Convergence and divergence of the photoregulation of pigmentation and cellular morphology in Fremyella diplosiphon

Photosynthetic pigment accumulation and cellular and filament morphology are regulated reversibly by green light (GL) and red light (RL) in the cyanobacterium Fremyella diplosiphon during complementary chromatic adaptation (CCA). The photoreceptor RcaE (regulator of chromatic adaptation), which appears to function as a light-responsive sensor kinase, controls both of these responses. Recent findings indicate that downstream of RcaE, the signaling pathways leading to light-dependent changes in morphology or pigment synthesis and/or accumulation branch, and utilize distinct molecular components. We recently reported that the regulation of the accumulation of the GL-absorbing photosynthetic accessory protein phycoerythrin (PE) and photoregulation of cellular morphology are largely independent, as many mutants with severe PE accumulation defects do not have major disruptions in the regulation of cellular morphology. Furthermore, morphology can be disrupted under GL without impacting GL-dependent PE accumulation. Most recently, however, we determined that the disruption of the cpeR gene, which encodes a protein that is known to function as an activator of PE synthesis under GL, results in disruption of cellular morphology under GL and RL. Thus, apart from RcaE, CpeR is only the second known regulator to impact morphology under both light conditions in F. diplosiphon.     

Morphogenes bolA and mreB mediate the photoregulation of cellular morphology during complementary chromatic acclimation in Fremyella diplosiphon

Photoregulation of pigmentation during complementary chromatic acclimation (CCA) is well studied in Fremyella diplosiphon; however, mechanistic insights into the CCA‐associated morphological changes are still emerging. F. diplosiphon cells are rectangular under green light (GL), whereas cells are smaller and spherical under red light (RL). Here, we investigate the role of morphogenes bolA and mreB during CCA using gene expression and gene function analyses. The F. diplosiphon bolA gene is essential as its complete removal from the genome was unsuccessful. Depletion of bolA resulted in slow growth, morphological defects and the accumulation of high levels of reactive oxygen species in a partially segregated ΔbolA strain. Higher expression of bolA was observed under RL and was correlated with lower expression of mreB and mreC genes in wild type. In a ΔrcaE strain that lacks the red‐/green‐responsive RcaE photoreceptor, the expression of bolA and mre genes was altered under both RL and GL. Observed gene expression relationships suggest that mreB and mreC expression is controlled by RcaE‐dependent photoregulation of bolA expression. Expression of F. diplosiphon bolA and mreB homologues in Escherichia coli demonstrated functional conservation of the encoded proteins. Together, these studies establish roles for bolA and mreB in RcaE‐dependent regulation of cellular morphology.     

Characterization of green mutants in Fremyella diplosiphon provides insight into the impact of phycoerythrin deficiency and linker function on complementary chromatic adaptation

Functions of phycobiliprotein (PBP) linkers are less well studied than other PBP polypeptides that are structural components or required for the synthesis of the light-harvesting phycobilisome (PBS) complexes. Linkers serve both structural and functional roles in PBSs. Here, we report the isolation of a phycoerythrin (PE) rod-linker mutant and a novel PE-deficient mutant in Fremyella diplosiphon. We describe their phenotypic characterization, including light-dependent photosynthetic pigment accumulation and photoregulation of cellular morphology. PE-linker protein CpeE and a novel protein impact PE accumulation, and thus PBS function, primarily under green light conditions.     

Temporal dynamics of changes in reactive oxygen species (ROS) levels and cellular morphology are coordinated during complementary chromatic acclimation in Fremyella diplosiphon

Fremyella diplosiphon alters the phycobiliprotein composition of its light-harvesting complexes, i.e., phycobilisomes, and its cellular morphology in response to changes in the prevalent wavelengths of light in the external environment in a phenomenon known as complementary chromatic acclimation (CCA). The organism primarily responds to red light (RL) and green light (GL) during CCA to maximize light absorption for supporting optimal photosynthetic efficiency. Recently, we found that RL-characteristic spherical cell morphology is associated with higher levels of reactive oxygen species (ROS) compared to growth under GL where lower ROS levels and rectangular cell shape are observed. The RL-dependent association of increased ROS levels with cellular morphology was demonstrated by treating cells with a ROS-scavenging antioxidant which resulted in the observation of GL-characteristic rectangular morphology under RL. To gain additional insights into the involvement of ROS in impacting cellular morphology changes during CCA, we conducted experiments to study the temporal dynamics of changes in ROS levels and cellular morphology during transition to growth under RL or GL. Alterations in ROS levels and cell morphology were found to be correlated with each other at early stages of acclimation of low white light-grown cells to growth under high RL or cells transitioned between growth in RL and GL. These results provide further general evidence that significant RL-dependent increases in ROS levels are temporally correlated with changes in morphology toward spherical. Future studies will explore the light-dependent mechanisms by which ROS levels may be regulated and the direct impacts of ROS on the observed morphology changes.     

CpcF-dependent regulation of pigmentation and development in Fremyella diplosiphon

Cyanobacteria harvest light for photosynthesis using photosynthetic light-harvesting complexes called phycobilisomes (PBSs). Lyases are enzymes responsible for covalent attachment of light-absorbing chromophores to the phycobiliproteins (PBPs) contained in PBSs. We isolated a pigmentation mutant in the filamentous cyanobacterium Fremyella diplosiphon and determined that it possesses an insertional mutation in cpcF, which encodes one component of a heterodimeric phycocyanin lyase. Here, we discuss the implications of the mutation in cpcF on light-dependent pigmentation and morphology responses characteristic of complementary chromatic adaptation in F. diplosiphon. Although cpcF encodes a phycocyanin lyase, significant decreases in the levels of all classes of PBPs are associated with CpcF deficiency in F. diplosiphon. Notably, CpcF deficiency has a limited effect on the shape of F. diplosiphon cells, but significantly impacts filament length. Possible mechanisms for the broad impact of CpcF deficiency on pigmentation and filament morphology are discussed.     

A novel role for a TonB-family protein and photoregulation of iron acclimation in Fremyella diplosiphon

Photosynthetic organisms display adaptations to changes in light and nutrient availability. Iron, which is required for the function of photosynthetic photosystems and other important biochemical processes, is an essential mineral that consequently impacts not only overall photosynthetic efficiency, but also the physiology of organisms in general. Our recent study represents the first functional characterization of a cyanobacterial TonB protein. TonB proteins classically are membrane proteins that support the transport of iron and vitamin B12 into cells. TonB proteins thus generally serve a critical role in organismal iron acclimation. We recently identified FdTonB, a TonB-family protein, in the filamentous freshwater cyanobacterium Fremyella diplosiphon. FdTonB contains conserved TonB residues and domains, as well as novel protein domains. Our recent study, however, supports a novel function for this protein in the photoregulation of morphology, rather than iron acclimation, in F. diplosiphon. Our detailed investigations into the responses of SF33 wild-type and ΔtonB mutant strains did not support a role for FdTonB in organismal responses to iron limitation. However, close examination of our recent results did highlight a novel interaction between light and iron acclimation in F. diplosiphon.Key words: cellular morphology, complementary chromatic adaptation, cyanobacteria, iron, phycobiliprotein, photomorphogenesis, photoregulation, siderophores     

Photoregulation of cellular morphology during complementary chromatic adaptation requires sensor-kinase-class protein RcaE in Fremyella diplosiphon

We used wild-type UTEX481; SF33, a shortened-filament mutant strain that shows normal complementary chromatic adaptation pigmentation responses; and FdBk14, an RcaE-deficient strain that lacks light-dependent pigmentation responses, to investigate the molecular basis of the photoregulation of cellular morphology in the cyanobacterium Fremyella diplosiphon. Detailed microscopic and biochemical analyses indicate that RcaE is required for the photoregulation of cell and filament morphologies of F. diplosiphon in response to red and green light.     

<Emphasis Type="Italic">Fremyella diplosiphon</Emphasis> as a Biodiesel Agent: Identification of Fatty Acid Methyl Esters via Microwave-Assisted Direct In Situ Transesterification

Increasing concerns on environmental and economic issues linked to fossil fuel use has driven great interest in cyanobacteria as third-generation biofuel agents. In this study, the biodiesel potential of a model photosynthetic cyanobacterium, Fremyella diplosiphon, was identified by fatty acid methyl esters (FAME) via direct transesterification. Total lipids in wild type (Fd33) and halotolerant (HSF33-1 and HSF33-2) strains determined by gravimetric analysis yielded 19% cellular dry weight (CDW) for HSF33-1 and 20% CDW for HSF33-2, which were comparable to Fd33 (18% CDW). Gas chromatography-mass spectrometry detected a high ratio of saturated to unsaturated FAMEs (2.48–2.61) in transesterified lipids, with methyl palmitate being the most abundant (C16:0). While theoretical biodiesel properties revealed high cetane number and oxidative stability, high cloud and pour point values indicated that fuel blending could be a viable approach. Significantly high FAME abundance in total transesterified lipids of HSF33-1 (40.2%) and HSF33-2 (69.9%) relative to Fd33 (25.4%) was identified using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry, indicating that robust salt stress response corresponds to higher levels of extractable FAME. Alkanes, a key component in conventional fuels, were present in F. diplosiphon transesterified lipids across all strains confirming that natural synthesis of these hydrocarbons is not inhibited during biodiesel production. While analysis of photosynthetic pigments and phycobiliproteins did not reveal significant differences, FAME abundance varied significantly in wild type and halotolerant strains indicating that photosynthetic pathways are not the sole factors that determine fatty acid production. We characterize the potential of F. diplosiphon for biofuel production with FAME yields in halotolerant strains higher than the wild type with no loss in photosynthetic pigmentation.     

Source-to-sink relationship between green leaves and green pseudobulbs of C<Subscript>3</Subscript> orchid in regulation of photosynthesis

In this paper, photosynthetic characteristics of green leaves (GL) and green pseudobulbs (GPSB) of C₃ orchid Oncidium Golden Wish were first studied. Light saturation for photosynthesis and maximum photosynthetic rates (P _max) were significantly higher in GL than in GPSB. The results of the optimal PSII quantum yield (F_v/F_m ratio), electron transport rate (ETR), the effective photochemical quantum yield (ΔF/F_m′) and nonphotochemical quenching (NPQ) of Chl fluorescence revealed that GPSB had lower light utilization than that of GL. Significantly higher photosynthetic pigments were found in GL than in GPSB. Alteration of source/sink ratio had no impact on all photosynthetic parameters for both GL and GPSB after a short term of 3 days or even a long term of 2 weeks of treatments although there were significant decreases in GL carbohydrate concentration of GL-darkened plants by the end of the day. However, decreases of all photosynthetic parameters of GL were observed in GL-darkened plants after 4 weeks of treatment compared to those of fully illuminated (FI) and GPSB-darkened plants. These results indicate that the level of carbohydrates in GL plays an important role in regulating their photosynthesis. Due to their lower photosynthetic capacities, GPSB function mainly as sinks. Darkening GPSB up to 2 weeks did not affect their own P _max and the P _max of GL and thus, did not result in significant decreases of total carbohydrate concentration of GPSB. As GPSB store a large amount of carbohydrates, it could also act as a source when the level of carbohydrates decreased. Thus, GL could depend on GPSB carbohydrates to regulate their photosynthesis when their source capacity was removed. However, 4 weeks after treatments, photosynthetic capacities of GL were significantly lower in GL- and GPSB-darkened plants than in FI plants, which could be due to the lower total soluble and insoluble sugar concentrations of both GL and GPSB in these plants.     

A plasmid sequence from Rhizobium leguminosarum 300 contains homology to sequences near the octopine TL-DNA right border

Summary The DNA sequence from a Rhizobium leguminosarum 300 (RL300) plasmid that contains homology to the T_c-DNA of Agrobacterium tumefaciens is described. The RL300 sequence has 78% homology to a 359 bp sequence in the T_c-DNA of pTi15955. The RL300 homology starts approximately 100 bp from the 24 bp border sequence of the T_L-DNA and ends approximately 3 bp from an IS66 homolog in the T_c-DNA. An unusual feature of the RL300 homology is the presence of 81 bp direct repeats with T_c-DNA homology, separated by 201 bp. One end of each direct repeat has a 12 bp palindrome. Four cloned sequences of RL300 with homology to the T DNA region were hybridized to plasmid lysates of RL300 derivatives to determine the source of each plasmid. The sequenced homolog, originally on pRH228, was isolated from pRL7JI; the other 3 homologs were isolated from the transmissable plasmids pRL7JI (pRH235) and pRL8JI (pRH235 and pRH236).     

The β(3) -integrin ligand of Borrelia burgdorferi is critical for infection of mice but not ticks

P66 is a Borrelia burgdorferi surface protein with β₃ integrin binding and channel forming activities. In this study, the role of P66 in mammalian and tick infection was examined. B. burgdorferiΔp66 strains were not infectious in wild‐type, TLR2^?/?‐ or MyD88^?/?‐deficient mice. Strains with p66 restored to the chromosome restored near wild‐type infectivity, while complementation with p66 on a shuttle vector did not restore infectivity. Δp66 mutants are cleared quickly from the site of inoculation, but analyses of cytokine expression and cellular infiltrates at the site of inoculation did not reveal a specific mechanism of clearance. The defect in these mutants cannot be attributed to nutrient limitation or an inability to adapt to the host environment in vivo as Δp66 bacteria were able to survive as well as wild type in dialysis membrane chambers in the rat peritoneum. Δp66 bacteria were able to survive in ticks through the larva to nymph moult, but were non‐infectious in mice when delivered by tick bite. Independent lines of evidence do not support any increased susceptibility of the Δp66 strains to factors in mammalian blood. This study is the first to define a B. burgdorferi adhesin as essential for mammalian, but not tick infection.     

Distinct leaf developmental and gene expression responses to light quantity depend on blue-photoreceptor or plastid-derived signals,and can occur in the absence of phototropins

Leaf palisade cell development and the composition of chloroplasts respond to the fluence rate of light to maximise photosynthetic light capture while minimising photodamage. The underlying light sensory mechanisms are probably multiple and remain only partially understood. Phototropins (PHOT1 and PHOT2) are blue light receptors regulating responses which are light quantity-dependent and which include the control of leaf expansion. Here we show that genes for proteins in the reaction centres show long-term responses in wild type plants, and single blue photoreceptor mutants, to light fluence rate consistent with regulation by photosynthetic redox signals. Using contrasting intensities of white or broad-band red or blue light, we observe that increased fluence rate results in thicker leaves and greater number of palisade cells, but the anticlinal elongation of those cells is specifically responsive to the fluence rate of blue light. This palisade cell elongation response is still quantitatively normal in fully light-exposed regions of phot1 phot2 double mutants under increased fluence rate of white light. Plants grown at high light display elevated expression of RBCS (for the Rubisco small subunit) which, together with expected down-regulation of LHCB1 (for the photosynthetic antenna primarily of photosystem II), is also observed in phot double mutants. We conclude that an unknown blue light photoreceptor, or combination thereof, controls the development of a typical palisade cell morphology, but phototropins are not essential for either this response or acclimation-related gene expression changes. Together with previous evidence, our data further demonstrate that photosynthetic (chloroplast-derived) signals play a central role in the majority of acclimation responses.     

Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion

Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA‐C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using β‐rec/six system to be able to delete all genes within each family (up to a quadruple ΔchsA/C/B/G mutant in family 1 and a quadruple ΔcsmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs‐activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ΔchsA, ΔchsB and ΔchsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs‐activity in the ΔchsG and ΔchsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ΔcsmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ΔcsmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (ΔcsmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus.     

Reduced susceptibility to Fusarium head blight in Brachypodium distachyon through priming with the Fusarium mycotoxin deoxynivalenol

The fungal cereal pathogen Fusarium graminearum produces deoxynivalenol (DON) during infection. The mycotoxin DON is associated with Fusarium head blight (FHB), a disease that can cause vast grain losses. Whilst investigating the suitability of Brachypodium distachyon as a model for spreading resistance to F. graminearum, we unexpectedly discovered that DON pretreatment of spikelets could reduce susceptibility to FHB in this model grass. We started to analyse the cell wall changes in spikelets after infection with F. graminearum wild‐type and defined mutants: the DON‐deficient Δtri5 mutant and the DON‐producing lipase disruption mutant Δfgl1, both infecting only directly inoculated florets, and the mitogen‐activated protein (MAP) kinase disruption mutant Δgpmk1, with strongly decreased virulence but intact DON production. At 14 days post‐inoculation, the glucose amounts in the non‐cellulosic cell wall fraction were only increased in spikelets infected with the DON‐producing strains wild‐type, Δfgl1 and Δgpmk1. Hence, we tested for DON‐induced cell wall changes in B. distachyon, which were most prominent at DON concentrations ranging from 1 to 100 ppb. To test the involvement of DON in defence priming, we pretreated spikelets with DON at a concentration of 1 ppm prior to F. graminearum wild‐type infection, which significantly reduced FHB disease symptoms. The analysis of cell wall composition and plant defence‐related gene expression after DON pretreatment and fungal infection suggested that DON‐induced priming of the spikelet tissue contributed to the reduced susceptibility to FHB.     

A Mutant of Arabidopsis thaliana lpar;L.) Heynh. with Modified Control of Aspartate Kinase by Threonine

Mutagenesis and subsequent selection of Arabidopsis thaliana plantlets on a growth inhibitory concentration of lysine has led to the isolation of lysine-resistant mutants. The ability to grow on 2 m M lysine has been used to isolate mutants that may contain an aspartate kinase with altered regulatory-feedback properties. One of these mutants (RL 4) was characterized by a relative enhancement of soluble lysine. The recessive monogenic nuclear transmission of the resistance trait was established. It was associated with an aspartate kinase less sensitive to feedback inhibition by threonine. Two mutants (RLT 40 and RL 4) in Arabidopsis, characterized by an altered regulation of aspartate kinase, were crossed to assess the effects of the simultaneous presence of these different aspartate kinase forms. A double mutant (RLT40 × RL4) was isolated and characterized by two feedback-desensitized isozymes of aspartate kinase to, respectively, lysine and threonine but no threonine and/or lysine overproduction was observed. Genetical analysis of this unique double aspartate kinase mutant indicated that both mutations were located on chromosome 2, but their loci (ak1and ak2) were found to be unlinked.     

Photoregulation of morphogenesis of tobacco plants transformed with the gene of human interlenkin-18

The effects of blue light (BL), green light (GL), and red light (RL) on morphogenesis photoregulation of transgenic tobacco (Nicotiana tabacum L.) plants containing a copy of human interleukin-18 gene were studied. Wild-type and transgenic (Il1 No.87–1 and Il18 No.7–11) lines and derived calluses were used. Photomorphogenesis of transgenic lines under GL and RL differed substantially from that of initial line at early stages of morphogenesis; this was expressed in hypocotyl lengthening under GL and cotyledon enhanced expansion under RL. Growth responses to light quality were related to changes in the levels of zeatin, IAA, and ABA. Thus, hypocotyl lengthening and increased cotyledon area under GL occurred at the elevated level of IAA and reduced level of ABA. Growth of callus cells in transgenic lines during zero passage differed from wild-type calluses only in darkness. The data obtained indicate that tobacco plant transformation with the human interleukin-18 gene changed functioning of the photoregulation systems at early developmental stages. This phenomenon is evidently explained by the pleiotropic effects of the gene inserted.     

A Trichoderma atroviride stress‐activated MAPK pathway integrates stress and light signals

Cells possess stress‐activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.