Artificially designed synthetic promoter for a high level of salt induction using a cis-engineering approach.

This work aimed to design a synthetic salt-inducible promoter using a cis-engineering approach. The designed promoter (PS) comprises a minimal promoter sequence for basal-level expression and upstream cis-regulatory elements (CREs) from promoters of salinity-stress-induced genes. The copy number, spacer lengths, and locations of CREs were manually determined based on their occurrence within native promoters. The initial activity profile of the synthesized PS promoter in transiently transformed N. tabacum leaves shows a seven-fold, five-fold, and four-fold increase in reporter GUS activity under salt, drought, and abscisic acid stress, respectively, at the 24-h interval, compared to the constitutive CaMV35S promoter. Analysis of gus expression in stable Arabidopsis transformants showed that the PS promoter induces over a two-fold increase in expression under drought or abscisic acid stress and a five-fold increase under salt stress at 24- and 48-h intervals, compared to the CaMV35S promoter. The promoter PS exhibits higher and more sustained activity under salt, drought, and abscisic acid stress compared to the constitutive CaMV35S.

Isolation and functional characterization of cold-induced gene (AmCIP) promoter from Ammopiptanthus mongolicus.

AmCIP is a dehydrin-like protein which involved in abiotic stress tolerance in xerophytes evergreen woody plant A. mongolicus. AmCIP could be induced in the cotyledon and radicle during cold acclimation. To further elucidate the regulation of the upstream region of the gene, we isolated and characterized the promoter of AmCIP. Herein, a 1115 bp 5'-flanking region of AmCIP genomic DNA was isolated and cloned by genome walking from A. mongolicus and the segment sequence was identified as "PrAmCIP" promoter. Analysis of the promoter sequence revealed the presences of some basic cis-acting elements, which were related to various environmental stresses and plant hormones. GUS histochemical staining of transgene tobacco showed that PrAmCIP was induced by 4℃, 55℃, NaCl, mannitol and ABA, whereas it could hardly drive GUS gene expression under normal conditions. Furthermore, we constructed three deletion fragments and genetically transformed them into Arabidopsis thaliana. GUS histochemical staining showed that the MYCATERD1 element of the CP7 fragment (-189 âˆ¼ -1) may be a key element in response to drought. In conclusion, we provide an inducible promoter, PrAmCIP, which can be applied to the development of transgenic plants for abiotic stresse tolerance.

A rapid and efficient Agrobacterium-mediated transient transformation system in grape berries.

Transient transformation is extremely useful for rapid in vivo assessment of gene function, especially for fruit-related genes. Grape berry, while an important fruit crop, is recalcitrant to transient transformation, due to the high turgor pressure in its mesocarp cells that limits the ability of Agrobacterium to penetrate into the tissue. It is urgent to establish a simple transient transformation system for rapid analysis of gene function. In this study, different injection methods, grape genotypes, and developmental stages were tested in order to develop a rapid and efficient Agrobacterium-mediated transient transformation methodology for grape berries. Two injection methods, namely punch injection and direct injection, were evaluated using the ß-glucuronidase (GUS) gene and by x-gluc tissue staining and 4-methylumbelliferyl-ß-D-glucuronide fluorescence analysis. The results indicated that there were no significant differences on transformation effects between the two methods, but the latter was more suitable because of its simplicity and convenience. Six grape cultivars ('Hanxiangmi', 'Moldova', 'Zijixin', 'Jumeigui', 'Shine-Muscat', and 'A17') were tested for transient transformation. 'Hanxiangmi', 'Moldova', and 'Zijixin' grape berries were not suitable for agroinfiltration due to frequently fruit cracking, browning, and formation of scar skin. The fruit integrity rates of 'Jumeigui', 'Shine-Muscat', and 'A17' berries were all above 80%, and GUS activity was detected in the berries of the three cultivars 3-14 days after injection with the Agrobacterium culture, while higher GUS activities were observed in the 'Jumeigui' berries. The levels of GUS activity in injected berries at 7-8 weeks after full blooming (WAFB) were more than twice at 6 WAFB. In subsequent assays, the over-expression of MYB transcription factor VvMYB44 via transient transformation accelerated the anthocyanin accumulation and fruit coloring through raising the expression levels of VvLAR1, VvUFGT, VvLDOX, VvANS, and VvDFR, which verified the effectiveness of this transformation system. These experiments finally identified the reliable grape cultivars and suitable operational approach for transient transformation and further indicated that this Agrobacterium-mediated transient transformation system was efficient and suitable for the elucidation of gene function in grape berries.

Genome-Wide Investigation of Apyrase (APY) Genes in Peanut (Arachis hypogaea L.) and Functional Characterization of a Pod-Abundant Expression Promoter AhAPY2-1p.

Peanut (Arachis hypogaea L.) is an important food and feed crop worldwide and is affected by various biotic and abiotic stresses. The cellular ATP levels decrease significantly during stress as ATP molecules move to extracellular spaces, resulting in increased ROS production and cell apoptosis. Apyrases (APYs) are the nucleoside phosphatase (NPTs) superfamily members and play an important role in regulating cellular ATP levels under stress. We identified 17 APY homologs in A. hypogaea (AhAPYs), and their phylogenetic relationships, conserved motifs, putative miRNAs targeting different AhAPYs, cis-regulatory elements, etc., were studied in detail. The transcriptome expression data were used to observe the expression patterns in different tissues and under stress conditions. We found that the AhAPY2-1 gene showed abundant expression in the pericarp. As the pericarp is a key defense organ against environmental stress and promoters are the key elements regulating gene expression, we functionally characterized the AhAPY2-1 promoter for its possible use in future breeding programs. The functional characterization of AhAPY2-1P in transgenic Arabidopsis plants showed that it effectively regulated GUS gene expression in the pericarp. GUS expression was also detected in flowers of transgenic Arabidopsis plants. Overall, these results strongly suggest that APYs are an important future research subject for peanut and other crops, and AhPAY2-1P can be used to drive the resistance-related genes in a pericarp-specific manner to enhance the defensive abilities of the pericarp.

Identification of TCP family in moso bamboo (Phyllostachys edulis) and salt tolerance analysis of PheTCP9 in transgenic Arabidopsis.

MAIN CONCLUSION: Bioinformatic analysis of moso bamboo TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors reveals their conservation and variation as well as the probable biological functions in abiotic stress response. Overexpressing PheTCP9 in Arabidopsis thaliana illustrates it may exhibit a new vision in different aspects of response to salt stress. Plant specific TCPs play important roles in plant growth, development and stress response, but studies of TCP in moso bamboo are limited. Therefore, in this study, a total of 40 TCP genes (PheTCP1 ~ 40) were identified and characterized from moso bamboo genome and divided into three different subfamilies, namely, 7 in TEOSINTE BRANCHED 1 / CYCLOIDEA (TB1/CYC), 14 in CINCINNATA (CIN) and 19 in PROLIFERATING CELL FACTOR (PCF). Subsequently, we analyzed the gene structures and conserved domain of these genes and found that the members from the same subfamilies exhibited similar exon/intron distribution patterns. Selection pressure and gene duplication analysis results indicated that PheTCP genes underwent strong purification selection during evolution. There were many cis-elements related to phytohermone and stress responsive existing in the upstream promoter regions of PheTCP genes, such as ABRE, CGTCA-motif and ARE. Subcellular localization experiments showed that PheTCP9 was a nuclear localized protein. As shown by ß-glucuronidase (GUS) activity, the promoter of PheTCP9 was significantly indicated by salt stress. PheTCP9 was significantly induced in the roots, stems and leaves of moso bamboo. It was also significantly induced by NaCl solution. Overexpressing PheTCP9 increased the salt tolerance of transgenic Arabidopsis. Meanwhile, H2O2 and malondialdehyde (MDA) contents were significantly lower in PheTCP9 over expression (OE) transgenic Arabidopsis than WT. Catalase (CAT) activity, K+/Na+ ratio as well as CAT2 expression level was also much improved in transgenic Arabidopsis than WT under salt conditions. In addition, PheTCP9 OE transgenic Arabidopsis held higher survival rates of seedlings than WT under NaCl conditions. These results showed the positive regulation functions of PheTCP9 in plants under salt conditions.

Stability of Transgene Inheritance in Progeny of Field-Grown Pear Trees over a 7-Year Period.

Breeding woody plants is a very time-consuming process, and genetic engineering tools have been used to shorten the juvenile phase. In addition, transgenic trees for commercial cultivation can also be used in classical breeding, but the segregation of transgenes in the progeny of perennial plants, as well as the possible appearance of unintended changes, have been poorly investigated. We studied the inheritance of the uidA gene in the progeny of field-grown transgenic pear trees for 7 years and the physical and physiological parameters of transgenic seeds. A total of 13 transgenic lines were analyzed, and the uidA gene segregated 1:1 in the progeny of 9 lines and 3:1 in the progeny of 4 lines, which is consistent with Mendelian inheritance for one and two transgene loci, respectively. Rare and random deviations from the Mendelian ratio were observed only for lines with one locus. Transgenic seeds' mass, size, and shape varied slightly, despite significant fluctuations in weather conditions during cultivation. Expression of the uidA gene in the progeny was stable. Our study showed that the transgene inheritance in the progeny of pear trees under field conditions occurs according to Mendelian ratio, does not depend on the environment, and the seed vigor of transgenic seeds does not change.

Expression Patterns of DNA Methylation and Demethylation Genes during Plant Development and in Response to Phytohormones.

DNA methylation and demethylation precisely and effectively modulate gene expression during plant growth and development and in response to stress. However, expression profiles of genes involved in DNA methylation and demethylation during plant development and their responses to phytohormone treatments remain largely unknown. We characterized the spatiotemporal expression patterns of genes involved in de novo methylation, methyl maintenance, and active demethylation in roots, shoots, and reproductive organs using ß-glucuronidase (GUS) reporter lines. Promoters of DNA demethylases were generally more highly active at the mature root tissues, whereas the promoters of genes involved in DNA methylation were more highly active at fast-growing root tissues. The promoter activity also implies that methylation status in shoot apex, leaf primordia, floral organs, and developing embryos is under tight equilibrium through the activity of genes involved in DNA methylation and demethylation. The promoter activity of DNA methylation and demethylation-related genes in response to various phytohormone treatments revealed that phytohormones can alter DNA methylation status in specific and redundant ways. Overall, our results illustrate that DNA methylation and demethylation pathways act synergistically and antagonistically in various tissues and in response to phytohormone treatments and point to the existence of hormone-linked methylome regulation mechanisms that may contribute to tissue differentiation and development.

The durum wheat annexin, TdAnn6, improves salt and osmotic stress tolerance in Arabidopsis via modulation of antioxidant machinery.

TdAnn6 is a gene encoding an annexin protein in durum wheat (Triticum durum). The function of TdAnn6 in plant response to stress is not yet clearly understood. Here, we isolated TdAnn6 and characterized it in genetically modified Arabidopsis thaliana. Expressing TdAnn6 in Arabidopsis coincided with an improvement in stress tolerance at germination and seedling stages. In addition, TdAnn6-expressing seedling antioxidant activities were improved with lower level of malondialdehyde, and enhanced transcript levels of six stress-related genes during salt/osmotic stresses. Under greenhouse conditions, the TdAnn6 plants exhibited increased tolerance to salt or drought stress. To deepen our understanding of TdAnn6 function, we isolated a 1515-bp genomic fragment upstream of its coding sequence, designated as PrTdAnn6. The PrTdAnn6 promoter was fused to the ß-glucuronidase reporter gene and transferred to Arabidopsis. By histochemical GUS staining, GUS activity was detected in the roots, leaves, and floral organs, but no activity was detected in the seeds. Furthermore, we noticed a high stimulation of promoter activity when A. thaliana seedlings were exposed to NaCl, mannitol, ABA, GA, and cold conditions. This cross-talk between tissue-specific expression and exogenous stress stimulation may provide additional layers of regulation for salt and osmotic stress responses in crops.

Functional analysis of TmHKT1;4-A2 promoter through deletion analysis provides new insight into the regulatory mechanism underlying abiotic stress adaptation.

MAIN CONCLUSION: Bioinformatic, molecular, and biochemical analysis were performed to get more insight into the regulatory mechanism by which TmHKT1;4-A2 is regulated. HKT transporters from different plant species have been shown to play important role in plant response to salt. In previous work, TmHKT1;4-A2 gene from Triticum monococcum has been characterized as a major gene for Nax1 QTL (Tounsi et al. Plant Cell Physiol 57:2047-2057, 2016). So far, little is known about its regulatory mechanism. In this study, the promoter region of TmHKT1;4-A2 (1400 bp) was isolated and considered as the full-length promoter (PA2-1400). In silico analysis revealed the presence of important cis-acting elements related to abiotic stresses and phytohormones. Interestingly, our real-time RT-PCR analysis provided evidence that TmHKT1;4-A2 is regulated not only by salt stress but also by osmotic, heavy metal, oxidative, and hormones stresses. In transgenic Arabidopsis plants, TmHKT1;4-A2 is strongly active in vascular tissues of roots and leaves. Through 5'-end deletion analysis, we showed that PA2-1400 promoter is able to drive strong GUS activity under normal conditions and in response to different stresses compared to PA2-824 and PA2-366 promoters. These findings provide new information on the regulatory mechanism of TmHKT1;4-A2 and shed more light on its role under different stresses.

The Rooting of Stem Cuttings and the Stability of uidA Gene Expression in Generative and Vegetative Progeny of Transgenic Pear Rootstock in the Field.

Adventitious rooting plays an important role in the commercial vegetative propagation of trees. Adventitious root formation is a complex biological process, but knowledge of the possible unintended effects induced by both the integration/expression of transgenes and in vitro conditions on the rooting is limited. The long-term stability of transgene expression is important both for original transformants of woody plants and its progeny. In this study, we used field-grown pear rootstock GP217 trees transformed with the reporter ß-glucuronidase (uidA) genes with and without intron and re-transformed with the herbicide resistance bar gene as model systems. We assessed the unintended effects on rooting of pear semi-hardwood cuttings and evaluated the stability of transgene expression in progeny produced by generative (seedlings) and vegetative (grafting, cutting) means up to four years. Our investigation revealed that: (1) The single and repeated transformations of clonal pear rootstocks did not result in unintended effects on adventitious root formation in cuttings; (2) stability of the transgene expression was confirmed on both generative and vegetative progeny, and no silenced transgenic plants were detected; (3) yearly variation in the gene expressions was observed and expression levels were decreased in extremely hot and dry summer; (4) the intron enhanced the expression of uidA gene in pear plants approximately two-fold compared to gene without intron. The current study provides useful information on transgene expression in progeny of fruit trees under natural environmental conditions.

Functional Analysis of the Marigold (Tagetes erecta) Lycopene ε-cyclase (TeLCYe) Promoter in Transgenic Tobacco.

Lycopene ε-cyclases (LCYEs) are key enzymes in carotenoid biosynthesis converting red lycopene to downstream lutein. The flowers of marigold (Tagetes erecta) have been superior sources to supply lutein. However, the transcriptional regulatory mechanisms of LCYe in lutein synthesis are still unclear in marigold. In this work, the expression pattern of the TeLCYe gene in marigold indicated that TeLCYe mainly expressed in floral organs. To gain a better understanding of the expression and regulatory mechanism of TeLCYe gene, the TeLCYe promoter was isolated, sequenced, and analyzed through bioinformatics tools. The results suggested that the sequence of TeLCYe promoter contained various putative cis-acting elements responsive to exogenous and endogenous factors. The full-length TeLCYe promoter and three 5'-deletion fragments were fused to the GUS reporter gene and transferred into tobacco to test the promoter activities. A strong GUS activity was observed in stems of seedlings, leaves of seedlings, middle stems, top leaves, petals, stamens, and stigmas in transgenic tobacco containing full-length TeLCYe promoter LP0-2086. Deletion of - 910 to - 429 bp 5' to ATG significantly increased the GUS activity in chloroplast-rich tissues and floral organs, while deletion occurring from 1170 to 910 bp upstream ATG decreased the TeLCYe promoter strength in stems of seedlings, leaves of seedlings, top leaves and sepals. Functional characterization of the full-length TeLCYe promoter and its' deletion fragments in stable transgenic tobacco indicated that the LP0-2086 contains some specific cis-acting elements, which might result in the high-level expression of in floral organs, and LP2-910 might contain some specific cis-acting elements which improved GUS activities in vegetable tissues.

Molecular cloning and functional characterization of a seed-specific VvßVPE gene promoter from Vitis vinifera.

MAIN CONCLUSION: The grapevine VvßVPE promoter is specifically expressed in the seed. The - 1306~- 1045 bp core region restricts expression in other tissues and organs. Vacuolar processing enzyme (VPE) is a cysteine proteinase regulating vacuolar protein maturation and executing programmed cell death (PCD) in plants. Vitis vinifera (Vv)ßVPE is a ß-type VPE showing seed-specific expression that processes seed proteins during ovule development. However, the regulation of the seed-specific gene expression is far from understood. In this study, we characterize VvßVPE promoter (pVvßVPE) from 12 seeded and seedless grape genotypes. 94.56% of the pVvßVPE coding sequence is consistent. Two ßVPE promoters were constructed and transformed into Arabidopsis thaliana via ß-glucuronidase (GUS) fused expression vectors, using cv. Pinot Noir and cv. Thompson as seed and seedless candidates. GUS staining in different tissues and organs revealed that VvßVPE expresses specifically in the embryo, including the cotyledon, hypocotyl and suspensor, but not in the leaf, stem, root or flowers of the seedling. Using promoter deletion analysis, we created four incomplete VvßVPE promoters and found each pVvßVPE deletion could drive GUS gene to express in seeds. Interestingly, seed specificity disappeared when the promoter missed the core - 1306~- 1045 bp region. All deletion promoters presenting various quantified GUS activities indicate that the region - 1704~- 1306 bp inhibits, and the region - 705~- 861 bp promotes gene expression of VvßVPE. Our results demonstrate that pVvßVPE is a seed-specific promoter in both seeded and seedless grapes. Moreover, the core region of pVvßVPE (- 1306~- 1045 bp) is the key one responsible for seed-specific expression.

Promoter of the TmHKT1;4-A1 gene of Triticum monococcum directs stress inducible, developmental regulated and organ specific gene expression in transgenic Arbidopsis thaliana.

HKT transporters which mediate Na+-specific transport or Na+-K+ co-transport, play an important role in protecting plants from salinity stress by preventing Na+-over-accumulation in leaves. In this work, a 1508-bp genomic fragment upstream of the TmHKT1;4-A1 translated sequence from Triticum monococcum has been isolated, cloned, and designated as the ''PrTmHKT1;4-A1'' promoter. Sequence analysis of ''PrTmHKT1;4-A1'' revealed the presence of cis-regulatory elements which could be required for abiotic stress and abscisic acid (ABA) responsiveness. The PrTmHKT1;4-A1 sequence was fused to the ß-glucuronidase gene and the resulting construct was transferred into Arabidopsis plants. Histochemical assays of stably transformed Arabidopsis plants showed that PrTmHKT1;4-A1 is active in this heterologous system. Under control conditions, GUS histochemical staining was observed significantly only in leaves of 20-day-old plants. Histological sections prepared at this stage and in leaves revealed activity localized in leaf veins (phloem and bundle sheath). In flowers, GUS activity was detected only in sepals. After 3 days of challenging the plants with salt, dehydration or ABA treatments, the PrTmHKT1;4-A1 transformed Arabidopsis plants showed a substantial increase in the GUS staining in leaves, compared to untransformed plants under the same conditions. Real time qPCR expression analysis of the uidA gene, showed that GusA transcripts were up-regulated by salt, dehydration, and ABA treatments. All together, these results showed that PrTmHKT1;4-A1 is an age-dependent, abiotic-stress-inducible, organ-specific and tissue-specific promoter in a heterologous dicot system.

Expression analysis of proteinase inhibitor-II under OsRGLP2 promoter in response to wounding and signaling molecules in transgenic Nicotiana benthamiana.

Proteinase inhibitor-II (PI-II) genes are important defense related genes that play critical regulatory roles in plant growth and development. In the present study, the expression of tomato PI-II gene was investigated under the control of a wound-inducible OsRGLP2 (Oryza sativa root germin like protein 2) promoter in transgenic tobacco plants after wounding, ABA and MeJA applications. Transcript level of target gene in transgenic plants was confirmed by quantitative real time PCR (qPCR). In response to ABA treatment at different concentrations, PI-II gene was strongly induced under OsRGLP2 promoter at higher concentration (100 µM), while considerable level of target gene expression was observed with MeJA application at 50 µM concentration. Upon wounding, relatively high PI-II gene expression was observed after 36-h treatment. Correspondingly, high GUS activity was detected at 36 h with histochemical assay and microscopic analysis in the vascular regions of leaves, stem and roots in wounded transgenic plants. This inducibility of PI-II gene by wounding, ABA and MeJA indirectly indicates its role in plant defense mechanism against biotic and abiotic stresses. Moreover, it was also suggested that ABA and MeJA dependent signaling pathways are involved in stimulation of PI-II gene. To the best of our knowledge, this is the first report describing the induction of PI-II gene under the regulation of OsRGLP2 promoter under stress conditions. The results of present research are useful for potential role of PI-II gene to improve stress tolerance in transgenic crops. Thus, efficacy of this gene can potentially be exploited to test the responses of different plants to various environmental stresses.

Exploring the Functions of 9-Lipoxygenase (DkLOX3) in Ultrastructural Changes and Hormonal Stress Response during Persimmon Fruit Storage.

Lipoxygenase (LOX) initiates the hydroperoxidation of polyunsaturated fatty acids and is involved in multiple physiological processes. In this study, investigation of various microscopic techniques showed that the fruit peel cellular microstructure of the two persimmon cultivars differed after 12 days of storage, resulting in fruit weight loss and an increased number and depth of microcracks. Analysis of subcellular localization revealed that greater amounts of DkLOX3-immunolabelled gold particles accumulated in "Fupingjianshi" than in "Ganmaokui" during storage. In addition, the expression of DkLOX3 was positively up-regulated by abscisic acid (ABA), concomitant with the promotion of ethylene synthesis and loss of firmness, and was suppressed by salicylic acid (SA), concomitant with the maintenance of fruit firmness, inhibition of ethylene production and weight loss. In particular, the expression of DkLOX3 differed from the ethylene trajectory after methyl jasmonate (MeJA) treatment. Furthermore, we isolated a 1105 bp 5' flanking region of DkLOX3 and the activity of promoter deletion derivatives was induced through various hormonal treatments. Promoter sequence cis-regulatory elements were analysed, and two conserved hormone-responsive elements were found to be essential for responsiveness to hormonal stress. Overall, these results will provide us with new clues for exploring the functions of DkLOX3 in fruit ripening and hormonal stress response.

Isolation and characterization of a novel pollen-specific promoter in maize (Zea mays L.).

ZmSTK2_USP, located on the long arm of chromosome 4, belongs to the serine/threonine kinase gene in maize. The sequence analysis of 2100 bp upstream from the start codon ATG has shown that it contains cis-element motifs and two types of anther/pollen-specific promoter elements (GTGA and AGAAA), suggesting that it is the pollen-specific promoter. To investigate the function of ZmSTK2_USP promoter, the GUS gene fusion system was employed. In proZmSTK2_USP-GUS genetically modified plants, GUS activity was detected in mature pollen grains and pollen tubes but not found in other floral and vegetative tissues. These results show that proZmSTK2_USP is the pollen-specific promoter and drives pollen-specific activity during the middle stage of pollen development until pollen maturation.

Salt tolerance function of the novel C2H2-type zinc finger protein TaZNF in wheat.

The expression profile chip of the wheat salt-tolerant mutant RH8706-49 was investigated under salt stress in our laboratory. Results revealed a novel gene induced by salt stress with unknown functions. The gene was named as TaZNF (Triticum aestivum predicted Dof zinc finger protein) because it contains the zf-Dof superfamily and was deposited in GenBank (accession no. KF307327). Further analysis showed that TaZNF significantly improved the salt-tolerance of transgenic Arabidopsis. Various physiological indices of the transgenic plant were improved compared with those of the control after salt stress. Non-invasive micro-test (NMT) detection showed that the root tip of transgenic Arabidopsis significantly expressed Na(+) excretion. TaZNF is mainly localized in the nucleus and exhibited transcriptional activity. Hence, this protein was considered a transcription factor. The TaZNF upstream promoter was then cloned and was found to contain three salts, one jasmonic acid methyl ester (MeJA), and several ABA-responsive elements. The GUS staining and quantitative results of different tissues in the full-length promoter in the transgenic plants showed that the promoter was not tissue specific. The promoter activity in the root, leaf, and flower was enhanced after induction by salt stress. Moreover, GUS staining and quantitative measurement of GUS activity showed that the promoter sequence contained the positive regulatory element of salt and MeJA after their respective elements were mutated in the full-length promoter. RNA-Seq result showed that 2727 genes were differentially expressed; most of these genes were involved in the metabolic pathway and biosynthesis of secondary metabolite pathway.

Finding new elicitors that induce resistance in rice to the white-backed planthopper Sogatella furcifera.

Herein we report a new way to identify chemical elicitors that induce resistance in rice to herbivores. Using this method, by quantifying the induction of chemicals for GUS activity in a specific screening system that we established previously, 5 candidate elicitors were selected from the 29 designed and synthesized phenoxyalkanoic acid derivatives. Bioassays confirmed that these candidate elicitors could induce plant defense and then repel feeding of white-backed planthopper Sogatella furcifera.

An active Catharanthus roseus desacetoxyvindoline-4-hydroxylase-like gene and its transcriptional regulatory profile.

BACKGROUND: Desacetoxyvindoline-4-hydroxylase is a key enzyme in the biosynthesis of vindoline, the important intermediate leading to vinblastine and vincristine in Catharanthus roseus. RESULTS: A d4h-like gene has been isolated from C. roseus C20hi cells based on an EST sequence from the Suppression Subtractive Hybridization cDNA library. The full length cDNA of d4h-like was 1427 bp encoding 372 amino acids. It had 66% identities and 80% positives with d4h at the amino acid level. It belonged to 2-oxoglutarate dependent oxygenase superfamily as d4h did. Real-time quantitative PCR analysis revealed that d4h-like was expressed high in roots, flowers and C20hi cells, very low in leaves and stems. Methyl jasmonate could significantly increase the accumulation of d4h-like transcripts. 2,4-D inhibited its expression. An approximate 2,910 bp of 5'-promoter region of d4h-like was obtained, fused to GUS reporter gene and analyzed with fluorescence quantitative assays using transient expression in C. roseus cell suspensions, indicating that d4h-like promoter could drive GUS gene expression in vivo. CONCLUSION: These results suggest that d4h-like is closely related with d4h in the genetic evolution but with different transcriptional expression profiles. It may be revolved in the hormone-independency of C20hi cells.

Qualitative and quantitative analysis of the Nictaba promoter activity during development in Nicotiana tabacum.

Nictaba is a genuine jasmonate inducible lectin expressed in the leaves and roots of Nicotiana tabacum cv. Samsun NN. Although the jasmonate pathway is generally highly conserved among plant species, recent research showed that it is followed by plant specific downstream processes. Previously the Nictaba promoter activity was studied in the model plant Arabidopsis thaliana, using the ß-glucuronidase (GUS) as a gene reporter system. In this paper the promoter activity of Nictaba was analyzed in N. tabacum plants stably expressing a promoter-GUS fusion construct. Both histochemical and fluorometric techniques were used to follow Nictaba promoter activity during the development of the tobacco plants. GUS staining was predominantly detected in the cotyledons, the leaves and the roots during the youngest plant stages. As the plants grow older GUS staining was mostly present in the older leaves. A detailed comparative analysis was made of the GUS staining results obtained in transgenic Arabidopsis and tobacco lines.