Characterization of a zebra mutant of rice with increased susceptibility to light stress

The rice zebra mutant TCM248 is a single recessive mutant. This mutant develops transverse-striped leaves with green and white sectors under alternate light/dark growth conditions. Mutants that were grown under a higher light intensity during the light period showed a more intense striped phenotype. The white tissues contained abnormal chloroplasts with few internal membrane structures, while the green tissues in the mutants contained normal chloroplasts. The white tissue contained only trace amounts of Chls and carotenoids, and mRNA accumulation of nuclear genes encoding chloroplast proteins (rbcS, cab) was strongly suppressed compared to that in the wild type plants. A series of growth condition shift experiments demonstrated that the mutant displayed the striped phenotype only if it was exposed to the alternate light/dark growth conditions during a limited stage of early leaf development. These data suggest that the zebra gene is involved in the acquisition of photoprotective capacity of the plants and that this gene functions at an early stage of chloroplast differentiation.     

A leaf shape mutant provides insight into PINOID Serine/Threonine Kinase function in cucumber(Cucumis sativus L.)

Optimizing leaf shape is a major challenge in efforts to develop an ideal plant type. Cucumber leaf shapes are diverse; however, the molecular regulatory mechanisms underlying leaf shape formation are unknown. In this study, we obtained a round leaf mutant(rl) from an ethyl methanesulfonate-induced mutagenesis population. Genetic analysis revealed that a single recessive gene, rl, is responsible for this mutation. A modified Mut Map analysis combined linkage mapping identified a single nucleotide polymorphism within a candidate gene,Csa1 M537400, as the mutation underlying the trait.Csa1 M537400 encodes a PINOID kinase protein involved in auxin transport. Expression of Csa1 M537400 was significantly lower in the rl mutant than in wild type, and it displayed higher levels of IAA(indole-3-acetic acid) in several tissues. Treatment of wild-type plants with an auxin transport inhibitor induced the formation of round leaves,similar to those in the rl mutant. Altered expression patterns of several auxin-related genes in the rl mutant suggest that rl plays a key role in auxin biosynthesis,transport, and response in cucumber. These findings provide insight into the molecular mechanism underlying the regulation of auxin signaling pathways in cucumber,and will be valuable in the development of an ideal plant type.     

QTL-seq identifies an early flowering QTL located near Flowering Locus T in cucumber

Key message

Next-generation sequencing enabled a fast discovery of a major QTL controlling early flowering in cucumber, corresponding to the FT gene conditioning flowering time in Arabidopsis.

Abstract

Next-generation sequencing technologies are making it faster and more efficient to establish the association of agronomic traits with molecular markers or candidate genes, which is the requirement for marker-assisted selection in molecular breeding. Early flowering is an important agronomic trait in cucumber (Cucumis sativus L.), but the underlying genetic mechanism is unknown. In this study, we identified a candidate gene for early flowering QTL, Ef1.1 through QTL-seq. Segregation analysis in F₂ and BC₁ populations derived from a cross between two inbred lines “Muromskij” (early flowering) and “9930” (late flowering) suggested quantitative nature of flowering time in cucumber. Genome-wide comparison of SNP profiles between the early and late-flowering bulks constructed from F₂ plants identified a major QTL, designated Ef1.1 on cucumber chromosome 1 for early flowering in Muromskij, which was confirmed by microsatellite marker-based classical QTL mapping in the F₂ population. Joint QTL-seq and traditional QTL analysis delimited Ef1.1 to an 890 kb genomic region. A cucumber gene, Csa1G651710, was identified in this region, which is a homolog of the FLOWERING LOCUS T (FT), the main flowering switch gene in Arabidopsis. Quantitative RT-PCR study of the expression level of Csa1G651710 revealed significantly higher expression in early flowering genotypes. Data presented here provide support for Csa1G651710 as a possible candidate gene for early flowering in the cucumber line Muromskij.     

A New Glabrous Gene (csgl3) Identified in Trichome Development in Cucumber (Cucumis sativus L.)

Spines or trichomes on the fruit of cucumbers enhance their commercial value in China. In addition, glabrous mutants exhibit resistance to aphids and therefore their use by growers can reduce pesticide residues. Previous studies have reported two glabrous mutant plants containing the genes, csgl1 and csgl2. In the present study, a new glabrous mutant, NCG157, was identified showing a gene interaction effect with csgl1 and csgl2. This mutant showed the glabrous character on stems, leaves, tendrils, receptacles and ovaries, and there were no spines or tumors on the fruit surface. Inheritance analysis showed that a single recessive gene, named csgl3, determined the glabrous trait. An F₂ population derived from the cross of two inbred lines 9930 (a fresh market type from Northern China that exhibits trichomes) and NCG157 (an American processing type with glabrous surfaces) was used for genetic mapping of the csgl3 gene. By combining bulked segregant analysis (BAS) with molecular markers, 18 markers, including two simple sequence repeats (SSR), nine insertion deletions (InDel) and seven derived cleaved amplified polymorphism sequences (dCAPs), were identified to link to the csgl3 gene. All of the linked markers were used as anchor loci to locate the csgl3 gene on cucumber chromosome 6. The csgl3 gene was mapped between the dCAPs markers dCAPs-21 and dCAPs-19, at genetic distances of 0.05 cM and 0.15 cM, respectively. The physical distance of this region was 19.6 kb. Three markers, InDel-19, dCAPs-2 and dCAPs-11, co-segregated with csgl3. There were two candidate genes in the region, Csa6M514860 and Csa6M514870. Quantitative real-time PCR showed that the expression of Csa6M514870 was higher in the tissues of 9930 than that of NCG157, and this was consistent with their phenotypic characters. Csa6M514870 is therefore postulated to be the candidate gene for the development of trichomes in cucumber. This study will facilitate marker-assisted selection (MAS) of the smooth plant trait in cucumber breeding and provide for future cloning of csgl3.     

Map-Based Cloning of zb7 Encoding an IPP and DMAPP Synthase in the MEP Pathway of Maize

IspH is a key enzyme in the last step of the methyl-D-erythritol-4-phosphate (MEP) pathway. Loss of function of IspH can often result in complete yellow or albino phenotype in many plants. Here, we report the characterization of a recessive mutant of maize, zebra7 (zb7), showing transverse green/yellow striped leaves in young plants. The yellow bands of the mutant have decreased levels of chlorophylls and carotenoids with delayed chloroplast development. Low temperature suppressed mutant phenotype, while alternate light/dark cycle or high temperature enlarged the yellow section. Map-based cloning demonstrated that zb7 encodes the IspH protein with a mis-sense mutation in a conserved region. Transgenic silencing of Zb7 in maize resulted in complete albino plantlets that are aborted in a few weeks, confirming that Zb7 is important in the early stages of maize chloroplast development. Zb7 is constitutively expressed and its expression subject to a 16-h light/8-h dark cycle regulation. Our results suggest that the less effective or unstable IspH in zb7 mutant, together with its diurnal expression, are mechanistically accounted for the zebra phenotype. The increased IspH mRNA in the leaves of zb7 at the late development stage may explain the restoration of mutant phenotype in mature stages.     

Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.     

Genetic analysis and identification of a candidate gene associated with in vitro regeneration ability of cucumber

Key message

Candidate genes associated with in vitro regeneration were identified in cucumber.

Abstract

The ability to regenerate shoots or whole plants from differentiated plant tissues is essential for plant transformation. In cucumber (Cucumis sativus L.), regeneration ability varies considerably across accessions, but the genetic mechanism has not yet been demonstrated. In the present study, 148 recombinant inbred lines and a core collection were examined to identify candidate genes involved in cucumber regeneration. Four QTL for cotyledon regeneration that explained 9.7–16.6% of the phenotypic variation in regeneration were identified on cucumber chromosomes 1, 3, and 6. The loci Fcrms1.1 and Fcrms₊1.1 were consistently detected in the same genetic interval on two regeneration media. A genome-wide association study revealed 18 SNPs (??log(p)?>?5) significantly associated with cotyledon regeneration. Three candidate genes in this region were identified. RT-PCR analyses revealed that Csa1G642540 was significantly more highly expressed in genotypes with high cotyledon regeneration rates than in those with low regeneration. The Csa1G642540 CDS driven by its native promoter was transformed into cucumber line 9110Gt; molecular analyses showed that the T-DNA had integrated into the genomes of 8.6% of regenerated plantlets. The seeds from T₀ plants expressing Csa1G642540 were tested for regeneration from cotyledon explants, and the segregate ratio in regeneration frequency is 3:1. The AT3G44110.1, the homologue gene of Csa1G642540 in Arabidopsis, has been reported as PM H⁺-ATPase activity regulation, integrating flowering signals and enlarging meristem function. These results demonstrate that Csa1G642540 might play an important role in regeneration in cucumber and could serve as a selectable marker for regeneration from cotyledons.

     

High-resolution mapping of the dull fruit skin gene D in cucumber (Cucumis sativus L.)

Dull/glossy fruit skin is a highly valuable external quality trait that affects the market value of cucumbers. In this study, genetic analysis showed that one single dominant gene, D (dull fruit skin), determines the dull fruit skin trait in cucumber. By combining bulked segregant analysis with 11 published polymorphic molecular markers on chromosome 5, the D/d gene was preliminarily mapped between markers SCZ69 and SSR16203, at genetic distances of 0.3 and 0.6 cM, respectively. Subsequently, a larger F2 (S06 × S94) population (842 individuals in total) was used for high-resolution mapping of the D/d gene. Finally, the D/d gene was fine-mapped between markers SSR37 and SSR112, at a physical distance of 244.9 kb (containing 31 candidate genes), using eight newly developed polymorphic simple sequence repeat (SSR) markers between SCZ69 and SSR16203. Based on semi-quantitative RT-PCR analysis, the possible candidate gene D was identified as Csa016880 or Csa016887. Meanwhile, validity analysis of the markers SSR37 and SSR112 was performed with 72 dull/glossy fruit lines, and showed that the two co-dominant SSR markers could be used for marker-assisted selection of the dull/glossy fruit trait in cucumber breeding. Moreover, this study will be helpful for cloning of the D gene in cucumber.     

Evaluation and precise mapping of QFhb.nau-2B conferring resistance against Fusarium infection and spread within spikes in wheat (Triticum aestivum L.)

Leaf color mutants are valuable resources for studying regulatory mechanisms of photosynthetic pigment metabolism. In this study, a chlorophyll-deficient golden leaf mutant lcm1 of Chinese cabbage was identified from its wild-type “FT” by ethyl methanesulfonate (EMS) mutagenesis. The phenotype of the golden leaf mutant lcm1 was that the leaves remained golden throughout development. Pigment measurements showed that the chlorophyll content of the mutant lcm1 was less than that of the wild-type “FT”. There were no obvious grana lamellae observed in the chloroplast microstructure of the mutant lcm1. Genetic analysis revealed that the mutant lcm1 phenotype was controlled by a single recessive nuclear gene, lcm1 (BrChlH). The MutMap method and Kompetitive Allele Specific PCR genotyping were used to predict that Bra006208 encoding a Mg-chelatase H subunit, the candidate gene for the mutant lcm1. The 4249-bp lcm1 gene had five exons and a single nucleotide substitution (G to A) in the third exon that resulted in an amino acid substitution (A to V). This SNP2523351 occurred in the highly conserved CobN-Mg_chel domain of the LCM1 (BrCHLH) protein. This study suggests that lcm1 gene plays an important role in the function of CHLH and provides a solid foundation for further studies on the development of plant leaf color.

     

The evolutionarily conserved tetratrico peptide repeat protein pale yellow green7 is required for photosystem I accumulation in Arabidopsis and copurifies with the complex

Pale yellow green7-1 (pyg7-1) is a photosystem I (PSI)-deficient Arabidopsis (Arabidopsis thaliana) mutant. PSI subunits are synthesized in the mutant, but do not assemble into a stable complex. In contrast, light-harvesting antenna proteins of both photosystems accumulate in the mutant. Deletion of Pyg7 results in severely reduced growth rates, alterations in leaf coloration, and plastid ultrastructure. Pyg7 was isolated by map-based cloning and encodes a tetratrico peptide repeat protein with homology to Ycf37 from Synechocystis. The protein is localized in the chloroplast associated with thylakoid membranes and copurifies with PSI. An independent pyg7 T-DNA insertion line, pyg7-2, exhibits the same phenotype. pyg7 gene expression is light regulated. Comparison of the roles of Ycf37 in cyanobacteria and Pyg7 in higher plants suggests that the ancient protein has altered its function during evolution. Whereas the cyanobacterial protein mediates more efficient PSI accumulation, the higher plant protein is absolutely required for complex assembly or maintenance.     

The chloroplast ycf7 (petL) open reading frame of Chlamydomonas reinhardtii encodes a small functionally important subunit of the cytochrome b6f complex.

The small chloroplast open reading frame ORF43 (ycf7) of the green unicellular alga Chlamydomonas reinhardtii is cotranscribed with the psaC gene and ORF58. While ORF58 has been found only in the chloroplast genome of C.reinhardtii, ycf7 has been conserved in land plants and its sequence suggests that its product is a hydrophobic protein with a single transmembrane alpha helix. We have disrupted ORF58 and ycf7 with the aadA expression cassette by particle-gun mediated chloroplast transformation. While the ORF58::aadA transformants are indistinguishable from wild type, photoautotrophic growth of the ycf7::aadA transformants is considerably impaired. In these mutant cells, the amount of cytochrome b6f complex is reduced to 25-50% of wild-type level in mid-exponential phase, and the rate of transmembrane electron transfer per b6f complex measured in vivo under saturating light is three to four times slower than in wild type. Under subsaturating light conditions, the rate of the electron transfer reactions within the b6f complex is reduced more strongly in the mutant than in the wild type by the proton electrochemical gradient. The ycf7 product (Ycf7) is absent in mutants deficient in cytochrome b6f complex and present in highly purified b6f complex from the wild-type strain. Ycf7-less complexes appear more fragile than wild-type complexes and selectively lose the Rieske iron-sulfur protein during purification. These observations indicate that Ycf7 is an authentic subunit of the cytochrome b6f complex, which is required for its stability, accumulation and optimal efficiency. We therefore propose to rename the ycf7 gene petL.     

Chloroplast development in Arabidopsis thaliana requires the nuclear-encoded transcription factor sigma B

Development of plastids into chloroplasts, the organelles of photosynthesis, is triggered by light. However, little is known of the factors involved in the complex coordination of light-induced plastid gene expression, which must be directed by both nuclear and plastid genomes. We have isolated an Arabidopsis mutant, abc1, with impaired chloroplast development, which results in a pale green leaf phenotype. The mutated nuclear gene encodes a sigma factor, SigB, presumably for the eubacterial-like plastid RNA polymerase. Our results provide direct evidence that a nuclear-derived prokaryotic-like SigB protein, plays a critical role in the coordination of the two genomes for chloroplast development.     

Variegation mutants and mechanisms of chloroplast biogenesis

Variegated plants typically have green‐ and white‐sectored leaves. Cells in the green sectors contain normal‐appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development.     

Oryza sativa Chloroplast Signal Recognition Particle 43 (OscpSRP43) Is Required for Chloroplast Development and Photosynthesis

A rice chlorophyll-deficient mutant w67 was isolated from an ethyl methane sulfonate (EMS)–induced IR64 (Oryza sativa L. ssp. indica) mutant bank. The mutant exhibited a distinct yellow-green leaf phenotype in the whole plant growth duration with significantly reduced levels of chlorophyll and carotenoid, impaired chloroplast development and lowered capacity of photosynthesis compared with the wild-type IR64. Expression of a number of genes associated with chlorophyll metabolism, chloroplast biogenesis and photosynthesis was significantly altered in the mutant. Genetic analysis indicated that the yellow-green phenotype was controlled by a single recessive nuclear gene located on the short arm of chromosome 3. Using map-based strategy, the mutation was isolated and predicted to encode a chloroplast signal recognition particle 43 KD protein (cpSRP43) with 388 amino acid residuals. A single base substitution from A to T at position 160 resulted in a premature stop codon. OscpSRP43 was constitutively expressed in various organs with the highest level in the leaf. Functional complementation could rescue the mutant phenotype and subcellular localization showed that the cpSRP43:GFP fusion protein was targeted to the chloroplast. The data suggested that Oryza sativa cpSRP43 (OscpSRP43) was required for the normal development of chloroplasts and photosynthesis in rice.     

拟南芥叶绿体分裂突变体pdl37的基因鉴定与分析

pd137是经甲基磺酸乙脂（ethyl methane sulphonate, EMS）诱变并通过筛选得到的一个拟南芥叶绿体分裂突变体。该突变体的叶绿体表型与野生型相比有很大差异： 叶绿体面积显著增大, 细胞中叶绿体数量明显减少。遗传分析显示pd137的突变表型受隐性单基因控制。本研究通过遗传作图将该突变基因粗定位于拟南芥2号染色体的分子标记CH2-13.70和CH2-16.0区间内。该区间内已知的与叶绿体分裂相关的基因只有FtsZ2-1。对FtsZ2-1基因的测序结果显示pd137突变体的FtsZ2-1基因第505位碱基发生了无义突变, 使蛋白质翻译提前终止。该突变还严重影响了FtsZ2-1基因的mRNA水平。转基因互补实验进一步验证了该突变体表型是由于FtsZ2-1基因突变引起。本项工作为研究叶绿体分裂的机制提供了新材料和一些有用的线索。     

Expression of NADPH-Protochlorophyllide oxidoreductase gene in fully green leaves of cucumber

Photoreduction of protochlorophyllide a to chlorophyllide a that is catalyzed by NADPH-protochlorophyllide oxidoreductase (NPR) is only a light-dependent step in overall processes of biosynthesis of Chl in angiosperms. Unlike many other plants, in fully green leaves of cucumber the expression of only a single NPR gene of cucumber was positively regulated by light and developmental age of leaf, and also by diurnal and circadian rhythms. The single NPR gene was shown to be involved in Chl synthesis throughout of the vegetative growth of cucumber (in cotyledons and fully green leaves).