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Heat shock transcription factor (HSF) plays an essential role on the increased tolerance against heat stress by regulating the expression of heat-responsive genes. In this study, a HSF gene, CarHSFB2, was isolated and characterized in chickpea. CarHSFB2 was a nuclear protein with a predicted polypeptide of 267 amino acids and encoded by a single/low copy genes. Phylogenetic analysis showed that CarHSFB2 belonged to the class B HSFs. It had little or no any transcription activation activity due to lack of aromatic, hydrophobic, and acidic amino acid (AHA) motifs. CarHSFB2 showed different expression patterns among different developmental processes (leaf senescence, developing seed, and embryo of germinating seed). It was induced by the stress of heat, salt, wound and drought, and the treatment of HO, IAA, and GA3, respectively, while inhibited by 6-BA. However, the other stress and chemical treatments (cold, ABA, MeJA, Et, and SA) had no obvious effect on its expression. Overexpression of CarHSFB2 in Arabidopsis seedlings showed the increased tolerance to drought and heat stress. Additionally, stress-responsive genes, RD22, RD26, and RD29A, showed significantly higher expression levels in transgenic Arabidopsis seedlings than in the wild type (WT) under drought stress, whereas HsfA2, HsfB2a, and HsfA7a in transgenic Arabidopsis seedlings were markedly accumulated in transcript level than in the WT under heat stress. All these results indicate that CarHSFB2, a class B HSF, positively functions in different developmental processes and various stress responses, especially in positive response to heat and drought stresses, in chickpea. [ABSTRACT FROM AUTHOR]

Cotton is one of the most economically important cultivated crops. It is the major source of natural fiber for the textile industry and an important target for genetic modification for both biotic stress and herbicide tolerance. Therefore, the characterization of genes and regulatory regions that might be useful for genetic transformation is indispensable. The isolation and characterization of new regulatory regions is of great importance to drive transgene expression in genetically modified crops. One of the major drawbacks in cotton production is pest damage; therefore, the most promising, cost-effective, and sustainable method for pest control is the development of genetically resistant cotton lines. Considering this scenario, our group isolated and characterized the promoter region of a MCO (multicopper oxidase) from Gossypium hirsutum, named GhAO-like1 (ascorbate oxidase-like1). The quantitative expression, together with the in vivo characterization of the promoter region reveals that GhAO-like1 has a flower- and fruit-specific expression pattern. The GUS activity is mainly observed in stamens, as expected considering that the GhAO-like1 regulatory sequence is enriched in cis elements, which have been characterized as a target of reproductive tissue specific transcription factors. Both histological and quantitative analyses in Arabidopsis thaliana have confirmed flower (mainly in stamens) and fruit expression of GhAO-like1. In the present paper, we isolated and characterized both in silico and in vivo the promoter region of the GhAO-like1 gene. The regulatory region of GhAO-like1 might be useful to confer tissue-specific expression in genetically modified plants. [ABSTRACT FROM AUTHOR]

Garden asparagus ( Asparagus officinalis L.) is a dioecious species whose male and female flowers are found in separate unisexual individuals. A region called the M-locus, located on a pair of homomorphic sex chromosomes, controls sexual dimorphism in asparagus. To date, no sex determining gene has been isolated from asparagus. To identify more genes involved in flower development in asparagus, subtractive hybridization library of male flowers in asparagus was constructed by suppression subtraction hybridization. A total of 107 expressed sequence tags (ESTs) were identified. BLASTX analysis showed that the library contained several genes that could be related to flower development. The expression patterns of seven selected genes believed to be involved in the development of asparagus male flower were further analyzed by semi-quantitative or real-time reverse-transcription polymerase chain reaction (RT-PCR). Results showed that AOEST, AOEST, and AOEST were strongly expressed in the male flower stage, whereas no transcript level of AOEST was detected in the female flower stage. The expression levels of AOEST, AOEST, AOEST, and AOEST in the male flower stage were also higher than those in the female flower stage, although these transcripts were also expressed in other tissues. The identified genes can provide a strong starting point for further studies on the underlying molecular differences between the male and female flowers of asparagus. [ABSTRACT FROM AUTHOR]

Cherry plum is a popular ornamental tree worldwide and most cultivars are selected for purple foliage. Here, we report the investigation of molecular mechanism underlying red pigmentation in purple-leaf plum ‘Ziyeli’ (Prunus cerasifera Ehrhar f. atropurpurea (Jacq.) Rehd.), which shows red color pigmentation in fruit (flesh and skin) and foliage. Six anthocyanin-activating MYB genes, designated PcMYB10.1 to PcMYB10.6, were isolated based on RNA-Seq data from leaves of cv. Ziyeli. Of these PcMYB10 genes, five (PcMYB10.1 through PcMYB10.5) show distinct spatial and temporal expression patterns, while the PcMYB10.6 gene is highly expressed in all the purple-coloured organs of cv. Ziyeli. Constitutive activation of PcMYB10.6 is closely related to red pigmentation in the leaf, fruit (flesh and skin), and sepal. However, the PcMYB10.6 activation cannot induce red pigmentation in the petal of cv. Ziyeli during late stages of flower development due to due to a lack of expression of PcUFGT. The inhibition of red pigmentation in the petal of cherry plum could be attributed to the high-level expression of PcANR that directs anthocyanidin flux to proanthocyanidin biosynthesis. In addition, PcMYB10.2 is highly expressed in fruit and sepal, but its expression cannot induce red pigmentation. This suggests the PcMYB10 gene family in cherry plum may have diverged in function and PcMYB10.2 plays little role in the regulation of red pigmentation. Our study provides for the first time an example of constitutive activation of an anthocyanin-activating MYB gene in Prunus although its underlying mechanism remains unclear. [ABSTRACT FROM AUTHOR]

Key message: Conditions for the isolation and transfection of maize nucellus protoplasts were established. We demonstrated its utilization for protein expression, localization, protein-protein interaction, and the investigation of PCD-related processes. Abstract: Plant protoplasts are an important and versatile cell system that is widely used in the analysis of gene characterization and diverse signaling pathways. Programmed cell death (PCD) occurs throughout the life of plants from embryogenesis to fertilization. The maize nucellus undergoes typical PCD during development of the embryo sac. The nucellus protoplast shows potential for use in research of PCD-related processes. No studies have reported previously the isolation and transfection of nucellus protoplasts. In this study, conditions for the isolation and transfection of maize nucellus protoplasts were established. The maize protoplast system can be used for protein expression, localization, and protein-protein interaction. We applied this system to investigate PCD-related processes. Quantitative real-time PCR analysis revealed that transient expression of MADS29 in the maize nucellus protoplast increases Cys-protease gene transcript level. In addition, β-glucuronidase and luciferase activity assays showed that MADS29 could enhance the promoter activities of the Cys-protease gene. Thus, we demonstrated the potential of a highly efficient maize nucellus protoplast system for transient gene expression and investigation of PCD-related processes. [ABSTRACT FROM AUTHOR]

Gibberellins (GAs) are important in the floral regulatory networks of angiosperm plants. Several lines of evidence suggest that GAs also play a pivotal role in conifer male and female cone development. To gain new insights into the GA metabolism pathway in conifer trees and the role of GA metabolism in male and female cone development, we identified GA metabolism genes in Pinus tabuliformis. These included one PtCPS gene, one PtKS gene, one PtKO gene, TWO PtKAO genes, one PtGA20ox gene, two PtGA3ox genes and 12 PtGA2ox genes. According to phylogenetic analysis, the GA biosynthesis pathway evolved after the divergence of mosses from ferns, but the GA-deactivating gene family underwent divided expansion after divergence of the angiosperms from gymnosperms. However, the active sites of all GA metabolism enzymes were conserved during the evolution of land plants. During male and female cone development of P. tabuliformis, the expression of most of the PtGA2ox genes, especially PtGA2ox10, was higher than GA biosynthesis genes. However, the expression of PtKAO1 in cones peaked at a very early developmental stage. The expression pattern of GA metabolism genes indicated that GAs play different roles at the early and late stages of cone development. [ABSTRACT FROM AUTHOR]

Engineering of plant protection requires well-characterized tissue-specific promoters for the targeted expression of insecticidal resistance genes. Herein, we describe the isolation of five different fragments of promoters of three distinct flower-specific cotton ( Gossypium hirsutum) genes. Expression analyses of the three genes GhPME-like1, GhβGal-like1 and GhPL-like1 revealed that they are expressed highly in flowers buds ranging from 4 to 12 mm in size. Several putative regulatory cis-elements were identified in the promoter regions, including elements involved in the control of tissue-specific gene expression in pollen grains and fruits. In vivo analyses of these promoters were performed using the heterologous plant system Arabidopsis thaliana by fusing them with the gene uidA (GUS). GUS staining in Arabidopsis tissues revealed that their expression was restricted to anthers, with the majority of expression in pollen grains and in the upper portion of the carpels and siliques. A comparison between a CaMV35S::GUS constitutive promoter and the promoters isolated in this study revealed that the cotton promoters were more active and were specific to flowers and fruits, which are organs that are preferentially attacked by important pest insects such as the boll weevil ( Anthonomus grandis). The activity of the promoters was also confirmed using transient expression assays in flower buds of G. hirsutum. The promoters of GhPME-like1, GhβGal-like1 and GhPL-like1 are specific to reproductive tissues and could represent important biotechnological tools for controlling insect pests, in particular the cotton boll weevil, which attacks floral and fruit tissues. [ABSTRACT FROM AUTHOR]

Abstract: The WUSCHEL (WUS)-related homeobox (WOX) gene family plays an important role in coordinating gene transcription in the early phases of embryogenesis. In this study, we isolated and characterized WOX5 from common wheat and its relatives Triticum monococcum, Triticum urartu, Aegilops speltoides, Aegilops searsii, Aegilops sharonensis, Aegilops longissima, Aegilops bicornis, Aegilops tauschii, and Triticum turgidum. The size of the characterized WOX5 alleles ranged from 1029 to 1038bp and encompassed the complete open reading frame (ORF) as well as 5′ upstream and 3′ downstream sequences. Domain prediction analysis showed that the putative primary structures of wheat WOX5 protein include the highly conserved homeodomain besides the WUS-box domain and the EAR-like domain, which is/are present in some members of the WOX protein family. The full-length ORF was subcloned into a prokaryotic expression vector pET30a, and an approximate 26-kDa protein was successfully expressed in Escherichia coli BL21 (DE3) cells with IPTG induction. The WOX5 genes from wheat-related species exhibit a similar structure to and high sequence similarity with WOX5 genes from common wheat. The degree of divergence and phylogenetic tree analysis among WOX5 alleles suggested the existence of three homoeologous copies in the A, B, or D genome of common wheat. Quantitative PCR results showed that TaWOX5 was primarily expressed in the root and calli induced by auxin and cytokinin, indicating that TaWOX5 may play a role related to root formation or development and is associated with hormone regulation in somatic embryogenesis. [Copyright &y& Elsevier]

Transgene spreading is a major concern in cultivating genetically modified (GM) corn. Cross-pollination may cause the spread of transgenes from GM cornfields to conventional fields. Occasionally, seed lot contamination, volunteers, mixing during sowing, harvest, and trade can also lead to transgene escape. Obviously, new biological confinement technologies are highly desired to mitigate transgene spreading in addition to physical separation and isolation methods. In this study, we report the development of a built-in containment method to mitigate transgene spreading in corn. In this method, an RNAi cassette for suppressing the expression of the nicosulfuron detoxifying enzyme CYP81A9 and an expression cassette for the glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene G10 were constructed and transformed into corn via Agrobacterium-mediated transformation. The GM corn plants that were generated were found to be sensitive to nicosulfuron but resistant to glyphosate, which is exactly the opposite of conventional corn. Field tests demonstrated that GM corn plants with silenced CYP81A9 could be killed by applying nicosulfuron at 40 g/ha, which is the recommended dose for weed control in cornfields. This study suggests that this built-in containment method for controlling the spread of corn transgenes is effective and easy to implement. [ABSTRACT FROM AUTHOR]

Drought is the major abiotic stress restricting plant growth. To gain insight into the regulatory mechanisms for drought stress tolerance, we performed a genetic screen for identifying Arabidopsis ( Arabidopsis thaliana) drought-resistant mutants. A drought-resistant mutant vrm1 was isolated because of its increased root growth and fresh weight under drought stress. Compared with the wild type (WT), the vrm1 mutant was more resistant to drought stress. The vrm1 plants showed the higher levels of free proline and soluble sugars and the lower relative stomatal conductance as well as the lower water loss rate than the WT under drought stress. Moreover, the higher transcription levels of drought stress-responsive genes NCED3, RD22, RD29A, and COR15A were detected in vrm1 plants subjected to drought and mannitol stresses as compared with WT plants. These results suggest that increased drought resistance of the vrm1 mutant was associated with, at least in part, the higher expression of these drought stress-responsive genes. [ABSTRACT FROM AUTHOR]

Abstract: Ethylene-response factors (ERFs) play an important role in regulating gene expression in plant responses to biotic and abiotic stresses. In this study, a new ERF transcription factor, GmERF7, was isolated from soybean. Sequence analysis showed that GmERF7 contained an AP2/ERF domain with 58 amino acids, two putative nuclear localization signal (NLS) domains, an acidic amino acid-rich transcriptional activation domain and a conserved N-terminal motif [MCGGAI(I/L)]. The expression of GmERF7 was induced by drought, salt, methyl jasmonate (MeJA), ethylene (ETH) and abscisic acid (ABA) treatments. However, the expression of GmERF7 decreased under cold treatment. GmERF7 localized to the nucleus when transiently expressed in onion epidermal cells. Furthermore, GmERF7 protein bound to the GCC-box element in vitro and activated the expression of the β-glucuronidase (GUS) reporter gene in tobacco leaves. Activities of GmERF7 promoter (GmERF7P) upregulated in tobacco leaves with 10h drought, salt and ETH treatments. However, activities of GmERF7P decreased with 10h cold and ABA treatments. Overexpression of GmERF7 in tobacco plants led to higher levels of chlorophyll and soluble carbohydrates and a lower level of malondialdehyde compared with wild-type tobacco plants under salt stress conditions, which indicated that GmERF7 enhanced salt tolerance in transgenic plants. [Copyright &y& Elsevier]