Showing papers in "Plant Cell Reports in 2012"
TL;DR: The recent progress made in unraveling how ethylene promotes germination and acts as an antagonist of ABA is summarized and far less is known about jasmonates in seeds is summarized.
Abstract: Appropriate responses of seeds and fruits to environmental factors are key traits that control the establishment of a species in a particular ecosystem. Adaptation of germination to abiotic stresses and changing environmental conditions is decisive for fitness and survival of a species. Two opposing forces provide the basic physiological mechanism for the control of seed germination: the increasing growth potential of the embryo and the restraint weakening of the various covering layers (seed envelopes), including the endosperm which is present to a various extent in the mature seeds of most angiosperms. Gibberellins (GA), abscisic acid (ABA) and ethylene signaling and metabolism mediate environmental cues and in turn influence developmental processes like seed germination. Cross-species work has demonstrated that GA, ABA and ethylene interact during the regulation of endosperm weakening, which is at least partly based on evolutionarily conserved mechanisms. We summarize the recent progress made in unraveling how ethylene promotes germination and acts as an antagonist of ABA. Far less is known about jasmonates in seeds for which we summarize the current knowledge about their role in seeds. While it seems very clear that jasmonates inhibit germination, the results obtained so far are partly contradictory and depend on future research to reach final conclusions on the mode of jasmonate action during seed germination. Understanding the mechanisms underlying the control of seed germination and its hormonal regulation is not only of academic interest, but is also the ultimate basis for further improving crop establishment and yield, and is therefore of common importance.
269 citations
TL;DR: This study indicates that SlERF5 is mainly involved in the responses to abiotic stress in tomato, and is a member of the ethylene responsive factor (ERF) superfamily.
Abstract: A novel member of the AP2/ERF transcription factor family, SlERF5, was identified from a tomato mature leaf cDNA library screen. The complete DNA sequence of SlERF5 encodes a putative 244-amino acid DNA-binding protein which most likely acts as a transcriptional regulator and is a member of the ethylene responsive factor (ERF) superfamily. Analysis of the deduced SlERF5 protein sequence showed that it contained an ERF domain and belonged to the class III group of ERFs proteins. Expression of SlERF5 was induced by abiotic stress, such as high salinity, drought, flooding, wounding and cold temperatures. Over-expression of SlERF5 in transgenic tomato plants resulted in high tolerance to drought and salt stress and increased levels of relative water content compared with wild-type plants. This study indicates that SlERF5 is mainly involved in the responses to abiotic stress in tomato.
205 citations
TL;DR: The current status of understanding the genomic and epigenomic changes that take place under in vitro conditions is discussed and it is hoped that a precise and comprehensive knowledge of the molecular basis of these variations and acquisition of developmental cell fate would help to devise strategies to improve the totipotency and embryogenic capability in recalcitrant species and genotypes.
Abstract: In vitro cell and tissue-based systems have tremendous potential in fundamental research and for commercial applications such as clonal propagation, genetic engineering and production of valuable metabolites. Since the invention of plant cell and tissue culture techniques more than half a century ago, scientists have been trying to understand the morphological, physiological, biochemical and molecular changes associated with tissue culture responses. Establishment of de novo developmental cell fate in vitro is governed by factors such as genetic make-up, stress and plant growth regulators. In vitro culture is believed to destabilize the genetic and epigenetic program of intact plant tissue and can lead to chromosomal and DNA sequence variations, methylation changes, transposon activation, and generation of somaclonal variants. In this review, we discuss the current status of understanding the genomic and epigenomic changes that take place under in vitro conditions. It is hoped that a precise and comprehensive knowledge of the molecular basis of these variations and acquisition of developmental cell fate would help to devise strategies to improve the totipotency and embryogenic capability in recalcitrant species and genotypes, and to address bottlenecks associated with clonal propagation.
198 citations
TL;DR: The results presented here provide a fundamental clue for cloning specific function genes in further studies and applications and semi-quantitative RT-PCR showed variable stress responses in subgroup III.
Abstract: WRKY transcription factors participate in diverse physiological and developmental processes in plants. They have highly conserved WRKYGQK amino acid sequences in their N-termini, followed by the novel zinc-finger-like motifs, Cys2His2 or Cys2HisCys. To date, numerous WRKY genes have been identified and characterized in a number of herbaceous species. Survey and characterization of WRKY genes in a ligneous species would facilitate a better understanding of the evolutionary processes and functions of this gene family. In this study, 104 poplar WRKY genes (PtWRKY) were identified in the latest poplar genome sequence. According to their structural features, the predicted members were divided into the previously defined groups I–III, as described in rice. In addition, chromosomal localization of the genes demonstrated that there might be WRKY gene hot spots in 2.3 Mb regions on chromosome 14. Furthermore, approximately 83% (86 out of 104) WRKY genes participated in gene duplication events, including 69% (29 out of 42) gene pairs which exhibited segmental duplication. Using semi-quantitative RT-PCR, the expression patterns of subgroup III genes were investigated under different stresses [cold, drought, salinity and salicylic acid (SA)]. The data revealed that these genes presented different expression levels in response to various stress conditions. Expression analysis exhibited PtWRKY76 gene induced markedly in 0.1 mM SA or 25% PEG-6000 treatment. The results presented here provide a fundamental clue for cloning specific function genes in further studies and applications. Key message This study identified 104 poplar WRKY genes and demonstrated WRKY gene hot spots on chromosome 14. Furthermore, semi-quantitative RT-PCR showed variable stress responses in subgroup III.
176 citations
TL;DR: Evaluated genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume, and IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act 11 genes were the best combined to study abiotic stress.
Abstract: Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (Cq) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (β-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.
144 citations
TL;DR: This review addresses methods of obtaining secondary metabolites from plant cell suspension and hairy root cultures and their exudates, particularly the physiological mechanisms of secondary metabolites release and trafficking.
Abstract: This review addresses methods of obtaining secondary metabolites from plant cell suspension and hairy root cultures and their exudates, particularly the physiological mechanisms of secondary metabolites release and trafficking The efficiency for product recovery of metabolites can be increased by various methods, based on the principle of continuous product release into the cultivation medium The most common methods for metabolite recovery are elicitation, influencing membrane permeability, and in situ product removal The biosynthetic pathways can be influenced by cultivation conditions, transformation, or application of elicitors The membrane permeability can be altered through the application of chemical or physical treatments Product removal can be greatly increased through a two-phase system and the introduction of absorbents into the cultivation medium In this review, we describe some improved approaches that have proven useful in these efforts
136 citations
TL;DR: It is suggested that ZmSNAC1 functions as a stress-responsive transcription factor in positive modulation of abiotic stress tolerance, and may have applications in the engineering of drought-tolerant crops.
Abstract: NAC proteins are plant-specific transcription factors that play essential roles in stress responses. However, only little information regarding stress-related NAC genes is available in maize. In this study, a maize NAC gene, ZmSNAC1, was cloned and functionally characterized. Expression analysis revealed that ZmSNAC1 was strongly induced by low temperature, high-salinity, drought stress, and abscisic acid (ABA) treatment, but downregulated by salicylic acid treatment. Subcellular localization experiments in Arabidopsis protoplast cells indicated that ZmSNAC1 was localized in the nucleus. Transactivation assays demonstrated that ZmSNAC1 functioned as a transcriptional activator. Overexpression of ZmSNAC1 in Arabidopsis led to hypersensitivity to ABA and osmotic stress at the germination stage, but enhanced tolerance to dehydration compared to wild-type seedlings. These results suggest that ZmSNAC1 functions as a stress-responsive transcription factor in positive modulation of abiotic stress tolerance, and may have applications in the engineering of drought-tolerant crops. Key message ZmSNAC1 functioned as a stress-responsive transcription factor in response to abiotic stresses, and might be useful for crop tolerance improvement.
132 citations
TL;DR: Results show that OsMTP1 is a bivalent cation transporter localized in the cell membrane, which is necessary for efficient translocation of Zn, Cd and other heavy metals, and maintain ion homeostasis in plant.
Abstract: Rice (Oryza sativa L. ‘Nipponbare’) cDNA subtractive suppression hybridization (SSH) libraries constructed using cadmium (Cd)-treated seedling roots were screened to isolate Cd-responsive genes. A cDNA clone, encoding the rice homolog of Metal Tolerance Protein (OsMTP1), was induced by Cd treatment. Plant MTPs belong to cation diffusion facilitator (CDF) protein family, which are widespread in bacteria, fungi, plants, and animals. OsMTP1 heterologous expression in yeast mutants showed that OsMTP1 was able to complement the mutant strains’ hypersensitivity to Ni, Cd, and Zn, but not other metals including Co and Mn. OsMTP1 expression increased tolerance to Zn, Cd, and Ni in wild-type yeast BY4741 during the exponential growth phase. OsMTP1 fused to green fluorescent protein was localized in onion epidermal cell plasma membranes, consistent with an OsMTP1 function in heavy metal transporting. OsMTP1 dsRNAi mediated by transgenic assay in rice seedlings resulted in heavy metal sensitivity and changed the heavy metal accumulation in different organs of mature rice under low-concentration heavy metal stress. Taken together, our results show that OsMTP1 is a bivalent cation transporter localized in the cell membrane, which is necessary for efficient translocation of Zn, Cd and other heavy metals, and maintain ion homeostasis in plant.
131 citations
TL;DR: Interestingly, the fact that a R2R3 MYB transcription factor named BoPAP1 was extremely up-regulated in the purple kale and induced by low temperature attracted the attention, and evidences strongly suggest that BoP AP1 may play an important role in activating the anthocyanin structural genes for the abundant anthOCyanin accumulation in the Purple kale.
Abstract: The purple kale (Brassica Oleracea var. acephala f. tricolor) is a mutation in kales, giving the mutant phenotype of brilliant purple color in the interior. Total anthocyanin analysis showed that the amount of anthocyanins in the purple kale was up to 1.73 mg g−1 while no anthocyanin was detected in the white kale. To elucidate the molecular mechanism of the anthocyanin biosynthesis in the purple kale, we analyzed the expression of structural genes and some transcription factors associated with anthocyanin biosynthesis in the purple cultivar “Red Dove” and the white cultivar “White Dove”. The result showed that nearly all the anthocyanin biosynthetic genes showed higher expression levels in the purple cultivar than in the white cultivar, especially for DFR and ANS, they were barely detected in the white cultivar. Interestingly, the fact that a R2R3 MYB transcription factor named BoPAP1 was extremely up-regulated in the purple kale and induced by low temperature attracted our attention. Further sequence analysis showed that BoPAP1 shared high similarity with AtPAP1 and BoMYB1. In addition, the anthocyanin accumulation in the purple kale is strongly induced by the low temperature stress. The total anthocyanin contents in the purple kale under low temperature were about 50-fold higher than the plants grown in the greenhouse. The expression of anthocyanin biosynthetic genes C4H, F3H, DFR, ANS and UFGT were all enhanced under the low temperature. These evidences strongly suggest that BoPAP1 may play an important role in activating the anthocyanin structural genes for the abundant anthocyanin accumulation in the purple kale.
118 citations
TL;DR: A number of strategies to increase protein yields are outlined and recent achievements in algal protein production including biopharmaceuticals such as vaccines, antibodies, hormones and enzymes with implications on health-related approaches are summarized.
Abstract: Chlamydomonas reinhardtii has many advantages compared with traditional systems for the molecular farming of recombinant proteins. These include low production costs, rapid scalability at pilot level, absence of human pathogens and the ability to fold and assemble complex proteins accurately. Currently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its usefulness for biotechnological applications. However, several factors affect the level of recombinant protein expression in Chlamydomonas such as enhancer elements, codon dependency, sensitivity to proteases and transformation-associated genotypic modification. The present review outlines a number of strategies to increase protein yields and summarizes recent achievements in algal protein production including biopharmaceuticals such as vaccines, antibodies, hormones and enzymes with implications on health-related approaches. The current status of bioreactor developments for algal culture and the challenges of scale-up and optimization processes are also discussed.
113 citations
TL;DR: The positive role of ZmHSP16.9 in transgenic tobacco conferred tolerance to heat and oxidative stresses by increased seed germination rate, root length, and antioxidant enzyme activities compared with WT plants, and Sequence alignments and phylogenetic analysis reveal this to be cytosolic class I sHSP.
Abstract: Various organisms produce HSPs in response to high temperature and other stresses. The function of heat shock proteins, including small heat shock protein (sHSP), in stress tolerance is not fully explored. To improve our understanding of sHSPs, we isolated ZmHSP16.9 from maize. Sequence alignments and phylogenetic analysis reveal this to be a cytosolic class I sHSP. ZmHSP16.9 expressed in root, leaf and stem tissues under 40 °C treatment, and was up-regulated by heat stress and exogenous H2O2. Overexpression of ZmHSP16.9 in transgenic tobacco conferred tolerance to heat and oxidative stresses by increased seed germination rate, root length, and antioxidant enzyme activities compared with WT plants. These results support the positive role of ZmHSP16.9 in response to heat stress in plant.
TL;DR: The results indicate that trans-resveratrol production decreases as the initial cell density increases for a constant elicitor concentration in Monastrell suspension cultured cells treated with cyclodextrins individually or in combination with methyljasmonate; the decrease observed in cell cultures elicited with CyclodextRins alone is far more drastic than those observed in the combined treatment.
Abstract: In this work, the effect of different inducing factors on trans-resveratrol extracellular production in Monastrell grapevine suspension cultured cells is evaluated. A detailed analysis provides the optimal concentrations of cyclodextrins, methyljasmonate and UV irradiation dosage, optimal cell density, elicitation time and sucrose content in the culture media. The results indicate that trans-resveratrol production decreases as the initial cell density increases for a constant elicitor concentration in Monastrell suspension cultured cells treated with cyclodextrins individually or in combination with methyljasmonate; the decrease observed in cell cultures elicited with cyclodextrins alone is far more drastic than those observed in the combined treatment. trans-Resveratrol extracellular production observed by the joint use of cyclodextrins and methyljasmonate (1,447.8 ± 60.4 μmol trans-resveratrol g−1 dry weight) is lower when these chemical compounds are combined with UV light short exposure (669.9 ± 45.2 μmol trans-resveratrol g−1 dry weight). Likewise, trans-resveratrol production is dependent on levels of sucrose in the elicitation medium with the maximal levels observed with 20 g l−1 sucrose and the joint action of cyclodextrins and 100 μM methyljasmonate. The sucrose concentration did not seem to limit the process although it affects significantly the specific productivity since the lowest sucrose concentration is 10 g l−1, the highest productivity is reached (100.7 ± 5.8 μmol trans-resveratrol g−1 dry weight g−1 sucrose) using cyclodextrins and 25 μM methyljasmonate.
TL;DR: This research provides the first report of producing a plant-derived vaccine at scale under cGMP regulations in an academic setting and an important step for plant-produced vaccines to become a commercial reality.
Abstract: Despite the success in expressing a variety of subunit vaccine proteins in plants and the recent stride in improving vaccine accumulation levels by transient expression systems, there is still no plant-derived vaccine that has been licensed for human use. The lack of commercial success of plant-made vaccines lies in several technical and regulatory barriers that remain to be overcome. These challenges include the lack of scalable downstream processing procedures, the uncertainty of regulatory compliance of production processes, and the lack of demonstration of plant-derived products that meet the required standards of regulatory agencies in identity, purity, potency and safety. In this study, we addressed these remaining challenges and successfully demonstrate the ability of using plants to produce a pharmaceutical grade Norwalk virus (NV) vaccine under current Good Manufacture Practice (cGMP) guidelines at multiple gram scales. Our results demonstrate that an efficient and scalable extraction and purification scheme can be established for processing virus-like particles (VLPs) of NV capsid protein (NVCP). We successfully operated the upstream and downstream NVCP production processes under cGMP regulations. Furthermore, plant-derived NVCP VLP demonstrates the identity, purity, potency and safety that meet the preset release specifications. This material is being tested in a Phase I human clinical trial. This research provides the first report of producing a plant-derived vaccine at scale under cGMP regulations in an academic setting and an important step for plant-produced vaccines to become a commercial reality.
TL;DR: The results suggest that ICE1–CBF cold-response pathway is conserved in tea plants, and two components of this pathway, CsICE1 and CsCBF1, play roles in cold responses inTea plants.
Abstract: C-repeat/dehydration-responsive element binding factors (CBFs) can induce the expression of a suite of cold-responsive genes to increase plant cold tolerance, and inducer of CBF expression 1 (ICE1) is a major activator for CBF. In the present study, we isolated the full-length cDNAs of ICE1 and CBF from Camellia sinensis, designated as CsICE1 and CsCBF1, respectively. The deduced protein CsICE1 contains a highly conserved basic helix-loop-helix (bHLH) domain and C-terminal region of ICE1-like proteins. CsCBF1 contains all conserved domains of CBFs in other plant species and can specifically bind to the C-repeat/dehydration-responsive element (CRT/DRE) as confirmed by electrophoretic mobility shift assay. The transcription of CsICE1 had no apparent alteration after chilling treatment (4°C). CsCBF1 expression was not detected in normal temperature (20°C) but was induced immediately and significantly by low temperature (4°C). Our results suggest that ICE1–CBF cold-response pathway is conserved in tea plants. CsICE1 and CsCBF1, two components of this pathway, play roles in cold responses in tea plants.
TL;DR: Analysis of transcript differences between libraries from the early and late seedling developmental stages demonstrated that starch and sucrose metabolism and phenylpropanoid biosynthesis may be the dominant metabolic events during tuberous root formation and plant hormones probably play critical roles in regulation of this developmental process.
Abstract: The tuberous root of radish is an important vegetable, but insufficient transcriptomic and genomic data are currently available to understand the molecular mechanisms underlying tuberous root formation and development. High-throughput transcriptomic sequencing is essential to generate a large transcript sequence data set for gene discovery and molecular marker development. In this study, a total of 107.3 million clean reads were generated using Illumina paired-end sequencing technology. De novo assembly generated 61,554 unigenes with an average length of 820 bp. Based on a sequence similarity search with known proteins or nucleotides, 85.51 % (52,634), 90.18 % (55,507) and 54 % (33,242) consensus sequences showed homology with sequences in the Nr, Nt and Swiss-Prot databases, respectively. Of these annotated unigenes, 21,109 and 17,343 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. A total of 27,809 unigenes were assigned to 123 pathways in the Kyoto Encyclopedia of Genes and Genomes database. Analysis of transcript differences between libraries from the early and late seedling developmental stages demonstrated that starch and sucrose metabolism and phenylpropanoid biosynthesis may be the dominant metabolic events during tuberous root formation and plant hormones probably play critical roles in regulation of this developmental process. In total, 14,641 potential EST-SSRs were identified among the unigenes, and 12,733 primer pairs for 2,511 SSR were obtained. Summarily, this study gave us a clue to understand the radish tuberous root formation and development, and also provided us with a valuable sequence resource for novel gene discovery and marker-assisted selective breeding in radish. De novo assembled and characterized the radish tuberous root transcriptome; explored the mechanism of radish tuberous root formation; development of EST-SSR markers in radish.
TL;DR: Overexpression of CBP60g in Arabidopsis enhanced the defense response, hypersensitivity to abscisic acid and tolerance to drought stress, and serves as a molecular link that positively regulates ABA- and SA-mediated pathways in plants.
Abstract: Calmodulin-binding proteins (CBPs) have been known to be involved in both biotic and abiotic stress responses. Recently, two closely related CBPs, Arabidopsis SAR Deficient 1 and CBP60g, were found to belong to a new family of transcription factors that regulate salicylic acid (SA) biosynthesis triggered by microbe-associated molecular patterns. In this study, we found that overexpression of CBP60g in Arabidopsis caused elevated SA accumulation, increased expression of the defense genes, and enhanced resistance to Pseudomonas syringae. In addition to the enhanced defense response, the CBP60g overexpression lines showed hypersensitivity to abscisic acid (ABA) and enhanced tolerance to drought stress. We also found that treatment with ABA and drought stress leads to a higher expression level of the ICS1 gene, which encodes isochorismate synthase, in the CBP60g overexpression lines than in the wild-type control plants. Our results suggest that CBP60g serves as a molecular link that positively regulates ABA- and SA-mediated pathways in plants. Key message Overexpression of CBP60g in Arabidopsis enhanced the defense response, hypersensitivity to abscisic acid and tolerance to drought stress.
TL;DR: The goal of this review is to piece together the findings concerning MAPK cascades in ABA signaling by reviewing the available data and questions and further perspectives of the roles played by MAPK cascade in A BA signaling.
Abstract: Abscisic acid (ABA) is a universal hormone in higher plants and plays a major role in various aspects of plant stress, growth, and development. Mitogen-activated protein kinase (MAPK) cascades are key signaling modules for responding to various extracellular stimuli in plants. The available data suggest that MAPK cascades are involved in some ABA responses, including antioxidant defense, guard cell signaling, and seed germination. Some MAPK phosphatases have also been demonstrated to be implicated in ABA responses. The goal of this review is to piece together the findings concerning MAPK cascades in ABA signaling. Questions and further perspectives of the roles played by MAPK cascades in ABA signaling are also furnished.
TL;DR: The SV threshold, space-determined hypothesis and D-bottleneck are proposed to interpret the loss of the regenerability through a long period of plant tissue culture (PTC), which consider natural selection theory to be fit for cell population in vitro.
Abstract: Plants regenerated from in vitro cultures possess an array of genetic and epigenetic changes. This phenomenon is known as ‘somaclonal variation’ and the frequency of somaclonal variation (SV) is usually elevated far beyond that expected in nature. Initially, the relationship between time in culture and detected SV was found to support the widespread belief that SV accumulates with culture age. However, a few studies indicated that older cultures yielded regenerants with less SV. What leads to this seemed contradiction? In this article, we have proposed a novel in vitro callus selection hypothesis, differentiation bottleneck (D-bottleneck) and dedifferentiation bottleneck (Dd-bottleneck), which consider natural selection theory to be fit for cell population in vitro. The results of multiplication races between the cells with the true-to-type phenotype and the deleterious cells determine the increase/decrease of SV frequencies in calli or regenerants as in vitro culture time goes on. The possibility of interpreting the complex situation of time-related SV by the evolutionary theory is discussed in this paper. In addition, the SV threshold, space-determined hypothesis and D-bottleneck are proposed to interpret the loss of the regenerability through a long period of plant tissue culture (PTC).
TL;DR: The transformation procedure, from initiating IEs to planting putative transgenic plantlets in the glasshouse, was completed within 11–16 weeks, and was approximately threefold more efficient than the previously reported best sorghum transformation system.
Abstract: A highly efficient microprojectile transformation system for sorghum (Sorghum bicolor L.) has been developed by using immature embryos (IEs) of inbred line Tx430. Co-bombardment was performed with the neomycin phosphotransferase II (nptII) gene and the green fluorescent protein (gfp) gene, both under the control of the maize ubiquitin1 (ubi1) promoter. After optimization of both tissue culture media and parameters of microprojectile transformation, 25 independent transgenic events were obtained from 121 bombarded IEs. The average transformation frequency (the total number of independent transgenic events divided by the total number of bombarded IEs) was 20.7% in three independent experiments. Transgenic events were confirmed by both PCR screening and Southern hybridization of genomic DNA from primary transgenics (T0). More than 90% of transformants were fertile and displayed normal morphology in a containment glasshouse. Co-transformation rate of the nptII and gfp genes was 72% in these experiments. The segregation of nptII and gfp in T1 progenies was observed utilizing fluorescence microscopy and geneticin selection of seedlings indicating both were inherited in the T1 generation. The transformation procedure, from initiating IEs to planting putative transgenic plantlets in the glasshouse, was completed within 11–16 weeks, and was approximately threefold more efficient than the previously reported best sorghum transformation system.
TL;DR: It is suggested that Al induces lignification in the roots of Al-sensitive rice varieties, probably through an increase in H2O2 accumulation.
Abstract: The effects of aluminum (Al) on root elongation, lipid peroxidation, hydrogen peroxide (H2O2) accumulation, antioxidant levels, antioxidant enzymatic activity, and lignin content in the roots of the Al-tolerant rice variety azucena and the Al-sensitive variety IR64 were investigated. Treatment with Al induced a greater decrease in root elongation and a greater increase in H2O2 and lipid peroxidation as determined by the total thiobarbituric acid-reactive substance (TBARS) level in IR64 than in azucena. Azucena had significantly higher levels of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and glutathione peroxidase GSH POD activity compared with IR64. The concentrations of reduced glutathione (GSH) and ascorbic acid, and the GSH/GSSG ratio (reduced vs. oxidized glutathione) were also higher in azucena than in IR64 in the presence of Al. The addition of 1 mg/L GSH improved root elongation in both varieties and decreased H2O2 production under Al stress. By contrast, treatment with buthionine sulfoximine, a specific inhibitor of GSH synthesis, decreased root elongation in azucena and stimulated H2O2 production in both varieties. Moreover, Al treatment significantly increased the cytoplasmic activity of peroxidase (POD) as well as the levels of POD bound ionically and covalently to cell walls in the Al-sensitive variety. The lignin content was also increased. Treatment with exogenous H2O2 also increased the lignin content and decreased root elongation in IR64. These results suggest that Al induces lignification in the roots of Al-sensitive rice varieties, probably through an increase in H2O2 accumulation.
TL;DR: A new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids has been proposed and elucidates significant contribution of DO XP pathway to withanolide biosynthesis.
Abstract: Withanolides are pharmaceutically important C28-phytochemicals produced in most prodigal amounts and diversified forms by Withania somnifera. Metabolic origin of withanolides from triterpenoid pathway intermediates implies that isoprenogenesis could significantly govern withanolide production. In plants, isoprenogenesis occurs via two routes: mevalonate (MVA) pathway in cytosol and non-mevalonate or DOXP/MEP pathway in plastids. We have investigated relative carbon contribution of MVA and DOXP pathways to withanolide biosynthesis in W. somnifera. The quantitative NMR-based biosynthetic study involved tracing of 13C label from 13C1-d-glucose to withaferin A in withanolide producing in vitro microshoot cultures of the plant. Enrichment of 13C abundance at each carbon of withaferin A from 13C1-glucose-fed cultures was monitored by normalization and integration of NMR signal intensities. The pattern of carbon position-specific 13C enrichment of withaferin A was analyzed by a retro-biosynthetic approach using a squalene-intermediated metabolic model of withanolide (withaferin A) biosynthesis. The pattern suggested that both DOXP and MVA pathways of isoprenogenesis were significantly involved in withanolide biosynthesis with their relative contribution on the ratio of 25:75, respectively. The results have been discussed in a new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids.
TL;DR: It is concluded that CdPCS1 encodes functional PCS and may be part of metal detoxification mechanism of the heavy metal accumulating plant C.demersum.
Abstract: Phytochelatin synthase (PCS), the key enzyme involved in heavy metal detoxification and accumulation has been used from various sources to develop transgenic plants for the purpose of phytoremediation. However, some of the earlier studies provided contradictory results. Most of the PCS genes were isolated from plants that are not potential metal accumulators. In this study, we have isolated PCS gene from Ceratophyllum
demersum cv. L. (CdPCS1), a submerged rootless aquatic macrophyte, which is considered as potential accumulator of heavy metals. The CdPCS1 cDNA of 1,757 bp encodes a polypeptide of 501 amino acid residues and differs from other known PCS with respect to the presence of a number of cysteine residues known for their interaction with heavy metals. Complementation of cad1-3 mutant of Arabidopsis deficient in PC (phytochelatin) biosynthesis by CdPCS1 suggests its role in the synthesis of PCs. Transgenic tobacco plants expressing CdPCS1 showed several-fold increased PC content and precursor non-protein thiols with enhanced accumulation of cadmium (Cd) and arsenic (As) without significant decrease in plant growth. We conclude that CdPCS1 encodes functional PCS and may be part of metal detoxification mechanism of the heavy metal accumulating plant C.
demersum. Heterologous expression of PCS gene from C.
demersum complements Arabidopsis
cad1-3 mutant and leads to enhanced accumulation of Cd and As in transgenic tobacco.
TL;DR: This work uses the grape as a model to study the ABA perception mechanism in fruit trees and suggests that VvPP2C4 is the major PP2C involved in ABA Perception in leaves and roots, and VvRCAR6 and V vRCAR5 respectively, are the major receptors involved in aBA perception in these organs.
Abstract: Molecular control mechanisms for abiotic stress tolerance are based on the activation and regulation of specific stress-related genes. The phytohormone abscisic acid (ABA) is a key endogenous messenger in a plant’s response to such stresses. A novel ABA binding mechanism which plays a key role in plant cell signaling cascades has recently been uncovered. In the absence of ABA, a type 2C protein phosphatase (PP2C) interacts and inhibits the kinase SnRK2. Binding of ABA to the PYR/PYLs receptors enables interaction between the ABA receptor and the PP2C protein, and abrogates the SnRK2 inactivation. The active SnRK2 is then free to activate the ABA-responsive element Binding Factors which target ABA-dependent gene expression. We used the grape as a model to study the ABA perception mechanism in fruit trees. The grape ABA signaling cascade consists of at least seven ABA receptors and six PP2Cs. We used a yeast two-hybrid system to examine physical interaction in vitro between the grape ABA receptors and their interacting partners, and found that twenty-two receptor-PP2C interactions can occur. Moreover, quantifying these affinities by the use of the LacZ reporter enables us to show that VvPP2C4 and VvPP2C9 are the major binding partners of the ABA receptor. We also tested in vivo the root and leaf gene expression of the various ABA receptors and PP2Cs in the presence of exogenic ABA and under different abiotic stresses such as high salt concentration, cold and drought, and found that many of these genes are regulated by such abiotic environmental factors. Our results indicate organ specificity in the ABA receptor genes and stress specificity in the VvPP2Cs. We suggest that VvPP2C4 is the major PP2C involved in ABA perception in leaves and roots, and VvRCAR6 and VvRCAR5 respectively, are the major receptors involved in ABA perception in these organs. Identification, characterization and manipulation of the central players in the ABA signaling cascades in fruit trees is likely to prove essential for improving their performance in the future.
TL;DR: Compared with drought-sensitive cultivar, the drought-resistant cultivar could maintain greater expansin activity and cell wall extension, which was contributive to its resultant faster growth under water stress.
Abstract: Expansin protein is a component of the cell wall generally accepted to be the key regulator of cell wall extension during plant growth. Plant hormones regulate expansin gene expression as well as plant growth during drought stress. However, the relationship between expansin and plant hormone is far from clear. Here, we studied the involvement of expansin in plant cell growth mediated by the hormones indole-3-acetic acid (IAA) and abscisic acid (ABA) under osmotic stress which was induced by polyethylene glycol (PEG)-6000. Wheat coleoptiles from a drought-resistant cultivar HF9703 and a drought-sensitive cultivar 921842 were used to evaluate cell growth and expansin activity. Osmotic stress induced the accumulation of ABA. ABA induced expansin activity mainly by enhancing expansin expression, since ABA induced cell wall basification via decreasing plasma membrane H+-ATPase activity, which was unfavorable for expansin activity. Although ABA induced expansin activity and cell wall extension, treatment with exogenous ABA and/or fluridone (FLU, an ABA inhibitor) suggested that ABA was involved in the coleoptile growth inhibition during osmotic stress. IAA application to detached coleoptiles also enhanced coleoptile growth and increased expansin activity, but unlike ABA, IAA-induced expansin activity was mainly due to the decrease of cell wall pH by increasing plasma membrane H+-ATPase activity. Compared with drought-sensitive cultivar, the drought-resistant cultivar could maintain greater expansin activity and cell wall extension, which was contributive to its resultant faster growth under water stress.
TL;DR: A novel sugarcane Sc-Dir gene, DIRd subfamily, which is highly stalk-specific expression and involved in the response to artificial stresses of drought, salts, and oxidatives is identified.
Abstract: Dirigent and dirigent-like family proteins contain a number of proteins involved in lignification or in the response to pathogen infection and abiotic stress in plants. In the present study, a full-length cDNA sequence of a dirigent-like gene designated ScDir (GenBank Accession Number JQ622282) was obtained from sugarcane based on the stem full-length cDNA library. The ScDir gene was 819-bp long, including a 564-bp ORF encoding 187 amino acid residues. The protein N-terminus contained signal peptides at amino acid residues of 1–25 and transmembrane regions at 7–26 aa. A his-tagged ScDir protein with an estimated molecular mass of 27.4 kDa was expressed in Escherichia coli system. The expressed ScDir protein had increased the host cell’s tolerance to PEG and NaCl. When an endogenous GAPDH gene was used as internal control, results from real-time qPCR demonstrated that the ScDir mRNA amount in sugarcane stems was significantly higher than that in the roots, leaves and buds by 18.64 ± 0.48, 25,635.16 ± 2,966.03 and 721.50 ± 8.17-fold, respectively. The ScDir transcript levels in sugarcane seedling increased under H2O2, PEG or NaCl stress. The expression level of ScDir was significantly upregulated under PEG stress, and the highest level was observed at 12 h after stress. Thus, both the ScDir-hosted cell performance and the enhanced expressions in sugarcane imply that the ScDir gene is involved in the response to abiotic stresses of drought, salts and oxidation. The transcription of the ScDir gene is highly stem-specific, as revealed by real-time qPCR. Key message A novel sugarcane Sc-Dir gene, DIRd subfamily, which is highly stalk-specific expression and involved in the response to artificial stresses of drought, salts, and oxidatives.
TL;DR: Results show that, during the defensive response of soybean to viral infection, callose deposition at PD is mainly responsible for restricting the movement of the virus between cells and it occurs prior to the HR response.
Abstract: Callose is a β-l,3-glucan with diverse roles in the viral pathogenesis of plants. It is widely believed that the deposition of callose and hypersensitive reaction (HR) are critical defence responses of host plants against viral infection. However, the sequence of these two events and their resistance mechanisms are unclear. By exploiting a point inoculation approach combined with aniline blue staining, immuno-electron microscopy and external sphincters staining with tannic acid, we systematically investigated the possible roles of callose deposition during viral infection in soybean. In the incompatible combination, callose deposition at the plasmodesmata (PD) was clearly visible at the sites of inoculation but viral RNA of coat protein (CP-RNA) was not detected by RT-PCR in the leaf above the inoculated one (the upper leaf). In the compatible combination, however, callose deposition at PD was not detected at the site of infection but the viral CP-RNA was detected by RT-PCR in the upper leaf. We also found that in the incompatible combination the fluorescence due to callose formation at the inoculation point disappeared following the injection of 2-deoxy-d-glucose (DDG, an inhibitor of callose synthesis). At same time, in the incompatible combination, necrosis was observed and the viral CP-RNA was detected by RT-PCR in the upper leaf and HR characteristics were evident at the inoculation sites. These results show that, during the defensive response of soybean to viral infection, callose deposition at PD is mainly responsible for restricting the movement of the virus between cells and it occurs prior to the HR response.
TL;DR: The obtained unigenes will provide the foundation for research on functional genomics in Allium sativum and its closely related species, and fill the gap of the existing plant EST database.
Abstract: Garlic is widely used as a spice throughout the world for the culinary value of its flavor and aroma, which are created by the chemical transformation of a series of organic sulfur compounds. To analyze the transcriptome of Allium sativum and discover the genes involved in sulfur metabolism, cDNAs derived from the total RNA of Allium sativum buds were analyzed by Illumina sequencing. Approximately 26.67 million 90 bp paired-end clean reads were achieved in two libraries. A total of 127,933 unigenes were generated by de novo assembly and were compared with the sequences in public databases. Of these, 45,286 unigenes had significant hits to the sequences in the Nr database, 29,514 showed significant similarity to known proteins in the Swiss-Prot database and, 20,706 and 21,952 unigenes had significant similarity to existing sequences in the KEGG and COG databases, respectively. Moreover, genes involved in organic sulfur biosynthesis were identified. These unigenes data will provide the foundation for research on gene expression, genomics and functional genomics in Allium sativum. Key message The obtained unigenes will provide the foundation for research on functional genomics in Allium sativum and its closely related species, and fill the gap of the existing plant EST database.
TL;DR: The applicability of microsatellite markers in various genotyping applications, determining phylogenetic relationships and comparative mapping in several important grass species is demonstrated and the utility of markers in germplasm characterizations is demonstrated.
Abstract: The unavailability of microsatellite markers and saturated genetic linkage map has restricted the genetic improvement of foxtail millet [Setaria italica (L.) P. Beauv.], despite the fact that in recent times it has been documented as a new model species for biofuel grasses. With the objective to generate a good number of microsatellite markers in foxtail millet cultivar ‘Prasad’, 690 clones were sequenced which generated 112.95 kb high quality sequences obtained from three genomic libraries each enriched with different microsatellite repeat motifs. Microsatellites were identified in 512 (74.2%) of the 690 positive clones and 172 primer pairs (pp) were successfully designed from 249 (48.6%) unique SSR-containing clones. The efficacies of the microsatellite containing genomic sequences were established by superior primer designing ability (69%), PCR amplification efficiency (85.5%) and polymorphic potential (52%) in the parents of F2 mapping population. Out of 172 pp, functional 147 markers showed high level of cross-species amplification (~74%) in six grass species. Higher polymorphism rate and broad range of genetic diversity (0.30–0.69 averaging 0.58) obtained in constructed phylogenetic tree using 52 microsatellite markers, demonstrated the utility of markers in germplasm characterizations. In silico comparative mapping of 147 foxtail millet microsatellite containing sequences against the mapping data of sorghum (~18%), maize (~16%) and rice (~5%) indicated the presence of orthologous sequences of the foxtail millet in the respective species. The result thus demonstrates the applicability of microsatellite markers in various genotyping applications, determining phylogenetic relationships and comparative mapping in several important grass species.
TL;DR: Progress in the field of plant-derived peptides over the last 5 years is reviewed, addressing new challenges for diverse pathologies.
Abstract: Peptides have unique properties that make them useful drug candidates for diverse indications, including allergy, infectious disease and cancer. Some peptides are intrinsically bioactive, while others can be used to induce precise immune responses by defining a minimal immunogenic region. The limitations of peptides, such as metabolic instability, short half-life and low immunogenicity, can be addressed by strategies such as multimerization or fusion to carriers, to improve their pharmacological properties. The remaining major drawback is the cost of production using conventional chemical synthesis, which is also difficult to scale-up. Over the last 15 years, plants have been shown to produce bioactive and immunogenic peptides economically and with the potential for large-scale synthesis. The production of peptides in plants is usually achieved by the genetic fusion of the corresponding nucleotide sequence to that of a carrier protein, followed by stable nuclear or plastid transformation or transient expression using bacterial or viral vectors. Chimeric plant viruses or virus-like particles can also be used to display peptide antigens, allowing the production of polyvalent vaccine candidates. Here we review progress in the field of plant-derived peptides over the last 5 years, addressing new challenges for diverse pathologies.
TL;DR: The results suggest that OsCAD2 is largely responsible for monolignol biosynthesis in rice, which is similar to that indicated for the predominant role of other plant bona fide CAD protein to monoligne biosynthesis.
Abstract: Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step of monolignol biosynthesis. The rice genome contains 12 CAD-like genes, and whereas the proteins encoded by OsCAD2 and OsCAD7 are known to function in monolignol biosynthesis, the degree to which these enzymes contribute to this process and the involvement of the enzymes encoded by the remaining ten genes is unclear. This paper investigates the role of OsCAD2 and the nine other OsCAD-like proteins in monolignol biosynthesis. Among the OsCAD genes analyzed, OsCAD2, an enzyme belonging to the bona fide CAD phylogenetic group, was the most abundantly expressed gene in the uppermost internode, and was expressed at levels that were more than seven times greater than those of the second most abundantly expressed gene, OsCAD1. Promoter-GUS analysis of OsCAD2 (pCAD::GUS) in the internode, sheath, and roots revealed that GUS expression was strong in tissues that accumulated high levels of lignin. Furthermore, expression always preceded lignin accumulation, showing the tight correlation between OsCAD2 expression and monolignol biosynthesis. Additionally, expression of pCAD::GUS was well synchronized with that of rice caffeic acid 3-O-methyltransferase (OsCOMT::GUS), suggesting that the two enzymes function cooperatively during monolignol biosynthesis. Co-expression network analysis of eight OsCAD genes further revealed that, among the OsCAD genes, expression of OsCAD2 was most tightly associated with the transcription of lignin biosynthesis-related genes. These results suggest that OsCAD2 is largely responsible for monolignol biosynthesis in rice, which is similar to that indicated for the predominant role of other plant bona fide CAD protein to monolignol biosynthesis.