Plasma Membrane Vesicles From Cauliflower Meristematic Tissue And Their Role In Water Passage

Plasma Membrane Vesicles From Cauliflower Meristematic Tissue And Their Role In Water Passage | 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 Plasma Membrane Vesicles From Cauliflower Meristematic Tissue And Their Role In Water Passage Paula Garcia-Ibañez, Juan Nicolas-Espinosa, Micaela Carvajal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-29232/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Jan, 2021 Read the published version in BMC Plant Biology → Version 1 posted You are reading this latest preprint version Abstract Background Cauliflower ( Brassica oleracea L. var. botrytis ) inflorescences are composed mainly of meristematic tissue, which has a high cellular proliferation. This considerable cellular density makes the inflorescence an organ with a large proportion of membranes. However, little is known about the specific role of the lipid and protein composition of the plasma membrane present in this organ. Results In this work, we analyzed the lipids and proteins present in plasma membrane from two different stages of development of cauliflower inflorescence and compared them with leaf plasma membrane. For this purpose, plasma membrane vesicles were obtained by centrifugation for each sample and the vesicular diameter and osmotic permeability ( Pf ) were analyzed by dynamic light scattering and the stopped-flow technique, respectively. In addition, fatty acids and sterols were analyzed by gas chromatography and HPLC, respectively. The protein composition of the inflorescences and leaves was characterized by HPLC-ESI-QTOF-MS and the data obtained were compared with Brassicaceae proteins present in the UniProt database in relation to the presence of aquaporins determined by western blot analysis. The highest Pf value was found in 90 day inflorescences-derived plasma membrane vesicles (61.4 ± 4.14 µms − 1 ). For sterols and fatty acids, the concentrations varied according to the organ of origin. The protein profile revealed the presence of aquaporins from the PIP1 and PIP2 subfamilies in both inflorescences and leaves. Conclusion This study shows that the composition of the sterols, the degree of unsaturation of the fatty acids, and the proteins present in the membranes analyzed give them high functionality for water passage. This represents an important addition to the limited information available in this field. Plant Physiology and Morphology Plant Molecular Biology and Genetics plasma membrane aquaporin brassica osmotic permeability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Nowadays, crops from the Brassicaceae family are among the ones most cultivated, worldwide. Of these, the cauliflower ( Brassica oleracea L. var. botrytis ) stands out for its high production (24 million tonnes in 2014) and economic relevance [ 1 ]. The main reason for its great demand is the presence of diverse health-promoting bioactives - such as glucosinolates, polyphenols, or vitamin C - that add great nutritional value to its edible part [ 2 ]. However, little recent information on the phytochemistry of the cauliflower inflorescence molecular structures is available. Smyth [ 4 ] described how this inflorescence begins to develop with the formation of secondary meristems. After that, a continuous proliferation of meristematic tissue takes place, generating a highly branched compact pattern with a whitish color and spiral-like shape [ 4 ]. Following this, the immature inflorescence initiates its maturation, finally reaching total floral differentiation [ 5 ]. Although the intervention of genes related to the identity of the floral meristem, such as TFL1, LFY, AP1 , or CAL , has been studied [ 1 ], further comprehension of the biochemical mechanisms underlying this process is needed. In addition, Grevsen et al. [ 6 ] observed that environmental factors, mainly temperature, influence the generation of the inflorescences. As the main focus of the research to date has been the genetic control of the inflorescence development, little is known about the lipid and protein composition of the membranes present in it. However, a high cellular proliferation is present in the edible part of cauliflower, giving a high membrane concentration [ 7 ]. Biological membranes are relevant to cell functionality, due to their ability to create an adequate environment for diverse types of proteins, such as transmembrane proteins. The latter mainly determine the specific functionality of each type of membrane present in cells and control diverse constitutive functions, such as endocytosis and ion and water transport [ 8 ]. Among the important transmembrane proteins, aquaporins are one of the main protagonists, since they are responsible for the water transport through biological membranes [ 9 ]. In cauliflower meristematic tissue cells, it has been observed that there is a high abundance of aquaporins embedded in the vacuolar membrane, which allows swelling of growing cells, while maintaining the cellular turgor [ 10 ]. However, the functionality of these proteins can be affected by the lipids of the membrane in which they are embedded [ 11 , 12 ]. This suggests that the lipid composition of the membrane determines not only its physical characteristics but also the activity and functionality of the proteins present in it [ 13 ]. One of the main components of lipid membranes are sterols. These molecules have been linked with different functions, such as lipid packing, since they are able to interact with membrane proteins and fatty acids [ 14 ]. Furthermore, a high sterol (mainly stigmasterol, campesterol, and β-sitosterol in plant membranes) content has been related to an increment in membrane water permeability [ 15 ]. Other fundamental components of cellular membranes are fatty acids, since they contribute to the thickness, stability, and permeability, according to the proportions of saturated and unsaturated fatty acids [ 16 ]. Therefore, both the lipid and the protein composition are determinants of the membrane transport activity, giving each type of membrane specific functional characteristics. For the above reasons, and with the aim of determining the specific functions of the plasma membrane of the cauliflower meristematic tissue, in this work the presence of fatty acids, sterols, aquaporins, and other proteins was analyzed in two different stages of inflorescence development. To allow a comparison with vegetative tissue, leaf plasma membranes were also analyzed. Methods Plant material Fifty commercial cauliflower seeds (from Sakata Seed Iberica S.L.U., Valencia, Spain) were induced to germinate by imbibition with water and continuous aeration for 24 h. Then, the seeds were transplanted to vermiculite and were kept in darkness, at 28ºC and 60% relative humidity, for two days. The seedlings (5 days old) were transferred to the agricultural soil of an experimental farm (37°47'52.7"N, 0°52'00.7"W, 15 m asl, Murcia, Spain). The experiment was carried out from December to February with average temperatures and relative humidities of 17ºC and 60% (day), and 4ºC and 65% (night), under a semi-arid Mediterranean climate. The daily average temperature and relative humidity were recorded with dataloggers (AFORA S.A., Barloworld Scientific, Murcia, Spain). All plants were drip-irrigated with ¼-strength Hoagland nutrient solution. They were harvested at 70 and 90 days after transplanting. The intermediate leaves and inflorescences (15 of each) were sampled at random and weighed fresh in three technical replicates. After that, samples were kept in storage at 4ºC for one day until processing. Plasma membrane extraction Samples of fresh material (100 g) were sliced in small pieces and vacuum-infiltrated with a 1:1.6 (w/v) proportion of an extraction buffer (0.5 M sucrose, 1 mM DTT, 50 mM HEPES, 1.30 mM ascorbic acid, pH 7.5) and 0.5 g of PVP. After 10 min, samples were homogenized and filtered through a nylon mesh with a pore diameter of 100 µm. Then, the filtrate was centrifuged at 10000x g for 30 min, at 4ºC; the supernatants were collected and centrifuged for 35 min at 50000x g , at 4ºC. The pellet obtained was resuspended in 500 µl of a buffer containing 5 mM PBS and 0.5 M sucrose (pH 6.5) (FAB). Three different extractions per sample type were performed. Two milliliters of this microsomal fraction were introduced in a two-phase system composed of PEG-3350/Dextran-T500-6.3% (w/w), 5 mM KCl, 330 mM sucrose, 2.5 mM NaF, and 5 mM K 3 PO 4 (pH 7.8). The system was centrifuged for 5 min at 4000 xg . Then, the upper phase was collected and a wash was performed with a buffer containing 9 mM KCl, 0.2 M EGTA, 0.5 mM NaF, and 10 mM Tris-borate (pH 8.3). Then, a centrifugation at 55000 xg for 35 min, at 4ºC, was performed. The pellet obtained was resuspended in FAB. The final protein concentration was determined using an RC DC protein assay kit (BioRad, California, USA), with bovine serum albumin as the standard. Three different extractions per sample type were performed. Vesicle size The mean size of the vesicles obtained from different samples was determined by dynamic light scattering, using a Malvern ZetaSizer Nano XL (Malvern Instruments Ltd., Orsay, France) as described by Barrajón-Catalán et al. [ 17 ]. This instrument allows the analysis of particles with diameters from 1 nm to 3 µm. Stopped flow light scattering These measurements were performed in a PiStar (Applied Photophysics, Leatherhead, UK) spectrophotometer at 20ºC, as described in Maurel [ 9 ]. The kinetics of vesicle volume adjustment were monitored by dynamic light scattering at 90º and with a λ ex of 515 nm. Purified plasma membrane vesicles were subjected to a 100x dilution in a buffer with 30 mM KCl and 20 mM Tris-Mes (pH 8.3, 90 mOsmol kg − 1 H 2 O). For the measurement, the diluted vesicle preparation was mixed in a 1:1 proportion (v:v) with the same buffer supplemented with 540 mM sucrose (630 mOsmol kg − 1 H 2 O). In this way, an osmotic gradient of 270 mOsmol kg − 1 H 2 O was generated. The osmotic permeability ( Pf ) was calculated using this formula: Where K exp is the adjusted exponential velocity constant, V 0 is the mean vesicular volume, A v is the mean vesicular surface area, V w is the water molar mass, and C out is the external osmolarity. Lipids and sterols analysis Five hundred microliters of plasma membrane were mixed with a chloroform-methanol (1:2) mixture. As an internal standard for further sterol analysis, β-colestanol (20 µl, at 0.1 mg ml − 1 ) was added. Then, 0.25 ml of chloroform were added to the mixture before centrifugation at 10000 xg for 6 min. The resultant interphase, corresponding to the protein content, was collected for further analysis. The chloroformic phase was removed to another tube and evaporated with N 2 . For sterol analysis, 50 µl samples of the chloroformic phase were dried with N 2 and then acetylated using pyridine (50 µl) and Ac 2 O (100 µl). After 2 h, the solvents were evaporated with N 2 and 20 µl of ethyl acetate were added. Sterols and fatty acids were determined by gas chromatography, employing an HP5 capillary column (30 m x 0.25 mm x 0.25 µm). This was coupled to a flame ionization detector (FID). Helium was used as the mobile phase (1 ml min − 1 ) and a heat gradient was imposed: from 150 to 195ºC, increasing 3ºC per min, then from 195 to 220ºC at 2ºC per min, and from 220 to 300ºC at 6ºC per min. Proteomic analysis Samples (500 µl) were mixed with 100 µl of 50 mM ammonium bicarbonate (pH 8.3) with 0.01% Protease Max (Promega, Madison, USA). Then, the samples were reduced by adding 100 µl of 20 mM DTT at 56ºC, for 20 min. After that, alkylation was performed by incubation with 100 µl of 100 mM IAA for 30 min, at room temperature and in the dark. Digestion was performed by incubation with 1 µg of trypsin (1:100 w/w) for 3 h, at 37ºC. The samples were dried in a speed vacuum concentrator. The dry samples were resuspended in 20 µl of water/acetonitrile/formic acid (94.9:5:0.1). Then, they were injected onto an Agilent Advance Bio Peptide Mapping HPLC column (2.7 µm x 100 mm x 2.1 mm, Agilent technologies) thermostatted at 55ºC and with a flow rate of 0.4 ml/min. A mixture of water/acetonitrile/formic acid (10:89.9:0.1) was used as the eluent. For detection, an Agilent 6550 Q-TOF coupled with a dual electrospray (AJS-Dual ESI) was used. The experimental parameters were set in MassHunter Workstation Data Acquisition software (Agilent Technologies, Santa Clara, CA, USA), as described in Martínez-Ballesta et al. [ 18 ]. The data were processed with Spectrum Mill MS Proteomics Workbench (Agilent Technologies). The data obtained were compared with the information available in the UniProt database ( www.uniprot.org ) for the Brassicaceae family. Protein function and location were determined from the Gene Ontology database [ 19 ]. Gel electrophoresis and immunoblotting Plasma membrane isolated from cauliflower leaves and inflorescences was employed. Ten micrograms of protein per lane were loaded for 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as shown in Muries et al. [ 20 ]. Then, the proteins were transferred to a PVDF membrane and maintained for 20 min at 15 V in an electrophoretic transfer cell (Trans-Blot SD cell, BioRad, CA, USA), using Towing transfer buffer supplemented with 0.05% SDS [ 20 ]. Blocking solution (TBS containing 2% (w/v) skimmed dry milk) was applied to the membrane for 1 h at room temperature. After that, the membrane was again incubated for 1 h at room temperature, with TBS containing 0.05% Tween 20 and one of the selected antibodies. An antibody raised against the first 45 N-terminus residues of Arabidopsis thaliana PIP1;1 (dilution 1:3000, kindly provided by Prof. Dr. Anthony Schäffner) and another raised against 17 residues from the C-terminal peptide of PIP2;2 of A. thaliana (dilution 1:20000, kindly provided by Dr. Veronique Santoni) were used. Incubation was performed overnight at 4ºC. Goat anti-rabbit IgG coupled to horseradish peroxidase was employed as a secondary antibody (dilution 1:20000). A chemiluminescent signal was developed with West-Pico Super Signal substrate (Pierce, Rockford, IL, USA). The quantification was carried out using ImageJ software and by performing a densitometry analysis. Data analysis The statistical analysis comprised a one-way ANOVA followed by a Tukey HSD post hoc test, performed using RStudio (version 3.4.4.). Results Mean size of plasma membrane vesicles The results for the mean vesicle size (nm), represented in Fig. 1 , show significant differences between inflorescences and leaves and between the maturation stages ( p < 0.05). The mean vesicle size was greater for leaves-derived plasma membrane vesicles than for inflorescences-derived ones ( p < 0.05). The polydispersity data (Fig. 2 ) show that the variability in the size of inflorescences-derived plasma membrane vesicles was higher than for leaves-derived ones ( p < 0.05). Hence, vesicles obtained from leaves were more homogeneous in size. Osmotic water permeability Figure 3 shows that the osmotic water permeability ( Pf , µm s − 1 ) of the plasma membrane vesicles differed significantly between the two maturation stages of the inflorescences ( p < 0.05), those derived from 90-day inflorescences having the highest Pf (64.4 ± 4.14 µm s − 1 ). Lower values were obtained for leaves, with no significant differences between the two maturation stages ( p > 0.05). Lipid analysis Fatty acids The results of the fatty acids (% of total fatty acids) analysis are shown in Table 1 . The percentage of palmitoleic acid (C16:1) did not vary between 70 days and 90 days for inflorescences-derived plasma membrane vesicles ( p > 0.05). Furthermore, a similar percentage of palmitoleic acid was found in vesicles derived from 90-day leaves ( p > 0.05), but a significant decrease was observed for vesicles from 70-day leaves (~ 29–31% vs ~ 19%). For oleic acid (C18:1), similar percentages were found in vesicles derived from inflorescences at the two maturation stages ( p > 0.05). Surprisingly, the presence of oleic acid in leaves-derived vesicles greatly differed, the percentage in 70-day leaves being double that in inflorescences and almost 14-times higher when compared to 90-day leaves ( p < 0.05). For linoleic acid (C18:2), a statistically significant decrease was found between day 70 and day 90 for inflorescences-derived plasma membrane vesicles ( p < 0.05). However, the opposite was observed in leaves-derived vesicles, the content of linoleic acid being increased at 90-days ( p < 0.05). The linolenic acid (C18:3) percentage differed significantly among the four types of sample ( p < 0.05). Higher proportions were found in leaves, the vesicles from 70-day leaves having the highest percentage (~ 48%, vs ~ 44% for 90-day leaves). For inflorescences-derived vesicles, the proportion of linolenic acid was higher in 90-day samples than in those taken at 70 days ( p < 0.05, ~ 40% vs ~ 35%). Table 1 Fatty acid percentage, double bond index (DBI= ∑(unsaturated fatty acids x number of double bonds)), and percentage of monounsaturated fatty acids (MUFA), for plasma membrane from cauliflower inflorescences and leaves at 70 days and 90 days of development. The data are represented as the means (n = 3, where n = different plasma membrane extractions) ± SE. Different letters indicate statistically significant differences (p < 0.05, Tukey’s test) between treatments. Inflorescences Leaves % Fatty acids 70-day 90-day 70-day 90-day Palmitoleic acid (C16:1) 31.41 ± 0.44a 30.99 ± 1.22a 19.09 ± 0.4b 29.42 ± 0.75a Oleic cid (C18:1) 6.42 ± 0.7b 5.59 ± 0.78b 11.66 ± 0.51a 0.79 ± 0.07c Linoleic acid (C18:2) 26.71 ± 1.09a 22.66 ± 0.68b 21.24 ± 0.69b 25.35 ± 1.01a Linolenic acid (C18:3) 35.46 ± 0.91d 40.91 ± 0.8c 48.08 ± 0.64a 44.44 ± 0.51b MUFA 37.88 ± 0.26a 36.5 ± 0.45a 30.78 ± 0.27b 30.31 ± 0.47b DBI 166.28 ± 1.27c 173.74 ± 0.3bc 198.65 ± 3.8a 184.82 ± 3.62b The total percentages of monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) were also determined (Table 1 ). The MUFA percentages for inflorescences-derived samples were significantly higher ( p 0.05) in the double bond index (DBI) were found between the maturation stages for inflorescences-derived samples. However, the DBI was significantly higher ( p < 0.05) in samples from 70-day leaves than in those from 90-day leaves. Sterol content The sterol content (µg/mg of protein) of plasma membrane vesicles was also assessed (Table 2 ). The campesterol concentration showed a statistically significant increase in vesicles derived from 70-day inflorescences, being 3-fold higher than in those from 90-day ones ( p 0.05). For stigmasterol, no statistically significant differences were found between vesicles from inflorescences at the two maturation stages ( p > 0.05). However, in leaves-derived vesicles, the concentration of stigmasterol was higher for 90-day leaves (0.16 ± 0.07 vs 0.25 ± 0.09 µg/mg of protein, p < 0.05). A 2-fold, statistically significant increase in β-sitosterol was found in vesicles from 90-day inflorescences when compared to those of 70-day inflorescences ( p < 0.05). A similar difference was seen when comparing vesicles from 70-day leaves with those of 90-day leaves (0.95 ± 0.11 vs 2.14 ± 0.37 µg/mg of protein, p < 0.05). The stigmasterol/β-sitosterol ratio was also analysed. The highest ratio was found in vesicles derived from 70-day inflorescences, 2-times higher than for 90-day inflorescences and 5-times higher than for both 70-day and 90-day leaves ( p 0.05). Table 2 Sterol content (µg/mg of protein) of plasma membrane from cauliflower inflorescences and leaves at 70 days and 90 days of plant development. The data are represented as the means (n = 3, where n = different plasma membrane extractions) ± SE. Different letters indicate statistically significant differences (p < 0.05, Tukey’s test) between treatments. Inflorescences Leaves µg/mg of protein 70-day 90-day 70-day 90-day Campesterol 1.94 ± 0.39b 5.68 ± 0.64a 1.72 ± 0.46b 2.10 ± 0.35b Stigmasterol 0.53 ± 0.16a 0.60 ± 0.19a 0.16 ± 0.07c 0.25 ± 0.09b β-Sitosterol 0.79 ± 0.11b 1.71 ± 0.05a 0.95 ± 0.11b 2.14 ± 0.37a Stigmasterol/β-Sitosterol ratio 0.67 ± 0.08a 0.35 ± 0.1b 0.17 ± 0.02c 0.12 ± 0.03c Proteomic analysis and immunoblotting A proteomic analysis was performed with the samples from inflorescences and leaves in order to assess qualitatively the proteins present in the vesicles obtained. As shown in Fig. 4 , the proteins obtained were organized according to their cellular anatomical entity characteristics. In all samples studied (inflorescences and leaves at both maturation stages) membrane component related proteins were the most abundant (42–48%). A high percentage of the proteins identified were organelle related; most of them were components of organelles membranes (40–41%). Nevertheless, differences between membranes derived from inflorescences and those from leaves were observed. Cytoplasm proteins represented a higher percentage in the inflorescences (12%) than in the leaves (2%). Chloroplast proteins were identified in leaves samples, in contrast to inflorescences samples. Furthermore, a higher number of chloroplast proteins were identified in 70-day leaves samples than in those of 90-day leaves. Proteins were also classified by their molecular function (Fig. 5 ). They were grouped in nine functional clusters (catalytic, structural molecules, transporter, binding, translation factor, antioxidant, molecular function regulator, enzyme regulator, and nutrient reservoir activities). All the samples showed the same protein activity distribution, the binding proteins (38–40%) standing out as the main group, followed by catalytic activity proteins (32–35%), structural proteins (16–18%), and transport activity proteins (6–8%). A search focused on the aquaporins present in plasma membrane derived from cauliflower inflorescences and leaves was also performed. Aquaporin-related peptides were spotted in all sample types (Table 3 ). In both inflorescences and leaves, peptides corresponding to a wide group of PIP1 aquaporin subfamilies (PIP1;1, PIP1;2, PIP1;3, PIP1;4, and PIP1;5) were identified. Nevertheless, only in the inflorescence samples were peptides related to the PIP2 subfamily detected, PIP2;5 and PIP2;7. Special emphasis can be placed on PIP2;7, for which three different peptide fragments were detected, while only one peptide determined PIP2;5. Members of the Tonoplast Intrinsic Protein (TIP) subfamily were also identified in both types of sample, although this group of aquaporins is generally targeted to the vacuolar membrane. In particular, TIP1;2 and TIP2;1 were found in 70-day inflorescences and 90-day leaves, while only TIP1;2 peptides were detected in 90-day inflorescences and 70-day leaves samples (Table 3 ). Table 3 Aquaporin proteins identified in plasma membrane samples of cauliflower inflorescences and leaves. All protein sequences were retrieved from Brassica oleracea L. var. oleracea information in the NCBI and UniProt databases (ID). The symbols ‘+’ and ‘-’ indicate the presence or absence of the protein in the samples, respectively. Protein NCBI ID UniProt ID Inflorescences Leaves 70-day 90-day 70-day 90-day PIP1;1 XP_013604594.1 A0A0D3DUU2 + + + + PIP1;2 XP_013637020.1 A0A0D3C6I1 + + + + PIP1;3 XP_013600561.1 A0A0D3C6T1 + + + + PIP1;4 XP_013612790.1 A0A0D3D7M3 + + + + PIP1;5 XP_013599049.1 A0A0D3DFM5 + + + + PIP2;5 XP_013599897.1 A0A0D3DT38 + + - - PIP2;7 XP_013629883.1 A0A0D3BL75 + + - - TIP1;2 XP_013613430.1 A0A0D2ZPE6 + + + + TIP2;1 XP_013587105.1 A0A0D3CJP0 + - - + The results obtained from SDS-PAGE analysis of plasma membrane proteins from leaves and inflorescences are shown in Fig. 6 . The presence of two bands was detected; an upper band of 60 kDa, corresponding to dimeric (D) forms of PIPs, and a lower band of ca. 30 kDa, corresponding to the monomeric form (M). Two PIPs groups were analysed, PIP1 and PIP2. For PIP1, bands from samples of 70-day and 90-day inflorescences (23.4% and 15.7% D + M) were less dense than those of 70-day and 90-day leaves-derived plasma membrane proteins (31% and 28.9% D + M). For PIP2 aquaporins, much denser bands were found for 70-day (27.7% D + M) and 90-day inflorescences (61.4% D + M) when compared with 70-day and 90-day leaves-derived samples (5.2% and 5.7% D + M). Discussion The isolation of plasma membrane vesicles using the two-phase aqueous polymer technique [ 21 ] has been reported to produce homogeneous material in terms of yield and composition [ 22 ]. However, the vesicles isolated from plant tissues can vary depending on the type of plant [ 23 ], the organ, and the culture conditions [ 18 ]. In our work, the vesicles obtained from adult plants were bigger than those obtained previously from seedlings [ 18 ]. Furthermore, the vesicles obtained from inflorescences were smaller but more heterogeneous in size than those obtained from leaves. Also, the vesicles yield from inflorescences was double that from leaves (data not shown). Although these results, that could have been due to differences in cell size and tissue lignification, may be unimportant in a plant physiological study they could be important if an industrial application is considered [ 24 ]. The main function of the plasma membrane is the regulation of the passage of diverse molecules and water through it. The Pf usually is the parameter chosen to describe water fluxes across the plant membranes that are driven by the osmolarity gradient [ 25 ]. The plasma membrane vesicles derived from broccoli leaves by Martínez-Ballesta et al. [ 26 ] had Pf values similar to the ones obtained in our work. Nevertheless, the Pf values obtained for the plasma membrane vesicles derived from 70-day and 90-day inflorescences were 1.6- and 2.5-times higher, respectively, than those of vesicles from 90-day leaves. Since little or no information concerning Pf in protoplasts or vesicles derived from Brassica inflorescences exists, these results shed light on this matter. Similar values of Pf have been reported for plasma membrane vesicles and protoplasts obtained from pepper roots (30 and 40 µm s − 1 ) [ 27 ]. Furthermore, Pf values as high as 540 µm s − 1 have been found in plasma membrane from Beta vulgaris roots [ 28 ]. This suggests that the water osmotic permeability in inflorescences might be similar to that in roots, due to their requirement for water to maintain turgor. Indeed, a relationship between cell turgor in meristematic tissue and cellular division has been reported [ 29 ]. One of the main structural components affecting the physical characteristics of biological membranes are fatty acids. The proportions of different saturated and unsaturated fatty acids may affect the permeability of the bilayer [ 30 ]. In our study, the plasma membrane vesicles obtained from cauliflower had a high proportion of unsaturated fatty acids, which provides greater fluidity [ 31 ]. In leaf plasma membranes from other species - such as broccoli, Cakile maritima L., and Brassica napus L. - linolenic acid (C18:3) was a minor component [ 23 ]. However, the proportion of this fatty acid was greater in plasma membrane vesicles produced from cauliflower leaves and inflorescences. This difference in fatty acids distribution might have a protective effect against temperature changes, since previous work was carried out in a crop chamber but we grew cauliflowers in the field; an increase in linolenic acid (C18:3) was found in peach fruits under low-temperature stress [ 32 ]. Also, a greater degree of unsaturation produces looser packing of the polyunsaturated carbon chains, decreasing the interaction with other molecules and allowing deeper penetration of water into the bilayer [ 33 ]; this could be related to the higher Pf . But, the fact that Pf was higher in 70-d inflorescences must be related to the aquaporins presence. In our vesicles derived from cauliflower leaves the oleic acid (C18:1) proportion was lower than that reported for Brassica oleracea L. var. italica in Chalbi et al. [ 23 ]. When comparing the data of B. oleracea var. italica leaves [ 23 ] with our work, a higher RUFA was obtained in cauliflower, due to the low percentage of oleic acid (5.95 ± 0.13 and 89.1 ± 5.76 for 70 and 90-day leaves vs 1.19 ± 0.18 in broccoli leaves). In the same way, the DBI was also affected by the different proportions of unsaturated fatty acids. In our analysis, the highest DBI was found in 70-day leaves, since the percentage of linolenic acid (C18:3) was higher in this sample than in B. oleracea var. italica (48.08 ± 0.64% vs 5.52 ± 0.8%). In previous studies of soy ( Glycine max L.) plasma membrane, a rise in oleic acid (C18:1) and a decrease in linoleic acid (C18:2) and linolenic acid (C18:3) were observed, increasing membrane rigidity [ 34 ]. Since the plants studied in Chalbi et al. [ 23 ] were grown in a controlled environment chamber, the lipid proportions might be quite different from those of plants cultivated in the field, where the climatic conditions, such as temperature and humidity, are highly variable. Sterols also contribute to the bilayer permeability [ 22 ]. Campesterol has been studied regarding its contribution to increasing the spatial organization of the lipid bilayer and, thus, its order [ 35 ]. Furthermore, it has been linked with a decrease in ionic permeability through lipid membranes [ 36 ]. In our work, plasma membrane from 90-day cauliflower inflorescences had the highest content of campesterol per mg of protein. This might be due to the dual function of campesterol, as a structural component and also the precursor of brassinosteroid hormones that are required for normal plant development [ 37 ]. As has been reported in Arabidopsis thaliana , brassinosteroids help root meristem growth [ 38 ]. The fact that the sitosterol/stigmasterol ratio was much higher in the plasma membranes of inflorescences, mainly in the young ones (70 d), could have contributed to the increase in Pf . In fact, sitosterol has been pointed out as the main regulator of water permeability though membranes along with aquaporins [ 22 , 39 ]. By means of the proteomic analysis performed by HPLC-ESI-QTOF-MS, the whole batch of identified proteins in each sample were analysed and categorized according to their cellular location (Fig. 4 ). However, a few proteins could not be assigned to defined categories in each fraction. Although a high percentage of plasma membrane proteins was identified in all samples (42–48%), a notable presence of organelle proteins was also found (40–41%). This could be considered contamination of our plasma membrane samples, in particular by organelle endomembranes. In the inflorescences the percentage of cytoplasm proteins found was higher than in leaves. This could be related to the higher cellular density in cauliflower inflorescences [ 7 ]. In addition, as we mentioned before, chloroplast-located proteins were identified only in samples from leaves. The presence of these proteins in leaves reflects the photosynthetic activity of this organ, whereas photosynthesis does not take place in inflorescences. Furthermore, the abundance of chloroplast proteins was greater in 70-day leaves (19.6%) than in 90-day leaves (4.1%), which could be related to the greater photosynthetic activity of the young leaves [ 40 ]. In addition, we categorized proteins based on their functional category (Fig. 5 ) [ 19 ]. The presence of different transporter activity proteins (6–8%) was detected; these comprised transmembrane ion transporters and ion channels, also located mainly in the plasma membrane. The number of detected proteins revealed that the abundance decreased as the maturity of the tissues sampled increased, being highest in young inflorescences, lower in mature inflorescences, much lower in young leaves, and lowest in mature leaves. In addition, the structural protein group was represented generally by structural constituents of ribosomes. There is a possibility that these proteins are associated with different membranes, including the plasma membrane, since it has been reported that proteins from ribosomes come from association with the endoplasmic reticulum (ER) and the ER is involved in plasma membrane turnover [ 41 ]. Table 3 shows the aquaporins identified in plasma membrane extracts of cauliflower inflorescences and leaves. As PIPs aquaporins showed a very conservative structure, the digestion by trypsine enzyme and solubilisation is very similar in all of them. Although the knowledge of the sequences of all B. oleracea aquaporins was very useful to investigate the presence of MIPs isoforms, and the analysis of peptides revealed five PIP1 isoforms (PIP1;1, PIP1;2, PIP1;3, PIP1;4, and PIP1;5) (Table 3 ), due to the high homology within the PIP1 subfamily, we could not identify isoforms unambiguously. Also, two TIPs were identified in our plasma membrane samples. Since TIPs are usually located in the tonoplast [ 42 ], their presence could be related with vacuolar contamination. However, the fact that TIPs have been found located in the plasma membrane in pea cotyledons [ 43 ] points to the possibility that TIPs were present in our plasma membrane. Additionally, PIP2 subfamily proteins were found only in inflorescences (Table 3 ), in particular PIP2;5 and PIP2;7. Of these, only PIP2;7 was unambiguously identified; this aquaporin has been shown to be expressed mainly in B. oleracea flowers [ 44 ]. Furthermore, when PIP2;7 was overexpressed in A. thaliana roots the hydraulic conductivity increased six-fold, showing the important role of PIP2;7 in water transport. Further information about plasma membrane aquaporins in cauliflower was obtained from immunoblotting. The results show that PIP1 was present in both leaves and inflorescences, although its density in inflorescences was lower. The greater presence of PIP1 proteins in leaves could be explained by its regulatory role in CO 2 transport [ 45 ]. This CO 2 transport would be indispensable for the photosynthetic activity in leaves, whereas in inflorescences the constant cell division and growth would produce CO 2 that would need to be carried to the leaves. In addition, the other PIP1 members have been postulated as O 2 transport facilitators; in particular, PIP1;3 in tobacco plants [ 46 ]. In addition, the results for PIP2 show a greater density in samples from inflorescences - which mirrors the information obtained in the proteomic assay, in which PIP2 could only be detected in those samples (Fig. 6 ). The exclusive identification of PIP2 proteins in inflorescences (PIP2;1, PIP2;2, PIP2;5, and PIP2;7) could be related to the elevated demand for water in cauliflower inflorescences, to maintain nutrient uptake and turgor [ 47 ]. The localization of PIP2 in plasma membrane samples from inflorescences (Fig. 6 ) could also explain the differences observed in Pf ; the 90-day inflorescences showed the highest Pf , in accordance with the higher concentration of PIP2 found in these samples (Fig. 3 ). The non-detection of PIP2 in leaves could indicate that the function of PIPs is performed here by TIPs or that only limited water transport across the lipid bilayer occurs. However, this aspect needs to be investigated further. Conclusions In summary, this study shows that the inflorescence of cauliflower, rich in meristematic tissue, has a large amount of plasma membrane per gram of tissue. This membrane is characterized by a low degree of unsaturation, which could increase the rigidity decreasing water transport, but the high content in sitosterol (highly correlated with water passage) would compensate this fact. In relation to this, the high presence of aquaporins in inflorescences, especially PIP2;5 and PIP2;7, indicates a potential role of aquaporins in the water transport required for the continuous development of the meristematic tissue. Furthermore, the fact that the aquaporins contribution to water transport in inflorescences must be higher than in leaves, with a potential correlation with the stage of development, provides to sterols and aquaporins a specific role in development. Our work highlights the need for further research on specific aquaporins in relation to adult plant development under natural conditions. Abbreviations DBI: Double bond index; ER: Endoplasmic reticulum; FID: Flame ion detector; HPLC: High Performance Liquid Chromatography; MUFA: Monounsaturated fatty acids; PIP: Plasma membrane intrinsic proteins; PUFA: Polyunsaturated fatty acids; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS: Tween 20 blocking solution; TIP: Tonoplast intrinsic protein Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding This work was funded by the CDTI, Spain (BIOTAGUT) and by the Spanish Ministerio de Ciencia, Innovación y Universidades (AGL2016-80247-C2-1-R). P. García-Ibañez was funded by a grant from the Fundación Séneca-CARM, Spain (21273/FPI/19). Authors' contributions MCA contributed to the conception and design of this work. PGI carried out the experiments and JNE performed the analytical work of proteomics. PGI and JNE prepared figures and tables, and prepared the first draft of the manuscript. MCA contributed to manuscript revisions, reads and approved the submitted version. MCA obtained the funding. All authors have read and approved the manuscript. Acknowledgements The authors thank Dr. D. Walker for the correction of the English in the manuscript. References Thorwarth P, Yousef EAA, Schmid KJ. Genomic prediction and association mapping of curd-related traits in gene bank accessions of cauliflower. 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Grosjean K, Mongrand S, Beney L, Simon-Plas F, Gerbeau-Pissot P. Differential effect of plant lipids on membrane organization specificities of phytosphingolipids and phytosterols. J Biol Chem. 2015;290:5810–25. Benz R, Cros D. Influence of sterols on ion transport through lipid bilayer membranes. BBA - Biomembr. 1978;506:265–80. Zullo MAT, Bajguz A. The Brassinosteroids family – structural diversity of natural compounds and their precursors. In: Brassinosteroids: Plant Growth and Development. 2019. Chaiwanon J, Wang ZY. Spatiotemporal brassinosteroid signaling and antagonism with auxin pattern stem cell dynamics in Arabidopsis roots. Curr Biol. 2015;25:1031–42. López-Pérez L, Fernández-García N, Olmos E, Carvajal M. The phi thickening in roots of broccoli plants: An acclimation mechanism to salinity? Int J Plant Sci. 2007;168:1141–9. David MM, Coelho D, Barrote I, Correia MJ. Leaf age effects on photosynthetic activity and sugar accumulation in droughted and rewatered Lupinus albus plants. Aust J Plant Physiol. 1998;25:299–306. Staehelin LA. The plant ER: A dynamic organelle composed of a large number of discrete functional domains. Plant J. 1997;11:1151–65. Chaumont F, Barrieu F, Herman EM, Chrispeels MJ. Characterization of a maize tonoplast aquaporin expressed in zones of cell division and elongation. Plant Physiol. 1998;117:1143–52. Robinson DG, Haschke HP, Hinz G, Hoh B, Maeshima M, Marty F. Immunological detection of tonoplast polypeptides in the plasma membrane of pea cotyledons. Planta. 1996;198:95–103. Diehn TA, Pommerrenig B, Bernhardt N, Hartmann A, Bienert GP. Genome-wide identification of aquaporin encoding genes in Brassica oleracea and their phylogenetic sequence comparison to Brassica crops and Arabidopsis. Front Plant Sci. 2015;6:1–20. Uehlein N, Sperling H, Heckwolf M, Kaldenhoff R. The Arabidopsis aquaporin PIP1;2 rules cellular CO 2 uptake. Plant Cell Environ. 2012;35:1077–83. Tyerman SD, Wignes JA, Kaiser BN. Root hydraulic and aquaporin responses to N availability. In: Plant Aquaporins. Signaling and Communication in Plants. Cham: Springer; 2017. pp. 207–36. Schumann C, Knoche M. Swelling of cell walls in mature sweet cherry fruit: factors and mechanisms. Planta. 2020;251:65. Cite Share Download PDF Status: Published Journal Publication published 07 Jan, 2021 Read the published version in BMC Plant Biology → Version 1 posted 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-29232","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":657156,"identity":"6f932046-18c4-4b6e-a764-83f3126d6f07","order_by":0,"name":"Paula Garcia-Ibañez","email":"","orcid":"","institution":"Centro de Edafologia y Biologia Aplicada del Segura","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"","lastName":"Garcia-Ibañez","suffix":""},{"id":657157,"identity":"670e8292-a576-4a8b-82b7-e071be6bb774","order_by":1,"name":"Juan Nicolas-Espinosa","email":"","orcid":"","institution":"Centro de Edafologia y Biologia Aplicada del Segura","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Nicolas-Espinosa","suffix":""},{"id":657158,"identity":"b26568a4-d09b-49ca-bcd2-b219be73e247","order_by":2,"name":"Micaela Carvajal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYPACNgZ+EAEGzMRqkWwgUQsDg8EBmBZCQLf98LPPPDV88sbHDx978OMPg7x8O/MDhg9/cGsxO5NmPJvnGJvhtjNp6Ya9bQyGGw6zGTDObMOj5QaDMeMMNjbGbTd4zCR4GxgSDJh5GJiBDDxa2D8zzvjHZr95Bv83yT9/GBLkm4Fa/uBz2A0eY4aPbWyJGyR42KR52BgSGA4DteALCrMzOcUMH/vYkmecSTM3lm2TAPvlYC8+vxw/vpkh4dsx235g0D1888dGXr7/8ENQ0BECx2AMCTB5gKAGBoYaItSMglEwCkbBiAUAnKVLFR51MUUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7321-4956","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Micaela","middleName":"","lastName":"Carvajal","suffix":""}],"badges":[],"createdAt":"2020-05-14 21:41:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-29232/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-29232/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-020-02778-6","type":"published","date":"2021-01-07T15:02:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1307989,"identity":"4a0a31f3-e9d0-400b-9dfb-11a066d0710a","added_by":"auto","created_at":"2020-06-11 16:38:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33473,"visible":true,"origin":"","legend":"Comparative size (nm) of plasma membrane vesicles obtained from 70- and 90-day cauliflower inflorescences and leaves. The data are represented as the means (n=3, where n=different plasma membrane extractions) ± SE. Different letters show statistically significant differences (p \u003c 0.05). ","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-29232/v1/fig1.png"},{"id":1307990,"identity":"738b835e-14b8-4175-b0aa-0defa697d3b3","added_by":"auto","created_at":"2020-06-11 16:38:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91218,"visible":true,"origin":"","legend":"Polydispersity curves of plasma membrane vesicles obtained from 70- and 90-day cauliflower inflorescences and leaves. The curves are mean values (n=3, where n=different plasma membrane extractions) ± SE.","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-29232/v1/fig2.png"},{"id":1307991,"identity":"f199667b-99da-427a-8c6e-19c111dbcdbd","added_by":"auto","created_at":"2020-06-11 16:38:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30715,"visible":true,"origin":"","legend":"Osmotic water permeability (Pf, µm s-1) of plasma membrane vesicles obtained from 70-day and 90-day cauliflower inflorescences and leaves. The data are the means (n=3, where n=different plasma membrane extractions) ± SE. Different letters indicate statistically significant differences (p \u003c 0.05, Tukey’s test) between treatments.","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-29232/v1/fig3.png"},{"id":1307992,"identity":"354f22ff-9767-4ad3-8d12-13cabf35d87e","added_by":"auto","created_at":"2020-06-11 16:38:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":142615,"visible":true,"origin":"","legend":"Classification of plasma membrane proteins identified in cauliflower, according to their cellular location. The proteins identified were classified based on eight categories described in the Gene Ontology database [19]. The number and proportion of proteins classified in each category are shown. (A) 70-day inflorescences, (B) 90-day inflorescences, (C) 70-day leaves, and (D) 90-day leaves. ","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-29232/v1/fig4.png"},{"id":1307993,"identity":"fe1f1c0e-d121-4607-92f9-e48f7ab69363","added_by":"auto","created_at":"2020-06-11 16:38:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":153060,"visible":true,"origin":"","legend":"Functional classification of plasma membrane proteins identified in cauliflower. The proteins identified were classified based on nine categories described in the Gene Ontology database [19]. The number and proportion of proteins classified in each category are shown. (A) 70-day inflorescences, (B) 90-day inflorescences, (C) 70-day leaves, and (D) 90-day leaves. ","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-29232/v1/fig5.png"},{"id":1307994,"identity":"fe2d526e-0ea1-439e-8350-df37d2e3b82b","added_by":"auto","created_at":"2020-06-11 16:38:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":128820,"visible":true,"origin":"","legend":"Immunoblotting analysis for PIP1 and PIP2 aquaporins present in cauliflower plasma membrane vesicles. A: plasma membrane from 70-day inflorescences, B: plasma membrane from 90-day inflorescences, C: plasma membrane from 70-day leaves, D: plasma membrane from 90-day leaves. ","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-29232/v1/fig6.png"},{"id":13541308,"identity":"72bef612-d1be-4c77-9e25-9ef36e918fe3","added_by":"auto","created_at":"2021-09-17 01:50:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1025694,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-29232/v1/99975b05-143e-43f0-86b4-6db6dc5b9f8e.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePlasma Membrane Vesicles From Cauliflower Meristematic Tissue And Their Role In Water Passage\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eNowadays, crops from the \u003cem\u003eBrassicaceae\u003c/em\u003e family are among the ones most cultivated, worldwide. Of these, the cauliflower (\u003cem\u003eBrassica oleracea\u003c/em\u003e L. var. \u003cem\u003ebotrytis\u003c/em\u003e) stands out for its high production (24\u0026nbsp;million tonnes in 2014) and economic relevance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The main reason for its great demand is the presence of diverse health-promoting bioactives - such as glucosinolates, polyphenols, or vitamin C - that add great nutritional value to its edible part [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, little recent information on the phytochemistry of the cauliflower inflorescence molecular structures is available. Smyth [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] described how this inflorescence begins to develop with the formation of secondary meristems. After that, a continuous proliferation of meristematic tissue takes place, generating a highly branched compact pattern with a whitish color and spiral-like shape [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Following this, the immature inflorescence initiates its maturation, finally reaching total floral differentiation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although the intervention of genes related to the identity of the floral meristem, such as \u003cem\u003eTFL1, LFY, AP1\u003c/em\u003e, or \u003cem\u003eCAL\u003c/em\u003e, has been studied [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], further comprehension of the biochemical mechanisms underlying this process is needed. In addition, Grevsen et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] observed that environmental factors, mainly temperature, influence the generation of the inflorescences.\u003c/p\u003e \u003cp\u003eAs the main focus of the research to date has been the genetic control of the inflorescence development, little is known about the lipid and protein composition of the membranes present in it. However, a high cellular proliferation is present in the edible part of cauliflower, giving a high membrane concentration [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Biological membranes are relevant to cell functionality, due to their ability to create an adequate environment for diverse types of proteins, such as transmembrane proteins. The latter mainly determine the specific functionality of each type of membrane present in cells and control diverse constitutive functions, such as endocytosis and ion and water transport [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the important transmembrane proteins, aquaporins are one of the main protagonists, since they are responsible for the water transport through biological membranes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In cauliflower meristematic tissue cells, it has been observed that there is a high abundance of aquaporins embedded in the vacuolar membrane, which allows swelling of growing cells, while maintaining the cellular turgor [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the functionality of these proteins can be affected by the lipids of the membrane in which they are embedded [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This suggests that the lipid composition of the membrane determines not only its physical characteristics but also the activity and functionality of the proteins present in it [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne of the main components of lipid membranes are sterols. These molecules have been linked with different functions, such as lipid packing, since they are able to interact with membrane proteins and fatty acids [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, a high sterol (mainly stigmasterol, campesterol, and β-sitosterol in plant membranes) content has been related to an increment in membrane water permeability [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Other fundamental components of cellular membranes are fatty acids, since they contribute to the thickness, stability, and permeability, according to the proportions of saturated and unsaturated fatty acids [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, both the lipid and the protein composition are determinants of the membrane transport activity, giving each type of membrane specific functional characteristics. For the above reasons, and with the aim of determining the specific functions of the plasma membrane of the cauliflower meristematic tissue, in this work the presence of fatty acids, sterols, aquaporins, and other proteins was analyzed in two different stages of inflorescence development. To allow a comparison with vegetative tissue, leaf plasma membranes were also analyzed.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eFifty commercial cauliflower seeds (from Sakata Seed Iberica S.L.U., Valencia, Spain) were induced to germinate by imbibition with water and continuous aeration for 24\u0026nbsp;h. Then, the seeds were transplanted to vermiculite and were kept in darkness, at 28\u0026ordm;C and 60% relative humidity, for two days. The seedlings (5 days old) were transferred to the agricultural soil of an experimental farm (37\u0026deg;47'52.7\"N, 0\u0026deg;52'00.7\"W, 15\u0026nbsp;m asl, Murcia, Spain). The experiment was carried out from December to February with average temperatures and relative humidities of 17\u0026ordm;C and 60% (day), and 4\u0026ordm;C and 65% (night), under a semi-arid Mediterranean climate. The daily average temperature and relative humidity were recorded with dataloggers (AFORA S.A., Barloworld Scientific, Murcia, Spain). All plants were drip-irrigated with \u0026frac14;-strength Hoagland nutrient solution. They were harvested at 70 and 90 days after transplanting. The intermediate leaves and inflorescences (15 of each) were sampled at random and weighed fresh in three technical replicates. After that, samples were kept in storage at 4\u0026ordm;C for one day until processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasma membrane extraction\u003c/h2\u003e \u003cp\u003eSamples of fresh material (100\u0026nbsp;g) were sliced in small pieces and vacuum-infiltrated with a 1:1.6 (w/v) proportion of an extraction buffer (0.5\u0026nbsp;M sucrose, 1\u0026nbsp;mM DTT, 50\u0026nbsp;mM HEPES, 1.30\u0026nbsp;mM ascorbic acid, pH 7.5) and 0.5\u0026nbsp;g of PVP. After 10\u0026nbsp;min, samples were homogenized and filtered through a nylon mesh with a pore diameter of 100\u0026nbsp;\u0026micro;m. Then, the filtrate was centrifuged at 10000x\u003cem\u003eg\u003c/em\u003e for 30\u0026nbsp;min, at 4\u0026ordm;C; the supernatants were collected and centrifuged for 35\u0026nbsp;min at 50000x\u003cem\u003eg\u003c/em\u003e, at 4\u0026ordm;C. The pellet obtained was resuspended in 500\u0026nbsp;\u0026micro;l of a buffer containing 5\u0026nbsp;mM PBS and 0.5\u0026nbsp;M sucrose (pH 6.5) (FAB). Three different extractions per sample type were performed. Two milliliters of this microsomal fraction were introduced in a two-phase system composed of PEG-3350/Dextran-T500-6.3% (w/w), 5\u0026nbsp;mM KCl, 330\u0026nbsp;mM sucrose, 2.5\u0026nbsp;mM NaF, and 5\u0026nbsp;mM K\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (pH 7.8). The system was centrifuged for 5\u0026nbsp;min at 4000\u003cem\u003exg\u003c/em\u003e. Then, the upper phase was collected and a wash was performed with a buffer containing 9\u0026nbsp;mM KCl, 0.2\u0026nbsp;M EGTA, 0.5\u0026nbsp;mM NaF, and 10\u0026nbsp;mM Tris-borate (pH 8.3). Then, a centrifugation at 55000\u003cem\u003exg\u003c/em\u003e for 35\u0026nbsp;min, at 4\u0026ordm;C, was performed. The pellet obtained was resuspended in FAB. The final protein concentration was determined using an RC DC protein assay kit (BioRad, California, USA), with bovine serum albumin as the standard. Three different extractions per sample type were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eVesicle size\u003c/h2\u003e \u003cp\u003eThe mean size of the vesicles obtained from different samples was determined by dynamic light scattering, using a Malvern ZetaSizer Nano XL (Malvern Instruments Ltd., Orsay, France) as described by Barraj\u0026oacute;n-Catal\u0026aacute;n et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This instrument allows the analysis of particles with diameters from 1\u0026nbsp;nm to 3\u0026nbsp;\u0026micro;m.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStopped flow light scattering\u003c/h2\u003e \u003cp\u003eThese measurements were performed in a PiStar (Applied Photophysics, Leatherhead, UK) spectrophotometer at 20\u0026ordm;C, as described in Maurel [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The kinetics of vesicle volume adjustment were monitored by dynamic light scattering at 90\u0026ordm; and with a λ\u003csub\u003eex\u003c/sub\u003e of 515\u0026nbsp;nm. Purified plasma membrane vesicles were subjected to a 100x dilution in a buffer with 30\u0026nbsp;mM KCl and 20\u0026nbsp;mM Tris-Mes (pH 8.3, 90 mOsmol kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e H\u003csub\u003e2\u003c/sub\u003eO). For the measurement, the diluted vesicle preparation was mixed in a 1:1 proportion (v:v) with the same buffer supplemented with 540\u0026nbsp;mM sucrose (630 mOsmol kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e H\u003csub\u003e2\u003c/sub\u003eO). In this way, an osmotic gradient of 270 mOsmol kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e H\u003csub\u003e2\u003c/sub\u003eO was generated. The osmotic permeability (\u003cem\u003ePf\u003c/em\u003e) was calculated using this formula:\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" style=\"width: 233px;\"\u003e\u003c/p\u003e \u003cp\u003eWhere K\u003csub\u003eexp\u003c/sub\u003e is the adjusted exponential velocity constant, V\u003csub\u003e0\u003c/sub\u003e is the mean vesicular volume, A\u003csub\u003ev\u003c/sub\u003e is the mean vesicular surface area, V\u003csub\u003ew\u003c/sub\u003e is the water molar mass, and C\u003csub\u003eout\u003c/sub\u003e is the external osmolarity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLipids and sterols analysis\u003c/h2\u003e \u003cp\u003eFive hundred microliters of plasma membrane were mixed with a chloroform-methanol (1:2) mixture. As an internal standard for further sterol analysis, β-colestanol (20\u0026nbsp;\u0026micro;l, at 0.1\u0026nbsp;mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was added. Then, 0.25\u0026nbsp;ml of chloroform were added to the mixture before centrifugation at 10000\u003cem\u003exg\u003c/em\u003e for 6\u0026nbsp;min. The resultant interphase, corresponding to the protein content, was collected for further analysis. The chloroformic phase was removed to another tube and evaporated with N\u003csub\u003e2\u003c/sub\u003e. For sterol analysis, 50\u0026nbsp;\u0026micro;l samples of the chloroformic phase were dried with N\u003csub\u003e2\u003c/sub\u003e and then acetylated using pyridine (50\u0026nbsp;\u0026micro;l) and Ac\u003csub\u003e2\u003c/sub\u003eO (100\u0026nbsp;\u0026micro;l). After 2\u0026nbsp;h, the solvents were evaporated with N\u003csub\u003e2\u003c/sub\u003e and 20\u0026nbsp;\u0026micro;l of ethyl acetate were added. Sterols and fatty acids were determined by gas chromatography, employing an HP5 capillary column (30\u0026nbsp;m x 0.25\u0026nbsp;mm x 0.25\u0026nbsp;\u0026micro;m). This was coupled to a flame ionization detector (FID). Helium was used as the mobile phase (1\u0026nbsp;ml min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and a heat gradient was imposed: from 150 to 195\u0026ordm;C, increasing 3\u0026ordm;C per min, then from 195 to 220\u0026ordm;C at 2\u0026ordm;C per min, and from 220 to 300\u0026ordm;C at 6\u0026ordm;C per min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProteomic analysis\u003c/h2\u003e \u003cp\u003eSamples (500\u0026nbsp;\u0026micro;l) were mixed with 100\u0026nbsp;\u0026micro;l of 50\u0026nbsp;mM ammonium bicarbonate (pH 8.3) with 0.01% Protease Max (Promega, Madison, USA). Then, the samples were reduced by adding 100\u0026nbsp;\u0026micro;l of 20\u0026nbsp;mM DTT at 56\u0026ordm;C, for 20\u0026nbsp;min. After that, alkylation was performed by incubation with 100\u0026nbsp;\u0026micro;l of 100\u0026nbsp;mM IAA for 30\u0026nbsp;min, at room temperature and in the dark. Digestion was performed by incubation with 1\u0026nbsp;\u0026micro;g of trypsin (1:100 w/w) for 3\u0026nbsp;h, at 37\u0026ordm;C. The samples were dried in a speed vacuum concentrator. The dry samples were resuspended in 20\u0026nbsp;\u0026micro;l of water/acetonitrile/formic acid (94.9:5:0.1). Then, they were injected onto an Agilent Advance Bio Peptide Mapping HPLC column (2.7\u0026nbsp;\u0026micro;m x 100\u0026nbsp;mm x 2.1\u0026nbsp;mm, Agilent technologies) thermostatted at 55\u0026ordm;C and with a flow rate of 0.4\u0026nbsp;ml/min. A mixture of water/acetonitrile/formic acid (10:89.9:0.1) was used as the eluent. For detection, an Agilent 6550 Q-TOF coupled with a dual electrospray (AJS-Dual ESI) was used. The experimental parameters were set in MassHunter Workstation Data Acquisition software (Agilent Technologies, Santa Clara, CA, USA), as described in Mart\u0026iacute;nez-Ballesta et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The data were processed with Spectrum Mill MS Proteomics Workbench (Agilent Technologies).\u003c/p\u003e \u003cp\u003eThe data obtained were compared with the information available in the UniProt database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.uniprot.org\" target=\"_blank\"\u003ewww.uniprot.org\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e) for the \u003cem\u003eBrassicaceae\u003c/em\u003e family. Protein function and location were determined from the Gene Ontology database [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGel electrophoresis and immunoblotting\u003c/h2\u003e \u003cp\u003ePlasma membrane isolated from cauliflower leaves and inflorescences was employed. Ten micrograms of protein per lane were loaded for 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as shown in Muries et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Then, the proteins were transferred to a PVDF membrane and maintained for 20\u0026nbsp;min at 15\u0026nbsp;V in an electrophoretic transfer cell (Trans-Blot SD cell, BioRad, CA, USA), using Towing transfer buffer supplemented with 0.05% SDS [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Blocking solution (TBS containing 2% (w/v) skimmed dry milk) was applied to the membrane for 1\u0026nbsp;h at room temperature. After that, the membrane was again incubated for 1\u0026nbsp;h at room temperature, with TBS containing 0.05% Tween 20 and one of the selected antibodies. An antibody raised against the first 45\u0026nbsp;N-terminus residues of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e PIP1;1 (dilution 1:3000, kindly provided by Prof. Dr. Anthony Sch\u0026auml;ffner) and another raised against 17 residues from the C-terminal peptide of PIP2;2 of \u003cem\u003eA. thaliana\u003c/em\u003e (dilution 1:20000, kindly provided by Dr. Veronique Santoni) were used. Incubation was performed overnight at 4\u0026ordm;C. Goat anti-rabbit IgG coupled to horseradish peroxidase was employed as a secondary antibody (dilution 1:20000). A chemiluminescent signal was developed with West-Pico Super Signal substrate (Pierce, Rockford, IL, USA). The quantification was carried out using ImageJ software and by performing a densitometry analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis comprised a one-way ANOVA followed by a Tukey HSD \u003cem\u003epost hoc\u003c/em\u003e test, performed using RStudio (version 3.4.4.).\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMean size of plasma membrane vesicles\u003c/h2\u003e \u003cp\u003eThe results for the mean vesicle size (nm), represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, show significant differences between inflorescences and leaves and between the maturation stages (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). The mean vesicle size was greater for leaves-derived plasma membrane vesicles than for inflorescences-derived ones (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The polydispersity data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) show that the variability in the size of inflorescences-derived plasma membrane vesicles was higher than for leaves-derived ones (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). Hence, vesicles obtained from leaves were more homogeneous in size.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOsmotic water permeability\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the osmotic water permeability (\u003cem\u003ePf\u003c/em\u003e, \u0026micro;m s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of the plasma membrane vesicles differed significantly between the two maturation stages of the inflorescences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), those derived from 90-day inflorescences having the highest \u003cem\u003ePf\u003c/em\u003e (64.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.14\u0026nbsp;\u0026micro;m s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Lower values were obtained for leaves, with no significant differences between the two maturation stages (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLipid analysis\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eFatty acids\u003c/h2\u003e \u003cp\u003eThe results of the fatty acids (% of total fatty acids) analysis are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The percentage of palmitoleic acid (C16:1) did not vary between 70 days and 90 days for inflorescences-derived plasma membrane vesicles (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Furthermore, a similar percentage of palmitoleic acid was found in vesicles derived from 90-day leaves (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), but a significant decrease was observed for vesicles from 70-day leaves (~\u0026thinsp;29\u0026ndash;31% vs\u0026thinsp;~\u0026thinsp;19%). For oleic acid (C18:1), similar percentages were found in vesicles derived from inflorescences at the two maturation stages (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Surprisingly, the presence of oleic acid in leaves-derived vesicles greatly differed, the percentage in 70-day leaves being double that in inflorescences and almost 14-times higher when compared to 90-day leaves (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For linoleic acid (C18:2), a statistically significant decrease was found between day 70 and day 90 for inflorescences-derived plasma membrane vesicles (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the opposite was observed in leaves-derived vesicles, the content of linoleic acid being increased at 90-days (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The linolenic acid (C18:3) percentage differed significantly among the four types of sample (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Higher proportions were found in leaves, the vesicles from 70-day leaves having the highest percentage (~\u0026thinsp;48%, vs\u0026thinsp;~\u0026thinsp;44% for 90-day leaves). For inflorescences-derived vesicles, the proportion of linolenic acid was higher in 90-day samples than in those taken at 70 days (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ~\u0026thinsp;40% vs\u0026thinsp;~\u0026thinsp;35%).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFatty acid percentage, double bond index (DBI= \u0026sum;(unsaturated fatty acids x number of double bonds)), and percentage of monounsaturated fatty acids (MUFA), for plasma membrane from cauliflower inflorescences and leaves at 70 days and 90 days of development. The data are represented as the means (n\u0026thinsp;=\u0026thinsp;3, where n\u0026thinsp;=\u0026thinsp;different plasma membrane extractions)\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. Different letters indicate statistically significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Tukey\u0026rsquo;s test) between treatments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eInflorescences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eLeaves\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e% Fatty acids\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e70-day\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e90-day\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e70-day\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e90-day\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePalmitoleic acid (C16:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOleic cid (C18:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLinoleic acid (C18:2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLinolenic acid (C18:3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMUFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDBI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e166.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e173.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e198.65\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e184.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.62b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe total percentages of monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) were also determined (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The MUFA percentages for inflorescences-derived samples were significantly higher (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05) when compared to leaves. As well, no statistically significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the double bond index (DBI) were found between the maturation stages for inflorescences-derived samples. However, the DBI was significantly higher (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in samples from 70-day leaves than in those from 90-day leaves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003eSterol content\u003c/h2\u003e \u003cp\u003eThe sterol content (\u0026micro;g/mg of protein) of plasma membrane vesicles was also assessed (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The campesterol concentration showed a statistically significant increase in vesicles derived from 70-day inflorescences, being 3-fold higher than in those from 90-day ones (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No differences in the campesterol concentration were found between vesicles derived from 70- and 90-day leaves (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). For stigmasterol, no statistically significant differences were found between vesicles from inflorescences at the two maturation stages (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, in leaves-derived vesicles, the concentration of stigmasterol was higher for 90-day leaves (0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 vs 0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u0026nbsp;\u0026micro;g/mg of protein, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A 2-fold, statistically significant increase in β-sitosterol was found in vesicles from 90-day inflorescences when compared to those of 70-day inflorescences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A similar difference was seen when comparing vesicles from 70-day leaves with those of 90-day leaves (0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 vs 2.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u0026nbsp;\u0026micro;g/mg of protein, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The stigmasterol/β-sitosterol ratio was also analysed. The highest ratio was found in vesicles derived from 70-day inflorescences, 2-times higher than for 90-day inflorescences and 5-times higher than for both 70-day and 90-day leaves (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A statistically significant difference in the stigmasterol/β-sitosterol ratio was not found between 70-day and 90-day leaves (0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 vs 0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u0026nbsp;\u0026micro;g/mg of protein, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSterol content (\u0026micro;g/mg of protein) of plasma membrane from cauliflower inflorescences and leaves at 70 days and 90 days of plant development. The data are represented as the means (n\u0026thinsp;=\u0026thinsp;3, where n\u0026thinsp;=\u0026thinsp;different plasma membrane extractions)\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. Different letters indicate statistically significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Tukey\u0026rsquo;s test) between treatments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eInflorescences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eLeaves\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e\u0026micro;g/mg of protein\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e70-day\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e90-day\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e70-day\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e90-day\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCampesterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStigmasterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-Sitosterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStigmasterol/β-Sitosterol ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eProteomic analysis and immunoblotting\u003c/h2\u003e \u003cp\u003eA proteomic analysis was performed with the samples from inflorescences and leaves in order to assess qualitatively the proteins present in the vesicles obtained. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the proteins obtained were organized according to their cellular anatomical entity characteristics. In all samples studied (inflorescences and leaves at both maturation stages) membrane component related proteins were the most abundant (42\u0026ndash;48%). A high percentage of the proteins identified were organelle related; most of them were components of organelles membranes (40\u0026ndash;41%). Nevertheless, differences between membranes derived from inflorescences and those from leaves were observed. Cytoplasm proteins represented a higher percentage in the inflorescences (12%) than in the leaves (2%). Chloroplast proteins were identified in leaves samples, in contrast to inflorescences samples. Furthermore, a higher number of chloroplast proteins were identified in 70-day leaves samples than in those of 90-day leaves.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProteins were also classified by their molecular function (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). They were grouped in nine functional clusters (catalytic, structural molecules, transporter, binding, translation factor, antioxidant, molecular function regulator, enzyme regulator, and nutrient reservoir activities). All the samples showed the same protein activity distribution, the binding proteins (38\u0026ndash;40%) standing out as the main group, followed by catalytic activity proteins (32\u0026ndash;35%), structural proteins (16\u0026ndash;18%), and transport activity proteins (6\u0026ndash;8%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA search focused on the aquaporins present in plasma membrane derived from cauliflower inflorescences and leaves was also performed. Aquaporin-related peptides were spotted in all sample types (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In both inflorescences and leaves, peptides corresponding to a wide group of PIP1 aquaporin subfamilies (PIP1;1, PIP1;2, PIP1;3, PIP1;4, and PIP1;5) were identified. Nevertheless, only in the inflorescence samples were peptides related to the PIP2 subfamily detected, PIP2;5 and PIP2;7. Special emphasis can be placed on PIP2;7, for which three different peptide fragments were detected, while only one peptide determined PIP2;5. Members of the Tonoplast Intrinsic Protein (TIP) subfamily were also identified in both types of sample, although this group of aquaporins is generally targeted to the vacuolar membrane. In particular, TIP1;2 and TIP2;1 were found in 70-day inflorescences and 90-day leaves, while only TIP1;2 peptides were detected in 90-day inflorescences and 70-day leaves samples (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAquaporin proteins identified in plasma membrane samples of cauliflower inflorescences and leaves. All protein sequences were retrieved from \u003cem\u003eBrassica oleracea\u003c/em\u003e L. var. \u003cem\u003eoleracea\u003c/em\u003e information in the NCBI and UniProt databases (ID). The symbols \u003cb\u003e\u0026lsquo;+\u0026rsquo;\u003c/b\u003e and \u003cb\u003e\u0026lsquo;-\u0026rsquo;\u003c/b\u003e indicate the presence or absence of the protein in the samples, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNCBI ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eUniProt ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eInflorescences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eLeaves\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e70-day\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e90-day\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e70-day\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e90-day\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePIP1;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_013604594.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A0D3DUU2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePIP1;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_013637020.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A0D3C6I1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePIP1;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_013600561.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A0D3C6T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePIP1;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_013612790.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A0D3D7M3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePIP1;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_013599049.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A0D3DFM5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePIP2;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_013599897.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A0D3DT38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePIP2;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_013629883.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A0D3BL75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTIP1;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_013613430.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A0D2ZPE6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTIP2;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP_013587105.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA0A0D3CJP0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe results obtained from SDS-PAGE analysis of plasma membrane proteins from leaves and inflorescences are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The presence of two bands was detected; an upper band of 60\u0026nbsp;kDa, corresponding to dimeric (D) forms of PIPs, and a lower band of ca. 30\u0026nbsp;kDa, corresponding to the monomeric form (M). Two PIPs groups were analysed, PIP1 and PIP2. For PIP1, bands from samples of 70-day and 90-day inflorescences (23.4% and 15.7% D\u0026thinsp;+\u0026thinsp;M) were less dense than those of 70-day and 90-day leaves-derived plasma membrane proteins (31% and 28.9% D\u0026thinsp;+\u0026thinsp;M). For PIP2 aquaporins, much denser bands were found for 70-day (27.7% D\u0026thinsp;+\u0026thinsp;M) and 90-day inflorescences (61.4% D\u0026thinsp;+\u0026thinsp;M) when compared with 70-day and 90-day leaves-derived samples (5.2% and 5.7% D\u0026thinsp;+\u0026thinsp;M).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eThe isolation of plasma membrane vesicles using the two-phase aqueous polymer technique [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] has been reported to produce homogeneous material in terms of yield and composition [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the vesicles isolated from plant tissues can vary depending on the type of plant [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the organ, and the culture conditions [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In our work, the vesicles obtained from adult plants were bigger than those obtained previously from seedlings [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, the vesicles obtained from inflorescences were smaller but more heterogeneous in size than those obtained from leaves. Also, the vesicles yield from inflorescences was double that from leaves (data not shown). Although these results, that could have been due to differences in cell size and tissue lignification, may be unimportant in a plant physiological study they could be important if an industrial application is considered [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main function of the plasma membrane is the regulation of the passage of diverse molecules and water through it. The \u003cem\u003ePf\u003c/em\u003e usually is the parameter chosen to describe water fluxes across the plant membranes that are driven by the osmolarity gradient [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The plasma membrane vesicles derived from broccoli leaves by Mart\u0026iacute;nez-Ballesta et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] had \u003cem\u003ePf\u003c/em\u003e values similar to the ones obtained in our work. Nevertheless, the \u003cem\u003ePf\u003c/em\u003e values obtained for the plasma membrane vesicles derived from 70-day and 90-day inflorescences were 1.6- and 2.5-times higher, respectively, than those of vesicles from 90-day leaves. Since little or no information concerning \u003cem\u003ePf\u003c/em\u003e in protoplasts or vesicles derived from \u003cem\u003eBrassica\u003c/em\u003e inflorescences exists, these results shed light on this matter. Similar values of \u003cem\u003ePf\u003c/em\u003e have been reported for plasma membrane vesicles and protoplasts obtained from pepper roots (30 and 40\u0026nbsp;\u0026micro;m s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, \u003cem\u003ePf\u003c/em\u003e values as high as 540\u0026nbsp;\u0026micro;m s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e have been found in plasma membrane from \u003cem\u003eBeta vulgaris\u003c/em\u003e roots [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This suggests that the water osmotic permeability in inflorescences might be similar to that in roots, due to their requirement for water to maintain turgor. Indeed, a relationship between cell turgor in meristematic tissue and cellular division has been reported [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne of the main structural components affecting the physical characteristics of biological membranes are fatty acids. The proportions of different saturated and unsaturated fatty acids may affect the permeability of the bilayer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In our study, the plasma membrane vesicles obtained from cauliflower had a high proportion of unsaturated fatty acids, which provides greater fluidity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In leaf plasma membranes from other species - such as broccoli, \u003cem\u003eCakile maritima\u003c/em\u003e L., and \u003cem\u003eBrassica napus\u003c/em\u003e L. - linolenic acid (C18:3) was a minor component [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the proportion of this fatty acid was greater in plasma membrane vesicles produced from cauliflower leaves and inflorescences. This difference in fatty acids distribution might have a protective effect against temperature changes, since previous work was carried out in a crop chamber but we grew cauliflowers in the field; an increase in linolenic acid (C18:3) was found in peach fruits under low-temperature stress [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Also, a greater degree of unsaturation produces looser packing of the polyunsaturated carbon chains, decreasing the interaction with other molecules and allowing deeper penetration of water into the bilayer [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]; this could be related to the higher \u003cem\u003ePf\u003c/em\u003e. But, the fact that \u003cem\u003ePf\u003c/em\u003e was higher in 70-d inflorescences must be related to the aquaporins presence. In our vesicles derived from cauliflower leaves the oleic acid (C18:1) proportion was lower than that reported for \u003cem\u003eBrassica oleracea\u003c/em\u003e L. var. italica in Chalbi et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. When comparing the data of \u003cem\u003eB. oleracea\u003c/em\u003e var. italica leaves [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] with our work, a higher RUFA was obtained in cauliflower, due to the low percentage of oleic acid (5.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 and 89.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.76 for 70 and 90-day leaves vs 1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 in broccoli leaves). In the same way, the DBI was also affected by the different proportions of unsaturated fatty acids. In our analysis, the highest DBI was found in 70-day leaves, since the percentage of linolenic acid (C18:3) was higher in this sample than in \u003cem\u003eB. oleracea\u003c/em\u003e var. italica (48.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64% vs 5.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8%). In previous studies of soy (\u003cem\u003eGlycine max\u003c/em\u003e L.) plasma membrane, a rise in oleic acid (C18:1) and a decrease in linoleic acid (C18:2) and linolenic acid (C18:3) were observed, increasing membrane rigidity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Since the plants studied in Chalbi et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] were grown in a controlled environment chamber, the lipid proportions might be quite different from those of plants cultivated in the field, where the climatic conditions, such as temperature and humidity, are highly variable.\u003c/p\u003e \u003cp\u003eSterols also contribute to the bilayer permeability [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Campesterol has been studied regarding its contribution to increasing the spatial organization of the lipid bilayer and, thus, its order [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, it has been linked with a decrease in ionic permeability through lipid membranes [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In our work, plasma membrane from 90-day cauliflower inflorescences had the highest content of campesterol per mg of protein. This might be due to the dual function of campesterol, as a structural component and also the precursor of brassinosteroid hormones that are required for normal plant development [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. As has been reported in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, brassinosteroids help root meristem growth [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The fact that the sitosterol/stigmasterol ratio was much higher in the plasma membranes of inflorescences, mainly in the young ones (70 d), could have contributed to the increase in \u003cem\u003ePf\u003c/em\u003e. In fact, sitosterol has been pointed out as the main regulator of water permeability though membranes along with aquaporins [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBy means of the proteomic analysis performed by HPLC-ESI-QTOF-MS, the whole batch of identified proteins in each sample were analysed and categorized according to their cellular location (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, a few proteins could not be assigned to defined categories in each fraction. Although a high percentage of plasma membrane proteins was identified in all samples (42\u0026ndash;48%), a notable presence of organelle proteins was also found (40\u0026ndash;41%). This could be considered contamination of our plasma membrane samples, in particular by organelle endomembranes. In the inflorescences the percentage of cytoplasm proteins found was higher than in leaves. This could be related to the higher cellular density in cauliflower inflorescences [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, as we mentioned before, chloroplast-located proteins were identified only in samples from leaves. The presence of these proteins in leaves reflects the photosynthetic activity of this organ, whereas photosynthesis does not take place in inflorescences. Furthermore, the abundance of chloroplast proteins was greater in 70-day leaves (19.6%) than in 90-day leaves (4.1%), which could be related to the greater photosynthetic activity of the young leaves [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, we categorized proteins based on their functional category (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The presence of different transporter activity proteins (6\u0026ndash;8%) was detected; these comprised transmembrane ion transporters and ion channels, also located mainly in the plasma membrane. The number of detected proteins revealed that the abundance decreased as the maturity of the tissues sampled increased, being highest in young inflorescences, lower in mature inflorescences, much lower in young leaves, and lowest in mature leaves. In addition, the structural protein group was represented generally by structural constituents of ribosomes. There is a possibility that these proteins are associated with different membranes, including the plasma membrane, since it has been reported that proteins from ribosomes come from association with the endoplasmic reticulum (ER) and the ER is involved in plasma membrane turnover [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the aquaporins identified in plasma membrane extracts of cauliflower inflorescences and leaves. As PIPs aquaporins showed a very conservative structure, the digestion by trypsine enzyme and solubilisation is very similar in all of them. Although the knowledge of the sequences of all \u003cem\u003eB. oleracea\u003c/em\u003e aquaporins was very useful to investigate the presence of MIPs isoforms, and the analysis of peptides revealed five PIP1 isoforms (PIP1;1, PIP1;2, PIP1;3, PIP1;4, and PIP1;5) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), due to the high homology within the PIP1 subfamily, we could not identify isoforms unambiguously. Also, two TIPs were identified in our plasma membrane samples. Since TIPs are usually located in the tonoplast [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], their presence could be related with vacuolar contamination. However, the fact that TIPs have been found located in the plasma membrane in pea cotyledons [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] points to the possibility that TIPs were present in our plasma membrane.\u003c/p\u003e \u003cp\u003eAdditionally, PIP2 subfamily proteins were found only in inflorescences (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), in particular PIP2;5 and PIP2;7. Of these, only PIP2;7 was unambiguously identified; this aquaporin has been shown to be expressed mainly in \u003cem\u003eB. oleracea\u003c/em\u003e flowers [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Furthermore, when PIP2;7 was overexpressed in \u003cem\u003eA. thaliana\u003c/em\u003e roots the hydraulic conductivity increased six-fold, showing the important role of PIP2;7 in water transport.\u003c/p\u003e \u003cp\u003eFurther information about plasma membrane aquaporins in cauliflower was obtained from immunoblotting. The results show that PIP1 was present in both leaves and inflorescences, although its density in inflorescences was lower. The greater presence of PIP1 proteins in leaves could be explained by its regulatory role in CO\u003csub\u003e2\u003c/sub\u003e transport [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This CO\u003csub\u003e2\u003c/sub\u003e transport would be indispensable for the photosynthetic activity in leaves, whereas in inflorescences the constant cell division and growth would produce CO\u003csub\u003e2\u003c/sub\u003e that would need to be carried to the leaves. In addition, the other PIP1 members have been postulated as O\u003csub\u003e2\u003c/sub\u003e transport facilitators; in particular, PIP1;3 in tobacco plants [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, the results for PIP2 show a greater density in samples from inflorescences - which mirrors the information obtained in the proteomic assay, in which PIP2 could only be detected in those samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The exclusive identification of PIP2 proteins in inflorescences (PIP2;1, PIP2;2, PIP2;5, and PIP2;7) could be related to the elevated demand for water in cauliflower inflorescences, to maintain nutrient uptake and turgor [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The localization of PIP2 in plasma membrane samples from inflorescences (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) could also explain the differences observed in \u003cem\u003ePf\u003c/em\u003e; the 90-day inflorescences showed the highest \u003cem\u003ePf\u003c/em\u003e, in accordance with the higher concentration of PIP2 found in these samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The non-detection of PIP2 in leaves could indicate that the function of PIPs is performed here by TIPs or that only limited water transport across the lipid bilayer occurs. However, this aspect needs to be investigated further.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn summary, this study shows that the inflorescence of cauliflower, rich in meristematic tissue, has a large amount of plasma membrane per gram of tissue. This membrane is characterized by a low degree of unsaturation, which could increase the rigidity decreasing water transport, but the high content in sitosterol (highly correlated with water passage) would compensate this fact. In relation to this, the high presence of aquaporins in inflorescences, especially PIP2;5 and PIP2;7, indicates a potential role of aquaporins in the water transport required for the continuous development of the meristematic tissue. Furthermore, the fact that the aquaporins contribution to water transport in inflorescences must be higher than in leaves, with a potential correlation with the stage of development, provides to sterols and aquaporins a specific role in development. Our work highlights the need for further research on specific aquaporins in relation to adult plant development under natural conditions.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eDBI: Double bond index; ER: Endoplasmic reticulum; FID: Flame ion detector; HPLC: High Performance Liquid Chromatography; MUFA: Monounsaturated fatty acids; PIP: Plasma membrane intrinsic proteins; PUFA: Polyunsaturated fatty acids; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS: Tween 20 blocking solution; TIP: Tonoplast intrinsic protein\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAvailability of data and material\u003c/strong\u003e \u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e \u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by the CDTI, Spain (BIOTAGUT) and by the Spanish Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades (AGL2016-80247-C2-1-R). P. Garc\u0026iacute;a-Iba\u0026ntilde;ez was funded by a grant from the Fundaci\u0026oacute;n S\u0026eacute;neca-CARM, Spain (21273/FPI/19).\u003c/p\u003e \u003ch2\u003eAuthors' contributions\u003c/h2\u003e \u003cp\u003eMCA contributed to the conception and design of this work. PGI carried out the experiments and JNE performed the analytical work of proteomics. PGI and JNE prepared figures and tables, and prepared the first draft of the manuscript. MCA contributed to manuscript revisions, reads and approved the submitted version. MCA obtained the funding. All authors have read and approved the manuscript.\u003c/p\u003e \u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors thank Dr. D. Walker for the correction of the English in the manuscript.\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cspan\u003eThorwarth P, Yousef EAA, Schmid KJ. 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Planta. 2020;251:65.\u003c/span\u003e \u003c/li\u003e\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"plasma membrane, aquaporin, brassica, osmotic permeability","lastPublishedDoi":"10.21203/rs.3.rs-29232/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-29232/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCauliflower (\u003cem\u003eBrassica oleracea\u003c/em\u003e L. var. \u003cem\u003ebotrytis\u003c/em\u003e) inflorescences are composed mainly of meristematic tissue, which has a high cellular proliferation. This considerable cellular density makes the inflorescence an organ with a large proportion of membranes. However, little is known about the specific role of the lipid and protein composition of the plasma membrane present in this organ.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this work, we analyzed the lipids and proteins present in plasma membrane from two different stages of development of cauliflower inflorescence and compared them with leaf plasma membrane. For this purpose, plasma membrane vesicles were obtained by centrifugation for each sample and the vesicular diameter and osmotic permeability (\u003cem\u003ePf\u003c/em\u003e) were analyzed by dynamic light scattering and the \u003cem\u003estopped-flow\u003c/em\u003e technique, respectively. In addition, fatty acids and sterols were analyzed by gas chromatography and HPLC, respectively. The protein composition of the inflorescences and leaves was characterized by HPLC-ESI-QTOF-MS and the data obtained were compared with \u003cem\u003eBrassicaceae\u003c/em\u003e proteins present in the UniProt database in relation to the presence of aquaporins determined by western blot analysis. The highest \u003cem\u003ePf\u003c/em\u003e value was found in 90\u0026nbsp;day inflorescences-derived plasma membrane vesicles (61.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.14 \u0026micro;ms\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). For sterols and fatty acids, the concentrations varied according to the organ of origin. The protein profile revealed the presence of aquaporins from the PIP1 and PIP2 subfamilies in both inflorescences and leaves.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study shows that the composition of the sterols, the degree of unsaturation of the fatty acids, and the proteins present in the membranes analyzed give them high functionality for water passage. This represents an important addition to the limited information available in this field.\u003c/p\u003e","manuscriptTitle":"Plasma Membrane Vesicles From Cauliflower Meristematic Tissue And Their Role In Water Passage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-06-11 16:38:27","doi":"10.21203/rs.3.rs-29232/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a490dfbc-e29c-4494-a495-1b24c42e0414","owner":[],"postedDate":"June 11th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":118263,"name":"Plant Physiology and Morphology"},{"id":118264,"name":"Plant Molecular Biology and Genetics"}],"tags":[],"updatedAt":"2021-01-10T15:05:51+00:00","versionOfRecord":{"articleIdentity":"rs-29232","link":"https://doi.org/10.1186/s12870-020-02778-6","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2021-01-07 15:02:15","publishedOnDateReadable":"January 7th, 2021"},"versionCreatedAt":"2020-06-11 16:38:27","video":"","vorDoi":"10.1186/s12870-020-02778-6","vorDoiUrl":"https://doi.org/10.1186/s12870-020-02778-6","workflowStages":[]},"version":"v1","identity":"rs-29232","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-29232","identity":"rs-29232","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}