Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Pollen ultrastructure in <Emphasis Type="Italic">Aristolochia manshuriensis</Emphasis> and <Emphasis Type="Italic">A. contorta</Emphasis> (Aristolochiaceae)

Pollen ultrastructure has been studied in two relict and rare species of the genus Aristolochia, A. contorta Bunge and A. manshuriensis Kom. (Aristolochiaceae). Both species have inaperturate, spheroidal, sometimes distally monocolpate or distally bicolpate pollen grains. The equatorial and polar axes of pollen grain in A. manshuriensis are 48.5 and 44.0 μm, respectively. The percentage of defective pollen grains in A. manshuriensis is 3.4%. The fossulate, perforated exine is up to 2.3 μm in thickness; the sexine and the nexine are almost equal in thickness. In A. contorta, the equatorial axis of pollen grain is 36.6 μm: the defectiveness percentage, 24.5%. The exine is verrucate, up to 0.3 μm in thickness, while the sexine is two to three times thicker than the nexine. The pollen germination experiments have shown that pollen of A. manshuriensis, in contrast to A. contorta, can germinate in 10–20% sucrose at 22°С. These data and the high percentage of pollen defectiveness in A. contorta indicate that the androecium function in this species is reduced. The reduction of the androecium function is evidenced by a small amount of pollen grains in anthers or empty anthers and a high percentage of defective pollen grains.     

Regulatory <Emphasis Type="Italic">cis</Emphasis>-elements on citrus peel-specific expressed gene, <Emphasis Type="Italic">CuCRTISO-like</Emphasis>, promoter respond to hormones and abiotic stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

We identified a peel-specific expressed gene in Citrus unshiu fruits by differentially expressed gene (DEG) analysis, which showed a homology with carotenoid isomerase-like genes identified from other plants and, therefore, designated as CuCRTISO-like. Here we determined the promoter sequence of CuCRTISO-like and analyzed histochemical GUS activity using transgenic Arabidopsis plants harboring CuCRTISO-like promoter-GUS gene constructs (pCRTL-Prom1~pCRTL-Prom5 lines). The promoter activity of CuCRTISO-like was detected in the cotyledon at 5 and 10 days after germination (DAG), young leaf, and anther, but not in the cotyledon at 15 DAG and mature leaf. Several cis-acting elements involved in hormones and abiotic stresses are located on the CuCRTISO-like promoter. Salicylic acid and ethylene treatments induced the GUS activity in the pCRTL-prom1 and pCRTL-Prom4 line, respectively. Treatment of drought and wounding stress induced the GUS activity in the pCRTL-Prom4 and pCRTL-Prom3 line, respectively. Heat stress treatment induced GUS activity more strongly as the promoter length decreased except for no GUS activity in the pCRTL-Prom5 line. The CuCRTISO-like expression during fruit maturation of C. unshiu showed a peel-specific expression pattern. Our results suggest that CuCRTISO-like promoter activity is regulated in a developmental and organ-specific manner, and responds to hormones and abiotic stresses.     

Semi-dwarf barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) <Emphasis Type="Italic">brh2</Emphasis> and <Emphasis Type="Italic">ari-l</Emphasis> mutants are deficient in a U-box E3 ubiquitin ligase

Lodging is the process where crop plants fall over and lie on the ground due to strong winds and heavy precipitation. This problem reduces yield and increases the risk of fungal infections and pre-harvest germination. In order to avoid lodging, plant breeders utilize short-culm mutants, which often have a robust culm that can support the weight of a heavy spike. In barley (Hordeum vulgare L.), thousands of short-culm mutants have been isolated in breeding programs around the world. Our long-term goal is to reveal the genetic network underlying culm length, with the objective to provide an enlarged repertoire of genes and alleles suitable for future breeding of lodging resistant barley. In the present work we studied a group of allelic brh2 and ari-l mutants, which have a relatively strong semi-dwarf phenotype and are phenotypically similar to previously identified mutants deficient in brassinosteroid signalling or metabolism. The Brh2 gene is located in the centromeric region of chromosome 4H and we applied a candidate gene approach to identify the gene. Brh2 is orthologous to TUD1 in rice (Orysa sativa L.), which encodes a U-box E3 ubiquitin ligase. We identified one missense mutation, one nonsense mutation and four deletions of the complete Brh2 gene. The mutants could respond to exogenously applied brassinolide, which suggests that the apparent brassinosteroid deficient phenotype of barley brh2 and ari-l mutants is related to brassinosteroid metabolism rather than signalling.     

<Emphasis Type="Italic">Rf5</Emphasis> is able to partially restore fertility to Honglian-type cytoplasmic male sterile <Emphasis Type="Italic">japonica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) lines

Three-line japonica hybrids have been developed mainly on Chinsurah Boro II (BT)-type cytoplasmic male sterile (CMS) lines of Oryza sativa L., but the unstable sterility of some BT-type CMS lines, and the threat of genetic vulnerability when using a single cytoplasm source, have inhibited their use in rice cultivation. Previously, the sterility of Honglian (HL)-type japonica CMS lines derived from common red-awned wild rice (Oryza rufipogon) has been proven to be more stable than that of BT-type japonica CMS lines. Here, we genetically characterized HL-type japonica CMS lines and the restorer-of-fertility (Rf) gene for breeding HL-type japonica hybrids. HL-type japonica CMS lines displayed stained abortive pollen grains, unlike HL-type indica CMS lines. The BT-type japonica restorer lines, which contain Rf, had different capabilities to restore HL-LiuqianxinA (HL-LqxA), an HL-type japonica CMS line, and the restorers for the HL-type japonica CMS lines could be selected from the preexisting BT-type japonica restorers in rice production. A genetic analysis showed that the restoration of normal fertility to HL-LqxA was controlled by a major gene and was affected by minor effector genes and/or modifiers. The major Rf in SiR2982, a BT-type japonica restorer, was mapped to a ~100-kb physical region on chromosome 10, and was demonstrated to be Rf5 (Rf1a) by sequencing. Furthermore, Rf5 partially restored fertility and had a dosage effect on HL-type japonica CMS lines. These results will be helpful for the development of HL-type japonica hybrids.     

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.

     

Structure and expression of the <Emphasis Type="Italic">TaGW7</Emphasis> in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

OsGW7 (also known as OsGL7) is homologous to the Arabidopsis thaliana gene that encodes LONGIFOLIA protein, which regulates cell elongation, and is involved in regulating grain length in rice. However, our knowledge on its ortholog in wheat, TaGW7, is limited. In this study, we identified and mapped TaGW7 in wheat, characterized its nucleotide and protein structures, predicted the cis-elements of its promoter, and analysed its expression patterns. The GW7 orthologs in barley (HvGW7), rice (OsGW7), and Brachypodium distachyon (BdGW7) were also identified for comparative analyses. TaGW7 mapped onto the short arms of group 2 chromosomes (2AS, 2BS, and 2DS). Multiple alignments indicated GW7 possesses five exons and four introns in all but two of the species analysed. An exon–intron junction composed of introns 3–4 and exons 4–5 was highly conserved. GW7 has a conserved domain (DUF 4378) and two neighbouring low complexity regions. GW7 was mainly expressed in wheat spikes and stems, in barley seedling crowns, and in rice anthers and embryo-sacs during early development. Drought and heat significantly increased and decreased GW7 expression in wheat, respectively. In barley, GW7 was significantly down-regulated in paleae and awns but up-regulated in seeds under drought treatment and down-regulated under Fusarium and stem rust inoculation. In rice, OsGW7 expression differed significantly under drought treatments. Collectively, these results provide insights into GW7 structure and expression in wheat, barley and rice. The GW7 sequence structure and expression data are the foundation for manipulating GW7 and uncovering its roles in plants.     

<Emphasis Type="Italic">VpUR9</Emphasis>, a novel RING-type ubiquitin ligase gene from <Emphasis Type="Italic">Vitis pseudoreticulata</Emphasis>, is involved in powdery mildew response in transgenic <Emphasis Type="Italic">V. vinifera</Emphasis> plants

Ubiquitination plays important roles in disease resistance in plants. We report the identification and functional characterization of the RING-type ubiquitin ligase gene VpUR9 from Chinese wild Vitis pseudoreticulata accession Baihe-35-1. VpUR9, encodes 164 amino acids and possesses a RING conserved motif. It is homologously cloned from the cDNA library of the high powdery mildew (Erysiphe necator [Schw.] Burr) resistant V. pseudoreticulata accession Baihe-35-1 inoculated with E. necator. The gene is induced in response to powdery mildew and salicylic acid. VpUR9 fused with FLAG-tag controlled by 35S promoter was transformed into 15 regenerated V. vinifera L. cv. Red Globe lines via Agrobacterium tumefaciens-mediated transformation. Twelve of these lines were confirmed by Western blot of FLAG-tag. As a result, the powdery mildew-resistance of Red Globe transformed with VpUR9 was repressed. Furthermore, the expression of some disease-resistant related genes (NPR1, PR1, PR10 and PAL) of the transgenic Red Globe declined compared with wild type grapes when inoculated with powdery mildew or salicylic acid. When treated with jasmonic acid methyl ester, its PR1 gene expression decreased, while the expressions of NPR1, PR10 and PAL all increased, contrasting with the wild type grape.     

<Emphasis Type="Italic">Sr33</Emphasis> and <Emphasis Type="Italic">Sr35</Emphasis> gene homolog identification in genomes of cereals related to <Emphasis Type="Italic">Aegilops tauschii</Emphasis> and <Emphasis Type="Italic">Triticum monococcum</Emphasis>

Using bioinformatics analysis, the homologs of genes Sr33 and Sr35 were identified in the genomes of Triticum aestivum, Hordeum vulgare, and Triticum urartu. It is known that these genes confer resistance to highly virulent wheat stem rust races (Ug99). To identify amino acid sites important for this resistance, the found homologs were compared with the Sr33 and Sr35 protein sequences. It was found that sequences S5DMA6 and E9P785 are the closest homologs of protein RGAle, a Sr33 gene product, and sequences M7YFA9 (CNL-C) and F2E9R2 are homologs of protein CNL9, a Sr35 gene product. It is assumed that the homologs of genes Sr33 and Sr35, which were obtained from the wild relatives of wheat and barley, can confer resistance to various forms of stem rust and can be used in the future breeding programs aimed at improvement of national wheat varieties.     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

Characterisation of a novel quantitative trait locus, <Emphasis Type="Italic">GN4</Emphasis>-<Emphasis Type="Italic">1</Emphasis>, for grain number and yield in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Key message

A novel QTL for grain number, GN4-1, was identified and fine-mapped to an ~ 190-kb region on the long arm of rice chromosome 4.

Abstract

Rice grain yield is primarily determined by three components: number of panicles per plant, grain number per panicle and grain weight. Among these traits, grain number per panicle is the major contributor to grain yield formation and is a crucial trait for yield improvement. In this study, we identified a major quantitative trait locus (QTL) responsible for rice grain number on chromosome 4, designated GN4-1 (a QTL for Grain Number on chromosome 4), using advanced segregating populations derived from the crosses between an elite indica cultivar ‘Zhonghui 8006’ (ZH8006) and a japonica rice ‘Wuyunjing 8’ (WYJ8). GN4-1 was delimited to an ~ 190-kb region on chromosome 4. The genetic effect of GN4-1 was estimated using a pair of near-isogenic lines. The GN4-1 gene from WYJ8 promoted accumulation of cytokinins in the inflorescence and increased grain number per panicle by ~ 17%. More importantly, introduction of the WYJ8 GN4-1 gene into ZH8006 increased grain yield by ~ 14.3 and ~ 11.5% in the experimental plots in 2014 and 2015, respectively. In addition, GN4-1 promoted thickening of the culm and may enhance resistance to lodging. These results demonstrate that GN4-1 is a potentially valuable gene for improvement of yield and lodging resistance in rice breeding.