidly upon elicitation and precedes that of the HRGP gene. In situ hybridization experiments demonstrate the presence of HRGP and peroxidase mRNAs in parsley tissue around fungal infection sites. Peroxidase mRNA accumulation is particularly sharply restricted to plant cells directly adjacent to fungal hyphae. These results provide further evidence for an important role of specific cell wall modifications in plant defense.
Elicitation of soybean cells causes a rapid insolubilization of two cell wall structural proteins, p33 and p100. Likewise, a short elicitation of 30 min rendered cell walls more refractory to enzyme digestion as assayed by the yield of protoplasts released. This effect could be ascribed to protein cross-linking because of its insensitivity to inhibitors of transcription (actinomycin D) and translation (cycloheximide) and its induction by exogenous H2O2. Moreover, the induced loss of protoplasts could be prevented by preincubation with DTT, which also blocks peroxidase-mediated oxidative cross-linking. The operation of protein insolubilization in plant defense was also demonstrated by its occurrence in the incompatible interaction but not in the compatible interaction between soybean and Pseudomonas syringae pv glycinea. Likewise, protein insolubilization was observed in bean during non-host hypersensitive resistance to the tobacco pathogen P. s. pv tabaci mediated by the hypersensitive resistance and pathogenicity (Hrp) gene cluster. Our data strongly suggest that rapid protein insolubilization leads to a strengthened cell wall, and this mechanism functions as a rapid defense in the initial stages of the hypersensitive response prior to deployment of transcription-dependent defenses.
When sedentary endoparasitic nematodes infect plants, they induce complex feeding sites within the root tissues of their host. To characterize cell wall changes induced within these structures at a molecular level, we studied the expression of an extensin gene (coding for a major structural cell wall protein) in nematode-infected tobacco roots. Extensin gene expression was observed to be induced very early upon infection. This induction was weak, transient, and probably due to wounding during penetration and migration of the tobacco cyst nematode Globodera tabacum ssp solanacea-rum. In contrast, high extensin gene expression was observed during the whole second larval stage (an ~2-week-long phase of establishment of the feeding site) of the root knot nematode Meloidogyne javanica. During later stages of this interaction, expression gradually decreased. Extensin gene expression was found in at least three different tissues of the gall. We propose that distinct mechanisms lead to induced expression in these different cell types. The significance of these results for the understanding of plant-nematode interactions as well as the function of structural cell wall proteins, such as extensin, is discussed.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
A 3.4-kilobase genomic DNA fragment from Pisum sativum L. containing the LegA gene, which encodes a major legumin storage protein, was transferred to Nicotiana plumbaginifolia using an Agrobacterium tumefaciens strain containing the Bin 19 binary vector system. Northern hybridisation analysis of legA-transformed plants demonstrated that legumin-specific RNA was present in developing seeds but not in developing leaves. Legumin protein was immunologically detected in the mature seeds of legA-transformed plants, and was present as the correct-size protein composed of disulphide-bonded polypeptides. It is concluded that the transferred pea genomic fragment contains all the information necessary for seed-specific expression of the legA gene, and for correct processing of the primary transcript and the precursor legumin protein.
Lea genes code for mRNAs and proteins that are late embryogenesis abundant in higher plant seed embryos. They appear to be ubiquitous in higher plants and may be induced to high levels of expression in other tissues and at other times of ontogeny by ABA and/or desiccation. Presented here are the genomic and cDNA sequences for 6 of these genes from cotton seed embryos and the derived amino acid sequences of the corresponding proteins.
The Lea genes contain the standard sequence features of eucaryotic genes (TATA box and poly (A) addition sequences) and have 1 or more introns. Sequences differences between cDNA and genomic DNA confirm the existence of small multigene families for several Lea genes. The amino acid composition and sequence for the Lea proteins are unusual. Five are extremely hydrophilic, four contain no cys or trp and 4 have sequence domains that suggest amphiphilic helical structures. Hypothetical functions in desiccation survival, based on amino acid sequence, are discussed.
Following wounding of carrot (Daucus carota L.) roots, the activity of a nuclear factor (EGBF-1) that binds a 5'-region of the carrot extensin gene declines to undetectable levels within 48 h. Mixing of nuclear extracts from wounded roots with nuclear extracts from unwounded roots has demonstrated the existence of a wound-induced inhibitor of EGBF-1. Inhibition of EGBF-1 DNA-binding activity by nuclear extracts from wounded roots is shown to be specific for EGBF-1, and to be destroyed by heat treatment. In addition, inhibition is saturable and occurs rapidly. Active EGBF-1 can be reconstituted from its inhibited state by renaturation of proteins from mixed extracts following denaturation by boiling in sodium dodecyl sulfate and 2-mercaptoethanol, and electrophoretic separation, indicating that inhibition is dependent upon the reversible interaction of EGBF-1 with a titratable factor. However, EGBF-1 activity could not be detected in nuclear extracts from wounded roots following denaturation and electrophoretic separation. Inhibitory activity was not detectable in nuclear extracts from roots that had been trated with ethylene. The action of the inhibitor indicates one possible mechanism for the control of EGBF-1 activity in carrot roots following wounding.
Experiments conducted in vitro using the electrophoretic mobility shift assay have shown that a single region of the extensin gene of carrot (Daucus carota L.) interacts with a protein factor designated Extensin Gene Binding Factor-1 (EGBF-1) present in nuclear extracts obtained from carrot roots. This interaction is sequence-specific as judged by the failure of other plant gene sequences to compete with the extensin gene for EGBF-1 binding. The EGBF-1 activity is organspecific, not being expressed in nuclear extracts obtained from carrot leaves or stems. Both ethylene treatment and wounding of roots are shown to have a controlling influence on the expression of EGBF-1 activity in nuclear extracts. These results demonstrate that at least three distinct signals: ethylene treatment, wounding, and development, are important in determining the activity of EGBF-1 in nuclear extracts, and indicate a role for EGBF-1 in stress-related signal transduction and the regulation of extensin-gene expression.
A family of cross-hybridising cDNA clones has been isolated from a cDNA library produced with poly(A)+ RNA from the roots of oilseed-rape (Brassica napus L.). The clones were selected as abundantly expressed in root by differential screening of the root cDNA library with cDNA probes prepared from root, green leaf, etiolated leaf and developing seed. mRNA species corresponding to the selected abundant clones were expressed in roots at levels of at least 400 times those in other organs, as shown by Northern blot analysis and RNase protection assays. Complete nucleotide sequence determination of the cDNA clones showed that they encoded proteins homologous to carrot extensin and were the products of at least three different genes. An extensin gene, designated extA, was obtained from an oilseed rape (B. napus L.) genomic library screened with a cDNA species encoding a protein expressed abundantly in roots. The gene is a member of a multigene family, consisting of about 3 members per haploid genome with strong homology to the probe, and a further 20 or so members with weaker homology. The isolated gene, although not identical to the cDNA probe, was also found to be specifically expressed in roots, and was transcribed into a mRNA species approximately 1300 nucleotides in size. A single transcription start was identified by S1 mapping. The complete nucleotide sequence of the extA gene and its flanking regions has been determined and shown to encode a protein homologous to carrot and tomato extensins.
Nicotiana plumbaginifolia Viv. harbors a single extensin gene, although related hydroxyproline-rich sequences are present in the genome. Northern analysis showed that the gene is highly expressed in roots and to a lesser extent in stems. Expression in leaves is low but mRNA levels are increased upon infection with the incompatible bacterium Pseudomonas syringae. Extensin transcript levels in leaves were slightly enhanced after wounding and salicylic acid treatment. In-situ hybridization experiments showed high accumulation of extensin mRNA in cells which, at certain stages of development, require reinforcement of their cell walls. The cortical cells in stem nodes and roots, which are put under severe mechanical stress by adjacent developing tissues, tend to express the gene to high levels. Immunolocalization of the extensin protein in stems and roots demonstrated a close association of the protein with lignin deposition. Mature tissues contained more extensin than younger tissues. The extensin promoter was fused to the -glucuronidase gene.
Infection of the stem base of sunflower (Helianthus annuus L.) plants by Sclerotini sclerotiorum (Lib.) de Bary induces the accumulation of at least four RNA species in stems and leaves (2.5, 1.6, 0.56 and 0.135 kb) which hybridize to a genomic clone of hydroxyproline-rich glycoproteins (HRGPs) from carrot. This induction is observed as early as 2 days after infection in a tolerant line (PS3B), while it occurred only after 3 days in a susceptible one (HA89). It appears that the tolerance is associated to an early increase of extensin hybridizable RNA; steady state levels of these transcripts could be used as a molecular marker of sunflower tolerance to the stem base white mold. Oxalic acid, the major toxin produced by Sclerotinia sclerotiorum, induces a differential accumulation of the same HRGP transcripts in the two lines, in an organo-specific way. Only the 1.6- and the 0.135-kb RNA species are representative of the tolerance of PS3B to both the toxin and the fungus. Besides its role as toxin in this host-pathogen interaction, oxalic acid also appears to play the role of an elicitor as inducer of the accumulation of HRGP transcripts.
The isolation of high molecular weight DNA from whole organisms (or large tissue masses) is improved by the disruption of tissues by freeze-grinding and the rapid introduction of organic deproteinizers.
The organs and tissues where the Brassica napus extA extensin gene is expressed have been identified. The extA gene with 3.75 kb of 5' flanking sequence was transferred to tobacco via disarmed Agrobacterium tumefaciens vectors and transgenic plants regenerated. The gene was found to be inactive in transgenic tobacco leaf, but was active as measured by RNA transcript assays in both stem and root tissues. To determine the cell-specific expression pattern of the extA gene, a promoter-reporter gene fusion construct was made consisting of 1.0 kb of 5' extA sequence fused to the coding region of the glucuronidase (GUS) gene. This fusion construct was introduced into B. napus via Agrobacterium rhizogenes, and expression of GUS in transgenic rape hairy roots was examined. GUS activity was only seen in the vascular tissues of the rape root, and was found to be specifically localised in the phloem.
A fast and efficient method for the isolation of RNA from plant tissues is described. Tuber tissue is homogenized in a guanidine hydrochloride-containing buffer followed by direct extraction with phenol/chloroform. The RNA is precipitated from the aqueous phase, washed with 3 M sodium acetate and 70% ethanol, and finally dissolved in water. The yield of RNA is up to 500 micrograms/g of tissue and several tests indicate intact and nondegraded RNA. This method can be adapted to a small-scale version by the use of 1.5-ml tubes, allowing rapid isolation of RNA from a larger number of samples. Finally, this method is of particular use for isolating RNA from tissues with a high polysaccharide and nuclease content such as wounded potato tubers.
A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
A broad host range cloning vehicle that can be mobilized at high frequency into Gram-negative bacteria has been constructed from the naturally occurring antibiotic resistance plasmid RK2. The vehicle is 20 kilobase pairs in size, encodes tetracycline resistance, and contains two single restriction enzyme sites suitable for cloning. Mobilization is effected by a helper plasmid consisting of the RK2 transfer genes linked to a ColE1 replicon. By use of this plasmid vehicle, a gene bank of the DNA from a wild-type strain of Rhizobium meliloti has been constructed and established in Escherichia coli. One of the hybrid plasmids in the bank contains a DNA insert of approximately 26 kilobase pairs which has homology to the nitrogenase structural gene region of Klebsiella pneumoniae.
The bean grp1.8 full-length promoter is specifically active in vascular tissue during normal development of tobacco. Deletion of a negative regulatory element resulted in ectopic activity of the promoter in cortical cells of hypocotyls, roots and stems. A 169 bp fragment (-205 to -36) of the grp1.8 promoter conferred vascular-specific expression to CaMV 35S minimal promoters whereas a 141 bp fragment (-205 to -64) strongly activated these minimal promoters both in vascular and cortical cells. These experiments defined a new regulatory element (VSE) that is essential for vascular-specific expression and is located between -64 and -36. The 141 bp grp1.8 promoter sequence had enhancer-like properties as it was active in both orientations. A 24 bp sequence (bp -119 to -96, corresponding to the SE1 regulatory element) enhanced expression from several minimal promoters strongly but unspecifically, whereas a 26 bp sequence (-98 to -73, corresponding to the RSE regulatory element) induced vascular-specific expression. Thus, the grp1.8 promoter is regulated by a combinatorial mechanism that can integrate the action of different, non-additively acting regulatory elements into vascular-specific expression.
Homologous hydroxyproline-rich glycoproteins (HRGPs) of the plant extracellular matrix include extensins, repetitive proline-rich proteins (RPRPs), some nodulins, gum arabic glycoprotein (GAGP), arabinogalactan-proteins (AGPs), and chimeric proteins such as potato lectin which contain an extensin module fused to a lectin. The key to the role of HRGPs in cell wall self-assembly and cell extension lies in their chemistry, which is dependent on extensive post-translational modifications (PTMs): hydroxylation, glycosylation, and cross-linking. Repetitive peptide motifs characterize HRGPs. One or more repetitive peptide motifs and their variants, singly or in combination, may constitute functional sites involved in various aspects of cell wall assembly, as follows:
Rules for the post-translational modifications are emerging:
Their protistan origin obscures the phylogenetic affinities of a single extensin-HRGP family due to their sequence divergence. We propose a phylogenetic series ranging from the minimally glycosylated basic RPRPs to the highly glycosylated acidic AGPs. Furthermore, based on similarities between dicots and gymnosperm extensins, and their marked difference from graminaceous monocot extensins, graminaceous monocot and dicot lines may have diverged as early as the progymnosperms. The origin of the embryophytes (land plants) is an even more open question, the nearest extant algal relatives being unknown. The Charophyta, frequently suggested as ancestral to the embryophytes, seem unlikely as the Charalean cell wall of Chara and Nitella lacks hydroxyproline.
Plant bZIP proteins exhibit a relaxed DNA-binding specificity for DNA sequence motifs containing an ACGT core. Gel mobility shift experiments employing ten different recombinant plant bZIP proteins demonstrated that nucleotides flanking the ACGT core affected binding specificity and identified three different types of ACGT elements: G-box, CACGTG; C-box, GACGTC; and A-box, TACGTA, motifs. These ten different bZIP proteins could be categorized into three groups according to their qualitative and quantitative specificity for G-box and C-box elements. Dissociation constant values (Kd values) of these bZIP proteins for high affinity G-box and C-box elements and reciprocal competition gel mobility shift assays confirmed our classification scheme. Group 1 proteins exhibit a stronger binding affinity for G-box elements, group 2 proteins bind both G-box and C-box motifs with comparable binding affinity, whereas the group 3 proteins display a stronger binding affinity for C-box oligonucleotides. Studies using a panel of G-box and C-box oligonucleotides differing in their flanking sequences identified high affinity binding sites. All ten plant bZIP proteins examined, except TGA1a, exhibited type A G-box binding activity preferring class I G-box elements. In contrast to the situation observed for G-box elements, C-box motifs displayed a very much more stringent flanking nucleotide requirement for binding activity. Protein/DNA binding experiments using scanning mutants of a high affinity G-box element and G-box/C-box hybrid elements demonstrated that bZIP protein binding activity depends upon the affinity of protein dimer subunits for ACGT half-sites. Information provided by our systematic analysis of plant bZIP DNA binding specificity can be used to identify high affinity binding sites for the plant bZIP proteins studied here. Assuming that only high affinity bZIP binding sites are likely to function in vivo, identification of these sites will allow us to predict which genes are activated by a particular bZIP protein.
We have analysed the expression of the endogenous extensin genes in Brassica napus, using northern hybridisation and dot blotting. In the unstressed plant, the extA gene is only expressed in the root, expression in the leaf, petiole and stem being absent. We have found that wounding dramatically alters this normal pattern of expression. Expression in wounded leaf is seen after 36 h, in wounded petioles after 11 h and in wounded stem after 17 h. Differences in the amount of extensin mRNA accumulated are also seen: wounded petiole accumulating extensin message to a level higher than the leaf or the stem. Inhibitors of ethylene biosynthesis greatly delay the onset of accumulation of extensin mRNA in wounded tissues. Wounding the root causes the level of extensin message to decline with time, until levels below the limit of non-specific hybridisation are reached 11 h after wounding. Thus, application of the wounding stimulus results in the accumulation of extensin gene transcripts to different degrees and at different times in the aerial parts of the plant, and results in a decline in the same transcripts in the roots. Extensin transcript accumulation as a result of wounding is also dependent on the age of the tissue; high levels of message are seen in old wounded leaves, while expression in young wounded leaves is absent.
The bean grp1.8 gene is specifically expressed in vascular tissue. Monomers and multimers of a 28 bp regulatory element of the grp1.8 promoter (vs-1) specifically activated both the -82 CaMV 35S and the -76/grp1.8 minimal promoters in vascular tissue of transgenic tobacco plants. vs-1 partially overlaps with a negative regulatory element in the grp1.8 promoter that is necessary for restriction of gene expression to vascular tissue. Nuclear extracts from tobacco and tomato cells contain a factor that binds to vs-1 in vitro. To study the molecular basis of xylem-specific expression mediated by the vs-1 promoter element, a gene was isolated from tomato encoding a protein that binds to vs-1 in vitro. This protein, designated VSF-1, contains a bZIP motif close to the C-terminus. Mutated vs-1 elements were no longer bound by VSF-1 and also failed to activate the minimal -82 CaMV 35S promoter in vivo. Transient expression of VSF-1 in protoplasts stimulated vs-1 dependent activation of the -76/grp1.8 minimal promoter. Binding studies and use of a polyclonal antiserum against VSF-1 provided further evidence that vs-1 is a potential in vivo target site, as VSF-1 was a part of the observed complex formed between vs-1 and nuclear protein extract. vs-1 does not contain the 5'-ACGT-3' core sequence that is part of known plant bZIP protein binding sites or another palindromic sequence. Based on the unusual binding specificity and a characteristic amino acid sequence in the bZIP domain we propose that VSF-1 and the partially homologous PosF21, a bZIP protein from Arabidopsis, belong to a new family of plant bZIP proteins.
C/EBP is a sequence-specific DNA binding protein that regulates gene expression in certain mammalian cells. The region of the C/EBP polypeptide required for specific recognition of DNA is related in amino acid sequence to other regulatory proteins, including the Fos and Jun transforming proteins. It has been proposed that these proteins bind DNA via a bipartite structural motif, consisting of a dimerization interface termed the "leucine zipper" and a DNA contact surface termed the "basic region." An evaluation of the properties of conserved amino acids within the basic region of 11 deduced protein sequences, coupled with the observation that they are located at an invariant distance from the leucine zipper, has led to the formulation of a "scissors-grip" model for DNA binding. The architectural features of this model are well suited for interaction with directly abutted, dyadsymmetric DNA sequences. Data supportive of the model were obtained with chemical probes of protein: DNA complexes.
Sequence and structure of 6 Lea proteins and their genes from cotton
- J Baker
- C Steele
- Dure
Baker J, Steele C, Dure L: Sequence and structure of 6 Lea proteins and their genes from cotton. Plant Mol Biol 11: 277– 291 (1988).