Morphology and physicochemical properties of melanin particles from the lichen <i>Lobaria pulmonaria</i>

Melanins are the pigments with diverse protective functions in a wide range of organisms. The ability of lichens to synthesize melanins is an important adaptation in response to adverse light conditions, including ultraviolet (UV) irradiation and high light. Previously, we demonstrated that melanization of the lichen Lobaria pulmonaria is accompanied by the accumulation of melanin granules in the upper cortex. However, very little information is available on the morphology and physicochemical properties of these particles. Here, for the first time, we used polyclonal antibodies against eumelanin to visualize melanin in the upper cortex of L. pulmonaria and confirm its identity. Using scanning electron microscopy, we showed that extracted melanins are large, spherical aggregates, with an average size of 230 nm. The aggregates had an irregular and unstable structure and displayed high adhesive properties, according to atomic force microscopy. Purification using size exclusion chromatography showed that melanins coeluted with polysaccharides. Purified particles were visualized as discrete structures (subaggregates) with an average size of 42 nm and displayed higher stability and lower adhesive properties than non-purified aggregates. In colloidal aqueous systems, melanin particles formed aggregates with particle sizes of 279 and 212 nm for the non-purified and purified samples, respectively, suggesting that the removal of the polysaccharide component during purification causes the condensation of the polyaromatic structure of melanin. Taken together, our results suggest that melanin particles extracted from UV-melanized thalli of the lichen L. pulmonaria comprise a supramolecular assembly of melanin and polysaccharides. Analysis of the size, ζ-potential, and polydispersity index suggests that melanin particles are capable of structural rearrangements that lead to changes in their nanomechanical properties. The ability of the melanin to bind to polysaccharides may enhance the structural robustness of mycobiont cell walls and increase the tolerance of lichen thalli to stressful environments.黑色素是一类在多种生物中发挥多样保护功能的色素。地衣合成黑色素的能力，是其应对包括紫外线（UV）辐照与强光在内的不良光照条件的重要适应性策略。此前我们已证实，肺衣（Lobaria pulmonaria）的黑色素化过程伴随着上皮层中黑色素颗粒的积累。然而，目前关于这类颗粒的形态学与理化性质的相关研究信息仍十分匮乏。本研究首次使用针对真黑色素（eumelanin）的多克隆抗体，对肺衣上皮层中的黑色素进行了可视化标记，并确证了其成分属性。通过扫描电子显微镜观察，我们发现提取得到的黑色素呈大型球状聚集体，平均粒径为230 nm。原子力显微镜分析显示，该聚集体结构不规则且稳定性欠佳，同时表现出较强的黏附特性。尺寸排阻色谱纯化结果表明，黑色素与多糖共洗脱。纯化后的颗粒呈现为离散的亚聚集体结构，平均粒径为42 nm，相较于未纯化的聚集体，其稳定性更高、黏附性更低。在胶体水溶液体系中，未纯化样品与纯化样品的黑色素颗粒分别形成粒径为279 nm和212 nm的聚集体，这表明纯化过程中多糖组分的去除会促使黑色素多芳香族结构发生凝聚。综合以上结果，我们认为从经紫外线诱导黑色素化的肺衣叶状体中提取的黑色素颗粒，是黑色素与多糖构成的超分子组装体。对粒径、ζ电势与多分散性指数的分析表明，黑色素颗粒能够发生结构重排，进而改变其纳米机械特性。黑色素与多糖结合的能力，可增强真菌共生体细胞壁的结构稳定性，并提升地衣叶状体对胁迫环境的耐受能力。