Spatial and temporal distribution of cystoliths in mulberry leaves and their formation under the influence of phytohormones 6-BA and ABA

Spatial and temporal distribution of cystoliths in mulberry leaves and their formation under the influence of phytohormones 6-BA and ABA | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Spatial and temporal distribution of cystoliths in mulberry leaves and their formation under the influence of phytohormones 6-BA and ABA Chao Yang, Qi Zhang, Peng Qian, Jiubo Liang, Lin Chen, Jianglian Yuan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3887434/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Mulberry ( Morus spp.) has been studied to gain insight into cystolith formation, which is primarily composed of calcium carbonate (CaCO 3 ) crystals and commonly found in mulberry leaves. However, the effects of phytohormones on cystolith formation in mulberry and the origin of carbon within these structures remain poorly understood. This study utilized scanning electron microscopy (SEM), plant tissue sections, and silver nitrate staining techniques to comprehensively analyze the morphology of cystoliths in mulberry. Additionally, the distribution pattern of cystoliths was investigated, and mulberry seedlings were treated with 6-Benzylaminopurine (6-BA) and Abscisic acid (ABA). The results revealed that 6-BA significantly enhanced cystolith accumulation, whereas ABA had suppressive effects on cystolith formation in mulberry leaves. Furthermore, the concentration of applied phytohormones positively correlated with the yield of cystoliths. Based on these results, it is postulated that these phytohormones may modulate carbon absorption in mulberry by influencing stomatal conductance, thereby regulating cystolith formation. This research offers valuable insights into the underlying mechanisms driving mulberry cystolith formation and contributes to the optimal utilization of mulberry resources. Cystoliths Mulberry Distribution patterns Phytohormone 6-Benzylaminopurine Abscisic acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Mulberry ( Morus spp.) is a member of the Moraceae family in the order Rosaceae. It is widely recognized as the main food source for silkworms ( Bombyx mori ), playing a crucial role in the sericulture industry and contributing substantially to the economy of various countries. Mulberry leaves are rich in proteins and carbohydrate, both essential for the growth and development of silkworms. Moreover, mulberry leaves are exceptionally rich in calcium (Zheng et al. 2017 ; Liang et al. 2015 ; Liu et al. 2003 ; Liu et al. 2009 ) due to the presence of unique cells that house two types of carbon-calcium crystals: CaCO 3 and Calcium oxalate (CaOX) (Katayama et al. 2007 ; Nagaoka et al. 2010 ; Wu et al. 2006 ). Scientific researches have shown that the presence of CaCO 3 crystals can stimulate the appetite of silkworms (Nagaoka et al. 2010 ). Furthermore, mulberry exhibits potential as an innovative source of animal feed or as a calcium supplement to meet the calcium requirements of animals. In 1839, Meyen made the first recorded observation of CaCO 3 crystals in Ficus elastica R. (Meyen 1839 ). These crystals form distinctive stalked bulbous structures that deposit in specific cells found in the leaves (Sugimura and Nitta 2007 ). In 1854, Weddel designated this unique structure “cystoliths”, while the specialized cells in which they develop were referred to as “lithocysts” (Weddell et al. 1854). Lithocysts exhibit clear differentiations from other plant cells, such as epidermal cells, projections, and parenchyma tissues (Nitta et al. 2006 ; Sugimura et al. 1999 ). The biomineralization process of CaCO 3 crystals relies on amorphous calcium carbonate (ACC) acting as a precursor substance, playing a crucial role in facilitating this process (Weiner et al. 2003 ; Su et al. 2013 ; Setoguchi et al. 1989 ). ACC serves as a transient storage and transportation form for CaCO 3 crystals due to its solubility, which is approximately ten times higher than that of CaCO 3 crystals (Levi-Kalisman et al. 2002 ; Sugimura and Nitta 2007 ; Weiner et al. 2003 ). Cystoliths are commonly observed in plant species of the Moraceae, Urticaceae, Ulmaceae, Euphorbiaceae, and Cannabaceae families (Setoguchi et al. 1989 ; Ajello 1941 ; Giannopoulos et al. 2019 ; Okazaki et al. 1991 ; Karabourniotis et al. 2020 ; Gal et al. 2012 ), particularly within the leaves of these plants. Since the early 20th century, researchers have extensively studied cystoliths using mulberry as a model system (Okazaki et al. 1986 ; Setoguchi et al. 1989 ; Watt et al. 1987 ). Mulberry leaves contain two distinct types of lithocysts, one with projection and the other without (Sugimura et al. 1999 ; Fujio 1971 ). The presence or absence of these lithocysts types has been correlated with the classification of mulberry species (Fujio 1971 ). In addition, extensive research has been conducted on the fundamental aspects of cystoliths. In the case of Morus alba cv. Minamisakari , the process of cystolith formation is initiated as the innermost cell wall separates from the cellular structure, gradually maturing into fully developed cystoliths (Sugimura and Nitta 2007 ). Cystoliths are formed through the interaction of CaCO 3 crystals with cellulose in the cell wall. During cystolith formation, pectin, another component of the cell wall, undergoes specific degradation and is ultimately absent in mature cystoliths (Katayama et al. 2008 ). Studies have revealed that the central regions of cystoliths consist of silicon dioxide (SiO 2 ), while the spheroid region primarily comprise CaCO 3 crystals, which accounts for up to 91.5% of their composition (Katayama et al. 2008 ; Nitta et al. 2006 ; Eschrich 1954 ). Histological experiments have shown that a single mature cystolith can accumulate approximately 48.8 ± 4.0 ng of calcium and around 120 ng of CaCO 3 crystals (Nitta et al. 2006 ; Sugimura et al. 1999 ; Sugimura and Nitta 2007 ). In the case of M. alba L. Minamisakari , cystoliths can reach a diameter of up to 40 µm. In their natural environment, fully mature leaves typically exhibit an average density of 23 cystoliths per square millimeter (Sugimura et al. 1999 ). A valuable model of cystoliths was established in 2006, providing researchers with a valuable tool for studying these unique structures (Nitta et al. 2006 ). The CaOX crystals in mulberry leaves, such as M. alba L. Minamisakari , typically exhibit a prismatic or druse-like morphology. It is worth noting that although the content of CaOX increases with leaf age, the overall mass of CaOX remains relatively constant per unit mass of mulberry leaf dry powder (Nagaoka et al. 2010 ). The presence of CaOX crystals is widespread in plants (Coté 2009 ; Mazen et al. 2004 ; Lersten and Horner 2011 ; He et al. 2014 ), observed in various photosynthetic tissues, including root, stem, leaf, flower, and seed (Franceschi and Horner 1980 ; Bouropoulos et al. 2001 ; Cerritos-Castro et al. 2022 ; Zhang et al. 2019 ; Franceschi and Nakata 2005 ). These crystals are primarily located within the vacuoles of specialized cells known as idioblasts (Cerritos-Castro et al. 2022 ; Zhang et al. 2019 ). As early as 2000, researchers established a connection between organismal calcification and carbon dioxide (CO 2 ) (Gattuso and Buddemeier 2000 ). This linkage revealed that the decomposition of CaOX crystals under conditions of carbon starvation from the atmosphere, induced by the phytohormone ABA or during drought, contributes to the generation of internal CO 2 . This internal CO 2 acts as a carbon source for plants and plays a vital role in photosynthesis, Tooulakou named this phenomenon alarm photosynthesis to describe the process of CaOX crystal dissolution (Tooulakou et al. 2016a , b ; Tooulakou et al. 2019 ). Researchers conducted cultivation experiments using various concentrations of calcium solution on Morus australis P., resulting in significant findings concerning the size of cystoliths and the average density of CaOX crystals (Wu et al. 2006 ). It has also been observed that the normal growth of mulberry is dependent on reaching a specific threshold of calcium concentration (Wu et al. 2006 ). Additionally, Werner noted in Morus niga L. and Ulmaceae species that epidermal cells surrounding normal lithocysts form small cystoliths, referred to as “Nebencystolithen” (Werner 1931 ). In recent years, hybrid mulberry, particularly Morus alba L. (Guisangyou12) variety, has emerged as a viable source of animal feed. This hybrid mulberry exhibits superior traits compared to its hybrid parents, including strong stress resistance, high yield, and excellent quality. Moreover, the widespread cultivation of hybrid mulberry, especially for herbaceous planting, enables mechanized harvesting while reducing labor and financial resources. Currently, the study of cystoliths in mulberry primarily relies on Japanese research using the M. alba L. Minamisakari variety as experimental material, which has not yet gained wide-scale planted. On the other hand, Guisangyou12 is an outstanding hybrid mulberry variety developed and promoted in China, with a cultivation area of 72,000 hectares, holding immense potential as an economically valuable plant species. Although significant physiological data exists regarding cystoliths in mulberry, there are still gaps in basic research in this area. This includes the unexplored influence of phytohormones on cystolith formation in mulberry and the sources of elements in cystoliths. Two phytohormones, ABA and 6-BA, have been found to significantly affect stomatal conductance in plant leaves. Therefore, this study aimed to investigate the spatial and temporal distribution patterns of cystoliths in mulberry leaves. Furthermore, it sought to explore the effects of different concentrations of ABA and 6-BA on cystolith formation using the experimental material of Guisangyou12 hybrid mulberry. The density and diameter of cystoliths was used as an indicator for detection. Conducting further fundamental research on mulberry cystoliths is crucial for the sustainable and beneficial utilization of mulberry resources. Materials and methods Plant materials and culture conditions Guisangyou12 ( M. alba ) hybrid mulberry grown in a natural environment were utilized to study the spatial and temporal distribution patterns of cystoliths in mulberry leaves for this research. Additional experimental materials were collected between August and October 2022. All plant materials were cultivated at the Mulberry Resource Center of Southwest University in Beibei, Chongqing, China. Prior to conducting experiments on effects of phytohormones and high calcium concentration on cystolith formation, Guisangyou12 hybrid mulberry seeds were stored in our laboratory and subjected to a meticulous cleaning process using aseptic water. Afterward, the seeds were placed on filter paper to remove excess moisture. Following this, they were soaked in aseptic water at 4°C for 48 hours before being transferred to sterile soil. The soil mixture consisted of a combination of humus soil, perlite, and vermiculite in a ratio of 5:1:1, respectively. During cultivation, the plants were exposed to a light intensity of 8000 lux, a temperature of 25°C, and a photoperiod set to a 16-hour light and 8-hour dark cycle. As shown in Fig. 3 a, leaf age was determined based on the length of the leaf, considering the 1st leaf age as when its length reached or exceeded 4.00 cm, and subsequent leaves were similarly categorized. Once the plants reached the 5th leaf stage, they were transferred to larger plastic pots, and cultivation was continued. Regular watering and fertilization practices were maintained throughout the entire growth period. Section preparation and observation The leaves were gently cleansed with water to remove any impurities, and excess moisture was carefully removed using filter paper. For sampling purposes, rectangular leaf sections measuring 0.8 cm in width and 3 cm in length were carefully taken from areas near the leaf veins, while leaf vein samples were collected 1 cm away from the petiole. Subsequently, the leaf samples were then immersed in a 5% para-formaldehyde solution (PFA) for 24 hours (48 hours for leaf veins) at 4°C. Afterward, any residual PFA was thoroughly rinsed off using a 0.1% phosphate-buffered saline solution (PBS). Subsequently, the samples were subjected to a dehydration process by gradually immersing them in ethanol, following a gradient starting from 50%, then progressing through 75%, 85%, 90%, 95%, and finally reaching 100%. Each step of the ethanol gradient lasted for 3 hours (5 hours for leaf veins) at a controlled temperature of 37°C. Finally, the samples were submerged in a mixture of 50% xylene and 50% ethanol, followed by pure xylene, for a period of 24 hours (48 hours for leaf veins) at room temperature. The samples were carefully transferred to embedding boxes containing a mixture of xylene and paraffin in varying proportions: 70% xylene + 30% paraffin, 30% xylene + 70% paraffin, and finally 100% paraffin. Each mixture was subjected to a soaking step at 70°C for 24 hours, ensuring complete infiltration of the plant tissue by the paraffin. The embedding process utilized clean and uncontaminated paraffin, and the samples were subsequently stored at -20°C. To obtain sections suitable for analysis, the embedded material was skillfully sectioned using a microtome (HM325 Thermo Fisher Scientific, Shanghai, China) to a thickness of 20 µm (10 µm for leaf veins). Following the sectioning process, the sections were flattened on deionized water at 37°C and carefully affixed to glass slides. Subsequently, the slides with sections were placed in a 70°C oven for 1.5 hours to facilitate secure adhesion. The sections were then subjected to deparaffinization by immersing them in xylene for 20 minutes, followed by a gradient of ethanol solutions (100%, 95%, 90%, 80%, 70%, and finally deionized water), with each step lasting 5 minutes. To eliminate any residual moisture, the slides were placed in a 37°C oven. The sections were swiftly stained with a 0.2% solution of Alizarin red S staining solution (ARS) pH 8.3 from Beyotime (Shanghai, China) for a duration of 8 minutes. Subsequently, the sections were rinsed until the glass slides were clean and free from excess stain. Once the staining process was complete, the sections were immediately examined under a Light Microscope (LM) (IX73 Olympus, Tokyo, Japan), where the CaCO 3 crystals appeared as purple-red structures. Histochemical localization of calcium deposition After the aforementioned pre-treatment of the leaves, the mulberry leaves underwent a meticulous process of dehydration and de-coloration until achieving a transparent white state. At this stage, the leaves were subjected to a 10-minute treatment with 5% Silver Nitrate Staining solution (SNS). Subsequently, the leaves were examined and photographed under a LM (IX73 Olympus, Tokyo, Japan) to visually detect the presence of CaCO 3 crystals, which were identifiable by their black appearance. Preparation and observation of mulberry leaf samples using SEM Following the leaf pre-processing as described in section “Section preparation and observation”, the leaves were carefully dried at 80°C until a constant weight was achieved. To facilitate observation, the samples were securely attached to a sample holder using conductive double-sided tape. The sample holder, with the mounted leaf samples, was subsequently introduced into a plasma sputtering device for the purpose of gold coating. Once the gold coating process was complete, the samples were positioned within a SEM (SU3500 Hitachi, Tokyo, Japan) for detailed observation and photography. Preparation of solutions and treatment procedure To initiate the experiment, precisely measured 0.5600 g of calcium chloride (CaCl 2 ) and subsequently diluted it with deionized water to a final volume of 1L, resulting in a 5 mM CaCl 2 solution. Furthermore, 13.22 mg of ABA and 11.24 mg of 6-BA were accurately measured and dissolved in 50 ml of ultra-pure water, producing a 1 mM solution. From this prepared solution, volumes of 0.25 ml, 0.50 ml, 2.50 ml, 5.00 ml, 12.50 ml, and 25.00 ml were extracted and then diluted with ultra-pure water to generate solutions with concentrations of 5 µM, 10 µM, 50 µM, 100 µM, 250 µM, and 500 µM, correspondingly. These solutions should be carefully sprayed onto all the leaves of the mulberry plant. This treatment regime is to be repeated every 24 hours for a total of 10 repetitions. Preparation and observation of stomata using light microscope After the application of phytohormones and obtaining the leaves, the abaxial surface of mulberry leaves was promptly and delicately peeled off using tweezers, aiming to acquire a single layer of epidermal cells to the greatest extent possible. Subsequently, the obtained sample was examined under a LM (IX73 Olympus, Tokyo, Japan). Data processing and analysis The ImageJ was used to count the total number of cystoliths in a defined unit field of view measuring 3.5 mm by 2.6 mm. For measuring the diameter of the cystoliths, the Olympus Olyvia was employed. To analyze the collected data and generate graphical representations, GraphPad Prism 9 was utilized. This comprehensive approach facilitated the processing, analysis, and visualization of data concerning cystoliths density and diameter. The results are reported as the mean ± standard deviation (SD). To determine the significant difference between two groups, one way analysis of variance (ANOVA) test followed by post hoc multiple comparison Bonferroni test were used. A p-value less than 0.05 was considered statistically significant. Results Two types of lithocysts were identified in mulberry leaves In order to observe these lithocysts, a set of methods was developed based on previous research (Nitta et al. 2006 ; Sugimura et al. 1999 ; Wu et al. 2006 ; Sugimura et al. 1998 ; Sugimura et al. 2001 ). SEM and plant tissue sections were utilized to examine the adaxial surface of various mulberry species. The SEM observations revealed that cystoliths containing both types of lithocysts were distinguishable from neighboring mulberry cells. In mature leaves of M. notabilis (Chuansang), lithocysts with projection were observed (Fig. 1 a-c), whereas lithocysts without projection were observed in M. alba (Husang32) (Fig. 1 d-f). Specifically, in Chuansang, the projection took the form of a hook-like structure (Fig. 1 a and b). Lithocysts extended above the adaxial side of leaf surface, connecting to the epidermal cells, while the cystoliths with an inconspicuous stalk appeared as a spherical structure (Fig. 1 c). Conversely, in Husang32, lithocysts still formed a cap-like structure on the leaf surface but lacked cystoliths projection (Fig. 1 c and e). In this case, the cystoliths were stalked spherical structures, with the lithocysts embedded within the leaf tissue, surrounded by both epidermal cells and palisade tissue (Fig. 1 f). Furthermore, the spherical regions of both cystoliths types were able to be stained purple-red with ARS, while the stalked regions remained unstained (Fig. 1 c and f). The presence of cystoliths in mulberry leaves varies according to their development stages and position To conduct morphological observations, the 1st to the 21th leaves (Fig. 3 a) of the mulberry of M. alba (Guisangyou12) were individually collected from their natural environment. In the young leaves, lithocysts were present but without any cystoliths (Fig. 2 a- 1 ). In the early stages, the lithocysts appeared as cap-like protrusions on the leaf surface (Fig. 2 a- 2 ). As the leaves mature and expand, the lithocysts underwent changes in shape. The cystoliths started to emerge as prominent tips, gradually growing into spherical structures within the lithocysts. They extended towards the central region of the lithocysts and started to develop stalks (Fig. 2 a- 3 ). Eventually, at the end of stalks, they continued to enlarge, filling the lithocysts and forming bulbous subcellular structures known as cystoliths (Fig. 2 a- 4 to a- 6 ). Cystoliths at different growth stages were observable on the 3rd (Fig. 2 b- 1 ) and 7th leaves (Fig. 2 b- 2 ). In the 3rd leaf, numerous lithocysts (Fig. 2 a- 2 to a- 4 ) in the early stage of cystolith growth were observed, alongside a few lithocysts without cystoliths (Fig. 2 a- 1 ) and more developed lithocysts (Fig. 2 a- 5 ). However, in the 7th leaf, no lithocysts in the early growth stage were detected, with nearly all lithocysts being relatively mature. Upon leaf maturation, the diameter of cystoliths gradually increased with age (Fig. 3 c). Notably, there was a significant difference in cystolith diameter between the 5th and 11th leaves. The smallest cystolith measured 16.79 µm, while the largest measured 55.59 µm. However, in the 20th and 21st leaves, the diameter range of cystolith was relatively narrow, with a minimum diameter of 68.24 µm and the maximum diameter of 86.98 µm (Fig. 3 c). The density of cystoliths exhibited a gradual increase from the 5th leaf to the 14th leaf, followed by a gradual decreased from the 14th leaf to the 21st leaf (Fig. 3 d). In the 14th leaf, the density of cystoliths reached its highest value at 15 per square millimeter, whereas the 21st leaf had a density of 11 per square millimeter (Fig. 3 d). Interestingly, in the 5th leaf, numerous lithocysts were observed that remained unstained by SNS. These lithocysts primarily existed at a considerable distance from the leaf veins, while almost all lithocysts near the leaf veins were stained by SNS (Fig. 3 b- 1 ). We cultivated mulberry using a 5 mM CaCl 2 solution to thoroughly investigate the presence of lithocysts without cystoliths in the 5th leaf grown in its natural environment (Fig. 3 b- 1 ). The results revealed that even in young leaves, lithocysts were stained with SNS, and some of cystoliths filled the entirety of the lithocysts (Fig. 4 b- 1 ). However, not all lithocysts exhibited this characteristic (Fig. 4 b- 2 ). Furthermore, we also conducted investigations on mature leaves from 20 different mulberry species in their natural growth environment. We found that cystoliths did not completely occupy the lithocysts (Supplemental Fig. 1). Additionally, the density of cystoliths decreased with the age of mulberry leaves cultured in a 5 mM CaCl 2 solution. Notably, the 3rd leaf exhibited the highest density with 23 cystoliths per unit area (Fig. 4 c). Moreover, we also observed the formation of Nebencystolithen, which are small cystoliths that appear near the lithocysts. These Nebencystolithen first became visible at the 6th leaf stage, and their density gradually increased with the leaf age (Fig. 4 a). The 9th leaf was chosen as the experimental material to investigate the variation in cystoliths density across different positions on the adaxial surface, based on their distance from leaf midvein (Fig. 5 a). The findings revealed that cystoliths density was highest and most stable in positions closer to the leaf midvein (Fig. 5 b). These results were consistent across other mulberry varieties, such as Guisangyou62, Kangqing283 × Kangqing10, and Yuesang51 (Supplemental Fig. 2). In addition, CaOX crystals were observed in the bundle sheath cells, arranged in straight lines (Fig. 5 c-d). Opposite effects of phytohormone 6-BA and ABA on mulberry cystoliths formation To investigate the influence of phytohormone on mulberry cystoliths formation, 3-month-old greenhouse-grown mulberry seedlings were subjected to varying concentrations of 6-BA and ABA. A rectangular sampling site measuring 0.8 cm in width and 3 cm in length was defined within a stable region near the leaf midvein (Fig. 5 a). The results indicated that as the concentration of ABA increased, plant growth was suppressed (Fig. 6 a and Supplemental Table 1), and cystoliths density and diameter decreased in both the 4th (Fig. 6 b and Supplemental Tables 2 and 3) and 6th (Fig. 6 c and Supplemental Tables 2 and 3) leaves. For the 6th leaf of mulberry, a concentration of 5 µM ABA did not have a discernible impact on cystoliths density. However, higher concentrations of ABA (10 µM to 500 µM) significantly inhibited cystoliths density (Fig. 6 b and Supplemental Table 2). Notably, the effect of ABA on the 6th leaf was more pronounced compared to the 4th leaf, except for 5 µM ABA concentration. Additionally, there was a concentration-dependent decrease in cystoliths density for the 4th leaf with increasing ABA concentrations (Supplemental Table 2). On the other hand, lower concentrations of 6-BA stimulated mulberry growth, with the most significant promotion observed at a concentration of 50 µM 6-BA. However, higher concentrations of 6-BA (250 µM and 500 µM) suppressed the growth of mulberry (Fig. 7 a and Supplemental Table 1). The density and diameter of cystoliths increased with increasing concentrations of 6-BA in both the 6th (Fig. 7 b and Supplemental Tables 2 and 3) and 8th (Fig. 7 c and Supplemental Tables 2 and 3) leaves. The effect of 6-BA on the 8th leaf was more significant compared to the 6th leaf, except for 500 µM 6-BA concentration (Supplemental Table 2). Importantly, significant changes in stomata were observed after the phytohormone treatment of mulberry leaves. ABA caused stomatal closure, while 6-BA induced stomatal opening (Supplemental Fig. 3). Discussions Regulation of cystolith formation in mulberry leaves Since the early 20th century, mulberry plants have served as a model organism for studying cystoliths. In this study, two types of cystoliths with and without projection across different mulberry varieties were observed. During leaf development in mulberry, cystoliths initiated their formation in young leaves and gradually matured within the lithocysts. As the leaves expanded, these cystoliths manifested as egg-shaped protrusions on the leaf surface. These findings are consistent with previous research conducted by Nitta, Fujio and Sugimura et al. (Nitta et al. 2006 ; Fujio 1971 ; Sugimura et al. 1999 ). Furthermore, interestingly, we observed few cystoliths present in the 1st to the 3rd leaves, and many lithocysts in the 5th leaf that remained unstained by SNS. This disparity can be attributed to the maturity of the leaves and the development of lithocysts within the mulberry foliage. It is possible that insufficient CaCO 3 crystals deposition within the lithocysts may be responsible for this phenomenon, as outlined by previous studies (Sugimura et al. 1999 ). It is worth noting that young leaves lack a well-established system for calcium deposition since they are heterotrophic and rely on carbohydrate import from other regions of the mulberry plant. Conversely, mature leaves are autotrophic and serve as the primary source for carbon metabolites and mineral transport (Fellows and Geiger 1974 ; Turgeon 1989 ; Giannopoulos et al. 2019 ; Sugimura et al. 1998 ). The size of the cystoliths exhibited variation based on the calcium content in the environment (Wu et al. 2006 ). Only in environments with exceptionally high calcium concentrations, partial cystoliths completely fill the lithocysts, resulting in the formation of Nebencystolithen in mature mulberry leaves (Okazaki 1984 ). In addition, the excessive accumulation of cystoliths in mulberry leaves could lead to pathological symptoms such as smoke spots on the surface of mulberry leaves, seriously affecting both the quality and yield of mulberry leaves (Liu et al. 2006 ). Overall, under normal growth conditions, cystoliths generally do not completely occupy the entire lithocysts. We hypothesize that the formation of cystoliths is strictly regulated by mulberry plant, as complete filling of the lithocysts could be detrimental to their growth and development. Apoplastic transport of calcium in mulberry cystoliths In the 9th leaf of mulberry, a higher density of cystoliths was observed in the vicinity of the leaf midvein compared to the leaf edge. Additionally, the presence of CaOX crystals was detected within the leaf veins. However, mulberry plants do not form CaOX crystals under conditions of very low calcium concentrations. In lithocysts lacking CaCO 3 crystals deposition, only stem-like structures were formed (Wu et al. 2006 ). It is well-established that CaCO 3 crystals is the main component of cystoliths, as indicated by previous studies (Nitta et al. 2006 ; Sugimura et al. 1999 ; Sugimura et al. 2001 ; Setoguchi et al. 1989 ). The transport of calcium from the environment into the plant occurs through the xylem and vascular bundles (Katayama et al. 2007 ; Gilliham et al. 2011 ; Franceschi 1989 ). This calcium is then transported into the lithocysts for cystolith formation. The transportation of calcium ion (Ca 2+ ), relies on driving forces such as transpiration pull (Broadley et al. 2003 ) and osmosis (Gilliham et al. 2011 ), facilitated by the vascular bundles. We then propose that cystoliths initially form near the leaf veins due to the transport of Ca 2+ through the xylem and vascular bundles. Carbon contribution to cystoliths from atmospheric CO 2 via stomatal pathways in mulberry Cystoliths primarily consist of CaCO 3 crystals, with a portion of the carbon potentially originating from atmospheric CO 2 (Fig. 8 ). Previous research has demonstrated that in various plants, the carbon found in CaOX crystals originates from the breakdown of ascorbic acid (Nakata 2003 ; Webb 1999 ; Nakata and McConn 2007 ). Interestingly, under conditions of carbon starvation, such as in Amaranthus hybridus L., CaOX crystals can even decompose to produce CO 2 as a means of fulfilling the plant’s most fundamental survival state, referred to as alarm photosynthesis (Tooulakou et al. 2016a , b ; Giannopoulos et al. 2019 ). In the context of this study, the application of ABA to mulberry leaves resulted in a decrease in cystolith density and diameter with increasing ABA concentration. Conversely, when 6-BA was applied to mulberry leaves, the opposite effect was observed. We proposed that the closure of stomata in the leaves was induced by ABA(Supplemental Fig. 3), as supported by previous studies (Tooulakou et al. 2016b ; Acharya 2009 ; Zhang You et al. 2023 ; Shen et al. 2006 ). As shown in Fig. 8 , this process of stomatal closure has implications for the carbon uptake of mulberry, and potentially indirectly impacts cystolith formation. It is possible that cystoliths in mulberry leaves undergo decomposition akin to alarm photosynthesis, providing a source of carbon to meet the minimum growth requirements of the mulberry. Despite strong inhibition of mulberry growth at high concentrations of the phytohormone ABA, the integrity of cystoliths within the leaves remains unaffected. Thus, it is likely that the carbon constituents found in mulberry cystoliths originate from endogenous sources within the plant, such as ascorbic acid. However, further experimental evidence is needed to support this hypothesis. Moreover, the formation and decomposition of these cystoliths are under strict regulation by mulberry plant itself. In contrast, the application of 6-BA, known for its ability to promote stomatal opening (Supplemental Fig. 3) (Acharya 2009 ; Farber et al. 2016 ; Song et al. 2006 ), exhibited the opposite effects, resulting in an increase in both cystolith density and diameter at higher 6-BA concentrations. It is worth noting that airborne CO 2 to form ACC, which subsequently dehydrates to form CaCO 3 crystals. Mulberry plants are widely recognized for their high rates of carbon fixation, and a portion of this carbon is involved in the formation of the cystoliths. Therefore, it is the specific presence of cystoliths in mulberry, rather than in many other plant species, that can be attributed to the presence of CaOX crystals. We postulate that the carbon within the mulberry cystoliths comes not only from internal sources but also potentially from atmospheric CO 2 absorbed through the stomata (Fig. 8 ). This dual source of carbon could potentially account for the presence of both CaOX crystals and CaCO 3 crystals in mulberry. However, it is important to note that this hypothesis remains tentative and necessitates further data and experimental confirmation. Conclusions In the present study, we examined the distribution patterns of cystoliths in mulberry plants, considering leaf position and age variations. Our findings indicate a strong correlation between cystolith growth and leaf maturity. Initially, cystoliths appear near the leaf veins and, under normal growth conditions, do not occupy the entire lithocysts. Notably, the formation of cystoliths in mulberry is influenced by the phytohormones ABA and 6-BA, which potentially impact the stomatal conductance of mulberry leaves. This research presents novel insights into the carbon source within cystoliths, thereby improving our understanding of the formation mechanisms underlying mulberry cystoliths and contributing to the optimal utilization of mulberry resources. Abbreviations CaCO 3 Calcium carbonate CO 2 Carbon dioxide SEM Scanning electron microscopy SNS Silver nitrate staining solution 6-BA 6-benzylaminopurine CaCl 2 Calcium chloride ABA Abscisic acid Ca 2+ Calcium ion CaOX Calcium oxalate PFA Para-formaldehyde solution ACC Amorphous calcium carbonate PBS Phosphate-buffered saline solution ARS Alizarin red S staining solution LM Light microscope Declarations Acknowledgements This work was supported by the research grants from the National Key R&D Program of China (Grant No. 2022YFD1201602), Innovation Research 2035 Pilot Plan of Southwest University (Grant SWU-XDZD22008), and the Chongqing Research Program of Basic Research and Frontier Technology (Grant No. cstc2021yszx-jcyj0004). Authorship contribution statement Chao Yang, Jiubo Liang and Ningjia He conceived and designed the project; Qi Zhang and Lin Chen performed the experiments; Jianglian Yuan and Peng Qian planted the mulberry seedlings; Chao Yang and Qi Zhang analyzed data; Chao Yang wrote the manuscript and Ningjia He revised the manuscript. All authors read and approved the manuscript. Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose. References Acharya BR, Assmann, Sarah M (2009) Hormone interactions in stomatal function. Plant Mol Biol 69 (4):451-462. https://doi.org/10.1007/s11103-008-9427-0 Ajello L (1941) Cytology and Cellular Interrelations of Cystolith Formation in Ficus elastica . Am J Bot 28 (7):589-594. https://doi.org/10.2307/2437007 Bouropoulos N, Weiner S, Addadi L (2001) Calcium oxalate crystals in tomato and tobacco plants: morphology and in vitro interactions of crystal-associated macromolecules. 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Food Sci 38:159-163. https://doi.org/10.7506/spkx1002-6630-201708025 Supplementary Files SupplementalData.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 20 Feb, 2024 Reviewers invited by journal 20 Feb, 2024 Editor invited by journal 19 Feb, 2024 Editor assigned by journal 17 Feb, 2024 First submitted to journal 14 Feb, 2024 Editorial decision: Major revisions 09 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3887434","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273947177,"identity":"c64b76b4-7122-4766-ae95-79bb08b312cc","order_by":0,"name":"Chao Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYLACxgYGBjZm5sMPPhjYyBGvhY+9Lc1wRkGaMfFa5HjOGEjzfDicSFC1wfGzh1/83GEnxyaRYGBsY8CcwMB++OgGvFrO5KVZ9p5JNgZqSXicY8CWx8CTlnYDr5YDOWbGjG3MiW0SCQeMcwx4ihkkeMzwazn/BqSlHqglsUHawgBIEtRyI8f4MWPb4cQ2nsMM0gwGBoS1SN54Y8bY23bcmI29jc2wxyDBmI2QX/jO5xh/+NlWLSffzP/5wY8//+X42Q8fw6tF4QADmwSKCBs+5SAg38DA/IGQolEwCkbBKBjhAADbmkrJJ/CdbQAAAABJRU5ErkJggg==","orcid":"","institution":"Southwest University","correspondingAuthor":true,"prefix":"","firstName":"Chao","middleName":"","lastName":"Yang","suffix":""},{"id":273947178,"identity":"14bedf37-cae6-4268-9853-6a567537c907","order_by":1,"name":"Qi Zhang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Zhang","suffix":""},{"id":273947179,"identity":"ac5bbf97-6205-46e3-b052-d9c6e2f49a75","order_by":2,"name":"Peng Qian","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Qian","suffix":""},{"id":273947180,"identity":"64406bcf-9ef0-4bbb-a2e3-f2b67ca1c532","order_by":3,"name":"Jiubo Liang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Jiubo","middleName":"","lastName":"Liang","suffix":""},{"id":273947181,"identity":"f6eaee93-3eb8-43af-bab7-a6cda45e80fa","order_by":4,"name":"Lin Chen","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Chen","suffix":""},{"id":273947182,"identity":"6c617c93-5d6f-45b2-99f3-4f8c3894d289","order_by":5,"name":"Jianglian Yuan","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Jianglian","middleName":"","lastName":"Yuan","suffix":""},{"id":273947183,"identity":"e7426c9f-fdcd-450a-977b-f72840f6d043","order_by":6,"name":"Ningjia He","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Ningjia","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2024-01-22 08:46:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3887434/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3887434/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51503623,"identity":"38f4d5e6-5ecd-4084-a6ca-147b713cc4a0","added_by":"auto","created_at":"2024-02-22 18:15:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":88305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of two types of lithocysts in mulberry. \u003c/strong\u003eLM and SEM images of cystoliths and lithocysts in mature mulberry leaves from \u003cem\u003eMorus alba\u003c/em\u003e (Husang32) and \u003cem\u003eMorus notabilis\u003c/em\u003e (Chuansang) grown in a natural environment. \u003cstrong\u003ea and b\u003c/strong\u003e SEM images illustrating lithocysts with projections in the leaves of Chuansang; \u003cstrong\u003ed and e\u003c/strong\u003e SEM images showing lithocysts without projections in the leaves of Husang32; \u003cstrong\u003ea and d\u003c/strong\u003e SEM images capturing the adaxial surface of mulberry leaves; \u003cstrong\u003eb and e\u003c/strong\u003e SEM images displaying cross-sectional views of mulberry leaves; \u003cstrong\u003ec and f\u003c/strong\u003e LM images presenting cross sections of mulberry leaves stained with ARS, where the purple-red areas indicate the presence of CaCO\u003csub\u003e3\u003c/sub\u003e crystals. The observed leaves were obtained from Husang32 (\u003cstrong\u003ec\u003c/strong\u003e) and Chuansang (\u003cstrong\u003ef\u003c/strong\u003e). CP, cystoliths with projection; S, stalk; C, cystoliths; L, lithocysts; EP, epidermal cells; P, palisade tissue; AD, adaxial surface; CA, cap-like protrusions; SP, spongy tissue; Scale bar = 50 µm\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887434/v1/7bf16ff33a515498ca418e61.jpg"},{"id":51503615,"identity":"f8d320ed-ccc2-47cc-a320-429e8ed290fb","added_by":"auto","created_at":"2024-02-22 18:15:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108093,"visible":true,"origin":"","legend":"\u003cp\u003eProcess of cystoliths formation in mulberry grown in a natural environment. a LM photographs presenting cross sections of mulberry leaves stained with ARS, where the purple-red areas indicate the presence of CaCO\u003csub\u003e3\u003c/sub\u003e crystals. a-1 Lithocysts without cystoliths exist in the leaf; a-2 Cystoliths form cap-like protrusion and stalks within lithocysts; a-3 Stalks extend towards the central region of lithocysts and cystoliths expand at the end of stalks; a-4 to 6 Cystoliths grow gradually within lithocysts. L, lithocysts; S, stalk; CA, cap-like protrusion; C, cystoliths. Scale bar = 100 µm; b Cystoliths localizations (indicated by black spots) within lithocysts stained with SNS in mulberry leaves at different stages: 3th (b-1), and 7th (b-2). The arrow in Fig. b corresponds to Fig. a. Scale bar = 200 µm\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887434/v1/f3d4b91aa04e3eaffa21bb6f.jpg"},{"id":51503618,"identity":"a462292e-2434-404d-bdae-a51ccfce87b8","added_by":"auto","created_at":"2024-02-22 18:15:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":69498,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in cystoliths density and diameter with leaf age in mulberry grown in a natural environment. a Overview of a mulberry seedling used in this study. The numbers shown in leaves denote their respective age; b Localization of cystoliths (indicated by black spots) within lithocysts stained with SNS in mulberry leaves at different stages: 5th (b-1), 12th (b-2), 18th (b-3), and 21th (b-4). The black arrows indicate lithocysts without accumulated CaCO\u003csub\u003e3\u003c/sub\u003e crystals in Fig. b-1. Scale bar = 500 µm; c and d Variation in cystoliths diameter (c) and density (d) of throughout leaf aging. Error bars represent the mean ± SD (c n = 12, n represents the number of cystoliths; d n = 7, n denotes the number of samples per unit area of cystoliths)\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887434/v1/d5ae0a15a28fb990277800bc.jpg"},{"id":51503612,"identity":"a7828eb2-bc5b-4ce3-abbd-2dc67681f963","added_by":"auto","created_at":"2024-02-22 18:15:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135247,"visible":true,"origin":"","legend":"\u003cp\u003eObservation of mulberry cultured in a 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e solution. a Localization of cystoliths (indicated by black spots) within lithocysts stained with SNS in mulberry leaves at different stages: 6th (a-1), 8th (a-2), 10th (a-3), and 12th (a-4). The formation of Nebencystolithen (marked by black arrows) occurs in the neighboring plant cells surrounding lithocysts. Scale bar = 200 µm; b LM photographs presenting cross sections of mulberry leaves stained with ARS, where the purple-red areas indicate the presence of CaCO\u003csub\u003e3\u003c/sub\u003e crystals. b-1 Cystoliths fill the entire lithocysts; b-2 Cystoliths are not fully covered with the entire lithocysts. Scale bar = 100 µm; c Variation in cystoliths density throughout leaf aging in mulberry. Error bars represent the mean ± SD (n = 7, n indicates the number of samples per unit area of cystoliths)\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887434/v1/d0ad5e16d861b118fe7110a2.jpg"},{"id":51503619,"identity":"4ef370cc-746e-4ac4-a8a3-fcca2ece0bb5","added_by":"auto","created_at":"2024-02-22 18:15:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":125439,"visible":true,"origin":"","legend":"\u003cp\u003eFormation of cystoliths in different parts of mulberry leaves and identification of CaOX crystals in the veins of mulberry leaves. a The four sampling locations on the 9th leaf of mulberry grown in a natural environment. Each rectangle measures 0.8 cm wide and 3 cm long, numbered as 1, 2, 3, and 4; b Variation in cystoliths density with leaf position, as shown in Fig. a; Error bars represent the mean ± SD (n = 7, n shows the number of samples per unit area of cystoliths). Significance differences were determined by one way analysis of variance (ANOVA) test followed by post hoc multiple comparison Bonferroni test. Different letters denote statistically significant differences between the means (P \u0026lt; 0.05); c and d LM photographs cross sections of mulberry leaf veins. The black arrows indicate the presence of CaOX crystals within bundle sheath cell. c Scale bar = 100 µm; d Scale bar = 20 µm\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887434/v1/8264c99875ced82e956c6318.jpg"},{"id":51503617,"identity":"1422ed74-6b60-42e0-a185-26ce7be63edb","added_by":"auto","created_at":"2024-02-22 18:15:21","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":72415,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of cystoliths formation in mulberry by ABA. a Images of mulberry treated with varying concentrations of ABA. Scale bar = 10 cm; b and c Variation in cystoliths density observed in the 4th (b) and 6th (c) leaves of mulberry; Error bars represent the mean ± SD (n = 26, n represents the number of samples per unit area of cystoliths). Significance differences were determined by one way analysis of variance (ANOVA) test followed by post hoc multiple comparison Bonferroni test. Different letters denote statistically significant differences between the means (P \u0026lt; 0.05\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887434/v1/34f110196c61945c6c228a06.jpg"},{"id":51503614,"identity":"e2b87c48-a6cf-4509-8db7-ceda4512d7d5","added_by":"auto","created_at":"2024-02-22 18:15:20","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":75227,"visible":true,"origin":"","legend":"\u003cp\u003ePromotion of cystoliths formation in mulberry by 6-BA. a Images of mulberry treated with varying concentrations of 6-BA. Scale bar = 10 cm; b and c Variation in cystoliths density observed in the 6th (b) and 8th (c) leaves of mulberry; Error bars represent the mean ± SD (n = 26, n indicates the number of samples per unit area of cystoliths). Significance differences were determined by one way analysis of variance (ANOVA) test followed by post hoc multiple comparison Bonferroni test. Different letters denote statistically significant differences between the means (P \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887434/v1/7958ae8fd60614c297c6b3cc.jpg"},{"id":51503622,"identity":"b4d73185-5ac4-472c-ab36-c61834c4d0ae","added_by":"auto","created_at":"2024-02-22 18:15:21","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":79146,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the influence of phytohormones 6-BA and ABA on the formation of cystoliths in mulberry leaves. The phytohormones 6-BA and ABA have distinct effects on stomatal regulation. ABA promotes stomatal closure, preventing the entry of atmospheric CO\u003csub\u003e2\u003c/sub\u003e through stomata and triggering a respiration process known as alarm photosynthesis in mulberry leaves (indicated by the red arrow). Meanwhile, CaCO\u003csub\u003e3\u003c/sub\u003e crystals are hydrolyzed to produce ACC, which is subsequently decomposed into CO\u003csub\u003e2\u003c/sub\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e. A portion of the CO\u003csub\u003e2\u003c/sub\u003e is transported to other plant cell to participate in photosynthesis; On the other hand, 6-BA facilitates the entry of atmospheric CO\u003csub\u003e2\u003c/sub\u003e into the leaves, where it combines with Ca\u003csup\u003e2+\u003c/sup\u003e to contribute to the formation of ACC and cystoliths (indicated by the black arrow)\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3887434/v1/84a63df34c71803d864b5389.jpg"},{"id":51504227,"identity":"fbb3892a-2ab7-4b8e-9a37-8024e15540d9","added_by":"auto","created_at":"2024-02-22 18:23:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":995646,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3887434/v1/e8183bf2-3d29-46fb-b3d9-0e7e7e32f8ac.pdf"},{"id":51503624,"identity":"aec244a4-9181-486f-bd80-890ba3bab221","added_by":"auto","created_at":"2024-02-22 18:15:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1387628,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalData.docx","url":"https://assets-eu.researchsquare.com/files/rs-3887434/v1/a518d158fe9335b57f120c25.docx"}],"financialInterests":"","formattedTitle":"Spatial and temporal distribution of cystoliths in mulberry leaves and their formation under the influence of phytohormones 6-BA and ABA","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMulberry (\u003cem\u003eMorus\u003c/em\u003e spp.) is a member of the Moraceae family in the order Rosaceae. It is widely recognized as the main food source for silkworms (\u003cem\u003eBombyx mori\u003c/em\u003e), playing a crucial role in the sericulture industry and contributing substantially to the economy of various countries. Mulberry leaves are rich in proteins and carbohydrate, both essential for the growth and development of silkworms. Moreover, mulberry leaves are exceptionally rich in calcium (Zheng et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liang et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) due to the presence of unique cells that house two types of carbon-calcium crystals: CaCO\u003csub\u003e3\u003c/sub\u003e and Calcium oxalate (CaOX) (Katayama et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Nagaoka et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Scientific researches have shown that the presence of CaCO\u003csub\u003e3\u003c/sub\u003e crystals can stimulate the appetite of silkworms (Nagaoka et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Furthermore, mulberry exhibits potential as an innovative source of animal feed or as a calcium supplement to meet the calcium requirements of animals.\u003c/p\u003e \u003cp\u003eIn 1839, Meyen made the first recorded observation of CaCO\u003csub\u003e3\u003c/sub\u003e crystals in \u003cem\u003eFicus elastica\u003c/em\u003e R. (Meyen \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1839\u003c/span\u003e). These crystals form distinctive stalked bulbous structures that deposit in specific cells found in the leaves (Sugimura and Nitta \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In 1854, Weddel designated this unique structure \u0026ldquo;cystoliths\u0026rdquo;, while the specialized cells in which they develop were referred to as \u0026ldquo;lithocysts\u0026rdquo; (Weddell et al. 1854). Lithocysts exhibit clear differentiations from other plant cells, such as epidermal cells, projections, and parenchyma tissues (Nitta et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sugimura et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The biomineralization process of CaCO\u003csub\u003e3\u003c/sub\u003e crystals relies on amorphous calcium carbonate (ACC) acting as a precursor substance, playing a crucial role in facilitating this process (Weiner et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Setoguchi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). ACC serves as a transient storage and transportation form for CaCO\u003csub\u003e3\u003c/sub\u003e crystals due to its solubility, which is approximately ten times higher than that of CaCO\u003csub\u003e3\u003c/sub\u003e crystals (Levi-Kalisman et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Sugimura and Nitta \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Weiner et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Cystoliths are commonly observed in plant species of the Moraceae, Urticaceae, Ulmaceae, Euphorbiaceae, and Cannabaceae families (Setoguchi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Ajello \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1941\u003c/span\u003e; Giannopoulos et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Okazaki et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Karabourniotis et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), particularly within the leaves of these plants. Since the early 20th century, researchers have extensively studied cystoliths using mulberry as a model system (Okazaki et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Setoguchi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Watt et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1987\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMulberry leaves contain two distinct types of lithocysts, one with projection and the other without (Sugimura et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Fujio \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). The presence or absence of these lithocysts types has been correlated with the classification of mulberry species (Fujio \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). In addition, extensive research has been conducted on the fundamental aspects of cystoliths. In the case of \u003cem\u003eMorus alba cv. Minamisakari\u003c/em\u003e, the process of cystolith formation is initiated as the innermost cell wall separates from the cellular structure, gradually maturing into fully developed cystoliths (Sugimura and Nitta \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Cystoliths are formed through the interaction of CaCO\u003csub\u003e3\u003c/sub\u003e crystals with cellulose in the cell wall. During cystolith formation, pectin, another component of the cell wall, undergoes specific degradation and is ultimately absent in mature cystoliths (Katayama et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Studies have revealed that the central regions of cystoliths consist of silicon dioxide (SiO\u003csub\u003e2\u003c/sub\u003e), while the spheroid region primarily comprise CaCO\u003csub\u003e3\u003c/sub\u003e crystals, which accounts for up to 91.5% of their composition (Katayama et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Nitta et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Eschrich \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1954\u003c/span\u003e). Histological experiments have shown that a single mature cystolith can accumulate approximately 48.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 ng of calcium and around 120 ng of CaCO\u003csub\u003e3\u003c/sub\u003e crystals (Nitta et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sugimura et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Sugimura and Nitta \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In the case of \u003cem\u003eM. alba L. Minamisakari\u003c/em\u003e, cystoliths can reach a diameter of up to 40 \u0026micro;m. In their natural environment, fully mature leaves typically exhibit an average density of 23 cystoliths per square millimeter (Sugimura et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). A valuable model of cystoliths was established in 2006, providing researchers with a valuable tool for studying these unique structures (Nitta et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe CaOX crystals in mulberry leaves, such as \u003cem\u003eM. alba L. Minamisakari\u003c/em\u003e, typically exhibit a prismatic or druse-like morphology. It is worth noting that although the content of CaOX increases with leaf age, the overall mass of CaOX remains relatively constant per unit mass of mulberry leaf dry powder (Nagaoka et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The presence of CaOX crystals is widespread in plants (Cot\u0026eacute; \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mazen et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Lersten and Horner \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; He et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), observed in various photosynthetic tissues, including root, stem, leaf, flower, and seed (Franceschi and Horner \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Bouropoulos et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Cerritos-Castro et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Franceschi and Nakata \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). These crystals are primarily located within the vacuoles of specialized cells known as idioblasts (Cerritos-Castro et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As early as 2000, researchers established a connection between organismal calcification and carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) (Gattuso and Buddemeier \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). This linkage revealed that the decomposition of CaOX crystals under conditions of carbon starvation from the atmosphere, induced by the phytohormone ABA or during drought, contributes to the generation of internal CO\u003csub\u003e2\u003c/sub\u003e. This internal CO\u003csub\u003e2\u003c/sub\u003e acts as a carbon source for plants and plays a vital role in photosynthesis, Tooulakou named this phenomenon alarm photosynthesis to describe the process of CaOX crystal dissolution (Tooulakou et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Tooulakou et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResearchers conducted cultivation experiments using various concentrations of calcium solution on \u003cem\u003eMorus australis\u003c/em\u003e P., resulting in significant findings concerning the size of cystoliths and the average density of CaOX crystals (Wu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). It has also been observed that the normal growth of mulberry is dependent on reaching a specific threshold of calcium concentration (Wu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Additionally, Werner noted in \u003cem\u003eMorus niga\u003c/em\u003e L. and Ulmaceae species that epidermal cells surrounding normal lithocysts form small cystoliths, referred to as \u0026ldquo;Nebencystolithen\u0026rdquo; (Werner \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1931\u003c/span\u003e). In recent years, hybrid mulberry, particularly \u003cem\u003eMorus alba\u003c/em\u003e L. (Guisangyou12) variety, has emerged as a viable source of animal feed. This hybrid mulberry exhibits superior traits compared to its hybrid parents, including strong stress resistance, high yield, and excellent quality. Moreover, the widespread cultivation of hybrid mulberry, especially for herbaceous planting, enables mechanized harvesting while reducing labor and financial resources. Currently, the study of cystoliths in mulberry primarily relies on Japanese research using the \u003cem\u003eM. alba L. Minamisakari\u003c/em\u003e variety as experimental material, which has not yet gained wide-scale planted. On the other hand, Guisangyou12 is an outstanding hybrid mulberry variety developed and promoted in China, with a cultivation area of 72,000 hectares, holding immense potential as an economically valuable plant species.\u003c/p\u003e \u003cp\u003eAlthough significant physiological data exists regarding cystoliths in mulberry, there are still gaps in basic research in this area. This includes the unexplored influence of phytohormones on cystolith formation in mulberry and the sources of elements in cystoliths. Two phytohormones, ABA and 6-BA, have been found to significantly affect stomatal conductance in plant leaves. Therefore, this study aimed to investigate the spatial and temporal distribution patterns of cystoliths in mulberry leaves. Furthermore, it sought to explore the effects of different concentrations of ABA and 6-BA on cystolith formation using the experimental material of Guisangyou12 hybrid mulberry. The density and diameter of cystoliths was used as an indicator for detection. Conducting further fundamental research on mulberry cystoliths is crucial for the sustainable and beneficial utilization of mulberry resources.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and culture conditions\u003c/h2\u003e \u003cp\u003eGuisangyou12 (\u003cem\u003eM. alba\u003c/em\u003e) hybrid mulberry grown in a natural environment were utilized to study the spatial and temporal distribution patterns of cystoliths in mulberry leaves for this research. Additional experimental materials were collected between August and October 2022. All plant materials were cultivated at the Mulberry Resource Center of Southwest University in Beibei, Chongqing, China. Prior to conducting experiments on effects of phytohormones and high calcium concentration on cystolith formation, Guisangyou12 hybrid mulberry seeds were stored in our laboratory and subjected to a meticulous cleaning process using aseptic water. Afterward, the seeds were placed on filter paper to remove excess moisture. Following this, they were soaked in aseptic water at 4\u0026deg;C for 48 hours before being transferred to sterile soil. The soil mixture consisted of a combination of humus soil, perlite, and vermiculite in a ratio of 5:1:1, respectively. During cultivation, the plants were exposed to a light intensity of 8000 lux, a temperature of 25\u0026deg;C, and a photoperiod set to a 16-hour light and 8-hour dark cycle. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, leaf age was determined based on the length of the leaf, considering the 1st leaf age as when its length reached or exceeded 4.00 cm, and subsequent leaves were similarly categorized. Once the plants reached the 5th leaf stage, they were transferred to larger plastic pots, and cultivation was continued. Regular watering and fertilization practices were maintained throughout the entire growth period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSection preparation and observation\u003c/h2\u003e \u003cp\u003eThe leaves were gently cleansed with water to remove any impurities, and excess moisture was carefully removed using filter paper. For sampling purposes, rectangular leaf sections measuring 0.8 cm in width and 3 cm in length were carefully taken from areas near the leaf veins, while leaf vein samples were collected 1 cm away from the petiole. Subsequently, the leaf samples were then immersed in a 5% para-formaldehyde solution (PFA) for 24 hours (48 hours for leaf veins) at 4\u0026deg;C. Afterward, any residual PFA was thoroughly rinsed off using a 0.1% phosphate-buffered saline solution (PBS). Subsequently, the samples were subjected to a dehydration process by gradually immersing them in ethanol, following a gradient starting from 50%, then progressing through 75%, 85%, 90%, 95%, and finally reaching 100%. Each step of the ethanol gradient lasted for 3 hours (5 hours for leaf veins) at a controlled temperature of 37\u0026deg;C. Finally, the samples were submerged in a mixture of 50% xylene and 50% ethanol, followed by pure xylene, for a period of 24 hours (48 hours for leaf veins) at room temperature. The samples were carefully transferred to embedding boxes containing a mixture of xylene and paraffin in varying proportions: 70% xylene\u0026thinsp;+\u0026thinsp;30% paraffin, 30% xylene\u0026thinsp;+\u0026thinsp;70% paraffin, and finally 100% paraffin. Each mixture was subjected to a soaking step at 70\u0026deg;C for 24 hours, ensuring complete infiltration of the plant tissue by the paraffin. The embedding process utilized clean and uncontaminated paraffin, and the samples were subsequently stored at -20\u0026deg;C. To obtain sections suitable for analysis, the embedded material was skillfully sectioned using a microtome (HM325 Thermo Fisher Scientific, Shanghai, China) to a thickness of 20 \u0026micro;m (10 \u0026micro;m for leaf veins). Following the sectioning process, the sections were flattened on deionized water at 37\u0026deg;C and carefully affixed to glass slides. Subsequently, the slides with sections were placed in a 70\u0026deg;C oven for 1.5 hours to facilitate secure adhesion. The sections were then subjected to deparaffinization by immersing them in xylene for 20 minutes, followed by a gradient of ethanol solutions (100%, 95%, 90%, 80%, 70%, and finally deionized water), with each step lasting 5 minutes. To eliminate any residual moisture, the slides were placed in a 37\u0026deg;C oven. The sections were swiftly stained with a 0.2% solution of Alizarin red S staining solution (ARS) pH 8.3 from Beyotime (Shanghai, China) for a duration of 8 minutes. Subsequently, the sections were rinsed until the glass slides were clean and free from excess stain. Once the staining process was complete, the sections were immediately examined under a Light Microscope (LM) (IX73 Olympus, Tokyo, Japan), where the CaCO\u003csub\u003e3\u003c/sub\u003e crystals appeared as purple-red structures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHistochemical localization of calcium deposition\u003c/h2\u003e \u003cp\u003eAfter the aforementioned pre-treatment of the leaves, the mulberry leaves underwent a meticulous process of dehydration and de-coloration until achieving a transparent white state. At this stage, the leaves were subjected to a 10-minute treatment with 5% Silver Nitrate Staining solution (SNS). Subsequently, the leaves were examined and photographed under a LM (IX73 Olympus, Tokyo, Japan) to visually detect the presence of CaCO\u003csub\u003e3\u003c/sub\u003e crystals, which were identifiable by their black appearance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and observation of mulberry leaf samples using SEM\u003c/h2\u003e \u003cp\u003eFollowing the leaf pre-processing as described in section \u0026ldquo;Section preparation and observation\u0026rdquo;, the leaves were carefully dried at 80\u0026deg;C until a constant weight was achieved. To facilitate observation, the samples were securely attached to a sample holder using conductive double-sided tape. The sample holder, with the mounted leaf samples, was subsequently introduced into a plasma sputtering device for the purpose of gold coating. Once the gold coating process was complete, the samples were positioned within a SEM (SU3500 Hitachi, Tokyo, Japan) for detailed observation and photography.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of solutions and treatment procedure\u003c/h2\u003e \u003cp\u003eTo initiate the experiment, precisely measured 0.5600 g of calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e) and subsequently diluted it with deionized water to a final volume of 1L, resulting in a 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e solution. Furthermore, 13.22 mg of ABA and 11.24 mg of 6-BA were accurately measured and dissolved in 50 ml of ultra-pure water, producing a 1 mM solution. From this prepared solution, volumes of 0.25 ml, 0.50 ml, 2.50 ml, 5.00 ml, 12.50 ml, and 25.00 ml were extracted and then diluted with ultra-pure water to generate solutions with concentrations of 5 \u0026micro;M, 10 \u0026micro;M, 50 \u0026micro;M, 100 \u0026micro;M, 250 \u0026micro;M, and 500 \u0026micro;M, correspondingly. These solutions should be carefully sprayed onto all the leaves of the mulberry plant. This treatment regime is to be repeated every 24 hours for a total of 10 repetitions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and observation of stomata using light microscope\u003c/h2\u003e \u003cp\u003eAfter the application of phytohormones and obtaining the leaves, the abaxial surface of mulberry leaves was promptly and delicately peeled off using tweezers, aiming to acquire a single layer of epidermal cells to the greatest extent possible. Subsequently, the obtained sample was examined under a LM (IX73 Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData processing and analysis\u003c/h2\u003e \u003cp\u003eThe ImageJ was used to count the total number of cystoliths in a defined unit field of view measuring 3.5 mm by 2.6 mm. For measuring the diameter of the cystoliths, the Olympus Olyvia was employed. To analyze the collected data and generate graphical representations, GraphPad Prism 9 was utilized. This comprehensive approach facilitated the processing, analysis, and visualization of data concerning cystoliths density and diameter. The results are reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). To determine the significant difference between two groups, one way analysis of variance (ANOVA) test followed by post hoc multiple comparison Bonferroni test were used. A p-value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTwo types of lithocysts were identified in mulberry leaves\u003c/h2\u003e \u003cp\u003eIn order to observe these lithocysts, a set of methods was developed based on previous research (Nitta et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sugimura et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sugimura et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Sugimura et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). SEM and plant tissue sections were utilized to examine the adaxial surface of various mulberry species. The SEM observations revealed that cystoliths containing both types of lithocysts were distinguishable from neighboring mulberry cells. In mature leaves of \u003cem\u003eM. notabilis\u003c/em\u003e (Chuansang), lithocysts with projection were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c), whereas lithocysts without projection were observed in \u003cem\u003eM. alba\u003c/em\u003e (Husang32) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f). Specifically, in Chuansang, the projection took the form of a hook-like structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b). Lithocysts extended above the adaxial side of leaf surface, connecting to the epidermal cells, while the cystoliths with an inconspicuous stalk appeared as a spherical structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Conversely, in Husang32, lithocysts still formed a cap-like structure on the leaf surface but lacked cystoliths projection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and e). In this case, the cystoliths were stalked spherical structures, with the lithocysts embedded within the leaf tissue, surrounded by both epidermal cells and palisade tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Furthermore, the spherical regions of both cystoliths types were able to be stained purple-red with ARS, while the stalked regions remained unstained (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe presence of cystoliths in mulberry leaves varies according to their development stages and position\u003c/h2\u003e \u003cp\u003eTo conduct morphological observations, the 1st to the 21th leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) of the mulberry of \u003cem\u003eM. alba\u003c/em\u003e (Guisangyou12) were individually collected from their natural environment. In the young leaves, lithocysts were present but without any cystoliths (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the early stages, the lithocysts appeared as cap-like protrusions on the leaf surface (Fig.\u0026nbsp;\u0026lt;link rid=\"fig3\"\u0026gt;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u0026lt;/link\u0026gt;\u003c/span\u003ea-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As the leaves mature and expand, the lithocysts underwent changes in shape. The cystoliths started to emerge as prominent tips, gradually growing into spherical structures within the lithocysts. They extended towards the central region of the lithocysts and started to develop stalks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Eventually, at the end of stalks, they continued to enlarge, filling the lithocysts and forming bulbous subcellular structures known as cystoliths (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e to a-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Cystoliths at different growth stages were observable on the 3rd (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and 7th leaves (Fig.\u0026nbsp;\u0026lt;link rid=\"fig3\"\u0026gt;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u0026lt;/link\u0026gt;\u003c/span\u003eb-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the 3rd leaf, numerous lithocysts (Fig.\u0026nbsp;\u0026lt;link rid=\"fig3\"\u0026gt;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u0026lt;/link\u0026gt;\u003c/span\u003ea-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e to a-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) in the early stage of cystolith growth were observed, alongside a few lithocysts without cystoliths (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and more developed lithocysts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, in the 7th leaf, no lithocysts in the early growth stage were detected, with nearly all lithocysts being relatively mature.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon leaf maturation, the diameter of cystoliths gradually increased with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Notably, there was a significant difference in cystolith diameter between the 5th and 11th leaves. The smallest cystolith measured 16.79 \u0026micro;m, while the largest measured 55.59 \u0026micro;m. However, in the 20th and 21st leaves, the diameter range of cystolith was relatively narrow, with a minimum diameter of 68.24 \u0026micro;m and the maximum diameter of 86.98 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The density of cystoliths exhibited a gradual increase from the 5th leaf to the 14th leaf, followed by a gradual decreased from the 14th leaf to the 21st leaf (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In the 14th leaf, the density of cystoliths reached its highest value at 15 per square millimeter, whereas the 21st leaf had a density of 11 per square millimeter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Interestingly, in the 5th leaf, numerous lithocysts were observed that remained unstained by SNS. These lithocysts primarily existed at a considerable distance from the leaf veins, while almost all lithocysts near the leaf veins were stained by SNS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe cultivated mulberry using a 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e solution to thoroughly investigate the presence of lithocysts without cystoliths in the 5th leaf grown in its natural environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results revealed that even in young leaves, lithocysts were stained with SNS, and some of cystoliths filled the entirety of the lithocysts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, not all lithocysts exhibited this characteristic (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, we also conducted investigations on mature leaves from 20 different mulberry species in their natural growth environment. We found that cystoliths did not completely occupy the lithocysts (Supplemental Fig.\u0026nbsp;1). Additionally, the density of cystoliths decreased with the age of mulberry leaves cultured in a 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e solution. Notably, the 3rd leaf exhibited the highest density with 23 cystoliths per unit area (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Moreover, we also observed the formation of Nebencystolithen, which are small cystoliths that appear near the lithocysts. These Nebencystolithen first became visible at the 6th leaf stage, and their density gradually increased with the leaf age (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThe 9th leaf was chosen as the experimental material to investigate the variation in cystoliths density across different positions on the adaxial surface, based on their distance from leaf midvein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The findings revealed that cystoliths density was highest and most stable in positions closer to the leaf midvein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). These results were consistent across other mulberry varieties, such as Guisangyou62, Kangqing283 \u0026times; Kangqing10, and Yuesang51 (Supplemental Fig.\u0026nbsp;2). In addition, CaOX crystals were observed in the bundle sheath cells, arranged in straight lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOpposite effects of phytohormone 6-BA and ABA on mulberry cystoliths formation\u003c/h2\u003e \u003cp\u003eTo investigate the influence of phytohormone on mulberry cystoliths formation, 3-month-old greenhouse-grown mulberry seedlings were subjected to varying concentrations of 6-BA and ABA. A rectangular sampling site measuring 0.8 cm in width and 3 cm in length was defined within a stable region near the leaf midvein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The results indicated that as the concentration of ABA increased, plant growth was suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and Supplemental Table\u0026nbsp;1), and cystoliths density and diameter decreased in both the 4th (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and Supplemental Tables\u0026nbsp;2 and 3) and 6th (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec and Supplemental Tables\u0026nbsp;2 and 3) leaves. For the 6th leaf of mulberry, a concentration of 5 \u0026micro;M ABA did not have a discernible impact on cystoliths density. However, higher concentrations of ABA (10 \u0026micro;M to 500 \u0026micro;M) significantly inhibited cystoliths density (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and Supplemental Table\u0026nbsp;2). Notably, the effect of ABA on the 6th leaf was more pronounced compared to the 4th leaf, except for 5 \u0026micro;M ABA concentration. Additionally, there was a concentration-dependent decrease in cystoliths density for the 4th leaf with increasing ABA concentrations (Supplemental Table\u0026nbsp;2). On the other hand, lower concentrations of 6-BA stimulated mulberry growth, with the most significant promotion observed at a concentration of 50 \u0026micro;M 6-BA. However, higher concentrations of 6-BA (250 \u0026micro;M and 500 \u0026micro;M) suppressed the growth of mulberry (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and Supplemental Table\u0026nbsp;1). The density and diameter of cystoliths increased with increasing concentrations of 6-BA in both the 6th (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb and Supplemental Tables\u0026nbsp;2 and 3) and 8th (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and Supplemental Tables\u0026nbsp;2 and 3) leaves. The effect of 6-BA on the 8th leaf was more significant compared to the 6th leaf, except for 500 \u0026micro;M 6-BA concentration (Supplemental Table\u0026nbsp;2). Importantly, significant changes in stomata were observed after the phytohormone treatment of mulberry leaves. ABA caused stomatal closure, while 6-BA induced stomatal opening (Supplemental Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussions","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRegulation of cystolith formation in mulberry leaves\u003c/h2\u003e \u003cp\u003eSince the early 20th century, mulberry plants have served as a model organism for studying cystoliths. In this study, two types of cystoliths with and without projection across different mulberry varieties were observed. During leaf development in mulberry, cystoliths initiated their formation in young leaves and gradually matured within the lithocysts. As the leaves expanded, these cystoliths manifested as egg-shaped protrusions on the leaf surface. These findings are consistent with previous research conducted by Nitta, Fujio and Sugimura et al. (Nitta et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Fujio \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Sugimura et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, interestingly, we observed few cystoliths present in the 1st to the 3rd leaves, and many lithocysts in the 5th leaf that remained unstained by SNS. This disparity can be attributed to the maturity of the leaves and the development of lithocysts within the mulberry foliage. It is possible that insufficient CaCO\u003csub\u003e3\u003c/sub\u003e crystals deposition within the lithocysts may be responsible for this phenomenon, as outlined by previous studies (Sugimura et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). It is worth noting that young leaves lack a well-established system for calcium deposition since they are heterotrophic and rely on carbohydrate import from other regions of the mulberry plant. Conversely, mature leaves are autotrophic and serve as the primary source for carbon metabolites and mineral transport (Fellows and Geiger \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Turgeon \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Giannopoulos et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sugimura et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe size of the cystoliths exhibited variation based on the calcium content in the environment (Wu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Only in environments with exceptionally high calcium concentrations, partial cystoliths completely fill the lithocysts, resulting in the formation of Nebencystolithen in mature mulberry leaves (Okazaki \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). In addition, the excessive accumulation of cystoliths in mulberry leaves could lead to pathological symptoms such as smoke spots on the surface of mulberry leaves, seriously affecting both the quality and yield of mulberry leaves (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverall, under normal growth conditions, cystoliths generally do not completely occupy the entire lithocysts. We hypothesize that the formation of cystoliths is strictly regulated by mulberry plant, as complete filling of the lithocysts could be detrimental to their growth and development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eApoplastic transport of calcium in mulberry cystoliths\u003c/h2\u003e \u003cp\u003eIn the 9th leaf of mulberry, a higher density of cystoliths was observed in the vicinity of the leaf midvein compared to the leaf edge. Additionally, the presence of CaOX crystals was detected within the leaf veins. However, mulberry plants do not form CaOX crystals under conditions of very low calcium concentrations. In lithocysts lacking CaCO\u003csub\u003e3\u003c/sub\u003e crystals deposition, only stem-like structures were formed (Wu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). It is well-established that CaCO\u003csub\u003e3\u003c/sub\u003e crystals is the main component of cystoliths, as indicated by previous studies (Nitta et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sugimura et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Sugimura et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Setoguchi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). The transport of calcium from the environment into the plant occurs through the xylem and vascular bundles (Katayama et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Gilliham et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Franceschi \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). This calcium is then transported into the lithocysts for cystolith formation. The transportation of calcium ion (Ca\u003csup\u003e2+\u003c/sup\u003e), relies on driving forces such as transpiration pull (Broadley et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and osmosis (Gilliham et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), facilitated by the vascular bundles. We then propose that cystoliths initially form near the leaf veins due to the transport of Ca\u003csup\u003e2+\u003c/sup\u003e through the xylem and vascular bundles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCarbon contribution to cystoliths from atmospheric CO\u003csub\u003e2\u003c/sub\u003e via stomatal pathways in mulberry\u003c/h2\u003e \u003cp\u003eCystoliths primarily consist of CaCO\u003csub\u003e3\u003c/sub\u003e crystals, with a portion of the carbon potentially originating from atmospheric CO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Previous research has demonstrated that in various plants, the carbon found in CaOX crystals originates from the breakdown of ascorbic acid (Nakata \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Webb \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Nakata and McConn \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Interestingly, under conditions of carbon starvation, such as in \u003cem\u003eAmaranthus hybridus\u003c/em\u003e L., CaOX crystals can even decompose to produce CO\u003csub\u003e2\u003c/sub\u003e as a means of fulfilling the plant\u0026rsquo;s most fundamental survival state, referred to as alarm photosynthesis (Tooulakou et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Giannopoulos et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the context of this study, the application of ABA to mulberry leaves resulted in a decrease in cystolith density and diameter with increasing ABA concentration. Conversely, when 6-BA was applied to mulberry leaves, the opposite effect was observed. We proposed that the closure of stomata in the leaves was induced by ABA(Supplemental Fig.\u0026nbsp;3), as supported by previous studies (Tooulakou et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e; Acharya \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zhang You et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, this process of stomatal closure has implications for the carbon uptake of mulberry, and potentially indirectly impacts cystolith formation. It is possible that cystoliths in mulberry leaves undergo decomposition akin to alarm photosynthesis, providing a source of carbon to meet the minimum growth requirements of the mulberry. Despite strong inhibition of mulberry growth at high concentrations of the phytohormone ABA, the integrity of cystoliths within the leaves remains unaffected. Thus, it is likely that the carbon constituents found in mulberry cystoliths originate from endogenous sources within the plant, such as ascorbic acid. However, further experimental evidence is needed to support this hypothesis. Moreover, the formation and decomposition of these cystoliths are under strict regulation by mulberry plant itself.\u003c/p\u003e \u003cp\u003eIn contrast, the application of 6-BA, known for its ability to promote stomatal opening (Supplemental Fig.\u0026nbsp;3) (Acharya \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Farber et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), exhibited the opposite effects, resulting in an increase in both cystolith density and diameter at higher 6-BA concentrations. It is worth noting that airborne CO\u003csub\u003e2\u003c/sub\u003e to form ACC, which subsequently dehydrates to form CaCO\u003csub\u003e3\u003c/sub\u003e crystals. Mulberry plants are widely recognized for their high rates of carbon fixation, and a portion of this carbon is involved in the formation of the cystoliths. Therefore, it is the specific presence of cystoliths in mulberry, rather than in many other plant species, that can be attributed to the presence of CaOX crystals.\u003c/p\u003e \u003cp\u003eWe postulate that the carbon within the mulberry cystoliths comes not only from internal sources but also potentially from atmospheric CO\u003csub\u003e2\u003c/sub\u003e absorbed through the stomata (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This dual source of carbon could potentially account for the presence of both CaOX crystals and CaCO\u003csub\u003e3\u003c/sub\u003e crystals in mulberry. However, it is important to note that this hypothesis remains tentative and necessitates further data and experimental confirmation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, we examined the distribution patterns of cystoliths in mulberry plants, considering leaf position and age variations. Our findings indicate a strong correlation between cystolith growth and leaf maturity. Initially, cystoliths appear near the leaf veins and, under normal growth conditions, do not occupy the entire lithocysts. Notably, the formation of cystoliths in mulberry is influenced by the phytohormones ABA and 6-BA, which potentially impact the stomatal conductance of mulberry leaves. This research presents novel insights into the carbon source within cystoliths, thereby improving our understanding of the formation mechanisms underlying mulberry cystoliths and contributing to the optimal utilization of mulberry resources.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.934900542495479%\"\u003e\n \u003cp\u003eCaCO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.9746835443038%\"\u003e\n \u003cp\u003eCalcium carbonate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.573236889692586%\"\u003e\n \u003cp\u003eCO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.517179023508135%\"\u003e\n \u003cp\u003eCarbon dioxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.934900542495479%\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.9746835443038%\"\u003e\n \u003cp\u003eScanning electron microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.573236889692586%\"\u003e\n \u003cp\u003eSNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.517179023508135%\"\u003e\n \u003cp\u003eSilver nitrate staining solution\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.934900542495479%\"\u003e\n \u003cp\u003e6-BA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.9746835443038%\"\u003e\n \u003cp\u003e6-benzylaminopurine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.573236889692586%\"\u003e\n \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.517179023508135%\"\u003e\n \u003cp\u003eCalcium chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.934900542495479%\"\u003e\n \u003cp\u003eABA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.9746835443038%\"\u003e\n \u003cp\u003eAbscisic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.573236889692586%\"\u003e\n \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.517179023508135%\"\u003e\n \u003cp\u003eCalcium ion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.934900542495479%\"\u003e\n \u003cp\u003eCaOX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.9746835443038%\"\u003e\n \u003cp\u003eCalcium oxalate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.573236889692586%\"\u003e\n \u003cp\u003ePFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.517179023508135%\"\u003e\n \u003cp\u003ePara-formaldehyde solution\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.934900542495479%\"\u003e\n \u003cp\u003eACC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.9746835443038%\"\u003e\n \u003cp\u003eAmorphous calcium carbonate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.573236889692586%\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.517179023508135%\"\u003e\n \u003cp\u003ePhosphate-buffered saline solution\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.934900542495479%\"\u003e\n \u003cp\u003eARS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.9746835443038%\"\u003e\n \u003cp\u003eAlizarin red S staining solution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.573236889692586%\"\u003e\n \u003cp\u003eLM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.517179023508135%\"\u003e\n \u003cp\u003eLight microscope\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the research grants from the National Key R\u0026amp;D Program of China (Grant No. 2022YFD1201602), Innovation Research 2035 Pilot Plan of Southwest University (Grant SWU-XDZD22008), and the Chongqing Research Program of Basic Research and Frontier Technology (Grant No. cstc2021yszx-jcyj0004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChao Yang, Jiubo Liang and Ningjia He conceived and designed the project; Qi Zhang and Lin Chen performed the experiments; Jianglian Yuan and\u0026nbsp;Peng Qian\u0026nbsp;planted the mulberry seedlings; Chao Yang and Qi Zhang analyzed data; Chao Yang wrote the manuscript and Ningjia He revised the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcharya BR, Assmann, Sarah M (2009) Hormone interactions in stomatal function. Plant Mol Biol 69 (4):451-462. https://doi.org/10.1007/s11103-008-9427-0\u003c/li\u003e\n\u003cli\u003eAjello L (1941) Cytology and Cellular Interrelations of Cystolith Formation in \u003cem\u003eFicus elastica\u003c/em\u003e. Am J Bot 28 (7):589-594. https://doi.org/10.2307/2437007\u003c/li\u003e\n\u003cli\u003eBouropoulos N, Weiner S, Addadi L (2001) Calcium oxalate crystals in tomato and tobacco plants: morphology and in vitro interactions of crystal-associated macromolecules. 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Plant Soil 444 (1):299-314. https://doi.org/10.1007/s11104-019-04283-8\u003c/li\u003e\n\u003cli\u003eZhang You, Shiyuan Guo, Qiao Li, Yanjun Fang, Panpan Huang, Ju C, Wang C (2023) The CBL1/9-CIPK1 calcium sensor negatively regulates drought stress by phosphorylating the PYLs ABA receptor. Nature communications 14 (1):5886. https://doi.org/10.1038/s41467-023-41657-0\u003c/li\u003e\n\u003cli\u003eZheng S, Zeng W, Han L, Liu C, Yu M, Xiang Z, Zhao A (2017) Comprehensive evaluation of nutritional quality of leaves from 45 mulberry germplasms and varieties. Food Sci 38:159-163. https://doi.org/10.7506/spkx1002-6630-201708025\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"brazilian-journal-of-botany","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brjb","sideBox":"Learn more about [Brazilian Journal of Botany](https://www.springer.com/journal/40415)","snPcode":"40415","submissionUrl":"https://www.editorialmanager.com/brjb/default2.aspx","title":"Brazilian Journal of Botany","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cystoliths, Mulberry, Distribution patterns, Phytohormone, 6-Benzylaminopurine, Abscisic acid","lastPublishedDoi":"10.21203/rs.3.rs-3887434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3887434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMulberry (\u003cem\u003eMorus\u003c/em\u003e spp.) has been studied to gain insight into cystolith formation, which is primarily composed of calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e) crystals and commonly found in mulberry leaves. However, the effects of phytohormones on cystolith formation in mulberry and the origin of carbon within these structures remain poorly understood. This study utilized scanning electron microscopy (SEM), plant tissue sections, and silver nitrate staining techniques to comprehensively analyze the morphology of cystoliths in mulberry. Additionally, the distribution pattern of cystoliths was investigated, and mulberry seedlings were treated with 6-Benzylaminopurine (6-BA) and Abscisic acid (ABA). The results revealed that 6-BA significantly enhanced cystolith accumulation, whereas ABA had suppressive effects on cystolith formation in mulberry leaves. Furthermore, the concentration of applied phytohormones positively correlated with the yield of cystoliths. Based on these results, it is postulated that these phytohormones may modulate carbon absorption in mulberry by influencing stomatal conductance, thereby regulating cystolith formation. This research offers valuable insights into the underlying mechanisms driving mulberry cystolith formation and contributes to the optimal utilization of mulberry resources.\u003c/p\u003e","manuscriptTitle":"Spatial and temporal distribution of cystoliths in mulberry leaves and their formation under the influence of phytohormones 6-BA and ABA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-22 18:15:12","doi":"10.21203/rs.3.rs-3887434/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-02-20T22:09:09+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-20T14:53:31+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Brazilian Journal of Botany","date":"2024-02-19T19:01:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-17T05:37:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Brazilian Journal of Botany","date":"2024-02-15T01:08:20+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revisions","date":"2024-02-09T13:20:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"brazilian-journal-of-botany","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brjb","sideBox":"Learn more about [Brazilian Journal of Botany](https://www.springer.com/journal/40415)","snPcode":"40415","submissionUrl":"https://www.editorialmanager.com/brjb/default2.aspx","title":"Brazilian Journal of Botany","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d42eee13-1b42-4243-8a84-5e006c3d4719","owner":[],"postedDate":"February 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-10-03T11:14:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-22 18:15:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3887434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3887434","identity":"rs-3887434","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}