Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli.

Results

By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l^?1 in E. coli under the shake-flask conditions.

Conclusions

Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system.

     

A novel 9-bp insertion detected in steroid 21-hydroxylase gene (CYP21A2): prediction of its structural and functional implications by computational methods

Background

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia (CAH). Detection of underlying mutations in CYP21A2 gene encoding steroid 21-hydroxylase enzyme is helpful both for confirmation of diagnosis and management of CAH patients. Here we report a novel 9-bp insertion in CYP21A2 gene and its structural and functional consequences on P450c21 protein by molecular modeling and molecular dynamics simulations methods.

Methods

A 30-day-old child was referred to our laboratory for molecular diagnosis of CAH. Sequencing of the entire CYP21A2 gene revealed a novel insertion (duplication) of 9-bp in exon 2 of one allele and a well-known mutation I172N in exon 4 of other allele. Molecular modeling and simulation studies were carried out to understand the plausible structural and functional implications caused by the novel mutation.

Results

Insertion of the nine bases in exon 2 resulted in addition of three valine residues at codon 71 of the P450c21 protein. Molecular dynamics simulations revealed that the mutant exhibits a faster unfolding kinetics and an overall destabilization of the structure due to the triple valine insertion was also observed.

Conclusion

The novel 9-bp insertion in exon 2 of CYP21A2 genesignificantly lowers the structural stability of P450c21 thereby leading to the probable loss of its function.     

Origin of mound-field landscapes: a multi-proxy approach combining contemporary vegetation, carbon stable isotopes and phytoliths

Background and aims

Seasonally flooded South American savannas harbor different kinds of mound-field landscapes of largely unknown origin. A recent study used soil carbon-isotope depth profiles and other proxies to infer vegetation history in murundu landscapes in Brazil. Results suggested that differential erosion, not building-up processes (e.g., termite mounds), produced mounds. We tested this approach to inferring mound origin in a mound-field landscape in French Guiana.

Methods

We examined carbon-isotope depth profiles of soil organic matter, phytolith profiles and contemporary vegetation composition in mounds and inter-mounds.

Results

Relative abundance of C3 and C4 plants across habitats was very different from that in murundu landscapes; C3 plants were better represented in inter-mounds than on mounds. Habitat differences in C3/C4 distribution were subtler than in murundu landscapes, limiting inference of vegetation history based on carbon isotopes. Still, carbon-isotope and phytolith depth profiles gave similar pictures of vegetation history, both favoring a building-up hypothesis, corroborating other evidence that these mounds are vestiges of ancient agricultural raised fields.

Conclusions

Carbon-isotope depth profiles are unlikely to be adequate for deciphering origin of mound-field landscapes from vegetation history in seasonally flooded savannas. Including data on current vegetation and phytoliths makes inferences more robust.     

Loss of all three calreticulins, CRT1, CRT2 and CRT3, causes enhanced sensitivity to water stress in Arabidopsis

Key message

The calreticulin triple knockout mutant shows growth defects in response to abiotic stress.

Abstract

The endoplasmic reticulum (ER) is an essential organelle that is responsible for the folding and maturation of proteins. During ER stress, unfolded protein aggregates accumulate in the cell, leading to the unfolded protein response (UPR). The UPR up-regulates the expression of ER-stress-responsive genes encoding calreticulin (CRT), an ER-localized Ca²⁺-binding protein. To understand the function of plant CRTs, we generated a triple knockout mutant, t123, which lacks CRT1, CRT2 and CRT3 and examined the roles of calreticulins in abiotic stress tolerance. A triple knockout mutant increased sensitivity to water stress which implies that calreticulins are involved in the Arabidopsis response to water stress. We identified that the cyclophilin AtCYP21-2, which is located in the ER, was specifically enhanced in the t123 mutants. Seed germination of the atcyp21-1 mutant was retarded by water stress. Taken together, these results suggest that regulatory proteins that serve to protect plants from water stress are folded properly in part with the help of calreticulins. The AtCYP21-2 may also participate in this protein-folding process in association with calreticulins.     

OsMLO12, encoding seven transmembrane proteins,is involved with pollen hydration in rice

Key message

MLO mediates pollen hydration.

Abstract

Hydration is the first step in pollen germination. However, the process is not well understood. OsMLO12 is highly expressed in mature pollen grains; plants containing alleles caused by transfer DNA insertions do not produce homozygous progeny. Reciprocal crosses between wild-type and OsMLO12/osmlo12 plants showed that the mutant alleles were not transmitted through the male gametophyte. Microscopic observations revealed that, although mutant grains became mature pollen with three nuclei, they did not germinate in vitro or in vivo due to a failure in hydration. The OsMLO12 protein has seven transmembrane motifs, with an N-terminal extracellular region and a C-terminal cytosolic region. We demonstrated that the C-terminal region mediates a calcium-dependent interaction with calmodulin. Our findings suggest that pollen hydration is regulated by MLO12, possibly through an interaction with calmodulin in the cytosol.     

The plasma membrane-localised Ca2+-ATPase ACA8 plays a role in sucrose signalling involved in early seedling development in Arabidopsis

Key message

Arabidopsis Ca ²⁺ -ATPase ACA8 plays a role in sucrose signalling during early seedling development by integrating developmental signals with carbon source availability.

Abstract

Calcium (Ca²⁺) is an essential signal transduction element in eukaryotic organisms. Changes in the levels of intracellular Ca²⁺ affect multiple developmental processes in plants, including cell division, polar growth, and organogenesis. Here, we report that the plasma-membrane-localised Arabidopsis Ca²⁺-ATPase ACA8 plays a role in sucrose signalling during early seedling development. Disruption of the ACA8 gene elevated the expression of genes that encode transporters for Ca²⁺ efflux. The seedlings that carried a T-DNA insertion mutation in ACA8 experienced water stress during early development. This response was unrelated to inadequate osmoregulatory responses and was most likely caused by disruption of cell membrane integrity and severe ion leakage. In addition, aca8-1 seedlings displayed a significant decline in photosynthetic performance and arrested root growth after removal of sucrose from the growth medium. The two phenomena resulted from impaired photosynthesis, reduced cell proliferation in the root meristem and the sucrose control of cell-cycle events. All of the stress-response phenotypes were rescued when expression of ACA8 was restored in aca8-1 mutant. Taken together, our results indicate that ACA8-mediated Ca²⁺ signalling contributes to modulate early seedling development and coordinates root development with nutrient availability.     

Genetic evidence that Arabidopsis ALTERED ROOT ARCHITECTURE encodes a putative dehydrogenase involved in homoserine biosynthesis

Key message

Genetic and molecular analysis of an Arabidopsis root development mutant identified a putative dehydrogenase gene involved in homoserine biosynthesis.

Abstract

In higher plants, homoserine (Hse) is derived from aspartate (Asp) and is an important intermediate for production of methionine (Met), threonine (Thr), and isoleucine (Ile). In Arabidopsis, six enzymes involved in the biosynthesis of Hse from Asp have been well characterized. It is not known, however, whether there exist other enzymes involved in this process. In this work, we characterized an Arabidopsis mutant, ara (a ltered r oot a rchitecture), with a short primary root and an increased number of lateral roots. Genetic and molecular analysis indicated that the ARA gene encodes a protein with a D-isomer specific 2-hydroxyacid dehydrogenase domain. ARA is expressed in all plant organs and is localized in the cell periphery. The ara mutant phenotypes can be rescued by exogenously applied Hse, Met, Ile and 2-oxobutanoate. Based on the results presented here, we propose that the ARA protein may be a dehydrogenase involved in homoserine biosynthesis.     

Identification and phylogenetic analysis ofDictyostelium discoideumkinesin proteins

Background

Kinesins constitute a large superfamily of motor proteins in eukaryotic cells. They perform diverse tasks such as vesicle and organelle transport and chromosomal segregation in a microtubule- and ATP-dependent manner. In recent years, the genomes of a number of eukaryotic organisms have been completely sequenced. Subsequent studies revealed and classified the full set of members of the kinesin superfamily expressed by these organisms. ForDictyostelium discoideum, only five kinesin superfamily proteins (Kif's) have already been reported.

Results

Here, we report the identification of thirteen kinesin genes exploiting the information from the raw shotgun reads of theDictyostelium discoideumgenome project. A phylogenetic tree of 390 kinesin motor domain sequences was built, grouping theDictyosteliumkinesins into nine subfamilies. According to known cellular functions or strong homologies to kinesins of other organisms, four of theDictyosteliumkinesins are involved in organelle transport, six are implicated in cell division processes, two are predicted to perform multiple functions, and one kinesin may be the founder of a new subclass.

Conclusion

This analysis of theDictyosteliumgenome led to the identification of eight new kinesin motor proteins. According to an exhaustive phylogenetic comparison,Dictyosteliumcontains the same subset of kinesins that higher eukaryotes need to perform mitosis. Some of the kinesins are implicated in intracellular traffic and a small number have unpredictable functions.     

QTL mapping for salt tolerance and domestication-related traits in Vigna marina subsp. oblonga,a halophytic species

Key message

QTL mapping in F ₂ population [ V. luteola × V. marina subsp. oblonga ] revealed that the salt tolerance in V. marina subsp. oblonga is controlled by a single major QTL.

Abstract

The habitats of beach cowpea (Vigna marina) are sandy beaches in tropical and subtropical regions. As a species that grows closest to the sea, it has potential to be a gene source for breeding salt-tolerant crops. We reported here for the first time, quantitative trait loci (QTLs) mapping for salt tolerance in V. marina. A genetic linkage map was constructed from an F₂ population of 120 plants derived from an interspecific cross between V. luteola and V. marina subsp. oblonga. The map comprised 150 SSR markers. The markers were clustered into 11 linkage groups spanning 777.6 cM in length with a mean distance between the adjacent markers of 5.59 cM. The F_2:3 population was evaluated for salt tolerance under hydroponic conditions at the seedling and developmental stages. Segregation analysis indicated that salt tolerance in V. marina is controlled by a few genes. Multiple interval mapping consistently identified one major QTL which can explain about 50 % of phenotypic variance. The flanking markers may facilitate transfer of the salt tolerance allele from V. marina subsp. oblonga into related Vigna crops. The QTL for domestication-related traits from V. marina are also discussed.     

Blue-light irradiation up-regulates the ent-kaurene synthase gene and affects the avoidance response of protonemal growth in Physcomitrella patens

Main conclusion

We report a novel physiological response to blue light in the moss Physcomitrella patens . Blue light regulates ent -kaurene biosynthesis and avoidance response to protonemal growth.

Abstract

Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurenoic acid via ent-kaurene. While the moss Physcomitrella patens has part of the GA biosynthetic pathway, from geranylgeranyl diphosphate to ent-kaurenoic acid, no GA is found in this species. Caulonemal differentiation in a P. patens mutant with a disrupted bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase (PpCPS/KS) gene is suppressed under red light, and is recovered by application of ent-kaurene and ent-kaurenoic acid. This indicates that derivatives of ent-kaurenoic acid, not GAs, might act as endogenous developmental regulators. Here, we found unique responses in the protonemal growth of P. patens under unilateral blue light, and these regulators were involved in the responses. When protonemata of the wild type were incubated under blue light, the chloronemal filaments grew in the opposite direction to the light source. Although this avoidance was not observed in the ent-kaurene deficient mutant, chloronemal growth toward a blue-light source in the mutant was suppressed by application of ent-kaurenoic acid, and the growth was rescued to that in the wild type. Expression analysis of the PpCPS/KS gene showed that the mRNA level under blue light was rapidly increased and was five times higher than under red light. These results suggest that regulators derived from ent-kaurenoic acid are strongly involved not only in the growth regulation of caulonemal differentiation under red light, but also in the light avoidance response of chloronemal growth under blue light. In particular, growth under blue light is regulated via the PpCPS/KS gene.     

Cloning,characterization and functional analysis of the role MhNCED3, a gene encoding 9-cis-epoxycarotenoid dioxygenase in Malus hupehensis Rehd., plays in plant tolerance to osmotic and Cd2+ stresses

Aims

Abscisic acid (ABA) plays an important role in the stress tolerance of seedlings and 9-cis-epoxycarotenoid dioxygenase (NCED) is considered to be the rate-limiting enzyme involved in ABA biosynthesis. However, the genes encoding NCED in M. hupehensis Rehd. have not been reported.

Methods

In this study, a gene encoding NCED, MhNCED3, was isolated from the roots of M. hupehensis Rehd. Its functions were investigated in M. hupehensis Rehd. seedlings and transgenic Arabidopsis lines under various abiotic stresses.

Results

The expression of MhNCED3 in M. hupehensis Rehd. roots was differentially induced by dehydration, chilling, salt and cadmium stresses and ABA biosynthesis was highly correlated with MhNCED3 expression. Ectopic expression of MhNCED3 successfully complemented the phenotypic defects of the 129B08/nced3 mutant. Furthermore, overexpression of MhNCED3, when it was transformed into the wild type (WT) seedling resulted in enhanced tolerance to osmotic and cadmium stresses compared to the normal WT seedling. The transgenic lines displayed higher rates of seed germination, improved growth and developmental status, reduced water loss/oxidative damage, lowered apoptosis rates and increased ABA accumulation. Furthermore, the higher antioxidant enzyme activities detected in the transgenic lines were probably responsible for the decrease in oxidative damage and apoptosis rates.

Conclusions

Overall, MhNCED3 played a significant role in enhancing plant tolerance to abiotic stresses through the regulation of endogenous ABA biosynthesis.     

The Arabidopsis kinesin gene AtKin-1 plays a role in the nuclear division process during megagametogenesis

Key message

Atkin - 1 , the only Kinesin-1 member of Arabidopsis thaliana , plays a role during female gametogenesis through regulation of nuclear division cycles.

Abstract

Kinesins are microtubule-dependent motor proteins found in eukaryotic organisms. They constitute a superfamily that can be further classified into at least 14 families. In the Kinesin-1 family, members from animal and fungi play roles in long-distance transport of organelles and vesicles. Although Kinesin-1-like sequences have been identified in higher plants, little is known about their function in plant cells, other than in a recently identified Kinesin-1-like protein in a rice pollen semi-sterile mutant. In this study, the gene encoding the only Kinesin-1 member in Arabidopsis, AtKin-1 was found to be specifically expressed in ovules and anthers. AtKin-1 loss-of-function mutants showed substantially aborted ovules in siliques, and this finding was supported by complementation testing. Reciprocal crossing between mutant and wild-type plants indicated that a defect in AtKin-1 results in partially aborted megagametophytes, with no observable effects on pollen fertility. Further observation of ovule development in the mutant pistils indicated that the enlargement of the megaspore was blocked and nuclear division arrested at the one-nucleate stage during embryo sac formation. Our data suggest that AtKin-1 plays a role in the nuclear division cycles during megagametogenesis.