Showing papers in "Plant Cell Reports in 2015"

Seed priming: state of the art and new perspectives.

Abstract: Priming applied to commercial seed lots is widely used by seed technologists to enhance seed vigour in terms of germination potential and increased stress tolerance. Priming can be also valuable to seed bank operators who need improved protocols of ex situ conservation of germplasm collections (crop and native species). Depending on plant species, seed morphology and physiology, different priming treatments can be applied, all of them triggering the so-called ‘pre-germinative metabolism’. This physiological process takes place during early seed imbibition and includes the seed repair response (activation of DNA repair pathways and antioxidant mechanisms), essential to preserve genome integrity, ensuring proper germination and seedling development. The review provides an overview of priming technology, describing the range of physical–chemical and biological treatments currently available. Optimised priming protocols can be designed using the ‘hydrotime concept’ analysis which provides the theoretical bases for assessing the relationship between water potential and germination rate. Despite the efforts so far reported to further improve seed priming, novel ideas and cutting-edge investigations need to be brought into this technological sector of agri-seed industry. Multidisciplinary translational research combining digital, bioinformatic and molecular tools will significantly contribute to expand the range of priming applications to other relevant commercial sectors, e.g. the native seed market.

Advances in the understanding of cuticular waxes in Arabidopsis thaliana and crop species

Abstract: The aerial parts of plants are covered with a cuticle, a hydrophobic layer consisting of cutin polyester and cuticular waxes that protects them from various environmental stresses. Cuticular waxes mainly comprise very long chain fatty acids and their derivatives such as aldehydes, alkanes, secondary alcohols, ketones, primary alcohols, and wax esters that are also important raw materials for the production of lubricants, adhesives, cosmetics, and biofuels. The major function of cuticular waxes is to control non-stomatal water loss and gas exchange. In recent years, the in planta roles of many genes involved in cuticular wax biosynthesis have been characterized not only from model organisms like Arabidopsis thaliana and saltwater cress (Eutrema salsugineum), but also crop plants including maize, rice, wheat, tomato, petunia, Medicago sativa, Medicago truncatula, rapeseed, and Camelina sativa through genetic, biochemical, molecular, genomic, and cell biological approaches. In this review, we discuss recent advances in the understanding of the biological functions of genes involved in cuticular wax biosynthesis, transport, and regulation of wax deposition from Arabidopsis and crop species, provide information on cuticular wax amounts and composition in various organs of nine representative plant species, and suggest the important issues that need to be investigated in this field of study.

Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system

Abstract: Technologies to achieve the specific and precise knockout of genes are critical for understanding gene functions and fundamental biological processes. Targeted genome editing as a new and efficient method to mutate genes has been rapidly used in many organisms. Compared with the earlier systems, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 nuclease (Cas9) is an easier and more efficient system, and it has been widely used in recent years (Gaj et al. 2013). The Cas9 endonuclease is dictated by a 20-base pair (bp) sequence at the 50 end of the single-guide RNA (sgRNA), which then acts as a guide to the specific site of the genome, where Cas9 is able to cleave double-stranded DNA, leading to deletion, insertion, or substitution at the target sites (Sander and Joung 2014). Since 2013, CRISPR/Cas9 has been successfully applied by transient expression and/or stable transgenic lines in several plant species, such as Arabidopsis, Nicotiana benthamiana, rice, wheat, maize, and tomato (Brooks et al. 2014; Jiang et al. 2013; Li et al. 2013; Miao et al. 2013; Nekrasov et al. 2013; Shan et al. 2013). Moreover, the mutations generated in the primary transgenic plants by the CRISPR/Cas9 system can be stably transmitted to the next generation (Brooks et al. 2014; Feng et al. 2014). Thus, the CRISPR/Cas9 system is becoming a powerful tool for genome editing in plants, whereas the reports of the usage and efficiency of the CRISPR/Cas9 system-mediated plant genome engineering are still limited. Potato is a very important crop for world food security. Environmental changes and extended cultivation regions are challenges for potato cultivars. Understanding the functions of genes will help us to improve agronomic traits using molecular engineering technologies. Since cultivated potatoes are tetraploid, the functional approach of genes by molecular genetics is very difficult. Similar to other plant species in which the CRISPR/Cas9 has been successfully used, gene transformation in potato is efficient, and the genome sequence of double-haploid DM and diploid RH is available, which made potato an ideal candidate for this genome editing system (The Potato Genome Sequencing Consortium 2011). Here, we used the CRISPR/Cas9 system to produce knockouts of genes in potato successfully, which will provide an excellent foundation for future gene function studies. In order to construct a CRISPR/Cas9 plasmid which would function efficiently in potato, we first cloned a native promoter for potato U6 RNAs (StU6P) from DM Communicated by N. Stewart.

Suberin: biosynthesis, regulation, and polymer assembly of a protective extracellular barrier

Abstract: Suberin is a lipid-phenolic biopolyester deposited in the cell walls of certain boundary tissue layers of plants, such as root endodermis, root and tuber peridermis, and seed coats. Suberin serves as a protective barrier in these tissue layers, controlling, for example, water and ion transport. It is also a stress-induced anti-microbial barrier. The suberin polymer contains a variety of C16–C24 chain-length aliphatics, such as ω-hydroxy fatty acids, α,ω-dicarboxylic fatty acids, and primary fatty alcohols. Suberin also contains high amounts of glycerol and phenolics, especially ferulic acid. In addition, non-covalently linked waxes are likely associated with the suberin polymer. This review focusses on the suberin biosynthetic enzymes identified to date, which include β-ketoacyl-CoA synthases, fatty acyl reductases, long-chain acyl-CoA synthetases, cytochrome P450 monooxygenases, glycerol 3-phosphate acyltransferases, and phenolic acyltransferases. We also discuss recent advances in our understanding of the transport of suberin components intracellularly and to the cell wall, polymer assembly, and the regulation of suberin deposition.

The interaction between nitrogen availability and auxin, cytokinin, and strigolactone in the control of shoot branching in rice (Oryza sativa L.)

Abstract: Nitrogen availability and cytokinin could promote shoot branching in rice, whereas auxin and strigolactone inhibited it. The interaction between nitrogen availability and the three hormones is discussed. Rice shoot branching is strongly affected by nitrogen availability and the plant hormones auxin, cytokinin, and strigolactone; however, the interaction of them in the regulation of rice shoot branching remains a subject of debate. In the present study, nitrogen and the three hormones were used to regulate rice tiller bud growth in the indica rice variety Yangdao 6. Both nitrogen and CK promoted shoot branching in rice, whereas auxin and SL inhibited it. We used HPLC to determine the amounts of endogenous IAA and CK, and we used quantitative real-time PCR analysis to quantify the expression levels of several genes. Nitrogen enhanced the amount of CK by promoting the expression levels of OsIPTs in nodes. In addition, both nitrogen and CK downregulated the expression of genes related to SL synthesis in root and nodes, implying that the inhibition of SL synthesis by nitrogen may occur at least partially through the CK pathway. SL did not significantly reduce the amount of CK or the expression levels of OsIPT genes, but it did significantly reduce the amount of auxin and the auxin transport capacity in nodes. Auxin itself inhibited CK synthesis and promoted SL synthesis in nodes rather than in roots. Furthermore, we found that CK and SL quickly reduced and increased the expression of FC1 in buds, respectively, implying that FC1 might be a common target for the CK and SL pathways. Nitrogen and auxin delayed expression change patterns of FC1, potentially by changing the downstream signals for CK and SL.

Silicon improves salt tolerance by increasing root water uptake in Cucumis sativus L.

Abstract: Silicon enhances root water uptake in salt-stressed cucumber plants through up-regulating aquaporin gene expression. Osmotic adjustment is a genotype-dependent mechanism for silicon-enhanced water uptake in plants. Silicon can alleviate salt stress in plants. However, the mechanism is still not fully understood, and the possible role of silicon in alleviating salt-induced osmotic stress and the underlying mechanism still remain to be investigated. In this study, the effects of silicon (0.3 mM) on Na accumulation, water uptake, and transport were investigated in two cucumber (Cucumis sativus L.) cultivars (‘JinYou 1’ and ‘JinChun 5’) under salt stress (75 mM NaCl). Salt stress inhibited the plant growth and photosynthesis and decreased leaf transpiration and water content, while added silicon ameliorated these negative effects. Silicon addition only slightly decreased the shoot Na levels per dry weight in ‘JinYou 1’ but not in ‘JinChun 5’ after 10 days of stress. Silicon addition reduced stress-induced decreases in root hydraulic conductivity and/or leaf-specific conductivity. Expressions of main plasma membrane aquaporin genes in roots were increased by added silicon, and the involvement of aquaporins in water uptake was supported by application of aquaporin inhibitor and restorative. Besides, silicon application decreased the root xylem osmotic potential and increased root soluble sugar levels in ‘JinYou 1.’ Our results suggest that silicon can improve salt tolerance of cucumber plants through enhancing root water uptake, and silicon-mediated up-regulation of aquaporin gene expression may in part contribute to the increase in water uptake. In addition, osmotic adjustment may be a genotype-dependent mechanism for silicon-enhanced water uptake in plants.

Microalgal lipid droplets: composition, diversity, biogenesis and functions

Abstract: Lipid droplet is the major site of neutral lipid storage in eukaryotic cells, and increasing evidence show its involvement in numerous cellular processes such as lipid homeostasis, signaling, trafficking and inter-organelle communications. Although the biogenesis, structure, and functions of lipid droplets have been well documented for seeds of vascular plants, mammalian adipose tissues, insects and yeasts, relative little is known about lipid droplets in microalgae. Over the past 5 years, the growing interest of microalgae as a platform for biofuel, green chemicals or value-added polyunsaturated fatty acid production has brought algal lipid droplets into spotlight. Studies conducted on the green microalga Chlamydomonas reinhardtii and other model microalgae such as Haematococcus and Nannochloropsis species have led to the identification of proteins associated with lipid droplets, which include putative structural proteins different from plant oleosins and animal perilipins, as well as candidate proteins for lipid biosynthesis, mobilization, trafficking and homeostasis. Biochemical and microscopy studies have also started to shed light on the role of chloroplasts in the biogenesis of lipid droplets in Chlamydomonas.

Overexpression of EaDREB2 and pyramiding of EaDREB2 with the pea DNA helicase gene (PDH45) enhance drought and salinity tolerance in sugarcane (Saccharum spp. hybrid)

Abstract: EaDREB2 overexpressed in sugarcane enhanced tolerance to drought and salinity. When co-transformed with plant DNA helicase gene, DREB2 showed greater level of salinity tolerance than in single-gene transgenics. Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and can potentially cause up to 50 % yield loss. DREB proteins play a vital regulatory role in abiotic stress tolerance in plants. We previously reported that expression of EaDREB2 is enhanced by drought stress in Erianthus arundinaceus. In this study, we have isolated the DREB2 gene from E. arundinaceus, transformed one of the most popular sugarcane variety Co 86032 in tropical India with EaDREB2 through Agrobacterium-mediated transformation, pyramided the EaDREB2 gene with the gene coding for PDH45 driven by Port Ubi 2.3 promoter through particle bombardment and evaluated the V1 transgenics for soil deficit moisture and salinity stresses. Soil moisture stress was imposed at the tillering phase by withholding irrigation. Physiological, molecular and morphological parameters were used to assess drought tolerance. Salinity tolerance was assessed through leaf disc senescence and bud sprout assays under salinity stress. Our results indicate that overexpression of EaDREB2 in sugarcane enhances drought and salinity tolerance to a greater extent than the untransformed control plants. This is the first report of the co-transformation of EaDREB2 and PDH45 which shows higher salinity tolerance but lower drought tolerance than EaDREB2 alone. The present study seems to suggest that, for combining drought and salinity tolerance together, co-transformation is a better approach.

Sp-miR396a-5p acts as a stress-responsive genes regulator by conferring tolerance to abiotic stresses and susceptibility to Phytophthora nicotianae infection in transgenic tobacco

Abstract: Overexpression of Sp-miR396a-5p in tobacco increased tolerance to salt, drought, cold stress and susceptibility to Phytophthora nicotianae infection. MicroRNA396 (miR396) is one of the conserved microRNA families in plants, and it targeted growth-regulating factors (GRFs) family. The GRF transcription factors are associated with growth and stress responses. However, the molecular mechanisms of miR396 responding to environmental stresses are elusive. The purpose of this study was to explore the function of tomato miR396a-5p (Sp-miR396a-5p) in Solanaceae responses to abiotic and biotic stresses. We showed that Sp-miR396a-5p transcript levels were up-regulated under salt and drought stresses and down-regulated after Phytophthora infestans (P. infestans) infection. Consistently, overexpression of Sp-miR396a-5p in tobacco enhanced its tolerance to salt, drought and cold stresses. Additionally, the expression of Sp-miR396a-5p was found to be down-regulated under pathogen-related biotic stress. Tobacco plants overexpressing Sp-miR396a-5p showed increased susceptibility to Phytophthora nicotianae (P. nicotianae) infection. Physiological analysis indicated that Sp-miR396a-5p overexpression enhanced osmoregulation and decreased production of reactive oxygen species (ROS). Furthermore, four Sp-miR396a-5p target genes, NtGRF1, NtGRF3, NtGRF7 and NtGRF8, were down-regulated in these plants. Our results suggested that Sp-miR396a-5p plays critical roles in both abiotic stresses through targeting NtGRF7-regulated expression of osmotic stress-responsive genes and pathogen infection via the regulatory networks of NtGRF1 and NtGRF3.

Overexpression of a Miscanthus lutarioriparius NAC gene MlNAC5 confers enhanced drought and cold tolerance in Arabidopsis.

Abstract: MLNAC5 functions as a stress-responsive NAC transcription factor gene and enhances drought and cold stress tolerance in transgenic Arabidopsis via the ABA-dependent signaling pathway. NAC transcription factors (TFs) play crucial roles in plant responses to abiotic stress. Miscanthus lutarioriparius is one of Miscanthus species native to East Asia. It has attracted much attention as a bioenergy crop because of its superior biomass productivity as well as wide adaptability to different environments. However, the functions of stress-related NAC TFs remain to be elucidated in M. lutarioriparius. In this study, a detailed functional characterization of MlNAC5 was carried out. MlNAC5 was a member of ATAF subfamily and it showed the highest sequence identity to ATAF1. Subcellular localization of MlNAC5-YFP fusion protein in tobacco leaves indicated that MlNAC5 is a nuclear protein. Transactivation assay in yeast cells demonstrated that MlNAC5 functions as a transcription activator and its activation domain is located in the C-terminus. Overexpression of MlNAC5 in Arabidopsis had impacts on plant development including dwarfism, leaf senescence, leaf morphology, and late flowering under normal growth conditions. Furthermore, MlNAC5 overexpression lines in Arabidopsis exhibited hypersensitivity to abscisic acid (ABA) and NaCl. Moreover, overexpression of MlNAC5 in Arabidopsis significantly enhanced drought and cold tolerance by transcriptionally regulating some stress-responsive marker genes. Collectively, our results indicated that MlNAC5 functions as an important regulator during the process of plant development and responses to salinity, drought and cold stresses.

Activated expression of AtWRKY53 negatively regulates drought tolerance by mediating stomatal movement.

Abstract: Key message AtWRKY53 is an early factor in drought response and activated expression of AtWRKY53 reg- ulates stomatal movement via reduction of H2O2 con- tent and promotion of starch metabolism in guard cells. Abstract Drought is one of the most serious environ- mental factors limiting the productivity of agricultural crops worldwide. However, the mechanisms underlying drought tolerance in plants remain unclear. AtWRKY53 belongs to the group III of WRKY transcription factors. In this study, we observed both the mRNA and protein products of this gene are rapidly induced under drought conditions. Phenotypic analysis showed AtWRKY53 over- expression lines were hypersensitive to drought stress compared with Col-0 plants. The results of stomatal movement assays and abscisic acid (ABA) content detec- tion indicated that the impaired stomatal closure of 53OV lines was independent of ABA. Further analysis found that WRKY53 regulated stomatal movement via reducing the H2O2 content and promoting the starch metabolism in guard cells. The results of quantitative real-time reverse transcriptase PCR showed that the expression levels of CAT2, CAT3 and QQS were up-regulated in 53OV lines. Chromatin immunoprecipitation assays demonstrated that AtWRKY53 can directly bind to the QQS promoter se- quences, thus led to increased starch metabolism. In sum- mary, our results indicated that the activated expression of AtWRKY53 inhibited stomatal closure by reducing H2O2 content and facilitated stomatal opening by promoting starch degradation.

Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice

Abstract: Frequency of CRISPR/Cas9-mediated targeted mutagenesis varies depending on Cas9 expression level and culture period of rice callus. Recent reports have demonstrated that the CRISPR/Cas9 system can function as a sequence-specific nuclease in various plant species. Induction of mutation in proliferating tissue during embryogenesis or in germline cells is a practical means of generating heritable mutations. In the case of plant species in which cultured cells are used for transformation, non-chimeric plants can be obtained when regeneration occurs from mutated cells. Since plantlets are regenerated from both mutated and non-mutated cells in a random manner, any increment in the proportion of mutated cells in Cas9- and guide RNA (gRNA)-expressing cells will help increase the number of plants containing heritable mutations. In this study, we examined factors affecting mutation frequency in rice calli. Following sequential transformation of rice calli with Cas9- and gRNA- expression constructs, the mutation frequency in independent Cas9 transgenic lines was analyzed. A positive correlation between Cas9 expression level and mutation frequency was found. This positive relationship was observed regardless of whether the transgene or an endogenous gene was used as the target for CRISPR/Cas9-mediated mutagenesis. Furthermore, we found that extending the culture period increased the proportion of mutated cells as well as the variety of mutations obtained. Because mutated and non-mutated cells might proliferate equally, these results suggest that a prolonged tissue culture period increases the chance of inducing de novo mutations in non-mutated cells. This fundamental knowledge will help improve systems for obtaining non-chimeric regenerated plants in many plant species.

The Brachypodium distachyon BdWRKY36 gene confers tolerance to drought stress in transgenic tobacco plants.

Abstract: The expression of BdWRKY36 was upregulated by drought treatment. BdWRKY36 -overexpressing transgenic tobacco increased drought tolerance by controlling ROS homeostasis and regulating transcription of stress related genes. WRKY transcription factor plays important roles in plant growth, development and stress response. However, the function of group IIe WRKYs is less known. In this study, we cloned and characterized a gene of group IIe WRKY, designated as BdWRKY36, from Brachypodium distachyon. Transient expression of BdWRKY36 in onion epidermal cell suggested its localization in the nucleus. Transactivation assay revealed that the C-terminal region, instead of full length BdWRKY36, had transcriptional activity. BdWRKY36 expression was upregulated by drought. Overexpression of BdWRKY36 in transgenic tobacco plants resulted in enhanced tolerance to drought stress. Physiological–biochemical indices analyses showed that BdWRKY36-overexpressing tobacco lines had lesser ion leakage (IL) and reactive oxygen species (ROS) accumulation, but higher contents of chlorophyll, relative water content (RWC) and activities of antioxidant enzyme than that in control plants under drought condition. Meanwhile expression levels of some ROS-scavenging and stress-responsive genes were upregulated in BdWRKY36-overexpressing tobacco lines under drought stress. These results demonstrate that BdWRKY36 functions as a positive regulator of drought stress response by controlling ROS homeostasis and regulating transcription of stress related genes.

PtrWRKY73, a salicylic acid-inducible poplar WRKY transcription factor, is involved in disease resistance in Arabidopsis thaliana

Abstract: A salicylic acid-inducible WRKY gene, PtrWRKY73, from Populus trichocarpa , was isolated and characterized. Overexpression of PtrWRKY73 in Arabidopsis thaliana increased resistance to biotrophic pathogens but reduced resistance against necrotrophic pathogens. WRKY transcription factors are commonly involved in plant defense responses. However, limited information is available about the roles of the WRKY genes in poplar defense. In this study, we isolated a salicylic acid (SA)-inducible WRKY gene, PtrWRKY73, from Populus trichocarpa, belonging to group I family and containing two WRKY domains, a D domain and an SP cluster. PtrWRKY73 was expressed predominantly in roots, old leaves, sprouts and stems, especially in phloem and its expression was induced in response to treatment with exogenous SA. PtrWRKY73 was localized to the nucleus of plant cells and exhibited transcriptional activation. Overexpression of PtrWRKY73 in Arabidopsis thaliana resulted in increased resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae (PstDC3000), but more sensitivity to the necrotrophic fungal pathogen Botrytis cinerea. The SA-mediated defense-associated genes, such as PR1, PR2 and PAD4, were markedly up-regulated in transgenic plants overexpressing PtrWRKY73. Arabidopsis non-expressor of PR1 (NPR1) was not affected, whereas a defense-related gene PAL4 had reduced in PtrWRKY73 overexpressor plants. Together, these results indicated that PtrWRKY73 plays a positive role in plant resistance to biotrophic pathogens but a negative effect on resistance against necrotrophic pathogens.

ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox

Abstract: Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 ·− , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA–GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

Overexpression of CuZnSOD from Arachis hypogaea alleviates salinity and drought stress in tobacco

Abstract: Overexpression of CuZnSOD gene from Arachis hypogaea demonstrating its involvement in abiotic stress tolerance. Abiotic stress is accompanied by the formation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radicals, causing extensive cellular damage and inhibition of photosynthesis that limit the plant productivity. The level of ROS in cells needs to be tightly regulated and the toxic effects of ROS are countered by enzymatic as well as non-enzymatic antioxidant systems. The superoxide dismutase is the first enzyme involved in the detoxification of ROS and converts superoxide (O 2 ·− ) radicals to H2O2. A full-length cDNA clone encoding a CuZnSOD, named AhCuZnSOD, was isolated from the salt tolerant cell lines of Arachis hypogaea, stably thriving at 200 mM NaCl. The cell line showed higher transcript accumulation under multiple abiotic stresses, including drought, salinity, cold and oxidative stress treatment. The functional role of AhCuZnSOD in alleviation of abiotic stress was assessed by its overexpression in transgenic tobacco plants. The T1 transgenic plants showed improved tolerance to salinity and dehydration stress as indicated by higher seed germination and better chlorophyll content. The transgenic plants survived under longer periods of water deficiency and salinity stress and displayed improved recovery after rehydration compared to the wild type (WT) plants. The enhanced level of the transgene correlated with higher relative water content, less electrolyte damage, less malondialdehyde, higher antioxidant enzyme activity, H2O2 and O 2 ·− accumulation under stress conditions compared to WT plants. Our results substantiate that increased levels of SOD activity brought about by overexpression of AhCuZnSOD gene may play an important role in ameliorating oxidative injury induced by various environmental stresses.

Chrysanthemum WRKY gene CmWRKY17 negatively regulates salt stress tolerance in transgenic chrysanthemum and Arabidopsis plants

Abstract: CmWRKY17 was induced by salinity in chrysanthemum, and it might negatively regulate salt stress in transgenic plants as a transcriptional repressor. WRKY transcription factors play roles as positive or negative regulators in response to various stresses in plants. In this study, CmWRKY17 was isolated from chrysanthemum (Chrysanthemum morifolium). The gene encodes a 227-amino acid protein and belongs to the group II WRKY family, but has an atypical WRKY domain with the sequence WKKYGEK. Our data indicated that CmWRKY17 was localized to the nucleus in onion epidermal cells. CmWRKY17 showed no transcriptional activation in yeast; furthermore, luminescence assay clearly suggested that CmWRKY17 functions as a transcriptional repressor. DNA-binding assay showed that CmWRKY17 can bind to W-box. The expression of CmWRKY17 was induced by salinity in chrysanthemum, and a higher expression level was observed in the stem and leaf compared with that in the root, disk florets, and ray florets. Overexpression of CmWRKY17 in chrysanthemum and Arabidopsis increased the sensitivity to salinity stress. The activities of superoxide dismutase and peroxidase and proline content in the leaf were significantly lower in transgenic chrysanthemum than those in the wild type under salinity stress, whereas electrical conductivity was increased in transgenic plants. Expression of the stress-related genes AtRD29, AtDREB2B, AtSOS1, AtSOS2, AtSOS3, and AtNHX1 was reduced in the CmWRKY17 transgenic Arabidopsis compared with that in the wild-type Col-0. Collectively, these data suggest that CmWRKY17 may increase the salinity sensitivity in plants as a transcriptional repressor.

Constitutive expression of Arabidopsis MYB transcription factor, AtMYB11, in tobacco modulates flavonoid biosynthesis in favor of flavonol accumulation

Abstract: Heterologous expression of AtMYB11 , a flavonol-specific transcription factor from Arabidopsis , in tobacco modulates flavonoid biosynthesis, however, with a lower efficiency as compared to its paralogs AtMYB12 and AtMYB111. Transcriptional regulation is the most important means for controlling flavonoid biosynthesis under temporal and spatial cues. In Arabidopsis, three functionally redundant MYB transcription factors (AtMYB11, AtMYB111 and AtMYB12) have been characterized as flavonol-specific regulators which positively modulate expression of biosynthetic genes involved in flavonol biosynthesis. Based on expression of AtMYB111 and AtMYB12 in heterologous systems, studies suggest that these transcription factors can be used to develop plants with enhanced flavonol biosynthesis. The potential of AtMYB11 to activate flavonol biosynthesis in a heterologous system has not yet been studied. In this study, the regulatory potential of AtMYB11 has been studied in Nicotiana tabacum by developing transgenic plants constitutively expressing AtMYB11. Our analysis using leaf and petal tissues of the transgenic plants indicates that AtMYB11 enhances flavonol and chlorogenic acid (CGA) biosynthesis in tobacco through up-regulation of the biosynthetic genes. Activation of flavonol biosynthesis in tobacco by AtMYB11 is not as pronounced as with AtMYB12 or AtMYB111. Taken together, these results reveal a differential regulatory mechanism in plants for modulating flavonol biosynthesis. This study demonstrated that AtMYB11 can be strategically used for enhancing the health beneficial flavonols in species other than Arabidopsis.

Role of HXXXD-motif/BAHD acyltransferases in the biosynthesis of extracellular lipids

Abstract: Terrestrial plants have evolved specific adaptations to preserve water and protect themselves from their environment. Such adaptations range from secondary metabolites and specialized structures that conduct water and nutrients, to cell wall modifications (i.e., cuticle and suberin) that prevent dehydration and provide a physical barrier to pathogens. Both the plant cuticle and suberized cell walls contain a lipid polymer framework embedded with waxes, and constitute a promising target for controlled genetic modification to improve desirable agronomic traits. Recent advances in genomic and molecular techniques coupled with the development of robust analytical methods have accelerated progress in comprehending these intractable lipid polymers. Gene products characterized in the wax, cutin and suberin pathways include a subset of HXXXD/BAHD family enzymes that catalyze acyl transfer reactions between CoA-activated hydroxycinnamic acid derivatives and hydroxylated aliphatics. This review highlights our current understanding of HXXXD/BAHD acyltransferases in extracellular lipid biosynthesis and discusses the chemical, ultrastructural and physiological ramifications of impairing the expression of BAHD acyltransferase-encoding genes related to cutin and suberin synthesis.

Identification of TaWD40D, a wheat WD40 repeat-containing protein that is associated with plant tolerance to abiotic stresses.

Abstract: TaWD40D that encodes a member of WD40 family proteins is a novel gene involved in the wheat response to abiotic stress. TaWD40D functions as a positive regulator of plant responses to salt stress and osmotic stress in plant. Abiotic stresses can severely affect plant growth and crop productivity. WD40 repeat-containing proteins play a key role in protein–protein or protein–DNA interactions by acting as scaffolding molecules and promoting protein activity. In this study, a stress-inducible gene, TaWD40D, was identified from Chinese spring wheat (Triticum aestivum L.). TaWD40D encodes a protein containing seven WD40 domains. Subcellular localization in Nicotiana benthamiana mesophyll cells and Arabidopsis root cells showed the presence of TaWD40D in the cytoplasm and nucleus. Heterologous overexpression of TaWD40D in Arabidopsis greatly increased plant tolerance to abscisic acid (ABA), salt stress, and osmotic stress during seed germination and seedling development. The expression patterns of two genes from the SOS pathway (SOS2 and SOS3) and three ABA genes (ABI2, RAB18 and DREB2A) functioning in ABA-dependent and ABA-independent pathways were altered in the transgenic lines overexpressing TaWD40D under the treatments. Notably, the basal level of the ABI2 expression was substantially increased in the TaWD40D overexpression lines. The down-regulation of TaWD40D in wheat by virus-induced gene silencing resulted in a decreased relative water content and less vigorous growth compared to non-silenced lines. Our results suggest that TaWD40D functions as a positive regulator of plant responses to salt stress and osmotic stress that could be utilized for the genetic improvement of stress tolerance in crop plants.

Dendrobium micropropagation: a review

Abstract: Dendrobium is one of the largest and most important (ornamentally and medicinally) orchid genera. Tissue culture is now an established method for the effective propagation of members of this genus. This review provides a detailed overview of the Dendrobium micropropagation literature. Through a chronological analysis, aspects such as explant, basal medium, plant growth regulators, culture conditions and final organogenic outcome are chronicled in detail. This review will allow Dendrobium specialists to use the information that has been documented to establish, more efficiently, protocols for their own germplasm and to improve in vitro culture conditions based on the optimized parameters detailed in this review. Not only will this expand the use for mass propagation, but will also allow for the conservation of important germplasm. Information on the in vitro responses of Dendrobium for developing efficient protocols for breeding techniques based on tissue culture, such as polyploidization, somatic hybridization, isolation of mutants and somaclonal variants and for synthetic seed and bioreactor technology, or for genetic transformation, is discussed in this review. This is the first such review on this genus and represents half a decade of literature dedicated to Dendrobium micropropagation.

The AtLRK10L1.2, Arabidopsis ortholog of wheat LRK10, is involved in ABA-mediated signaling and drought resistance.

Abstract: Key message The loss-of-function mutants of theArabidopsisorthologue of the wheatLRK10gene shows ABA-insensitive and drought stress-sensitive phenotypes, suggesting that LRK10L1.2 is positively involved in ABA signaling.

Expression differences of anthocyanin biosynthesis genes reveal regulation patterns for red pear coloration

Abstract: Key message This research reveals the different expression patterns of anthocyanin biosynthesis enzyme genes and transcription factors in six red-skinned pear cultivars with different genetic backgrounds.

Methane-rich water induces cucumber adventitious rooting through heme oxygenase1/carbon monoxide and Ca(2+) pathways.

Abstract: Methane-rich water triggered adventitious rooting by regulating heme oxygenase1/carbon monoxide and calcium pathways in cucumber explants. Heme oxygenase1/carbon monoxide (HO1/CO) and calcium (Ca2+) were reported as the downstream signals in auxin-induced cucumber adventitious root (AR) formation. Here, we observed that application of methane-rich water (MRW; 80 % saturation) obviously induced AR formation in IAA-depleted cucumber explants. To address the universality, we checked adventitious rooting in soybean and mung bean explants, and found that MRW (50 and 10 % saturation, respectively) exhibited the similar inducing results. To further determine if the HO1/CO system participated in MRW-induced adventitious rooting, MRW, HO1 inducer hemin, its activity inhibitor zinc protoporphyrin IX (ZnPP), and its catalytic by-products CO, bilirubin, and Fe2+ were used to detect their effects on cucumber adventitious rooting in IAA-depleted explants. Subsequent results showed that MRW-induced adventitious rooting was blocked by ZnPP and further reversed by 20 % saturation CO aqueous solution. However, the other two by-products of HO1, bilirubin and Fe2+, failed to induce AR formation. Above responses were consistent with the MRW-induced increases of HO1 transcript and corresponding protein level. Further molecular evidence indicted that expression of marker genes, including auxin signaling-related genes and cell cycle regulatory genes, were modulated by MRW alone but blocked by the cotreatment with ZnPP, the latter of which could be significantly rescued by the addition of CO. By using the Ca2+-channel blocker and Ca2+ chelator, the involvement of Ca2+ pathway in MRW-induced adventitious rooting was also suggested. Together, our results indicate that MRW might serve as a stimulator of adventitious rooting, which was partially mediated by HO1/CO and Ca2+ pathways.

Development of high oleic oil crop platform in flax through RNAi-mediated multiple FAD2 gene silencing

Abstract: Simultaneous gene silencing of both FAD2 genes in high linoleic acid flax leads to high level of oleic acid, which is stable across multiple generations. High oleic oil is one of the preferred traits in oil crop engineering due to its stability and multiple applications as an industrial feedstock. Flax possesses two isoforms of FAD2 enzymes that desaturate monounsaturated oleic acid to polyunsaturated linoleic acid. These two enzymes are encoded by two FAD2 genes. By simultaneous gene silencing both FAD2 genes in high linoleic acid flax, Linola, high level of oleic acid up to 80 % was achieved in 69 silencing lines. The high oleic trait was stable across multiple generations with oleic acid reaching up to 77 % in homozygote T3 progeny. The RNAi-mediated gene-silencing approach generated high oleic linseed oil, as well as a high oleic platform that can be exploited for further fatty acid engineering.

Arabidopsis AtERF71/HRE2 functions as transcriptional activator via cis-acting GCC box or DRE/CRT element and is involved in root development through regulation of root cell expansion.

Abstract: Key message AtERF71/HRE2 binds to GCC box or DRE/CRT as transcription activator and plays an important role in root development via root cell expansion regulation.

Transcriptional feedback regulation of YUCCA genes in response to auxin levels in Arabidopsis

Abstract: The IPyA pathway, the major auxin biosynthesis pathway, is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels. The phytohormone auxin plays an important role in plant growth and development, and levels of active free auxin are determined by biosynthesis, conjugation, and polar transport. Unlike conjugation and polar transport, little is known regarding the regulatory mechanism of auxin biosynthesis. We discovered that expression of genes encoding indole-3-pyruvic acid (IPyA) pathway enzymes is regulated by elevated or reduced active auxin levels. Expression levels of TAR2, YUC1, YUC2, YUC4, and YUC6 were downregulated in response to synthetic auxins [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] exogenously applied to Arabidopsis thaliana L. seedlings. Concomitantly, reduced levels of endogenous indole-3-acetic acid (IAA) were observed. Alternatively, expression of these YUCCA genes was upregulated by the auxin biosynthetic inhibitor kynurenine in Arabidopsis seedlings, accompanied by reduced IAA levels. These results indicate that expression of YUCCA genes is regulated by active auxin levels. Similar results were also observed in auxin-overproduction and auxin-deficient mutants. Exogenous application of IPyA to Arabidopsis seedlings preincubated with kynurenine increased endogenous IAA levels, while preincubation with 2,4-D reduced endogenous IAA levels compared to seedlings exposed only to IPyA. These results suggest that in vivo conversion of IPyA to IAA was enhanced under reduced auxin levels, while IPyA to IAA conversion was depressed in the presence of excess auxin. Based on these results, we propose that the IPyA pathway is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels.

Downregulation of transcription factor aflR in Aspergillus flavus confers reduction to aflatoxin accumulation in transgenic maize with alteration of host plant architecture

Abstract: We report success of host-induced gene silencing in downregulation of aflatoxin biosynthesis in Aspergillus flavus infecting maize transformed with a hairpin construct targeting transcription factor aflR. Infestation of crops by aflatoxin-producing fungi results in economic losses as well as negative human and animal health effects. Currently, the control strategies against aflatoxin accumulation are not effective to the small holder farming systems in Africa and this has led to widespread aflatoxin exposure especially in rural populations of sub-Saharan Africa that rely on maize as a staple food crop. A recent strategy called host-induced gene silencing holds great potential for developing aflatoxin-resistant plant germplasm for the African context where farmers are unable to make further investments other than access to the germplasm. We transformed maize with a hairpin construct targeting the aflatoxin biosynthesis transcription factor aflR. The developed transgenic maize were challenged with an aflatoxigenic Aspergillus flavus strain from Eastern Kenya, a region endemic to aflatoxin outbreaks. Our results indicated that aflR was downregulated in A. flavus colonizing transgenic maize. Further, maize kernels from transgenic plants accumulated significantly lower levels of aflatoxins (14-fold) than those from wild type plants. Interestingly, we observed that our silencing cassette caused stunting and reduced kernel placement in the transgenic maize. This could have been due to “off-target” silencing of unintended genes in transformed plants by aflR siRNAs. Overall, this work indicates that host-induced gene silencing has potential in developing aflatoxin-resistant germplasm.

DREB1A overexpression in transgenic chickpea alters key traits influencing plant water budget across water regimes.

Abstract: We demonstrate the role of DREB1A transcription factor in better root and shoot partitioning and higher transpiration efficiency in transgenic chickpea under drought stress Chickpea (Cicer arietinum L.) is mostly exposed to terminal drought stress which adversely influences its yield. Development of cultivars for suitable drought environments can offer sustainable solutions. We genetically engineered a desi-type chickpea variety to ectopically overexpress AtDREB1A, a transcription factor known to be involved in abiotic stress response, driven by the stress-inducible Atrd29A promoter. From several transgenic events of chickpea developed by Agrobacterium-mediated genetic transformation, four single copy events (RD2, RD7, RD9 and RD10) were characterized for DREB1A gene overexpression and evaluated under water stress in a biosafety greenhouse at T6 generation. Under progressive water stress, all transgenic events showed increased DREB1A gene expression before 50 % of soil moisture was lost (50 % FTSW or fraction of transpirable soil water), with a faster DREB1A transcript accumulation in RD2 at 85 % FTSW. Compared to the untransformed control, RD2 reduced its transpiration in drier soil and higher vapor pressure deficit (VPD) range (2.0–3.4 kPa). The assessment of terminal water stress response using lysimetric system that closely mimics the soil conditions in the field, showed that transgenic events RD7 and RD10 had increased biomass partitioning into shoot, denser rooting in deeper layers of soil profile and higher transpiration efficiency than the untransformed control. Also, RD9 with deeper roots and RD10 with higher root diameter showed that the transgenic events had altered rooting pattern compared to the untransformed control. These results indicate the implicit influence of rd29A::DREB1A on mechanisms underlying water uptake, stomatal response, transpiration efficiency and rooting architecture in water-stressed plants.

Biotechnology of oil palm: strategies towards manipulation of lipid content and composition.

Abstract: Oil palm is a major economic crop for Malaysia. The major challenges faced by the industry are labor shortage, availability of arable land and unstable commodity price. This has caused the industry to diversify its applications into higher value products besides increasing its yield. While conventional breeding has its limitations, biotechnology was identified as one of the tools for overcoming the above challenges. Research on biotechnology of oil palm began more than two decades ago leveraging a multidisciplinary approach involving biochemical studies, gene and promoter isolation, transformation vector construction and finally genetic transformation to produce the targeted products. The main target of oil palm biotechnology research is to increase oleic acid in the mesocarp. Other targets are stearic acid, palmitoleic acid, ricinoleic acid, lycopene (carotenoid) and biodegradable plastics. Significant achievements were reported for the biochemical studies, isolation of useful oil palm genes and characterization of important promoters. A large number of transformation constructs for various targeted products were successfully produced using the isolated oil palm genes and promoters. Finally transformation of these constructs into oil palm embryogenic calli was carried out while the regeneration of transgenic oil palm harboring the useful genes is in progress.