Fine mapping and candidate gene analysis of the novel thermo-sensitive genic male sterility tms9-1 gene in rice

Key message

Fine mapping of the novel thermo-sensitive genic male sterility locus tms9 - 1 in the traditional TGMS line HengnongS-1 revealed that the MALE STERILITY1 homolog OsMS1 is the candidate gene.

Abstract

Photoperiod-thermo-sensitive genic male sterility (P/TGMS) has been widely used in the two-line hybrid rice breeding system. HengnongS-1 is one of the oldest TGMS lines and is often used in indica two-line breeding programs in China. In this study, our genetic analysis showed that the TGMS gene in HengnongS-1 was controlled by a single recessive gene that was non-allelic with the other TGMS loci identified, including C815S, Zhu1S and Y58S. Using SSR markers and bulked segregant analysis, we located the TGMS locus on chromosome 9 and named the gene tms9-1. Fine mapping further narrowed the tms9-1 loci to a 162 kb interval between two dCAPS markers. Sequence analysis revealed that a T to C substitution results in an amino acid change in the tms9-1 candidate gene (Os09g27620) in HengnongS-1 as compared to Minghui63. Sequencing of other rice accessions, including six P/TGMS lines, seven indica varieties and nine japonica varieties, showed that this SNP was exclusive to HengnongS-1. With multiple sequence alignment and expression pattern analyses, the rice MALE STERILITY1 homolog OsMS1 gene was identified as the candidate gene for tms9-1. Therefore, our study identified a novel TGMS locus and will facilitate the functional identification of the tms9-1 gene. Moreover, the markers linked to the tms9-1 gene will provide useful tools for the development of new TGMS lines by marker-assisted selection in two-line hybrid rice breeding programs.     

Alteration of flower color in Iris germanica L. ‘Fire Bride’ through ectopic expression of phytoene synthase gene (crtB) from Pantoea agglomerans

Key message

Genetic modulation of the carotenogenesis in I. germanica ‘Fire Bride’ by ectopic expression of a crtB gene causes several flower parts to develop novel orange and pink colors.

Abstract

Flower color in tall bearded irises (Iris germanica L.) is determined by two distinct biochemical pathways; the carotenoid pathway, which imparts yellow, orange and pink hues and the anthocyanin pathway, which produces blue, violet and maroon flowers. Red-flowered I. germanica do not exist in nature and conventional breeding methods have thus far failed to produce them. With a goal of developing iris cultivars with red flowers, we transformed a pink iris I. germanica, ‘Fire Bride’, with a bacterial phytoene synthase gene (crtB) from Pantoea agglomerans under the control of the promoter region of a gene for capsanthin–capsorubin synthase from Lilium lancifolium (Llccs). This approach aimed to increase the flux of metabolites into the carotenoid biosynthetic pathway and lead to elevated levels of lycopene and darker pink or red flowers. Iris callus tissue ectopically expressing the crtB gene exhibited a color change from yellow to pink-orange and red, due to accumulation of lycopene. Transgenic iris plants, regenerated from the crtB-transgenic calli, showed prominent color changes in the ovaries (green to orange), flower stalk (green to orange), and anthers (white to pink), while the standards and falls showed no significant differences in color when compared to control plants. HPLC and UHPLC analysis confirmed that the color changes were primarily due to the accumulation of lycopene. In this study, we showed that ectopic expression of a crtB can be used to successfully alter the color of certain flower parts in I. germanica ‘Fire Bride’ and produce new flower traits.     

Different non-host resistance responses of two rice subspecies,japonica and indica,to Puccinia striiformis f. sp. tritici

Key message

Japonica and indica have different non-host resistance (NHR) abilities to Puccinia striiformis f. sp. tritici ( Pst ), and hydrogen peroxide (H ₂ O ₂ ) has a positive function in NHR to japonica against Pst.

Abstract

Non-host interactions between Puccinia striiformis f. sp. tritici (Pst) and two rice subspecies were characterized using 23 rice varieties, including 11 japonica and 12 indica. Results showed that the infected fungal structures were easily produced in the leaves of indica, whereas only several substomatal vesicles and primary infection hyphae were observed in the leaves of japonica. This result indicated that indica is less resistant or more susceptible to Pst than japonica. Hydrogen peroxide accumulated in the initial phase of japonica–Pst interaction but not in indica–Pst interaction. A set of reactive oxygen species (ROS)-related genes was also induced in response to Pst infection, suggesting that ROS activation is one of the major mechanisms of non-host resistance of rice to Pst.     

Arabidopsis Brassinosteroid-overproducing gulliver3-D/dwarf4-D mutants exhibit altered responses to Jasmonic acid and pathogen

Key message

Arabidopsis gulliver3 - D/dwarf4 - D displays growth-promoting phenotypes due to activation tagging of a key brassinosteroid biosynthetic gene DWARF4. In gul3-D/dwf4-D , the Jasmonate and Salicylate signaling pathways were relatively activated and suppressed, respectively.

Abstract

Energy allocation between growth and defense is elegantly balanced to achieve optimal development in plants. Brassinosteroids (BRs), steroidal hormones essential for plant growth, are regulated by other plant hormones, including auxin and jasmonates (JA); auxin stimulates the expression of a key brassinosteroid (BR) biosynthetic gene, DWARF4 (DWF4), whereas JA represses it. To better understand the interaction mechanisms between growth and defense, we isolated a fast-growing mutant, gulliver3-D (gul3-D), that resulted from the activation tagging of DWF4, and examined the response of this mutant to defense signals, including JA, Pseudomonas syringae pv. tomato (Pst DC3000) infection, and wounding. The degree of root growth inhibition following MeJA treatment was significantly decreased in gul3-1D/dwf4-5D relative to the wild type, suggesting that JA signaling is partially desensitized in gul3-1D. Quantitative RT-PCR analysis of the genes involved in JA and salicylic acid (SA) responses, including MYC2, PDF1.2, CORI3, PR1, and PR2, revealed that JA signaling was preferentially activated in gul3-1D, whereas SA signaling was suppressed. As a result, gul3-1D was more susceptible to a biotrophic pathogen, Pst DC3000. Based on our results, we propose a model in which BR and JA cooperate to balance energy allocation between growth and defense responses. In ambient conditions, BRs promote plant growth; however, when stresses trigger JA signaling, JA compromises BR signaling by downregulating DWF4 expression.     

Structural requirements of strigolactones for germination induction and inhibition of Striga gesnerioides seeds

Key message

Structure–activity relationship studies of strigolactones and Striga gesnerioides seed germination revealed strict structural requirements for germination induction and a new function of the plant hormones as germination inhibitors.

Abstract

Stereoisomers of the naturally occurring strigolactones, strigol, sorgolactone, orobanchol, sorgomol and 5-deoxystrigol, 36 in total, were prepared and screened for the ability to induce and/or inhibit the germination of Striga hermonthica and Striga gesnerioides seeds collected from mature plants that parasitized on sorghum and cowpea, respectively. All of the compounds induced S. hermonthica seed germination, albeit displayed differential activities. On the other hand, only a limited number of the compounds induced significant germination in S. gesnerioides, thus indicating strict structural requirements. Strigolactones inducing high germination in S. gesnerioides induced low germination in S. hermonthica. Strigolactones with the same configuration at C3a, C8b and C2′ as that in 5-deoxystrigol (9a) induced high germination of S. hermonthica seeds, but most of them inhibited the germination of S. gesnerioides. The differential response of S. gesnerioides to strigolactones may play an important role in the survival of the species. However, the compounds could be used as means of control if mixed cropping of cowpea and sorghum is adopted.     

Development of somatic hybrids Solanum × michoacanum Bitter. (Rydb.) (+) S. tuberosum L. and autofused 4x S. × michoacanum plants as potential sources of late blight resistance for potato breeding

Key message

Phytophthora infestans resistant somatic hybrids of S. × michoacanum (+) S. tuberosum and autofused 4 x S. × michoacanum were obtained. Our material is promising to introgress resistance from S. × michoacanum into cultivated potato background.

Abstract

Solanum × michoacanum (Bitter.) Rydb. (mch) is a wild diploid (2n = 2x = 24) potato species derived from spontaneous cross of S. bulbocastanum and S. pinnatisectum. This hybrid is a 1 EBN (endosperm balance number) species and can cross effectively only with other 1 EBN species. Plants of mch are resistant to Phytophthora infestans (Mont) de Bary. To introgress late blight resistance genes from mch into S. tuberosum (tbr), genepool somatic hybridization between mch and susceptible diploid potato clones (2n = 2x = 24) or potato cultivar Rywal (2n = 4x = 48) was performed. In total 18,775 calli were obtained from postfusion products from which 1,482 formed shoots. The Simple Sequence Repeat (SSR), Cleaved Amplified Polymorphic Sequences (CAPS) and Random Amplified Polymorphic DNA (RAPD) analyses confirmed hybrid nature of 228 plants and 116 autofused 4x mch. After evaluation of morphological features, flowering, pollen stainability, tuberization and ploidy level, 118 somatic hybrids and 116 autofused 4x mch were tested for late blight resistance using the detached leaf assay. After two seasons of testing three somatic hybrids and 109 4x mch were resistant. Resistant forms have adequate pollen stainability for use in crossing programme and are a promising material useful for introgression resistance from mch into the cultivated potato background.     

Phosphate starvation promoted the accumulation of phenolic acids by inducing the key enzyme genes in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots

Key message

Phosphate starvation increased the production of phenolic acids by inducing the key enzyme genes in a positive feedback pathway in Saliva miltiorrhiza hairy roots. SPX may be involved in this process.

Abstract

Salvia miltiorrhiza is a wildly popular traditional Chinese medicine used for the treatment of coronary heart diseases and inflammation. Phosphate is an essential plant macronutrient that is often deficient, thereby limiting crop yield. In this study, we investigated the effects of phosphate concentration on the biomass and accumulation of phenolic acid in S. miltiorrhiza. Results show that 0.124 mM phosphate was favorable for plant growth. Moreover, 0.0124 mM phosphate was beneficial for the accumulation of phenolic acids, wherein the contents of danshensu, caffeic acid, rosmarinic acid, and salvianolic acid B were, respectively, 2.33-, 1.02-, 1.68-, and 2.17-fold higher than that of the control. By contrast, 12.4 mM phosphate inhibited the accumulation of phenolic acids. The key enzyme genes in the phenolic acid biosynthesis pathway were investigated to elucidate the mechanism of phosphate starvation-induced increase of phenolic acids. The results suggest that phosphate starvation induced the gene expression from the downstream pathway to the upstream pathway, i.e., a feedback phenomenon. In addition, phosphate starvation response gene SPX (SYG1, Pho81, and XPR1) was promoted by phosphate deficiency (0.0124 mM). We inferred that SPX responded to phosphate starvation, which then affected the expression of later responsive key enzyme genes in phenolic acid biosynthesis, resulting in the accumulation of phenolic acids. Our findings provide a resource-saving and environmental protection strategy to increase the yield of active substance in herbal preparations. The relationship between SPX and key enzyme genes and the role they play in phenolic acid biosynthesis during phosphate deficiency need further studies.

     

A comparison study of Agrobacterium-mediated transformation methods for root-specific promoter analysis in soybean

Key message

Both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.

Abstract

An efficient genetic transformation system is crucial for promoter analysis in plants. Agrobacterium-mediated transformation is the most popular method to produce transgenic hairy roots or plants. In the present study, first, we compared the two different Agrobacterium rhizogenes-mediated hairy root transformation methods using either constitutive CaMV35S or the promoters of root-preferential genes, GmEXPB2 and GmPAP21, in soybean, and found the efficiency of in vitro hairy root transformation was significantly higher than that of in vivo transformation. We compared Agrobacterium rhizogenes-mediated hairy root and Agrobacterium tumefaciens-mediated whole plant transformation systems. The results showed that low-phosphorous (P) inducible GmEXPB2 and GmPAP21 promoters could not induce the increased expression of the GUS reporter gene under low P stress in both in vivo and in vitro transgenic hairy roots. Conversely, GUS activity of GmPAP21 promoter was significantly higher at low P than high P in whole plant transformation. Therefore, both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.     

The Vaccinium corymbosum FLOWERING LOCUS T-like gene (VcFT): a flowering activator reverses photoperiodic and chilling requirements in blueberry

Key message

The blueberry FLOWERING LOCUS T ( FT )-like gene ( VcFT ) cloned from the cDNA of a tetraploid, northern highbush blueberry ( Vaccinium corymbosum L.) is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.

Abstract

Blueberry is a woody perennial bush with a longer juvenile period than annual crops, requiring vernalization to flower normally. Few studies have been reported on the molecular mechanism of flowering in blueberry or other woody plants. Because FLOWERING LOCUS T (FT) from Arabidopsis thaliana plays a multifaceted role in generating mobile molecular signals to regulate plant flowering time, isolation and functional analysis of the blueberry (Vaccinium corymbosum L.) FT-like gene (VcFT) will facilitate the elucidation of molecular mechanisms of flowering in woody plants. Based on EST sequences, a 525-bpVcFT was identified and cloned from the cDNA of a tetraploid, northern highbush blueberry cultivar, Bluecrop. Ectopic expression of 35S:VcFT in tobacco induced flowering an average of 28 days earlier than wild-type plants. Expression of the 35S:VcFT in the blueberry cultivar Aurora resulted in an extremely early flowering phenotype, which flowered not only during in vitro culture, a growth stage when nontransgenic shoots had not yet flowered, but also in 6–10-week old, soil-grown transgenic plants, in contrast to the fact that at least 1 year and 800 chilling hours are required for the appearance of the first flower of both nontransgenic ‘Aurora’ and transgenic controls with the gusA. These results demonstrate that the VcFT is a functional floral activator and overexpression of the VcFT is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.     

Three new sterigmatocystin analogues from marine-derived fungus Aspergillus versicolor MF359

During the systematic screening of active compounds from marine-derived fungi, the extract of a strain of Aspergillus versicolor MF359 isolated from a marine sponge of Hymeniacidon perleve was identified for detailed chemical investigation. Three new secondary metabolites, named hemiacetal sterigmatocystin (1), acyl-hemiacetal sterigmatocystin (2), and 5-methoxydihydrosterigmatocystin (3), together with a known compound, aversin (4), were characterized. 1 represents a first structure of sterigmatocystin hemiacetal from nature. The antibacterial activities of these identified compounds were evaluated against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa. Compound 3 showed activity against S. aureus and B. subtilis with MIC values of 12.5 and 3.125 μg/mL, respectively.     

Characterization of a heavy-ion induced white flower mutant of allotetraploid Nicotiana tabacum

Key message

We characterized a white flower mutant of allotetraploid N. tabacum as a DFR-deficient mutant; one copy of DFR has a cultivar-specific frameshift, while the other was deleted by heavy-ion irradiation.

Abstract

In most plants, white-flowered mutants have some kind of deficiency or defect in their anthocyanin biosynthetic pathway. Nicotiana tabacum normally has pink petals, in which cyanidin is the main colored anthocyanidin. When a relevant gene in the cyanidin biosynthetic pathway is mutated, the petals show a white color. Previously, we generated white-flowered mutants of N. tabacum by heavy-ion irradiation, which is accepted as an effective mutagen. In this study, we determined which gene was responsible for the white-flowered phenotype of one of these mutants, cv. Xanthi white flower 1 (xwf1). Southern blot analysis using a DNA fragment of the dihydroflavonol 4-reductase (DFR) gene as a probe showed that the xwf1 mutant lacked signals that were present in wild-type genomic DNAs. Sequence analysis demonstrated that one copy of the DFR gene (NtDFR2) was absent from the genome of the xwf1 mutant. The other copy of the DFR gene (NtDFR1) contained a single-base deletion resulting in a frameshift mutation, which is a spontaneous mutation in cv. Xanthi. Introduction of NtDFR2 cDNA into the petal limbs of xwf1 by particle bombardment resulted in production of the pink-colored cells, whereas introduction of NtDFR1 cDNA did not. These results indicate that xwf1 is a DFR-deficient mutant. One copy of NtDFR1 harbors a spontaneous frameshift mutation, while the other copy of NtDFR2 was deleted by heavy-ion beam irradiation.     

New species and new combinations in the sematophyllum subpinnatum complex (Sematophyllaceae)

Three new tropical American species ofSematophyllum are described with relatively broad leaves and short upper leaf cells:S. cubense, S. cataractae, andS. oedophysidium. The Central AmericaS. apaloblastum andS. barnesii are new combinations.Sematophyllum galipense, S. amnigenum, andS. cochleatum are briefly discussed.     

Four new species of Smilax (Smilacaceae) from southeastern Asia

Described as new areSmilax biumbellata of the affinity ofS. nervomarginata,S. siamensis of the affinity of 5.perfoliata, and two others inSmilax sect.Macranthae:S. inversa andS. pachysandroides.Smilax inversa (characterized by strongly deflexed leaves and inflorescences on zigzag stems) andS. pachysandroides (with subsessile leaves on low simple stems) show no close resemblance to any previously known species of the section.     

Characterization of the sugar-O-methyltransferase LobS1 in lobophorin biosynthesis

Lobophorins A (1) and B (2) belong to a large group of spirotetronate natural products with potent antibacterial and antitumor activities. The cloning of the lobophorin biosynthesis gene cluster from the deep-sea-derived Streptomyces sp. SCSIO 01127 identified a sugar-O-methyltransferase-encoding gene lobS1. The lobS1 inactivation mutant accumulated two new lobophorin analogs 3 and 4, different from 1 and 2 by lacking the 4-methyl group at the terminal l-digitoxose, respectively. Biochemical experiments verified that LobS1 was a SAM-dependent sugar-O-methyltransferase that required divalent metal ions for better activity. Antibacterial assays revealed compounds 3 and 4 were generally less potent than compounds 1 and 2. These findings suggest that the methylation on the terminal digitoxose by LobS1 tailors lobophorin biosynthesis and highlights the importance of this methylation for antibacterial potence.     

Screening promoters for Anthurium transformation using transient expression

Key message

There are multiple publications on Anthurium transformation, yet a commercial product has not been achieved. This may be due to use of non-optimum promoters here we address this problem.

Abstract

Different promoters and tissue types were evaluated for transient β-glucuronidase (GUS) expression in Anthurium andraeanum Hort. ‘Marian Seefurth’ following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35S promoter from Cauliflower Mosaic Virus fused to a GUS reporter gene were bombarded into in vitro grown anthurium lamina, somatic embryos and roots. The number of GUS foci and the intensity of GUS expression were evaluated for each construct. Ubiquitin promoters from rice and maize resulted in the highest number of expressing cells in all tissues examined. Due to the slow growth of anthurium plants, development of transgenic anthurium plants takes years. This research has rapidly identified multiple promoters that express in various anthurium tissues facilitating the development of transformation vectors for the expression of desirable traits in anthurium plants.