<Emphasis Type="Italic">ALDH2</Emphasis> genes are negatively correlated with natural deastringency in Chinese PCNA persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.)

Chinese pollination-constant and non-astringent persimmon (C-PCNA) has important application values in the genetic improvement of PCNA for its trait of natural deastringency controlled by a single dominant gene. However, the key genes and the regulatory networks are still not fully understood. The process of C-PCNA natural deastringency may be associated with the acetaldehyde-mediated coagulation of soluble tannins, but the functions of ALDH2 genes related to the metabolism of acetaldehyde are not clear. In this work, three types of persimmon cultivars, ‘Eshi 1’ and ‘Luotian Tianshi’ (C-PCNA type), ‘Youhou’ (J-PCNA type), and ‘Mopanshi’ (non-PCNA type), were sampled. Two members of ALDH2 family genes, DkALDH2a and DkALDH2b, were isolated from ‘Eshi 1’ persimmon fruit. Gene expression patterns indicated that they may be involved in “coagulation effect”, which leads to natural deastringency in C-PCNA persimmon fruit. Transient expression in ‘Eshi 1’ leaves further demonstrated that their expression can reduce the consumption of soluble tannins and inhibit the astringency removal process. Therefore, DkALDH2a and DkALDH2b are negatively correlated with natural deastringency in C-PCNA persimmon.     

SSR-based molecular profiling of 237 persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.) germplasms using an <Emphasis Type="Italic">ASTRINGENCY</Emphasis>-linked marker

Pollination-constant non-astringent (PCNA) trait is desirable in persimmon production because it confers natural astringency loss in mature persimmon fruit. Expression of the PCNA trait requires six homozygous recessive PCNA (ast) alleles at the single ASTRINGENCY (AST) locus in hexaploid persimmon. When crossing non-PCNA accessions to breed PCNA offspring, knowledge of ast and non-PCNA (AST) allele dosage in the parental accessions is important, because more PCNA offspring can segregate from a non-PCNA parent with more ast and fewer AST alleles. Previously, we have demonstrated that a region linked to the AST locus has numerous fragment size polymorphisms with varying numbers of simple sequence repeats. Here, we reveal the polymorphisms in this region in a broad collection of persimmon germplasms. Among 237 accessions, we distinguished 21 AST- and 5 ast-linked fragments with different sizes. Based on the number of fragments detected per individual, we identified 21 non-PCNA accessions with three different ast alleles; by crossing these with a PCNA parent, we obtain PCNA offspring under autohexaploid inheritance. Furthermore, AST and ast allelic combination patterns in hexaploid persimmon were shown to be applicable to cultivar identification of non-PCNA accessions. We directly sequenced ast-linked fragments from 48 accessions with one-size peak of ast-linked fragment and found two distinctive groups of fragments based on single nucleotide polymorphisms. This result suggests that a bottleneck event occurred during ast allele development. We conclude that our fragment size profile can be used to accelerate PCNA breeding that uses non-PCNA parents and to study ast allele accumulation in persimmon.     

Characterization of a highly polymorphic region closely linked to the <Emphasis Type="Italic">AST</Emphasis> locus and its potential use in breeding of hexaploid persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.)

The pollination-constant, non-astringent (PCNA) type of persimmon is ideal for production because its fruits lose astringency at harvest regardless of seed formation. The PCNA trait in Japanese persimmons is controlled by a single locus, AST, and is recessive to the non-PCNA trait. Because cultivated persimmon is hexaploid, only the homozygous genotype with six recessive alleles is PCNA. A region tightly linked to AST has been used as a DNA marker for breeding. Three non-PCNA (A) alleles have been reported. Here, we show that the region linked to AST is highly polymorphic and includes microsatellites. By analyzing the size of PCR-amplified fragments, we distinguished 12 different A alleles from 14 non-PCNA cultivars and a Chinese PCNA ‘Luotian-tianshi.’ Then, using A fragment size, we assessed A allele inheritance in six non-PCNA × PCNA populations by analyzing segregation of each A allele in a population and segregation of progeny genotypes. By using A allele segregation analysis, we were able to estimate the copy number of each A allele in five non-PCNA parents but not in ‘Amahyakume.’ By analyzing progeny genotype segregation, we were able to estimate the ‘Amahyakume’ genotype. Our approach can be used not only for the selection of PCNA individuals in populations, but also for estimation of the copy number of A alleles in a possible non-PCNA parent. This would enable us to select non-PCNA parents with fewer A alleles, which would segregate more PCNA individuals in crosses with PCNA cultivars.     

<Emphasis Type="Italic">chr</Emphasis> genes from adaptive replicons are responsible for chromate resistance by <Emphasis Type="Italic">Burkholderia xenovorans</Emphasis> LB400

The chromate ion transporter (CHR) superfamily includes proteins that confer chromate resistance by extruding toxic chromate ions from cytoplasm. Burkholderia xenovorans strain LB400 encodes six CHR homologues in its multireplicon genome and has been reported as highly chromate-resistant. The objective of this work was to analyze the involvement of chr redundant genes in chromate resistance by LB400. It was found that B. xenovorans plant rhizosphere strains lacking the megaplasmid are chromate-sensitive, suggesting that the chr gene present in this replicon is responsible for the chromate-resistance phenotype of the LB400 strain. Transformation of a chromate-sensitive B. xenovorans strain with each of the six cloned LB400 chr genes showed that genes from ‘adaptive replicons’ (chrA1b and chr1NCb from chromosome 2 and chrA2 from the megaplasmid) conferred higher chromate resistance levels than chr genes from ‘central’ chromosome 1 (chrA1a, chrA6, and chr1NCa). An LB400 insertion mutant affected in the chrA2 gene displayed a chromate-sensitive phenotype, which was fully reverted by transferring the chrA2 wild-type gene, and partially reverted by chrA1b or chr1NCb genes. These data indicate that chr genes from adaptive replicons, mainly chrA2 from the megaplasmid, are responsible for the B. xenovorans LB400 chromate-resistance phenotype.     

Horizontal transfer of the C-termini of <Emphasis Type="Italic">tccC</Emphasis> genes in <Emphasis Type="Italic">Photorhabdus</Emphasis> and <Emphasis Type="Italic">Xenorhabdus</Emphasis>

The high molecular weight insecticidal toxin complexes (Tcs), including four toxin-complex loci (tca, tcb, tcc and tcd), were first identified in Photorhabdus luminescens W14. Each member of tca, tcb or tcc is required for oral toxicity of Tcs. However, the sequence sources of the C-termini of tccC3, tccC4, tccC6 and tccC7 are unknown. Here, we performed a whole genome survey to identify the orthologs of Tc genes, and found 165 such genes in 14 bacterial genomes, including 40 genes homologous to tccC1-7 in P. luminescens TT01. The sequence sources of the C-termini of tccC2-6 were determined by sequence analysis. Further phylogenetic investigations suggested that the C-termini of 6 tccC genes experienced horizontal gene transfer events.     

An ATP-Binding Cassette Transporter Gene from <Emphasis Type="Italic">Cucumis sativus</Emphasis> L., <Emphasis Type="Italic">CsABC19</Emphasis>, Is Involved in Propamocarb Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A propamocarb-responsive gene named CsABC19 was isolated from a cucumber cultivar ‘D0351’ using a homologous cloning strategy. The full-length cDNA of CsABC19 was 921 bp with a complete ORF encoding 306 amino acids. Quantitative real-time PCR analysis revealed that CsABC19 was induced in the root, stem, leaf, and fruit by propamocarb and the expression levels of CsABC19 seemed to be different in different tissues. Further functional analysis showed that CsABC19 transgenic Arabidopsis plants appeared better growth performance under propamocarb stress and lower propamocarb residues. Our findings suggest that CsABC19 plays a crucial role in plant responses to propamocarb stress and also provide new clues for the mechanism regulation of the responses to propamocarb stress in cucumber.     

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.

     

Evolutionary divergence of the rye <Emphasis Type="Italic">Pm17</Emphasis> and <Emphasis Type="Italic">Pm8</Emphasis> resistance genes reveals ancient diversity

Key message

We have isolated a novel powdery mildew resistance gene in wheat that was originally introgressed from rye. Further analysis revealed evolutionary divergent history of wheat and rye orthologous resistance genes.

Abstract

Wheat production is under constant threat from a number of fungal pathogens, among them is wheat powdery mildew (Blumeria graminis f. sp. tritici). Deployment of resistance genes is the most economical and sustainable method for mildew control. However, domestication and selective breeding have narrowed genetic diversity of modern wheat germplasm, and breeders have relied on wheat relatives for enriching its gene pool through introgression. Translocations where the 1RS chromosome arm was introgressed from rye to wheat have improved yield and resistance against various pathogens. Here, we isolated the Pm17 mildew resistance gene located on the 1RS introgression in wheat cultivar ‘Amigo’ and found that it is an allele or a close paralog of the Pm8 gene isolated earlier from ‘Petkus’ rye. Functional validation using transient and stable transformation confirmed the identity of Pm17. Analysis of Pm17 and Pm8 coding regions revealed an overall identity of 82.9% at the protein level, with the LRR domains being most divergent. Our analysis also showed that the two rye genes are much more diverse compared to the variants encoded by the Pm3 gene in wheat, which is orthologous to Pm17/Pm8 as concluded from highly conserved upstream sequences in all these genes. Thus, the evolutionary history of these orthologous loci differs in the cereal species rye and wheat and demonstrates that orthologous resistance genes can take different routes towards functionally active genes. These findings suggest that the isolation of Pm3/Pm8/Pm17 orthologs from other grass species, additional alleles from the rye germplasm as well as possibly synthetic variants will result in novel resistance genes useful in wheat breeding.

     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

Pyr-miR171f-targeted <Emphasis Type="Italic">PyrSCL6</Emphasis> and <Emphasis Type="Italic">PyrSCL22</Emphasis> genes regulate shoot growth by responding to IAA signaling in pear

MicroRNA171 (miR171) is a highly conserved miRNA family, crucial for plant growth and development, and has been reported in Arabidopsis thaliana and tomato (Solanum lycopersicum), but the role of miR171 has not been explored in pear. In this study, an effort was made to decipher the mechanism underlying dwarf in ‘Zhongai 3’, of which the shoot length and shoot growth rate during the growing season were much less than those of the vigorous cultivar ‘Zaosu’, and the same for the indole-3-acetic acid (IAA) content in shoot tips after May 22, 2016. We identified a member of the miR171 family, which was most sensitive to IAA and targeted two genes conformed by 5′-RACE, and we named Pyr-miR171f. The two targets were named as PyrSCL6 and PyrSCL22, and contained a GRAS-conserved domain and encoded nucleus proteins. Quantitative RT-PCR analysis revealed that Pyr-miR171f was more abundant in ‘Zaosu’ shoot tips than in ‘Zhongai 3’ shoot tips, whereas the PyrSCL6 and PyrSCL22 mRNAs were more abundant in ‘Zhongai 3’ shoot tips than in ‘Zaosu’ shoot tips. The abundance of Pyr-miR171f and PyrSCL6 and PyrSCL22 mRNAs increased, but the trends were opposite between Pyr-miR171f and its target mRNAs in tissue culture seedlings treated by IAA. Our results suggest that IAA-induced miR171f negatively regulates the IAA signaling cascade via the GRAS pathway to maintain apical dominance. This work reveals a role for the miR171-SCL pathway in the dwarfing of ‘Zhongai 3’, and provides a theoretical basis for dwarf pear breeding.     

Functional Analysis of <Emphasis Type="Italic">VvBG1</Emphasis> During Fruit Development and Ripening of Grape

β-glucosidase (BG) was believed to take part in abscisic acid (ABA) synthesis via hydrolysis of ABA glucose ester to release active ABA during plant growth and development. However, there is no genetic evidence available to indicate the role of genes during fruit ripening. Here, the expression patterns of three genes (VvBG1, VvBG2, and VvBG3) encoding β-glucosidase were analyzed during grape fruit development, and it was found that β-glucosidase activity increased in grape fruit in response to various stresses. Furthermore, to verify the function of β-glucosidase during fruit ripening, heterogeneous expression of the VvBG1 gene in strawberry fruit was validated, and the results showed that the VvBG1 over-expression increased β-glucosidase and promoted the fruit ripening process in strawberry. In addition, we found that ABA contents increased in the VvBG1 over-expression of strawberry fruit, which induced fruit anthocyanin, soluble solid accumulation, and fruit softening. Moreover, genes related to coloring (CHS, CHI, F3H, and UFGT), softening (PG1, PL1, and EXP1), and aroma (SAAT, and QR) were up-regulated. This work will elucidate the specific roles of VvBGs in the synthesis of ABA and provide some new insights into the ABA-controlled grape ripening mechanism.