Microsoft Word - 1murphy.docx CHEMICAL ENGINEERING TRANSACTIONS VOL. 58, 2017 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors: Remigio Berruto, Pietro Catania, Mariangela Vallone Copyright © 2017, AIDIC Servizi S.r.l. ISBN 978-88-95608-52-5; ISSN 2283-9216 Morpho-physiologic Traits in Two Sage Taxa Grown under Different Irrigation Regime Francesca Grisafia, Elisabetta Oddob, Antonella Maggiob, Antonina Panarisia, Maria Panarisia a Dipartimento di Scienze Agrarie e Forestali, Università di Palermo, Italy b Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Università di Palermo, Italy francesca.grisafi@unipa.it Sage is an important aromatic crop, extensively cultivated worldwide. Drought stress affects yield and composition of secondary metabolites such as fatty acids, essential oils, antioxidants, changing the essential oil composition profile with respect to the ISO 9909 standard. Drought tolerance may differ among genotypes, so we compared the response of Salvia officinalis L. and S. officinalis cv. ‘Maxima’ grown under two different irrigation regimes to highlight differences in leaf growth, water potential, gas exchange and essential oil composition. Potted plants were grown in the greenhouse at 100% or 50% of field water capacity for three months. Monthly measurements of 3rd node leaf length, width and area were taken using ImageJ software. Midday leaf water potential was measured using a pressure chamber and leaf stomatal conductance, transpiration and net photosynthesis were measured using a portable infrared gas analyzer. Essential oil composition was determined by gas chromatography and mass spectrometry. Drought stress reduced leaf area in both taxa, but the effect was less pronounced in S. officinalis than in S. officinalis cv. ‘Maxima’ (-30%). Leaf water potential was slightly lower in the latter cultivar than in S. officinalis under water deficit. In irrigated S. officinalis cv. ‘Maxima’ plants transpiration and stomatal conductance rates were more than twice those of S. officinalis, while under water deficit the difference between the taxa was not significant. Interestingly, net photosynthesis in S. officinalis was about twice that measured in S. officinalis cv. ‘Maxima’, both in irrigated and in stressed plants. Furthermore, in S. officinalis water deficit resulted in a slight reduction of photosynthetic rate, while in S. officinalis cv. ‘Maxima’ the reduction was around 50%. Both taxa were affected by drought stress, responding with a reduction in leaf expansion and in transpiration, in order to reduce water loss. However, S. officinalis appeared to have a greater efficiency, maintaining higher levels of carbon assimilation. 1. Introduction The genus Salvia (Lamiaceae, tribe Mentheae) is one of the largest and most important aromatic and medicinal genera of the Lamiaceae family and comprises about 900 species, widespread throughout the world (Walker et al., 2007), displaying a remarkable diversity in growth forms, secondary compounds, floral morphology and pollination biology. The essential oils produced by the leaves are acknowledged worldwide because of their beneficial uses: there are a great number of literature reports on analyses of essential oils from plants of this genus and morphological and genetic variations are also observed according to their geographical origin. As its Latin gender name Salvia means ‘‘to cure’’ and species name ‘‘officinalis’’ means medicinal, it is clear that sage has a historical reputation of promoting health and treating ailments (Russo et al., 2013). In Ancient Rome, it was even called the sacred plant (Durling et al., 2007; Kamatou et al., 2008). Water deficit is an environmental stress experienced by many plants that strongly impairs production (Passioura 1996). Water stress causes large morphological or physiological changes across a range of temporal and spatial scales (Chaves et al. 2002) including reduced expansion of aerial organs, maintenance of root growth, decrease of transpiration and photosynthesis, accumulation of osmotica ( Hummel et al., 2010). Drought tolerance may differ with genotype, and both environmental and genetic factors may influence essential oil composition, as reported for three of the many cultivars of sage (Böszörményi et al., 2009). The DOI: 10.3303/CET1758117 Please cite this article as: Grisafi F., Oddo E., Maggio A., Panarisi A., Panarisi M., 2017, Morpho-physiologic traits in two sage taxa grown under different irrigation regime, Chemical Engineering Transactions, 58, 697-702 DOI: 10.3303/CET1758117 697 sage cultivar ‘Maxima’ is very decorative, strongly scented, with large silver-grey leaves and to the best of our knowledge, there are no published data on its response to stress and essential oil composition. The aim of this preliminary work was to compare growth, water relations, photosynthesis and main essential oil composition of Salvia officinalis L. and S. officinalis cv. ‘Maxima’ grown under two different irrigation regimes. 2. Materials and Methods Plants of Salvia officinalis L. and Salvia officinalis cv. ‘Maxima’, obtained from cuttings and on average 25 cm high, were grown in 2 L pots in garden soil in a greenhouse of the Department of Agricultural and Forest Sciences, Palermo, Italy. Plants were watered with tap water twice a week to 100% (control, C) or 50% (moderate water deficit, MWD) of field water capacity for three months, from March to May 2016. Soluble fertilizer was dissolved in the irrigation water once every 3 weeks throughout the experiment. The effect of the different water regimes on plant growth traits was followed monthly: from the beginning of the experiment, photographs of healthy, completely expanded young leaves (3rd node from the top of the shoot) were taken, flattening leaves between two glass slides and including a section of graph paper as reference. Leaf length, width and area were measured on 12 leaves per treatment using ImageJ software. At the end of the experiment, in May, physiological traits such as leaf water potential and gas exchange parameters were measured on three plants per treatment. Leaf water potential (ΨL) was measured the day after irrigation on leaves collected at 11 AM using a pressure chamber (SKPM 1400, Skye Instruments). Non-destructive measurements of leaf stomatal conductance to water vapour (gs), transpiration (E) and net photosynthesis (NP) were measured on 9 healthy, completely expanded 3rd node leaves per treatment between 11 and 12 AM using a portable infrared gas analyzer (HCM- 1000, Walz). All gas exchange data were expressed on a leaf area basis. All the measurements were conducted on sunny days in the greenhouse under ambient temperature and light conditions. Air-dried leaf samples were crushed, then hydrodistilled for 3 h using a Clevenger-type apparatus. Oils were dried over anhydrous sodium sulphate and stored under N2 at -20°C. Samples were prepared with a GERSTEL MultiPurpose Sampler MPS equipped with a Cooled Agitator. Analysis was performed in a 7000C Triple Quadrupole GC/MS System, fitted with a fused silica Agilent HP-5 capillary column (10 m x 0.25 mm i.d.; 0.25 μm film thickness), coupled to a Agilent Mass Selective Detector; Diluted samples (1/100 v/v, in n- hexane) of 1 μL were injected at 250 °C automatically and in the splitless mode; transfer line temperature, 295 °C. MassHunter Workstation Software was used for chromatogram analysis. Most constituents were identified by gas chromatography by comparison of their retention indices with those of the literature or with those of authentic compounds available in our laboratories. Further identification was made by comparison of their mass spectra with those stored in NIST 02 and Wiley 275 Libraries or with mass spectra from literature (Adams, 2012) and home-made library. Component relative concentrations were calculated based on GC peak areas without using correction factors. Results were plotted using Microsoft Office Excel 97-2003. Data are expressed as mean ± S.D. Data were analysed with one-way analysis of variance (ANOVA), using the software package SigmaPlot 12 (Systat Software, Inc., San Jose, USA). Fisher’s LSD test was used to compare means when ANOVA was significant (P < 0.05). 3. Results and Discussion Plant growth was measured on the basis of leaf length, width and leaf area. In March, at the beginning of the experiment, 3rd node leaf area was on average 10 cm2 for S. officinalis and 18 cm2 for S. officinalis cv. ‘Maxima’. Leaf area decreased during the experiments in both species, independently from irrigation regime (Figure 1). At the end of the experiment, leaves of the 3rd node of S. officinalis had a 50% decrease in leaf area, while this decrease was slightly larger for S. officinalis var. ‘Maxima’ (55%). When comparing control plants and MWD plants, after one month, leaf area was reduced about 30 % in S. officinalis, and 12 % in S. officinalis var. ‘Maxima’. At the end of the experiment, leaf area was not significantly different among treatments in S. officinalis, while S. officinalis var. ‘Maxima’ under 50% water irrigation had a 30% reduction in leaf area. Correspondingly, leaf length was reduced about 50% from the beginning to the end of the experiment in both taxa and all treatments, while leaf width was not reduced as much in S. officinalis cv. ‘Maxima’ as in S. officinalis (data not shown). Exposure of plants to extreme stress conditions such as drought will initiate a diverse set of physiological, morphological, and developmental changes in order to survive (Gomes et al., 2010; Ozkur et al., 2009) and the response of plants to drought varies greatly depending on genotype and stress severity (Verslues et al., 2006). The depressive effect of drought on plant growth we observed under moderate water stress is in agreement with other reports for Salvia officinalis, with reductions in shoot length, leaf area, and leaf number (Bettaieb et al., 2009, Mansori et al., 2016). 698 The leaf water potential values measured at the end of the experimental period showed small though not significant differences, probably due to the low number of replicates. However, on average, C plants reached less negative ΨL values than MWD plants in both taxa (Figure 2). Figure 1. Changes in leaf area of 3rd node leaves in plants of S. officinalis and S. officinalis cv. ‘Maxima’ irrigated at 100% (C) and 50% (MWD) of field capacity. Data are reported as mean ± SD (n=12). Figure 2. Leaf water potential measured at the end of the experiment (May) at 11 AM on samples of S. officinalis and S. officinalis cv. ‘Maxima’ irrigated at 100% (C) and 50% (MWD) of field capacity. Data are reported as mean ± SD (n=3). Figure 3: Leaf stomatal conductance measured at the end of the experiment (May) between 11 and 12 AM on samples of S. officinalis and S. officinalis cv. ‘Maxima’ irrigated at 100% (C) and 50% (MWD) of field capacity. Data are reported as mean ± SD (n=9). 0 5 10 15 20 25 S. offi ci nal i s C S. offi ci nal i s MWD S. maxi ma C S. maxi ma MWD L e a f a re a , c m 2 March Apri l May 0 10 20 30 40 50 60 70 80 90 S. offi ci nal i s C S. offi ci nal i s MWD S. maxi ma C S. maxi ma MWD S to m a ta l c o n d u ct a n ce , m m o l / m 2 s 699 The most negative ΨL was reached by S. officinalis var. ‘Maxima’, -0.923 MPa. The ∆ΨL between C and MWD plants belonging to the same taxa was slightly larger in S. officinalis var. ‘Maxima’ (0.115 MPa) than in S. officinalis (0.104 MPa). Measured values were in all cases above the level of turgor loss point reported in the literature for S. officinalis saplings, -1.26 MPa (Savi et al., 2017). Though leaf water potential values were similar in both taxa, gas exchange traits differed greatly, especially when comparing C treatments. S. officinalis var. ‘Maxima’ C showed the highest levels of stomatal conductance (Figure 3). When comparing the taxa, gs values of S. officinalis var. ‘Maxima’ were twice those of S. officinalis, both under 100% and 50% irrigation. The reduced irrigation regime led to a 50% reduction in gs in S. officinalis MDW, and a slightly smaller reduction in S. officinalis var. ‘Maxima’ MDW (45%), indicating stomatal closure as an early response to stress, which aided in maintaining relatively constant ΨL. A similar pattern was recorded for leaf transpiration (Figure 4), with S. officinalis var. ‘Maxima’ C showing transpiration rates 2.5 times greater than S. officinalis C. However, differences in E among S. officinalis C, S. officinalis MWD and S. officinalis var. ‘Maxima’ MWD were not significant. Stomatal conductance is often used as a proxy for photosynthetic capacity, based on the assumption that higher gs allows a greater uptake of CO2 into the leaves. In this study, however, higher gs did not correspond with higher photosynthetic rates: in S. officinalis NP was almost twice that of S. officinalis var. ‘Maxima’ under both irrigation regimes (Figure 5). Figure 4: Leaf transpiration measured at the end of the experiment (May) between 11 and 12 AM on samples of S. officinalis and S. officinalis cv. ‘Maxima’ irrigated at 100% (C) and 50% (MWD) of field capacity. Data are reported as mean ± SD (n=9). Figure 5: Net photosynthesis measured at the end of the experiment (May) between 11 and 12 AM on samples of S. officinalis and S. officinalis cv. ‘Maxima’ irrigated at 100% (C) and 50% (MWD) of field capacity. Data are reported as mean ± SD (n=9). In both taxa the reduced irrigation regime led to a decrease in NP rates. This reduction was small but significant (-20%) in S. officinalis MWD compared to S. officinalis C, while it was quite more consistent in S. officinalis var. ‘Maxima’ MWD compared to S. officinalis var. ‘Maxima’ C (-40%).During the experiment, all 0 2 4 6 8 10 12 S. offi ci nal i s C S. offi ci nal i s MWD S. maxi ma C S. maxi ma MWD N e t P h o to sy n th e si s, μ m o l / m 2 s 700 plants were exposed to some degree of water deficit, as the irrigation schedule did not guarantee a constant replenishment of water lost by evapotranspiration especially in the month of May. However, the effects of the two different watering regimes were evident both from the morpho-physiological data and from the appearance of the whole plants (Figure 6), where leaves of the MWD plants were deeply wilted and folded, particularly in S. officinalis cv. ‘Maxima’. Leaf wilting can act as a defence mechanism against excessive water loss and excessive radiation (Pérez-Estrada et al., 2000, Savi et al., 2016). Figure 6: Appearance of representative samples after three months of irrigation supplied twice a week at 100% and 50% of field capacity. (A) S. officinalis, (B) S. officinalis cv. ‘Maxima’. Pictures were taken immediately before one of the scheduled irrigations. Analysis of essential oils showed the presence of about a hundred components, of which the most abundant are presented in Table 1. There were qualitative and quantitative differences between S. officinalis and S. officinalis cv. ‘Maxima’ under 100% irrigation regime. Mild water deficit did not affect the qualitative and quantitative composition of oils in S. officinalis, while in S. officinalis cv. ‘Maxima’ there were greater differences between the two treatments. Comparing S. officinalis and S. officinalis cv. ‘Maxima’ MDW, a similar qualitative composition was found, but noteworthy quantitative differences. Table 1: Main essential oil composition of plants of S. officinalis and S. officinalis cv. ‘Maxima’ irrigated at 100% (C) and 50% (MWD) of field capacity. S. officinalis C S. officinalis MWD S. Maxima C S. Maxima MWD RT RTcorrected KI Compound Area Sum % Area Sum % Area Sum % Area Sum % 12.70 10.64 933 α-thujene 3.81 0.84 3.32 4.51 13.50 11.44 946 α-pinene 5.76 6.28 0.14 4.03 14.00 11.94 954 camphene - - 7.27 0.12 15.45 13.39 974 sabinene 2.65 1.58 0.02 8.19 18.80 16.74 1020 α-terpinene 0.54 0.97 9.14 0.68 19.20 17.14 1026 p-cymene 11.88 12.32 0.18 9.82 24.49 22.44 1097 linalool 3.23 3.98 0.03 7.41 25.29 23.24 1105 trans-sabinene hydrate 7.29 6.54 0.09 3.24 27.09 25.04 1121 cis-pinene hydrate 10.44 9.81 0.85 9.94 28.69 26.64 1134 camphor 8.61 8.33 0.31 3.43 45.39 43.33 1305 undecanal 4.04 4.63 0.74 1.73 51.19 49.13 1400 1,7-di-epi-a-cedrene 1.40 1.39 2.85 1.01 55.54 53.48 1477 γ-muurolene 2.22 2.35 0.28 8.42 58.29 56.23 1525 eugenyl acetate 2.81 3.52 0.32 - 78.93 76.87 1935 methyl hexadecanoate 5.10 6.74 - 1.90 A B 701 4. Conclusions Our results indicated that water deficit caused a significant reduction of plant growth. Both Salvia officinalis and Salvia officinalis cv. ‘Maxima’ are affected by drought stress, responding with a significant reduction in leaf expansion and in transpiration, in order to reduce water loss. However, S. officinalis exhibited a greater photosynthetic efficiency, maintaining higher levels of carbon assimilation also under mild water deficit. Essential oil composition differed between the two genotypes and was influenced by drought stress more in Salvia officinalis cv. ‘Maxima’ than in Salvia officinalis. 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