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.     

Endophytic microbes <Emphasis Type="Italic">Bacillus</Emphasis> sp. LZR216-regulated root development is dependent on polar auxin transport in <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Key message

Endophytic microbes Bacillus sp. LZR216 isolated from Arabidopsis root promoted Arabidopsis seedlings growth. It may be achieved by promoting the lateral root growth and inhibiting the primary root elongation.

Abstract

Plant roots are colonized by an immense number of microbes, including epiphytic and endophytic microbes. It was found that they have the ability to promote plant growth and protect roots from biotic and abiotic stresses. But little is known about the mechanism of the endophytic microbes-regulated root development. We isolated and identified a Bacillus sp., named as LZR216, of endophytic bacteria from Arabidopsis root. By employing a sterile experimental system, we found that LZR216 promoted the Arabidopsis seedlings growth, which may be achieved by promoting the lateral root growth and inhibiting the primary root elongation. By testing the cell type-specific developmental markers, we demonstrated that Bacillus sp. LZR216 increases the DR5::GUS and DR5::GFP expression but decreases the CYCB1;1::GUS expression in Arabidopsis root tips. Further studies indicated that LZR216 is able to inhibit the meristematic length and decrease the cell division capability but has little effect on the quiescent center function of the root meristem. Subsequently, it was also shown that LZR216 has no significant effects on the primary root length of the pin2 and aux1-7 mutants. Furthermore, LZR216 down-regulates the levels of PIN1-GFP, PIN2-GFP, PIN3-GFP, and AUX1-YFP. In addition, the wild-type Arabidopsis seedlings in the present of 1 or 5 µM NPA (an auxin transport inhibitor) were insensitive to LZR216-inhibited primary root elongation. Collectively, LZR216 regulates the development of root system architecture depending on polar auxin transport. This study shows a new insight on the ability of beneficial endophytic bacteria in regulating postembryonic root development.

     

Antipodal cells persist through fertilization in the female gametophyte of Arabidopsis

Key message

Extended antipodal life-span.

Abstract

The female gametophyte of most flowering plants forms four cell types after cellularization, namely synergid cell, egg cell, central cell and antipodal cell. Of these, only the antipodal cells have no established functions, and it has been proposed that in many plants including Arabidopsis, the antipodal cells undergo programmed cell death during embryo sac maturation and prior to fertilization. Here, we examined the expression of female gametophyte-specific fluorescent reporters in mature embryo sacs of Arabidopsis, and in developing seeds shortly after fertilization. We observed expression of the fluorescence from the reporter genes in the three antipodal cells in the mature stage embryo sac, and continuing through the early syncytial endosperm stages. These observations suggest that rather than undergoing programmed cell death and degenerating at the mature stage of female gametophyte as previously supposed, the antipodal cells in Arabidopsis persist beyond fertilization, even when the other cell types are no longer present. The results support the concept that the Arabidopsis female gametophyte at maturity should be considered to be composed of seven cells and four cell types, rather than the previously prevailing view of four cells and three cell types.     

AtHSP17.8 overexpression in transgenic lettuce gives rise to dehydration and salt stress resistance phenotypes through modulation of ABA-mediated signaling

Key message

Transgenic Arabidopsis and lettuce plants overexpressing AtHSP17.8 showed ABA-hypersensitive but abiotic stress-resistant phenotypes. ABA treatment caused a dramatic induction of early ABA-responsive genes in AtHSP17.8 -overexpressing transgenic lettuce.

Abstract

Plant small heat shock proteins function as chaperones in protein folding. In addition, they are involved in responses to various abiotic stresses, such as dehydration, heat and high salinity in Arabidopsis. However, it remains elusive how they play a role in the abiotic stress responses at the molecular level. In this study, we provide evidence that Arabidopsis HSP17.8 (AtHSP17.8) positively regulates the abiotic stress responses by modulating abscisic acid (ABA) signaling in Arabidopsis, and also in lettuce, a heterologous plant when ectopically expressed. Overexpression of AtHSP17.8 in both Arabidopsis and lettuce leads to hypersensitivity to ABA and enhanced resistance to dehydration and high salinity stresses. Moreover, early ABA-responsive genes, ABI1, ABI5, NCED3, SNF4 and AREB2, were rapidly induced in AtHSP17.8-overexpressing transgenic Arabidopsis and lettuce. Based on these data, we propose that AtHSP17.8 plays a crucial role in abiotic stress responses by positively modulating ABA-mediated signaling in both Arabidopsis and lettuce. Moreover, our results suggest that stress-tolerant lettuce can be engineered using the genetic and molecular resources of Arabidopsis.     

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.     

Arabidopsis Brassinosteroid-overproducing gulliver3-D/dwarf4-D mutants exhibit altered responses to Jasmonic acid and pathogen

Key message

Arabidopsis gulliver3 - D/dwarf4 - D displays growth-promoting phenotypes due to activation tagging of a key brassinosteroid biosynthetic gene DWARF4. In gul3-D/dwf4-D , the Jasmonate and Salicylate signaling pathways were relatively activated and suppressed, respectively.

Abstract

Energy allocation between growth and defense is elegantly balanced to achieve optimal development in plants. Brassinosteroids (BRs), steroidal hormones essential for plant growth, are regulated by other plant hormones, including auxin and jasmonates (JA); auxin stimulates the expression of a key brassinosteroid (BR) biosynthetic gene, DWARF4 (DWF4), whereas JA represses it. To better understand the interaction mechanisms between growth and defense, we isolated a fast-growing mutant, gulliver3-D (gul3-D), that resulted from the activation tagging of DWF4, and examined the response of this mutant to defense signals, including JA, Pseudomonas syringae pv. tomato (Pst DC3000) infection, and wounding. The degree of root growth inhibition following MeJA treatment was significantly decreased in gul3-1D/dwf4-5D relative to the wild type, suggesting that JA signaling is partially desensitized in gul3-1D. Quantitative RT-PCR analysis of the genes involved in JA and salicylic acid (SA) responses, including MYC2, PDF1.2, CORI3, PR1, and PR2, revealed that JA signaling was preferentially activated in gul3-1D, whereas SA signaling was suppressed. As a result, gul3-1D was more susceptible to a biotrophic pathogen, Pst DC3000. Based on our results, we propose a model in which BR and JA cooperate to balance energy allocation between growth and defense responses. In ambient conditions, BRs promote plant growth; however, when stresses trigger JA signaling, JA compromises BR signaling by downregulating DWF4 expression.     

Four new species of Smilax (Smilacaceae) from southeastern Asia

Described as new areSmilax biumbellata of the affinity ofS. nervomarginata,S. siamensis of the affinity of 5.perfoliata, and two others inSmilax sect.Macranthae:S. inversa andS. pachysandroides.Smilax inversa (characterized by strongly deflexed leaves and inflorescences on zigzag stems) andS. pachysandroides (with subsessile leaves on low simple stems) show no close resemblance to any previously known species of the section.     

PP2A mediates lateral root development under NaCl-induced osmotic stress throughout auxin redistribution in Arabidopsis thaliana

Aim

Auxin plays an important role in modulating root system architecture. The effect of salinity on root development has been extensively studied; however, evidence on how salinity affects lateral root development and its underlying molecular mechanism is scarce. Here, we analyzed the role of protein phosphatase PP2A activity in auxin redistribution during Arabidopsis root system adaptation under NaCl-induced osmotic stress.

Method

Arabidopsis Col-0 and DR5::UidA seedlings were grown in MS media containing NaCl alone or in combination with the auxin transport inhibitor naphthylphthalamic acid, the synthetic auxin α-Naphthaleneacetic acid or the phosphatase inhibitor Okadaic acid. After 8 days, primary root length and lateral root number in seedlings were quantified and the auxin distribution was analyzed.

Results

Promotion of primary root shortening and lateral root development induced by osmotic stress correlated with an increase in active auxin content and a >50 % reduction in protein phosphatase type 2A (PP2A) activity. Moreover, the observed effects on seedlings under osmotic stress are more pronounced with the PP2A inhibitor Okadaic acid.

Conclusion

Our data suggest PP2A is a positive regulator of osmotic stress-induced root system architecture modulation, involving auxin redistribution in Arabidopsis thaliana.     

Arabidopsis CDPK6 phosphorylates ADF1 at N-terminal serine 6 predominantly

Key message

We found that Arabidopsis AtADF1 was phosphorylated by AtCDPK6 at serine 6 predominantly and the phosphoregulation plays a key role in the regulation of ADF1-mediated depolymerization of actin filaments.

Abstract

Since actin-depolymerizing factor (ADF) is highly conserved among eukaryotes, it is one of the key modulators for actin organization. In plants, ADF is directly involved in the depolymerization of actin filaments, and therefore important for F-actin-dependent cellular activities. The activity of ADF is tightly controlled through a number of molecular mechanisms, including phosphorylation-mediated inactivation of ADF. To investigate Arabidopsis ADF1 phosphoregulation, we generated AtADF1 phosphorylation site-specific mutants. Using transient expression and stable transgenic approaches, we analyzed the ADF1 phosphorylation mutants in the regulation of actin filament organizations in plant cells. By in vitro phosphorylation assay, we showed that AtADF1 is phosphorylated by AtCDPK6 at serine 6 predominantly. Chemically induced expression of AtCDPK6 can negatively regulate the wild-type AtADF1 in depolymerizing actin filaments, but not those of the mutants AtADF1(S6A) and AtADF1(S6D). These results demonstrate a regulatory function of Arabidopsis CDPK6 in the N-terminal phosphorylation of AtADF1.     

Cosuppression of RBCS3B in Arabidopsis leads to severe photoinhibition caused by ROS accumulation

Key message

Cosuppression of an Arabidopsis Rubisco small subunit gene RBCS3B at Arabidopsis resulted in albino or pale green phenotypes which were caused by ROS accumulation

Abstract

As the most abundant protein on Earth, Rubisco has received much attention in the past decades. Even so, its function is still not understood thoroughly. In this paper, four Arabidopsis transgenic lines (RBCS3B-7, 18, 33, and 35) with albino or pale green phenotypes were obtained by transformation with a construct driving expression of sense RBCS3B, a Rubisco small subunit gene. The phenotypes produced in these transgenic lines were found to be caused by cosuppression. Among these lines, RBCS3B-7 displayed the most severe phenotypes including reduced height, developmental arrest and plant mortality before flowering when grown under normal light on soil. Chloroplast numbers in mesophyll cells were decreased compared to WT, and stacked thylakoids of chloroplasts were broken down gradually in RBCS3B-7 throughout development. In addition, the RBCS3B-7 line was light sensitive, and PSII activity measurement revealed that RBCS3B-7 suffered severe photoinhibition, even under normal light. We found that photoinhibition was due to accumulation of ROS, which accelerated photodamage of PSII and inhibited the repair of PSII in RBCS3B-7.     

Ectopic expression of a novel Ser/Thr protein kinase from cotton (Gossypium barbadense), enhances resistance to Verticillium dahliae infection and oxidative stress in Arabidopsis

Key message

Overexpression of a cotton defense-related gene GbSTK in Arabidopsis resulted in enhancing pathogen infection and oxidative stress by activating multiple defense-signaling pathways.

Abstract

Serine/threonine protein kinase (STK) plays an important role in the plant stress-signaling transduction pathway via phosphorylation. Most studies about STK genes have been conducted with model species. However, their molecular and biochemical characterizations have not been thoroughly investigated in cotton. Here, we focused on one such member, GbSTK. RT-PCR indicated that it is induced not only by Verticillium dahliae Kleb., but also by signaling molecules. Subcellular localization showed that GbSTK is present in the cell membrane, cytoplasm, and nucleus. Overexpression of GbSTK in Arabidopsis resulted into the enhanced resistance to V. dahliae. Moreover, Overexpression of GbSTK elevated the expression of PR4, PR5, and EREBP, conferring on transgenic plants enhanced reactive oxygen species scavenging capacity and oxidative stress tolerance. Our results suggest that GbSTK is active in multiple defense-signaling pathways, including those involved in responses to pathogen infection and oxidative stress.     

Sequence and functional analyses of the aldehyde dehydrogenase 7B4 gene promoter in Arabidopsis thaliana and selected Brassicaceae: regulation patterns in response to wounding and osmotic stress

Key message

The core promoter of the antiquitin ALDH7B4 gene was compared between selected Brassicaceae. Conserved cis elements controlling osmotic stress and wound-induced expression were identified and analysed in Arabidopsis thaliana leaves and seeds.

Abstract

Aldehyde dehydrogenases metabolise a wide range of aliphatic and aromatic aldehydes, which become cytotoxic at high levels. Family 7 aldehyde dehydrogenase genes, often described as antiquitins or turgor-responsive genes in plants, are broadly conserved across all domains. Despite the high conservation of the plant ALDH7 proteins and their importance in stress responses, their regulation has not been investigated. Here, we compared ALDH7 genes of different Brassicaceae and found that, in contrast to the gene organisation and protein coding sequences, similarities in the promoter sequences were limited to the first few hundred nucleotides upstream of the translation start codon. The function of this region was studied by isolating the core promoter of the Arabidopsis thaliana ALDH7B4 gene, taken as model. The promoter was found to be responsive to wounding in addition to salt and dehydration stress. Cis-acting elements involved in stress responsiveness were analysed and two conserved ACGT-containing motifs proximal to the translation start codon were found to be essential for the responsiveness to osmotic stress in leaves and in seeds. The integrity of an upstream ACGT motif and a dehydration-responsive element/C-repeat—low temperature-responsive element was found to be necessary for ALDH7B4 expression in seeds and induction by salt, dehydration and ABA in leaves. The comparison of the gene expression in selected Arabidopsis mutants demonstrated that osmotic stress-induced ALDH7B4 expression in leaves and seeds involves both ABA- and lipid-signalling components.