Fine mapping of CscpFtsY, a gene conferring the yellow leaf phenotype in cucumber (Cucumis sativus L.).

BACKGROUND: Leaf color mutants are ideal materials to study pigment metabolism and photosynthesis. Leaf color variations are mainly affected by chlorophylls (Chls) and carotenoid contents and chloroplast development in higher plants. However, the regulation of chlorophyll metabolism remains poorly understood in many plant species. The chloroplast signal-recognition particle system is responsible for the insertion of the light-harvesting chlorophyll a/b proteins (LHCPs) to thylakoid membranes, which controls the chloroplast development as well as the regulation of Chls biosynthesis post-translationally in higher plants. RESULTS: In this study, the yellow leaf cucumber mutant, named yl, was found in an EMS-induced mutant library, which exhibited a significantly reduced chlorophyll content, abnormal chloroplast ultrastructure and decreased photosynthetic capacity. Genetic analysis demonstrated that the phenotype of yl was controlled by a recessive nuclear gene. Using BSA-seq technology combined with the map-based cloning method, we narrowed the locus to a 100 kb interval in chromosome 3. Linkage analysis and allelism test validated the candidate SNP residing in CsaV3_3G009150 encoding one homolog of chloroplast signal-recognition particle (cpSRP) receptor in Arabidopsis, cpFtsY, could be responsible for the yellow leaf phenotype of yl. The relative expression of CscpFtsY was significantly down-regulated in different organs except for the stem, of yl compared with that in the wild type (WT). Subcellular localization result showed that CscpFtsY located in the chloroplasts of mesophyll cells. CONCLUSIONS: The yl mutant displayed Chls-deficient, impaired chloroplast ultrastructure with intermittent grana stacks and significantly decreased photosynthetic capacity. The isolation of CscpFtsY in cucumber could accelerate the progress on chloroplast development by cpSRP-dependant LHCP delivery system and regulation of Chls biosynthesis in a post-translational way.

Physiological analysis and transcriptome sequencing of a delayed-green leaf mutant 'Duojiao' of ornamental crabapple (Malus sp.).

Malus spectabilis 'Duojiao' is a spontaneous delayed-green leaf color mutant of M. spectabilis 'Riversii' and has chloroplasts with irregularly arranged vesicles and indistinct stromal lamellae. The yellow leaves of mutant have less chlorophyll (Chl), carotenoids, and flavonoids. Measurement of photosynthetic gas exchange indicated that the mutant has lower photosynthetic activity than 'Riversii' plants. Transcriptome sequencing with the Illumina platform was used to characterize differences in gene expression between the leaves of plants with yellow and green colors and elucidate the molecular mechanisms responsible for variation in leaf color in ornamental crabapple. In the comparison group of mutant yellow leaves and the maternal green leaves, 1848 differentially significant expressed genes (DEGs) were annotated by transcriptomic analysis. Many DEGs and transcription factors were identified related to chloroplast development, Chl synthesis and degradation, photosynthesis, carotenoid biosynthesis, flavonoid biosynthesis and other pathways related to plant leaf color formation. Among these, the Chl biosynthesis-related coproporphyrinogen gene, oxidative decarboxylase gene, and Chl a oxygenase gene were down-regulated, indicating that Chl biosynthesis was blocked. GLK1, which regulates chloroplast development, was down-regulated in yellow leaves. Parallel experiments showed that the content of the Chl synthesis precursors, protoporphyrinogen IX, chlorophyllide a, and chlorophyllide b and the activity of chlorophyllogen III oxidase and chlorophyllide a oxygenase in the yellow leaves of 'Duojiao' were lower than those in the green leaves of 'Riversii'. Thus, leaf color formation is greatly affected by Chl metabolism and chloroplast development. The reliability of the RNA-sequencing data was confirmed by quantitative real-time PCR analysis with 12 selected DEGs. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01248-7.

Involvement of CsERF2 in leaf variegation of Cymbidium sinense 'Dharma'.

MAIN CONCLUSION: CsERF2, an ethylene response factor, plays a role in leaf variegation. Leaf variegation is a main ornamental characteristic in Cymbidium sinense (C. sinense). However, the mechanisms of leaf color variegation remain largely unclear. In the present study, we analyzed the cytological and physiological features, as well as molecular analyses of leaves from wild-type (WT) and leaf variegation mutants of Cymbidium sinense 'Dharma'. Chloroplasts with typical and functional structures were discovered in WT and green sectors of the mutants leaves (MG), but not in yellow sectors of the mutant leaves (MY). The activities of key enzymes involved in chlorophyll (Chl) degradation and their substrate contents were significantly increased in MY. Genes related to Chl degradation also showed a significant up-regulation in MY. Transcriptomic analysis showed that the expression of all identified ethylene response factors (ERFs) was significantly up-regulated, and the 1-aminocyclopropane-1-carboxylic acid (ACC) content in MY was significantly higher compared with MG. QRT-PCR analysis validated that the expression levels of genes related to Chl degradation could be positively affected by ethylene (ETH) treatment. Stable overexpression of CsERF2 in Nicotiana tabacum (N. tabacum) led to a decrease in Chl content and abnormal chloroplast. Transcriptomic analysis and qRT-PCR results showed that the KEGG pathway related to chloroplast development and function showed significant change in transgenic N. tabacum. Therefore, the leaf color formation of C. sinense was greatly affected by chloroplast development and Chl metabolism. CsERF2 played an important role in leaf variegation of C. sinense.

The identification of key candidate genes mediating yellow seedling lethality in a Lilium regale mutant.

Leaf color mutants are ideal materials for exploring plant photosynthesis mechanisms, chlorophyll biosynthetic pathways and chloroplast development. The yellow seedling lethal mutant lrysl1 was discovered from self-bred progenies of Lilium regale; however, the mechanism of leaf color mutation remains unclear. In this study, the ultrastructural and physiological features and de novo RNA-Seq data of a L. regale leaf color mutant and wild-type L. regale were investigated. Genetic analysis indicated that the characteristics of the lrysl1 mutant were controlled by a recessive nuclear gene. The chlorophyll a, chlorophyll b and carotenoid contents in the mutant leaves were lower than those in the wild-type leaves. Furthermore, the contents of the chlorophyll precursors aminolevulinic acid (ALA), porphobilinogen (PBG), protoporphyrin IX (ProtoIX), Mg-protoporphyrin IX (Mg-ProtoIX), and protochlorophyll (Pchl) decreased significantly in mutant leaves. Transcriptome data from the mutant and wild type showed that a total of 892 differentially expressed genes were obtained, of which 668 and 224 were upregulated genes and downregulated genes in the mutant, respectively. Almost all genes in the photosynthesis pathway and chlorophyll biosynthetic pathway were downregulated in the mutant, which corroborated the differences in the physiological features mentioned above. Further research indicated that the chloroplasts of the mutant leaves exhibited an abnormal morphology and distribution and that the expression of a gene related to chloroplast development was downregulated. It was concluded that abnormal chloroplast development was the main cause of leaf color mutation in the mutant lrysl1 and that LrGLK was a gene related to chloroplast development in L. regale. This research provides a foundation for further research on the mechanism by which LrGLK regulates chloroplast development in L. regale.

A lil3 chlp double mutant with exclusive accumulation of geranylgeranyl chlorophyll displays a lethal phenotype in rice.

BACKGROUND: Phytyl residues are the common side chains of chlorophyll (Chl) and tocopherols. Geranylgeranyl reductase (GGR), which is encoded by CHLP gene, is responsible for phytyl biosynthesis. The light-harvesting like protein LIL3 was suggested to be required for stability of GGR and protochlorophyllide oxidoreductase in Arabidopsis. RESULTS: In this study, we isolated a yellow-green leaf mutant, 637ys, in rice (Oryza sativa). The mutant accumulated majority of Chls with unsaturated geranylgeraniol side chains and displayed a yellow-green leaf phenotype through the whole growth period. The development of chloroplasts was suppressed, and the major agronomic traits, especially No. of productive panicles per plant and of spikelets per panicle, dramatically decreased in 637ys. Besides, the mutant exhibited to be sensitive to light intensity and deficiency of tocopherols without obvious alteration in tocotrienols in leaves and grains. Map-based cloning and complementation experiment demonstrated that a point mutation on the OsLIL3 gene accounted for the mutant phenotype of 637ys. OsLIL3 is mainly expressed in green tissues, and its encoded protein is targeted to the chloroplast. Furthermore, the 637ys 502ys (lil3 chlp) double mutant exclusively accumulated geranylgeranyl Chl and exhibited lethality at the three-leaf stage. CONCLUSIONS: We identified the OsLIL3 gene through a map-based cloning approach. Meanwhile, we demonstrated that OsLIL3 is of extreme importance to the function of OsGGR, and that the complete replacement of phytyl side chain of chlorophyll by geranylgeranyl chain could be fatal to plant survival in rice.

The Reason for Growth Inhibition of Ulmus pumila 'Jinye': Lower Resistance and Abnormal Development of Chloroplasts Slow Down the Accumulation of Energy.

Ulmus pumila 'Jinye', the colorful leaf mutant of Ulmus pumila L., is widely used in landscaping. In common with most leaf color mutants, U. pumila 'Jinye' exhibits growth inhibition. In this study, U. pumila L. and U. pumila 'Jinye' were used to elucidate the reasons for growth inhibition at the physiological, cellular microstructural, and transcriptional levels. The results showed that the pigment (chlorophyll a, chlorophyll b, and carotenoids) content of U. pumila L. was higher than that of U. pumila 'Jinye', whereas U. pumila 'Jinye' had a higher proportion of carotenoids, which may be the cause of the yellow leaves. Examination of the cell microstructure and RNA sequencing analysis showed that the leaf color and growth inhibition were mainly due to the following reasons: first, there were differences in the structure of the thylakoid grana layer. U. pumila L. has a normal chloroplast structure and clear thylakoid grana slice layer structure, with ordered and compact thylakoids. However, U. pumila 'Jinye' exhibited the grana lamella stacking failures and fewer thylakoid grana slice layers. As the pigment carrier and the key location for photosynthesis, the close stacking of thylakoid grana could combine more chlorophyll and promote efficient electron transfer promoting the photosynthesis reaction. In addition, U. pumila 'Jinye' had a lower capacity for light energy absorption, transformation, and transportation, carbon dioxide (CO2) fixation, lipopolysaccharide biosynthesis, auxin synthesis, and protein transport. The genes related to respiration and starch consumption were higher than those of U. pumila L., which indicated less energy accumulation caused the growth inhibition of U. pumila 'Jinye'. Finally, compared with U. pumila 'Jinye', the transcription of genes related to stress resistance all showed an upward trend in U. pumila L. That is to say, U. pumila L. had a greater ability to resist adversity, which could maintain the stability of the intracellular environment and maintain normal progress of physiological metabolism. However, U. pumila 'Jinye' was more susceptible to changes in the external environment, which affected normal physiological metabolism. This study provides evidence for the main cause of growth inhibition in U. pumila 'Jinye', information for future cultivation, and information on the mutation mechanism for the breeding of colored leaf trees.

Candidate Genes for Yellow Leaf Color in Common Wheat (Triticum aestivum L.) and Major Related Metabolic Pathways according to Transcriptome Profiling.

The photosynthetic capacity and efficiency of a crop depends on the biosynthesis of photosynthetic pigments and chloroplast development. However, little is known about the molecular mechanisms of chloroplast development and chlorophyll (Chl) biosynthesis in common wheat because of its huge and complex genome. Ygm, a spontaneous yellow-green leaf color mutant of winter wheat, exhibits reduced Chl contents and abnormal chloroplast development. Thus, we searched for candidate genes associated with this phenotype. Comparative transcriptome profiling was performed using leaves from the yellow leaf color type (Y) and normal green color type (G) of the Ygm mutant progeny. We identified 1227 differentially expressed genes (DEGs) in Y compared with G (i.e., 689 upregulated genes and 538 downregulated genes). Gene ontology and pathway enrichment analyses indicated that the DEGs were involved in Chl biosynthesis (i.e., magnesium chelatase subunit H (CHLH) and protochlorophyllide oxidoreductase (POR) genes), carotenoid biosynthesis (i.e., ß-carotene hydroxylase (BCH) genes), photosynthesis, and carbon fixation in photosynthetic organisms. We also identified heat shock protein (HSP) genes (sHSP, HSP70, HSP90, and DnaJ) and heat shock transcription factor genes that might have vital roles in chloroplast development. Quantitative RT-PCR analysis of the relevant DEGs confirmed the RNA-Seq results. Moreover, measurements of seven intermediate products involved in Chl biosynthesis and five carotenoid compounds involved in carotenoid-xanthophyll biosynthesis confirmed that CHLH and BCH are vital enzymes for the unusual leaf color phenotype in Y type. These results provide insights into leaf color variation in wheat at the transcriptional level.

The Rice YL4 Gene Encoding a Ribosome Maturation Domain Protein Is Essential for Chloroplast Development.

Chloroplast RNA splicing and ribosome maturation (CRM) domain proteins are a family of plant-specific proteins associated with RNA binding. In this study, we have conducted a detailed characterization of a novel rice CRM gene (LOC_Os04g39060) mutant, yl4, which showed yellow-green leaves at all the stages, had fewer tillers, and had a decreased plant height. Map-based cloning and CRISPR/Cas9 editing techniques all showed that YL4 encoded a CRM domain protein in rice. In addition, subcellular localization revealed that YL4 was in chloroplasts. YL4 transcripts were highly expressed in all leaves and undetectable in roots and stems, and the mutation of YL4 affected the transcription of chloroplast-development-related genes. This study indicated that YL4 is essential for chloroplast development and affects some agronomic traits.

Screening and Validation of Internal Reference Genes for Quantitative Real-Time PCR Analysis of Leaf Color Mutants in Dendrobium officinale.

Leaf color mutants (LCMs) are important resources for studying diverse metabolic processes such as chloroplast biogenesis and differentiation, pigments' biosynthesis and accumulation, and photosynthesis. However, in Dendrobium officinale, LCMs are yet to be fully studied and exploited due to the unavailability of reliable RGs (reference genes) for qRT-PCR (quantitative real-time reverse transcription PCR) normalization. Hence, this study took advantage of previously released transcriptome data to select and evaluate the suitability of ten candidate RGs, including Actin (Actin), polyubiquitin (UBQ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-α (EF1α), ß-tubulin (ß-TUB), α-tubulin (α-TUB), 60S ribosomal protein L13-1 (RPL13AD), aquaporin PIP1-2 (PIP1-2), Intima protein (ALB3) and Cyclin (CYCB1-2) for normalizing leaf color-related genes' expression levels via qRT-PCR. Stability rankings analysis via common software Best-Keeper, GeNorm, and NormFinder disclosed that all ten genes met the requirements of RGs. Of them, EF1α exhibited the highest stability and was selected as the most reliable. The reliability and accuracy of EF1α were confirmed through qRT-PCR analysis of fifteen chlorophyll pathway-related genes. The expression patterns of these genes via EF1α normalization were consistent with the results by RNA-Seq. Our results offer key genetic resources for the functional characterization of leaf color-related genes and will pave the way for molecular dissection of leaf color mutations in D. officinale.

Phenotypic characterization of the wheat temperature-sensitive leaf color mutant and physical mapping of mutant gene by reduced-representation sequencing.

Few available leaf color mutants in crops have greatly limited the understanding of photosynthesis mechanisms, leading to few accomplishments in crop yield improvement via enhanced photosynthetic efficiency. Here, a noticeable albino mutant, CN19M06, was identified. A comparison between CN19M06 and the wild type CN19 at different temperatures showed that the albino mutant was temperature-sensitive and produced leaves with a decreased chlorophyll content at temperatures below 10 °C. Genetic analysis suggested that the albinism was controlled by one recessive nuclear gene named TSCA1, which was putatively assigned to the region of 718.1-729.8 Mb on chromosome 2AL using bulked-segregant analysis and double-digest restriction site-associated DNA. Finally, molecular linkage analysis physically anchored TSCA1 to a narrowed region of 718.8-725.3 Mb with a 6.5 Mb length on 2AL flanked by InDel 18 and InDel 25 with 0.7 cM genetic interval. Among the 111 annotated functional genes in the corresponding chromosomal region, only TraesCS2A01G487900 of the PAP fibrillin family was both related to chlorophyll metabolism and temperature sensitivity; therefore, it was considered the putative candidate gene of TSCA1. Overall, CN19M06 has great potential for exploring the molecular mechanism of photosynthesis and monitoring temperature changes in wheat production.

Slym1 control the color etiolation of leaves by facilitating the decomposition of chlorophyll in tomato.

Photosynthesis, as an important biological process of plants, produces organic substances for plant growth and development. Although the molecular mechanisms of photosynthesis had been well investigated, the relationship between chlorophyll synthesis and photosynthesis remains largely unknown. The leaf-color mutant was an ideal material for studying photosynthesis and chlorophyll synthesis, which had been seldom investigated in tomato. Here, we obtained a yellow leaf tomato mutant ym (The mutant plants from the line of zs4) in field. Transmission electron microscopy (TEM) and photosynthetic parameters results demonstrated that chloroplast's structure was obviously destroyed and photosynthetic capacity gets weak. The mutant was hybridized with the control to construct the F2 segregation population for sequencing. Slym1 gene, controlling yellow mutant trait, was identified using Bulked Segregation Analysis. Slym1 was up-regulated in the mutant and Slym1 was located in the nucleus. The genes associated with photosynthesis and chlorophyll synthesis were down-regulated in Slym1-OE transgenic tomato plants. The results suggested that Slym1 negatively regulate photosynthesis. Photosynthetic pigment synthesis related genes HEMA, HEMB1, CHLG and CAO were up-regulated in Slym1 silencing plants. The redundant Slym1 binding the intermediate proteins MP resulting in hindering the interaction between MP and HY5 due to the Slym1 with a high expression level in ym mutant, lead to lots of the HY5 with unbound state accumulates in cells, that could accelerate the decomposition of chlorophyll. Therefore, the yellow leaf-color mutant ym could be used as an ideal material for further exploring the relationship between leaf color mutant and photosynthesis and the specific mechanism.

Comparative Transcriptome Profiling Analysis Reveals the Adaptive Molecular Mechanism of Yellow-Green Leaf in Rosa beggeriana 'Aurea'.

Rosa beggeriana 'Aurea' is a yellow-green leaf (yl) mutant and originated from Rosa beggeriana Schrenk by 60Co-γ irradiation, which is an important ornamental woody species. However, the molecular mechanism of the yl mutant remains unknown. Herein, comparative transcriptome profiling was performed between the yl type and normal green color type (WT) by RNA sequencing. A total of 3,372 significantly differentially expressed genes (DEGs) were identified, consisting of 1,585 upregulated genes and 1,787 downregulated genes. Genes that took part in metabolic of biological process (1,090), membrane of cellular component (728), catalytic (1,114), and binding of molecular function (840) were significantly different in transcription level. DEGs involved in chlorophyll biosynthesis, carotenoids biosynthesis, cutin, suberine, wax biosynthesis, photosynthesis, chloroplast development, photosynthesis-antenna proteins, photosystem I (PSI) and photosystem II (PSII) components, CO2 fixation, ribosomal structure, and biogenesis related genes were downregulated. Meanwhile, linoleic acid metabolism, siroheme biosynthesis, and carbon source of pigments biosynthesis through methylerythritol 4-phosphate (MEP) pathways were upregulated. Moreover, a total of 147 putative transcription factors were signification different expression, involving NAC, WRKY, bHLH, MYB and AP2/ERF, C2H2, GRAS, and bZIP family gene. Our results showed that the disturbed pigments biosynthesis result in yl color by altering the ratio of chlorophylls and carotenoids in yl mutants. The yl mutants may evoke other metabolic pathways to compensate for the photodamage caused by the insufficient structure and function of chloroplasts, such as enhanced MEP pathways and linoleic acid metabolism against oxidative stress. This research can provide a reference for the application of leaf color mutants in the future.

Molecular and Photosynthetic Performance in the Yellow Leaf Mutant of Torreya grandis According to Transcriptome Sequencing, Chlorophyll a Fluorescence, and Modulated 820 nm Reflection.

To study the photosynthetic energy mechanism and electron transfer in yellow leaves, transcriptomics combined with physiological approaches was used to explore the mechanism of the yellow leaf mutant Torreya grandis 'Merrillii'. The results showed that chlorophyll content, the maximal photochemical efficiency of PSII (Fv/Fm), and the parameters related to the OJ phase of fluorescence (φEo, φRo) were all decreased significantly in mutant-type T. grandis leaves. The efficiency needed for an electron to be transferred from the reduced carriers between the two photosystems to the end acceptors of the PSI (δRo) and the quantum yield of the energy dissipation (φDo) were higher in the leaves of mutant-type T. grandis compared to those in wild-type leaves. Analysis of the prompt fluorescence kinetics and modulated 820 nm reflection showed that the electron transfer of PSII was decreased, and PSI activity was increased in yellow T. grandis leaves. Transcriptome data showed that the unigenes involved in chlorophyll synthesis and the photosynthetic electron transport complex were downregulated in the leaves of mutant-type T. grandis compared to wild-type leaves, while there were no observable changes in carotenoid content and biosynthesis. These findings suggest that the downregulation of genes involved in chlorophyll synthesis leads to decreased chlorophyll content, resulting in both PSI activity and carotenoids having higher tolerance when acting as photo-protective mechanisms for coping with chlorophyll deficit and decrease in linear electron transport in PSII.

Fine Mapping and Transcriptome Analysis of Virescent Leaf Gene v-2 in Cucumber (Cucumis sativus L.).

Leaf color mutants are the ideal materials to explore the pathways of chlorophyll metabolism, chloroplast development and photosynthesis system. In this study, a new virescent leaf mutant 104Y was identified by spontaneous mutation, whose cotyledon and upper five true leaves were yellow color. The yellow true leaves gradually turned green from top to bottom with increased chlorophyll contents. Genetic analysis indicated that the virescent leaf was controlled by one single recessive gene v-2, which was accurately mapped into 36.0-39.7 Mb interval on chromosome 3 by using BSA-seq and linkage analysis. Fine mapping analysis further narrowed v-2 into 73-kb genomic region including eight genes with BC1 and F2 populations. Through BSA-seq and cDNA sequencing analysis, only one nonsynonymous mutation existed in the Csa3G890020 gene encoding auxin F-box protein was identified, which was predicted as the candidate gene controlling virescent leaf. Comparative transcriptome analysis and quantitative real-time PCR analysis revealed that the expression level of Csa3G890020 was not changed between EC1 and 104Y. However, RNA-seq analysis identified that the key genes involved in chlorophyll biosynthesis and auxin signaling transduction network were mainly down-regulated in 104Y compared with EC1, which indicated that the regulatory functions of Csa3G890020 could be performed at post-transcriptional level rather than transcriptional level. This is the first report to map-based clone an auxin F-box protein gene related to virescent leaf in cucumber. The results will exhibit a new insight into the chlorophyll biosynthesis regulated by auxin signaling transduction network.

The AabHLH35 Transcription Factor Identified from Anthurium andraeanum is Involved in Cold and Drought Tolerance.

Anthurium andraeanum Lind. is a popular potted and cut-flower plant with an attractive spathe and foliage. It is native to tropical rainforest areas and is able to blossom throughout the year under suitable conditions. However, various abiotic stresses seriously restrict the ornamental value of A. andraeanum and increase the costs of cultivation. A dark green (dg) leaf color mutant of A. andraeanum 'Sonate', which accumulates high levels of anthocyanin, has shown increased vigor and tolerance to stresses during cultivation and is, thus, an ideal germplasm for studying stress tolerance in this species. Here, we show that the anthocyanin content in dg mutant plants at different stages of leaf development was higher than in wild-type (WT) plants, and the ability to tolerate under low-temperature (LT, 14 °C) stress was stronger in dg than in WT plants. RNA-Seq of cDNA libraries from young leaves of dg and WT identified AabHLH35 as a differentially expressed gene (DEG) that was significantly up-regulated in dg. Furthermore, heterologous expression of AabHLH35 improved tolerance to cold and drought stresses in Arabidopsis. These results have built an important molecular foundation for further study of stress tolerance in A. andraeanum.

Fine Mapping of CsVYL, Conferring Virescent Leaf Through the Regulation of Chloroplast Development in Cucumber.

Leaf color mutants in higher plants are ideal materials for investigating the structure and function of photosynthetic system. In this study, we identified a cucumber vyl (virescent-yellow leaf) mutant in the mutant library, which exhibited reduced pigment contents and delayed chloroplast development process. F2 and BC1 populations were constructed from the cross between vyl mutant and cucumber inbred line 'Hazerd' to identify that the vyl trait is controlled by a simply recessive gene designated as CsVYL. The CsVYL gene was mapped to a 3.8 cM interval on chromosome 4 using these 80 F2 individuals and BSA (bulked segregation analysis) approach. Fine genetic map was conducted with 1542 F2 plants and narrowed down the vyl locus to an 86.3 kb genomic region, which contains a total of 11 genes. Sequence alignment between the wild type (WT) and vyl only identified one single nucleotide mutation (C→T) in the first exon of gene Csa4G637110, which encodes a DnaJ-like zinc finger protein. Gene Expression analysis confirmed the differences in transcription level of Csa4G637110 between wild type and mutant plants. Map-based cloning of the CsVYL gene could accelerate the study of chloroplast development and chlorophyll synthesis of cucumber.

Comparative profiling of microRNAs and their effects on abiotic stress in wild-type and dark green leaf color mutant plants of Anthurium andraeanum 'Sonate'.

MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including Anthurium andraeanum. To identify miRNAs and their target genes in A. andraeanum and study their responses to abiotic stresses, we conducted deep-sequencing of two small RNA (sRNA) libraries prepared from young leaves of wild-type (WT) and dark green (dg) leaf color mutant plants of A. andraeanum 'Sonate'. A total of 53 novel miRNAs were identified, 32 of which have been annotated to 18 miRNA families. 10 putative miRNAs were found to be differentially expressed in WT and dg, among which two miRNAs were significantly up-regulated and eight down-regulated in dg relative to WT. One differentially expressed miRNA, Aa-miR408, was dramatically up-regulated in dg. qRT-PCR analysis and heterologous expression of Aa-miR408 in Arabidopsis under different stress treatments suggest that Aa-miR408 is involved in abiotic stress responses in A. andraeanum. Our results provide a foundation for further dissecting the roles of miRNAs and their targets in regulating abiotic stress tolerance in A. andraeanum.