<Emphasis Type="Italic">SnRK2</Emphasis> Homologs in <Emphasis Type="Italic">Gossypium</Emphasis> and <Emphasis Type="Italic">GhSnRK2.6</Emphasis> Improved Salt Tolerance in Transgenic Upland Cotton and <Emphasis Type="Italic">Arabidopsis</Emphasis>

SnRK2s are a large family of plant-specific protein kinases, which play important roles in multiple abiotic stress responses in various plant species. But the family in Gossypium has not been well studied. Here, we identified 13, 10, and 13 members of the SnRK2 family from Gossypium raimondii, Gossypium arboreum, and Gossypium hirsutum, respectively, and analyzed the locations of SnRK2 homologs in chromosomes based on genome data of cotton species. Phylogenetic tree analysis of SnRK2 proteins showed that these families were classified into three groups. All SnRK2 genes were comprised of nine exons and eight introns, and the exon distributions and the intron phase of homolog genes among different cotton species were analogous. Moreover, GhSnRK2.6 was overexpressed in Arabidopsis and upland cotton, respectively. Under salt treatment, overexpressed Arabidopsis could maintain higher biomass accumulation than wild-type plants, and GhSnRK2.6 overexpression in cotton exhibited higher germination rate than the control. So, the gene GhSnRK2.6 could be utilized in cotton breeding for salt tolerance.     

Transformation of bahiagrass (<Emphasis Type="Italic">Paspalum notatum</Emphasis> Flugge)

Bahiagrass (Paspalum notatum Flugge), a forage species widely used in the southeastern United States, and from Central Mexico to Argentina, was targeted for improvement through genetic engineering. Embryogenic callus, initiated from germinating seedlings, was bombarded with a vector containing the bar selectable marker/reporter gene that confers resistance to phosphinothricin (glufosinate) herbicide (trade names Liberty, Ignite and Finale). Thirty-two transgenic plants were recovered. These plants were identified by the polymerase chain reaction (PCR) and verified by Southern analysis. Transgenic plants with bar, as well as non-transgenic plants without bar, regenerated from bombarded callus and selected with glufosinate, developed strong and stable resistance to glufosinate during selection. This unusual resistance in non-transgenic plants has persisted for over a year and is passed on to new tillers. The development of resistance in non-transgenic cells reduced the herbicide selection efficiency and made it necessary to identify transgenic plants by PCR where the 32 transgenic plants were recovered from 674 glufosinate-resistant plants, giving a very low selection efficiency.     

First microsatellite markers for <Emphasis Type="Italic">Paspalum plicatulum</Emphasis> (Poaceae) characterization and cross-amplification in different <Emphasis Type="Italic">Paspalum</Emphasis> species of the Plicatula group

Background

Paspalum plicatulum is a perennial rhizomatous grass with natural diploid and polyploid cytotypes. It is a member of Plicatula, which has historically been recognized as a highly complex group containing species of ecological, ornamental and forage importance. The complex nature of the P. plicatulum genome makes it a challenging species for genetic research. This study aimed to develop and characterize microsatellite molecular markers in P. plicatulum and to evaluate their transferability to other Plicatula group species.

Findings

Microsatellite sequences were identified from three enriched libraries from P. plicatulum. Specific primers were designed, and 25 displayed polymorphism when screened across 48 polyploid Paspalum spp. genotypes. The number of bands per locus ranged from 2 to 17, with a mean of 8.65. Private bands for each species were identified; the highest number of private bands was observed for P. plicatulum in 52% of the loci analyzed. The mean polymorphism information content of all loci was 0.69, and the mean discriminatory power was 0.82. Microsatellite markers were satisfactorily cross-amplified for the eight tested Plicatula-group Paspalum species, with P. atratum exhibiting the highest transferability rate (89.86%). STRUCTURE and Discriminant Analysis of Principal Components separated accessions into three groups but did not reveal separation of the accessions according to species.

Conclusions

This study describes the first microsatellite markers in P. plicatulum, which are polymorphic, efficient for the detection and quantification of genetic variation, and show high transferability into other species of the Plicatula group. This set of markers can be used in future genetic and molecular studies necessary for the proper development of conservation and breeding programs. Private bands within the markers can be used to assist in species identification.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

Genome-wide identification and evolution of <Emphasis Type="Italic">TC1</Emphasis>/<Emphasis Type="Italic">Mariner</Emphasis> in the silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>) genome

TC1/Mariner transposons belong to class II transposable elements (TEs) that use DNA-mediated “cut and paste” mechanism to transpose, and they have been identified in almost all organisms. Although silkworm (Bombyx mori) has a large amount of TC1/Mariner elements, the genome wide information of this superfamily in the silkworm is unknown. In this study, we have identified 2670 TC1/Mariner (Bmmar) elements in the silkworm genome. All the TEs were classified into 22 families by means of fgclust, a tool of repetitive sequence classification, seven of which was first reported in this study. Phylogenetic and structure analyses based on the catalytic domain (DDxD/E) of transposase sequences indicated that all members of TC1/Mariner were grouped into five subgroups: Mariner, Tc1, maT, DD40D and DD41D/E. Of these five subgroups, maT rather than Mariner possessed most members of TC1/Mariner (51.23%) in the silkworm genome. In particular, phylogenetic analysis and structure analysis revealed that Bmmar15 (DD40D) formed a new basal subgroup of TC1/Mariner element in insects, which was referred to as bmori. Furthermore, we concluded that DD40D appeared to intermediate between mariner and Tc1. Finally, we estimated the insertion time for each copy of TC1/Mariner in the silkworm and found that most of members were dramatically amplified during a period from 0 to 1 mya. Moreover, the detailed functional data analysis showed that Bmmar1, Bmmar6 and Bmmar9 had EST evidence and intact transposases. These implied that TC1/Mariner might have potential transpositional activity. In conclusion, this study provides some new insights into the landscape, origin and evolution of TC1/Mariner in the insect genomes.     

<Emphasis Type="Italic">Rhizobium</Emphasis> strains in the biological control of the phytopathogenic fungi <Emphasis Type="Italic">Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>) <Emphasis Type="Italic">rolfsii</Emphasis> on the common bean

Aims

To identify Rhizobium strains’ ability to biocontrol Sclerotium rolfsii, a fungus that causes serious damage to the common bean and other important crops, 78 previously isolated rhizobia from common bean were assessed.

Methods

Dual cultures, volatiles, indole-acetic acid (IAA), siderophore production and 16S rRNA sequencing were employed to select strains for pot and field experiments.

Results

Thirty-three antagonistic strains were detected in dual cultures, 16 of which were able to inhibit ≥84% fungus mycelial growth. Antagonistic strains produced up to 36.5 μg mL^?1 of IAA, and a direct correlation was verified between IAA production and mycelium inhibition. SEMIA 460 inhibited 45% of mycelial growth through volatile compounds. 16S rRNA sequences confirmed strains as Rhizobium species. In pot condition, common bean plants grown on S. rolfsii-infested soil and inoculated with SEMIA 4032, 4077, 4088, 4080, 4085, or 439 presented less or no disease symptoms. The most efficient strains under field conditions, SEMIA 439 and 4088, decreased disease incidence by 18.3 and 14.5% of the S. rolfsii-infested control.

Conclusions

Rhizobium strains could be strong antagonists towards S. rolfsii growth. SEMIA 4032, 4077, 4088, 4080, 4085, and 439 are effective in the biological control of the collar rot of the common bean.

     

Identification of a novel <Emphasis Type="Italic">SPLIT</Emphasis>-<Emphasis Type="Italic">HULL</Emphasis> (<Emphasis Type="Italic">SPH</Emphasis>) gene associated with hull splitting in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Key message

The split-hull phenotype caused by reduced lemma width and low lignin content is under control of SPH encoding a type-2 13-lipoxygenase and contributes to high dehulling efficiency.

Abstract

Rice hulls consist of two bract-like structures, the lemma and palea. The hull is an important organ that helps to protect seeds from environmental stress, determines seed shape, and ensures grain filling. Achieving optimal hull size and morphology is beneficial for seed development. We characterized the split-hull (sph) mutant in rice, which exhibits hull splitting in the interlocking part between lemma and palea and/or the folded part of the lemma during the grain filling stage. Morphological and chemical analysis revealed that reduction in the width of the lemma and lignin content of the hull in the sph mutant might be the cause of hull splitting. Genetic analysis indicated that the mutant phenotype was controlled by a single recessive gene, sph (Os04g0447100), which encodes a type-2 13-lipoxygenase. SPH knockout and knockdown transgenic plants displayed the same split-hull phenotype as in the mutant. The sph mutant showed significantly higher linoleic and linolenic acid (substrates of lipoxygenase) contents in spikelets compared to the wild type. It is probably due to the genetic defect of SPH and subsequent decrease in lipoxygenase activity. In dehulling experiment, the sph mutant showed high dehulling efficiency even by a weak tearing force in a dehulling machine. Collectively, the results provide a basis for understanding of the functional role of lipoxygenase in structure and maintenance of hulls, and would facilitate breeding of easy-dehulling rice.

     

Drastic anthocyanin increase in response to <Emphasis Type="Italic">PAP1</Emphasis> overexpression in <Emphasis Type="Italic">fls1</Emphasis> knockout mutant confers enhanced osmotic stress tolerance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

pap1 - D/fls1ko double mutant plants that produce substantial amounts of anthocyanin show tolerance to abiotic stress.

Abstract

Anthocyanins are flavonoids that are abundant in various plants and have beneficial effects on both plants and humans. Many genes in flavonoid biosynthetic pathways have been identified, including those in the MYB-bHLH-WD40 (MBW) complex. The MYB gene Production of Anthocyanin Pigment 1 (PAP1) plays a particularly important role in anthocyanin accumulation. PAP1 expression in many plant systems strongly increases anthocyanin levels, resulting in a dark purple color in many plant organs. In this study, we generated double mutant plants that harbor fls1ko in the pap1-D background (i.e., pap1-D/fls1ko plants), to examine whether anthocyanins can be further enhanced by blocking flavonol biosynthesis under PAP1 overexpression. We also wanted to examine whether the increased anthocyanin levels contribute to defense against osmotic stresses. The pap1-D/fls1ko mutants accumulated higher anthocyanin levels than pap1-D plants in both control and sucrose-treated conditions. However, flavonoid biosynthesis genes were slightly down-regulated in the pap1-D/fls1ko seedlings as compared to their expression in pap1-D seedlings. We also report the performance of pap1-D/fls1ko seedlings in response to plant osmotic stresses.

     

<Emphasis Type="Italic">Cantharellus</Emphasis> sect. <Emphasis Type="Italic">Amethystini</Emphasis> in Asia

In this contribution on the genus Cantharellus in Asia, C. subvaginatus is described from the Republic of Korea as a close relative to the Chinese C. vaginatus, which is here reported for the first time from India. Both species are here placed in Cantharellus subg. Cantharellus sect. Amethystini, together with the Indian C. pseudoformosus (syn.: C. umbonatus) and the Malayan C. subamethysteus. As such, Asia has suddenly become the continent with the highest diversity for Amethystini. Species delimitation in sect. Amethystini is molecularly supported by a combined phylogenetic analysis of rDNA sequences obtained for LSU and ITS and additionally suggests the existence of a still undescribed species in North America. Character variability is discussed for all known members of Amethystini, including atypical specimens of the North American C. lewisii that are morphologically more reminiscent of the South Korean C. subvaginatus.     

Enhanced <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> immune responses caused by heterologous plant genes from <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Background

Pinellia ternata is a Chinese traditional medicinal herb, used to cure diseases including insomnia, eclampsia and cervical carcinoma, for hundreds of years. Non-self-recognition in multicellular organisms can initiate the innate immunity to avoid the invasion of pathogens. A design for pathogen independent, heterosis based, fresh resistance can be generated in F₁ hybrid was proposed.

Results

By library functional screening, we found that P. ternata genes, named as ptHR375 and ptHR941, were identified with the potential to trigger a hypersensitive response in Nicotiana benthamiana. Significant induction of ROS and Callose deposition in N. benthamiana leaves along with activation of pathogenesis-related genes viz.; PR-1a, PR-5, PDF1.2, NPR1, PAL, RBOHB and ERF1 and antioxidant enzymes was observed. After transformation into N. benthamiana, expression of pathogenesis related genes was significantly up-regulated to generate high level of resistance against Phytophthora capsici without affecting the normal seed germination and morphological characters of the transformed N. benthamiana. UPLC-QTOF-MS analysis of ptHR375 transformed N. benthamiana revealed the induction of Oxytetracycline, Cuelure, Allantoin, Diethylstilbestrol and 1,2-Benzisothiazol-3(2H)-one as bioactive compounds. Here we also proved that F₁ hybrids, produced by crossing of the ptHR375 and ptHR941 transformed and non-transformed N. benthamiana, show significant high levels of PR-gene expressions and pathogen resistance.

Conclusions

Heterologous plant genes can activate disease resistance in another plant species and furthermore, by generating F₁ hybrids, fresh pathogen independent plant immunity can be obtained. It is also concluded that ptHR375 and ptHR941 play their role in SA and JA/ET defense pathways to activate the resistance against invading pathogens.

     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.