Isolation and functional characterization of a novel stress inducible promoter from pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> L)

The present study deals with isolation and characterization of a novel hybrid-proline-rich protein gene (CcHyPRP) promoter from pigeonpea. Real time PCR analysis revealed that CcHyPRP expression was strongly induced by dehydration, salt, Abscisic acid (ABA) and Salicylic acid (SA) treatments. The CcHyPRP promoter, isolated by genome-walking method, contained 1112 bp and showed the presence of various cis -regulatory elements necessary for tissue specific expression and stress responsiveness. Different 5′ deletions of the promoter were generated and were used to drive the expression of β-glucuronidase reporter gene (gusA) in Arabidopsis thaliana. Histochemical and fluorometric assays confirmed that GUS expression driven by the full-length fragment (1112 bp) was higher when compared to different deletion fragments. Under normal conditions, GUS expression was predominantly detected in the roots and hypocotyls of transformants, while under mannitol, NaCl, ABA and SA treatment conditions higher GUS expression levels were observed in the roots and leaves. However, the GUS expression was mostly confined to the roots of transformants carrying 477 and 300 bp promoter regions. The results amply indicate that CcHyPRP promoter is regulated by different stress factors, and as such the promoter can be deployed in genetic engineering of crop plants for enhanced abiotic stress tolerance.     

Genome-wide comparative analysis of <Emphasis Type="Italic">LEAFY</Emphasis> promoter sequence in angiosperms

Regulation of the flowering mechanism is influenced by many environmental factors. Dissecting the regulatory processes upstream of the LFY (LEAFY) gene will help us to understand the molecular mechanisms of floral induction. In total, 53 LFY sequences were identified in 37 species. Among the 53 selected LFY promoters and after eliminating the short sequences, 47 LFY promoters were analyzed. Comparative genome studies for LFY promoters among plants showed that TATA-box existed in all herbaceous plants. The 1345-bp promoter sequence upstream to hickory LFY gene was cloned and analyzed, together with functional studies. The result of sequence alignment showed that the region of the hickory LFY promoter has only two conserved auxin response elements (AuxRE), whereas other plants had four. The positions of AuxRE in hickory and walnut were the same, but they were different from the positions from other plants. Furthermore the sequence analysis showed that the promoter have TATA-box and CAAT-box motifs. Deletion analysis of these motifs did not block β-glucuronidase (GUS) activity during the transient expression assay, suggesting that it may be a TATA-less promoter. Low temperature and light significantly induced the full-length promoter to increase about two folds of the GUS enzymatic activity, suggesting these environmental factors induced flowering in hickory.     

Characterization of <Emphasis Type="Italic">VvPAL-like</Emphasis> promoter from grapevine using transgenic tobacco plants

A 2000-bp 5′-flanking region of VvPAL-like was isolated from ‘Summer Black’ grapevine by PCR amplification, named pVvPAL-like. To gain a better understanding of the expression and regulatory mechanism of VvPAL-like, a chimeric expression unit consisting of the β-glucuronidase (GUS) reporter gene under the control of a 2000-bp fragment of the VvPAL-like promoter was transformed into tobacco via Agrobacterium tumefaciens. Histochemical staining showed that the full-length promoter directs efficient expression of the reporter gene in cotyledons and hypocotyls, stigma, style, anthers, pollen, ovary, trichomes, and vascular bundles of transgenic plants. A series of 5′ progressive deletions of the promoter revealed the presence of a negative regulatory region (?424 to ?292) in the VvPAL-like promoter. Exposure of the transgenic tobacco plants to various abiotic stresses demonstrated that the full-length construct could be induced by light, copper (Cu), abscisic acid (ABA), indole-3-acetic (IAA), methyl jasmonate (MeJA) (N-1-naphthylphthalamic acid), ethylene, and drought. Furthermore, the ethylene-responsive region was found to be located in the ?1461/?930 fragment, while the element(s) for the MeJA-responsive expression may be present in the ?424/?292 region in the VvPAL-like promoter. These findings will help us to better understand the molecular mechanisms by which VvPAL-like participates in biosynthesis of flavonoids and stress responses.     

Regulatory <Emphasis Type="Italic">cis</Emphasis>-elements on citrus peel-specific expressed gene, <Emphasis Type="Italic">CuCRTISO-like</Emphasis>, promoter respond to hormones and abiotic stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

We identified a peel-specific expressed gene in Citrus unshiu fruits by differentially expressed gene (DEG) analysis, which showed a homology with carotenoid isomerase-like genes identified from other plants and, therefore, designated as CuCRTISO-like. Here we determined the promoter sequence of CuCRTISO-like and analyzed histochemical GUS activity using transgenic Arabidopsis plants harboring CuCRTISO-like promoter-GUS gene constructs (pCRTL-Prom1~pCRTL-Prom5 lines). The promoter activity of CuCRTISO-like was detected in the cotyledon at 5 and 10 days after germination (DAG), young leaf, and anther, but not in the cotyledon at 15 DAG and mature leaf. Several cis-acting elements involved in hormones and abiotic stresses are located on the CuCRTISO-like promoter. Salicylic acid and ethylene treatments induced the GUS activity in the pCRTL-prom1 and pCRTL-Prom4 line, respectively. Treatment of drought and wounding stress induced the GUS activity in the pCRTL-Prom4 and pCRTL-Prom3 line, respectively. Heat stress treatment induced GUS activity more strongly as the promoter length decreased except for no GUS activity in the pCRTL-Prom5 line. The CuCRTISO-like expression during fruit maturation of C. unshiu showed a peel-specific expression pattern. Our results suggest that CuCRTISO-like promoter activity is regulated in a developmental and organ-specific manner, and responds to hormones and abiotic stresses.     

Effects of transgenic expression of <Emphasis Type="Italic">Brevibacterium linens</Emphasis> methionine gamma lyase (MGL) on accumulation of <Emphasis Type="Italic">Tylenchulus semipenetrans and key aminoacid contents</Emphasis> in <Emphasis Type="Italic">Carrizo citrange</Emphasis>

Alpha-dioxygenases (α-DOX) catalyzing the primary oxygenation of fatty acids to oxylipins were recently found in plants. Here, the biological roles of the pepper α-DOX (Ca-DOX) gene, which is strongly induced during non-host pathogen infection in chili pepper, were examined. Virus-induced gene silencing demonstrated that down-regulation of Ca-DOX enhanced susceptibility to bacterial pathogens and suppressed the hypersensitive response via the suppression of pathogenesis-related genes such as PR4, proteinase inhibitor II and lipid transfer protein (PR14). Ca-DOX-silenced pepper plants also exhibited more retarded growth with lower epidermal cell numbers and reduced cell wall thickness than control plants. To better understand regulation of Ca-DOX, transgenic Arabidopsis plants harboring the β-glucuronidase (GUS) reporter gene driven from a putative Ca-DOX promoter were generated. GUS expression was significantly induced upon avirulent pathogen infection in transgenic Arabidopsis leaves, whereas GUS induction was relatively weak upon virulent pathogen treatment. After treatment with plant hormones, early and strong GUS expression was seen after treatment of salicylic acid, whereas ethylene and methyl jasmonate treatments produced relatively weak and late GUS signals. These results will enable us to further understand the role of α-DOX, which is important in lipid metabolism, defense responses, and growth development in plants.     

Structural and functional analysis of new plant promoter pro-SmAMP1 from <Emphasis Type="Italic">Stellaria media</Emphasis>

The nucleotide sequence of a fragment of the promoter region of pro-SmAMP1 gene, having a length of 1257 bp and encoding antifungal peptides, was determined in chickweed (Stellaria media (L.) Vill.). Computer analysis of the nucleotide sequence revealed a number of cis-elements that are typical strong plant promoters. Five 5′-deletion variants were created taking into account the distribution of cis-elements:–1235,–771,–714,–603, and–481 bp of pro-SmAMP1 gene promoter, which were fused to the coding region of the uidA reporter gene in pCambia1381Z plant expression vector. The efficacy of pro-SmAMP1 promoter deletion variants was determined by transient expression in plants of Nicotiana benthamiana and using sequential generations of transgenic Nicotiana tabacum plants. It was found that the levels of GUS reporter protein activity in the extracts from transgenic and agroinfiltrated plants using all deletion variants of pro-SmAMP1 gene promoter were 3–5 times higher than those of 35S CaMV viral promoter. The highest activity of GUS protein was observed in the leaves of transgenic tobacco plants and closely correlated with the mRNA level of encoding gene. The levels of GUS activity did not differ significantly among 11 independent homozygous lines of T₂ generation of N. tabacum plants with different deletion variants of pro-SmAMP1 promoter. The results give reason to assume that all deletion variants of pro-SmAMP1 promoter provide stable and high level of expression of controlled genes. The shortest deletion variant–481 bp of pro-SmAMP1 promoter should be viewed as a potentially strong plant promoter for the genetic engineering of plants.     

Transgenic Analysis Reveals 5′ Abbreviated <Emphasis Type="Italic">Os</Emphasis>RGLP2 Promoter(s) as Responsive to Abiotic Stresses

Germins and germin-like proteins are ubiquitous, expressed at various developmental stages and in response to various abiotic and biotic stresses. In this study, to functionally validate the OsRGLP2 promoter, 5′ deletion analysis of the promoter sequences was performed and the deletion fragments fused with the β-glucuronidase (GUS) and green fluorescent protein reporter genes were used for transient expression in tobacco as well as for generating stable transgenic Arabidopsis plants. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the P-1063 and P-565 when subjected to abiotic stresses. Histochemical analysis of transgenic Arabidopsis plants revealed expression of reporter gene in root, leaf and stem sections of plants harboring P-1063 and P-565. Real-time qPCR analysis of transiently expressed tobacco leaves and transgenic Arabidopsis plants subjected to several abiotic stresses supported histochemical data and showed that P-565 responded to all the stresses to which the full-length promoter was responsive. The data suggest that P-565 may be a good alternative to full-length promoter region that harbors the necessary cis-elements in providing stable and high level of expression in response to wound, salt and temperature stresses.     

Isolation and characterization of the 4-coumarate:coenzyme A ligase (4CL1) promoter from <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

The most important enzyme of the phenylpropanoid pathway, 4-coumarate:coenzyme A ligase (4CL), is encoded by several homologous genes including 4CL1. The 4CL1 promoter is a tissue-specific gene expression element, particularly active in the secondary xylem or older stems. In this study, the 1127 bp 5′- upstream region of the 4CL1 coding sequence from Eucalyptus camaldulensis, Euc4CL1, was isolated and characterized. Essential putative cis-elements in the Euc4CL1 promoter included: a TATA-box at ?22/?28 position, two CCAAT-boxes at ?256/?260 and ?277/?281 positions, respectively, an AC-element at ?328/?336 and A-boxes at ?115/?120 and ?990/?995 positions. To investigate the effect of the Euc4CL1 promoter on gene expression, a plant transformation vector, pEuc4CL1p, containing the reporter gene for β-glucuronidase (GUS) under the control of Euc4CL1 promoter was constructed based on the pBI101 backbone and introduced in tobacco plants. Stable expression of the GUS gene in transgenic lines was analysed by a histochemical GUS assay. The results indicated the specific expression of the GUS gene in the stem xylem cells of transgenic tobacco lines was controlled by the Euc4CL1 promoter. The observations suggest the isolated Euc4CL1 promoter is a potential candidate for driving the expression of a foreign gene in plant xylem tissues.     

Functional analysis of a cryptic promoter from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> reveals bidirectionality

Cryptic promoter elements play a significant role in evolution of plant gene expression patterns and are prospective tools for creating gene expression systems in plants. In a previous report, a 452 bp promoter fragment designated as cryptic root-specific promoter (AY601849) was identified immediately upstream to T-DNA insertion, in the intergenic region between divergent genes SAHH1 and SHMT4, in T-DNA tagged mutant M57 of Arabidopsis thaliana. In silico analysis of 452 bp promoter revealed typical eukaryotic promoter architecture, presence of root-specific motifs and other cis-regulatory motifs responsible for the spatial and temporal expression. GUS expression driven by 452 bp in M57 was developmentally as well as light-regulated. The AT-rich 452 bp promoter does not show homology to any known sequences. The 452 bp promoter was further proved cryptic and detailed molecular characterization of the promoter carried out through serial 5′ and 3′ deletion analysis, by cloning the promoter fragments upstream to promoter-less GUS vector. A 279 bp fragment obtained by deleting 173 bp from 5′ end of 452 bp was capable of driving root-specific expression, similar to that of full-length promoter. Further, root tip-specific, root-specific and core-regulatory motifs for root-specific expression were identified at positions 173–227, 251–323 and 408–452 bp, respectively, from the 5′ end of 452 bp. The 452 bp promoter was equally functional in inverse orientation, hence bidirectional and symmetric. In heterologous systems, such as Brassica juncea and Oryza sativa, the promoter activity was not significant since GUS was not visually detected in transient assays.     

Heat shock responsiveness analysis of <Emphasis Type="Italic">Athsp70b</Emphasis> upstream region

A 1431-bp upstream fragment of Athsp70b was cloned via PCR amplification and expressed in onion epidermis by particle bombardment. Furthermore, the progressive deletions of the Athsp70b upstream fragment linked to the β-glucuronidase (GUS) coding region were performed. Then, a stable GUS expression was analyzed in tobacco BY2 cells and Arabidopsis. Our present results showed that about a 500-bp region upstream ATG of Athsp70b is suitable to confer heat inducibility to the GUS reporter gene in plants and around 116 bp contain nonperfect heat-sensitive element. This promoter responds to heat, salicylic acid, and benzyladenine. GUS staining was mainly observed in the vascular tissues and root tips, implying that Athsp70b is related to water transportation.     

A Novel <Emphasis Type="Italic">PhLRR</Emphasis> Gene Promoter is Sufficient for Engineering Male Sterility in Petunia

Tissue-specific promoters can drive genes specifically expressed in the target organs and have been widely used in plant molecular breeding. In this study, a 1.2-kb promoter region of an anther-specific gene PhLRR from Petunia hybrida “Fantasy” was isolated and fused to the β-glucuronidase (GUS) gene. The pPhLRR::GUS vector was heterogeneously transformed into tobacco in which the GUS staining was only detected in the early development stage of anthers and no GUS expression in any other three floral whirls or vegetative organs was observed. It is very different from other well-studied anther-specific promoters which drive genes specifically expressed in the later development stage of anthers or only in the pollens. Furthermore, the pPhLRR::Barnase was introduced into petunia and induced complete male sterility without influencing the ornamental characteristics or the female fertility in transformed plants. These results indicate that PhLRR promoter is a new kind of petunia anther-specific promoter and could be taken as a valuable tool in ornamental plant breeding.     

Promoters pro-SmAMP1 and pro-SmAMP2 from Wild Plant <Emphasis Type="Italic">Stellaria media</Emphasis> for the Biotechnology of Dicotyledons

Comparative results of the studied effectiveness of two new promoters, pro-SmAMP1 and pro- SmAMP2, from chickweed (Stellaria media L.) in various types of cultivated plants with transient expression and in stable transformants are given. The effectiveness of the promoters was evaluated through the expression of the reporter uidA gene by measuring the activity of its GUS protein product. It was found that the deletion variant (442 bp) of the pro-SmAMP1 promoter was significantly stronger in plants of Nicotiana benthamiana (Domin) with transient expression than the deletion variant (455 bp) of the pro-SmAMP2 promoter. The effectiveness of these short deletion variants of both promoters under transient expression in the plants of rapeseed (Brassica napus L.) and sugar beet (Beta vulgaris L.) was comparable with that of the viral CaMV35S promoter. The functionality of the pro-SmAMP2 promoter in the calluses of common flax plants (Linum usitatissimum L.) was shown. In the homozygous lines of transgenic tobacco plants (Nicotiana tabacum L.), all deletion variants of the pro-SmAMP1 promoter and the shortest version of pro-SmAMP2 were twice as strong as the CaMV35S viral promoter. The effectiveness of short variants of both promoters from the chickweed in controlling the gene encoding neomycin phosphotransferase II in the transgenic plants of tobacco and arabidopsis (Arabidopsis thaliana L.) growing on media supplemented with recommended concentrations of kanamycin are not inferior to the duplicated 2хCaMV35S viral promoter. The obtained experimental data show that short deletion variants of pro-SmAMP1 (442 bp) and pro-SmAMP2 (455 bp) promoters may be recommended as strong constitutive promoters for use in the biotechnology of crop plants.