**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We wish to thank the Provincia di Genova dei Frati Minori Cappuccini for the kind consultation of the ancient texts: Vincenzo Celesia, Selva Botanica, Genova 1892 (1MANOSSXX-64) and Zaccaria Boverio, De sacris ritibus ... , Napoli 1626 (ISIL: IT-GE0039). We also thank Elena Mora (DISTAV Botanical Garden) for her technical help during plant cultivation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

**Martina Tomasella 1, Sara Natale 1, Francesco Petruzzellis 1,2, Sara Di Bert 1, Lorenzo D'Amico 3,4, Giuliana Tromba 3 and Andrea Nardini 1,\***

**Abstract:** (1) Recent studies suggested that stem photosynthesis could favor bark water uptake and embolism recovery when stem segments are soaked in water under light conditions, but evidence for this phenomenon in drought-resistant Mediterranean species with photosynthetic stems is missing. (2) Embolism recovery upon immersion in water for 2 h–4 h under light was assessed (i) via a classical hydraulic method in leafless *Fraxinus ornus* and *Olea europaea* branch segments stressed to xylem water potentials (Ψxyl) inducing ca. 50% loss of hydraulic conductivity (PLC) and (ii) via X-ray micro-CT imaging of the stem segments of drought-stressed potted *F. ornus* saplings. Hydraulic recovery was also assessed in vivo in intact drought-stressed *F. ornus* saplings upon soil re-irrigation. (3) Intact *F. ornus* plants recovered hydraulic function through root water uptake. Conversely, the soaked stem segments of both species did not refill embolized conduits, although Ψxyl recovered to pre-stress levels (between −0.5 MPa and −0.2 MPa). (4) We hypothesize that xylem embolism recovery through bark water uptake, even in light conditions, may not be a common phenomenon in woody plants and/or that wounds caused by cutting short stem segments might inhibit the refilling process upon soaking.

**Keywords:** stem photosynthesis; hydraulic recovery; soaking; X-ray micro-CT; bark water uptake; embolism

#### **1. Introduction**

Most terrestrial plants rely on root-level water absorption to maintain their hydration status. However, water can also be absorbed from the surface of aboveground plant organs under particular biophysical conditions, i.e., when liquid water is wetting plant surfaces or the water potential of the surrounding atmospheric boundary layer is higher than that of cells [1]. This occurs when water vapor pressure in the air is at (or close to) saturation and, most importantly, when liquid water forms or falls on a plant surface due to fog, rain or snow melting, allowing local rehydration and partial xylem tension relief, especially when plants are experiencing a soil water deficit [2].

Water uptake through leaves has been widely observed in many plant lineages [3,4]. Leaf trichomes, depending on their density, composition and structure, are the major media for water absorption in a Mediterranean (*Quercus ilex*) and a temperate (*Fagus sylvatica*) species [5,6]. In addition, in two species without leaf trichomes or hydathodes, open stomata have been observed to play a major role in water uptake over cuticles when exposed to fog [7]. Leaf water absorption can also be involved in the recovery of leaf [8] and stem [9,10] hydraulic functions after drought- or frost-induced xylem embolism.

**Citation:** Tomasella, M.; Natale, S.; Petruzzellis, F.; Di Bert, S.; D'Amico, L.; Tromba, G.; Nardini, A. No Evidence for Light-Induced Embolism Repair in Cut Stems of Drought-Resistant Mediterranean Species under Soaking. *Plants* **2022**, *11*, 307. https://doi.org/10.3390/ plants11030307

Academic Editors: Milan S. Stankovic, Paula Baptista and Petronia Carillo

Received: 20 December 2021 Accepted: 21 January 2022 Published: 24 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The stems of woody plants, even when covered with suberized tissue, are also able to absorb water when the bark becomes wet, allowing partial xylem tension relief [11]. There is some evidence that leafless dehydrated branches soaked in water for at least some hours partially recover their xylem hydraulic function in a conifer (*Sequoia sempervirens*, [12]) and in an angiosperm (*Salix matsudana*, [13]). Xylem hydraulic recovery would require radial water movement from the phloem to the xylem through parenchyma rays, as proven in a dying experiment on wetted stems [11]. Liu et al. [13] soaked short *S. matsudana* stem segments in water and observed faster embolism repair (within 2 h) when under light conditions compared to dark conditions, where partial refilling nevertheless occurred. The enhanced refilling effect under light was ascribed to the sugars produced by stem photosynthesis, which provide the driving (osmotic) force for the process. In fact, embolism repair can occur as the result of a water potential gradient between the xylem apoplast, where sugars are accumulated, and parenchyma cells surrounding the conduits [14–16]. Independently of the mechanisms involved in the process, such soaking experiments on short leafless stem segments on other woody species performing stem photosynthesis are underrepresented, and they might constitute an interesting experimental model to investigate the biology of post-drought hydraulic recovery.

In the past decade, some methodological issues have arisen regarding stem hydraulic vulnerability assessment and the study of embolism recovery via classic hydraulic methods due to the possible overestimation of embolism rates, which may be generated when cutting xylem under tension [17]. At the same time, prolonged xylem relaxation prior to hydraulic measurements, which is suggested to avoid artefactual embolism appearance, can favor refilling, leading to the underestimation of embolism levels [18]. In well-established sample preparation procedures for hydraulic measurements, stems are often kept under water prior to hydraulic measurements, even for long time intervals, based on the principle that the very low pressure head upon immersion would avoid embolism dissolution [19]. However, the above-mentioned soaking experiments in short leafless branch segments [12,13] indicated that the active accumulation of solutes at the wood parenchyma–conduit interface drives water into the apoplast and might refill the conduits under soaking. If this happens, soaking stems for a prolonged period would induce overestimation of xylem hydraulic conductivity.

These controversies can be overcome using X-ray micro-computed tomography (micro-CT), an important tool to visualize embolized xylem conduits at a proper resolution and to quantify in vivo xylem vulnerability, as well as possible hydraulic recovery in intact plants [20,21]. Therefore, this method has been exploited to validate destructive hydraulic methods (e.g., [22,23]) and related sample preparation procedures [24].

In this study, we hypothesized that bark water uptake in the presence of light would induce the recovery of hydraulic function in two embolism-resistant Mediterranean angiosperm species performing stem photosynthesis, namely, *Fraxinus ornus* and *Olea europaea*. We tested this hypothesis both with a classical hydraulic method in dehydrated leafless cut branches of adult plants of both species, and in vivo through X-ray micro-CT imaging of segments of drought-stressed potted young saplings of *F. ornus*. There is evidence suggesting that *O. europaea* is capable of partial hydraulic recovery following drought-induced xylem embolism [18], making this species a good candidate to investigate the eventual process of conduit refilling under soaking conditions. Similarly, intact *F. ornus* plants have already been reported to recover hydraulic function when the soil is re-watered after substantial drought-induced loss of xylem hydraulic conductivity [25], but in vivo evidence is missing. For this reason, we additionally tested the hydraulic recovery capability of this species through root water uptake via micro-CT imaging of intact potted plants.

#### **2. Results**

The stem segments used for the soaking experiments, i.e., 2-year-old *F. ornus* and 1-year-old *O. europaea* branch portions, as well as the 1-year-old stem portions of *F. ornus* saplings, showed a relatively high capability of performing photosynthesis (Figure 1). In particular, the maximum quantum yield of PSII (Fv/Fm) in the outer bark was about 0.8 in all three different samples. Similar values were also measured in the outer wood and in the longitudinal section of the wood (sapwood + pith) of the 1-year-old branch and stem segments of both species. Lower (but still relatively high) Fv/Fm values were measured in the outer wood (0.65) and in the longitudinal section of the wood (0.49) of *F. ornus* 2-year-old branch portions.

#### *2.1. Hydraulic Measurements*

Some native embolism was detected in the hydrated branches of *F. ornus* and *O. europaea* (H samples), with PLC averaging 17% and 5%, respectively (Figure 2). After dehydration on the bench, PLC at the target Ψxyl (−3.7 MPa and −4.4 MPa in *F. ornus* and *O. europaea*, respectively) significantly increased to 56 ± 4% in *F. ornus* and to 54 ± 10% in *O. europaea*. After the soaking treatment in distilled water for 2 h (S2h samples), Ψxyl increased to prestress levels, averaging −0.46 MPa in *F. ornus* and −0.33 MPa in *O. europaea*, but stem PLC did not recover in either species. Moreover, prolonged immersion in water (four hours, S4h stems) did not induce xylem hydraulic recovery in *F. ornus,* albeit xylem tension further and significantly decreased with respect to the S2h samples (Ψxyl = −0.19 ± 0.06 MPa, *p* < 0.05).

**Figure 2.** Percentage loss of xylem hydraulic conductivity (PLC, (**a**,**b**)) and xylem water potential (Ψxyl, (**c**,**d**)) measured in *F. ornus* (**a**,**c**) and *O. europaea* (**b**,**d**) in hydrated (H, *n* = 5), drought-stressed (D, *n* = 5–6) and soaked (for 2 and 4 h, S2h and S4h, respectively, *n* = 6–7) stems. Note the different scales for Ψxyl between the two species. The 4 h soaking treatment was performed only for *F. ornus*. Different letters denote statistically significant differences among groups (*p* < 0.05).

#### *2.2. Micro-CT Analyses*

The micro-CT experiment on the potted plants of *F. ornus* proved the capability of xylem hydraulic recovery in intact plants through root water uptake 24 h after soil rehydration to field capacity. In fact, the embolized vessel area (EVA) was around 40–50% in drought-stressed (Dpot) plants, which reached Ψxyl of −3.50 MPa, and it was restored to pre-stress control (Cpot) values in the two re-irrigated (Rpot) plants (6% and 21% in the two scanned plants, Figure 3), which relieved Ψxyl to −0.25 MPa and −0.70 MPa.

The consecutive micro-CT scans performed on the 2-year-old *F. ornus* drought-stressed (D) plants after each progressive sample preparation step are shown in Figure S1. The first cut at the base of the stem did not significantly increase the embolized sapwood area (Aembol) or EVA. However, the second cut made to obtain the final D segment significantly increased EVA from 44 ± 7% (measured in the shoots cut at the base) to 63 ± 11%, with an overall increase of 27% compared to the intact plant (Figure S2).

The stem segments did not refill their embolized conduits after the 2 h soaking treatment under light, as the values of Aembol and EVA (1.07 ± 0.06% and 58 ± 1%, respectively) were similar to those calculated for D stem segments (*p* > 0.05; Figure 4 and Figure S3).

**Figure 3.** Transverse reconstructed images of the 1-year-old stem portion obtained with X-ray micro-CT in well-hydrated (Cpot, (**a**,**d**)), drought-stressed (Dpot, (**b**,**e**)) and re-irrigated (Rpot, (**c**,**f**)) intact *F. ornus* plants. Ψxyl = xylem water potential, Aembol = percentage of embolized sapwood area, EVA = percentage of embolized vessel area; all calculated excluding the immature sapwood close to the cambium (see arrow delimiting mature/immature sapwood in (**a**)).

**Figure 4.** Percentage of embolized sapwood area (Aembol, (**a**)) and percentage of embolized vessel area (EVA, (**b**)) measured in micro-CT transverse reconstructed images of *F. ornus* stem segments. D = segments of drought-stressed plants (*n* = 3). Soaking = segments of additional D plants scanned upon soaking in water for2h(*n* = 3). Values are means ± SE. n.s. = difference not significant among groups.

#### **3. Discussion**

#### *No Evidence for Hydraulic Recovery in Stem Segments upon Soaking*

The in vivo micro-CT analyses showed that *F. ornus* plants can recover xylem hydraulic function through root water uptake after experiencing substantial embolism levels under drought, confirming the previous classical hydraulic measurements of cut stems [25]. In addition, embolism recovery occurred even though plants were still experiencing overall negative xylem pressure, supporting previous observations. However, the leafless stem segments of *F. ornus* and *O. europaea* that reached about 50% PLC (i.e., the same target PLC used in the pot re-irrigation experiment) did not recover xylem hydraulics after immersion in water under light conditions (Figure 2). This outcome was additionally validated by the micro-CT scans of the *F. ornus* cut stems, obtained from the drought-stressed potted saplings (Figure 4 and Figure S2).

The hydraulic measurements of *F. ornus* branches (Figure 2) were in agreement with the micro-CT measurements of intact plants (Figure 3), confirming the lack of cutting artifacts. Likewise, the PLC values obtained in our study for *O. europaea* at the target Ψxyl were consistent with the vulnerability curves obtained for the same species both via hydraulic measurements and X-ray micro-CT scans [26,27], making us confident that sample preparation artefacts were avoided. However, with the methodological test performed at the micro-CT beamline on the dehydrated saplings of *F. ornus*, we aimed to examine if the sample preparation procedure used to obtain the final stem segments for soaking would induce additional non-native embolism. The progressive cuts made on the stem of the potted saplings increased the embolized vessel area (Figure S1 and Figure S2). In this test, we first made a cut underwater at the base of the shoot, and then a second cut was made at a distal position (ca. 20 cm from the previous one) to obtain a stem segment in the basal part of the stem. Given that the maximum vessel length of the saplings (40 cm on average) was higher than the obtained stem segment, it is likely that air entry upon cutting under substantial xylem tension induced embolism in the stem portion where the micro-CT scans were made (at 8 cm–9 cm from the first cut). Given that we aimed to perform the soaking experiment on the lower 1-year-old stem portion and that the plants were relatively small, we were forced to obtain the stem segment in that proximal stem region. With this simple test, we underline that experiments with in vivo imaging techniques should be further exploited to better identify the possible artefacts of sample preparation. However, the results of our cutting test do not influence the outcome of the soaking experiment because the samples scanned prior (D) or after (Soaking) immersion in water (Figure 4) were prepared with the same procedure.

The results of the soaking experiments contradict those obtained for *S. matsudana* stem segments soaked in water under light conditions, where bark water uptake contributed to refill about 75% of the previously embolized vessels after 2 h of immersion [13]. Here, we also showed that *F. ornus* and *O. europaea* stems can perform photosynthesis both at wood and bark levels (Figure 1), which should provide additional sugars to enhance the process of water uptake by generating an osmotic gradient.

There is evidence that local xylem tension relief is important to refill xylem conduits [28,29]. In *S. sempervirens*, partial hydraulic recovery was observed upon soaking when branch water potential slightly increased with respect to dehydrated conditions, but it was still quite negative (ca. −4 MPa, [12]). Similarly, recovery under substantial tension occurred in *Picea glauca* leafy twigs exposed to fog [10], suggesting possible localized hydraulic isolation in the xylem. Xylem tension in our two study species was even relaxed to Ψxyl above −0.5 MPa (Figure 2). These values are comparable to those measured in the intact potted plants of *F. ornus*, which recovered xylem hydraulics when re-irrigated in pots (Figure 3). Therefore, we note that the residual xylem tension of the cut stems was comparable to that of intact plants, and it was expected to promote the refilling of conduits through bark water uptake.

Earles et al. [12] suggested that tall trees, such as redwoods, which, due to their height, must develop low Ψxyl even under well-hydrated conditions, may profit from bark water

uptake during prolonged fog periods to partially restore xylem functionality. It is suggested that trees adapted to climates with prolonged periods of rain and/or fog [30] and trees at the timberline with crowns covered with snow that melts in spring [9,31] may have evolved bark characteristics that favor water uptake and subsequent hydraulic recovery. Indeed, radial water uptake may depend on outer (rhytidome) and/or inner (phloem) bark characteristics. For example, bark porosity and density influence hygroscopicity [32], while the hydrophobicity of cork cells in the outer bark limits water absorption [33,34]. It is possible that species adapted to Mediterranean climates, commonly not exposed to prolonged fogs/rainy periods, may not have evolved such bark characteristics that would favor radial water uptake. However, we might rule out this hypothesis because, although we did not directly measure bark water uptake, the marked Ψxyl relief upon soaking indicated substantial stem rehydration (Figure 2b). Alternatively, sample preparation/experimental conditions could have been responsible for the lack of hydraulic recovery. Previous experiments have highlighted the role of phloem in xylem hydraulic recovery [35]. In particular, stem girdling in *O. europaea* prevented embolism refilling upon branch rehydration, while hydraulic recovery was observed in samples with intact phloem [18]. Hence, it is possible that the wounds caused by the preparation of stem segments for soaking had similar inhibitory effects on the refilling process. Tests on different species could help improve our understanding as to whether refilling through bark water uptake might be species specific, as well as the possible limits of the soaking method when applied to cut stem segments.

#### **4. Materials and Methods**

#### *4.1. Plant Material*

The experiment was carried out on two Mediterranean tree species, namely, manna ash (*Fraxinus ornus* L.) and olive (*Olea europaea* L.), between mid-June and the end of July 2021. In order to test the hydraulic recovery capability upon soaking under light conditions, embolism was measured both with a classical hydraulic conductivity apparatus and in vivo via micro-CT, but on different plant material. Hydraulic measurements were performed on sun-exposed branches taken from several *F. ornus* trees and one *O. europaea* tree growing in the Botanical Garden of the University of Trieste (Italy, 45◦39 40.9 N, 13◦47 40.1 E). For micro-CT experiments, 2-year-old and 1-year-old *F. ornus* saplings provided by a local public nursery (Vivai Pascul, Regional Forestry Service, Tarcento, Italy) were transplanted in 3.4 L and 1 L pots in March 2020 and 2021, respectively. Pots were filled with a lightweight substrate for green roof installations (for the 2-year-old plants) or with red soil (for the 2-year-old plants) sampled from a vineyard in the Italian Karst (Duino-Aurisina, Italy). Plants were grown in a greenhouse of the University of Trieste, supplied with tap water twice a day to soil field capacity through a clock irrigation system.

To check the capability of the study species to perform bark and wood photosynthesis, some stem segments were analyzed with an imaging PAM chlorophyll fluorometer (Photon Systems Instruments, Brno, Czech Republic) to obtain the maximum quantum yield of PSII (Fv/Fm). Stem segments of approx. 2 cm length were dark adapted for 1 h, longitudinally sectioned and positioned on a plate with a layer of paper towels, keeping them hydrated through partial immersion in a film of water. Outer bark, outer xylem and xylem longitudinal section (sapwood + pith) were measured (see Figure 1a).

#### *4.2. Hydraulic Measurements*

*F. ornus* and *O. europaea* branches were collected in the late afternoon and rehydrated overnight while covered with a black plastic bag after cutting the basal 5 cm underwater. In the early morning, full hydration was checked to ensure that xylem water potential (Ψxyl) was above −0.3 MPa. A group of hydrated branches was used for hydraulic measurements to check possible residual embolism (hydrated group, H, *n* = 5). The remaining branches were dehydrated on the bench, covering several leaves with cling film and aluminum foil to stop transpiration and favor equilibration between leaf and stem xylem in order to measure Ψxyl with a pressure chamber (mod. 1505D, PMS Instrument Co., Albany, OR, USA). For olive, before bench dehydration, about 15 cm of the 2-year-old stem portions selected for hydraulic measurements and soaking treatment were deprived of leaf blades by cutting them at the insertion point at the petiole and sealing the cut section with impermeable glue (Super Attack, Loctite). Branches were dehydrated until reaching the target Ψxyl, fixed to -(3.5–3.9) MPa for *F. ornus* and to -(4.2–4.7) MPa for *O. Europaea,* based on preliminary assessments. At that point, a group of branches was immediately processed for hydraulic conductivity measurements (dehydration group, D, *n* = 5–6), while a third group was subjected to the soaking treatment as follows. Stem segments, obtained as described below, were sealed at the cut ends with wax tape (Parafilm® M), kept under water and then immersed in a white rectangular bowl containing distilled water, lying horizontally 1 cm below the water level. Stems were rotated by 180◦ every hour to allow whole stem light exposure. A LED panel (red/blue 96/24) was mounted above the bowl to produce a photosynthetic photon flux density (PPFD) at the water level of about 400 μmol m−2 s−1. According to Liu et al. [13], most of the embolized vessels of *S. matsudana* refilled already 2 h after branch segment immersion in light conditions. Therefore, duration of soaking was fixed to 2 h for both species (S2h group, *n* = 6). For *F. ornus,* a second set of branches was immersed for 4 h to check if longer rehydration times were needed for hydraulic recovery (S4h group, *n* = 7). After soaking, branch segments were prepared for hydraulic measurements as described below, while a 3 cm adjacent stem segment was taken for Ψxyl measurements, performed with a dew point hygrometer (WP4-C, Meter Group, Inc., Pullman, WA, USA). To this aim, the segment was quickly wiped with paper towel, cut longitudinally in half, placed in a sample holder and measured upon Ψxyl stabilization (reached in 30 min–40 min).

Measurements were performed on 3 cm–4 cm long segments of H, D, S2h and S4h stems. For all samples, the base of the branch was trimmed under clean tap water at a distance higher than the maximum vessel length. Maximum vessel length, determined with the "air method" described by Wang et al. [36], averaged 29 cm and 45 cm in *F. ornus* and *O. europaea* branches, respectively. Then, a selected ~15 cm long segment was cut and kept under water. S2h and S4h segments were sealed at both ends with Parafilm and soaked as described above. For hydraulic conductivity measurements, a shorter segment was obtained; the bark was removed from both cut ends, and several thin sharp slides were made with a razorblade at both ends to obtain a 3 cm–4 cm long segment; and the basal end was inserted in a hydraulic apparatus (see [25]).

Xylem hydraulic conductance was measured gravimetrically under a water head of 3.5 kPa, perfusing stems with filtered (0.45 μm) and degassed mineral water added with 10 mM KCl [37]. Hydraulic conductance was measured before (initial hydraulic conductance, *k*i) and after (maximum hydraulic conductance, *k*max), flushing the sample at high pressure (0.15 MPa) for 3 min to remove xylem embolism. Xylem hydraulic conductivity (*K*) was calculated as

$$\mathbf{K} = \mathbf{k} \times \mathbf{L} / \mathbf{A} \tag{1}$$

where L is the length of the segment, and A is the sapwood area, calculated as the average sapwood areas measured at the cut ends of the sample.

Percentage loss of hydraulic conductivity (PLC) was then calculated as

$$\text{PLC} = 100 \times \left[1 - \left(k\_{\text{i}} \, k\_{\text{max}} \, ^{-1} \right) \right] \tag{2}$$

#### *4.3. Micro-CT Scans and Image Processing*

Micro-CT scans were performed at the SYRMEP beamline of the Elettra Synchrotron light source (Trieste, Italy).

To check the capability of *F. ornus* to recover xylem embolism after soil rewetting, four 3-year-old saplings were dehydrated in pots to reach the target Ψxyl (−3.5 MPa) on 17–21 July 2020. The 2-year-old stem portion of two plants was scanned right after reaching the target Ψxyl (Dpot plants), while the other two were scanned 24 h after re-irrigation to soil

field capacity (recovery, Rpot plants). Two additional well-watered plants were measured as controls (Cpot). In all plants, Ψxyl was measured prior to scanning.

The soaking experiment was performed on potted *F. ornus* saplings on 1 August 2021. All plants were dehydrated by withholding irrigation for about 5 days. Three of them were scanned to detect the embolism level at the target Ψxyl (D plants). These plants were also used to tests the sample preparation effect on embolism formation, scanning the stem at the same point in three different steps: (i) the intact plant, (ii) the plant cut underwater right above the root collar after immersing the pot (sealed in a plastic bag) in water and (iii) the final stem segment obtained by cutting the stem 14 cm–21 cm above the previous cut, keeping the sample underwater. The length of the segment depended on the length of the 2-year-old stem segment portion, while the scan was always 8 cm–9 cm above the basal cut. After every cut, the cut surface was tightly sealed underwater with Parafilm. An additional scan was performed after cutting the stem segment a few mm above the scanned region in order to observe the fully embolized xylem.

The remaining three plants were prepared in the same way as the D plants, but they were only scanned after soaking the stem segment (obtained at step iii) in water for 2 h (S2h samples) as described above for branches measured with the hydraulic apparatus. This was carried out to avoid the possibility of multiple scans inhibiting cellular activity and possible related processes involved in water uptake and hydraulic recovery [38].

Before micro-CT scanning, all samples were quickly wrapped in cling film and Parafilm to avoid water loss and fixed to the sample holder. The CT studies were performed in propagation-based phase contrast modality using an Orca Flash 4.0 SCMOS, coupled with a 17 μm GGG scintillator, as a detector. The sample was placed 15 cm from the detector, and the pixel size was set at 2.1 μm. The experiment was performed in white beam mode, and 1.0 mm of silica was applied, resulting in a mean X-ray energy of about 22 keV. For each scan, 1800 projections were acquired during the sample rotation over 180◦.

The slice reconstructions were performed using SYRMEP Tomo Project (STP) software [39]. A phase retrieval pre-processing algorithm [40] was applied prior to the conventional filtered back-projection algorithm to increase the image contrast.

Reconstructed images were processed using ImageJ (https://imagej.nih.gov/ij/, accessed on 30 November 2021). Due to the larger diameter of the stems compared to the field of view (ca. 4 mm × 4 mm), analyses were conducted on about one-quarter of the stem section, excluding the immature xylem next to the vascular cambium (see Figure 3, Figure 4, Figure S1 and Figure S2). The embolized sapwood area (Aembol) was calculated by dividing the embolized pixel area by the analyzed sapwood area, expressed as percentage. The embolized vessel area (EVA) was calculated by dividing Aembol by the Aembol after the final cut above the scanned region (representing the percentage of sapwood area occupied by conduits), expressed as percentage.

#### *4.4. Statistics*

Statistical analyses were carried out with R (R Core Team, 2017). Boxplot panels were obtained with the "ggplot2" package in R. Bar charts were prepared with SigmaPlot (v. 12.0, Systat Software Inc., Berkshire, UK). For PLC and Ψxyl, the one-way ANOVA test (response variable ~f(treatment)) through the *aov* function was applied, followed by Tukey's HSD post hoc test (only for significant ANOVA, *p* < 0.05) through the *TukeyHSD* function in the "stats" package after checking for normality of residuals and homogeneity of variances. When homogeneity of variance assumption was violated, generalized least squares (GLS) models were calculated with the *gls* function, including a "varIdent" variance structure, in the "nlme" R package [41], followed by Tukey's HSD post hoc analysis (for significant tests), with *p*-values adjusted using the Bonferroni–Holm method. The effect of progressive cuttings on Aembol and EVA in the micro-CT test experiment was tested using linear mixed models (LMMs) through the *lme* function. Specifically, one LMM was fitted by separately setting Aembol or EVA as the response variable and by setting the cutting stage as the

explanatory one, with the plant replicate as the random effect. Pairwise comparisons were performed through *lsmeans* function in R package "emmeans" [42].

#### **5. Conclusions**

In this work, we tested the hypothesis that the immersion of photosynthesizing stem segments in distilled water for some hours in the presence of light would induce hydraulic recovery through bark water uptake in the embolized branches of two droughtresistant Mediterranean tree species (*F. ornus* and *O. europaea*). Hydraulic recovery was previously reported for the soaked stem segments of *S. matsudana*, and it was related to the generation of an osmotic gradient [13]. However, for the two species analyzed in this study, which are known to be capable of hydraulic repair under moderate tension (additionally demonstrated here by an in vivo micro-CT analysis of the potted saplings of *F. ornus*), refilling by bark water uptake did not occur. Our data suggest that refilling does not commonly occur in cut stems soaked under water, thus suggesting that this common procedure in hydraulic measurements is not likely to produce artefactual results. Clearly, further soaking studies under light conditions should be performed on photosynthesizing stems of other species to better understand both the potential and the limits of this process, as well as the possible consequences in terms of the accuracy of hydraulic measurements of xylem embolism.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/plants11030307/s1, Figure S1: X-ray micro-CT transverse images of stems of drought-stressed 2-year-old *F. ornus* saplings, Figure S2: Cutting effect on embolism formation in *F. ornus* potted saplings visualized with micro-CT. Figure S3: X-ray micro-CT transverse images of 2-year-old *F. ornus* stem segments after the soaking treatment (S2h).

**Author Contributions:** Conceptualization and experimental design, A.N., M.T. and S.N.; methodology, M.T., S.N. and A.N.; formal analysis, M.T. and S.N.; investigation, M.T., S.N., F.P., S.D.B., L.D. and G.T.; data curation, M.T.; visualization, M.T.; writing—original draft preparation, M.T. and A.N.; writing—review and editing, A.N.; supervision, A.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** The beamtime access to the SYRMEP beamline was granted by Elettra Sincrotrone Trieste (proposal no. 20195384 and no. 20210284).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the Supplementary Materials.

**Acknowledgments:** We thank the "Direzione centrale risorse agroalimentari, forestali e ittiche–area foreste e territorio" of the "Regione autonoma Friuli Venezia Giulia" and the public nursery Vivai Pascul (Tarcento, Italy) for providing us with the potted plant material.

**Conflicts of Interest:** The authors declare no conflict of interest.

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### *Article* **The INRAE Centre for Vegetable Germplasm: Geographically and Phenotypically Diverse Collections and Their Use in Genetics and Plant Breeding**

**Jérémy Salinier †, Véronique Lefebvre, Didier Besombes, Hélène Burck, Mathilde Causse , Marie-Christine Daunay, Catherine Dogimont , Juliette Goussopoulos, Christophe Gros, Brigitte Maisonneuve, Louis McLeod, Fatiha Tobal and Rebecca Stevens \***

> Unité de Génétique et Amélioration des Fruits et Légumes, INRAE, 84140 Montfavet, France<EMAIL_ADDRESS>(J.S<EMAIL_ADDRESS>(V.L<EMAIL_ADDRESS>(D.B<EMAIL_ADDRESS>(H.B<EMAIL_ADDRESS>(M.C<EMAIL_ADDRESS>(M.-C.D<EMAIL_ADDRESS>(C.D<EMAIL_ADDRESS>(J.G<EMAIL_ADDRESS>(C.G<EMAIL_ADDRESS>(B.M<EMAIL_ADDRESS>(L.M<EMAIL_ADDRESS>(F.T.)

**\*** Correspondence<EMAIL_ADDRESS>
† Current address: Unité Mixte de Recherche Peuplements Végétaux et Bioagresseurs en Milieu Tropical, CIRAD, 97410 Saint-Pierre, France.

**Abstract:** The French National Research Institute for Agriculture, Food and the Environment (INRAE) conserves and distributes five vegetable collections as seeds: the aubergine\* (in this article the word aubergine refers to eggplant), pepper, tomato, melon and lettuce collections, together with their wild or cultivated relatives, are conserved in Avignon, France. Accessions from the collections have geographically diverse origins, are generally well-described and fixed for traits of agronomic or scientific interest and have available passport data. In addition to currently conserving over 10,000 accessions (between 900 and 3000 accessions per crop), the centre maintains scientific collections such as core collections and bi- or multi-parental populations, which have also been genotyped with SNP markers. Each collection has its own merits and highlights, which are discussed in this review: the aubergine collection is a rich source of crop wild relatives of *Solanum*; the pepper, melon and lettuce collections have been screened for resistance to plant pathogens, including viruses, fungi, oomycetes and insects; and the tomato collection has been at the heart of genome-wide association studies for fruit quality traits and environmental stress tolerance.

**Keywords:** genetic resources; Solanaceae; *Cucumis*; *Lactuca*; diversity; vegetables; genebank

#### **1. Introduction**

The evolutionary history of vegetable crops is fascinating because it coincides with the birth and development of agriculture and the world history of human migrations and land discovery. For example, the wild ancestor of aubergine (eggplant), *Solanum insanum*, originated in Asia after ancestral species spread from Northeastern Africa two million years ago [1]. Domesticated chili pepper, *Capsicum annuum*, originated in Central–East Mexico more than 6500 years ago [2] and the process of tomato domestication started in Ecuador and Peru with *Solanum pimpinellifolium* before finishing with modern-sized fruit in Mexico [3]. Melon, *Cucumis melo*, was domesticated in both Africa and Asia before arriving in Europe [4,5]. The ancestor of lettuce was domesticated in Southwest Asia, and then some primitive forms were identified in Egypt around 4500 BC. With the Greek and Roman civilisations, lettuce spread rapidly through the Mediterranean, then Western Europe, and became known in America from the late fifteenth century [6–8]. Today's fruit and vegetables bear little resemblance to their undomesticated wild relatives: for example, the *Lactuca* ancestors of lettuce resemble a weed, with spines under the leaves, bitterness

**Citation:** Salinier, J.; Lefebvre, V.; Besombes, D.; Burck, H.; Causse, M.; Daunay, M.-C.; Dogimont, C.; Goussopoulos, J.; Gros, C.; Maisonneuve, B.; et al. The INRAE Centre for Vegetable Germplasm: Geographically and Phenotypically Diverse Collections and Their Use in Genetics and Plant Breeding. *Plants* **2022**, *11*, 347. https://doi.org/ 10.3390/plants11030347

Academic Editors: Milan S. Stankovic, Paula Baptista and Petronia Carillo

Received: 6 December 2021 Accepted: 21 January 2022 Published: 27 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and small seeds [9]. The domestication process for the five crops has changed the size of the edible part, total yield and also fruit shape and colour, ripening and plant architecture [10].

The domestication of plants has been at the expense of the plant's resistance to abiotic and biotic stresses [11] and latterly selection has also changed the organoleptic quality of the product [12]. The conservation of crop wild relatives (CWR) and knowledge of the evolutionary history of species mean that sources of genes for quality, resistance to pests and pathogens or adaptation to environmental conditions can be fully or partially restored by plant breeders, and knowledge at the genomic level facilitates their use. However, the accessibility and exchange of genetic resources is increasingly complicated, being regulated by international laws: exchange or collection of live material can no longer occur spontaneously. This means that ex-situ conservation is necessary, along with clear documentation (including phytosanitary documents, passport data and material transfer agreements) relating to the stored germplasm, particularly since the ITPGRFA (International Treaty on Plant Genetic Resources for Food and Agriculture) and the Nagoya protocol, adopted in 2010 for fair and equitable sharing of genetic resources [13]. These facts explain the importance of vegetable genebanks as a means of conserving and characterising phytogenetic resources.

Vegetable genebanks from around the world, a non-exhaustive list of which is included in Supplementary Table S1, conserve part of the diversity found in species of local and international importance. In France, a network called 'RARe' regroups the French Biological Resource Centres for agricultural, environmental and life sciences (https://www.agrobrcrare.org/ (accessed on 5 December 2021)). The vegetable genebank situated in Avignon in the south east of France, the INRAE Centre for Vegetable Germplasm—or Centre de Ressources Biologiques Légumes in French—conserves seeds for five vegetable crops. Three of these are from the Solanaceae family: aubergine is a staple food for much of Africa and Asia, pepper is an important crop worldwide, used fresh or dried as a spice, and tomato is one of the world's most produced fruit vegetables and used fresh or transformed in sauces and conserves. Melon (from the Cucurbitaceae family), grown worldwide in a diversity of forms and consumed as a sweet or non-sweet fruit or vegetable, and lettuce (from the Asteraceae family) complete the list of species conserved and are important for nutritional diversification [14].  The need for plants to suit new environmental or agricultural conditions is proof of their value.

The aim of this paper is to unite the information on the five collections conserved by the INRAE Centre for Vegetable Germplasm in a single article, which has not been accomplished previously. The collections are at the heart of much of the research carried out within the Genetics and Breeding of Fruit and Vegetables research unit and form the basis of many collaborations with both research institutes and plant breeders. We will include the history of the genebank's constitution, the characteristics of the material now present, the methods of conservation, the descriptors used, current databases and finally a spotlight on how the material has been used in different research programmes.

#### **2. Overview and Origins of the Five Collections**

Each collection has developed independently over different periods, having been built up by individual researchers working on different research programmes. Generally, the collections started in the 1960s and 1970s and grew rapidly at the end of the last century; introductions have mostly slowed since 2010 (Figure 1). The origins of accessions seen in Figure 2 are closely linked with the domestication/diversification centres for each crop.

**Figure 1.** Number of available accessions for each collection by year of introduction: each crop appears with a different colour (see legend). Note that the introduced accessions that have since been 'lost' do not appear.

**Figure 2.** Supposed geographic M49 region of origin or of breeding of the accessions using the colour code shown in the legend. Each square corresponds to 20 accessions. Note that sometimes the recorded origin is that of the collection, even if the accession comes from a foreign country.

Table 1 shows the number of accessions by geographic region of origin based on the M49 regions of the United Nations Statistics Division (standard country or area codes for statistical use) and Supplementary Figure S1 shows the number of accessions by precise country of origin and by continent. There are two points of note on the origin of an accession: firstly, the origin can correspond to the donating genebank's origin when historical passport data are not available, and, secondly, some accessions resulting from breeding programmes mixing parents of different origins are listed as being of unknown origin, which was seen as preferable to using the local geographic origin: this is the case for both aubergine and lettuce.

**Table 1.** Number of accessions originating from the different regions of the United Nations Statistical Division. Note that sometimes the recorded origin is that of the original collection, even if the accession was introduced from a foreign country. The colour scale ranges from dark (most represented region) to light (least represented region).

1 Not applicable for modern commercial cultivars (600 cultivated lettuces) because, for a given cultivar, breeding can be carried out in different countries.

The collections are rich in phenotypic diversity: many lines have been fixed for morphological characteristics often related to fruit size, shape or colour. Some of the visual diversity of a selection of our collections is shown in Figure 3 and includes wild or other cultivated species. The morphological diversity can be used to classify the collection into groups based on morphological data such as fruit colour and shape (Supplementary Figure S2), which is useful in certain cases—for example, for breeding or for comparing with genotyping data.

Table 2 summarises the number of accessions for the main individual species and the number of crop wild relatives, and Supplementary Figure S3 provides extra information on the number of accessions present in the collections for individual species.

**Figure 3.** Illustrations of examples of the phenotypic diversity present in the five collections. (**Top**) aubergine collection, from left to right—MM 01560: KOPEK PUTIH/TS58/X452; MM 00133: DE BUKAREST = BUCA; MM 00500: ZEBRINA LONGUE; MM 01737: AUBERGINE LOCAL OP10 (ABIDJAN); MM 00662: BIRM/S.0657; MM 01192: DIEGO ANTSINANARA (MARCHE). (**Second from top**) pepper collection, from left to right—PM0235: CHILI ICONO; PM0802: SAFI; PM0658: BOTIJILLO TINTO; PM1216: EX HD(YWx702)11; PM0611: MULATO ROQUE; PM1409: VA-NIA; PM0225: PEPERONE DI LUCCA. (**Middle line**) tomato collection, from left to right—T300077: LA1274 *S. peruvianum*; T300165: LA1447: *S. cheesmanii*; T101431: Marmande Jaune; T101560: Poivron des Andes; T102434: EA2679; T102446: EA2751. (**Second from bottom**) melon collection, from left to right—Unknown; ME0857: MR-1; ME2343: ACEM; ME2369: DAMIAN GUA; ME1038: SVI 0024; wild African accession. (**Bottom**) lettuce collection, from left to right—LC0251: butterhead cv Grosse brune têtue; LC0152: Batavia, cv craquante d'Ecully; LC0206: oak leaf, cv Feuille de chêne espagnole; Production of seed in insect-proof tunnel; LC0858: *L. virosa*, LS360.

**Table 2.** Summary of taxonomy of the collections for the accessions whose taxonomy has been identified.

1 includes 91 *Cucumis melo* subsp. agrestis (wild melon).

#### **3. Summary of the Individual Collections**

Each collection is described in detail below with information on geographic diversity, taxonomy and scientific resources available: core collections are available for aubergine, pepper and tomato [15,16]. Many mapping progenies have been created for research purposes and are available for collaborative projects.

(i). **The aubergine collection** (**2388 accessions**) contains two introductory peaks: one from 1991, which corresponds to the scientific collaboration with the taxonomist Richard N. Lester of the University of Birmingham, UK, and the second in 2004, when the collection of *Solanum* species related to aubergine were transferred from the University of Birmingham to INRAE [17]. More recently, we have introduced new aubergine accessions from the H2020 G2P-SOL project (http://www.g2p-sol.eu/ (accessed on 5 December 2021)). Most of the aubergine accessions originate from Africa and Asia. The aubergine collection is notable amongst the five collections because it includes a large proportion of crop wild relatives (26%). This number of CWR is rare in other germplasm centres, mostly because of the difficulties in maintaining them. The collection includes more than 1000 accessions principally from Africa (related cultivated species including *Solanum macrocarpon* L., *Solanum aethiopicum* L., *Solanum scabrum Mill.).* These species are indigenous African leafy vegetables and/or fruits: *S. aethiopicum* and *S. macrocarpon* provide a usable secondary gene pool for the improvement of *S. melongena.* Approximately 500 accessions, representing over a hundred wild species, the majority related to cultivated aubergine, complete the collection, as well as accessions of other Solanaceae of interest (*Atropa*, *Datura*, *Lycium*, *Nicandra*, *Physalis* and *Withania*) [18]. A word of caution must be added about the taxonomy of *Solanum* species. The taxonomic classification of aubergine dates back to the work of RN Lester in the 1990s; the nomenclature has evolved since the early 2000s and the taxonomic status of several taxa is unclear [19,20].

The aubergine scientific collection contains five double-haploid populations and F1, F2 and backcrosses of six biparental progenies. A total of 706 aubergine accessions from our collection were supplied to the G2P-SOL project, of which 106 are included in the final core collection from the project. The project has and will supply low- and high-density genotyping data, which extends the knowledge available on the whole collection.

#### **4. Collection Management**

The genebank follows typical procedures for seed storage, multiplication and distribution [45]. An overview of the processes and procedures is provided in Figure 4, with an indication given regarding the number of accessions or samples concerned by each process for a typical year. These figures are specific to the INRAE Centre for Vegetable Germplasm.

(i). Conservation of seed stocks

The genebank focuses on medium- and long-term storage (4 ◦C and 50% relative humidity for ~15 years or −20 ◦C and 50% relative humidity for >30 years [46]) with procedures for checking germination rates following long-term storage (after one year and then every 5 or 10 years depending on the results). Safety duplicates are held at a genebank centre based in Montpellier, France.

(ii). Regeneration of seed stocks

Regeneration by seed multiplication is performed mostly by controlled self-pollination in insect-proof greenhouses (three plants per accession, two for lettuce). For accessions that are regenerated as wild populations, i.e., those accessions that were harvested from a mixed genepool that potentially contains many rare alleles and/or accessions that are self-incompatible, we use isolation fields with natural pollinators. We aim for a minimum of 600 seeds per accession. Twenty seeds are used in germination tests.

The management of a collection of wild relatives of *Solanum* is complex: problems arise at numerous levels—for example, difficulty in obtaining accessions, misidentification, poor germination, physiological problems with flowering, pollination and fruit set [47]. Many wild accessions missing from the collection are endangered because of the eradication of their natural habitat, notably in East Africa [48]. Many species cannot be regenerated in the short culture periods available in the greenhouses or fields in Avignon; this is particularly true for the majority of *Solanum* species native to Australia and many species of tropical origin. New culture conditions for those species that are difficult to regenerate have been investigated in a recent project (https://www6.paca.inrae.fr/gafl\_eng/Partnershipsand-Projects/Closed-Projects/SESAM (accessed on 5 December 2021)) in which we have

varied the photoperiod, growing season or watering regime to obtain success in saving some accessions.

**Figure 4.** Current procedures for management of the collections at the INRAE Vegetable Germplasm Centre, including the major missions of introduction, multiplication, description, conservation and distribution and with an estimation of the number of samples or accessions involved. The multiplications are mostly carried out by self-pollination. Illustrations Camille Ulrich—Copyright INRAE.

#### (iii). Descriptions

Collections are systematically characterised using botanic and primary descriptors (fruit, flower, plant and leaf characteristics). A large percentage of the collections has been described using descriptors from either the International Plant Genetic Resources Institute, the International Union for the Protection of New Varieties in Plants or with in-house descriptors. The supplementary dataset includes the list of descriptors used for each collection (Supplementary Figure S4) and the proportion of accessions that have been described. Fruit diversity and colour are a particular focus for descriptions [23,26,49–51] and we are now establishing techniques for phenotyping root system architecture [52]. Specific ways of describing wild lettuce and aubergine, which have characteristics quite different to the cultivated species, have also been put into place (Supplementary Figure S4). Cultivated lettuce has been described for bolting and flowering characteristics; the heading characters were scored on progeny, either in the field for cultivars bred for outdoor culture, or under plastic tunnels for cultivars bred for protected cultures (Supplementary Figure S4). For the five collections, secondary descriptors include criteria such as pest and pathogen resistance (Supplementary Figure S4). Fixing of traits of agronomic interest (for example, fruit colour or shape) has been carried out for most of the accessions when possible, in order to complete phenotypic gaps in the collections.

(iv). Networks and sub-collections

Each INRAE collection is wholly or partially regenerated and/or described in partnership with private breeding companies, the French organisation 'Variety and Seed Study and Control Group' (GEVES) and the CIRAD. With the INRAE Centre for Vegetable Germplasm, a different subset of these partners forms three national networks: (i) the Fruit Solanaceae network (created in 1996), (ii) the melon network (since 1997) and (iii) the *Lactuca* network (created in 2020). Each network partner not only contributes to the network by multiplying seeds and describing accessions each year but also shares extra varieties or accessions with the network's members. The French national collection (approximately 100 accessions of patrimonial value) is defined by the network members and includes accessions from INRAE and the network's collections. The national collection is freely available to all parties and the public and appears in the databases (see below). In addition, the aubergine national collection has been submitted to the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA), to encourage the open exchange of plant genetic resources for food use.

#### (v). Seed and data sharing

Online local databases are used for storing passport data, descriptions and information on seeds (seed batch date, seed quantity, exchange, storage conditions and germination rate). Our local databases are linked to national or European databases where part of the collections are found (for the moment often only the national collection). These databases contain accession names and passport data as a minimum and currently include Siregal (https://urgi.versailles.inra.fr/siregal/siregal/grc.do (accessed on 5 December 2021)), where phenotyping data are also available, Florilège (http://florilege.arcad-project.org/fr (accessed on 5 December 2021)), ECPGR (https://www.ecpgr.cgiar.org/ (accessed on 5 December 2021)) and Eurisco (https://eurisco.ipk-gatersleben.de/ (accessed on 5 December 2021)). As many changes are occurring in the management of these databases and the links between them, we have provided a list of our accessions and passport data in Supplementary Table S2. This includes the national, network and INRAE collections. Please contact us for the distribution conditions specific to each accession.

Seeds can be ordered via our local website: https://www6.paca.inrae.fr/gafl\_eng/ Vegetable-Germplasm-Centre/Use-our-genetic-resources (accessed on 5 December 2021). A charge is made for sending accessions, which are provided with a phytosanitary passport or certificate and MTA as necessary.

We estimate that approximately 2000–3000 samples are sent annually to partners outside the research unit, of which around 50% are for private research companies, nearly 20% are for international and national research laboratories and around 5% go to members of the general public, associations and amateurs. The remaining 25% of samples are supplied to other INRAE laboratories.

#### **5. The Collections as Material for Scientific Study**

The genetic resources are used by INRAE, research institutes, universities and breeding companies. The collections are currently used or studied for the following themes.

#### (i). Domestication and structure of the collections

Solanaceae genetic resources conserved at INRAE have been used to investigate domestication scenarios by analysing transcriptome data from aubergine, pepper and tomato with their close wild relatives to show both common and species-specific demographic changes, the latter being particularly true for aubergine [53]. Geographic isolation has played a role in determining the genetic structure of aubergine populations; there is also evidence for outcrossing in aubergine between wild and cultivated gene pools [20,54–56].

In pepper, 1352 accessions of our collection, representing 11 *Capsicum* species with wide geographic diversity, were genotyped using 28 microsatellite markers. This collection was assigned to six clusters, with three clusters for *C. annuum* and three additional clusters separating the other main species, including the cultivated species and wild relatives, according to their taxonomic classification (*C. frutescens*/*C. chinense*, *C. baccatum*, *C. pubescens*). The three *C. annuum* clusters were significantly distinct for plant and fruit descriptors corresponding to cultivar types, showing that the genetic structure of cultivated pepper has been affected by human selection in primary as well as secondary diversification centres [21]. A

total of 869 accessions from our collection, together with more than 9000 accessions from nine other collections worldwide, were genotyped with more than 26,000 GBS-derived SNPs in the G2P-SOL project. This study revealed a reticulate interbreeding history in *C. annuum* and a differentiation between two major genepools (i.e., European and Central American/Asian). It also demonstrated that approximately 80% of the accessions we maintain at INRAE are not represented in the main world collections [26].

In tomato, where the phenotypic effects of domestication have been well documented [57,58], it has been shown that domestication has affected gene expression within gene regulatory networks: changes in gene co-expression levels were associated with lower nucleotide diversity, often because of the fixation of useful mutations during domestication [53,59]. Tomato also appears to have been domesticated in a two-step process: first in South America and then in Mesoamerica [60]. A similar reduction in nucleotide diversity has been seen in candidate genes involved in meristem development in cultivated compared to wild tomato: around half the genes analysed revealed footprints of selection and polymorphisms putatively involved in fruit size variation by showing negative Tajima's D and a reduction in nucleotide diversity in cultivated tomato [61,62].

Genotyping of 713 melon accessions has given information on the collection's genetic subdivisions and led to the hypothesis that melon has probably been domesticated at least twice [63]. More recently, one hundred accessions of our collection, together with more than 1000 accessions worldwide, have been re-sequenced: the data confirmed the two domestications in Africa and Asia and suggested the occurrence of a third domestication event in India [5]. Iconographical and textual sources suggest the presence of melon in the Mediterranean basin dating back to antiquity and are illustrated by two melon types in Italy in the late medieval times: melons of the Chate group, the likely ancestor of our sweet melons, and elongated and unsweet snake melons, still appreciated in East and North Africa [64]. Complementary studies have shown the genetic diversity available within melon cultigroups [65,66] and the phenotypic diversity, which is greater in cultivated melon than wild melon [51].

#### (ii). Resistance to plant pests and pathogens

In aubergine, the collections are of particular interest in screening for *Ralstonia solanacearum* resistance and mapping populations have been created to this end: *R. solanacearum* (bacterial wilt, southern wilt or potato brown rot) causes major yield losses in the tropics and subtropics but is also a threat in temperate climates [67–69]. Numerous strains or phylotypes exist, and, whereas one aubergine variety may be resistant or partially resistant to one strain, it is often not resistant to all strains. The aubergine collection is a source of resistance genes against *Ralstonia*: from a subset of 10 genotypes, none controlled all strains but some are able to control the most virulent strains (six accessions were totally resistant to six strains) [70–73]. Crosses between resistant and susceptible parents have generated recombinant inbred lines [72] for QTL mapping related to resistance in different environments; a major resistance gene, *ERs1*, has been identified. Further sequencing and QTL mapping for resistance have shown that both broad-spectrum and strain-specific QTLs exist and that, by combining three or more QTLs, large-spectrum resistance could be obtained [74,75]. Remarkably, aubergine accession Ceylan SM 164 was resistant to all six strains. Aubergine accessions SM6, Surya and AG91-25, as well as pepper accessions CA8 and MC4, were moderately to highly resistant to all six strains [76].

The pepper germplasm has been shown to include a diversity of genetic backgrounds favourable to resistance durability to pests and pathogens. In the case of potato virus Y, the evaluation of a core collection of *Capsicum annuum* landraces showed breakdown frequencies of given resistance alleles that varied from 0 to 53% [15]. In the same core collection, genome-wide association studies detected SNPs associated with the presence of the virus at inoculation and/or systemically [27]. Screening of 1179 accessions of the pepper collection for quantitative resistance to *P. capsici* revealed 26 new sources of resistance [77] and furthermore a key conserved QTL cluster has been shown to exhibit broad-spectrum resistance to *P. capsici* [78]. In a similar way, double-haploid lines obtained from the F1 hybrid of a resistant accession (H3) crossed with a susceptible accession (Vania) revealed QTLs for resistance to powdery mildew due to *Leveillula taurica* [79]. Other double-haploid progenies have been used to study the genetics of resistance to viruses such as potato virus Y, tobacco mosaic virus, tomato spotted wilt virus, cucumber mosaic virus and pepper veinal mottle virus [80–84], and *P. capsici* [85]. Within the G2P-SOL project, the final core collection is to be phenotyped for agronomic traits and biotic stress resistance, including to *Phytophthora capsici*, *Leveillula taurica*, cucumber mosaic virus and *Meloidogyne incognita* by INRAE and *Verticilium dahliae*, pepper mild mottle virus, *Fusarium oxysporum* and tomato spotted wilt virus by other partners.

The melon collection has been extensively evaluated for resistance to pests and pathogens, including several viruses (cucumber mosaic virus; potyviruses such as the zucchini yellow mosaic virus; and the whitefly-transmitted begomoviruses, melon chlorotic mosaic virus, tomato leaf curl New Delhi virus and watermelon chlorotic stunt virus); fungi such as Fusarium wilt and downy and powdery mildew, and also insects [86–91]. QTLs have been detected for both whitefly and aphid resistance in a biparental population: a major QTL affecting aphid behaviour and biotic potential co-localises with the *Vat* gene, although resistance is polygenic [32,92–95]. In melon, an example of a success story is the cloning of the *Vat* gene, which was shown to be a CC-NBS-LRR gene mediating both resistance to aphid infestation and virus infection using *Aphis gossypii* as a vector [94,96].

The lettuce collection in particular has been a target for screening for resistance to plant diseases including viruses, oomycetes, bacteria, nematodes and fungi (summarised in Table 3) [42,97–100]. Breeding for resistance in lettuce is a priority and many resistance gene sources have been reported, particularly in wild lettuce species, for potyvirus, *B. lactucae* and *Meloidogyne incognita* [100–102]. When resistance is found in wild species, the compatibility with *L. sativa* determines the success of breeding programmes: several species are compatible with *L. sativa* (*L. serriola*, *L. saligna* and *L. virosa*) and can therefore be used, but the crosses are only straightforward with *L. serriola*. The hybrids with *L. virosa* are sterile and often show physiological disorders such as necrosis or stunted plants, which are difficult to eliminate in any progenies that are obtained [101]. Resistant genes from *L. virosa* have been introgressed into a butterhead background to produce lines with new resistance to *Bremia* [103] and two potyviruses [43,44]. More than 400 lettuce accessions have also been tested with three stimulators of plant defences; some interesting protection against *B. lactucae* was obtained with a few cultivars [104].

#### (iii). Floral biology and crossing compatibility

The outcrossing potential of aubergine was investigated using 23 populations of wild weedy aubergine [54]. Controlled crosses with cultivated aubergine resulted in seed set and viable F1 progeny. The exerted stigmas of wild aubergines are likely to promote outcrossing under natural conditions, highlighting the risk of growing Bt transgenic plants in Southern India, where wild and cultivated aubergines coexist.

Variability in the melon germplasm collection (497 accessions of *C. melo*) has been used to validate a locus controlling sex determination in melon: within C. melo, most plants are monoecious (single sex flowers) or andromonoecious (male and hermaphrodite flowers). This trait is under the control of a recessive locus and a single-nucleotide polymorphism in the CmACS7 gene, which co-segregates with the sex determination phenotype of the flowers [53]. The insertion of a transposon, which epigenetically controls the expression of the CmWIP1 transcription factor, was shown to co-segregate with the gynoecious (female flowers only) phenotype [41]. Together with advances in the understanding of sex determination in plants, these studies have provided markers of interest for breeders, facilitating F1 seed production in melon.

The crossing potential of seven accessions of *L. virosa* with 10 lettuce cultivars was compared by using in vitro cultures of immature embryos or harvesting mature seeds. Few F1 progenies were obtained but, after backcrossing to lettuce and self-pollination, some lines with virus resistance were obtained [106].

**Table 3.** Screening of the lettuce collection for resistance to pests and pathogens.

#### (iv). Fruit quality and abiotic stress tolerance

In pepper, the collections have been used to show that the gene encoding Capsanthin Capsorubin Synthase is responsible for the yellow colour in pepper [50]. Following work on sensory traits in an intraspecific RIL population [49], several core collections of tomato have been used to evaluate the genetics of fruit quality traits [16,107–110]. An example is the genetics of fruit metabolite content—including sugars, vitamin C, amino acids and volatiles. These traits, using two different genotyped core collections, one composed mostly of cherry tomato accessions and the other including breeding material, have been shown to be heritable and under the control of multiple QTLs. The studies also pinpointed candidate genes for traits such as fruit malate content and phenylpropanoid volatile production [16,108]. The GWAS studies have paved the way towards predictive genomic selection, which can be used on crops such as tomato: this could be particularly useful for polygenic traits such as fruit quality [111]. The tomato scientific collections have also been evaluated for potential adaptation to environmental stress—in particular, under conditions of limited water [107,112], elevated temperature [113] and, for the MAGIC population, multiple stress conditions [114], revealing candidate genes involved in stress responses.

#### (v). Selection and breeding

The creation of new vegetable varieties has mostly stopped at INRAE and is now the domain of private plant breeders. However, some of our past creations are well known. Crosses of certain aubergine introductions combined with INRAE breeding in traditional populations allowed the creation of the first French F1 hybrids, registered in the official catalogue in 1973: F1 Bonica with purple globular fruits and F1 Baluroi with mediumlength purple fruits. In aubergine, crop wild relatives offer numerous possibilities for the improvement of *S. melongena* [48], including providing resistance to pests and pathogens: the challenge is creating interspecific hybrids (sexual or somatic) and research has been dedicated to this area over recent decades [115–118].

Crop wild relatives are also particularly good as rootstocks [119]. The Solanaceae family contains many candidates that can be used as rootstocks for cultivated aubergine and tomato. Part of the collection has been screened to find species having a good grafting affinity with aubergine and a number of candidates identified [120,121].

In pepper, our best-known varieties are Lamuyo F1 hybrid (the first pepper hybrid in the world) and Alby (cumulating five resistance genes) as well as the F1 sweet pepper hybrid Ulysse. In tomato, Montfavet H63-5 is an F1 hybrid widely grown in France, and elsewhere, in the 1970s. Recent tomato varieties include Garance and Jouvance (good colour and flavour, firm, eight disease resistance genes) and Terradou (processing tomato, high soluble solids).

The Margot (1988) melon was the first variety to integrate the *Vat* gene for aphid resistance, and breeders have incorporated the resistance into many varieties, currently including more than 50% of Charentais varieties. Our genetic resources are still used for breeding through our private/public networks and particularly the melon network: only the genetic diversity within the species *C. melo* is usable—there are currently no inter-specific crosses.

For lettuce breeding, resistance to Lettuce mosaic virus (LMV), identified in *L. virosa* PIVT1398 [42], was introduced into butterhead lines by INRAE [101]. The NILs with the *Mo3* locus were also resistant to another potyvirus: lettuce Italian necrotic virus (LINV) [44]. Some NILs with the *Mo3* resistance gene have been made available for research and breeding [43].

#### **6. Conclusions and Perspectives**

An overview of the highlights of each of the five collections with the scientific data and resources that have been produced is presented in Figure 5. The availability of genotyping data and phenotypic data has enhanced the usefulness and long-term perspectives of the collections, although phenotyping is still to be carried out on the collections: for example, root descriptors are mostly absent and little information is available on the adaptation of the collections to the environment. Projects including phenotyping or genotyping also allow us to improve the collections by eliminating duplicates or by using introductions to cover gaps. We mainly aim at being complementary to other collections in the world, but there is a need to know which accessions are complementary, and this shows the importance of having an inventory of genetic resources (passport data and descriptors) or genotyping data of collections, which also presents several advantages:

For the longer-term perspectives for our genetic resource collections at a local level, we can list as priorities:

We are continuing our efforts to reduce the cost of conservation, guarantee the longterm preservation of crop resources and increase the knowledge on these resources to facilitate their use in research and breeding projects.

**Figure 5.** Collection highlights, total number of accessions, screening data and scientific resources available for each of the five collections.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/plants11030347/s1, Figure S1: Number of accessions and their origins by country; Figure S2: Morphological groups (aubergine—fruit colour; pepper—fruit shape, colour and pungency; tomato fruit shape and colour; melon—cultigroups; and lettuce—cultigroups); Figure S3: Number of accessions and their taxa or species; Figure S4: Qualitative and quantitative descriptor lists used for each of the five crops and the proportion of accessions that have been described. Table S1: Non-exhaustive list of vegetable genebanks worldwide; Table S2: List of the accessions for each collection with passport data (Excel file).

**Author Contributions:** Conceptualisation, V.L., M.C., M.-C.D., C.D., B.M. and R.S.; analysis, J.S.; resources, D.B., H.B., J.G., C.G., L.M. and F.T.; data curation, J.S., J.G. and B.M.; writing—original draft preparation, J.S. and R.S.; writing—coordination of review and editing, R.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was in part funded by grants received from IBiSA (Infrastructures en Biologie Santé et Agronomie). The SESAM project was funded by a grant received from the French 'Ministère de l'Agriculture et de l'Alimentation' and the French 'Groupement national interprofessionnel des semences et plants'.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank all those who have participated in the buildingup, conservation, maintenance and characterisation of these collections over the years and those who have generously donated or shared material.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Article* **The Anti-Inflammatory Response of** *Lavandula luisieri* **and** *Lavandula pedunculata* **Essential Oils**

**Monica Zuzarte 1,2,3,\*, Cátia Sousa 2,4,5, Carlos Cavaleiro 4,6, Maria Teresa Cruz 2,4,5 and Lígia Salgueiro 4,6**

**Abstract:** Portuguese lavenders remain undervalued in global markets due to the lack of high-quality end-products and scarcity of scientific-based studies validating their bioactive potential. Moreover, chemical variability is frequent in these species, and can compromise both safety and efficacy. In the present study, the anti-inflammatory potential of *L. luisieri* and *L. pedunculata*, two highly prevalent species in Portugal, was assessed and correlated with their chemical variability. Representative samples with distinct chemical profiles were selected to assess the anti-inflammatory effect on LPSstimulated macrophages. *L. luisieri* essential oil with low quantities of necrodane derivatives was the most potent at inhibiting NO production. Interestingly, the essential oil was more effective than its main compounds (1,8-cineole and fenchone), assessed alone or in combination. Our results also demonstrated a significant effect of the oil on the expression of the inflammatory proteins (iNOS and pro-IL-1β) and on the NF-κB pathway. Overall, this study highlights the impact of chemical variability on oils' efficacy by showing distinct effects among the chemotypes. We also identify *L. luisieri* essential oil, with low quantities of necrodane derivatives, as the most promising in the mitigation of the inflammatory response, thus corroborating its traditional uses and paving the way for the development of herbal medicinal products.

**Keywords:** essential oil; iNOS; interleukin; lavenders; NF-κB

#### **1. Introduction**

In recent years, there has been a growing demand for natural products, with the global market impact of aromatic and medicinal plants increasing mainly in the health sector [1]. Several industries have perceived this trend and are searching for bioactive and biodegradable products that concomitantly present a safe profile to humans and animals [2]. Lamiaceae is among the families that comprise a higher number of aromatic plants with commercial interest [3]. For example, several lavender products, such as essential oils, fresh or dried flowers, and landscape plants, mainly from four *Lavandula* species, namely *L. stoechas*, *L. angustifolia*, *L. latifolia*, and *L.* x *intermedia* (a hybrid between the two last species), are highly appreciated. These species are extremely valuable and recognized in global markets and, in some cases, regulated by international ISO standards [4,5]. Nevertheless, the genus comprises many other species that remain undervalued and have no commercial value. For example, the Portuguese lavenders *L. luisieri* and *L. pedunculata* remain unbranded despite their economic potential, high prevalence, and popularity among

**Citation:** Zuzarte, M.; Sousa, C.; Cavaleiro, C.; Cruz, M.T.; Salgueiro, L. The Anti-Inflammatory Response of *Lavandula luisieri* and *Lavandula pedunculata* Essential Oils. *Plants* **2022**, *11*, 370. https://doi.org/ 10.3390/plants11030370

Academic Editors: Milan S. Stankovic, Paula Baptista and Petronia Carillo

Received: 31 December 2021 Accepted: 26 January 2022 Published: 29 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

local communities [6]. A lack of both scientific-based studies validating their traditional applications and high-quality end-products is partially responsible for this scenario. Indeed, the chemical variability of lavenders is high [7–9] and compromises their bioactive potential. Therefore, a well-defined chemical profile and validated bioactive potential are necessary to fulfil the requirements of competitive markets and guarantee efficacy and safety profiles.

*L. luisieri* has been traditionally used as a expectorant, stimulant, spasmolytic, and laxative, for relieving headaches and migraines, and for its disinfectant properties [7,10]. In addition, *L. pedunculata* has been applied to treat bronchitis, cough, asthma, headaches, anxiety, insomnia, strokes, and dyspepsia, and as an analgesic [11–13]. Several of these conditions are associated with an inflammatory response and their relief or even treatment may be related to the anti-inflammatory properties of these aromatic plants. Although the anti-inflammatory potential of other lavender species, namely *L. angustifolia*, *L. stoechas*, *L. multifida* and *L. viridis* [14–18], is known, information on *L. luisieri* and *L. pedunculata* essential oils is sparse. Only two studies were carried out on *L. luisieri* essential oils, one showing its effect on primary human chondrocytes and the intestinal cell line [19], and another highlighting its potential to reduce cytokines and chemokines in human acute monocytic leukemia cells (THP-1) [20]. Taking this into account, and the fact that chronic inflammation has been associated with aging and related diseases [21–23] that greatly impact the quality of life, it is imperative to search for effective and safe anti-inflammatory therapeutic strategies. Therefore, using an *in vitro* model of lipopolysaccharide (LPS)-stimulated macrophages, the present study aimed to evaluate the anti-inflammatory potential of these essential oils with distinct chemical profiles. For the most promising oil, the effect of the main compounds, alone or in combination, was also investigated, and its mechanism of action underlying the pharmacological effects, namely the nuclear factor kappa B (NF-κB) signaling pathway, was further explored. Our results demonstrate relevant antiinflammatory properties for the chemotype of *L. luisieri* essential oil with low quantities of necrodane derivatives, thus paving the way for the development of new plant-based anti-inflammatory therapeutics.

#### **2. Results**

#### *2.1. Essential Oil Characterization*

Samples of *L. luisieri* and *L. pedunculata* were collected throughout Portugal and their essential oils were further analyzed. The main chemical compounds present in the different samples allowed the identification of two chemotypes for *L. luisieri* and three for *L. pedunculata*. *L. luisieri* chemotypes were distinguished by the amounts of necrodane derivatives, with some samples presenting low concentrations of these compounds (6.9 ± 4.6%) and others showing high amounts (24.9 ± 2.4%). For *L. pedunculata*, differences occurred in the concentrations of three main compounds, with samples rich in fenchone (36.0% ± 12.9), 1,8-cineol (32.9% ± 8.3) or camphor (37.5% ± 8.1). The main compounds of representative samples of the identified chemotypes are listed in Table 1. Overall, the essential oils from both species were obtained with a yield of 0.8–1.10% and were characterized by high contents of oxygen-containing monoterpenes (54.9–80.3%). Regarding *L. luisieri*, the concentrations of 1,8-cineole, fenchone and necrodane derivatives differed among samples, with sample A1 characterized by low amounts of necrodane derivatives and high amounts of 1,8-cineole and fenchone, whereas in sample A2, necrodane derivatives occurred in higher amounts. For *L. pedunculata*, three main compounds (1,8-cineole, fenchone and camphor) were always identified, but their concentrations differed significantly between samples, with B1 rich in 1,8-cineole, B2 in fenchone, and B3 in camphor.

**Table 1.** Main compounds of *Lavandula luisieri* and *Lavandula pedunculata* essential oils.

Compounds listed in order of elution from the SPB-1 column. RI SPB-1: GC-retention indices relative to C9–C23 n-alkanes on the SPB-1 column. RI SW 10: GC-retention indices relative to C9–C23 n-alkanes on the SupelcoWax-10 column. A1 and A2—representative samples of *L. luisieri* chemotypes; B1–B3—representative samples of *L. pedunculata* chemotypes. (-)—not detected.

#### *2.2. Nitric Oxide Scavenging Potential of Lavandula luisieri and Lavandula pedunculata Essential Oils*

The antioxidant potential of the essential oils towards reactive nitrogen species (RNS) was assessed using an *in chemico* nitrite-scavenging assay. Overall, the essential oils were ineffective and no nitric oxide (NO) scavenging effect was observed for all the tested concentrations (Figure 1). Indeed, nitrite values remained very similar to those of the NO donor, SNAP, in both *L. luisieri* (Figure 1A,B) and *L. pedunculata* (Figure 1C–E) chemotypes.

**Figure 1.** NO scavenging potential of *Lavandula* spp. essential oils. Effect of *L. luisieri* essential oil with (**A**) low (sample A1) or (**B**) high (sample A2) amounts of necrodane derivatives and *L. pedunculata* rich in (**C**) 1,8-cineole (sample B1), (**D**) fenchone (sample B2) and (**E**) camphor (sample B3). Different concentrations of essential oils (0.08–0.64 μL/mL) were incubated with the NO donor, SNAP (100 mM), in culture medium for 3 h. Results are expressed as percentage of NO release triggered by SNAP (positive control, dark grey bars). Each value represents the mean ± SEM of three experiments, performed in duplicate (## *p* < 0.01, ### *p* < 0.001, compared to control).

#### *2.3. Effect of Lavandula luisieri and Lavandula pedunculata Essential Oils on Macrophages' Viability*

To evaluate the anti-inflammatory activity of the essential oils, we first evaluated their effect on cell viability of murine macrophages stimulated with LPS (Figure 2), in order to select non-toxic concentrations. For all the essential oils, a range of concentrations varying from 0.08 to 0.64 μL/mL was tested. Overall, the essential oils were devoid of toxicity, except for the highest concentration of *L. luisieri* essential oil rich in necrodane derivatives (sample A2), which decreased cell viability by more than 70% compared to LPS-treated cells (Figure 2B). The vehicle, DMSO, did not interfere with macrophages' viability compared to control cells (Figure S1A).

**Figure 2.** The effect of *Lavandula* spp. essential oils on cell viability. Effect of *L. luisieri* essential oil with (**A**) low (sample A1) or (**B**) high (sample A2) amounts of necrodane derivatives and *L. pedunculata* rich in (**C**) 1,8-cineole (sample B1), (**D**) fenchone (sample B2) and (**E**) camphor (sample B3) on macrophages' viability. Cells were maintained in culture medium (control), or incubated with 1 μg/mL LPS or with LPS in the presence of different concentrations of the oil (0.08–0.64 μL/mL), for 24 h. Results are expressed as percentage of MTT reduction by cells treated with LPS. Each value represents the mean ± SEM of three experiments, performed in duplicate (\* *p* < 0.05, \*\* *p* < 0.01 and \*\*\*\* *p* < 0.0001, compared to LPS). The dotted line represents the threshold (70% of maximal viability) below which cytotoxicity is recognized, in agreement with standard ISO 10993-5 [24].

#### *2.4. Effect of Lavandula luisieri and Lavandula pedunculata Essential Oils on NO Production*

Using an *in vitro* model of LPS-stimulated macrophages referred to previously, the non-toxic concentrations of the essential oils determined in the previous section were used to assess the effect of the oils with distinct chemical profiles on LPS-induced NO production. Macrophages produce residual levels of nitrites (stable metabolite of NO). However, when stimulated with LPS, NO production increases considerably when compared to control. *L. luisieri* essential oils having both low amounts of necrodane derivatives (sample A1) and high amounts of necrodane derivatives (sample A2), significantly decreased LPS-induced NO production in a concentration-dependent manner (Figure 3A,B, respectively). However, sample A1 was much more effective in decreasing NO production (Figure 3A) than sample A2 (Figure 3B) at the concentrations tested without compromising cell viability (Figure 2A). Regarding *L. pedunculata* essential oils, the samples with high amounts of 1,8-cineole (B1; Figure 3C) and high amounts of camphor (B3; Figure 3E) were able to inhibit NO production induced by LPS, in a concentration-dependent manner, with the latter being more active at the lower concentration tested. By comparison, the sample with high amounts of fenchone (B2; Figure 3D) significantly inhibited NO production, but only at the higher concentrations tested (0.32 and 0.64 μL/mL). The vehicle, DMSO, did not interfere with LPS-induced NO production when compared to LPS-treated cells (Figure S1B).

**Figure 3.** Inhibition of NO production by *Lavandula* spp. essential oils. Effect of *L. luisieri* essential oil with (**A**) low (sample A1) or (**B**) high (sample A2) amounts of necrodane derivatives and *L. pedunculata* rich in (**C**) 1,8-cineole (sample B1), (**D**) fenchone (sample B2) and (**E**) camphor (sample B3) on NO production induced by LPS in macrophages. Cells were maintained in culture medium (control), or incubated with 1 μg/mL LPS, or with LPS in the presence of different concentrations of the oil (0.08–0.64 μL/mL), for 24 h. Nitrite concentration was determined from a sodium nitrite standard curve and the results are expressed as a percentage of NO production by cells treated with LPS. Each value represents the mean ± SEM of three independent experiments, performed in duplicate (#### *p* < 0.0001, compared to control; \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 and \*\*\*\* *p* < 0.0001, compared to LPS).

Overall, regarding the ability of the essential oils to inhibit NO production, *L. luisieri* essential oil with low amounts of necrodane derivatives (sample A1) was the most potent (IC50 = 0.07 μL/mL; concentration range within the 95% confidence interval: 0.06 to 0.08 μL/mL).

#### *2.5. Effect of 1,8-Cineole and Fenchone on NO Production*

As *L. luisieri* essential oil with low amounts of necrodane derivatives (sample A1) was the most potent in inhibiting NO production induced by LPS in macrophages, its main compounds (1,8-cineole and fenchone) were also assessed to determine if the observed effect was due to these compounds. Our results showed that 1,8-cineole was not able to decrease LPS-induced NO production at non-toxic concentrations (Figure 4B,C). By comparison, fenchone significantly decreased NO production at the highest non-toxic concentrations tested (Figure 4E,F). Strikingly, when both compounds were combined in the same proportions as they occurred in the essential oil (33.9% 1,8-cineole and 18.2% fenchone), no effect was observed in LPS-induced NO production at non-toxic concentrations (Figure 4G,H). Notably, the effective concentrations for 1,8-cineole, fenchone, and the mixture were higher (≥0.64 μL/mL) in comparison to those tested for *L. luisieri* essential oil (sample A1), which suggests that other compounds are responsible for the bioactive potential of this essential oil, or that synergisms may occur between compounds.

**Figure 4.** Effect of 1,8-cineole, fenchone, and their mixture on NO production. Chemical structure of (**A**) 1,8-cineole and (**D**) fenchone. Effect of 1,8-cineole (**B**,**C**), fenchone (**E**,**F**) and 1,8-cineole combined with fenchone (**G**,**H**) on NO production and cell viability, respectively. Cells were maintained in culture medium (control), or incubated with 1 μg/mL LPS, or with LPS in the presence of different concentrations of the compounds (0.08– 2.5 μL/mL), for 24 h. Nitrite concentration was determined from a sodium nitrite standard curve and the results are expressed as a percentage of NO production by cells treated with LPS. Cell viability results are expressed as percentage of MTT reduction by cells treated with LPS. Each value represents the mean ± SEM of three experiments, performed in duplicate (#### *p* < 0.0001, compared to control; \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\*\* *p* < 0.0001, compared to LPS). The dotted line in cell viability graphs represents the threshold (70% of maximal viability) below which cytotoxicity is recognized, in agreement with standard ISO 10993-5 [24].

#### *2.6. Effect of Lavandula luisieri Essential on the Expression of Inflammatory Mediators*

As *L. luisieri* rich in 1,8-cineole and fenchone was the most potent essential oil tested and was more active than its major compounds assessed alone or in combination, we sought to confirm its anti-inflammatory potential. Thus, the effect on the levels of inflammatory proteins, namely inducible nitric oxide synthase (iNOS), ciclooxigenase-2 (COX-2), and the immature form of interleukin—1β (pro-IL-1β), was assessed.

In untreated cells (control) and in cells treated with the essential oil, without LPS stimulation, iNOS, COX-2 and pro-IL-1β were not detected or were slightly expressed (Figure 5). However, after LPS stimulation for 24 h, the levels of these mediators significantly increased (Figure 5). Nevertheless, when macrophages were concomitantly treated with LPS and *L. luisieri* essential oil (0.32 μL/mL), iNOS levels significantly decreased compared to LPStreated cells (Figure 5A,B), corroborating the previous results demonstrating NO inhibition (Figure 3A). However, *L. luisieri* essential oil did not interfere with LPS-induced COX-2 levels (Figure 5A,C). Interestingly, the essential oil significantly increased the levels of pro-IL-1β induced by LPS (Figure 5A,D), thus suggesting that the conversion of pro-IL-1β into its mature form IL-1β may be inhibited.

**Figure 5.** Inhibitory effect of *Lavandula luisieri* essential oil (EO) on LPS-induced inflammatory proteins. (**A**) Representative Western blots of iNOS, COX-2 and pro-IL-1β. Quantification of (**B**) iNOS, (**C**) COX-2 and (**D**) pro-IL-1β protein levels. Cells were maintained in culture medium (control), or incubated with 1 μg/mL LPS, or incubated with the essential oil (0.32 μL/mL) alone or simultaneously with 1 μg/mL LPS, for 24 h. Results are expressed as percentage of protein levels relative to LPS. Each value represents the mean ± SEM from at least 3 experiments (## *p* < 0.01; ### *p* < 0.001, compared to control; \* *p* < 0.05, \*\* *p* < 0.01, compared to LPS). Each value represents the mean ± SEM from three experiments.

#### *2.7. Effect of L. luisieri Essential Oil on NF-κB/p65 Nuclear Translocation*

A complex network of intracellular signaling pathways and transcription factors tightly regulate the expression of pro-inflammatory molecules. Among the signaling cascades, the NF-κB assumes a decisive role during inflammation. In basal conditions, NF-κB is retained in the cytoplasm by its inhibitor, IκB. Upon a specific stimulus, phosphorylation and, consequently, proteasomal degradation of IκB occurs. Then, free NF-κB dimers, composed of p65 and p50, rapidly translocate into the nucleus where transcription of target genes, such as *NOS2* and *COX-2*, is promoted [25]. To assess the effect of the oil on this signaling pathway, immunocytochemistry for the NF-κB subunit, p65, was performed. As expected, in untreated cells, immunoreactivity for NF-κB/p65 was exclusively located in the cytoplasm (Figure 6). However, after LPS treatment, NF-κB/p65 immunoreactivity was mostly observed in the nucleus (Figure 6). Treatment with *L. luisieri* essential oil (0.32 μL/mL) clearly impaired nuclear translocation of NF-κB/p65, as shown in Figure 6. Moreover, pre-treatment with MG-132 (10 μM), a synthetic proteasome inhibitor peptide, fully inhibited NF-κB/p65 nuclear translocation, as immunoreactivity is only visible in the cytoplasm (Figure 6). The vehicle (DMSO) did not interfere with NF-κB/p65 nuclear translocation when compared to control cells (data not shown). These results suggest that *L. luisieri* essential oil decreased iNOS protein levels by interfering with NF-κB/p65 nuclear translocation.

**Figure 6.** Inhibitory effect of *Lavandula luisieri* essential oil (EO) on NF-κB/p65 nuclear translocation. Macrophages were incubated on μ-slides and pre-treated with MG-132 (10 μM) for 1 h and, then, treated with 1 μg/mL LPS for 20 min, alone or in combination with the essential oil (0.32 μL/mL) or MG-132. Control cells (Ctrl) were left untreated. Immunofluorescence staining of NF-κB/p65 (green) was performed as detailed in the Materials and Methods section and DAPI (blue) was used as a counter stain. Representative images of each condition are shown. Scale bar: 20 μm.

#### **3. Discussion**

The Lamiaceae family comprises a high number of aromatic and medicinal plants with commercial interest. Among these species, lavenders stand out due to their high essential oil yield and pleasant odor, which are both important features for industrial applications. Indeed, the typical lavender scent is globally recognized and is included in many home and bath-care products. Lavender essential oil is considered a valuable raw-material in cosmetics and perfumes, and for flavoring [3,26]. Although the *Lavandula* genus comprises 39 wild essential oil bearing species, only four are commercially valued [3]. Nevertheless, some of the remaining species have economic potential mainly due to their bioactive properties. In fact, *L. luisieri* and *L. pedunculata* are widely used in traditional medicine by local communities, but scientific information validating these uses remains sparse. Moreover, local communities use these plants but are unaware of their chemical variability, which can compromise both safety and efficacy. Indeed, *L. luisieri* essential oils have a peculiar chemical composition due to the presence of necrodane derivatives that do not occur in other species [7,27–31] and, although these compounds are always present, significant variations among samples can occur [7]. Therefore, in the present work, two samples, one with low and another with high amounts of these compounds, were studied. Regarding *L. pedunculata*, the chemical composition of the essential oils from Portugal showed similarities with that reported for *L. stoechas* from other Mediterranean countries, namely from Spain [27], Greece [32] and Turkey [33]. Although *L. pedunculata* and *L. stoechas* are morphologically distinct, their essential oils present a similar chemical profile. This is interesting because *L. stoechas* is one of the four species with commercial value, thus suggesting that *L. pedunculata* can be used for the same purposes. Both species present the same major compounds, namely 1,8-cineole, fenchone and camphor, and the amount of these compounds can vary considerably. Therefore, three representative samples, with high amounts of each one of these compounds, were also selected.

As *L. luisieri* and *L. pedunculata* are used in traditional medicine to treat several conditions that share an inflammatory component, the present study aimed to validate the anti-inflammatory potential of these species considering their high chemical variability [7,8], which can affect oil quality and compromise both safety and bioactivity profiles. Inflammation is considered an important mechanism that maintains homeostasis, regardless of whether the insult is exogenous, as in the case of infections, or endogenous, as occurs, for instance, in metabolic disorders [34]. During this process, the first line of defense is provided by macrophages, which in the presence of a Toll-like receptor agonist such as microbial LPS, produce several pro-inflammatory mediators, including NO, COX-2, prostaglandins and cytokines, such as IL-1β [35]. Under normal conditions, the release of these pro-inflammatory mediators for a short period of time is of utmost importance, and aims to eradicate the harmful stimuli. However, the abnormal and sustained production of these mediators may result in damage to the host tissue, leading to a vicious circle that, if maintained over a long period, may evolve to chronic inflammation that has been associated with several aged-related diseases. One of the most important players in this process is the transcription factor NF-κB. Therefore, inhibition of NF-κB transcriptional activity, and its downstream mediators, represents a valuable therapeutic strategy for intervention in inflammation-based pathologies. In this context, the knowledge of phytochemicals' molecular mechanisms is a good strategy in the search for novel anti-inflammatory compounds. Taking into consideration the above, in this work we performed an initial screening assay using an *in vitro* inflammation model, specifically macrophages stimulated with LPS. Under these experimental conditions, LPS activates the pro-inflammatory transcription factor NF-κB/p65, which rapidly translocates into the nucleus to trigger the transcription of its target genes, such as *NOS2*, *COX-2* and *Il1*. Overall, the results achieved demonstrated that *L. luisieri* and *L. pedunculata* essential oils display anti-inflammatory potential, nonetheless presenting distinct efficacies and safety profiles depending on their chemical composition. In fact, *L. luisieri* essential oil with low amounts of necrodane derivatives was the most potent in mitigating the anti-inflammatory response, as assessed by the inhibition of NO

production evoked by LPS. Interestingly, the main compounds of the oil, namely 1,8-cineole and fenchone, were not the only compounds responsible for the observed effect, because they were much less effective than the oil, both alone and in combination, thus indicating that other minor compounds are responsible for the effect of the oil, or that synergistic effects among compounds may occur. In addition, the results achieved demonstrated that the observed anti-inflammatory effect was not related to a scavenging effect of the oil; therefore, the putative mechanism underlying the anti-inflammatory potential was further explored by addressing the effect of the oil on the expression of relevant inflammatory proteins. Our results clearly demonstrated that the anti-inflammatory potential ascribed to *L. luisieri* essential oil (with low amounts of necrodane derivatives) was achieved through inhibition of nuclear translocation of NF-κB/p65, which consequently leads to a decrease in iNOS protein levels and NO production. Interestingly, the essential oil also significantly increased the LPS induced expression of pro-IL-1β, the precursor of the potent pro-inflammatory cytokine IL-1β. It would be expected that pro-IL-1β levels were decreased because the essential oil compromised the nuclear translocation of NF-κB/p65. Nevertheless, we hypothesize that the observed accumulation indicates that pro-IL-1β is not being converted into IL-1β, thus suggesting an effect of the oil on the inflammasome, a proteolytic complex responsible for the maturation and secretion of the inflammatory and pyrogenic IL-1β. However, further studies should be performed to prove this hypothesis.

The results presented herein were corroborated by previous studies describing the anti-inflammatory potential of *L. luisieri* essential oil in both primary human chondrocytes and in an intestinal cell line [19], and in THP-1 cells [20]. The first study showed a decrease in iNOS levels and, consequently, in NO production, and reported that the effect was due to the inhibition of IκB phosphorylation and degradation, thus blocking NF-κB activation [19]. The latter pointed out a reduction in the cytokine tumor necrosis factor-α (TNFα) and the chemokine (C-C motif) ligand 2 (CCL2), following LPS stimulation. Regarding other lavender species, studies on *L. angustifolia* have frequently reported inhibitory effects on carrageenan-induced paw oedema [15], croton oil-induced ear oedema, and dextran-induced paw oedema models [36]. Moreover, decreases in the levels of TNF-α and IL-1β, and increases in IL-10, were also shown in a rat model of myocardial infarction [37]. *L. stoechas* also showed strong lipoxygenase inhibitory effects [14] and our group previously reported the ability of *L. viridis* essential oil to inhibit LPS-induced NO production through down-modulation of NF-κB-dependent *Nos2* transcription and, consequently, iNOS protein expression, in addition to a decrease in proteasomal activity, inhibition of *Il1b* and *Il6* transcription, and down-regulation of COX-2 levels [18]. Overall, these previous studies highlight the anti-inflammatory potential of lavender species, and the current study contributes by providing additional scientific information regarding less recognized species.

To conclude, our results indicate potent anti-inflammatory activity of *L. luisieri* essential oil via down-modulation of the NF-κB pathway, with significant inhibitions in major inflammatory mediators. In addition, the importance of well-defined chemical products is highlighted, because chemical variability compromises essential oils' quality and, consequently, their safety profile and efficacy. Moreover, our findings corroborate the traditional uses ascribed to these species and indicate that *L. luisieri*, with low amounts of necrodane derivatives, is the most promising for the development of safe anti-inflammatory agents, thus contributing to its industrial valorization.

#### **4. Materials and Methods**

#### *4.1. Plant Material*

Flowering parts of representative samples of *L. luisieri* (Rozeira) Rivas Mart. and *L. pedunculata* (Mill.) Cav. (Table 2) were collected. Voucher specimens were included in the Herbarium of the University of Coimbra (COI). Species authenticity was confirmed by Dr. Jorge Paiva, a taxonomist at the University of Coimbra, and plant names were checked at http://www.theplantlist.org accessed on 20 December 2021.

**Table 2.** Site of collection of *Lavandula* spp.

A1 and A2—representaive samples of *L. lusieri*; B1–B3—representative samples of *L. pedunculata*.

#### *4.2. Essential Oil Isolation and Analysis*

Flowering aerial parts of *L. luisieri* and *L. pedunculata* were submitted to hydrodistillation for 3 h in a Clevenger-type apparatus [38]. Essential oils were analyzed by gas chromatography (GC) on a Hewlett Packard 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with a HP GC ChemStation Rev. A.05.04 data handling system, equipped with a single injector and two flame ionization detectors (FID). A graphpak divider (Agilent Technologies, Part Number 5021-7148) was used for simultaneous sampling in two Supelco (Supelco Inc., Bellefont, PA, USA) fused silica capillary columns with different stationary phases: SPB-1 (polydimethylsiloxane; 30 m × 0.20 mm i.d., film thickness 0.20 μm), and SupelcoWax 10 (polyethylene glycol; 30 m × 0.20 mm i.d., film thickness 0.20 μm). The oven temperature program was: 70–220 ◦C (3 ◦C/min), 220 ◦C (15 min); injector temperature: 250 ◦C; detector carrier gas: He, adjusted to a linear velocity of 30 cm/s; splitting ratio 1:40; detector temperature: 250 ◦C. Gas chromatography-mass spectrometry (GC/MS) analyses were performed on a Hewlett Packard 6890 gas chromatograph fitted with a HP1 fused silica column (polydimethylsiloxane; 30 m × 0.25 mm i.d., film thickness 0.25 μm), interfaced with an Hewlett Packard Mass Selective Detector 5973 (Agilent Technologies, Palo Alto, CA, USA) operated by HP Enhanced ChemStation software, version A.03.00. GC parameters were as above; interface temperature: 250 ◦C; MS source temperature: 230 ◦C; MS quadrupole temperature: 150 ◦C; ionization energy: 70 eV; ionization current: 60 μA; scan range: 35–350 u; scans/s: 4.51. Retention indices (RIs) and mass spectra were used to identify volatile compounds. RIs were calculated by linear interpolation relative to retention times of a series of n-alkanes, and compared with those of authenticated samples from the database of the Laboratory of Pharmacognosy, Faculty of Pharmacy, University of Coimbra. Mass spectra were compared with reference spectra from a home-made library or from literature data [39,40]. Relative amounts of individual components were calculated based on GC peak areas without FID response factor correction.

#### *4.3. Nitric Oxide Scavenging Potential*

The essential oils' NO scavenging potential was evaluated using S-nitroso-N-acetyl-DL-penicillamine (SNAP) as a NO donor. NO production was measured using Griess reaction. Briefly, in 48-well plates, 300 μL of culture medium alone (control) or with different concentrations of the essential oils and 300 μM of SNAP were incubated for 3 h at 37 ◦C. After this period, equal volumes of the supernatants and Griess reagent [1% (*w*/*v*) sulphanilamide in 2.5% (*v*/*v*) phosphoric acid and 0.1% (*w*/*v*) naphthylethylenediamine dihydrochloride] were mixed and incubated in the dark, for 30 min, at room temperature. The absorbance was then read at 550 nm using a Biotek Synergy HT plate reader (Biotek, CA, USA). The concentration of nitrites accumulated in supernatants was calculated by interpolation of the absorbance of each sample in a standard curve of sodium nitrite. All experiments were performed in triplicate.

#### *4.4. Cell Culture and Treatments*

The mouse leukemic macrophage cell line, RAW 264.7 (ATCC—TIB-71), was cultured in Dulbecco's Modified Eagle Medium supplemented with 10% (*v*/*v*) non-inactivated Fetal Bovine Serum, 100 μg/mL streptomycin and 100 U/mL penicillin. The cell line was maintained at 37 ◦C, in a humidified atmosphere of 95% air and 5% CO2, and used after reaching 80–90% confluence. Cells were plated at 3 × 105 cells/well and left to stabilize for 12 h.

For cell treatments, essential oils, 1,8-cineol (extra pure; Merck, Darmstadt, Germany) and fenchone (99.9% purity; Fluka AG, Buchs, Switzerland) were initially diluted in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co., St. Louis, Mo, USA) at a 1:1 proportion and, then, serial dilutions were performed in culture medium. MG-132 (Z-Leu-Leu-Leu-CHO, Boston Biochem, Cambridge, MA, USA) was also dissolved in DMSO (Sigma-Aldrich Co). In both cases, the final concentration of DMSO did not exceed 0.1% (*v*/*v*). LPS from *Escherichia coli* 026:B6 (Sigma-Aldrich Co.) was dissolved in phosphate buffered saline (PBS). The concentrations of the essential oils, 1,8-cineol, fenchone, MG-132 and LPS, and the experimental treatment periods, are indicated in figures and/or figure legends.

#### *4.5. Cell Viability*

Assessment of cell viability was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. After the treatment period, a MTT solution (final concentration 0.5 mg/mL; Sigma-Aldrich Co.) was added to each well and further incubated at 37 ◦C for 15 min in a humidified atmosphere of 95% air and 5% CO2. Acidified isopropanol (0.04 N HCl in isopropanol) was added to each cell to dissolve formazan crystals and quantification was carried out using an Biotek Synergy HT plate reader (Biotek) at 570 and 620 nm (reference wavelength). A cell-free control was performed in order to exclude non-specific effects of the essential oils, 1,8-cineol and fenchone on MTT (data not shown). All experiments were performed in triplicate.

#### *4.6. Nitric Oxide Production*

NO production was measured as the amount of nitrite accumulation in the culture supernatants using the Griess reaction described in Section 4.3.

#### *4.7. Western Blotting*

Total cell extracts were obtained using RIPA buffer (50 mM Tris-HCL, pH 8.0, 1% Nonidet P-40, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS) and 2 mM ethylenediaminetetraacetic acid) freshly supplemented with 1 mM dithiothreitol (DTT), protease (Complete Mini, Roche Diagnostics, Mannheim, Germany) and phosphatase (PhosSTOP, Roche Diagnostics, Mannheim, Germany) inhibitor cocktails. The cell lysates were then sonicated (4 times, 40 μm peak to peak) in a Vibra Cell sonicator (Sonica and Material INC) and centrifuged for 10 min at 4 ◦C to remove nuclei and cell debris. Protein concentration in the total lysates was determined using the bicinchoninic acid kit (Sigma-Aldrich Co.) and cell lysates were denatured in sample buffer [0.125 mM Tris pH 6.8, 2% (*w*/*v*) SDS, 100 mM DTT, 10% glycerol and bromophenol blue] at 95 ◦C for 5 min. Equal amounts of proteins were then separated by SDS-PAGE and electrotransferred onto PDVF membranes. After blocking with 5% (*w*/*v*) non-fat milk in Tris-Buffered saline (TBS)-Tween 20 (0.1%) for 1 h, membranes were incubated overnight at 4 ◦C with the primary antibodies indicated in Table 3 and then with anti-rabbit (dilution 1:20,000; NIF1317, GE Healthcare, Chalfont St. Giles, UK) or anti-mouse (dilution 1:20,000; NIF1316, GE Healthcare, Chalfont St. Giles, UK) alkaline phosphatase-conjugated secondary antibodies. Immune complexes were detected with Enhanced ChemiFluorescent reagent (GE Healthcare) in the TyphoonTM FLA 9000 imaging system. Mouse anti-actin was used as loading control. Image analysis was performed with Image Quant TL software.

**Table 3.** List of primary antibodies used in Western blot assays.

#### *4.8. Immunocytochemistry*

To evaluate NF-κB/p65 nuclear translocation, immunocytochemistry was performed as previously described [41]. Briefly, at the end of the treatment period, cells were washed with ice-cold PBS pH = 7.4 and, then, fixed in 4% paraformaldehyde at room temperature, for 15 min. After fixing, cells were blocked with 5% Goat Serum, 0.3% Triton in PBS, pH = 7.4 for 1 h at room temperature. Then, slides were incubated with a rabbit monoclonal anti-NF-κB p65 (D14E12) XP® antibody (dilution 1:400; #8242, Cell Signaling Technology, Inc.) in 1% Bovine Serum Albumin in PBS (pH = 7.4) overnight at 4 ◦C and then incubated with anti-rabbit IgG (H+L) CFTM488A (dilution 1:400; SAB4600165, Biothium, Inc., Fremont, CA, USA) for 1 h at room temperature in the dark. The cells were counterstained with DAPI (dilution 1:1,000; Sigma-Aldrich D9542) to stain the nuclei. Specificity was confirmed in negative controls set up by omitting the primary antibody. Fluorescence images were obtained in a widefield fluorescence microscope (Axio Observer.Z1; Carl Zeiss, Germany) and images were acquired using Zen Black 2010 software.

#### *4.9. Statistical Analysis*

Results are expressed as mean ± SEM. Statistical analysis was performed with Graph-Pad Prism version 9 (GraphPad Software, San Diego, CA, USA), using a two-sided unpaired t-test for comparisons between a control and treated group or using one-way ANOVA with Dunnett's post-test for multiple comparisons to a control group. Results were considered statistically significant at *p* < 0.05.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants11030370/s1, Figure S1: Effect of DMSO on macrophages viability and NO production.

**Author Contributions:** Conceptualization, M.Z., M.T.C. and L.S.; methodology, M.Z., C.S. and C.C.; validation, M.Z. and C.S.; formal analysis, M.Z., C.C. and C.S.; investigation, M.Z. and C.S.; resources, M.T.C. and L.S.; writing—original draft preparation, M.Z.; writing—review and editing, C.S., C.C., M.T.C. and L.S.; visualization, M.Z.; supervision, M.T.C. and L.S.; project administration, M.Z. and L.S.; funding acquisition, M.T.C. and L.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financed by the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme, under project CENTRO-01-0145-FEDER-000012 (HealthyAging2020).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors thank Otília Vieira (CEDOC, NOVA Medical School, Portugal) for the kind gift of the mouse macrophage-like cell line Raw 264.7.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Review* **Critical Determinants in ER-Golgi Trafficking of Enzymes Involved in Glycosylation**

**Ning Zhang and Olga A. Zabotina \***

Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** All living cells generate structurally complex and compositionally diverse spectra of glycans and glycoconjugates, critical for organismal evolution, development, functioning, defense, and survival. Glycosyltransferases (GTs) catalyze the glycosylation reaction between activated sugar and acceptor substrate to synthesize a wide variety of glycans. GTs are distributed among more than 130 gene families and are involved in metabolic processes, signal pathways, cell wall polysaccharide biosynthesis, cell development, and growth. Glycosylation mainly takes place in the endoplasmic reticulum (ER) and Golgi, where GTs and glycosidases involved in this process are distributed to different locations of these compartments and sequentially add or cleave various sugars to synthesize the final products of glycosylation. Therefore, delivery of these enzymes to the proper locations, the glycosylation sites, in the cell is essential and involves numerous secretory pathway components. This review presents the current state of knowledge about the mechanisms of protein trafficking between ER and Golgi. It describes what is known about the primary components of protein sorting machinery and trafficking, which are recognition sites on the proteins that are important for their interaction with the critical components of this machinery.

**Keywords:** glycosyltransferases; ER-Golgi trafficking; mechanism of protein sorting; COPI and COPII complexes; sequences and motifs involved in trafficking

#### **1. Introduction**

All living cells generate structurally complex and compositionally diverse spectra of glycans and glycoconjugates, critical for organismal evolution, development, functioning, defense, and survival. The glycans attached to proteins and lipids determine their activity, solubility, subcellular localization, and structural organization in cells during normal and stressed conditions. Glycan-rich cell walls control cell growth and morphogenesis and protect them against environmental stresses. Glycosylation is the reaction that forms glycosidic linkages between activated sugar (donor substrate) and acceptor substrate (protein, lipid, polysaccharide, etc.). This reaction is performed by a large group of specialized enzymes, called glycosyltransferases, and broadly takes place in most organisms, such as yeast, humans, plants, etc. Because of the diversity of sugars and acceptor substrates, the resulting products of glycosylation present high variability and complexity in structures and functions.

Glycosyltransferases (GTs) are distributed among more than 130 gene families [1]. Most GTs are type II transmembrane proteins with several distinct domains: a short *N*terminal cytosolic tail, transmembrane domain (TMD), flexible stem region, and large catalytic domain. Another group of GTs comprises integral membrane proteins with multiple TMDs and a large catalytic domain frequently localized on the cytosolic side of the membrane. Based on the type of catalytic domain folds, the GTs are grouped into GT-A, GT-B, and GT-C [2,3]. Two tight β/α/β Rossmann domains form a central *β*-sheet in GT-A folding, and mostly GT-A type proteins contain DxD catalytic motifs that cooperate with

**Citation:** Zhang, N.; Zabotina, O.A. Critical Determinants in ER-Golgi Trafficking of Enzymes Involved in Glycosylation. *Plants* **2022**, *11*, 428. https://doi.org/10.3390/ plants11030428

Academic Editors: Milan S. Stankovic, Paula Baptista and Petronia Carillo

Received: 6 December 2021 Accepted: 1 February 2022 Published: 4 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

metal. Instead of being tightly associated, the two β/α/β Rossmann domains in GT-B are separate and form a cleft. GT-C proteins are predicted and found based on sequence and structure research and contain multiple hydrophobic helices. In addition, it was also found that the structure of the peptidoglycan glycosyltransferase of Aquifex aeolicus contains a lysozyme-like domain [4].

The primary types of glycosylation in various glycoconjugates are *N*-glycosylation and *O*-glycosylation. *N*-glycosylation is the formation of the glycosidic linkage between the amino group of an asparagine residue and the first sugar of the glycan. Asparagine in the Asn-X-Ser/Thr consensus sequence is the candidate for *N*-glycosylation, although not all Asn residues are glycosylated [5]. *N*-glycosylation is essential and usually impacts protein solubility, structure, and folding. It is also essential for protein localization and interactions with glycan-binding proteins. The glycan oligosaccharides in *N*-glycosylated proteins share the core sugar sequence and structure-Manα1-6(Manα1–3)Manβ1-4GlcNAcβ1-4GlcNAcβ1- Asn [6], which can be further branched with different sugars depending on the type of mature glycan synthesized. The *N*-glycosylation reactions take place in the endoplasmic reticulum (ER) and Golgi. The initiation of *N*-glycosylation occurs in the ER with the biosynthesis of the precursor oligosaccharide [7], which is later transferred to the peptide's Asn by the oligosaccharyltransferase (OST) complex.

At the end of the multistep process, the glycoprotein is transported to cis-Golgi, and the next steps of *N*-glycan processing continue in different Golgi cisternae. The GTs, such as medial-Golgi-localized *N*-acetylglucosaminyltransferase I (GnTI) [8,9], GnTII [10,11], trans-Golgi-localized Galactose-1-phosphate uridylyltransferase l (GALT) [12], *β*-galactosideα2,6-sialyltransferase l (ST6Gal-l) [13–15], and *β*-galactoside-α2,3-sialyltransferase-III (ST3Gal-llI) [16,17] are localized in distinct Golgi cisternae and responsible for subsequent steps of the synthesis of final glycan structures. It was demonstrated that most of these GTs form homo- and heterocomplexes, most likely to support the error-proof synthesis [14,18,19].

The *O*-glycosylation process is different in plant and mammalian cells. In mammalian type *O*-glycosylation, the glycan is attached to the hydroxyl group of the serine or threonine residue in a glycoprotein. *O*-glycosylation primarily occurs in Golgi and is also found in the cytoplasm and nucleus [20–22]. The first sugar, *N*-acetylgalactosamine (GalNAc), links to serine and threonine residues in Golgi and is called *O*-GalNAc. Multiple core sugar sequences are found in *O*-GalNAc-type glycosylation, and different biosynthesis steps are involved [6,23]. In plants, the main *O*-glycoproteins are the hydroxyproline-rich glycoprotein (HRGP) superfamily, including arabinogalactan proteins (AGPs), extensions (EXTs), and the repetitive Pro-rich proteins (PRPs) [24]. The type II arabino-3,6-galactans (AGs) get attached to noncontiguous Hyp residues in AGPs in Golgi [24]. In *A.thaliana*, eight Hyp-galactosyltransferases added the Gal to Hyp residues as the initiation steps of *O*glycosylation of AGPs [24,25]. The variation of AGPs depends on the complexity of galactan side chains attached to Hyp residues [24]. The *β* 1-6 galactosyltransferases are involved in the elongation of the side-chain backbone; other GTs, such as arabinosyltransferases, rhamnosyltransferases, and xylosyltransferases, further branch this galactan backbone [24]. The Ser residues and three to five contiguous Hyp residues are the candidate sites of *O*glycosylation in EXTs. The *β* 1-3-arabinosyltransferase attaches the first arabinofuranose reside to Hyp [24]. The Reduced Residual Arabinose 1–3 (RRA1-RRA3), Xyloglucanase 113 (XEG113), and Extensin Arabinose Deficient (ExAD) add the second, third and fourth Araf residues sequentially [24].

Cell-wall polysaccharides are synthesized in two locations: the Golgi and the plasma membrane. On the plasma membrane, most GTs involved in synthesizing polysaccharides are integral membrane proteins with multiple TMDs. For example, in plants, cellulose synthases (CESA) [3,26–28] are organized in multiprotein cellulose synthase complexes (CSCs) and synthesize cellulose microfibrils. These complexes are assembled in Golgi and delivered to the plasma membrane via cargo carriers. Multiple isoforms of CESA are identified: in primary plant cell wall synthesis, CESA1, CESA2, CESA3, CESA5, CESA6, and CESA9 assemble CSCs in Golgi [28,29]. At the same time, CESA4, CESA7, and CESA8

are involved in the biosynthesis of the secondary plant cell wall [28,29]. Other plant cell wall polysaccharides representing pectins and hemicelluloses are synthesized in Golgi by Golgi-localized GTs [3]. The latest studies suggest that Golgi-localized GTs are also organized in multiprotein complexes to synthesize polysaccharides. For example, *β* -1,4 xylosyltransferase (IRX) 9, IRX10, and IRX14 are xylosyltransferases and form the protein complex to synthesize the backbone of xylan [30–32]. The other seven GTs (cellulose synthase-like C4; xyloglucan xylosyltransferases XXT1, XXT2, and XXT5; galactosyltransferases XLT2 and MUR3 and fucosyltransferase FUT1) involved in xyloglucan biosynthesis were also shown to form heterocomplexes [3,33,34]. The homogalacturonan synthesizing galacturonosyltransferase (GAUT) 1 and GAUT7 proteins form a heterocomplex required to anchor catalytically active GAUT1 to Golgi [35].

#### **2. Main Components of the Secretory Pathway**

The trafficking of enzymes involved in glycosylation is essential for their proper delivery to the sites of their functioning in the cell, but it is still poorly understood. However, the information about trafficking processes available for various other proteins can also be applied to GTs. More information has recently become available concerning the primary components and routes of secretory pathways. The GTs and glycosidases involved in glycosylation, the focus of this review, most likely follow a similar secretory pathway and therefore are subjected to similar sorting mechanisms like other membrane proteins.

Thus, it has become clear that the coat protein complex I (COPI) -coated cargo carriers and the coat protein complex II (COPII)-coated cargo carriers mediate the trafficking path between the ER and the Golgi (Figure 1). One part of this trafficking pathway is the sorting signal in the protein sequence of GTs, which is recognized by the cargo receptor or COP coatomer to trigger the trafficking of proteins. The transmembrane region and *N*-terminal cytoplasmic domain of lipid phosphatase Sac1 are essential in the retention mechanism of phosphatidylinositol-3-phosphatase (Sac1) in Golgi [36]. The core components of the COPI-coated cargo carriers are the *α*-COP, *β*-COP, *β'*-COP, *γ*-COP, *δ*-COP, *ε*-COP, and *ζ*-COP subunit. The activation of the GTPase ADP-ribosylation factor 1 (Arf1) is a prerequisite for the assembly of the COPI coat [37–39]. Guanine nucleotide-exchange factors (GEFs) stimulate the GTPase Arf1 activation by exchanging GDP to GTP; then, the activated GTPase Arf1 embeds into the lipid membrane by using a myristoylated α-helix [40,41]. In turn, GTPase Arf1 recruits the coatomer complex with an inner coat and outer coat to transport protein and lipid cargo from the Golgi to the ER and between the Golgi cisternae [42,43]. COPII-coated cargo carriers deliver the cargo proteins from the ER to the Golgi, with the inner and outer layers of the protein lattice acting as the core component of COPII-coated cargo carriers [44–47]. The activation of the Ras-like small COPII coat Sar1 GTPase is the prerequisite of the assembly of the COPII coat [48]. Guanine nucleotideexchange factor Sec12 assists the exchange of the GDP to the GTP on Sar1 to recruit the coat protein Sec23-Sec24 inner layer of the protein lattice, and the heterotetramers of coat protein Sec13 and Sec31 as the outer layer of the protein lattice is recruited to continue the assembly of the COPII coat [44–47].

There are some differences in COPI and COPII transportation in plants compared to animals and yeast. In plant cells, the COPII and COPI-coated cargo carriers' transportation impacts plant growth, stress response, and protein transportation. In *A.thaliana*, multiple paralogs of COPI components have been discovered [49,50]. The silencing of the *β*1/2- COP gene enhanced the sensitivity of *A.thaliana* to salt stress [49,51]. Double mutant of *β*1/2-COP and a single mutant of *α*2-COP were dwarfed compared to *Col-0* [49,52], and the silencing of *β*1/2-COP or *α*2-COP or p24 protein altered the structure of the Golgi [49,52,53]. The knockout of *α*1-COP, *γ*-COP, and *ε*-COP resulted in a reduction in seed production due to altered pollen grain adherence and pollen tube germination [54]. The *β* -, *γ*-, and *δ*-COP proteins were shown to interact with each other and were localized in Golgi [55]. The *β* -, *γ*-, and *δ*-COP proteins were required to support the Golgi structure, and the silence of these genes caused the plant cell death [55]. The maintenance of the Golgi structure also