<Emphasis Type="Italic">LMI1</Emphasis>-like genes involved in leaf margin development of <Emphasis Type="Italic">Brassica napus</Emphasis>

In rapeseed (Brassica napus L.), leaf margins are variable and can be entire, serrate, or lobed. In our previous study, the lobed-leaf gene (LOBED-LEAF 1, BnLL1) was mapped to a 32.1 kb section of B. napus A10. Two LMI1-like genes, BnaA10g26320D and BnaA10g26330D, were considered the potential genes that controlled the lobed-leaf trait in rapeseed. In the present study, these two genes and another homologous gene (BnaC04g00850D) were transformed into Arabidopsis thaliana (L.) Heynh. plants to identify their functions. All three LMI1-like genes of B. napus produced serrate leaf margins. The expression analysis indicated that the expression level of BnaA10g26320D determined the difference between lobed- and entire-leaved lines in rapeseed. Therefore, it is likely that BnaA10g26320D corresponds to BnLL1.     

Genetic characterisation and fine mapping of a chlorophyll-deficient mutant (BnaC.ygl) in Brassica napus

A chlorophyll-deficient mutant with yellow-green leaves of Brassica napus was obtained by treatment with the chemical mutagen ethyl methanesulfonate. Compared with the wild type at seedling stage, the mutant displayed decreased total chlorophyll content, less granal stacks and granal membranes. Genetic analysis confirmed that the mutant phenotype was controlled by a recessive gene, which was designated as BnaC.ygl. Mapping of the gene was subsequently conducted in two populations with yellow-green leaves (population IBC₈ and IIBC₄, which comprised 3,472 and 5,288 individuals, respectively). Analysis on the public simple sequence repeat markers (SSR) showed that four SSR markers linked to BnaC.YGL gene displayed polymorphism. Based on the information of these SSR markers, the BnaC.YGL gene was mapped to the linkage group N17. From a survey of amplified fragment length polymorphism (AFLP), 15 of 47 AFLP markers were successfully converted into sequence characterised amplified region (SCAR) markers. BnY5 and CB10534, the closest flanking markers, were 0.32 and 0.03 cM away from the BnaC.YGL gene, respectively. And in the two populations, 18 makers cosegregated with BnaC.YGL. BLAST analysis revealed that the sequences of the makers displayed highly conserved homology with C06 of B. oleracea. The collinearity of makers to makers on N17 and on C06 showed that there might be an inversion occurring on the N17 group. These results are expected to accelerate the process of cloning the BnaC.YGL gene and facilitate the understanding of the biological processes of chloroplast development in Brassica napus.     

Genetics and fine mapping of a yellow-green leaf gene (<Emphasis Type="Italic">ygl</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis> L.)

Cabbage (Brassica oleracea var. capitata L.) is one of the most popular cultivated vegetables worldwide. Cabbage has rich phenotypic diversity, including plant height, head shape, head color, leaf shape and leaf color. Leaf color plays an important role in cabbage growth and development. At present, there are few reports on fine mapping of leaf color mutants in B. oleracea. In this study, a naturally occurring yellow-green leaf cabbage mutant (YL-1), derived from the self-pollinated progenies of the hybrid ‘Hosom’, was used for inheritance analysis and gene mapping. Segregation populations including F₂ and BC₁ were generated from the cross of two inbred lines, YL-1 and 01–20. Genetic analysis with the F₂ and BC₁ populations demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene, ygl-1. Insertion–deletion (InDel) markers, designed based on the parental re-sequencing data, were used for the preliminary mapping with BSA (bulked segregant analysis) method. A genetic map constructed with 15 InDels indicated that ygl-1 was located on chromosome C01. The ygl-1 gene is flanked by InDel markers ID2 and M8, with genetic distances of 0.4 cM and 0.35 cM, respectively. The interval distance between two markers is 167 kb. Thus, it enables us to locate the ygl-1 gene for the first time in B. oleracea. This study lays the foundation for candidate gene prediction and ygl-1gene cloning.     

A single nucleotide mutation of <Emphasis Type="Italic">IspF</Emphasis> gene involved in the MEP pathway for isoprenoid biosynthesis causes yellow-green leaf phenotype in rice

Key message

We identified IspF gene through yellow-green leaf mutant 505ys in rice. OsIspF was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. On expression levels of genes in this mutant, OsIspF itself and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase were all up-regulated, however, among eight genes associated with photosynthesis, only psaA, psaN and psbA genes for three reaction center subunits of photosystem obviously changed.

Abstract

Isoprenoids are the most abundant natural compounds in all organisms, which originate from the basic five-carbon units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, IPP and DMAPP are synthesized through two independent pathways, the mevalonic acid pathway in cytoplasm and the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids. The MEP pathway comprises seven enzymatic steps, in which IspF is the fifth enzyme. So far, no IspF gene has been identified in monocotyledonous plants. In this study, we isolated a leaf-color mutant, 505ys, in rice (Oryza sativa). The mutant displayed yellow-green leaf phenotype, reduced level of photosynthetic pigments, and arrested development of chloroplasts. By map-based cloning of this mutant, we identified OsIspF gene (LOC_Os02g45660) showing significant similarity to IspF gene of Arabidopsis, in which a missense mutation occurred in the mutant, resulting in an amino acid change in the encoded protein. OsIspF gene was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. Further, the mutant phenotype of 505ys was complemented by transformation with the wild-type OsIspF gene. Therefore, we successfully identified an IspF gene in monocotyledonous plants. In addition, real-time quantitative RT-PCR implied that a positive regulation could exist between the OsIspF gene and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase. At the same time, it also implied that the individual genes involved in the MEP pathway might differentially regulated expression levels of the genes associated with photosynthesis.

     

<Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll <Emphasis Type="Italic">a</Emphasis> for activity

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA ₆₈₀₃) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA ₆₈₀₃ gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA₆₈₀₃ was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA₆₈₀₃ was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA₆₈₀₃ is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.     

Genetic analysis and fine-mapping of a new rice mutant, <Emphasis Type="Italic">white and lesion mimic leaf1</Emphasis>

Rice (Oryza sativa L.) leaf color mutants are excellent models for studying chlorophyll biosynthesis and chloroplast development. In this study, we isolated a stable genetic white and lesion mimic leaf1 (wlml1) mutant from an ethyl methanesulfonate (EMS)-mutagenized population of the indica cultivar TN1. Compared with wild-type TN1, the wlml1 mutant had lower contents of chlorophyll and carotenoids, altered chloroplast ultrastructure, and altered regulation of genes associated with chlorophyll metabolism and chloroplast development. In addition, lesions formed on the leaves of wlml1 plants grown at 20 °C and genes related to disease resistance and antioxidant functions were up-regulated; by contrast, the mutant phenotype was partially suppressed at 28 °C. These findings indicated that WLML1 might play a role in chlorophyll metabolism and chloroplast development, as well as in biotic and abiotic stress responses. Genetic analysis showed that WLML1 was controlled by a recessive nuclear gene, and map-based cloning delimited WLML1 to a 159.7-kb region on chromosome 4 that includes 30 putative open reading frames. Based on these findings, the wlml1 mutant will be a good genetic material for further studies on chlorophyll metabolism and stress responses in rice.     

Salt tolerance conferred by expression of a global regulator IrrE from <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in oilseed rape

As salinity is a major threat to sustainable agriculture worldwide, cultivation of salt-tolerant crops becomes increasingly important. IrrE acts as a global regulator and a general switch for stress resistance in Deinococcus radiodurans. In this study, to determine whether the irrE gene can improve the salt tolerance of Brassica napus, we introduced the irrE gene into B. napus by the Agrobacterium tumefaciens-mediated transformation method. Forty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) analyses confirmed that the irrE gene had integrated into the plant genome. Northern as well as Western blot analyses revealed that the transgene was expressed at various levels in transgenic plants. Analysis for the T₁ progenies derived from four independent transformants showed that irrE had enhanced the salt tolerance of T₁ in the presence of 350 mM NaCl. Furthermore, under salt stress, transgenic plants accumulated more compatible solutes (proline) and a lower level of malondialdehyde (MDA), and they had higher activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). However, agronomic traits were not affected by irrE gene overexpression in the transgenic B. napus plants. This study indicates that the irrE gene can improve the salt tolerance of B. napus and represents a promising candidate for the development of crops with enhanced salt tolerance by genetic engineering.     

Identification of candidate genes of QTLs for seed weight in <Emphasis Type="Italic">Brassica napus</Emphasis> through comparative mapping among <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica</Emphasis>species

Background

Map-based cloning of quantitative trait loci (QTLs) in polyploidy crop species remains a challenge due to the complexity of their genome structures. QTLs for seed weight in B. napus have been identified, but information on candidate genes for identified QTLs of this important trait is still rare.

Results

In this study, a whole genome genetic linkage map for B. napus was constructed using simple sequence repeat (SSR) markers that covered a genetic distance of 2,126.4 cM with an average distance of 5.36 cM between markers. A procedure was developed to establish colinearity of SSR loci on B. napus with its two progenitor diploid species B. rapa and B. oleracea through extensive bioinformatics analysis. With the aid of B. rapa and B. oleracea genome sequences, the 421 homologous colinear loci deduced from the SSR loci of B. napus were shown to correspond to 398 homologous loci in Arabidopsis thaliana. Through comparative mapping of Arabidopsis and the three Brassica species, 227 homologous genes for seed size/weight were mapped on the B. napus genetic map, establishing the genetic bases for the important agronomic trait in this amphidiploid species. Furthermore, 12 candidate genes underlying 8 QTLs for seed weight were identified, and a gene-specific marker for BnAP2 was developed through molecular cloning using the seed weight/size gene distribution map in B. napus.

Conclusions

Our study showed that it is feasible to identify candidate genes of QTLs using a SSR-based B. napus genetic map through comparative mapping among Arabidopsis and B. napus and its two progenitor species B. rapa and B. oleracea. Identification of candidate genes for seed weight in amphidiploid B. napus will accelerate the process of isolating the mapped QTLs for this important trait, and this approach may be useful for QTL identification of other traits of agronomic significance.

     

Biogenesis of chlorophyll-binding proteins under iron stress in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The biogenesis of chlorophyll-binding proteins under iron stress has been investigated in vivo in a chlN deletion mutant of Synechocystis sp. PCC 6803. The chlN gene encodes one subunit of the light-independent protochlorophyllide reductase. The mutant is unable to synthesize chlorophyll in darkness, causing chlorophyll biosynthesis to become light dependent. When the mutant was propagated in darkness, essentially no chlorophyll and photosystems were detected. Upon return of the chlN deletion mutant to light, 77 K fluorescence emission spectra and oxygen evolution of greening cells under iron-sufficient or-deficient conditions were measured. The gradual blue shift of the photosystem I (PS I) peak upon greening under iron stress suggested the structural alteration of newly synthesized PS I. Furthermore, the rate of biogenesis of PS II was delayed under iron stress, which might be due to the presence of IsiA.     

Small RNA changes in synthetic <Emphasis Type="Italic">Brassica napus</Emphasis>

Main conclusion

Small RNAs and microRNAs were found to vary extensively in synthetic Brassica napus and subsequent generations, accompanied by the activation of transposable elements in response to hybridization and polyploidization.

Abstract

Resynthesizing B. napus by hybridization and chromosome doubling provides an approach to create novel polyploids and increases the usable genetic variability in oilseed rape. Although many studies have shown that small RNAs (sRNAs) act as important factor during hybridization and polyploidization in plants, much less is known on how sRNAs change in synthetic B. napus, particularly in subsequent generations after formation. We performed high-throughput sequencing of sRNAs in S₁–S₄ generations of synthetic B. napus and in the homozygous B. oleracea and B. rapa parent lines. We found that the number of small RNAs (sRNAs) and microRNAs (miRNAs) doubled in synthetic B. napus relative to the parents. The proportions of common sRNAs detected varied from the S₁ to S₄ generations, suggesting sRNAs are unstable in synthetic B. napus. The majority of miRNAs (67.2 %) were non-additively expressed in the synthesized Brassica allotetraploid, and 33.3 % of miRNAs were novel in the resynthesized B. napus. The percentage of miRNAs derived from transposable elements (TEs) also increased, indicating transposon activation and increased transposon-associated miRNA production in response to hybridization and polyploidization. The number of target genes for each miRNA in the synthesized Brassica allotetraploid was doubled relative to the parents, enhancing the complexity of gene expression regulation. The potential roles of miRNAs and their targets are discussed. Our data demonstrate generational changes in sRNAs and miRNAs in synthesized B. napus.

     

Interactions Between the Circadian Clock and Heme Oxygenase in the Retina of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The Drosophila retina has an autonomous peripheral circadian clock in which the expression of the gene encoding heme oxygenase (HO) is under circadian control with the ho mRNA peaking at the beginning of the day and in the middle of the night. The function of HO in the retina is unknown, but we observed that it regulates the circadian clock and protects photoreceptors against DNA damage. The decline in HO level increases and decreases the expression of the canonical clock genes period (per) and Clock (Clk), respectively. The opposite result was observed after increasing HO expression. Among three products of HO activity—carbon monoxide (CO), ferrous ions, and biliverdin—the latter has no effect on per and Clk expressions, but CO exerts the same effect as the increase of ho expression. This suggests that HO action on the clock is mediated by CO, which may affect Clk expression during the day and the level of per expression. While ho expression is not stimulated by nitric oxide (NO), NO has the same effect on the clock as HO, increasing Clk expression and decreasing the expression of per.