A betaine aldehyde dehydrogenase gene in quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis>): structure,phylogeny, and expression pattern

Betaine aldehyde dehydrogenase (BADH) is widely considered as a key enzyme in glycine betaine metabolism in higher plants. Several paralogous genes encoding different isozymes of BADH have been identified and characterized in some plants; however, until now, only limited information is available about BADH genes in quinoa (Chenopodium quinoa). Here, we report the molecular cloning, structural organization, phylogenetic evolution, and expression profile of a BADH gene (CqBADH1) from quinoa. The translated putative CqBADH1 protein included five conserved features of the ALDH Family 10. Comparisons between the cDNA and genomic sequences revealed that the CqBADH1 gene contained 15 exons and 14 introns. Comparative screening of introns in homologous genes demonstrated that the number and position of the BADH introns were highly conserved among the BADH genes in Amaranthaceae plants and in other more distantly related plant species. A phylogenetic analysis showed that CqBADH1 had the closest relationship with a protein from Atriplex canescens and belonged to the ALDH10 family. Expression profile analyses indicated that CqBADH1 was expressed only in root, and showed time-dependent expression profiles under NaCl-stress condition. Moreover, in quinoa, NaCl stress led to increased levels of CqBADH1 mRNA accompanied by the accumulation of glycine betaine. This is the first study to describe a BADH gene in quinoa.     

<Emphasis Type="Italic">Viola woosanensis</Emphasis>, a recurrent spontaneous hybrid between <Emphasis Type="Italic">V. ulleungdoensis</Emphasis> and <Emphasis Type="Italic">V. chaerophylloides</Emphasis> (Violaceae) endemic to Ulleung Island,Korea

Ulleung Island is an oceanic volcanic island in Korea, which has never been connected to the adjacent continent. Previous studies highlighted Ulleung Island as an excellent system to study the pattern and process of early stages of flowering plant evolutions on oceanic island. The predominant mode of speciation in flowering plants on Ulleung Island appears to be anagenesis. However, the potentially important role of hybrid speciation among incompletely reproductively isolated lineages cannot be ruled out. Viola woosanensis (Violaceae) is of purportedly hybrid origin between V. ulleungdoensis (i.e., formerly recognized as V. selkirkii in Ulleung Island) and V. chaerophylloides, based on morphology. To examine the origin of V. woosanensis, we sampled a total of 80 accessions, including V. woosanensis and its putative parental species and sequenced nrDNA ITS, and four highly variable chloroplast noncoding regions (trnL-trnF, rpl16 intron, atpF-atpH, and psbA-trnH). Representative species of Viola from Korea were also included in the phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference). Additive polymorphic sites in the nrDNA ITS regions were confirmed by cloning amplicons from representative species. The molecular data strongly supported the hybrid origin of V. woosanensis, and the maternal and paternal parent were determined to be V. ulleungdoensis and V. chaerophylloides, respectively. The presence of two parental ribotypes in V. woosanensis (with the exception in one population) was confirmed by cloning, suggesting V. woosanensis is primarily the F₁ generation. No trace of backcrossing and introgression with its parents was detected due to low fertility of hybrid species. We found a multiple and unidirectional hybrid origin of V. woosanensis. Additional studies are required to determine which factors contribute to asymmetric gene flow of Viola species in Ulleung Island.     

The opposite roles of <Emphasis Type="Italic">OsmiR408</Emphasis> in cold and drought stress responses in <Emphasis Type="Italic">Oryza sativa</Emphasis>

MiR408 is a conserved miRNA family in plants. Although AtmiR408 is generally regarded as participating in stress responses, it still remains obscure whether OsmiR408 modulates tolerance to environmental stress. In the current study, expression of Pre-OsmiR408 and OsmiR408 was found to be induced by cold stress, but repressed by drought stress in the rice cultivar “Kongyu 131”. By comparing the wild type and OsmiR408 transgenic lines, we found that OsmiR408 overexpression conferred enhanced cold tolerance at both the early seedling stage and the young seedling stage. On the other hand, the OsmiR408 transgenic lines exhibited decreased drought tolerance, which is further verified by greater water loss. We also predicted the putative target genes of OsmiR408 and verified the decreased expression of seven targets in OsmiR408 transgenic lines, including four phytocyanins and three atypical target genes. Among them, Os09g29390, a phytocyanin gene, and Os01g53880, an auxin responsive Aux/IAA gene, were down-regulated by cold treatment, which is opposite to the cold-induced expression of OsmiR408. Taken together, our results suggest opposite roles of OsmiR408 in plant responses to cold and drought stresses.     

Isocitrate dehydrogenase mutation in <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> results in virulence attenuation and immunoprotection in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss)</Emphasis>

Background

Vibrio anguillarum is an extracellular bacterial pathogen that is a causative agent of vibriosis in finfish and crustaceans with mortality rates ranging from 30% to 100%. Mutations in central metabolism (glycolysis and the TCA cycle) of intracellular pathogens often result in attenuated virulence due to depletion of required metabolic intermediates; however, it was not known whether mutations in central metabolism would affect virulence in an extracellular pathogen such as V. anguillarum.

Results

Seven central metabolism mutants were created and characterized with regard to growth in minimal and complex media, expression of virulence genes, and virulence in juvenile rainbow trout (Oncorhynchus mykiss). Only the isocitrate dehydrogenase (icd) mutant was attenuated in virulence against rainbow trout challenged by either intraperitoneal injection or immersion. Further, the icd mutant was shown to be immunoprotective against wild type V. anguillarum infection. There was no significant decrease in the expression of the three hemolysin genes detected by qRT-PCR. Additionally, only the icd mutant exhibited a significantly decreased growth yield in complex media. Growth yield was directly related to the abundance of glutamate. A strain with a restored wild type icd gene was created and shown to restore growth to a wild type cell density in complex media and pathogenicity in rainbow trout.

Conclusions

The data strongly suggest that a decreased growth yield, resulting from the inability to synthesize α-ketoglutarate, caused the attenuation despite normal levels of expression of virulence genes. Therefore, the ability of an extracellular pathogen to cause disease is dependent upon the availability of host-supplied nutrients for growth. Additionally, a live vaccine strain could be created from an icd deletion strain.

     

Secreted proteins produced by fungi associated with <Emphasis Type="Italic">Botryosphaeria</Emphasis> dieback trigger distinct defense responses in <Emphasis Type="Italic">Vitis vinifera</Emphasis> and <Emphasis Type="Italic">Vitis rupestris</Emphasis> cells

Grapevine trunk diseases (Eutypa dieback, esca and Botryosphaeria dieback) are caused by a complex of xylem-inhabiting fungi, which severely reduce yields in vineyards. Botryosphaeria dieback is associated with Botryosphaeriaceae. In order to develop effective strategies against Botryosphaeria dieback, we investigated the molecular basis of grapevine interactions with a virulent species, Neofusicoccum parvum, and a weak pathogen, Diplodia seriata. We investigated defenses induced by purified secreted fungal proteins within suspension cells of Vitis (Vitis rupestris and Vitis vinifera cv. Gewurztraminer) with putative different susceptibility to Botryosphaeria dieback. Our results show that Vitis cells are able to detect secreted proteins produced by Botryosphaeriaceae, resulting in a rapid alkalinization of the extracellular medium and the production of reactive oxygen species. Concerning early defense responses, N. parvum proteins induced a more intense response compared to D. seriata. Early and late defense responses, i.e., extracellular medium alkalinization, cell death, and expression of PR defense genes were stronger in V. rupestris compared to V. vinifera, except for stilbene production. Secreted Botryosphaeriaceae proteins triggered a high accumulation of δ-viniferin in V. vinifera suspension cells. Artificial inoculation assays on detached canes with N. parvum and D. seriata showed that the development of necrosis is reduced in V. rupestris compared to V. vinifera cv. Gewurztraminer. This may be related to a more efficient induction of defense responses in V. rupestris, although not sufficient to completely inhibit fungal colonization. Overall, our work shows a specific signature of defense responses depending on the grapevine genotype and the fungal species.     

<Emphasis Type="Italic">glyA</Emphasis> gene knock-out in <Emphasis Type="Italic">Escherichia coli</Emphasis> enhances L-serine production without glycine addition

In E. coli, glyA encodes for serine hydroxymethyltransferase (SHMT), which converts L-serine to glycine. When engineering L-serine-producing strains, it is therefore favorable to inactivate glyA to prevent L-serine degradation. However, most glyA knockout strains exhibit slow cell growth because of the resulting lack of glycine and C₁ units. To overcome this problem, we overexpressed the gcvTHP genes of the glycine cleavage system (GCV), to increase the C₁ supply before glyA was knocked out. Subsequently, the kbl and tdh genes were overexpressed to provide additional glycine via the L-threonine degradation pathway, thus restoring normal cell growth independent of glycine addition. Finally, the plasmid pPK10 was introduced to overexpress pgk, serA ^Δ197 , serC and serB, and the resulting strain E4G2 (pPK10) accumulated 266.3 mg/L of L-serine in a semi-defined medium without adding glycine, which was 3.18-fold higher than the production achieved by the control strain E3 (pPK10). This strategy can accordingly be applied to disrupt the L-serine degradation pathway in industrial production strains without causing negative side-effects, ultimately making L-serine production more efficient.     

Overexpression of soybean <Emphasis Type="Italic">GmERF9</Emphasis> enhances the tolerance to drought and cold in the transgenic tobacco

Ethylene response factors (ERFs) are widespread in plants, which are widely involved in plant response to biotic and abiotic stress. In this research, a soybean gene, GmERF9, was identified and the function was characterized. The results showed that GmERF9 contained a typical AP2/ERF binding domain and a putative nuclear localization signal sequence. The real-time fluorescence quantitative PCR (qPCR) revealed that the expression of GmERF9 could be induced by ethylene (ET), abscisic acid (ABA), drought, salt and cold stresses. GmERF9 protein could specifically bind to the GCC-box and activate the expression of the reporter gene in the yeast cells and tobacco leaves. Overexpression of GmERF9 enhanced the expression of pathogenesis-related (PR) genes, including PR1, PR2, Osmotin (PR5), and SAR8.2. Also, the overexpression of GmERF9 increased the accumulation of proline and soluble carbohydrate, and decreased the accumulation of malondialdehyde under drought and cold stresses in the transgenic tobacco compared to the wild type (WT) tobacco, which indicated that GmERF9 enhanced the tolerance to drought and cold stresses in the transgenic tobacco. In summary, the function of GmERF9 is involved in the response to environmental stresses for plants, which can be used as a candidate gene for genetic engineering of crops.     

Functional analysis of arabinofuranosidases and a xylanase of <Emphasis Type="Italic">Corynebacterium alkanolyticum</Emphasis> for arabinoxylan utilization in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-l-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-l-arabinofuranoside with k _cat values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.     

A reexamination on the deficiency of riboflavin accumulation in Malpighian tubules in larval translucent mutants of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A variety of insects accumulate high contents of riboflavin (vitamin B₂) in their Malpighian tubules (MTs). Although this process is known to be genetically controlled, the mechanism is not known. In the 1940s and the 1950s, several studies showed that riboflavin contents were low in the MTs of some Bombyx mori (silkworm) mutants with translucent larval skin mutations (e.g., w-3, od, oa, and otm) and that genes responsible for these translucent mutations also affected riboflavin accumulation in the MTs. Since the 2000s, it has been shown that the w-3 gene encodes an ABC transporter, whereas genes responsible for od, oa, and otm mutations encode for the biogenesis of lysosome-related organelles. These findings suggest that some genes of ABC transporters and biogenesis of lysosome-related organelles may control the accumulation of riboflavin in MTs. Therefore, we reexamined the effects that translucent mutations have on the accumulation of riboflavin in MTs by using the translucent and wild-type segregants in mutant strains to measure the specific effect that each gene has on riboflavin accumulation (independent of genomic background). We used nine translucent mutations (w-3^oe, oa, od, otm, Obs, oy, or, oh, and obt) even though the genes responsible for some of these mutations (Obs, oy, or, oh, and obt) have not yet been isolated. Through observation of larval MTs and measurements of riboflavin content using high-performance liquid chromatography, we found that the oa, od, otm, and or mutations were responsible for low contents of riboflavin in MTs, whereas the Obs and oy mutations did not affect riboflavin accumulation. This indicates that the molecular mechanism for riboflavin accumulation is similar but somewhat different than the mechanism responsible for uric acid accumulation in epidermal cells. We found that the genes responsible for oa, od, and otm mutations were consistent with those already established for uric acid accumulation in larval epidermis. This suggests that these three genes control riboflavin accumulation in MTs through a mechanism similar to that of uric acid accumulation, although we do not yet know why the or mutation also controls riboflavin accumulation.     

Silencing of <Emphasis Type="Italic">GbANS</Emphasis> reduces cotton resistance to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> through decreased ROS scavenging during the pathogen invasion process

Anthocyanins are secondary metabolites that play important roles in plant adaption to adverse environments. The anthocyanin biosynthetic pathway is conserved in high plants. Previous studies revealed the significant role of anthocyanins in natural-colorized cotton. However, little is known about the involvement of anthocyanins in the interaction of cotton and pathogen. In this study, a pathogen-induced gene was isolated from Gossypium barbadense that encodes an anthocyanidin synthase protein (GbANS) with dioxygenase structures. GbANS was preferentially expressed in colored tissue. Silencing of GbANS significantly reduced the production of anthocyanins, as well as the cotton’s resistance to Verticillium dahliae. Biochemical studies revealed that GbANS-silenced cotton accumulated more hydrogen peroxide compared to control plants during the V. dahliae invasion process. This accumulation of hydrogen peroxide corresponded with increased cell death around the invasion sites, which in turn accelerated the V. dahliae infection. Taken together, we found that GbANS contributes to the biosynthesis of anthocyanins in cotton and anthocyanins positively regulate cotton’s resistance to V. dahliae.     

A putative plastidial adenine nucleotide transporter,BRITTLE1-3, plays an essential role in regulating chloroplast development in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Differentiation from proplastids into chloroplasts is a light- and energy-dependent process. How this process is regulated is still poorly understood at the molecular level. We herein report a new putative plastidial adenine nucleotide transporter, BRITTLE1-3 (referred to as OsBT1-3), encoded by the rice (Oryza sativa) White Stripe Leaf 2 (WSL2) gene. Loss of OsBT1-3 function results in defective chloroplast biogenesis, severely reduced photosynthetic efficiency, and finally a white stripe leaf phenotype in the first four leaves. The expression levels of genes related to chlorophyll biosynthesis and photosynthesis are drastically reduced, accompanied with over accumulation of reactive oxygen species (ROS) in the wsl2 mutant. OsBT1-3 is targeted to the chloroplasts and it expresses in almost all tissues in plants, especially in young leaves. OsBT1-3 consists of 419 amino acids and exhibits features of all mitochondrial carrier proteins, including a typical transmembrane-spanning domain and a highly conserved sequence motif designated as the ‘mitochondrial energy transfer signatures’. Phylogenetic analysis shows that OsBT1-3 is a putative plastidial adenine nucleotide transporter and is most closely related to ZmBT1-2. Together, these observations suggest that the new putative adenine nucleotide transporter, OsBT1-3, plays an essential role in regulating chloroplast biogenesis and maintenance of ROS homeostasis during rice seedling de-etiolation.     

A <Emphasis Type="Italic">pqr2</Emphasis> mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the paraquat-resistant mutants that have been reported in plants, this study identified a novel A. thaliana mutant (pqr2) from an XVE inducible activation library based on its resistance to 2 μM paraquat. The pqr2 mutant exhibited a termination mutation in the exon of AT1G31830/PAR1/PQR2, encoded a polyamine uptake transporter AtPUT2/PAR1/PQR2. The PQR2 mutation could largely reduce superoxide accumulation and cell death in the pqr2 plants under paraquat treatment. Moreover, compared with wild type, the pqr2 mutant exhibited much reduced tolerance to putrescine, a classic polyamine compound, which confirmed that PQR2 encoded a defective polyamine transporter. Notably, co-treated with ABA and paraquat, both pqr2 mutant and wild type exhibited a lethal phenotype from seed germination, but the wild type like pqr2 mutant, could remain paraquat-resistance while co-treated with high dosage of Na₂WO₄, an ABA synthesis inhibitor. Gene expression analysis suggested that ABA signaling should widely regulate paraquat-responsive genes distinctively in wild type and pqr2 mutant. Hence, this study has for the first time reported about ABA negative effect on paraquat-resistance in A. thaliana, providing insight into the ABA signaling involved in the oxidative stress responses induced by paraquat in plants.     

Vetch aphid, <Emphasis Type="Italic">Megoura crassicauda</Emphasis> (Hemiptera: Aphididae), parasitism does not reduce the bean production of narrow-leaved vetch, <Emphasis Type="Italic">Vicia sativa</Emphasis> subsp. <Emphasis Type="Italic">nigra</Emphasis> (Fabaceae)

Megoura crassicauda Mordvilko (Hemiptera: Aphididae) is a dominant aphid species found on Vicia sativa subsp. nigra (L.) Ehrh. (Fabaceae) in the spring. Worker ants of Formica japonica, the dominant ant species attracted to the extrafloral nectaries of V. s. nigra, often attack ladybirds (Coccinella septempunctata), which are aphid enemies. However, the workers of F. japonica do not attack or exclude M. crassicauda, the non-myrmecophilous aphid. It appears that the “bodyguard” retained by the plant guards the plant’s herbivore by attacking the herbivores’ enemies, rather than guarding the plant itself. The relationship between V. s. nigra and M. crassicauda was observed in the field to examine and evaluate the cost of parasitism. Parasitism by M. crassicauda delayed flower bud formation markedly in V. s. nigra but did not kill the plants. V. s. nigra plants that were parasitized showed a net bean production similar to that of the non-parasitized controls. The parasitism rate of M. crassicauda increased when extrafloral nectaries were used by F. japonica. These results may indicate that M. crassicauda provides V. s. nigra with benefits by preventing other serious disadvantages.