Cucumber Chloroplast Research Articles

Chloroplast Pan-Genomes and Comparative Transcriptomics Reveal Genetic Variation and Temperature Adaptation in the Cucumber.

Although whole genome sequencing, genetic variation mapping, and pan-genome studies have been done on a large group of cucumber nuclear genomes, organelle genome information is largely unclear. As an important component of the organelle genome, the chloroplast genome is highly conserved, which makes it a useful tool for studying plant phylogeny, crop domestication, and species adaptation. Here, we have constructed the first cucumber chloroplast pan-genome based on 121 cucumber germplasms, and investigated the genetic variations of the cucumber chloroplast genome through comparative genomic, phylogenetic, haplotype, and population genetic structure analysis. Meanwhile, we explored the changes in expression of cucumber chloroplast genes under high- and low-temperature stimulation via transcriptome analysis. As a result, a total of 50 complete chloroplast genomes were successfully assembled from 121 cucumber resequencing data, ranging in size from 156,616-157,641 bp. The 50 cucumber chloroplast genomes have typical quadripartite structures, consisting of a large single copy (LSC, 86,339-86,883 bp), a small single copy (SSC, 18,069-18,363 bp), and two inverted repeats (IRs, 25,166-25,797 bp). Comparative genomic, haplotype, and population genetic structure results showed that there is more genetic variation in Indian ecotype cucumbers compared to other cucumber cultivars, which means that many genetic resources remain to be explored in Indian ecotype cucumbers. Phylogenetic analysis showed that the 50 cucumber germplasms could be classified into 3 types: East Asian, Eurasian + Indian, and Xishuangbanna + Indian. The transcriptomic analysis showed that matK were significantly up-regulated under high- and low-temperature stresses, further demonstrating that cucumber chloroplasts respond to temperature adversity by regulating lipid metabolism and ribosome metabolism. Further, accD has higher editing efficiency under high-temperature stress, which may contribute to the heat tolerance. These studies provide useful insight into genetic variation in the chloroplast genome, and established the foundation for exploring the mechanisms of temperature-stimulated chloroplast adaptation.

Impact of Light with Different Spectra on the Photosynthetic Activity of Cucumber Plants under Fusarium Wilt

The photosynthetic activity of 28-day-old cucumber plants of the Kustovy variety formed under LED illumination with the predominance of red light (RL) or far red light (FRL) and infected with the fungus Fusarium oxisporum sp. (F.ox.) was studied. The predominance of RL or FRL contributed to an increase in the content of chlorophyll and carotenoids per dry leaf weight compared to the plants grown under white light (WL). In the infected plants grown under WL and RL regimes, an increase in the total content of chlorophyll and carotenoids was observed relative to healthy plants, and a decrease in the same parameters for FRL was noted. In healthy cucumber leaves grown under RL and FRL regimes, an increase in the activity of photosystem (PS)1 and PS2 in comparison with WL was observed. Infection of cucumber plants grown under WL did not cause any changes in the functional parameters of PS2 against the background of a slight reduction in PS1 complexes. Under RL and FRL regimes, a decrease in the photochemical activity of PS 1 and PS 2 in infected leaves was noted. The activity of ascorbate peroxidase - one of antioxidant enzymes in chloroplasts decreased in healthy cucumber leaves under RL and FRL compared to WL and increased in infected leaves compared to the healthy ones under all light regimes, especially strongly under RL. The results obtained demonstrate different response mechanisms for cucumber chloroplasts to the infection with a fungal pathogen, depending on the light conditions of growing.

Influence of light intensity and its spectral composition on photosynthetic activity of cucumber Cucumis sativus under fusarium wilt

The responses of cucumber chloroplasts of the Kustovoi variety formed under lighting of different intensity (6000 and 11 000 lx) or under LED lighting with a predominance of red light and far red light to infection with fungus Fusarium oxysporum were studied. The amount of chlorophylls and carotenoids in chloroplasts formed at low light increased in 72 h after infection, and at high light a significant increase in pigment catabolism was observed. Under fusarium wilt, the violaxanthin cycle was not involved in the conditions of the studied light range, and the photochemical activity of chloroplasts did not depend much on the level of illumination. The predominance of red light or far red light caused an increase in both chlorophylls and carotenoids content in terms of the dry mass of the leaf compared to their content in plants grown on white light. Infection on white light and red light caused an increase in the total content of chlorophylls and carotenoids, and a decrease in these parameters relative to healthy plants was noted on far red light. Fusarium wilt led to a decrease in photochemical activity and electron transport of photosystem I (by 20 %) in chloroplasts on red light and far red light. A decrease in the functional efficiency of photosystem II was also observed, most likely due to irreversible changes in pigment-protein complexes with two types of LED lighting. Various mechanisms of the response of cucumber chloroplasts to pathogen infection are discussed, depending on the light conditions of the formation of photosynthetic membranes.

Revealing the Complete Chloroplast Genome of an Andean Horticultural Crop, Sweet Cucumber (Solanum muricatum), and Its Comparison with Other Solanaceae Species

Sweet cucumber (Solanum muricatum) sect. Basarthrum is a neglected horticultural crop native to the Andean region. It is naturally distributed very close to other two Solanum crops of high importance, potatoes, and tomatoes. To date, molecular tools for this crop remain undetermined. In this study, the complete sweet cucumber chloroplast (cp) genome was obtained and compared with seven Solanaceae species. The cp genome of S. muricatum was 155,681 bp in length and included a large single copy (LSC) region of 86,182 bp and a small single-copy (SSC) region of 18,360 bp, separated by a pair of inverted repeats (IR) regions of 25,568 bp. The cp genome possessed 87 protein-coding genes (CDS), 37 transfer RNA (tRNA) genes, eight ribosomal RNA (rRNA) genes, and one pseudogene. Furthermore, 48 perfect microsatellites were identified. These repeats were mainly located in the noncoding regions. Whole cp genome comparative analysis revealed that the SSC and LSC regions showed more divergence than IR regions. Similar to previous studies, our phylogenetic analysis showed that S. muricatum is a sister species to members of sections Petota + Lycopersicum + Etuberosum. We expect that this first sweet cucumber chloroplast genome will provide potential molecular markers and genomic resources to shed light on the genetic diversity and population studies of S. muricatum, which will allow us to identify varieties and ecotypes. Finally, the features and the structural differentiation will provide us with information about the genes of interest, generating tools for the most precise selection of the best individuals of sweet cucumber, in less time and with fewer resources.

Inheritance of chloroplast and mitochondrial genomes in cucumber revealed by four reciprocal F1 hybrid combinations

Both genomes in chloroplasts and mitochondria of plant cell are usually inherited from maternal parent, with rare exceptions. To characterize the inheritance patterns of the organelle genomes in cucumber (Cucumis sativus var. sativus), two inbred lines and their reciprocal F1 hybrids were analyzed using an next generation whole genome sequencing data. Their complete chloroplast genome sequences were de novo assembled, and a single SNP was identified between the parental lines. Two reciprocal F1 hybrids have the same chloroplast genomes with their maternal parents. Meanwhile, 292 polymorphic sites were identified between mitochondrial genomes of the two parental lines, which showed the same genotypes with their paternal parents in the two reciprocal F1 hybrids, without any recombination. The inheritance patterns of the chloroplast and mitochondria genomes were also confirmed in four additional cucumber accessions and their six reciprocal F1 hybrids using molecular markers derived from the identified polymorphic sites. Taken together, our results indicate that the cucumber chloroplast genome is maternally inherited, as is typically observed in other plant species, whereas the large cucumber mitochondrial genome is paternally inherited. The combination of DNA markers derived from the chloroplast and mitochondrial genomes will provide a convenient system for purity test of F1 hybrid seeds in cucumber breeding.

Introduction of Exogenous Glycolate Catabolic Pathway Can Strongly Enhances Photosynthesis and Biomass Yield of Cucumber Grown in a Low-CO2 Environment.

Carbon dioxide (CO2) is very important for photosynthesis of green plants. CO2 concentration in the atmosphere is relatively stable, but it drops sharply after sunrise due to the tightness of the greenhouse and the absorption of CO2 by vegetable crops. Vegetables in greenhouses are chronically CO2 starved. To investigate the feasibility of using genetic engineering to improve the photosynthesis and yield of greenhouse cucumber in a low CO2 environment, five genes encoding glyoxylate carboligase (GCL), tartronic semialdehyde reductase (TSR), and glycolate dehydrogenase (GlcDH) in the glycolate catabolic pathway of Escherichia coli were partially or completely introduced into cucumber chloroplast. Both partial pathway by introducing GlcDH and full pathway expressing lines exhibited higher photosynthetic efficiency and biomass yield than wild-type (WT) controls in low CO2 environments. Expression of partial pathway by introducing GlcDH increased net photosynthesis by 14.9% and biomass yield by 44.9%, whereas the expression of the full pathway increased seed yield by 33.4% and biomass yield by 59.0%. Photosynthesis, fluorescence parameters, and enzymatic measurements confirmed that the introduction of glycolate catabolic pathway increased the activity of photosynthetic carbon assimilation-related enzymes and reduced the activity of photorespiration-related enzymes in cucumber, thereby promoting the operation of Calvin cycle and resulting in higher net photosynthetic rate even in low CO2 environments. This increase shows an improvement in the efficiency of the operation of the photosynthetic loop. However, the utilization of cucumber of low concentration CO2 was not alleviated. This study demonstrated the feasibility of introducing the pathway of exogenous glycolate catabolic pathway to improve the photosynthetic and bio-yield of cucumber in a low CO2 environment. These findings are of great significance for high photosynthetic efficiency breeding of greenhouse cucumber.

The complete chloroplast genome sequence with a novel 24-bp deletion of a Korean solid green-type cucumber variety (Cucumis sativus var. sativus)

Cucumber (Cucumis sativus var. sativus) is one of the economically important vegetable crops. In this study, we characterized the complete chloroplast genome sequence of inbred line ID YHB bred from Korean solid green-type cucumber variety, through de novo assembly using next-generation sequencing. The chloroplast genome is 155,501 bp long and has typical quadripartite structures and gene contents as found in reported cucumber chloroplast genomes. Interestingly, sequence comparison revealed a novel 24-bp deletion present only in the chloroplast genome of the inbred line. Phylogenetic analysis confirmed that the inbred line was closely grouped with cucumber cultivars.

Melatonin Increases the Chilling Tolerance of Chloroplast in Cucumber Seedlings by Regulating Photosynthetic Electron Flux and the Ascorbate-Glutathione Cycle.

The aim of the study was to monitor the effects of exogenous melatonin on cucumber (Cucumis sativus L.) chloroplasts and explore the mechanisms through which it mitigates chilling stress. Under chilling stress, chloroplast structure was seriously damaged as a result of over-accumulation of reactive oxygen species (ROS), as evidenced by the high levels of superoxide anion (O2−) and hydrogen peroxide (H2O2). However, pretreatment with 200 μM melatonin effectively mitigated this by suppressing the levels of ROS in chloroplasts. On the one hand, melatonin enhanced the scavenging ability of ROS by stimulating the ascorbate–glutathione (AsA–GSH) cycle in chloroplasts. The application of melatonin led to high levels of AsA and GSH, and increased the activity of total superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) dehydroascorbate reductase (DHAR, EC 1.5.5.1), glutathione reductase (GR, EC1.6.4.2) in the AsA–GSH cycle. On the other hand, melatonin lessened the production of ROS in chloroplasts by balancing the distribution of photosynthetic electron flux. Melatonin helped maintain a high level of electron flux in the PCR cycle [Je(PCR)] and in the PCO cycle [Je(PCO)], and suppressed the O2-dependent alternative electron flux Ja(O2-dependent) which is one important ROS source. Results indicate that melatonin increased the chilling tolerance of chloroplast in cucumber seedlings by accelerating the AsA–GSH cycle to enhance ROS scavenging ability and by balancing the distribution of photosynthetic electron flux so as to suppress ROS production.

The complete chloroplast genome sequence of Cucumis sativus var. Hardwickii, the wild progenitor of cultivated cucumber

The complete chloroplast genome sequence of wild cucumber (Cucumis sativus var. hardwickii) was determined and characterized in this study. The genome is of 155 277 bp in length, containing a pair of inverted repeats regions (IRs) of 25 198 bp, which are separated by a large single-copy region of 86 618 bp and a small single-copy region of 18 263 bp. The wild cucumber chloroplast genome has 130 known genes, including 85 protein-coding genes, 8 ribosomal RNA genes, and 37 tRNA genes. Among these genes, 19 comprise one or two introns. There are 11 tRNA genes present in the IR of the chloroplast genome. Phylogenomic analysis showed that C. sativus var. hardwickii forms a cluster with other Cucumis species with strong bootstrap supports and is closely related to C. sativus var. sativus. This newly sequenced chloroplast genome sequence may provide useful genetic information to explore wild cucumber germplasms for cucumber breeding programs.

The role of putrescine in the regulation of proteins and fatty acids of thylakoid membranes under salt stress

Polyamines can alleviate the inhibitory effects of salinity on plant growth by regulating photosynthetic efficiency. However, little information is available to explain the specific mechanisms underlying the contribution of polyamines to salt tolerance of the photosynthetic apparatus. Here, we investigated the role of putrescine (Put) on the photosynthetic apparatus of cucumber seedlings under salt stress. We found that NaCl stress resulted in severe ion toxicity and oxidative stress in cucumber chloroplasts. In addition, salinity caused a significant increase in the saturated fatty acid contents of thylakoid membranes. Put altered unsaturated fatty acid content, thereby alleviating the disintegration of thylakoid grana lamellae and reducing the number of plastoglobuli in thylakoid membranes. BN-PAGE revealed Put up-regulated the expression of ATP synthase, CP47, D1, Qb, and psbA proteins and down-regulated CP24, D2, and LHCII type III in NaCl-stressed thylakoid membranes. qRT-PCR analysis of gene expression was used to compare transcript and protein accumulation among 10 candidate proteins. For five of these proteins, induced transcript accumulation was consistent with the pattern of induced protein accumulation. Our results suggest that Put regulates protein expression at transcriptional and translational levels by increasing endogenous polyamines levels in thylakoid membranes, which may stabilise photosynthetic apparatus under salt stress.

A tiling microarray for global analysis of chloroplast genome expression in cucumber and other plants.

Plastids are small organelles equipped with their own genomes (plastomes). Although these organelles are involved in numerous plant metabolic pathways, current knowledge about the transcriptional activity of plastomes is limited. To solve this problem, we constructed a plastid tiling microarray (PlasTi-microarray) consisting of 1629 oligonucleotide probes. The oligonucleotides were designed based on the cucumber chloroplast genomic sequence and targeted both strands of the plastome in a non-contiguous arrangement. Up to 4 specific probes were designed for each gene/exon, and the intergenic regions were covered regularly, with 70-nt intervals. We also developed a protocol for direct chemical labeling and hybridization of as little as 2 micrograms of chloroplast RNA. We used this protocol for profiling the expression of the cucumber chloroplast plastome on the PlasTi-microarray. Owing to the high sequence similarity of plant plastomes, the newly constructed microarray can be used to study plants other than cucumber. Comparative hybridization of chloroplast transcriptomes from cucumber, Arabidopsis, tomato and spinach showed that the PlasTi-microarray is highly versatile.

Effects of salt-tolerant rootstock grafting on ultrastructure, photosynthetic capacity, and H2O2-scavenging system in chloroplasts of cucumber seedlings under NaCl stress

Plant growth, photosynthetic parameters, chloroplast ultrastructure, and the ascorbate-glutathione cycle system in chloroplasts of self-grafted and rootstock-grafted cucumber leaves were investigated. Grafted plants were grown hydroponically and were exposed to 0, 50, and 100 mM NaCl concentrations for 10 days. Under NaCl stress, the hydrogen peroxide (H2O2) content in cucumber chloroplasts increased, the chloroplast ultrastructure was damaged, and the gas stomatal conductance, intercellular CO2 concentration, as well as shoot dry weight, plant height, stem diameter, leaf area, and leaf relative water content were inhibited, whereas these changes were less severe in rootstock-grafted plants. The activities of ascorbate peroxidase (APX; EC 1.11.1.11), glutathione reductase (GR; EC 1.6.4.2), and dehydroascorbate reductase (DHAR EC 1.8.5.1) were higher in the chloroplasts of rootstock-grafted plants compared with those of self-grafted plants under 50 and 100 mM NaCl. Similar trends were shown in leaf net CO2 assimilation rate and transpiration rate, as well as reduced glutathione content under 100 mM NaCl. Results suggest that rootstock grafting enhances the H2O2-scavenging capacity of the ascorbate–glutathione cycle in cucumber chloroplasts under NaCl stress, thereby protecting the chloroplast structure and improving the photosynthetic performance of cucumber leaves. As a result, cucumber growth is promoted.

Silicon supplementation ameliorated the inhibition of photosynthesis and nitrate metabolism by cadmium (Cd) toxicity in Cucumis sativus L.

The effects of silicon (Si) application on plant growth, pigments, photosynthetic parameters, chlorophyll a (Chl a) fluorescence parameters and nitrogen metabolism were studied in Cucumis sativus L. under cadmium (Cd) toxicity. Compared with the control, 100 μM CdCl 2 treatment caused dramatic accumulation of Cd in cucumber leaves, greatly induced chlorosis, and the transmission electron microscope (TEM) analysis indicated that Cd treatment cucumber chloroplast showed obvious swollen, thylakoids and chloroplast membrane were seriously damaged, and could not be observed clearly. Application of Si reversed the chlorosis, protected the chloroplast from disorganization, and significantly increased the pigments contents, which might be mainly responsible for the higher photosynthetic rate and accumulation of biomass under Cd stress. Further investigation of chlorophyll a fluorescence indicated that Cd treatment decreasing photosynthesis was not due to stomatal restriction, while was closely related integrity damage or function lost of the photosynthetic machinery which can be concluded from the higher intercellular CO 2 concentration (Ci) and lower F v/ F m and ΦPSII. Application of Si alleviated the inhibited level of photosynthesis and F v/ F m and ΦPSII by Cd, which might imply that Si plays important roles in protecting photosynthetic machinery from damaging. The Cd treatment also greatly inhibited the enzymes of nitrogen metabolism including nitrogen reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH), and Si supply decreased the inhibiting effects of Cd.

Effects of Exogenous Silicon on Photosynthetic Capacity and Antioxidant Enzyme Activities in Chloroplast of Cucumber Seedlings Under Excess Manganese

Effects of silicon on photosynthetic parameters and antioxidant enzymes of chloroplast in cucumber seedlings under excess Mn were studied. Compared with the control, excess Mn significantly inhibited net photosynthetic rate (Pn), stomatal conductance, as well as the maximum yield of the photosystem II photochemical reactions (Fv/Fm) and the quantum yield of photosysytem II electron transport (ΦPSII), application of Si reversed the negative effects of excess Mn. In the further investigation, it was obtained that application of Si significantly increased the activities of enzymes related with ascorbate-glutathione cycle including ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in cucumber chloroplast under excess Mn, this could be responsible for the lower accumulation of H 2O 2 and lower lipid peroxidation of chloroplast induced by Mn, and resulted in keeping higher photosynthesis.

Sequencing cucumber (Cucumis sativus L.) chloroplast genomes identifies differences between chilling-tolerant and -susceptible cucumber lines

Chilling injury in cucumber (Cucumis sativus L.) is conditioned by maternal factors, and the sequencing of its chloroplast genome could lead to the identification of economically important candidate genes. Complete sequencing of cucumber chloroplast (cp)DNA was facilitated by the development of 414 consensus chloroplast sequencing primers (CCSPs) from conserved cpDNA sequences of Arabidopsis (Arabidopsis thaliana L.), spinach (Spinacia oleracea L.), and tobacco (Nicotiana tabacum L.) cpDNAs, using degenerative primer technologies. Genomic sequence analysis led to the construction of 301 CCSPs and 72 cucumber chloroplast-specific sequencing primers (CSSPs), which were used for the complete sequencing of cpDNA of Gy14 (155 525 bp) and 'Chipper' (155 524 bp) cucumber lines, which are, respectively, susceptible and tolerant to chilling injury (4 degrees C for 5.5 h) in the first leaf stage. Comparative cpDNA sequence analyses revealed that 1 sequence span (located between genes trnK and rps16) and 2 nucleotides (located in genes atpB and ycf1) differed between chilling-susceptible and -tolerant lines. These sequence differences correspond to previously reported maternally inherited differences in chilling response between reciprocal F1 progeny derived from these lines. Sequence differences at these 3 cpDNA sites were also detected in a genetically diverse array of cucumber germplasm with different chilling responses. These and previously reported results suggest that 1 or several of these sequences could be responsible for the observed response to chilling injury in cucumber. The comprehensive sequencing of cpDNA of cucumber by CCSPs and CSSPs indicates that these primers have immediate applications in the analysis of cpDNAs from other dicotyledonous species and the investigation of evolutionary relationships.

The complete structure of the cucumber (Cucumis sativus L.) chloroplast genome: Its composition and comparative analysis

The complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.

Developmental changes in sub-plastidic distribution of chlorophyll biosynthetic intermediates in Cucumber ( Cucumis sativus L.)

Four-day-old etiolated cucumber seedlings (Cucumis sativus L.) were transferred to cool-white-fluorescent light (15 mumol m-2 s-1) for 1 h and 24 hours and etiochloroplasts and chloroplasts were isolated from developing cotyledons. Plastids were fractionated to stroma, envelope and thylakoid or inner membranes and the pigment contents of all these different fractions were analysed. In intact cucumber chloroplast protochlorophylide was present in significant amounts whereas protoporphyrin IX and Mg-protoporphyrin plus its monoester were present only in very small quantities. Out of the total chloroplastic protochlorophylide pool 1.0% was partitioned to envelope membranes and 99.0% was partitioned to thylakoids. Stroma had only trace amounts of protochlorophylide. In contrast to chloroplasts, etiochloroplasts had, besides protochlorophylide, significant amounts of other chlorophyll biosynthetic intermediates. In etiochloroplasts, protoporphyrin IX primarily partitioned to inner membranes (59.1%) followed by stroma (37.7%) and envelope (3.21%). The content of Mg-protoporphyrin IX plus its monoester in different subplastidic fractions was 74.4% for inner membranes, 22.58% for stroma and 3.02% for envelope. Protochlorophyllide primarily partitioned to inner membranes (95.79%), followed by envelope (4.15%) and, to a negligible extent (0.06%), into stroma. The sub-plastidic distribution of chlorophyll biosynthetic intermediates in etiochloroplasts was, therefore, different than that of chloroplasts. The significance of differential distribution of chlorophyll biosynthetic intermediates among thylakoids, envelope and stroma in developing and mature plastids is discussed in relation to chloroplast biogenesis.

Response of Antioxidative Enzymes in Cucumber Chloroplasts to Cadmium Toxicity

Solution culture experiments were conducted to investigate the effects of cadmium (Cd) toxicity on growth of cucumber seedlings, chlorophyll content, and net photosynthetic rate in leaves, activities of antioxidant enzymes, and contents of glutathione (GSH) and ascorbic acid (ASA) in purified intact chloroplasts. Results showed that lengths of main root and hypocotyl, weights of shoot and root, the root/shoot ratio, chlorophyll content, and net photosynthetic rate decreased significantly with increasing Cd level from 0 to 100 µmol L−1 in nutrient solution. Levels of malondialdehyde (MDA), H2O2, and O2 ·− in chloroplasts increased with increasing Cd concentration in solution. In the antioxidant defense system of chloroplasts, the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR), and the contents of GSH and ASA decreased with increasing Cd concentration in the nutrient solution. The results suggest that Cd interferes with the growth of cucumber seedlings by destroying chloroplast membrane integrity and decreasing chlorophyll content and net photosynthetic rate in leaves.

Two-dimensional crystallization and preliminary structure analysis of LHC-II from cucumber and spinach

Large and well-ordered two-dimensional (2D) crystals of the light-harvesting chlorophyll a/b protein complexes (LHC-II) from cucumber and spinach chloroplasts were produced by the so-called batch method. The twodimensional structures of these crystals were examined at about 1.5 nm resolution by electron microscopy and image processing. The projection maps showed that there were similar, but not identical, structure features between two different LHC-II complexes. A cmparison between 2D crystal formations of the two different LHC-II complexes was done and some factors affecting 2D crystallization of the membrane proteins were analyzed. The relations of the structures of the LHC-II complexes to their polypeptide components and Chl a/b ratio were also discussed.

In vitro processing of cucumber chloroplast tRNA(Leu)(CAA) precursor in a pea chloroplast soluble extract.

To study the sequential steps in the processing pathway of the chloroplast monocistronic intronless tRNA precursors, we examined cucumber chloroplast tRNA(Leu)(CAA) processing in a cucumber or pea chloroplast soluble extract. The tRNA(Leu)(CAA) precursor synthesized from SP6 RNA polymerase-directed transcription system, was used as a substrate. Incubation of the tRNA precursor with the pea extract resulted in processing of tRNA(Leu)(CAA) via 5'- and 3'-endonucleolytic cleavages followed by final trimming of extra 3' nucleotides by 3' exonuclease(s). No preferred order for endonucleolytic cleavages has been observed during the in vitro tRNA(Leu) processing and the simultaneous occurrence of the intermediates consisting of leader + tRNA(Leu) and tRNA(Leu) + trailer, indicate that either 5'- or 3'-endonucleolytic cleavage can occur as the first step in vitro.