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The globally distributed harmful algal blooms (HAB) species, Heterosigma akashiwo, has been found to exhibit ichthyotoxicity. Previous studies have shown that H. akashiwo achieves a competitive edge during bloom occurrences by inhibiting the growth of a coexisting diatom, Skeletonema costatum, through allelopathy. However, the specific allelopathic mechanisms underlying the allelopathic effects of H. akashiwo on S. costatum remain unknown. To bridge this gap, our study utilized a combination of quantitative real-time PCR and metabolomics to examine the allelopathic processes of H. akashiwo on S. costatum. Our results demonstrate that the growth of S. costatum is hindered when co-cultured with H. akashiwo (initial cell concentration, 2 × 104 cell/mL). Gene expression investigation showed a substantial reduction in the mRNA levels of cytochrome b6, ribulose bisphosphate carboxylase large chain, and silicon transporter in S. costatum when grown in co-culture conditions. Furthermore, metabolic pathway analysis suggested that the allelopathic effects of H. akashiwo disrupted several vital metabolic pathways in S. costatum, including a reduction in purine and pyrimidine metabolism and an increase in fatty acid biosynthesis. Our investigation has revealed the intricate and substantial involvement of allelopathy in the formation of H. akashiwo blooms, demonstrating the complexity of the allelopathic interaction between H. akashiwo and S. costatum. These insights also contribute significantly to our understanding of the dynamics within HAB species.

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The photoperiod, which is defined as the period of time within a 24-hour time frame that light is available, is an important environmental regulator of several physiological processes in phytoplankton, including harmful bloom-forming phytoplankton. The ichthyotoxic raphidophyte Heterosigma akashiwo is a globally distributed bloom-forming phytoplankton. Despite extensive studies on the ecological impact of H. akashiwo, the influence of the photoperiod on crucial biological processes of this species remains unclear. In this study, gene expression in H. akashiwo was analyzed over a 24-hour light-dark (14:10) treatment period. Approximately 36 % of unigenes in H. akashiwo were differentially expressed during this 24-hour treatment period, which is indicative of their involvement in the response to light-dark variation. Notably, the number of differentially expressed genes exhibited an initial increase followed by a subsequent decrease as the sampling time progressed (T0 vs. other time points). Unigenes associated with photosynthesis and photoprotection reached their peak expression levels after 2-4 h of illumination (T12-T14). In contrast, the expression of unigenes associated with DNA replication peaked at the starting point of the dark period (T0). Furthermore, although several unigenes annotated to photoreceptors displayed potential diel periodicity, genes from various photoreceptor families (such as phytochrome and cryptochrome) showed unique expression patterns. Collectively, our findings offer novel perspectives on the response of H. akashiwo to the light-dark cycle, serving as a valuable resource for investigating the physiology and ecology of this species.

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The raphidophyte Heterosigma akashiwo is a harmful algal species. The bloom of this organism has been associated with the massive mortality of fish in many coastal waters. To investigate the molecular mechanism of H. akashiwo blooms, having a reliable reference transcriptome of this species is essential. Therefore, in this study, a full-length transcriptome of H. akashiwo was obtained by single-molecule real-time sequencing. In total, 45.44 Gb subread bases were generated, and 16,668 unigenes were obtained after the sequencing data processing. A total of 8666 (52.00%) unigenes were successfully annotated using seven public databases. Among them, mostly phosphorus and nitrogen metabolism genes were detected. Moreover, there were 300 putative transcription factors, 4392 putative long non-coding RNAs, and 7851 simple sequence repeats predicted. This study provides a valuable reference transcriptome for understanding how H. akashiwo blooms at a molecular level.

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Harmful algae blooms (HABs) have increased in intensity and frequency worldwide, causing negative effects on public health and marine ecosystems. This study isolated and identified the bloom causing species and its associated algicidal bacterium during a phytoplankton bloom in coastal waters of Lianyungang, China. Morphological observations and DNA barcoding analysis indicate that the studied phytoplankton bloom was caused by the raphidophyte Heterosigma akashiwo, and the algicidal bacterium, strain LD-B1, was identified as a species belonging to the genus Pseudoalteromonas. Furthermore, the algicidal effects of strain LD-B1 against H. akashiwo were characterized; revealing strain LD-B1 show strong algicidal activity against H. akashiwo. After 48 h of bacterium culture addition, the algicidal rate reached up to 98.8% with a 1% final volume rate. Moreover, our findings indicate strain LD-B1's extracellular compounds involved in algicidal activity are likely not proteinaceous. These findings indicate that the isolated strain, LD-B1, is a promising algicidal bacterium to control H. akashiwo blooms.

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Nitrogen (N) is an essential nutrient for phytoplankton growth. There is ample evidence that N enrichment promotes harmful algae blooms (HABs) but molecular mechanisms regulating N-nutrient uptake and metabolism are not so clear, especially for the raphidophyte Heterosigma akashiwo, which forms ichthyotoxic HABs in many coastal waters. In this study, the utilization of three different chemical forms of N (nitrate, ammonium, and urea) by H. akashiwo CCMA 369 was investigated in batch culture conditions. Results showed that H. akashiwo grew well on all three N compounds, and the highest cell yield occurred in the NH4+ culture group. Reverse transcription quantitative PCR analysis revealed that the expression of high-affinity NO3- transporter (NIT), NH4+ transporter (AMT) and high-affinity urea active transporter (DUR3), were significantly up-regulated under N-limitation compared to the N-replete control. The mRNA levels of AMT and DUR3 also displayed a clear diel rhythm, with high levels at midnight. In addition, NH4+ addition (5 µM) did not depress the transcript abundance of any of the three N transporters. Compared with the co-occurring immobile diatom Skeletonema costatum, the high expression of AMT in dark period in H. akashiwo is consistent with its diel vertical migration behavior, which may promote N-nutrient acquisition from deeper layers and give advantages for H. akashiwo to form blooms.

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Sexual reproduction (SR), hallmarked by meiosis, is widespread in eukaryotes. In phytoplankton, SR has been observed in many lineages, but molecular information on SR or meiosis of harmful algal bloom (HAB) species is scarce. The raphidophyte Heterosigma akashiwo is a globally distributed and devastating HAB species, but molecular evidence of its SR or meiosis is lacking. Here, to address the gap of knowledge, the presence of meiotic genes in H. akashiwo were examined. Interestingly, seven meiosis-specific or related genes (SPO11, MND, RAD21, RAD51, MSH2, MSH6 and MEI2) were identified from H. akashiwo transcriptomes. Furthermore, expression patterns of these genes except MSH6 (excluded due to primer failure) were investigated using quantitative reverse-transcription PCR. Results showed that the examined genes exhibited similar diel rhythms, typically, highest in early dark period and then gradually decreasing until mid-day. In addition, the expression of these six genes was not higher in the stationary growth stage than in the exponential stage, as would be expected if meiosis was to form cysts, and their elevated expression in response to colchicine treatment (arresting cells in the G2/M transition) indicated a potential role of these genes in cell division and population growth in H. akashiwo. Consistent with this, we also found a morning to afternoon shift in the expression of these genes during the bloom of H. akashiwo. This study documents a part of the typical SR gene repertoire and its potential role in regulating cell division in H. akashiwo, offering candidates for population growth markers for bloom monitoring although its linkage to meiosis and SR remain to be studied further in the future.

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The interaction of cationic pyridinium porphyrins appending methylpyridyl, hydroxyphenyl, propionoxyphenyl or carboxyphenyl group at meso-20-position of porphyrin core with bovine serum albumin (BSA), was studied by the combination of absorption spectroscopy, surface-enhanced Raman spectroscopy (SERS), circular dichroism (CD) spectroscopy, fluorescence spectroscopy and synchronous spectroscopy. The spectral monitoring results indicate that the studied compounds could bind with the BSA molecule and the calculated binding constants show that the tetracationic porphyrin has higher binding affinity than those tricationic ones. The interactions between porphyrins and BSA employ an electrostatic binding mechanism and there was only one binding site which located on the surface of the protein molecule.

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The photoinduced electron transference (PET) interaction in porphyrin containing donor-acceptor (D-A) molecules is of great importance in nature and a significant part of the PET research has been devoted to the study of its mechanism ("through-space" or "through-bond") in these decades. Herein we synthesized a series of covalently linked porphyrin-anthraquinone dyads (Por-C(n)-AQ) bridged with flexible alkoxy chains at different lengths (n=1, 4, 10) and investigated their intramolecular PET using a combination of electronic absorption, steady-state fluorescence and decayed luminescence spectra. The experimental results show that the PET efficiency depends on the length of the flexible linkage between the porphyrin and anthraquinone moieties. Meanwhile, theoretical calculation applying the density functional theory (DFT) was also carried out to give the frontier orbital distribution and the optimized structures of these dyads. It is found that the orientation of the dyad with high PET efficiency is disadvantageous to π-π interaction. Thus, the PET of these dyads seemingly is best compatible with a "through-bond" (superexchange) mechanism.

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The synthesis and self-assembly behavior of porphyrin-polypyridyl ruthenium(II) hybrid, which consists of a flexible alkyl chain attached with two conjugated moieties is described. The electronic absorption spectrum and emission spectra show that the [C(8)-TPP-(ip)Ru(phen)(2)](ClO(4))(2), abbreviated as (C(8)ip)TPPC has optical properties. Scanning tunneling microscopy (STM) studies found that the pi-pi interaction and metal-ligand interaction allow (C(8)ip)TPPC to form self-assembled structure and have an edge-on orientation on the highly oriented pyrolytic graphite (HOPG) surface. The multidentate structure in (C(8)ip)TPPC molecules act as linkers between the molecules and form metal-ligand coordination, which forces the assembly process in the direction of stable columnar arrays. In addition, although the sample was stored for two months in ambient conditions, STM experiments showed that the order of (C(8)ip)TPPC self-assembly only slightly decreased which indicates that the self-assembled monolayer is stable. This work demonstrates that introducing a metal-ligand in the porphyrin-polypyridyl compound is a useful strategy to obtain novel surface assemblies.

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The interactions of 5,10,15-tris(1-methylpyridinium-4-yl)-20-(4-hydroxyphenyl)porphyrinatozinc(II) Zn[TMPyHP](3+) (2) along with Cu[TMPyHP](3+) (3), Co[TMPyHP](4+) (4), Mn[TMPyHP](4+) (5) and the free base porphyrin H(2)[TMPyHP](3+) (1) with duplex DNA have been studied by using a combination of absorption, fluorescence titration, surface-enhanced Raman spectroscopy (SERS), induced circular dichroism (ICD) spectroscopy, thermal DNA denaturation, viscosity measurements as well as gel electrophoresis experiment. Their binding modes and intrinsic binding constants (K(b)) to calf DNA (CT DNA) were comparatively studied and were found significantly influenced by different metals coordinated with the porphyrin plane. Except 3, which has four-coordination structure at the metal, all the metal derivatives showed non-intercalative DNA-binding mode and lower K(b) than the free base porphyrin 1, most probably due to the steric hindrance results from the axial ligands of the inserted metals which are five or six-coordination structures. Meanwhile, the insertion of metals into cationic porphyrin greatly removed the self-aggregation of the metal-free porphyrins, and thus fully enhanced the singlet oxygen ((1)O(2)) productivities in the DNA photocleavage experiments. Therefore, these metalloporphyrins have comparable DNA cleavage ability with the free base porphyrin.

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Four cationic porphyrin-anthraquinone (Por-AQ) hybrids differing in lengths of flexible alkyl linkage, 5-[4-(1-N-anthraquinonon-yl)-L-oxophenyl]-10,15,20-tris(N-methylpyridinium-4-yl)porphyrin triiodide, (L = acetyl, pentanoyl, octanoyl, undecanoyl, designed as [AQATMPyP]I3, [AQPTMPyP]I3, [AQOTMPyP]I3 and [AQUTMPyP]I3, respectively, see Fig. 1), were synthesized and their interactions with DNA were investigated. The results of spectroscopic, denaturation and viscosity measurements suggest that [AQATMPyP]I3 binds to DNA through non-intercalative mode while the other three hybrids with longer links bind via bis-intercalative mode. Ethidium bromide (EB) competition experiment was carried out to determine the binding constants (Kb) of these compounds for CT DNA, and [AQPTMPyP]I3 shows the largest Kb among these hybrids. The photocleavage mechanism and wavelength-dependent cleaving abilities of these hybrids to pBR322 plasmid DNA were also comparably investigated.

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Four tricationic pyridium porphyrins appending hydroxyphenyl, methoxyphenyl, propionoxyphenyl or carboxyphenyl group at meso-20-position of porphyrin core have been synthesized and their abilities to bind and cleave DNA have been investigated. Using a combination of absorption, fluorescence, circular dichroism (CD) spectra, thermal DNA denaturation as well as viscosity measurements, their binding modes and intrinsic binding constants (K(b)) to calf DNA (CT DNA) were comparatively studied and also compared with those of 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP). The results suggest that the K(b) values of these porphyrins are greatly influenced by the number of positive charges and steric hindrance. Theoretical calculations applying the density functional theory (DFT) have been carried out and explain their DNA-binding properties reasonably. The efficiency of DNA photocleavage by these porphyrins shows high dependence on the values of K(b).

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The DNA-binding affinities and DNA photocleavage abilities of cationic porphyrin, 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridiniumyl)porphyrin (CTMPyP), and its reference compound meso-tetrakis(N-methyl-4-pyridiniumyl)porphyrin (H2TMPyP) have been investigated. The DNA-binding behaviors of the two compounds in NaH2PO4 buffer were compared systematically by using absorption, fluorescence and circular dichroism (CD) spectra, thermal denaturation as well as viscosity measurements. The experimental results show that CTMPyP binds to DNA in an outside binding mode, while H2TMPyP in an intercalative mode. Photocleavage experiments reveal that both two compounds employ 1O2-mediated mechanism in cleaving DNA and H2TMPyP can cleave DNA more efficiently than CTMPyP. Theoretical calculations were carried out with the density functional theory (DFT), and the calculated results indicate that the character and energies of some frontier orbitals of CTMPyP are quite different from those of H2TMPyP. These theoretical results can be used to explain their different DNA-binding modes and affinities to a certain extent.

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A novel cationic porphyrin-anthraquinone (Por-AQ) hybrid has been synthesized and characterized. Using the combination of absorption titration, fluorescence spectra, circular dichroism (CD) as well as viscosity measurements, the binding properties of the hybrid to calf thymus (CT) DNA have been investigated compared with its parent porphyrin. The experimental results show that at low [Por]/[DNA] ratios, the parent porphyrin binds to DNA in an intercalative mode while the hybrid binds in a combined mode of outside binding (for porphyrin moiety) and partial intercalation (for anthraquinone). Ethidium bromide (EB) competition experiment determined the binding affinity constants (K(app)) of the compounds for CT DNA. Theoretical calculational results applying the density functional theory (DFT) can explain the different DNA binding behaviors reasonably. (1)O(2) was suggested to be the reactive species responsible for the DNA photocleavage of porphyrin moieties in both two compounds. The wavelength-depending cleavage activities of the compounds were also investigated.

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The electronic and fluorescence spectroscopic properties of a novel porphyrin-polypyridyl ruthenium(II) hybrid, [C(4)-TPP-(ip)Ru(phen)(2)](ClO(4))(2) (TPP=5,10,15,20-tetraphenylporphyrin, ip=imidazo[4,5-f][1,10]phenanthroline and phen=1,10-Phenanthroline), in which a polypyridyl ruthenium(II) moiety is linked to a porphyrin moiety by a butyl chain have been investigated and compared to its corresponding reference compounds. The studies of electronic absorption spectra have shown that there is an electronic interaction between the porphyrin moiety and the polypyridyl ruthenium(II) moiety in the hybrid. It can be found that intramolecular photoinduced electron and energy transfer processes may occur in the hybrid from the fluorescence spectra. When exciting in Soret band and Q band of porphyrin, the fluorescence quenching of the porphyrin moiety of the hybrid takes place due to electron transfer from the lowest singlet excited state (S(1)) to the appended polypyridyl rutherium(II) moiety, while the decay of S(2) (the second-excited singlet state) of the porphyrin moiety is mainly contributed to internal conversion to S(1). When exciting in MLCT band of the polypyridyl ruthenium(II) moiety, fluorescence corresponding to the polypyridyl ruthenium(II) moiety is quenched by intramolecular energy transfer from (3)MLCT of the ruthenium moiety to the lowest-energy triplet state localized on the porphyrin moiety.

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Coordination bonding driving supramolecular self-assembly of Zn(TPP) with Fe(p-ImEPTPP)Cl was investigated by fluorescence spectra and 1H NMR. The results indicate that an obvious fluorescence quenching observed from Zn(TPP) by addition of Fe (p-ImEPTPP)Cl is due to the coordination of the terminal imidazolyl group of Fe(p-ImEQTPP)Cl to Zn2+ of Zn(TPP). The change of fluorescence spectra of Zn(TPP)/Fe(p-ImEPTPP)Cl system by adding some Lewis bases, such as piperidine, imidazole and pyridine, as well as 1H NMR further supported this coordination.

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The fluorescence spectra of porphyrinatozinc(II)/iron(III) systems which consisted of tetraphenylporphyrinatozinc(II) and three kinds of imidazolyl-linked porphyrinatoiron(III) have been studied. An efficient fluorescence quenching of tetraphenylporphyrinatozinc(II) in the system was observed. Addition of a stronger organic base, such as piperdine, to the system can displace imidazolyl-linked porphyrinatoiron(III) and the fluorescence of the system restored partly. All these indicate the formation of porphyrinatozinc(II)/iron(III) supramolecular complex and coordination bonding formed by the coordination of imidazolyl group in imidazolyl-linked porphyrinatoiron(III) to Zn(II) in tetraphenyl-porphyrinatozinc(II) is the driving force of the supramolecular self-assembly. The association constants of the supramolecular complexes were calculated from the fluorescence spectroscopic titration data, and the differences among the association constants of the supramolecular complexes are discussed on the basis of the conformations which are dependent on the length of alkoxy chain linking imidazolyl group to porphyrinatoiron(III).

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