Castasterone Can be Biosynthesized from 28-homodolichosterone in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

We recently demonstrated the biosynthesis of 24-ethylidene brassinosteroids in Arabidopsis thaliana. To determine the physiological role of biosynthesis of 24-ethylidene brassinosteroids, metabolism of 28-homodolichosterone as the end product of 24-ethylidene brassinosteroids biosynthesis was examined by a crude enzyme solution prepared from A. thaliana. In wild-type plants, dolichosterone and castasterone were identified as enzyme products on GC-MS analysis. In a mutant where DWARF1 was overexpressed (35S-DWF1), the conversion rate of 28-homodolichosterone to castasterone was significantly increased. These results indicate that conversion of 28-homodolichosterone to castasterone is mediated by dolichosterone in Arabidopsis. In the root growth assay, inhibitory activity was enhanced in the order of castasterone > dolichosterone > 28-homodolichosterone, demonstrating that conversion of 28-homodolichosterone to castasterone via dolichosterone is a biosynthetic reaction that increases BR activity in Arabidopsis. Compared to Arabidopsis grown under dark conditions, light-grown Arabidopsis showed up-regulated DWARF1 expression, resulting in an increased conversion rate of 28-homodolichosterone to castasterone, suggesting that light is an important regulatory factor for the biosynthetic connection of 24-ethylidene brassinosteroids and 24-methyl brassinosteroids in A. thaliana. Consequently, 24-ethylidene brassinosteroids biosynthesis to generate 28-homodolichosterone is a lightregulated alternative route for synthesis of the biologically-active BRs, castasterone and brassinolide in Arabidopsis plants.     

Ectopic expression of sorbitol-6-phosphate 2-dehydrogenase gene from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis> enhances salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

d-Sorbitol-6-phosphate 2-dehydrogenase (S6PDH, E.C. 1.1.1.140) catalyzes the NADH-dependent conversion of d-fructose 6-phosphate (F6P) to d-sorbitol 6-phosphate (S6P). In this work, recombination and characterization of Haloarcula marismortui d-sorbitol-6-phosphate 2-dehydrogenase are reported. Haloarcula marismortui d-sorbitol-6-phosphate 2-dehydrogenase was expressed in P. pastoris and Arabidopsis thaliana. Enzyme assay indicated that HmS6PDH catalyzes the reduction of d-fructose 6-phosphate to d-sorbitol 6-phosphate and HmS6PDH activity was enhanced by NaCl. Furthermore, transgenic A. thaliana ectopic expressing HmS6PDH accumulate more sorbitol under salt stress. These results suggest that the ectopic expression of HmS6PDH in plants can facilitate future studies regarding the engineering and breeding of salt-tolerant crops.     

Light regulation of succinate dehydrogenase expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves

A potential mechanism of light regulation of the succinate dehydrogenase (SDH) expression in Arabidopsis thaliana leaves was studied. As was shown by dot-hybridization and polymerase chain reaction in real time (RT-PCR), the SDH mRNA level in wild-type Arabidopsis thaliana plants changed depending on light conditions. The level of SDH mRNA in darkness was higher than in the light. The analysis of Arabidopsis thaliana plants carrying the mutant genes of phytochromes A and B showed that phytochrome A was involved in the regulation of the SDH enzyme activity. The active form of phytochrome A suppressed the SDHI-2 gene expression, and that resulted in decreasing activity of SDH.     

The Role of MPK6 as Mediator of Ethylene/Jasmonic Acid Signaling in <Emphasis Type="Italic">Serendipita indica</Emphasis>-Colonized Arabidopsis Roots

Serendipita indica is an axenically cultivable fungus, which colonizes a broad range of plant species including the model plant Arabidopsis thaliana. Root colonization by this endophyte leads to enhanced plant fitness and performance and promotes resistance against different biotic and abiotic stresses. The involvement of MPK6 in this mutualistic interaction had been previously shown with an mpk6 A. thaliana mutant, which failed to respond to S. indica colonization. Here, we demonstrate that mpk6 roots are significantly less colonized by S. indica compared to wild-type roots and the foliar application of plant hormones, ethylene, or jasmonic acid, restores the colonization rate at least to the wild-type level. Further, hormone-treated mpk6 plants show typical S. indica-induced growth promotion effects. Moreover, expression levels of several genes related to plant defense and hormone signaling are significantly changed at different colonization phases. Our results demonstrate that the successful root colonization by S. indica depends on efficient suppression of plant immune responses. In A. thaliana, this process relies on intact hormone signaling in which MPK6 seems to play a pivotal role.     

Update on <Emphasis Type="Italic">transparent testa</Emphasis> mutants from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: characterisation of new alleles from an isogenic collection

Main conclusion

We present a comprehensive overview on flavonoid-related phenotypes of A. thaliana tt and tds mutants, provide tools for their characterisation, increase the number of available alleles and demonstrate that tds3 is allelic to tt12 and tds5 to aha10.Flavonoid biosynthesis is one of the best-studied secondary metabolite pathways in plants. In the model system Arabidopsis thaliana it leads to the synthesis of three phenolic compound classes: flavonol glycosides, anthocyanins and proanthocyanidins (PAs). PAs appear brown in their oxidised polymeric forms, and most A. thaliana mutants impaired in flavonoid accumulation were identified through screens for lack of this seed coat pigmentation. These mutants are referred to as transparent testa (tt) or tannin-deficient seed (tds). More than 20 mutants of these types have been published, probably representing most of the genes relevant for PA accumulation in A. thaliana. However, data about the genes involved in PA deposition or oxidation are still rather scarce. Also, for some of the known mutants it is unclear if they represent additional loci or if they are allelic to known genes. For the present study, we have performed a systematic phenotypic characterisation of almost all available tt and tds mutants and built a collection of mutants in the genetic background of the accession Columbia to minimise effects arising from ecotype variation. We have identified a novel tt6 allele from a forward genetic screen and demonstrated that tds3 is allelic to tt12 and tds5 to aha10.

     

Overexpression of UDP-glucose dehydrogenase from <Emphasis Type="Italic">Larix gmelinii</Emphasis> enhances growth and cold tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Uridine diphosphate glucose dehydrogenase (UGDH) plays an important role in biosynthesis of hemicellulose by catalyzing oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in biosynthesis of the plant cell wall. In this study, a UGDH ortholog referred to as LgUGDH was isolated from Larix gmelinii using PCR and rapid amplification of cDNA ends techniques. Real-time PCR shows that the LgUGDH gene was expressed primarily in larch stems in addition to its roots and leaves, and Southern blot analysis indicates that UGDH is encoded by two paralogous genes in L. gmelinii. Overexpression of LgUGDH increased the content of soluble sugars and hemicelluloses and enhanced vegetative growth and cold tolerance in transgenic Arabidopsis thaliana. These results reveal that L. gmelinii UGDH participates in sucrose/polysaccharide metabolism and cell wall biosynthesis and may be a good candidate gene for enhancing plant growth, cold tolerance, and hemicellulose content.     

Comparative analysis of nonlinear growth curve models for <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> rosette leaves

As a model organism, modeling and analysis of the phenotype of Arabidopsis thaliana (A. thaliana) leaves for a given genotype can help us better understand leaf growth regulation. A. thaliana leaves growth trajectories are to be nonlinear and the leaves contribute most to the above-ground biomass. Therefore, analysis of their change regulation and development of nonlinear growth models can better understand the phenotypic characteristics of leaves (e.g., leaf size) at different growth stages. In this study, every individual leaf size of A. thaliana rosette leaves was measured during their whole life cycle using non-destructive imaging measurement. And three growth models (Gompertz model, logistic model and Von Bertalanffy model) were analyzed to quantify the rosette leaves growth process of A. thaliana. Both graphical (plots of standardized residuals) and numerical measures (AIC, R² and RMSE) were used to evaluate the fitted models. The results showed that the logistic model fitted better in describing the growth of A. thaliana leaves compared to Gompertz model and Von Bertalanffy model, as it gave higher R² and lower AIC and RMSE for the leaves of A. thaliana at different growth stages (i.e., early leaf, mid-term leaf and late leaf).     

Peroxide-dependent oxidation reactions catalyzed by CYP191A1 from <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

Objectives

To find the catalytic activities of CYP191A1 from Mycobacterium smegmatis, in which functions of most P450s are unknown, by using a set of reductase systems, peroxides, and various substrates including fatty acids and human drugs.

Results

CYP191A1 was functionally expressed in Escherichia coli and purified. Its catalytic activities were examined with fatty acids, chromogenic and fluorogenic substrates, and several human P450 substrates, in the presence of six different types of electron transfer systems, such as rat NADPH-P450 reductase, Candida NADPH-P450 reductase, ferredoxin/ferredoxin reductase, putidaredoxin/putidaredoxin reductase, and peroxides (H₂O₂ and t-butyl hydroperoxide). The reactions catalyzed by CYP191A1 included the hydroxylation and O-dealkylation of several substrates.

Conclusions

CYP191A1 preferentially catalyzes the peroxide-dependent oxidation of various substrates over the reductase-dependent reaction. Its peroxygenase activity may be used an effective biocatalytic tool to synthesize the metabolites of drugs.

     

Overexpression of <Emphasis Type="Italic">AtGRDP2</Emphasis> gene in common bean hairy roots generates vigorous plants with enhanced salt tolerance

Proteins with glycine-rich repeats have been identified in plants, mammalians, fungi, and bacteria. Plant glycine-rich proteins have been associated to stress response. Previously, we reported that the Arabidopsis thaliana AtGRDP2 gene, which encodes a protein with a glycine-rich domain, plays a role in growth and development of A. thaliana and Lactuca sativa. In this study, we generated composite Phaseolus vulgaris plants that overexpress the AtGRDP2 gene in hairy roots generated by Agrobacterium rhizogenes. We observed that hairy roots harboring the AtGRDP2 gene developed more abundant and faster-growing roots than control hairy roots generated with the wild type A. rhizogenes. In addition, composite common bean plants overexpressing the AtGRDP2 gene in roots were more tolerant to salt stress showing increments in their fresh and dry weight. Our data further support the role of plant GRDP genes in development and stress response.     

Increase in Salt Tolerance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by <Emphasis Type="Italic">TaDi19</Emphasis>

The gene expression profile chip of salt-resistant wheat mutant RH8706-49 under salt stress was investigated. The overall length of the cDNA sequence of the probe was obtained using electronic cloning and RT-PCR. An unknown gene induced by salt was obtained, cloned, and named TaDi19 (Triticum aestivum drought-induced protein). No related report or research on the protein is available. qPCR analysis showed that gene expression was induced by many stresses, such as salt. Arabidopsis thaliana was genetically transferred using the overexpressing gene, which increased its salt tolerance. After salt stress, the transgenic plant demonstrated better physiological indicators (higher Ca²⁺ and lower Na⁺) than those of the wild-type plant. Results of non-invasive micro-test technology indicate that TaDi19-overexpressing A. thaliana significantly effluxed Na⁺ after salt treatment, whereas the wild-type plant influxed Na⁺. Chelating extracellular Ca²⁺ resulted in insignificant differences in salt tolerance between overexpressing and wild-type A. thaliana. Subcellular localization showed that the gene encoding protein was mainly located in the cell membrane and nucleus. TaDi19 was overexpressed in wild-type A. thaliana, and the transgenic lines were more salt-tolerant than the control A. thaliana. Thus, the wheat gene TaDi19 could increase the salt tolerance of A. thaliana.     

Expression of Human FGF18 by Fusion with Oleosin in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Seeds

Expression of recombinant human fibroblast growth factor 18 (hFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, but they are difficult, expensive. The expression of recombinant proteins fused to oleosin protein have distinct advantages, such as safety, ease, low cost. So we have expressed hFGF18 fused to oleosin protein in the oil bodies of Arabidopsis thaliana (A. thaliana) and screen the proliferation effect of NIH3T3 cells. The vector of oleosinhFGF18 fusion gene was constructed and transformed into wild A. thaliana. Transformed A. thaliana lines were obtained by the floral dip method and confirmed using polymerase chain reaction (PCR). The PCR results indicated that the oleosin-hFGF18 fusion gene was integrated into the A. thaliana genome. The oil bodies expression of oleosin-hFGF18 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biological activity showed that oil bodies expressing oleosin-hFGF18 could stimulate the proliferation of NIH3T3 cells.     

Brassinosteroid signaling modulates submergence-induced hyponastic growth in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The role of brassinosteroids (BRs) in hyponastic growth induced by submergence was investigated in Arabidopsis thaliana. Under flooding conditions, exogenously applied BRs increased hyponastic growth of rosette leaves. This hyponastic growth was reduced in a BR insensitive mutant (bri1-5), while it was increased in a BR dominant mutant (bes1-D). Further, expression of hypoxia marker genes, HRE1 and HRE2, was elevated in submerged bes1-D. These results indicate that BRs exert a positive action on hyponastic growth of submerged Arabidopsis leaves. Expression of ethylene biosynthetic genes, such as ACS6, ACS8 and ACO1, which are up-regulated by submergence, was also activated by application of BRs and in bes1-D. The enhanced hyponastic growth in submerged bes1-D was significantly reduced by application of cobalt ion, suggesting that BRs control hyponastic growth via ethylene, which seems to be synthesized by ACO6 and ACO8 followed by ACO1 in submerged leaves. A double mutant, bes1-Dxaco1-1, showed hyponastic growth activity similar to that seen in aco1-1, demonstrating that the BR signaling for regulation of hyponastic growth seems to be an upstream event in ethylene-induced hyponastic growth under submergence in Arabidopsis.     

Fatty acid ω-hydroxylases from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

Key message

Potato StCYP86A33 complements the Arabidopsis AtCYP86A1 mutant, horst - 1.

Abstract

Suberin is a cell-wall polymer that comprises both phenolic and aliphatic components found in specialized plant cells. Aliphatic suberin is characterized by bi-functional fatty acids, typically ω-hydroxy fatty acids and α,ω-dioic acids, which are linked via glycerol to form a three-dimensional polymer network. In potato (Solanum tuberosum L.), over 65 % of aliphatics are either ω-hydroxy fatty acids or α,ω-dioic acids. Since the biosynthesis of α,ω-dioic acids proceeds sequentially through ω-hydroxy fatty acids, the formation of ω-hydroxy fatty acids represents a significant metabolic commitment during suberin deposition. Four different plant cytochrome P450 subfamilies catalyze ω-hydroxylation, namely, 86A, 86B, 94A, and 704B; though to date, only a few members have been functionally characterized. In potato, CYP86A33 has been identified and implicated in suberin biosynthesis through reverse genetics (RNAi); however, attempts to express the CYP86A33 protein and characterize its catalytic function have been unsuccessful. Herein, we describe eight fatty acid ω-hydroxylase genes (three CYP86As, one CYP86B, three CYP94As, and a CYP704B) from potato and demonstrate their tissue expression. We also complement the Arabidopsis cyp86A1 mutant horst-1 using StCYP86A33 under the control of the Arabidopsis AtCYP86A1 promoter. Furthermore, we provide preliminary analysis of the StCYP86A33 promoter using a hairy root transformation system to monitor pStCYP86A33::GUS expression constructs. These data confirm the functional role of StCYP86A33 as a fatty acid ω-hydroxylase, and demonstrate the utility of hairy roots in the study of root-specific genes.