Light-Quality-Adapted Carotenoid Photoprotection in the Photosystem of Roseiflexus castenholzii.

The photosystem of filamentous anoxygenic phototroph Roseiflexus (Rfl.) castenholzii comprises a light-harvesting (LH) complex encircling a reaction center (RC), which intensely absorbs blue-green light by carotenoid (Car) and near-infrared light by bacteriochlorophyll (BChl). To explore the influence of light quality (color) on the photosynthetic activity, we compared the pigment compositions and triplet excitation dynamics of the LH-RCs from Rfl. castenholzii was adapted to blue-green light (bg-LH-RC) and to near-infrared light (nir-LH-RC). Both LH-RCs bind γ-carotene derivatives; however, compared to that of nir-LH-RC (12%), bg-LH-RC contains substantially higher keto-γ-carotene content (43%) and shows considerably faster BChl-to-Car triplet excitation transfer (10.9 ns vs 15.0 ns). For bg-LH-RC, but not nir-LH-RC, selective photoexcitation of Car and the 800 nm-absorbing BChl led to Car-to-Car triplet transfer and BChl-Car singlet fission reactions, respectively. The unique excitation dynamics of bg-LH-RC enhances its photoprotection, which is crucial for the survival of aquatic anoxygenic phototrophs from photooxidative stress.

Energy Transfer and Exciton Relaxation in B880-B800-RC Complex through Two-Dimensional Electronic Spectroscopy.

The light-harvesting (LH) and reaction center (RC) core complex of purple bacterium Roseiflexus castenholzii, B880-B800-RC, are different from those of the typical photosynthetic unit, (B850-B800)x-B880-RC. To investigate the excitation flowing dynamics in this unique complex, two-dimensional electronic spectroscopy is employed. The obtained time constants for the exciton relaxation in B880, exciton relaxation in B800, B800 → B880 energy transfer (EET), and B880 → closed RC EET are 43 fs, 177 fs, 1.9 ps, and 205 ps, respectively. These time constants result in an overall EET efficiency similar to that of the typical photosynthetic unit. Analysis of the oscillatory signals reveals that while several vibronic coherences are involved in the exciton relaxation process, only one prominent vibronic coherence, with a frequency of 27 cm-1 and coupled to the B880 electronic transition, may contribute to the B800 → B880 EET process.

Structural insights into the unusual core photocomplex from a triply extremophilic purple bacterium, Halorhodospira halochloris.

Halorhodospira (Hlr.) halochloris is a triply extremophilic phototrophic purple sulfur bacterium, as it is thermophilic, alkaliphilic, and extremely halophilic. The light-harvesting-reaction center (LH1-RC) core complex of this bacterium displays an LH1-Qy transition at 1,016 nm, which is the lowest-energy wavelength absorption among all known phototrophs. Here we report the cryo-EM structure of the LH1-RC at 2.42 Å resolution. The LH1 complex forms a tricyclic ring structure composed of 16 αßγ-polypeptides and one αß-heterodimer around the RC. From the cryo-EM density map, two previously unrecognized integral membrane proteins, referred to as protein G and protein Q, were identified. Both of these proteins are single transmembrane-spanning helices located between the LH1 ring and the RC L-subunit and are absent from the LH1-RC complexes of all other purple bacteria of which the structures have been determined so far. Besides bacteriochlorophyll b molecules (B1020) located on the periplasmic side of the Hlr. halochloris membrane, there are also two arrays of bacteriochlorophyll b molecules (B800 and B820) located on the cytoplasmic side. Only a single copy of a carotenoid (lycopene) was resolved in the Hlr. halochloris LH1-α3ß3 and this was positioned within the complex. The potential quinone channel should be the space between the LH1-α3ß3 that accommodates the single lycopene but does not contain a γ-polypeptide, B800 and B820. Our results provide a structural explanation for the unusual Qy red shift and carotenoid absorption in the Hlr. halochloris spectrum and reveal new insights into photosynthetic mechanisms employed by a species that thrives under the harshest conditions of any phototrophic microorganism known.

MdBT2 regulates nitrogen-mediated cuticular wax biosynthesis via a MdMYB106-MdCER2L1 signalling pathway in apple.

Cuticular waxes play important roles in plant development and the interaction between plants and their environment. Researches on wax biosynthetic pathways have been reported in several plant species. Also, wax formation is closely related to environmental condition. However, the regulatory mechanism between wax and environmental factors, especially essential mineral elements, is less studied. Here we found that nitrogen (N) played a negative role in the regulation of wax synthesis in apple. We therefore analysed wax content, composition and crystals in BTB-TAZ domain protein 2 (MdBT2) overexpressing and antisense transgenic apple seedlings and found that MdBT2 could downregulate wax biosynthesis. Furthermore, R2R3-MYB transcription factor 16-like protein (MdMYB106) interacted with MdBT2, and MdBT2 mediated its ubiquitination and degradation through the 26S proteasome pathway. Finally, HXXXD-type acyl-transferase ECERIFERUM 2-like1 (MdCER2L1) was confirmed as a downstream target gene of MdMYB106. Our findings reveal an N-mediated apple wax biosynthesis pathway and lay a foundation for further study of the environmental factors associated with wax regulatory networks in apple.

Carotenoid-Mediated Long-Range Energy Transfer in the Light Harvesting-Reaction Center Complex from Photosynthetic Bacterium Roseiflexus castenholzii.

The light harvesting-reaction center complex (LH-RC) of Roseiflexus castenholzii binds bacteriochlorophylls a (BChls a), B800 and B880, absorbing around 800 and 880 nm, respectively. We comparatively investigated the interband excitation energy transfer (EET) dynamics of the wild-type LH-RC (wt-LH-RC) of Rfl. castenholzii and its carotenoid (Car)-less mutant (m-LH-RC) and found that Car can boost the B800 → B880 EET rate from (2.43 ps)-1 to (1.75 ps)-1, accounting for 38% acceleration of the EET process. Interestingly, photoexcitation of wt-LH-RC at 800 nm induced pronounced excitation dynamics of Car despite the insufficient photon energy for direct Car excitation, a phenomenon which is attributed to the BChl-Car exciplex 1[B800(↑↑)···Car(↓↓)]*. Such an exciplex is suggested to play an essential role in promoting the B800 → B880 EET process, as corroborated by the recently reported cryo-EM structures of wt-LH-RC and m-LH-RC. The mechanism of Car-mediated EET will be helpful to deepen the understanding of the role of Car in bacterial photosynthesis.

New insights on the photocomplex of Roseiflexus castenholzii revealed from comparisons of native and carotenoid-depleted complexes.

In wild-type phototrophic organisms, carotenoids (Crts) are primarily packed into specific pigment-protein complexes along with (Bacterio)chlorophylls and play important roles in the photosynthesis. Diphenylamine (DPA) inhibits carotenogenesis but not phototrophic growth of anoxygenic phototrophs and eliminates virtually all Crts from photocomplexes. To investigate the effect of Crts on assembly of the reaction center-light-harvesting (RC-LH) complex from the filamentous anoxygenic phototroph Roseiflexus (Rfl.) castenholzii, we generated carotenoidless (Crt-less) RC-LH complexes by growing cells in the presence of DPA. Here, we present cryo-EM structures of the Rfl. castenholzii native and Crt-less RC-LH complexes with resolutions of 2.86 Å and 2.85 Å, respectively. From the high-quality map obtained, several important but previously unresolved details in the Rfl. castenholzii RC-LH structure were determined unambiguously including the assignment and likely function of three small polypeptides, and the content and spatial arrangement of Crts with bacteriochlorophyll molecules. The overall structures of Crt-containing and Crt-less complexes are similar. However, structural comparisons showed that only five Crts remain in complexes from DPA-treated cells and that the subunit X (TMx) flanked on the N-terminal helix of the Cyt-subunit is missing. Based on these results, the function of Crts in the assembly of the Rfl. castenholzii RC-LH complex and the molecular mechanism of quinone exchange is discussed. These structural details provide a fresh look at the photosynthetic apparatus of an evolutionary ancient phototroph as well as new insights into the importance of Crts for proper assembly and functioning of the RC-LH complex.

Carotenoid Single-Molecular Singlet Fission and the Photoprotection of a Bacteriochlorophyll b-Type Core Light-Harvesting Antenna.

Carotenoid (Car) in photosynthesis plays the major roles of accessary light harvesting and photoprotection, and the underlying structure-function relationship attracts continuing research interests. We have attempted to explore the dynamics of Car triplet excitation (3Car*) in the bacteriochlorophyll b (BChl b)-type light harvesting reaction center complex (LH1-RC) of photosynthetic bacterium Halorhodospira halochloris. We show that the LH1 antenna binds a single Car that was identified as a lycopene derivative. Although the Car is hardly visible in the LH1-RC stationary absorption, it shows up conspicuously in the triplet excitation profile with distinct vibronic features. This and the ultrafast formation of 3Car* on direct photoexcitation of Car unequivocally manifest the unimolecular singlet fission reaction of the Car. Moreover, the Car with even one molecule per complex is found to be rather effective in quenching 3BChl b*. The implications of different 3Car* formation mechanisms are discussed, and the self-photoprotection role of BChl b are proposed for this extremophilic species.

MdWRKY15 improves resistance of apple to Botryosphaeria dothidea via the salicylic acid-mediated pathway by directly binding the MdICS1 promoter.

Isochorismate synthase (ICS) plays an essential role in the accumulation of salicylic acid (SA) and plant disease resistance. Diseases caused by Botryosphaeria dothidea affect apple yields. Thus, it is important to understand the role of ICS1 in disease resistance to B. dothidea in apple. In this study, SA treatment enhanced the resistance to B. dothidea. MdICS1 was induced by B. dothidea and enhanced the resistance to B. dothidea. MdICS1 promoter analysis indicated that the W-box was vital for the response to B. dothidea treatment. MdWRKY15 was found to interact with the W-box using yeast one-hybrid screening. Subsequently, the interaction was confirmed by EMSA, yeast one-hybrid, ChIP-PCR, and quantitative PCR assays. Moreover, luciferase and GUS analysis further indicated that MdICS1 was transcriptionally activated by MdWRKY15. Finally, we found the function of MdWRKY15 in the resistance to B. dothidea was partially dependent on MdICS1 from the phenotype of transgenic apples and calli. In summary, we revealed that MdWRKY15 activated the transcription of MdICS1 by directly binding to its promoter to increase the accumulation of SA and the expression of disease-related genes, thereby resulting in the enhanced resistance to B. dothidea in the SA biosynthesis pathway.

MdWRKY46-Enhanced Apple Resistance to Botryosphaeria dothidea by Activating the Expression of MdPBS3.1 in the Salicylic Acid Signaling Pathway.

Salicylic acid (SA) is closely related to disease resistance of plants. WRKY transcription factors have been linked to the growth and development of plants, especially under stress conditions. However, the regulatory mechanism of WRKY proteins involved in SA production and disease resistance in apple is not clear. In this study, MdPBS3.1 responded to Botryosphaeria dothidea and enhanced resistance to B. dothidea. Electrophoretic mobility shift assays, yeast one-hybrid assays, and chromatin immunoprecipitation and quantitative PCR demonstrated that MdWRKY46 can directly bind to a W-box motif in the promoter of MdPBS3.1. Glucuronidase transactivation and luciferase analysis further showed that MdWRKY46 can activate the expression of MdPBS3.1. Finally, B. dothidea inoculation in transgenic apple calli and fruits revealed that MdWRKY46 improved resistance to B. dothidea by the transcriptional activation of MdPBS3.1. Viral vector-based transformation assays indicated that MdWRKY46 elevates SA content and transcription of SA-related genes, including MdPR1, MdPR5, and MdNPR1 in an MdPBS3.1-dependent way. These findings provide new insights into how MdWRKY46 regulates plant resistance to B. dothidea through the SA signaling pathway.

The R2R3 MYB transcription factor MdMYB30 modulates plant resistance against pathogens by regulating cuticular wax biosynthesis.

BACKGROUND: The MYB transcription factor family is one of the largest transcriptional factor families in plants and plays a multifaceted role in plant growth and development. However, MYB transcription factors involved in pathogen resistance in apple remain poorly understood. RESULTS: We identified a new MYB family member from apple, and named it MdMYB30. MdMYB30 was localized to the nucleus, and was highly expressed in young apple leaves. Transcription of MdMYB30 was induced by abiotic stressors, such as polyethylene glycol and abscisic acid. Scanning electron microscopy and gas chromatograph-mass spectrometry analyses demonstrated that ectopically expressing MdMYB30 in Arabidopsis changed the wax content, the number of wax crystals, and the transcription of wax-related genes. MdMYB30 bound to the MdKCS1 promoter to activate its expression and regulate wax biosynthesis. MdMYB30 also contributed to plant surface properties and increased resistance to the bacterial strain Pst DC3000. Furthermore, a virus-based transformation in apple fruits and transgenic apple calli demonstrated that MdMYB30 increased resistance to Botryosphaeria dothidea. Our findings suggest that MdMYB30 plays a vital role in the accumulation of cuticular wax and enhances disease resistance in apple. CONCLUSIONS: MdMYB30 bound to the MdKCS1 gene promoter to activate its transcription and regulate cuticular wax content and composition, which influenced the surface properties and expression of pathogenesis-related genes to resistance against pathogens. MdMYB30 appears to be a crucial element in the formation of the plant cuticle and confers apple with a tolerance to pathogens.

MdHIR4 transcription and translation levels associated with disease in apple are regulated by MdWRKY31.

KEY MESSAGE: Here we describe that the regulation of MdWRKY31 on MdHIR4 in transcription and translation levels associated with disease in apple. The phytohormone salicylic acid (SA) is a main factor in apple (Malus domestica) production due to its function in disease resistance. WRKY transcription factors play a vital role in response to stress. An RNA-seq analysis was conducted with 'Royal Gala' seedlings treated with SA to identify the WRKY regulatory mechanism of disease resistance in apple. The analysis indicated that MdWRKY31 was induced. A quantitative real-time polymerase chain reaction (qPCR) analysis demonstrated that the expression of MdWRKY31 was induced by SA and flg22. Ectopic expression of MdWRKY31 in Arabidopsis and Nicotiana benthamiana increased the resistance to flg22 and Pseudomonas syringae tomato (Pst DC3000). A yeast two-hybrid screen was conducted to further analyze the function of MdWRKY31. As a result, hypersensitive-induced reaction (HIR) protein MdHIR4 interacted with MdWRKY31. Biomolecular fluorescence complementation, yeast two-hybrid, and pull-down assays demonstrated the interaction. In our previous study, MdHIR4 conferred decreased resistance to Botryosphaeria dothidea (B. dothidea). A viral vector-based transformation assay indicated that MdWRKY31 evaluated the transcription of SA-related genes, including MdPR1, MdPR5, and MdNPR1 in an MdHIR4-dependent way. A GUS analysis demonstrated that the w-box, particularly w-box2, of the MdHIR4 promoter played a major role in the responses to SA and B. dothidea. Electrophoretic mobility shift assays, yeast one-hybrid assay, and chromatin immunoprecipitation-qPCR demonstrated that MdWRKY31 directly bound to the w-box2 motif in the MdHIR4 promoter. GUS staining activity and a protein intensity analysis further showed that MdWRKY31 repressed MdHIR4 expression. Taken together, our findings reveal that MdWRKY31 regulated plant resistance to B. dothidea through the SA signaling pathway by interacting with MdHIR4.

The MdWRKY31 transcription factor binds to the MdRAV1 promoter to mediate ABA sensitivity.

The phytohormone abscisic acid (ABA) is a major element involved in apple (Malus domestica) production because of its role in seed germination and early seedling development. The WRKY family, which is one of the largest families of transcription factors, plays an important role in ABA signaling in plants. However, the underlying molecular mechanisms of WRKY-mediated ABA sensitivity in apple are poorly understood. A genome-wide transcriptome analysis indicated that MdWRKY31 is a key factor induced by ABA. Quantitative real-time PCR showed that MdWRKY31 is induced by ABA in response to PEG4000, which is used to simulate drought. As a transcription factor, MdWRKY31 is localized in the nucleus. Ectopic expression of MdWRKY31 in Arabidopsis and Nicotiana benthamiana enhanced plant sensitivity to ABA. Overexpression of MdWRKY31 in apple roots and apple calli increased sensitivity to ABA, whereas repression of MdWRKY31 reduced sensitivity to ABA in the roots of 'Royal Gala'. Electrophoretic mobility shift assays, chromatin immunoprecipitation PCR, and yeast one-hybrid assays indicated that MdWRKY31 directly binds to the promoter of MdRAV1. Expression analyses of transgenic apple calli containing MdWRKY31 and pMdRAV1::GUS implied that MdWRKY31 represses the expression of MdRAV1. We also found that MdRAV1 binds directly to the promoters of MdABI3 and MdABI4 and repressed their expression. Our findings reveal a new important regulatory mechanism of MdWRKY31-MdRAV1-MdABIs in the ABA signaling pathway in apple.

Functional identification of apple on MdHIR4 in biotic stress.

In plants, hypersensitive-induced reaction (HIR) proteins are involved in stress responses, especially biotic stress. However, the potential molecular mechanisms of HIR-mediated biotic resistance in plants are rarely reported. We found that apple (Malus domestica) MdHIR4 was localized in the cell nucleus and membrane similar to AtHIR1 in Arabidopsis. Moreover, salicylic acid and the bacterial flagellin flg22 (a conserved, 22-amino acid motif), which are relevant to biotic stress, could induce MdHIR4 expression. Additionally, the transcription level of MdHIR4 was increased by Methyl jasmonate treatment. Ectopic expression of MdHIR4 in Arabidopsis and Nicotiana benthamiana reduced sensitivity to Methyl jasmonate and enhanced resistance to the bacterial pathogen Pst DC3000 (Pseudomonas syringae tomato DC3000). The interaction between MdHIR4 and AtJAZs proteins (AtJAZ3, AtJAZ4, and AtJAZ9) implied that MdHIR4 participated in the jasmonic acid (JA) signaling pathway. We found the expression of JA-related genes and PRs to change in transgenic plants, further demonstrating that MdHIR4 mediated biotic stress through the JA signaling pathway. Repressing the expression of MdHIR4 in apple leaves and calli increased resistance to Botryosphaeria dothidea by influencing the transcription of resistance-related genes. Our findings reveal the resistant function to biotic stress of MdHIR4 in transgenic plants, including Arabidopsis, tobacco, and apple, and identify the regulating mechanism of MdHIR4-related biotic resistance.

Apple AP2/EREBP transcription factor MdSHINE2 confers drought resistance by regulating wax biosynthesis.

MAIN CONCLUSION: This study showed that AP2/EREBP transcription factor MdSHINE2 functioned in mediating cuticular permeability, sensitivity to abscisic acid (ABA), and drought resistance by regulating wax biosynthesis. Plant cuticular wax plays crucial roles in protecting plants from environmental stresses, particularly drought stress. Many enzymes and transcription factors involved in wax biosynthesis have been identified in plant species. In this study, we identified an AP2/EREBP transcription factor, MdSHINE2 from apple, which is a homolog of AtSHINE2 in Arabidopsis. MdSHINE2 was constitutively expressed at different levels in various apple tissues, and the transcription level of MdSHINE2 was induced substantially by abiotic stress and hormone treatments. MdSHINE2-overexpressing Arabidopsis exhibited great change in cuticular wax crystal numbers and morphology and wax composition of leaves and stems. Moreover, MdSHINE2 heavily influenced cuticular permeability, sensitivity to abscisic acid, and drought resistance.