Overexpression of tomato GDP-l-galactose phosphorylase gene in tobacco improves tolerance to chilling stress

Key message

The overexpression of tomato GDP- l -galactose phosphorylase gene enhanced tolerance to chilling stress and reduced photoinhibition of photosystems I and II in transgenic tobacco.

Abstract

Chilling stress is a crucial factor that limits the geographical distribution and yield of chilling-sensitive plants. Ascorbate (AsA) protects plants by scavenging reactive oxygen species and reduces photoinhibition by promoting the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle to dissipate excess excitation energy. Possible mechanisms of AsA for plant photoprotection under chilling stress were investigated by isolating the tomato GDP-l-galactose phosphorylase gene (SlGGP) and producing transgenic tobacco plants with overexpression of SlGGP. The transgenic plants subjected to chilling stress accumulated less H₂O₂, demonstrated lower levels of ion leakage and malondialdehyde, and acquired higher net photosynthetic rate, higher maximum photochemical efficiency of PSII, and higher D1 protein content compared with the wild-type (WT) plants. The transgenic plants subjected to chilling stress also showed higher GDP-l-galactose phosphorylase activity, increased AsA content as well as ascorbate peroxidase and oxidizable P700 activities than WT plants. Thus, SlGGP overexpression is crucial in promoting AsA synthesis and alleviating photoinhibition of two photosystems.     

Functional analysis of the promoters of B-class MADS-box genes in London plane tree and their application in genetic engineering of sterility

Breeding flowerless and/or fruitless varieties are highly desirable for London plane tree because it can prevent pollen- and fruit-mediated environmental contamination. Floral tissue-specific cell ablation is an efficient method to create such sterile plants. Here we isolated and characterized APETALA3 (AP3)-like and PISTILLATA (PI)-like genes and the promoters of PaAP3 and PaPI, in London plane tree respectively. The promoter fragments were fused to GUS (β-glucuronidase) and BARNASE gene, respectively, and transformed into tobacco plants. In pPaAP3::GUS transgenic plants, the GUS activity could be detected in various organs, including leaves, stems and all floral organs. Furthermore, most tobacco plants transformed with pPaAP3::BARNASE were dead and the survivals showed abortion of inflorescence. In contrast, heterologous expression of pPaPI::GUS in tobacco plants led to specific GUS activity in the inner three whorls of flowers. Accordingly, tobacco plants transformed with pPaPI::BARNASE lack petal, stamen and pistil, with only sepal left. The results suggest that sterile lines of P. acerifolia may be obtained by genetic engineering with pPaPI::BARNASE construct, which might solve the problems of shedding fruit hairs and disseminative pollens, reducing air pollution and reducing the allergens that harmful to human health.     

Mutation of mpk6 enhances cadmium tolerance in Arabidopsis plants by alleviating oxidative stress

Background and aims

Cadmium (Cd) could activate activity of mitogen-activated protein kinase MPK6 in plants. In this study, we investigated the role of MPK6 in mediating Cd toxicity in plants.

Methods

The wild type Arabidopsis plants (WT) and the mpk6-2 mutants were subjected either 0 (Control) or 10 μM Cd treatment. Kinase activity of MPK6, nitric oxide (NO) level, Cd concentration, and oxidative stress were measured.

Results

In WT plants, Cd exposure rapidly stimulated kinase activity of MPK6. However, upon Cd exposure, mpk6-2 showed better growth than the WT. Although Cd-induced production of NO in roots was greater in WT than in mpk6-2, there was no difference in Cd concentration between the two plants. Nevertheless, the Cd-induced hydroperoxide burst, lipid peroxidation and loss of membrane integrity, were all more severe in the WT than in mpk6-2. Foliar applications of antioxidant ascorbic acid, vigorously improved the growth of both the WT and mpk6-2 under Cd exposure. Thereby the growth difference between these two plants was minimized.

Conclusions

Mutation of mpk6 enhances Cd tolerance in plants by alleviating oxidative stress, but did not affect cadmium accumulation in plants.     

Biochar application to a fertile sandy clay loam in boreal conditions: effects on soil properties and yield formation of wheat, turnip rape and faba bean

Background and aims

We studied the effect of different biochar (BC) application rates on soil properties, crop growth dynamics and yield on a fertile sandy clay loam in boreal conditions.

Methods

In a three-year field experiment conducted in Finland, the field was divided into three sub-experiments with a split-plot experimental design, one for each crop: wheat (Triticum aestivum), turnip rape (Brassica rapa), and faba bean (Vicia faba). The main plot factor was BC rate (0, 5 and 10 t DM ha^?1) and the sub-plot factor was the N-P-K fertiliser rate. Soil physico-chemical properties as well as plant development, yield components and quality were investigated.

Results

BC addition did not significantly affect the soil chemical composition other than the increased C and initially increased K contents. Increased soil moisture content was associated with BC application, especially at the end of the growing seasons. BC decreased the N content of turnip rape and wheat biomass in 2010, thus possibly indicating an initial N immobilisation. In dry years, the seed number per plant was significantly higher in faba bean and turnip rape when grown with BC, possibly due to compensation for decreased plant density and relieved water deficit. However, the grain yields and N uptake with BC addition were not significantly different from the control in any year.

Conclusions

Even though BC application to a fertile sandy clay loam in a boreal climate might have relieved transient water deficit and thereby supported yield formation of crops, it did not improve the yield or N uptake.     

Silencing NaTPI Expression Increases Nectar Germin,Nectarins, and Hydrogen Peroxide Levels and Inhibits Nectar Removal from Plants in Nature

Native flower visitors removed less nectar from trypsin proteinase inhibitor (TPI)-silenced Nicotiana attenuata plants (ir-pi) than from wild-type plants in four field seasons of releases, even when the nectar repellant, nicotine, was also silenced. Analysis of floral chemistry revealed no differences in the emission of the floral attractants benzylacetone and benzaldehyde or in the concentrations of nectar sugar and nicotine between wild-type and ir-pi flowers, suggesting that these two lines are equally able to attract insect visitors. TPI activity was found in all wild-type flower parts and was highest in anther heads, while TPI activity was not found in any parts of ir-pi flowers. The nectar of ir-pi flowers contained 3.6-fold more total proteins than the nectar of wild-type flowers. Proteomics analysis and hydrogen peroxide (H₂O₂) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins and about 1.5-fold more H₂O₂ compared with wild-type nectar. Field experiments with wild-type flowers supplemented with a solution containing sugar and glucose oxidase demonstrated a causal association between the accumulation of H₂O₂ and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H₂O₂ levels, which in turn reduces nectar removal by native insect floral visitors. The effect of silencing TPIs on nectar protein accumulation suggests an endogenous regulatory function for TPIs in N. attenuata flowers. The repellency of H₂O₂ to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators.Floral nectar is an innovative feature of plants that is thought to have evolved as a reward for pollen-transporting floral visitors. Sugars (e.g. Glc, Fru, and Suc), amino acids, and lipids (Baker and Baker, 1982, 1986) provide nutritional rewards that are essential for many pollinators. But nectar is also known to contain other compounds, such as volatile organic compounds (VOCs), alkaloids, phenolics, and nonprotein amino acids (Baker, 1977, 1978; Raguso, 2004; Kessler and Baldwin, 2007), which do not increase the nutritional value of nectar. Nectar is also exploited as a food source by nectar robbers and nectar-infesting microorganisms, which do not provide mutualistic services to the plant and are known to directly reduce a plant''s fitness either by competing with pollinators or by infesting reproductive organs (Traveset et al., 1998; Irwin and Brody, 1999; Maloof and Inouye, 2000; Farkas et al., 2007). Therefore, flowers must solve the dilemma of repelling nectar thieves or florivores that provide no pollination services while simultaneously attracting fitness-enhancing pollinators.Most of the defensive compounds in nectar have been reported to act selectively (i.e. only on antagonists). For example, the floral nectar of Catalpa speciosa contains iridoid glycosides that fend off nectar robbers but not the plant''s specific pollinators (Stephenson, 1981). Similarly, the presence of phenols in the floral nectar of Aloe vryheidensis lowers its palatability to generalist floral visitors like sunbirds or honey bees while not affecting the attractiveness of the nectar to a specialist bird, the dark-capped bulbul (Johnson et al., 2006). In its native habitat, Nicotiana attenuata (Solanaceae) maximizes its maternal and paternal reproductive success while repelling herbivores, florivores, and nectar robbers by producing a sophisticated blend of both repellants (nicotine) and attractants (benzylacetone) in its nectar and floral head space (Kessler et al., 2008) as well as by changing its floral phenology in response to herbivore attack, so as to switch from the use of night-active hawkmoth pollinators, which oviposit herbivores on the plants they pollinate, to day-active hummingbird pollinators, which do not (Kessler et al., 2010). While this sophisticated use of chemical attractants and repellants is likely a common solution to the dilemma, very little is known about the function of most secondary metabolites found in nectar (Thornburg, 2007).Similar chemically mediated strategies are used to solve a similar problem when plants use a combination of direct and indirect defenses to protect their leaves from herbivore attack (Halitschke et al., 2008). In N. attenuata, attack by the specialist herbivore Manduca sexta elicits a remarkable array of direct and indirect defenses, most of which are elicited by the jasmonate signaling pathway in response to herbivore-specific elicitors (Baldwin, 2001; Kessler and Baldwin, 2002; Wu and Baldwin, 2009). These herbivory-elicited responses include the accumulation of toxins and digestibility reducers, which function as direct defenses, as well as the release of a complicated blend of VOCs (Gaquerel et al., 2009), which repel further oviposition by M. sexta moths and attract predacious bugs that feed on M. sexta eggs or larvae, thereby functioning as an indirect defense (Kessler and Baldwin, 2001).Once herbivores start feeding on N. attenuata leaves, they are frequently repelled by a suite of locally and systemically elicited direct defenses (Steppuhn et al., 2008). Trypsin protease inhibitors (TPIs) are an effective component of this inducible defensive system that reduces the performance of folivores by targeting their main proteolytic digestive enzymes and is strongly induced by herbivore attack (van Dam et al., 2000; Glawe et al., 2003; Zavala et al., 2004b, 2008; Horn et al., 2005). However, in N. attenuata, the biosynthesis of TPIs incurs substantial fitness costs (Zavala et al., 2004a); silencing the TPI gene in N. attenuata abolishes the plant''s capacity to produce TPIs and allows it to grow faster, flower earlier, and produce more seed capsules compared with TPI-producing genotypes (Zavala et al., 2004a). Similarly, restoring TPI production by transforming an ecotype of N. attenuata naturally deficient in TPI production (Wu et al., 2007) reduces lifetime seed production (Zavala et al., 2004a). TPIs are not only restricted to leaves but accumulates in reproductive organs, where they may protect these fitness-valuable tissues against attack from florivores and microbes. Atkinson et al. (1993) and Johnson et al. (2007) elegantly demonstrated that TPIs dramatically accumulate in Nicotiana alata stigmas to become the most abundant protein in these tissues. PIs have been reported to accumulate in Solanum americanum seeds, where they were shown to play an important role in seed development (Suk-Fong et al., 2006). These studies highlight that while it is clear that TPIs occur at high levels in reproductive organs, their role in floral function has not been thoroughly explored.As part of a research program to study the defensive functions of nicotine and TPIs against folivores, we planted N. attenuata plants that had been transformed with RNA interference constructs to silence their nicotine (ir-pmt), TPI (ir-pi), or both (ir-pmt/pi) in the plant''s native habitats in Utah during four field seasons. Serendipitously, we noticed that the amount of nectar removed by the native community of floral visitors from ir-pmt/pi plants did not differ from that removed from wild-type plants, although we had recently discovered that silencing nicotine alone (ir-pmt) consistently increased nectar removal (Kessler and Baldwin, 2007). These observations suggested that silencing TPIs alone might impede nectar removal by the native community of floral visitors. During two field seasons (2007 and 2009), we compared the amount of nectar removed from wild-type plants and from TPI-silenced plants (ir-pi) and found that, indeed, less nectar was consistently removed from ir-pi plants. To understand these observations, we compared the floral chemistry of wild-type and ir-pi plants, including floral volatiles, nectar sugar, nicotine, and proteomes. We found that silencing TPIs increased the accumulation of nectar proteins, especially the nectar germin-like proteins (GLPs) and nectarins, which are known to participate in the nectar redox cycle and generate hydrogen peroxide (H₂O₂; Carter and Thornburg, 2000). Consistent with data on nectar proteins, we also found significantly higher levels of H₂O₂ in the nectar of ir-pi plants compared with those of wild-type plants. To test whether the differences in the accumulation of H₂O₂ in the nectar of ir-pi and wild-type plants could explain the nectar removal observations in the field, we experimentally increased H₂O₂ in the nectar of wild-type plants to the levels found in ir-pi nectar using a mixture of Glc oxidase (GOX) and Glc and compared nectar removal by the native community of floral visitors.     

Increased gibberellin contents contribute to accelerated growth and development of transgenic tobacco overexpressing a wheat ubiquitin gene

Key message

Overexpressing TaUb2 promoted stem growth and resulted in early flowering in transgenic tobacco plants. Ubiquitin are involved in the production, metabolism and proper function of gibberellin.

Abstract

The ubiquitin–26S proteasome system (UPS), in which ubiquitin (Ub) functions as a marker, is a post-translational regulatory system that plays a prominent role in various biological processes. To investigate the impact of different Ub levels on plant growth and development, transgenic tobacco (Nicotiana tabacum L.) plants were engineered to express an Ub gene (TaUb2) from wheat (Triticum aestivum L.) under the control of cauliflower mosaic virus 35S promoter. Transgenic tobacco plants overexpressing TaUb2 demonstrated an accelerated growth rate at early stage and an early flowering phenotype in development. The preceding expression of MADS-box genes also corresponded to the accelerated developmental phenotypes of the transgenic tobacco plants compared to that of wild-type (WT). Total gibberellin (GA) and active GA contents in transgenic tobacco plants were higher than those in WT at the corresponding developmental stages, and some GA metabolism genes were upregulated. Treatment with GA₃ conferred a similarly accelerated grown rate in WT plants to that of transgenic tobacco plants, while growth was inhibited when transgenic tobacco plants were treated with a GA biosynthesis inhibitor. Thus, the results suggest that Ub are involved in the production, metabolism and proper function of GA, which is important in the regulation of plant growth and development.     

Ammonium enhances the uptake, bioaccumulation, and tolerance of phenanthrene in cucumber seedlings

Aims

Phenanthrene is one of the ubiquitous, persistent organic pollutants commonly found in soil and sediments. The study will provide insight regarding the feasibility of nitrogen-assisted phytoremediation.

Methods

To study the effects of various nitrogen forms on cucumber seedling phenanthrene tolerance, hydroponic experiments were conducted in a greenhouse.

Results

Under phenanthrene stress, decreases in plant growth and biomass were more pronounced with a nitrate supply than with ammonium. In addition, phenanthrene concentrations in plants fed with ammonium were higher than those fed with nitrate. The reduction in plant protein and sugar, increases in nitrogen and phosphate concentrations, and increased activity of antioxidative enzymes may contribute to the phenanthrene stress response and adaptation. Higher peroxidase, superoxide dismutase, and catalase activities were found in ammonium-fed plants as compared to nitrate-fed plants under phenanthrene stress. Moreover, the reduction in soluble protein content and increases in phenanthrene transport and accumulation in non-photosynthetic organs may enable ammonium-fed plants to adapt more effectively to adverse conditions.

Conclusions

Overall, these results suggest that ammonium nutrition could provide a useful tool to improve the growth and adaption of plants under phenanthrene stress.     

Molecular cloning and characterization of a Brassica juncea yellow stripe-like gene, BjYSL7, whose overexpression increases heavy metal tolerance of tobacco

Key message

BjYSL7 encodes a plasma-localized metal–NA transporter and has transport Fe(II)–NA complexes activity. BjYSL7 is involved in the transport of Cd and Ni from roots to shoots.

Abstract

Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. In this study, we isolated a novel member of the yellow stripe-like (YSL) gene family BjYSL7 from the hyperaccumulator Brassica juncea. BjYSL7 is composed of 688 amino acids with 12 putative transmembrane domains and is over 90 % identical to TcYSL7 and AtYSL7. Real-time PCR analysis revealed that BjYSL7 mRNA was mainly expressed in the stem under normal condition. The expression of BjYSL7 was found to be up-regulated by 127.1-, 12.7-, and 3.4-fold in roots and 6.5-, 4.3-, and 2.8-fold in shoots under FeSO₄, NiCl₂, and CdCl₂ stresses, respectively. We have demonstrated that BjYSL7 is a Fe(II)–NA influx transporter by yeast functional complementation. Moreover, a BjYSL7::enhanced green fluorescent protein (EGFP) fusion localized to the plasma membrane of onion epidermal cells. The BjYSL7-overexpressing transgenic tobacco plants exhibited longer root lengths, lower relative inhibition rate of lengths and superior root hair development compared to that of wild-type (WT) plants in the presence of CdCl₂ and NiCl₂. Furthermore, the concentrations of Cd and Ni in shoots of BjYSL7-overexpressing plants are significantly higher than that of WT plants. Compared with WT plants, BjYSL7-overexpressing plants exhibited Fe concentrations that were higher in the shoots and seeds and lower in the roots. Taken together, these results suggest that BjYSL7 might be involved in the transport of Fe, Cd and Ni to the shoot and improving heavy metal resistance in plants.     

Overexpression of a novel salt stress-induced glycine-rich protein gene from alfalfa causes salt and ABA sensitivity in Arabidopsis

Key message

We cloned a novel salt stress-induced glycine-rich protein gene ( MsGRP ) from alfalfa. Its overexpression retards seed germination and seedling growth of transgenic Arabidopsis after salt and ABA treatments.

Abstract

Since soil salinity is one of the most significant abiotic stresses, salt tolerance is required to overcome salinity-induced reductions in crop productivity. Many glycine-rich proteins (GRPs) have been implicated in plant responses to environmental stresses, but the function and importance of some GRPs in stress responses remain largely unknown. Here, we report on a novel salt stress-induced GRP gene (MsGRP) that we isolated from alfalfa. Compared with some glycine-rich RNA-binding proteins, MsGRP contains no RNA recognition motifs and localizes in the cell membrane or cell wall according to the subcellular localization result. MsGRP mRNA is induced by salt, abscisic acid (ABA), and drought stresses in alfalfa seedlings, and its overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in Arabidopsis plants confers salinity and ABA sensitivity compared with WT plants. MsGRP retards seed germination and seedling growth of transgenic Arabidopsis plants after salt and ABA treatments, which implies that MsGRP may affect germination and growth through an ABA-dependent regulation pathway. These results provide indirect evidence that MsGRP plays important roles in seed germination and seedling growth of alfalfa under some abiotic stress conditions.     

Biochemical and kinetic characterization of GH43 β-d-xylosidase/α-l-arabinofuranosidase and GH30 α-l-arabinofuranosidase/β-d-xylosidase from rumen metagenome

The present study focuses on characterization of two hemicellulases, RuXyn1 and RuXyn2, from rumen bacterial metagenome and their capabilities for degradation of xylans. Glycosyl hydrolase (GH) family?43 ??-d-xylosidase/??-l-arabinofuranosidase RuXyn1 can hydrolyze p-nitrophenyl-??-d-xylopyranoside (pNPX), p-nitrophenyl-??-l-arabinofuranoside (pNPA), and xylo-oligosaccharide substrates, while GH30 1,5-??-l-arabinofuranosidase/??-d-xylosidase RuXyn2, the first ??-l-arabinofuranosidase assigned to this GH family, shows activities towards 1,5-??-l-arabinobiose and pNPX substrates but no activity for pNPA. Kinetic analysis for aryl-glycosides revealed that RuXyn2 had higher catalytic efficiency than RuXyn1 toward pNPX substrate. RuXyn1 shows high synergism with endoxylanase, elevating by 73% the reducing sugars released from brichwood xylans, and converted most intermediate xylo-oligosaccharide hydrolysate into xylose. The high xylose conversion capability of RuXyn1 suggests it has potential applications in enzymatic production of xylose and improvement of hemicellulose saccharification for production of biofuels. RuXyn2 shows no obviously synergistic effect in the endoxylanase-coupled assay for enzymatic saccharification of xylan. Further cosmid DNA sequencing revealed a neighboring putative GH43 ??-l-arabinofuranosidase RuAra1 and two putative GH3 ??-xylosidase/arabinosidases, RuXyn3 and RuXyn5, downstream of RuXyn2, indicating that this hemicellulase gene cluster may be responsible for production of end-product, xylose and arabinose, from hemicellulose biomass.     

Uptake and cellular distribution,in four plant species,of fluorescently labeled mesoporous silica nanoparticles

Key message

We report the uptake of MSNs into the roots and their movement to the aerial parts of four plant species and their quantification using fluorescence, TEM and proton-induced x - ray emission (micro - PIXE) elemental analysis.

Abstract

Monodispersed mesoporous silica nanoparticles (MSNs) of optimal size and configuration were synthesized for uptake by plant organs, tissues and cells. These monodispersed nanoparticles have a size of 20 nm with interconnected pores with an approximate diameter of 2.58 nm. There were no negative effects of MSNs on seed germination or when transported to different organs of the four plant species tested in this study. Most importantly, for the first time, a combination of confocal laser scanning microscopy, transmission electron microscopy and proton-induced X-ray emission (micro-PIXE) elemental analysis allowed the location and quantification MSNs in tissues and in cellular and sub-cellular locations. Our results show that MSNs penetrated into the roots via symplastic and apoplastic pathways and then via the conducting tissues of the xylem to the aerial parts of the plants including the stems and leaves. The translocation and widescale distribution of MSNs in plants will enable them to be used as a new delivery means for the transport of different sized biomolecules into plants.