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.     

Combinational transformation of three wheat genes encoding fructan biosynthesis enzymes confers increased fructan content and tolerance to abiotic stresses in tobacco

Key message

Seven kinds of transgenic tobacco plants transformed with combinations of three FBE genes were obtained. The transgenic plants transformed with Ta1-SST?+?Ta6-SFT genes appeared to have the highest fructan or soluble sugar content and the strongest salt tolerance.

Abstract

Fructan is thought to be one of the important regulators involved in plant tolerance to various abiotic stresses. In this study, wheat-derived genes, Ta1-SST, Ta6-SFT, and Ta1-FFT, encoding fructan biosynthesis enzymes (FBE) were isolated and cloned into vectors modified pBI121 or pZP211. Seven different combinations of the three target genes were transformed into tobacco plants through an Agrobacterium-mediated approach, and transgenic tobacco plants were identified by PCR, ELISA, and Southern blotting. Compared with tobacco plants transformed with other six combinations of the three target genes and with wild-type plants, the transgenic plants transformed with Ta1-SST?+?Ta6-SFT genes contained the highest fructan and soluble sugar content. All seven types of transgenic tobacco plants displayed a much higher level of tolerance to drought, low temperature, and high salinity compared with the wild type. Differences of drought and low temperature tolerance between the transgenic plants containing a single FBE gene and those harboring two or three FBE genes were not significant, but the salt tolerance level of the transgenic plants with different FBE gene combinations from high to low was: Ta1-SST?+?Ta6-SFT?>?Ta1-SST?+?Ta6-SFT?+?Ta1-FFT?>?Ta1-SST?+?Ta1-FFT?>?Ta1-SFT?+?Ta1-FFT?>?single FBE gene. These results indicated that the tolerances of the transgenic tobacco plants to various abiotic stresses were associated with the transformed target gene combinations and the contents of fructan and soluble sugar contained in the transgenic plants.     

A putative poplar PP2C-encoding gene negatively regulates drought and abscisic acid responses in transgenic Arabidopsis thaliana

Key message

Here we link for the first time a poplar gene with putative function in ABA signaling to the regulation of drought responses, providing a target for drought tolerance improvement in poplars.

Abstract

Populus species are valued for their fast growth and are cultivated widely. Many of the commonly used species and hybrids are, however, regarded as drought sensitive, which poses a problem for large-scale cultivation particularly in light of climate change-induced drought spells in areas of poplar growth. While many hundreds of drought-induced genes have been identified in Populus species, very little is known about the genes and the signaling process that leads to a drought response in these species. Based on sequence similarity, the poplar G059200 gene is a potential ortholog of AtPP2CA, an inhibitor of drought and abscisic acid (ABA) responses in Arabidopsis thaliana. To test if G059200 has a similar function, we generated transgenic A. thaliana plants overexpressing this gene. These transgenic lines exhibited reduced responses to exogenous ABA and reduced tolerance to osmotic stress. Finally, drought tolerance of plants was also significantly reduced. Taken together, these data provide evidences that G059200 acts as a negative regulator of ABA responses. The ability to negatively regulate drought stress responses suggests that G059200 may be targeted for drought tolerance breeding, for example, by identification of individuals harboring natural or induced loss-of-function alleles, or by RNA interference technology, to generate poplar plants with reduced activity of G059200.     

Enhanced accumulation of fatty acids and triacylglycerols in transgenic tobacco stems for enhanced bioenergy production

Key message

We report a novel approach for enhanced accumulation of fatty acids and triacylglycerols for utilization as biodiesel in transgenic tobacco stems through xylem-specific expression of Arabidopsis DGAT1 and LEC2 genes.

Abstract

The use of plant biomass for production of bioethanol and biodiesel has an enormous potential to revolutionize the global bioenergy outlook. Several studies have recently been initiated to genetically engineer oil production in seeds of crop plants to improve biodiesel production. However, the “food versus fuel” issues have also sparked some studies for enhanced accumulation of oils in vegetative tissues like leaves. But in the case of bioenergy crops, use of woody stems is more practical than leaves. Here, we report the enhanced accumulation of fatty acids (FAs) and triacylglycerols (TAGs) in stems of transgenic tobacco plants expressing Arabidopsis diacylglycerol acyltransferase 1 (DGAT1) and LEAFY COTYLEDON2 (LEC2) genes under a developing xylem-specific cellulose synthase promoter from aspen trees. The transgenic tobacco plants accumulated significantly higher amounts of FAs in their stems. On an average, DGAT1 and LEC2 overexpression showed a 63 and 80 % increase in total FA production in mature stems of transgenic plants over that of controls, respectively. In addition, selected DGAT1 and LEC2 overexpression lines showed enhanced levels of TAGs in stems with higher accumulation of 16:0, 18:2 and 18:3 TAGs. In LEC2 lines, the relative mRNA levels of the downstream genes encoding plastidic proteins involved in FA synthesis and accumulation were also elevated. Thus, here, we provide a proof of concept for our approach of enhancing total energy yield per plant through accumulation of higher levels of FAs in transgenic stems for biodiesel production.     

Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure

Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content.     

The poplar ARGOS-LIKE gene promotes leaf initiation and cell expansion,and controls organ size

We identified a Populus nigra auxin-regulated gene involved in organ size (PnARGOS)-LIKE, encoding one organ size related protein in black poplar. It is homologous to AtARGOS and AtARGOS-LIKE genes of Arabidopsis thaliana. ABRE-like, G-box, GATA and I-box motifs were discovered in the promoter region of the poplar ARGOS-LIKE gene. In wild type aspen (Populus tremula) plants, an ortholog of the PnARGOS-LIKE gene (PtrARGOS-LIKE) was noticeably expressed in actively dividing and expanding young leaves and calli, whereas its mRNA content increased in response to exogenous 6-benzylaminopurine, 1-naphthaleneacetic acid, and 24-epibrassinolide. Expression of the PtrARGOS-LIKE gene was reduced under a salinity treatment. In addition, we generated transgenic tobacco and aspen plants with an up-regulated expression of the PnARGOS-LIKE gene. A constitutive expression of the gene contributed to an increase in size of stems and leaves of the transgenic tobacco plants. In the transgenic aspen, a constitutive expression of the PnARGOS-LIKE gene promoted an increase in the frequency of leaf initiations and in leaf length and area. The size of transgenic tobacco and aspen leaves increased due to the enlargement of individual cells. The results show the significance of the PnARGOS-LIKE gene for control of leaf initiation and organ growth by cell expansion in poplar.     

Molecular characterization of rice sphingosine-1-phosphate lyase gene OsSPL1 and functional analysis of its role in disease resistance response

Key message

Our results indicate that overexpression of OsSPL1 in transgenic tobacco plants attenuated disease resistance and facilitated programmed cell death.

Abstract

Long-chain base phosphates including sphingosine-1-phosphate have been shown to act as signaling mediators in regulating programmed cell death (PCD) and stress responses in mammals. In the present study, we characterized a rice gene OsSPL1, encoding a putative sphingosine-1-phosphate lyase that is involved in metabolism of sphingosine-1-phosphate. Expression of OsSPL1 was down-regulated in rice plants after treatments with salicylic acid, benzothiadiazole and 1-amino cyclopropane-1-carboxylic acid, but was induced by infection with a virulent strain of Magnaporthe oryzae, the causal agent of rice blast disease. Transgenic tobacco lines with overexpression of OsSPL1 were generated and analyzed for the possible role of OsSPL1 in disease resistance response and PCD. The OsSPL1-overexpressing tobacco plants displayed increased susceptibility to infection of Pseudomonas syringae pv. tabaci (Pst), the causal agent of wildfire disease, showing severity of disease symptom and bacterial titers in inoculated leaves, and attenuated pathogen-induced expression of PR genes after infection of Pst as compared to the wild-type and vector-transformed plants. Higher level of cell death, as revealed by dead cell staining, leakage of electrolyte and expression of hypersensitive response indicator genes, was observed in the OsSPL1-overexpressing plants after treatment with fumonisin B1, a fungal toxin that induces PCD in plants. Our results suggest that OsSPL1 has different functions in regulating disease resistance response and PCD in plants.     

Enhancement of abiotic stress tolerance in poplar by overexpression of key Arabidopsis stress response genes, <Emphasis Type="Italic">AtSRK2C</Emphasis> and <Emphasis Type="Italic">AtGolS2</Emphasis>

Recent environmental issues have increased the demand for woody biomass as a renewable resource for industry and energy. For a stable supply of woody biomass, it is critical to decrease the effects of abiotic stresses, such as drought and salinity, which hinder plant growth. For the goal to develop practical stress-tolerant trees, we generated transgenic poplar plants (P. tremula × tremuloides), in which a key Arabidopsis regulatory factor involved in stress responses, SNF1-related protein kinase 2C (AtSRK2C), or galactinol synthase 2 (AtGolS2), was overexpressed. Both types of transgenic poplar plants displayed higher tolerance to abiotic stresses, in comparison with nontransgenic plants, indicating that AtSRK2C and AtGolS2 can function in the abiotic stress response pathway of poplar. We also examined the expression profiles of ten poplar genes putatively homologous to well-known Arabidopsis stress response genes and found that several of the poplar genes showed different responses to abiotic stress from their Arabidopsis counterparts. Whereas the overexpression of AtSRK2C in transgenic Arabidopsis plants was reported to upregulate the expression of endogenous genes, the overexpression of AtSRK2C or AtGolS2 in transgenic poplar did not. Taken together, our findings suggest that the details of the underlying molecular mechanisms of the abiotic stress response may differ, but that the key regulatory factors in Arabidopsis and poplar have common features and are effective molecular targets for further breeding to enhance abiotic stress tolerance in poplar.     

Differential regulation of cytokinin oxidase genes and cytokinin-induced auxin biosynthesis by cellular cytokinin level in transgenic poplars

Key message

The present work with transgenic poplar lines producing varying levels of trans -zeatin suggests the existence of a switching threshold for triggering ckx gene expression or suppressing cytokinin-induced auxin.

Abstract

Cytokinins have an important role in growth and developmental processes of plants. Transgenic plants with varying levels of cellular cytokinin are convenient tools for studying its role in morphogenetic as well as molecular responses. In this work, the transgenic lines producing either high level of cellular trans-zeatin (HX lines) or moderate level (MX lines) were compared with regard to their cytokinin oxidase activities and cellular auxin content. The HX lines showed typical cytokinin phenotypes including leafy shoots and spontaneous shoot formation on hormone free medium. In contrast, the MX lines did not show any striking phenotypes. However, in leaf disk culture on hormone free medium, they regenerated roots and subsequently formed shoots from the roots. Determination of cellular IAA content revealed a significant increase in the level in MX lines but not in HX lines. Of nine cytokinin oxidase genes (ckx) examined by qPCR, five were activated in HX lines but not in MX lines. Among them, ckx4 appeared to play a key role in maintaining cellular cytokinin level since it showed more than 1,000-fold increase in HX lines and in the leaf disks of untransformed control exposed to exogenous cytokinins. Although low level of cellular cytokinin did not induce the expression of ckx genes, it appeared to trigger cellular IAA biosynthesis.     

A precise and consistent assay for major wall polymer features that distinctively determine biomass saccharification in transgenic rice by near-infrared spectroscopy

Background

The genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance in bioenergy crops. As a result, it is important to develop a precise and rapid assay for the major wall polymer features that affect biomass saccharification in a large population of transgenic plants. In this study, we collected a total of 246 transgenic rice plants that, respectively, over-expressed and RNAi silenced 12 genes of the OsGH9 and OsGH10 family that are closely associated with cellulose and hemicellulose modification. We examined the wall polymer features and biomass saccharification among 246 transgenic plants and one wild-type plant. The samples presented a normal distribution applicable for statistical analysis and NIRS modeling.

Results

Among the 246 transgenic rice plants, we determined largely varied wall polymer features and the biomass enzymatic saccharification after alkali pretreatment in rice straws, particularly for the fermentable hexoses, ranging from 52.8 to 95.9%. Correlation analysis indicated that crystalline cellulose and lignin levels negatively affected the hexose and total sugar yields released from pretreatment and enzymatic hydrolysis in the transgenic rice plants, whereas the arabinose levels and arabinose substitution degree (reverse xylose/arabinose ratio) exhibited positive impacts on the hexose and total sugars yields. Notably, near-infrared spectroscopy (NIRS) was applied to obtain ten equations for predicting biomass enzymatic saccharification and seven equations for distinguishing major wall polymer features. Most of the equations exhibited high R ²/R ² _cv/R ² _ev and RPD values for a perfect prediction capacity.

Conclusions

Due to large generated populations of transgenic rice lines, this study has not only examined the key wall polymer features that distinctively affect biomass enzymatic saccharification in rice but has also established optimal NIRS models for a rapid and precise screening of major wall polymer features and lignocellulose saccharification in biomass samples. Importantly, this study has briefly explored the potential roles of a total of 12 OsGH9 and OsGH10 genes in cellulose and hemicellulose modification and cell wall remodeling in transgenic rice lines. Hence, it provides a strategy for genetic modification of plant cell walls by expressing the desired OsGH9 and OsGH10 genes that could greatly improve biomass enzymatic digestibility in rice.

     

Regulation of secondary xylem formation in young hybrid poplars by modifying the expression levels of the <Emphasis Type="Italic">PtaGLIMa</Emphasis> gene

Due to the importance of wood in many industrial applications, a tremendous amount of research has focused on the regulation of secondary xylem formation and wood properties. In this study, we performed functional analysis of PtaGLIM1a, a LIM gene that is predominantly expressed in the differentiation of secondary xylem of the hybrid poplar (Populus tremula × P. alba). With no growth retardation, transgenic poplar plants with increased and reduced expression levels of PtaGlim1a exhibited enhanced and diminished secondary growth, respectively, accompanied by a corresponding change in their lignin abundance. This study demonstrates that the wood-associated PtaGlim1a acts as a positive regulator of secondary xylem formation in poplar trees and could potentially be utilized in modifying the synthesis of plant secondary wall lignin.     

Heterologous expression of Arabidopsis C-repeat binding factor 3 (AtCBF3) and cold-regulated 15A (AtCOR15A) enhanced chilling tolerance in transgenic eggplant (Solanum melongena L.)

Key message

Our study shows that the expression of AtCBF3 and AtCOR15A improved the chilling tolerance in transgenic eggplant.

Abstract

In an attempt to improve chilling tolerance of eggplant (Solanum melongena L) plants, Arabidopsis C-repeat binding factor 3 (AtCBF3) and cold-regulated 15A (AtCOR15A) genes both driven by an Arabidopsis RESPONSIVE TO DESSICATION 29A promoter (AtRD29A) were transferred into the plants of eggplant cultivar Sanyueqie. Two independent homozygous transgenic lines were tested for their cold tolerance. The leaves of the transgenic plants in both lines withered much slower and slighter than the wild-type plants after exposure to cold stress treatment at 2 ± 1 °C. The gene expression of AtCBF3 and AtCOR15A was significantly increased as well as the proline content and the levels of catalase and peroxidase activities, while the relative electrical conductivity and the malondialdehyde content were remarkably decreased in the transgenic plants compared with the wild type at 4 ± 0.5 °C. The results showed that the expression of the exogenous AtCBF3 and AtCOR15A could promote the cold adaptation process to protect eggplant plants from chilling stress.