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Platycladus orientalis L. (Cupressaceae) has a lifespan of thousands of years. Ancient trees have very high scientific, economic and cultural values. The senescence of ancient trees is a new research area but is poorly understood. Leaves are the primary and the most sensitive organ of a tree. To understand leaf structural response to tree senescence in ancient trees, experiments investigating the morphology, anatomy and ultrastructure were conducted with one-year leaves of ancient P. orientalis (ancient tree >2,000 years) at three different tree senescent levels (healthy, sub-healthy and senescent) at the world’s largest planted pure forest in the Mausoleum of Yellow Emperor, Shaanxi Province, China. Observations showed that leaf structure significantly changed with the senescence of trees. The chloroplast, mitochondria, vacuole and cell wall of mesophyll cells were the most significant markers of cellular ultrastructure during tree senescence. Leaf ultrastructure clearly reflected the senescence degree of ancient trees, confirming the visual evaluation from above-ground parts of trees. Understanding the relationships between leaf structure and tree senescence can support decision makers in planning the protection of ancient trees more promptly and effectively by adopting the timely rejuvenation techniques before the whole tree irreversibly recesses.

Platycladus orientalis (Cupressaceae) is a native tree species used widely for landscaping and afforestation in arid and semiarid areas in Asia. Ancient P. orientalis trees not only have an important humanistic and historical value but also have a significant scientific value in aging mechanism research. Mesophyll protoplasts are an important material for studies of plant regeneration, transient gene expression, and senescence. Although an easy and effective technique of mesophyll protoplast isolation from mature trees is urgently needed, the isolation parameters have great material specificity. The young tissue of plants is an ideal material for protoplast preparation. In this study, we employed an orthogonal experiment and several single-variable experiments to determine the main factors influencing the successful isolation of mesophyll protoplasts and established an efficient technique for isolating mesophyll protoplasts from the young scale leaves of ancient P. orientalis. The optimal parameters for mesophyll protoplast isolation are as follows: mechanical homogenization of the leaf tissue, 1.5% Cellulase R-10, 0.4% Macerozyme R-10, 0.4% Pectolyase Y-23, 1.0% ligninase, 0.7 M mannitol (pH 5.8), and 16 h of incubation. Two centrifugations (100 × g for 3 min and 500 × g for 5 min) were repeated 2 times to obtain purified protoplast suspension. The yield and viability of protoplasts under optimal conditions were 4.8 × 10 6 g FW -1 (per gram fresh weight) and 82.5%, respectively. The results of flow cytometry analysis showed that the isolated protoplasts had ideal viability to meet the demands of further protoplast-based research.

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Tree aging is a new research area and has attracted research interest in recent years. Trees show extraordinary longevity; Platycladus orientalis L. (Cupressaceae) has a lifespan of thousands of years. Ancient trees are precious historical heritage and scientific research materials. However, tree aging and tree senescence have different definitions and are poorly understood. Since leaves are the most sensitive organ of a tree, we studied the structural response of leaves to tree age. Experiments investigating the leaf morphological structure, anatomical structure and ultrastructure were conducted in healthy P. orientalis at three different ages (ancient trees >2,000 years, 200 years < middle-aged trees <500 years, young trees <50 years) at the world’s largest planted pure forest in the Mausoleum of the Yellow Emperor, Shaanxi Province, China. Interestingly, tree age did not significantly impact leaf cellular structure. Ancient P. orientalis trees in forests older than 2,000 years still have very strong vitality, and their leaves still maintained a perfect anatomical structure and ultrastructure. Our observations provide new evidence for the unique pattern of tree aging, especially healthy aging. Understanding the relationships between leaf structure and tree age will enhance the understanding of tree aging.