Oil-bearing rose (Rosa damascena), locally known as Isparta gülü, is a member of the Rosaceae family and is an important essential oil-producing crop. It is highly valued for its essential oil, which is used in the perfumery, pharmaceutical, food and cosmetic industries. Oil-bearing rose has high economic value and is cultivated commercially in Türkiye, Bulgaria and Iran for its flowers, which are used for the production of rose oil, absolute and concrete. A high demand for good quality rose oil by different industries has caused an increase in oil-bearing rose cultivation and rose oil production. The blooming of the oil-bearing rose takes place once a year and continues for 25–30 days during May and June. Flowering from the bud stage to full blooming takes approximately 11–13 days, and the flower production rate reaches its height in early June (Baydar et al., 2013; Önder et al., 2022).

Flowering is an important aspect of the angiosperm life cycle allowing reproduction to take place. During flower development, significant changes occur in the polysaccharide levels and composition, color development, and production of primary and secondary metabolites within oil-bearing rose petals (Önder et al., 2022; Önder et al., 2023). The plant cell wall is a strong network of fibrils that are arranged to give each cell a consistent shape and a complex structure composed of a pectin matrix, proteins, tissue/organ-bound cellulosic microfibers and neutral non-cellulosic polysaccharides (McCann and Roberts, 1991; Carpita and Gibeaut, 1993). The plant cell wall shows great diversity in different species, cell types and even in subcellular cell wall domains (Derbyshire et al., 2007; Pelletier et al., 2010; Wolf et al., 2012). Despite the apparent diversity, pectins, hemicellulose, and cellulose form the main groups of polysaccharides in plant cell walls (Seymour and Gross, 1996; Brummell and Harpster, 2001; Yap et al., 2008). Pectins are structurally complex plant cell wall polysaccharides and have important roles in cell–cell adhesion. Although the mechanisms that regulate cell wall polysaccharides during flower development are not fully understood, information regarding changes in components, such as pectin, cellulose and uronic acid, has been obtained from different studies (O’Donoghue et al., 2002; Yap et al., 2008; Yang et al., 2021).

Changes in cell wall composition and structures of flowers occur due to the coordinated or interdependent action of a number of cell wall modifying enzymes. Cell wall-associated enzymes play important roles in cell wall development, remodeling and degradation during flower development (Yap et al., 2008; Zhang et al., 2019; Yang et al., 2021). Pectinases, such as polygalacturonase, pectin methyl esterase and pectin lyase, involved in these biochemical pathways, are among the enzymes that act on the pectin fractions in the cell wall. Pectin methyl esterase catalyzes pectin demethylation, breaking down calcium linkages in the acidic polysaccharide chain, thereby producing a substrate for polygalacturonase as well as cell separation for cell expansion (Brummell et al., 2004). During flower development, polygalacturonase affects pectin degradation and cell wall expansion by hydrolyzing pectic acid along with the main chain of polygalacturonic acid (Hadfeid and Bennet, 1998; Zhang et al., 2019). Pectin lyases degrade pectin polymers through a β-elimination mechanism yielding oligogalacturonides (Cao, 2012). Glucosidase enzymes, such as β-galactosidase, β-glucosidase and endoglucanase act on different carbohydrate substrates. β-galactosidase is the main glycosidase enzyme that removes the galactosyl component from the cell wall (Tateishi, 2008). β-glucosidase breaks β-glycosidic bonds, thereby breaking down polysaccharides and making sugars available (Opassiri et al., 2003; Mohapatra et al., 2010). Endoglucanase is an endo-hydrolase that acts on glucose polymers by cleaving glycosidic bonds. Its target in plant cell walls can be xyloglucans or mixed linkage glucans (Jara et al., 2019). The activities of the cell wall-related enzymes change during flower development, and these changes may cause major changes in petals (Panavas et al., 1998; Yap et al., 2008). In addition, the reduction of cell wall-related enzyme activities significantly delays the processes leading to senescence (Panavas et al., 1998). Any of these enzymes alone is not sufficient to cause changes in cell walls, but several isoforms of these enzymes and post-transcriptional regulatory mechanisms affect the overall process (Goulao et al., 2007).

Flower development is associated with the accumulation, solubilization and depolymerization of cell wall components, and the activities of different cell wall-related enzymes in flower development have been reported (Yap et al., 2008; Yang et al., 2021). However, the reported changes during flower development may be due to different cell wall modification mechanisms in different species (O’Donoghue et al., 2002; Yap et al., 2008), therefore, flowering plants need to be investigated individually. In addition, the flowers of various plants have commercial, industrial and aesthetic values and should be studied separately. To date, limited data are available on cell wall compounds and related enzyme activities in the petals of oil-bearing rose during flower development (Kumar et al., 2008). Therefore, the aim of the present study was to analyze changes in cell wall components and enzyme activities during flower development in oil-bearing rose petals. The findings can be used for a better understanding of the physiological processes associated with cell wall metabolism and regulation of flower development not only in oil-bearing rose but also in other flowering plant species.

The ash content of the petals was highest in stages I and II, and ash content decreased significantly at stage III, remained unchanged at stage IV and decreased further at stage V of flower development. ( Figure 2A ). NDF consists of cellulose, hemicellulose and lignin while ADF consists of cellulose and lignin fractions. The ADF and NDF contents were determined according to the nutritional fiber method, which is defined as the total biomass loss of raw biomass weight after hot water extraction. The changes in NDF and ADF contents were similar. The NDF content increased significantly from stage I to stage II of flower development, decreased at stage III and remained unchanged at the later stages of flower development ( Figure 2B ). While the highest NDF content was observed at stage II (25.94%), the lowest NDF content was observed at stage I (22.99%). The ADF content increased significantly at stage II (19.01%) and decreased significantly at stage IV ( Figure 2C ). The lowest ADF content was observed at stage I (16.82%) of flower development. The difference between the NDF and ADF contents is used to estimate the hemicellulose content of petals. The hemicellulose content of petals remained unchanged between stages I to IV but was higher at stage V compared to stage I of flower development ( Figure 2D ). Consequently, the highest (7.47%) and the lowest (6.17%) hemicellulose contents in petals were observed at stage V and stage I, respectively.

The total pectin content of petals increased steadily as flower development progressed, and total pectin content increased by 63%, 100%, 219% and 248% at stages II, III, IV and V, compared to stage I, respectively ( Figure 2E ). The pectin contents between stages II and III and stages IV and V were not significantly different from each other. The highest and the lowest total pectin contents were observed in stage V (94.4 mg g⁻¹) and in stage I (26.9 mg g⁻¹), respectively. The cellulose content was higher at early stages (I–III) but was lower at blooming stages (IV and V) of flower development. The cellulose content reached its highest level in stage II (167.8 mg g⁻¹), and its level gradually decreased from stage II to IV and remained the same in stages IV and V (98.4 mg g⁻¹) ( Figure 2F ). Uronic acids are components of polysaccharides and are present in varying amounts in different plant species. The uronic acid content exhibited marked fluctuations throughout flower development. The uronic acid content decreased from its highest level at stage I (8.0 mg g⁻¹) to its lowest level at stage III (3.8 mg g⁻¹), then increased at stage IV (8.0 mg g⁻¹). Though the uronic acid content slightly decreased at stage V, the amount of this decrease was not statistically significant ( Figure 2G ).

The plant cell wall is a solid structure consisting of interconnected networks of polysaccharides and proteins. During flower development, the composition and organization of cell wall polysaccharides alter. These changes are mediated by the activities of various cell wall-related enzymes during flower development. The activities of six cell wall-modifying enzymes were investigated in the petals of the oil-bearing rose flowers at five development stages, from green buds to fully opened flowers ( Figure 3 ). β-galactosidase activity increased from bud (stage I) to partially opened flower (stage IV) but decreased at full blooming (stage V) ( Figure 3A ). The highest β-galactosidase activity was observed at stage IV (6.73 U mg protein⁻¹) and the lowest activity at stage I (0.96 U mg protein⁻¹). β-glucosidase activity did not show an increase between stage I and II (16.0 and 32.4 U mg protein⁻¹) but significantly increased in stage III (205.9 U mg protein⁻¹) and IV (298.3 U mg protein⁻¹). At the full blooming stage (V), its activity decreased significantly to 145.5 U mg protein⁻¹ ( Figure 3B ). The endoglucanase activities in stages I and II of flower development were 297 and 773 U mg protein⁻¹, respectively. The activity in stages III and IV increased to 8097 and 14075 U mg protein⁻¹, respectively. Later, the activity decreased to 6566 U mg protein⁻¹ at stage V ( Figure 3C ). β-glucosidase and endoglucanase activities started to increase significantly at the stage of intensification of petal coloring and expansion (stage III), and the highest activity in both enzymes was observed in stage IV. Then, β-glucosidase and endoglucanase activities decreased significantly in stage V and fell below those measured at stage III.

Pectin lyase activity increased steadily and reached its highest level at stage IV of flower development ( Figure 3D ). However, at stage V, its activity decreased significantly to 47.1 U mg of protein⁻¹, approaching the activity level observed in stage II. The highest pectin lyase activity was in stage IV (86.4 U mg protein⁻¹), while the lowest activity was in stage I (23.7 U mg protein⁻¹). Pectin methyl esterase and polygalacturonase activities were very low in the first stages of flower development. Pectin methyl esterase activity differed from the other enzymes as it reached its highest activity in stage III (6.5 U mg protein⁻¹) and maintained its activity at stage IV (6.7 U mg protein⁻¹) of flower development ( Figure 3E ). Later, pectin methyl esterase activity decreased to 2.2 U mg protein⁻¹ at stage V. Polygalacturonase activity did not change significantly between stage I and II ( Figure 3F ), but its activity started increasing at stage III (2,966 U mg protein⁻¹) and reached its highest activity at stage IV (6,689 U mg protein⁻¹) of flower development. Later as with pectin methyl esterase activity, polygalacturonase activity decreased to 2,548 U mg protein⁻¹ at blooming (stage V)

Correlation analysis was carried out to determine the relationships between different cell wall components and cell wall-related enzymes during the flower development of oil-bearing rose. The results of this analysis for five flower development stages of oil-bearing rose are shown in Figure 4 . Of the 78 correlation coefficients, 14 were positively correlated and one was negatively correlated with each other. Ash content was negatively correlated with total pectin (−0.92) and positively correlated with cellulose (0.86) contents. NDF was positively correlated with ADF (0.91). β-glucosidase, endoglucanase and pectin lyase activities were positively correlated with the other cell wall enzymes. β-galactosidase and pectin methyl esterase activities were positively correlated with pectin lyase and endoglucanase, while polygalacturonase activity was positively correlated with endoglucanase and pectin lyase. Cellulose, hemicellulose and uronic acid contents did not show a significant correlation with any other parameter examined in the study.

Relationships between cell wall components, enzyme activities and flower development stages were further analyzed with principal coordinate analysis (PCA) and the results are shown in Figure 5 . The two principal components (PC) represented 81.81% of the total variation (59.22% and 22.59% for PC1 and PC2, respectively) observed in the study. The different flower development stages were divided into four main clusters. PCA showed that stages IV and V were closely related. The effects of flower development stages on cell wall components and enzyme activities were distributed along the PC1 axis in the order I > II > III > V > IV. Ash, cellulose and ADF content changed significantly at stage II of flower development and clustered on the left side of the PC1 axis. Significant changes occurred in NDF and hemicellulose contents at stage III of flower development. Cell wall enzyme activities were significantly affected in stages III and IV and clustered on the right side of the PC1 axis.

Flower development is related to the structure and alterations of the cell wall due to the activities of cell wall enzymes (Yang et al., 2021), and petal structures also differ among flowering plants. Cell wall structure has been studied in detail in carnation (Dianthus caryophyllus L.), sandersonia (Sandersonia aurantiaca Hook.), daylily (Hemerocallis citrina Baroni) and pigeon orchid (Dendrobium crumenatum Sw.) flowers. However, only daylily and pigeon orchid flower development from bud stage to blooming stages and associated changes in cell wall compositions have been reported (de Vetten and Huber, 1990; O’Donoghue et al., 2002; Yap et al., 2008; Yang et al., 2021). These studies showed that the content of cell wall polymers and activities of cell wall-related enzymes differ and these differences are associated with flower development. These studies suggested that flower development may be related to cell wall polymers and metabolism (O’Donoghue, 2006; Yap et al., 2008; Yang et al., 2021), therefore we aimed to determine the cell wall polymer content and cell wall-related enzyme activities of petals of oil-bearing rose from the bud to blooming stages.

Flower development is a complex process that leads to changes in the cell wall structure and polymer contents. In the study, the ash content of developing flowers decreased, and the contents of six cell wall components found in petals differed depending on the flower development stages. NDF is widely used in fiber characterization, as it contains important components of plant cell walls (cellulose, hemicellulose and lignin) and ADF contains cellulose and lignin (Ankom, 2017), thus it is possible to distinguish carbohydrates structurally and non-structurally (Skamarokhova et al., 2020). Hemicelluloses take part in the cross-linking of cellulose in cell walls and contribute to changes in primary cell wall structure (Brummell, 2006). The hemicellulose content of oil-bearing rose petals between the consecutive development stages essentially remained unchanged unlike ADF and NDF contents, which exhibited differences between the development stages. However, the hemicellulose content of pigeon orchid decreased steadily during flower development (Yap et al., 2008). Similarly, ash content decreased during flower development in Centaurea cyanus L., whereas the ash content was lower in the bud stage of Borage officinalis L. and increased during blooming (Fernandes et al., 2019). Ash content of plants shows high variability depending on physiological and morphological differences as well as genetic and environmental factors (Casler and Boe, 2003; Monti et al., 2008). Since the biomass of flowers can vary greatly due to phenological stage and cultural practices (Monti et al., 2008), the determination of ash content throughout flower development could help determine whether the weight increase of flowers is due to accumulation of cell wall components or due to accumulation of water. Petal area, fresh weight and relative water content increase throughout flower development in oil-bearing rose (Önder et al., 2022), but the very little variation found in ADF, NDF and hemicellulose contents and decreased ash content suggest that the weight increase of petals is mainly caused by accumulation of water and other solutes in oil-bearing rose.

Pectins are major components that mediate remodeling in cell walls (Yang et al., 2021), affect cell adhesion and form the structure of the middle lamella and primary cell wall in dicotyledons (Mohnen, 2008). The total pectin content of oil-bearing rose increased significantly during flower development ( Figure 2E ). Similarly, total pectin content increased steadily during the flower development of pigeon orchid. Pectin content was associated with flowering in Oncidium orchids and increased from the blooming development (B stage) to the blooming stage (C stage) (Wang et al., 2008). Cellulose plays an important role in cell wall stiffness by forming cellulose microfibrils that cross-link to the cell wall structure due to its high physical and chemical stability (Liu et al., 2021), therefore cellulose is part of flower development and blooming processes. In oil-bearing rose, the increase in cellulose content at the second stage of flower development and then its reduction at subsequent stages ( Figure 2F ) was similar to the development of goji berry (Liu et al., 2021) and blackberry fruits (Zhang et al., 2019). While the cellulose content showed a decreasing trend after stage II, total pectin content continued to increase during flower development. Similarly, cellulose content started to decrease after blooming, but total pectin content continued to increase until blooming in pigeon orchid (Yap et al., 2008). Uronic acid is the main component of pectin and hemicellulose and is used in flower bud formation and flower development processes (Chakraborty et al., 2022). While the loss of uronic acid continued from stage I to III, uronic acid content increased in stage IV in oil-bearing rose. During olive fruit development, polygalacturonase and pectin lyase enzyme activities increase and uronic acid acid content decrease as maturation progresses, resulting in cell wall degradation and decreased fruit flesh firmness (Diarte et al., 2022). In our study, we have also observed that polygalacturonase and pectin lyase enzyme activities increased until stage IV in which petal expansion becomes visible and blooming starts. Similarly, uronic acid content decreased between stage I and III, but unlike olive fruits, it increased at later stages of flower development and blooming. The significant increase in the uronic acid content after the third developmental stage also implies that the activities of the pectin metabolizing enzymes, which are involved in cell wall remodeling and cell wall breakdown are decreasing.

There are many factors that can regulate modifications of cell wall polymers, including glucosidases and pectinases. Glycosidases play important roles in the breakdown of cell wall polysaccharides and remodeling of the cell wall structure (Minic, 2008). In vitro activities of various glucosidases were measured and their activities changed significantly with flower development in oil-bearing rose. Among the cell wall related enzymes analyzed, β-glucosidase (16.03–298.28 U mg protein⁻¹), endoglucanase (297.17–14,075.06 U mg protein⁻¹), pectin lyase (23.69–86.44 U mg protein⁻¹) and polygalacturonase (248.79–6,688.77 U mg protein⁻¹) exhibited high activity throughout flower development in oil-bearing rose petals. The other enzymes, β-galactosidase (0.96–6.73 U mg protein⁻¹) and pectin methyl esterase (0.23–6.74 U mg protein⁻¹), had lower levels of activity. The activity of all cell wall-related enzymes was significantly lower at the full bloom period of flower development (stage V). β-galactosidase, β-glucosidase, endoglucanase, pectin lyase, pectin methyl esterase, and polygalacturonase activities were decreased by 27%, 51%, 53%, 46%, 67% and 62%, at stage V of flower development compared to stage IV, respectively.

β-galactosidase hydrolyzes glycosidic bonds and regulates cell wall flexibility, intercellular communication, and the mobility of enzymes within the cell wall matrix (Brummell and Harpster, 2001; Brummell, 2006). In our study, β-galactosidase activity increased steadily up to stage IV and decreased significantly at stage V. Similarly, β-galactosidase activity increased from the bud stage to the blooming stage and decreased significantly during senescence in sandersonia and daylily flowers (Panavas et al., 1998; O’Donoghue et al., 2002). On the other hand, β-galactosidase activity remained relatively constant until senescence in pigeon orchid (Yap et al., 2008). β-glucosidase is involved in many mechanisms, such as hydrolysis of glycolipids, production of odorant precursors, lignification and catabolism of the cell wall, activation of phytohormones, defense mechanism, flower development and growth (Mishra et al., 2014). In oil-bearing rose flowers, β-glucosidase activity was low during the bud stages (I and II), its activity peaked at stage IV and then decreased at stage V (full bloom stage). Similarly, β-glucosidase activity peaked at the petal separation stage during the flower development of tea (Camellia sinensis (L.) Kuntze) and decreased during the full bloom stage (Joshi and Gulati, 2011). β-glucosidase activity increased and the highest increase was observed at the full blooming stages (stages V and VI) of oil-bearing rose (Oka et al., 1999) and paper white (Narcissus papyraceus Ker-Gawl.) (Reuveni et al., 1999). However, in pigeon orchid, β-glucosidase activity reached its highest activity at the green bud stage and decreased with the blooming and senescence of flowers (Yap et al., 2008). Endoglucanase plays a role in cell separation, cell wall synthesis, remodeling and degradation (Liu et al., 2011; Jara et al., 2019). During the flower development of oil-bearing rose, endoglucanase activity was the highest at the partially opened flower (IV) and decreased significantly during the full bloom stage (V). Endoglucanase activity has been studied in detail during fruit development and ripening (Jara et al., 2019), but not during flower development. Endoglucanase showed significant activity during softening and ripening of different fruits, such as strawberry (Trainotti et al., 1999), tomato (Rose and Bennett, 1999), avocado (Fischer and Bennett, 1991) and pepper (Harpster et al., 1997).

Pectinases degrade different pectin types. For example, pectin lyase and pectin methyl esterase degrade pectin into short-chain pectin molecules and produce polygalacturonic and galacturonic acids (Yang et al., 2017). Polygalacturonase hydrolyzes pectic substances produced by the pectin methyl esterase, which leads to changes in cell wall properties, such as hardness, elasticity and permeability (Jeong et al., 2015). These events cause loosening of pectins, and structural remodeling and relaxation of the cell wall during cell expansion and cell development (Brummell et al., 2004; Jeong et al., 2015). The continuous increase in pectin lyase activity up to stage IV and a significant decrease in stage V ( Figure 3D ) in oil-bearing rose was similar to daylily flower development (Yang et al., 2021). In addition, the silencing of SlPL, which encodes the pectin lyase enzyme in tomatoes, increased fruit firmness and prolonged shelf life, indicating that pectin lyase changes the cell wall structure and affects firmness (Yang et al., 2017). The increase in pectin methylesterase activity was similar to the increase in polygalaturonase activity in oil-bearing rose. Both enzymes had the highest activity at stage IV and their activity dropped significantly at stage V. Pectin methylesterase activity peaked at blooming and then decreased till senescence in sandersonia flowers (O’Donoghue et al., 2002). However, pectin methyl esterase, pectin lyase and polygalacturanse activities in daylily increased throughout flower development and remained high after blooming (Yang et al., 2021). In blackberry fruits, pectin methylesterase and polygalaturonase activity were associated with tissue softening and cell wall relaxation (Zhang et al., 2019), which also occur during the blooming of oil-bearing rose flowers. The activities of some enzymes, such as endoglucosidase and polygalacturonose were very high. It should be noted that in vitro enzyme activity may not reflect the enzyme activity in planta due to substrate availability, preferential use of different isoforms, pH and ionic balance of cells and other enzyme activities in plant cell walls (Fry, 2004).

Multivariate analysis methods, such as correlation analysis with heat map and PCA showed the effects of flower developmental stages on cell wall polymers and cell wall enzyme activities. Correlation analysis only found a positive correlation between NDF and ADF, and a negative correlation between pectin and ash content during flower development of oil-bearing rose. However, cell wall-related enzymes showed positive correlations with each other, indicating that their activities during flower development are interlinked with each other. Although their correlation coefficients were not significant, pectin content showed positive and cellulose content negative correlations with the cell wall-related enzymes. PCA showed that stages III, IV and V of flower development were the most physiologically active development stages as revealed by the accumulation of cell wall polymers and enzyme activities.

Flower development starting from bud to blooming stages in oil-bearing rose is associated with changes in cell wall polymers and cell wall-related enzyme activities. In general, changes in cell wall polymers started to differ significantly at stage II. The petals of oil-bearing rose begin to open in stages III and IV, and, accordingly, the highest cell wall enzyme activities were observed in these stages, indicating that the activities of cell wall-related enzymes are a part of the flowering process. Cell wall structure and its modification is a complex process. To better understand cell wall formation and modification, interactions between non-enzymatic mechanisms and their regulation by cell wall-modifying enzymes may provide further insights into the chemistry and mechanisms of flower development in oil-bearing rose.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

SÖ and MT designed the experiments. SÖ, DÖ, SE and MM performed the experiments and analyzed the data. SÖ and MT wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.