Mutation in EMB1923 gene promoter is associated with chlorophyll deficiency in Chinese cabbage (Brassica campestris ssp. pekinensis)

Leaf color mutants are widespread in higher plants and can be used as markers in crop breeding or as important material in understanding the regulatory mechanisms of chlorophyll biosynthesis and chloroplast development. A stably inherited plant etiolated mutation (pem) was obtained from its wild‐type ‘FT’ (a doubled haploid line of the Chinese cabbage variety ‘Fukuda 50’) by combining ⁶⁰Co‐γ radiation and isolated microspore culture in Chinese cabbage. Compared to the wild‐type ‘FT’, the chlorophyll content in the pem mutant was decreased, the photosynthetic capacity was reduced and the chloroplast development was retarded. These physiological changes may lead to a reduction in growth and yield in the pem mutant line. Genetic analysis showed that the mutant phenotype was controlled by the single recessive nuclear pem gene. The pem gene was mapped to a 25.88 kb region on the A03 chromosome. Cloning and sequencing results showed that there was only one DNA sequence variation in this region, which was a 30 bp deletion on the promoter of Bra024218. Its homologous gene encodes EMBRYO DEFECTIVE 1923 (EMB1923) in Arabidopsis thaliana. We therefore predicted that Bra024218 was the mutated gene associated with etiolated leaves in Chinese cabbage. The pem mutant is a useful line for researching chloroplast development and the mechanism of leaf color mutation in Chinese cabbage.     

Isolation,characterization, and mapping of the stay green mutant in rice

Leaf color turns yellow during senescence due to the degradation of chlorophylls and photosynthetic proteins. A stay green mutant was isolated from the glutinous japonica rice Hwacheong-wx through N-methyl-N-nitrosourea mutagenesis. Leaves of the mutant remained green, while turning yellow in those of the wild-type rice during senescence. The stay green phenotype was controlled by a single recessive nuclear gene, tentatively symbolized as sgr(t). All the phenotypic characteristics of the mutant were the same as those of the wild-type lines except for the stay green trait. The leaf chlorophyll concentration of the mutant was similar to that of the wild-type before heading, but decreased steeply in the wild-type during grain filling, while very slowly in the mutant. However, no difference in photosynthetic activity was observed between the stay green mutant and the yellowing wild-type leaves, indicating that senescence is proceeding normally in the mutant leaves and that the mutation affects the rate of chlorophyll degradation during the leaf senescence. Using phenotypic and molecular markers, we mapped the sgr(t) locus to the long arm of chromosome 9 between RFLP markers RG662 and C985 at 1.8- and 2.1-cM intervals, respectively. Received: 29 April 2001 / Accepted: 17 July 2001     

A New GA-Insensitive Semidwarf Mutant of Rice (Oryza sativa L.) with a Missense Mutation in the SDG Gene

A semidwarf line of Indica rice, Xinguiai, was derived from the progeny of a cross between the double dwarf mutant Xinguiaishuangai and the wild-type variety Nanjing 6. The semidwarf phenotype was controlled by the semidwarf gene, sdg. The second sheath and shoot elongation responses of the dwarf mutant to exogenous gibberellin (GA₃) showed that sdg was insensitive to gibberellin (GA), and its endogenous GAs content was higher than that in wild-type cultivars. The SDG gene was cloned by a map-based cloning method and sequencing analysis revealed that the coding region of sdg had a single nucleotide substitution resulting in a single amino acid change from alanine to threonine. A cleaved amplified polymorphic sequence marker was designed according to sequences from mutant and wild-type materials. This sequence marker could be used to distinguish wild types and mutants, and thus, could be used for molecular marker-assisted selection. The dwarf phenotype of the sdg mutant was restored to a normal phenotype by introducing the wild-type SDG gene. Rice transformation experiments and GUS staining demonstrated that the SDG gene was predominantly expressed in vegetative organs.     

Cloning and molecular analysis of the Arabidopsis gene Terminal Flower 1

The Arabidopsis gene Terminal Flower 1 (TFL1) controls inflorescence meristem identity. A terminal flower (tfl1) mutant, which develops a terminal flower at the apex of the inflorescence, was induced by transformation with T-DNA. Using a plant DNA fragment flanking the integrated T-DNA as a probe, a clone was selected from a wild-type genomic library. Comparative sequence analysis of this clone with an EST clone (129D7T7) suggested the existence of a gene encoding a protein similar to that encoded by the cen gene which controls inflorescence meristem identity in Antirrhinum. Nucleotide sequences of the region homologous to this putative TFL1 gene were compared between five chemically induced tfl1 mutants and their parental wild-type ecotypes. Every mutant was found to have a nucleotide substitution which could be responsible for the tfl1 phenotype. This result confirmed that the cloned gene is TFL1 itself. In our tfl1 mutant, no nucleotide substitution was found in the transcribed region of the gene, and the T-DNA-insertion site was located at 458 bp downstream of the putative polyadenylation signal, suggesting that an element important for expression of the TFL1 gene exists in this area. Received: 14 November 1996 / Accepted: 29 November 1996     

ES5 is involved in the regulation of phosphatidylserine synthesis and impacts on early senescence in rice (Oryza sativa L.)

Leaf senescence, which affects plant growth and yield in rice, is an ideal target for crop improvement and remarkable advances have been made to identify the mechanism underlying this process. We have characterized an early senile mutant es5 (early leaf senescence 5) in rice exhibiting leaf yellowing phenotype after the 4-leaf stage. This phenotype was confirmed by the higher accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), the disintegration of chloroplasts, reduction in chlorophyll content and photosynthetic rate and up-regulation of senescence-associated genes (SAGs) like Osh36, OsI57, and OsI85. Positional cloning revealed that the es5 phenotype is the result of one base substitution in ES5, encoding phosphatidylserine synthase (PSS) family protein, which is involved in the base-exchange type reaction to synthesize the minor membrane phospholipid phosphatidylserine. Functional complementation of ES5 in the es5 plants completely restored the wild-type phenotype. Ultra-high-performance liquid chromatography (UHPLC) analysis showed that es5 plants had increased levels of phosphatidylserine (PS) and decreased level of phosphatidylcholine (PC). These results provide evidence about the role of PS in rice leaf senescence.

     

Ospapst1, a useful mutant for identifying seed purity and authenticity in hybrid rice

The stability and completeness of male sterility is still a challenge in some male sterile rice lines, especially those of photoperiod/thermo-sensitive genic male sterility (P/TGMS). Leaf color marker is a widely practiced approach to reduce the impact of self-pollinated seeds of male sterile lines. The papst1 is a leaf color mutant. The newly emerged leaves of papst1 are chlorosis and have an impaired photosynthesis. But the other agronomic traits, such as germination rate, duration of maturation and seed weight, are not changed. The papst1/PAPST1 F₁ showed the wild-type leaf phenotype. The papst1/PAPST1 F₂ progenies displayed an approximately 3:1 segregation ratio of WT phenotype:mutant phenotype (72: 28, χ² = 0.48, p > 0.05), suggesting that papst1 mutant phenotype is caused by a single repressive gene. Map-based cloning and sequencing analysis revealed that a point mutation was occurred in Os01 g16040 (OsPAPST1). Given these results, the Ospapst1 mutant is a useful mutant for identifying seed purity and authenticity in hybrid rice.     

GaHD1, a candidate gene for the Gossypium arboreum SMA-4 mutant,promotes trichome and fiber initiation by cellular H2O2 and Ca2+ signals

Cotton fibers are initiated from the epidermal cells of the ovule before or on the day of anthesis. Gossypium arboreum SMA-4 mutant contains recessive mutation (sma-4(ha)) and has the phenotypes of fibreless seeds and glabrous stems. In this study, fine mapping and alternative splicing analysis indicated a nucleotide substitution (AG?→?AC) at splicing site in a homeodomain-leucine zipper IV family gene (GaHD1) might cause gene A3S (Alternative 3′ splicing) mistake, suggested that GaHD1 was the candidate gene of sma-4(ha). Many genes related to the fiber initiation are identified to be differentially expressed in the mutant which could result in the blocked fiber initiation signals such as H₂O_2, or Ca in the mutant. Further comparative physiological analysis of H₂O₂ production and Ca²⁺ flux in the SMA-4 and wide type cotton confirmed that H₂O₂ and Ca were important fiber initiation signals and regulated by GaHD1. The in vitro ovule culture of the mutant with hormones recovered the fibered phenotype coupled with the restoration of these signals. Overexpressing of GaHD1 in Arabidopsis increased trichome densities on the sepal, leaf, and stem tissues while transient silencing of the GaHD1 gene in G. arboreum reduced the trichome densities. These phenotypes indicated that GaHD1 is the candidate gene of SMA-4 with a crucial role in acting upstream molecular switch of signal transductions for cotton trichome and fiber initiations.

     

Melanocortin 1 receptor (MC1R) polymorphisms’ influence on size and dermoscopic features of nevi

The melanocortin 1 receptor (MC1R) is a highly polymorphic gene. The loss‐of‐function MC1R variants (“R”) have been strongly associated with red hair color phenotype and an increased melanoma risk. We sequenced the MC1R gene in 175 healthy individuals to assess the influence of MC1R on nevus phenotype. We identified that MC1R variant carriers had larger nevi both on the back [p‐value = .016, adjusted for multiple parameters (adj. p‐value)] and on the upper limbs (adj. p‐value = .007). Specifically, we identified a positive association between the “R” MC1R variants and visible vessels in nevi [p‐value = .033, corrected using the FDR method for multiple comparisons (corrected p‐value)], dots and globules in nevi (corrected p‐value = .033), nevi with eccentric hyperpigmentation (corrected p‐value = .033), a high degree of freckling (adj. p‐value = .019), and an associative trend with presence of blue nevi (corrected p‐value = .120). In conclusion, the MC1R gene appears to influence the nevus phenotype.     

水稻黄绿叶突变体ygl-63的特征和基因定位

叶绿体的正常发育对于植物至关重要,突变体研究是探明叶绿体发育过程中基因功能的有效途径。叶色突变体已引起人们广泛的关注,通过对各种植物材料的研究,叶色突变的分子机制已取得一定进展,但远未被阐明,尤其在水稻当中。目前,已报道的水稻叶色突变体,主要表现为黄化、白化、亮绿、条斑条纹、温敏变色、转绿和转紫等。该研究使用甲基磺酸乙酯( EMS)处理粳稻日本晴,获得一份遗传稳定的突变体ygl-63,其整个生育期叶片均表现为黄绿色。通过测定ygl-63和野生型苗期叶片的叶绿素含量发现,ygl-63中叶绿素a、叶绿素b和总叶绿素含量与野生型相比分别下降了31.9％、42.2％和34.1％,同时叶绿素a/b值较野生型增加。这表明叶绿素含量的降低是导致ygl-63黄绿叶突变性状的主要原因,并且叶绿素b的降幅大于叶绿素a。在成熟后调查主要农艺性状发现ygl-63单株有效穗数和结实率分别减少8.9％和8.5％;千粒重增加10.4％;而株高,穗长和每穗着粒数和野生型相比差异并不显著。通过测量微量元素发现,ygl-63种子中的铁和锌含量较野生型显著降低,分别减少85.7％和64.8％。将ygl-63与正常绿色品种明恢63杂交获得F1和F2群体,进行遗传分析发现,ygl-63突变性状受1对隐性基因控制,通过基因定位,将该基因定位到水稻第11染色体长臂的分子标记InDel-3和InDel-5之间约2.4 cM范围内。该基因被认为是一个新的水稻叶色突变基因,暂命名为ygl-63( g)。所得结果为今后对ygl-63( g)基因的进一步研究奠定了基础。     

拟南芥角质层发育突变体中WBC11基因的分析与鉴定

从拟南芥(Arabidopsis thaliana L.)突变体库中筛选到一个发育突变体ku7fy1,其突变表型为叶片狭长,生长缓慢。该研究利用图位克隆技术和候选基因测序鉴定出ku7fy1角质层发育基因(white-brown complex11,WBC11)有一个点突变。对该突变体cDNA测序结果显示,WBC11基因的突变导致其第7个内含子在形成成熟mRNA时无法被正常剪切,使该突变体内WBC11的mRNA大量降解并在翻译时提前引入终止密码子。甲苯胺蓝染色实验显示,突变体叶片表面角质层有缺陷;遗传互补实验进一步证明,突变体ku7fy1中的突变基因是WBC11,ku7fy1表型是由WBC11突变造成的。     

Altered patterns of senescence and ripening in gf, a stay-green mutant of tomato (Lycopersicon esculentum Mill.)

The gf tomato mutant, which retains chlorophyll during ripening, has been found to be affected in leaf senescence. The leaves of the gfmutant show an absolute stay-green phenotype. As leaf senescence and fruit ripening proceed, there is a marked difference in chlorophyll content between wild-type and gf. In both attached and detached leaf studies, or after treatment with ethylene, the leaves withered and abscised in gf with only slight loss of chlorophyll and carotenoids. Total protein content declined and free amino acids increased during leaf senescence in wild-type and gf, but Western analysis showed that LHCII polypeptides were retained at higher levels in gf. Expression of senescence-related mRNAs increased normally in gf whereas those for cab, rbcS and rbcL declined in both mutant and wild-type. The mutant possesses enzyme activity for chlorophyllase, the formation of phaeophorbide a by the action of Mg-dechelatase and the oxygenolytic opening of the porphyrin macrocycle. Analysis of chlorophyll breakdown products in fruit indicated that gf, like other stay-green mutants, accumulates chlorophyllides a and b, but phaeophorbide a does not accumulate in vivo. This may indicate that, in the mutant, in vivo the action of phaeophorbide a-oxygenase is somehow presented, either by altered accessibility or transport of components required for thylakoid disassembly or the absence of another factor.     

Further insight into the role of <Emphasis Type="Italic">KAN1</Emphasis>, a member of <Emphasis Type="Italic">KANADI</Emphasis> transcription factor family in rice

The rice EMS-derived mutant leaf adaxialized 1 (lad1) was isolated based on its upward rolling leaf phenotype. Besides the adaxially rolled leaf, many other agronomic traits were also compromised in lad1. The rolling trait was characterized by a noticeable alteration of bulliform cells in the adaxial side of the leaves. Map-based cloning showed a single nucleotide substitution in the promoter region of the KAN1 gene in lad1 mutant. Further, over-expressing and CRISPR/cas9-edited knockdown transgenic plants confirmed that KAN1 was responsible for the mutant phenotype of lad1. Yeast two-hybrid and bimolecular fluorescence complementation assay demonstrated that KAN1 can interact with the auxin response factors ARF3, ARF7 and ARF15. Physiologically, the contents of auxin (IAA), abscisic acid (ABA), jasmonic acid (JA) and gibberellin (GA) were all significantly increased in the lad1 mutant. Moreover, the GA3 content dramatically decrease in wild-type, but increased in lad1 under IAA induction. Additionally, the expression levels of several IAA and GA biosynthesis and responsive-related genes and genes involved in leaf polarity determination were altered in lad1. Therefore, we hypothesized that KAN1/ARFs protein complexes act as auxin-dependent regulatory units that play a conserved role in leaf development.     

Isolation and characterization of a rice mutant with narrow and rolled leaves

Appropriate leaf shape has proved to be useful in improving photosynthesis and increasing grain yield. To understand the molecular mechanism of leaf morphogenesis, we identified a rice mutant nrl1, which was characterized by a phenotype of narrow and rolled leaves. Microscopic observation showed that the mutation significantly decreased the number of vascular bundles of leaf and stem. Genetic analysis revealed that the mutation was controlled by a single nuclear-encoded recessive gene. To isolate the nrl1 gene, 756 F₂ and F₃ mutant individuals from a cross of the nrl1 mutant with Longtepu were used and a high-resolution physical map of the chromosomal region around the nrl1 gene was made. Finally, the gene was mapped in 16.5 kb region between marker RL21 and marker RL36 within the BAC clone OSJNBa0027H05. Cloning and sequencing of the target region from the mutant showed that there was a 58 bp deletion within the second exon of the cellulose synthase-like D4 gene (TIGR locus Os12g36890). The nrl1 mutation was rescued by transformation with the wild-type cellulose synthase-like D4 gene. Accordingly, the cellulose synthase-like D4 gene was identified as the NRL1 gene. NRL1 was transcribed in various tissues and was mainly expressed in panicles and internodes. NAL7 and SLL1 were found to be upregulated, whereas OsAGO7 were downregulated in the nrl1 mutant. These findings suggested that there might be a functional association between these genes in regulating leaf development.     

Single base substitution in OsCDC48 is responsible for premature senescence and death phenotype in rice

A premature senescence and death 128 (psd128) mutant was isolated from an ethyl methane sulfonate‐induced rice IR64 mutant bank. The premature senescence phenotype appeared at the six‐leaf stage and the plant died at the early heading stage. psd128 exhibited impaired chloroplast development with significantly reduced photosynthetic ability, chlorophyll and carotenoid contents, root vigor, soluble protein content and increased malonaldehyde content. Furthermore, the expression of senescence‐related genes was significantly altered in psd128. The mutant trait was controlled by a single recessive nuclear gene. Using map‐based strategy, the mutation Oryza sativa cell division cycle 48 (OsCDC48) was isolated and predicted to encode a putative AAA‐type ATPase with 809 amino‐acid residuals. A single base substitution at position C2347T in psd128 resulted in a premature stop codon. Functional complementation could rescue the mutant phenotype. In addition, RNA interference resulted in the premature senescence and death phenotype. OsCDC48 was expressed constitutively in the root, stem, leaf and panicle. Subcellular analysis indicated that OsCDC48:YFP fusion proteins were located both in the cytoplasm and nucleus. OsCDC48 was highly conserved with more than 90% identity in the protein levels among plant species. Our results indicated that the impaired function of OsCDC48 was responsible for the premature senescence and death phenotype.     

Physiological character and molecular mapping of leaf-color mutant wyv1 in rice (Oryza sativa L.)

The seed of an excellent indica restorer line Jinhui10 (Oryza sativa L. ssp. indica) was treated by ethyl methanesulfonate (EMS); a leaf-color mutant displaying distinct phenotype throughout development grown in paddy field was identified from the progeny. The mutant leaf showed white-yellow at seedling stage and then turned to yellow-green at tillering stage, after that, virescent color appeared until to maturity. The mutant was thus temporarily designed as wyv1. The chlorophyll contents decreased significantly and the changing was consistent with the chlorotic level of wyv1 leaves. Chlorophyll fluorescence kinetic parameters measured at the seedling stage showed that co-efficiency of photochemical quenching (qP), actual photosystem II efficiency (ΦPS II), electron transport rate (ETR) and initial chlorophyll fluorescence level (Fo), net photosynthetic rate (Pn) and maximum photochemical efficiency (Fv / Fm) significantly decreased in severe chlorotic leaf of the mutant compared with that of wild type. However, no significant differences were observed for Pn and Fv/Fm between virescent leaf and normal green leaf. Genetic analysis suggested that the mutant phenotype was controlled by a single recessive nuclear gene which was finally mapped between SSR marker Y7 and Y6 on rice chromosome 3 based on F2 population of Xinong1A / wyv1. Genetic distances were 0.06 cM and 0.03 cM respectively, and the physical distance was 84 kb according to the sequence of indica rice 9311. The results must facilitate map-based cloning and functional analysis of WYV1 gene.     

The recessive phenotype of forked can be uncovered by heat shock in Drosophila

Heat shock uncovers the recessive forked phenotype when heterozygotes between f^36a and wild-type are heated during sensitive periods in pupal development. We call the phenocopy of a mutant in such a heterozygote a heterocopy. The heterocopy in f^36a/+ is virtually identical to the mutant phenotype; however, bristles on different parts of the body are affected during different sensitive periods. We discuss the hypothesis that the heat shock acts by affecting expression of the wild-type gene product corresponding to the mutant gene. The sensitive period for heterocopy induction in a specific tissue is proposed to correspond to the normal time of gene expression for the forked gene product in a particular tissue.     

Genetic and phenotypic characterization of cop1 mutants of Arabidopsis thaliana

Ten Arabidopsis lines that carry recessive mutations in the cop1 (constitutively photomorphogenic) locus have been isolated. These lines define at least four different alleles. All of the mutant lines produce dark-grown seedlings that mimic wild-type seedlings grown in the light. The phenotype of the dark-grown mutant seedlings includes: short hypocotyls, open and enlarged cotyledons, accumulation of anthocyanin, cell-type differentiation and chloroplast-like plastid differentiation in cotyledons. Moreover, in more prolonged dark-growth periods the mutants exhibit true leaf development that parallels that in light-grown siblings. The four mutant alleles represent two types of mutations: three alleles (cop 1-1, cop 1-2, and cop 1-3) have severely affected phenotypes whereas one allele (cop 1-4) has a less severe phenotype. Compared to the severe alleles, the cop 1-4 mutant has slightly longer hypocotyls in dark-grown seedlings and does not accumulate abnormal levels of anthocyanin. The cop1–1/cop1-4 hybrid seedlings are intermediate in many physiological properties under both dark- and light-growth conditions, relative to the two parents. These results may suggest that the extent of residual cop1 gene activity in the mutants dictates the degree to which the aberrant plant phenotype is expressed. Analysis of plants carrying both cop1 and hy, a mutation that results in a deficiency of active phyto-chrome, suggests that the cop1 gene product acts downstream of phytochrome. The differentiation of chloroplasts in the roots of light-grown cop1 plants but not in wild-type plants suggests that the wild-type cop1 gene product also normally plays a role in suppressing chloroplast development in the roots of light-grown plants. To aid the eventual molecular cloning of the cop1 locus, its chromosomal location has been mapped and a molecular marker that is located about 1 centimorgan away from the cop1 locus obtained.