Transcriptome Profiling of Cu Stressed Petunia Petals Reveals Candidate Genes Involved in Fe and Cu Crosstalk.

Copper (Cu) is an essential element for most living plants, but it is toxic for plants when present in excess. To better understand the response mechanism under excess Cu in plants, especially in flowers, transcriptome sequencing on petunia buds and opened flowers under excess Cu was performed. Interestingly, the transcript level of FIT-independent Fe deficiency response genes was significantly affected in Cu stressed petals, probably regulated by basic-helix-loop-helix 121 (bHLH121), while no difference was found in Fe content. Notably, the expression level of bHLH121 was significantly down-regulated in petals under excess Cu. In addition, the expression level of genes related to photosystem II (PSII), photosystem I (PSI), cytochrome b6/f complex, the light-harvesting chlorophyll II complex and electron carriers showed disordered expression profiles in petals under excess Cu, thus photosynthesis parameters, including the maximum PSII efficiency (FV/FM), nonphotochemical quenching (NPQ), quantum yield of the PSII (ΦPS(II)) and photochemical quenching coefficient (qP), were reduced in Cu stressed petals. Moreover, the chlorophyll a content was significantly reduced, while the chlorophyll b content was not affected, probably caused by the increased expression of chlorophyllide a oxygenase (CAO). Together, we provide new insight into excess Cu response and the Cu-Fe crosstalk in flowers.

Responses of Phragmites australis to copper stress: A combined analysis of plant morphology, physiology and proteomics.

Few relevant research attempts have been made to determine heavy metal resistance mechanisms of rhizomatous perennial plants. Thus, it is pertinent to investigate the physiological and biochemical changes in Phragmites australis under metal-stressed conditions to facilitate the development of strategies to enhance copper (Cu) tolerance. We measured parameters related to plant growth and development, metal translocation and physiological responses of P. australis subjected to Cu stress. In addition, the differentially expressed proteins (DEP) were evaluated using the isobaric tag for relative and absolute quantification (iTRAQ) system. A large amount of copper accumulates in the roots of P.australis, but the growth parameters were not sensitive to Cu. However, the high concentration of Cu reduced the content of chlorophyll a and chlorophyll b, and the expression of important photosynthesis proteins PsbD, PsbO and PsaA were all down-regulated, so photosynthesis was inhibited. In contrast, the content of ascorbic acid and proline both increased with the increase of copper stress. P.australis fixed a large amount of Cu in its roots, limiting the migration of Cu to other parts of the plant. Moreover, Cu stress can affect photosynthesis by inhibiting the activity of PSI, PSII and LHCII. In addition, P.australis synthesizes ascorbic acid through the D-mannose/L-galactose pathway, and synthesizes proline through the ornithine pathway. Ascorbic acid and proline can increase Cu tolerance and protect photosynthesis. These results provide a theoretical basis for understanding the tolerance and repair mechanisms of plants in response to heavy metal pollution.

A transporter Slr1512 involved in bicarbonate and pH-dependent acclimation mechanism to high light stress in Synechocystis sp. PCC 6803.

High light (HL) exposure leads to photoinhibition and excess accumulation of toxic reactive oxygen species (ROS) in photosynthetic organisms, negatively impacting the global primary production. In this study, by screening a mutant library, a gene related with bicarbonate transport, slr1512, was found involved in HL acclimation in model cyanobacterium Synechocystis sp. PCC 6803. Comparative growth analysis showed that the slr1512 knockout mutant dramatically enhanced the tolerance of Synechocystis towards long-term HL stress (200 µmol photons m-2 s-1) than the wild type, achieving an enhanced growth by ~1.95-folds after 10 d. The phenotype differences between Δslr1512 and the wild type were analyzed via absorption spectrum and chlorophyll a content measurement. In addition, the accessible bicarbonate controlled by slr1512 and decreased PSII activity were demonstrated, and they were found to be the key factors affecting the tolerance of Synechocystis against HL stress. Further analysis confirmed that intracellular bicarbonate can significantly affect the activity of photosystem II, leading to the altered accumulation of toxic ROS under HL. Finally, a comparative transcriptomics was applied to determine the differential responses to HL between Δslr1512 and the wild type. This work provides useful insights to long-term acclimation mechanisms towards HL and valuable information to guide the future tolerance engineering of cyanobacteria against HL.

Acuities into tolerance mechanisms via different bioassay during Brassicaceae-Alternaria brassicicola interaction and its impact on yield.

Heavy losses by dark leaf spot disease in oilseed Brassica have incited research towards identifying sources of genetic tolerance against causal pathogen, Alternaria brassicicola. Several morpho-molecular parameters were evaluated to test the performance of field mustard and rapeseed genotypes under artificial inoculation with this pathogen. During Brassica-Alternaria interaction, physio-biochemical defense response was witnessed in tolerant genotypes. Two tolerant genotypes (one for field mustard and one for rapeseed), i.e., EC250407 and EC1494 were identified. However, necrotic lesions were more prominent in susceptible genotypes with minimum chlorophyll (chlorophyll a, chlorophyll b and total chlorophyll) and carotenoids contents. Contrary to photosynthetic pigments, increase in total soluble protein (TSP) contents was observed with disease progression in susceptible genotypes. Tolerant genotypes of field mustard and rapeseed displayed remarkable increase in the activities of redox enzyme in infected leaves with least yield loss (6.47% and 5.74%) and disease severity index (DSI) of 2.9 and 2.1, respectively. However, yield/plant showed close association with other morpho-yield parameters, photosynthetic pigments and redox enzymes (superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD)) activities except silique length and TSP. Based on the results of morpho-biochemical analyses, redox enzymes and morphological parameters; their interplay is proposed to determine the tolerance outcome of the Brassica-A. brassicicola interaction.

Chloroplast development and genomes uncoupled signaling are independent of the RNA-directed DNA methylation pathway.

The Arabidopsis genome is methylated in CG and non-CG (CHG, and CHH in which H stands for A, T, or C) sequence contexts. DNA methylation has been suggested to be critical for seed development, and CHH methylation patterns change during stratification and germination. In plants, CHH methylation occurs mainly through the RNA-directed DNA methylation (RdDM) pathway. To test for an involvement of the RdDM pathway in chloroplast development, we analyzed seedling greening and the maximum quantum yield of photosystem II (Fv/Fm) in Arabidopsis thaliana seedlings perturbed in components of that pathway. Neither seedling greening nor Fv/Fm in seedlings and adult plants were affected in this comprehensive set of mutants, indicating that alterations in the RdDM pathway do not affect chloroplast development. Application of inhibitors like lincomycin or norflurazon inhibits greening of seedlings and represses the expression of photosynthesis-related genes including LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN1.2 (LHCB1.2) in the nucleus. Our results indicate that the LHCB1.2 promoter is poorly methylated under both control conditions and after inhibitor treatment. Therefore no correlation between LHCB1.2 mRNA transcription and methylation changes of the LHCB1.2 promoter could be established. Moreover, we conclude that perturbations in the RdDM pathway do not interfere with gun signaling.

The Alleviation of Photosynthetic Damage in Tomato under Drought and Cold Stress by High CO2 and Melatonin.

The atmospheric CO2 concentration (a[CO2]) is increasing at an unprecedented pace. Exogenous melatonin plays positive roles in the response of plants to abiotic stresses, including drought and cold. The effect of elevated CO2 concentration (e[CO2]) accompanied by exogenous melatonin on plants under drought and cold stresses remains unknown. Here, tomato plants were grown under a[CO2] and e[CO2], with half of the plants pre-treated with melatonin. The plants were subsequently treated with drought stress followed by cold stress. The results showed that a decreased net photosynthetic rate (PN) was aggravated by a prolonged water deficit. The PN was partially restored after recovery from drought but stayed low under a successive cold stress. Starch content was downregulated by drought but upregulated by cold. The e[CO2] enhanced PN of the plants under non-stressed conditions, and moderate drought and recovery but not severe drought. Stomatal conductance (gs) and the transpiration rate (E) was less inhibited by drought under e[CO2] than under a[CO2]. Tomato grown under e[CO2] had better leaf cooling than under a[CO2] when subjected to drought. Moreover, melatonin enhanced PN during recovery from drought and cold stress, and enhanced biomass accumulation in tomato under e[CO2]. The chlorophyll a content in plants treated with melatonin was higher than in non-treated plants under e[CO2] during cold stress. Our findings will improve the knowledge on plant responses to abiotic stresses in a future [CO2]-rich environment accompanied by exogenous melatonin.

Metabolome and transcriptome analyses reveal chlorophyll and anthocyanin metabolism pathway associated with cucumber fruit skin color.

BACKGROUND: Fruit skin color play important role in commercial value of cucumber, which is mainly determined by the content and composition of chlorophyll and anthocyanins. Therefore, understanding the related genes and metabolomics involved in composition of fruit skin color is essential for cucumber quality and commodity value. RESULTS: The results showed that chlorophyll a, chlorophyll b and carotenoid content in fruit skin were higher in Lv (dark green skin) than Bai (light green skin) on fruit skin. Cytological observation showed more chloroplast existed in fruit skin cells of Lv. A total of 162 significantly different metabolites were found between the fruit skin of the two genotypes by metabolome analysis, including 40 flavones, 9 flavanones, 8 flavonols, 6 anthocyanins, and other compounds. Crucial anthocyanins and flavonols for fruit skin color, were detected significantly decreased in fruit skin of Bai compared with Lv. By RNA-seq assay, 4516 differentially expressed genes (DEGs) were identified between two cultivars. Further analyses suggested that low expression level of chlorophyll biosynthetic genes, such as chlM, por and NOL caused less chlorophylls or chloroplast in fruit skin of Bai. Meanwhile, a predicted regulatory network of anthocyanin biosynthesis was established to illustrate involving many DEGs, especially 4CL, CHS and UFGT. CONCLUSIONS: This study uncovered significant differences between two cucumber genotypes with different fruit color using metabolome and RNA-seq analysis. We lay a foundation to understand molecular regulation mechanism on formation of cucumber skin color, by exploring valuable genes, which is helpful for cucumber breeding and improvement on fruit skin color.

C4 and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation.

Although biochemically related, C4 and crassulacean acid metabolism (CAM) systems are expected to be incompatible. However, Portulaca species, including P. oleracea, operate C4 and CAM within a single leaf, and the mechanisms behind this unique photosynthetic arrangement remain largely unknown. Here, we employed RNA-seq to identify candidate genes involved exclusively or shared by C4 or CAM, and provided an in-depth characterization of their transcript abundance patterns during the drought-induced photosynthetic transitions in P. oleracea. Data revealed fewer candidate CAM-specific genes than those recruited to function in C4 . The putative CAM-specific genes were predominantly involved in night-time primary carboxylation reactions and malate movement across the tonoplast. Analysis of gene transcript-abundance regulation and photosynthetic physiology indicated that C4 and CAM coexist within a single P. oleracea leaf under mild drought conditions. Developmental and environmental cues were shown to regulate CAM expression in stems, whereas the shift from C4 to C4 -CAM hybrid photosynthesis in leaves was strictly under environmental control. Moreover, efficient starch turnover was identified as part of the metabolic adjustments required for CAM operation in both organs. These findings provide insights into C4 /CAM connectivity and compatibility, contributing to a deeper understanding of alternative ways to engineer CAM into C4 crop species.

Structural features of the diatom photosystem II-light-harvesting antenna complex.

In photosynthesis, light energy is captured by pigments bound to light-harvesting antenna proteins (LHC) that then transfer the energy to the photosystem (PS) cores to initiate photochemical reactions. The LHC proteins surround the PS cores to form PS-LHC supercomplexes. In order to adapt to a wide range of light environments, photosynthetic organisms have developed a large variety of pigments and antenna proteins to utilize the light energy efficiently under different environments. Diatoms are a group of important eukaryotic algae and possess fucoxanthin (Fx) chlorophyll a/c proteins (FCP) as antenna which have exceptional capabilities of harvesting blue-green light under water and dissipate excess energy under strong light conditions. We have solved the structure of a PSII-FCPII supercomplex from a centric diatom Chaetoceros gracilis by cryo-electron microscopy, and also the structure of an isolated FCP dimer from a pennate diatom Phaeodactylum tricornutum by X-ray crystallography at a high resolution. These results revealed the oligomerization states of FCPs distinctly different from those of LHCII found in the green lineage organisms, the detailed binding patterns of Chl c and Fxs, a huge pigment network, and extensive protein-protein, pigment-protein, and pigment-pigment interactions within the PSII-FCPII supercomplex. These results therefore provide a solid structural basis for examining the detailed mechanisms of the highly efficient energy transfer and quenching processes in diatoms.

Comparative Proteomic Analysis Reveals That Chlorophyll Metabolism Contributes to Leaf Color Changes in Wucai ( Brassica campestris L.) Responding to Cold Acclimation.

Chlorophyll is a vital photosynthetic pigment that plays a key role in plant development, participating in light energy capture and energy conversion. In this study, a novel wucai ( Brassica campestris L.) germplasm with green outer leaves and yellow inner leaves at the adult stage (W7-2) was used to examine chlorophyll metabolism response to cold acclimation. A green leaf wucai genotype without leaf color changes named W7-1 was selected as the control to evaluate the chlorophyll metabolism changes of W7-2. Compared to W7-1, the contents of chlorophyll a (Chl a) and chlorophyll b (Chl b) in W7-2 were significantly reduced at five developmental stages (13, 21, 29, 37, and 45 days after planting (DAP)). An iTRAQ-based quantitative proteomic analysis was carried out at 21 and 29 DAP according to the leaf color changes in both of genotypes. 1409 proteins were identified, while 218 of them displayed differential accumulations between W7-2 and W7-1 during the two developmental stages. The differentially expressed proteins (DEPs) mainly assigned to chlorophyll biosynthesis, photosynthesis, carbohydrate metabolism, ribosome metabolism and posttranslational modification. Among these DEPs, NADPH-protochlorophyllide oxidoreductase (PORB) and Mg-protoporphyrin IX chelatase 1 (CHLI1) were the key enzymes participating in chlorophyll (Chl) biosynthesis, which was down-regulated at 21 DAP and up-regulated at 29 DAP in W7-2 compared with W7-1, respectively. The expression analysis of genes of three subunits of Mg-chelatase ( CHLI1, CHLD, and CHLH), Genomes Uncoupled 4 ( GUN4), and Thioredoxin ( TRX3) associated with chlorophyll metabolism also displayed significant down-regulation in W7-2. In particular, PORB showed significant up-regulation in W7-2, significantly affecting chlorophyll biosynthesis. Additionally, differences in chlorophyll metabolism between W7-2 and W7-1 were in terms of altered photosynthesis, carbohydrate, and energy metabolism. We found that the transcription levels of most photosynthesis proteins showed significantly lower levels, and the genes expression level, associated with carbohydrate and energy metabolism, were lower in W7-2 than in W7-1. Therefore, the present study results help understand the physiological and molecular mechanisms underlying leaf coloring responding to cold acclimation.

Differential molecular response of maize and Johnson grass against maize dwarf mosaic virus and bermuda grass southern mosaic virus.

Maize dwarf mosaic virus (MDMV) and bermuda grass southern mosaic virus (BgSMV) is the most important cereal potyvirus in Iran. Expression of some key genes in maize plants susceptible and tolerant to MDMV or BgSMV and gene expression profile of MDMV and BgSMV compatible or incompatible to Johnson grass plants were studied. Time points of 1, 9, 24 and 72 h after inoculation with both viruses were investigated as well. By analyzing the expression of the genes, it was identified that in maize infected by MDMV and BgSMV, the transcript levels of the peroxiredoxin, GLP, SAM, NPR1 and chlorophyll a-b binding genes were significantly higher in the tolerant than in susceptible plants during the entire experiment. In the BgSMV inoculated Johnson grass plants, some of the genes related to plant defense responses including NPR1, peroxiredoxin and SAM had higher expression level than the Johnson grass plants inoculated by MDMV. Important genes in maize tolerance like NPR1 and MT-LP, were analyzed by trilinear decomposition analysis and genes clustering. The upregulated expression of genes at one-hour post inoculation showed that the plant response to viruses was activated at the early stage of infection. Keywords: MDMV; BgSMV; gene expression; quantitative real-time PCR; trilinear decomposition analysis.

DNA fingerprinting and assessment of some physiological changes in Al-induced Bryophyllum daigremontianum clones.

Aluminum (Al) is one of the most important stress factors that reduce plant productivity in acidic soils. Present work thereby analyzed Al-induced genomic alterations in Bryophyllum daigremontianum clones using RAPD and ISSR markers, and investigated responding changes in photosynthetic pigment (chlorophyll a, b, a/b, total chlorophyll and carotenoid) contents and total soluble protein amounts in plant leaves. The main reason for the use of bulbiferous spurs originated clone plants was to increase reliability and acceptability of RAPD and ISSR techniques in DNA fingerprinting. Raised 40 clone plants were divided into five separate groups each with eight individuals and each experimental group was watered with 0 (control), 0 (acid control), 50, 100 and 200 µM AlCl3-containing Hoagland solutions on alternate days for two and a half months. All plant soils except control group were sprayed with 0.2% sulfuric acid following watering days and this contributed acidic characteristic (pH 4.8) to soil structure. Increase in Al concentrations were accompanied by an increase in total soluble protein amounts, a decrease in photosynthetic pigment contents, and with appearance, disappearance and intensity changes at RAPD and ISSR band profiles. Out of tested RAPD1-25 and ISSR1-15 primers, RAPD8, RAPD9, ISSR2 and ISSR7 primers produced reproducible band profiles that were distinguishable between treatment and control groups. Findings showed that RAPD and ISSR fingerprints have been useful biomarkers for investigation of plant genotoxicity, especially in clone plants. Moreover, if these fingerprints are integrated with other physiological parameters they could become more powerful tools in ecotoxicology.

Chlorophyll a/b binding-specificity in water-soluble chlorophyll protein.

We altered the chlorophyll (Chl) binding sites in various versions of water-soluble chlorophyll protein (WSCP) by amino acid exchanges to alter their preferences for either Chl a or Chl b. WSCP is ideally suited for this mutational analysis since it forms a tetrameric complex with only four identical Chl binding sites. A loop of 4-6 amino acids is responsible for Chl a versus Chl b selectivity. We show that a single amino acid exchange within this loop changes the relative Chl a/b affinities by a factor of 40. We obtained crystal structures of this WSCP variant binding either Chl a or Chl b. The Chl binding sites in these structures were compared with those in the major light-harvesting complex (LHCII) of the photosynthetic apparatus in plants to search for similar structural features involved in Chl a/b binding specificity.

An unusual role for the phytyl chains in the photoprotection of the chlorophylls bound to Water-Soluble Chlorophyll-binding Proteins.

Water-Soluble Chlorophyll Proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins which tetramerize upon binding four chlorophyll (Chl) molecules. The bound Chls are highly photostable, despite the lack of bound carotenoids known, in Chl-containing photosynthetic proteins, to act as singlet oxygen and Chl triplet (3Chl) quenchers. Although the physiological function of WSCPs is still unclear, it is likely to be related to their biochemical stability and their resistance to photodegradation. To get insight into the origin of this photostability, the properties of the 3Chl generated in WSCPs upon illumination were investigated. We found that, unlike the excited singlet states, which are excitonic states, the triplet state is localized on a single Chl molecule. Moreover, the lifetime of the 3Chl generated in WSCPs is comparable to that observed in other Chl-containing systems and is reduced in presence of oxygen. In contrast to previous observations, we found that WSCP actually photosensitizes singlet oxygen with an efficiency comparable to that of Chl in organic solvent. We demonstrated that the observed resistance to photooxidation depends on the conformation of the phytyl moieties, which in WSCP are interposed between the rings of Chl dimers, hindering the access of singlet oxygen to the oxidizable sites of the pigments.