Assessment of Drought and Zinc Stress Tolerance of Novel Miscanthus Hybrids and Arundo donax Clones Using Physiological, Biochemical, and Morphological Traits

Simple Summary Marginal land is characterized by low crop productivity and is sometimes additionally contaminated. Such marginal land however presents a large opportunity to produce non-food biomass from perennial grasses with low risks of Indirect Land Use Change (low ILUC). Miscanthus spp. and Arundo donax also known as giant reed are leading bioenergy crops due to their high biomass productivity, but yields can be limited by insufficient water supply or phytotoxic levels of heavy metals. Drought and heavy metals are the most serious abiotic stress and negatively affect crop growth and development. The current study was conducted to identify the most drought and heavy metal (Zn) tolerant hybrid among seven novel Miscanthus hybrids and seven Arundo clones. Based on the morpho-physiological and biochemical analysis, the M. sinensis × M. sacchariflorus hybrid GRC 10 and Arundo clone PC1 were the most drought and Zn stress tolerant. The findings of this study provide a foundation for further investigations of the molecular and physiological mechanisms and recommendations for the cultivation of GRC 10 hybrid line and Arundo PC1 in marginal land. Abstract High-yield potential perennial crops, such as Miscanthus spp. and Arundo donax are amongst the most promising sources of sustainable biomass for bioproducts and bioenergy. Although several studies assessed the agronomic performance of these species on diverse marginal lands, research to date on drought and zinc (Zn) resistance is scarce. Thus, the objective of this study was to investigate the drought and Zn stress tolerance of seven novel Miscanthus hybrids and seven Arundo clones originating from different parts of Italy. We subjected both species to severe drought (less than 30%), and Zn stress (400 mg/kg−1 of ZnSO4) separately, after one month of growth. All plants were harvested after 28 days of stress, and the relative drought and Zn stress tolerance were determined by using a set of morpho-physio-biochemical and biomass attributes in relation to stress tolerance indices (STI). Principal component analysis (PCA), hierarchical clustering analysis (HCA) and stress tolerance indices (STI) were performed for each morpho-physio-biochemical and biomass parameters and showed significant relative differences among the seven genotypes of both crops. Heatmaps of these indices showed how the different genotypes clustered into four groups. Considering PCA ranking value, Miscanthus hybrid GRC10 (8.11) and Arundo clone PC1 (11.34) had the highest-ranking value under both stresses indicating these hybrids and clones are the most tolerant to drought and Zn stress. In contrast, hybrid GRC3 (−3.33 lowest ranking value) and clone CT2 (−5.84) were found to be the most sensitive to both drought and Zn stress.


Introduction
The production of renewable energy from biomass crops has gained attention in European policies in recent decades for targeting the reduction of greenhouse gas emissions [1].Particularly among the energy crops, perennial biomass crops became the core point of research interest due to their high potential yield and lignocellulose biomass quality [2][3][4][5][6].Such perennial crops as Arundo donax also known as giant reed and genus Miscanthus, are traditionally used to produce bioenergy and bioethanol, bio-based products and anaerobic digestion [4,[7][8][9][10].To accomplish sustainable biomass for the bioeconomy, it is important to develop and identify genotypes that have better performance to abiotic stress tolerance and the ability to grow on underutilized marginal land to reduce the pressure on food production.In marginal lands, in particular, drought and heavy metals are two of the most limiting factors for crop production [11,12].However, both Arundo and Miscanthus are suitable energy crop species in temperate marginal land due to their outstanding resilience and photosynthetic capacity at low temperatures [13].Additionally, a prolonged drought in the summer months could limit the yields of these crops and threaten their survival [14].On the other hand, heavy metal (HM) concentration in soil has rapidly increased because of various natural processes and anthropogenic (industrial) activities [15][16][17].In Europe alone 137,000 km 2 of agricultural lands are contaminated with at least one or more heavy metals in higher concentrations than the threshold limit [18,19].Among heavy metals, zinc (Zn) can be found in high concentrations in agricultural soils, which can damage cell functions, it can displace other elements having similar charges, such as Fe (iron), and Mg (magnesium) and reduce plant growth and increase chlorosis in leaves [20][21][22].In the Earth's crust, the average content of Zn is 70 mg kg −1 [23], and it varies from 10 to 100 mg kg −1 in soils around the world [24].The availability of Zn for plant accumulation depends on its concentration in soil, the soil pH and soil clay fraction.Indeed, between 30 to 200 µg Zn g −1 dry mass (DM) is required as a micronutrient for most crop varieties to act in catalytic functions in several processes, like cell division, cell expansion, proteins, and carbohydrate metabolism [21,23].However, at high concentrations in the soil (above 200 µg Zn g −1 dry mass (DM)), Zn toxicity inhibits water uptake and nitrate assimilation, which induces leaf water content, stomatal conductance, transpiration, net photosynthesis and photosynthesis efficiency [22,25].The Zn toxicity threshold level widely depends on plant species, ranging from 100 to 500 mg (Zn) kg −1 (DM) [24].Toxic levels of heavy metals combined with drought adversely affect plant physiology through several mechanisms, including photosynthesis, leaf water content, growth inhibition, and ROS (reactive oxygen species), which damage cells and modify membrane lipids [26][27][28].This dual stress can be mitigated by several strategies, such as scavenging enzymes antioxidants, namely superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidases (PPO), and ascorbate peroxidase (APX), which can reduce the negative impact of ROS [29,30].Another survival strategy is to accumulate lower molecular weight organic solutes such as proline and phenols [31,32].Long-term drought circumstances and high Zn negatively affected many physiological processes supporting biomass growth in Miscanthus spp.and Arundo donax (giant reed) species, according to several prior research and recent findings [24,[33][34][35].These perennial energy crops have exhibited high tolerance and restoration capacity to HMs stress by detoxification and accumulation mechanisms [24,36].But to date, there is little known about the physiological and biochemical traits associated with drought and Zn stress tolerance among newly developed Miscanthus hybrids [37] and different clonal accessions of Arundo donax [4].
The main objectives of this study were to determine the drought and Zn stress conditions that (1) enabled discrimination between stress tolerant and susceptible Miscanthus hybrids and Arundo clones, as well as (2) to rank the responses using multiple traits of seven high-yielding Miscanthus hybrids and seven Arundo clones.

Plant Material, Growing Conditions and Experimental Design
The pot experiments were conducted from 2020 to 2022 in a growth chamber with a controlled environment at the laboratory of the Department of Sustainable Crop Production of the Università Cattolica del Sacro Cuore, Piacenza, Italy.All plant materials were collected from 4-years field trials in Piacenza (NW, Italy): rhizomes of Miscanthus hybrids were collected from a plot scale trials funded by the EU-BBI GRACE project [37] and clones of Arundo (Table 1) were collected from a self-funded field trials.Rhizomes of Miscanthus for each hybrid from dormant mother plants were washed, cut into 7-10 cm lengths (around 10 g fresh weight) with several buds, and planted at a depth of 5 cm in 4 L circular plastic pots with a commercial blend of peat-humus, soil and sand (3:1:1).Propagation of Arundo clones by single-node stem cuttings was described earlier [38].Briefly, the stems were cut and planted with the node at a depth of 1 cm below the surface in 4 L pots into the same compost described above.To increase the chances of Arundo stem survival, several stems per pot were planted and incubated in the dark for 10 days at a temperature of 25 • C (night 22 • C) and 55-60% humidity.After shoot emergence, a PPFD of 800 µmol m −2 s −1 was provided by light-emitting diodes (LED) in 16/8 h light/dark regimes for both species.Plants were watered to field water holding capacity (FWHC) every second day and fertilized weekly with a modified half-strength Hoagland's solution (pH 6.0, EC 1.1 dS m −1 ).One month after germination, plants of both species were subjected to drought and Zn stress.
Experiments were carried out in a completely randomized block design (CRBD) with four biological replicates.The plants of seven hybrid lines and seven clones of both crops were well-watered to maintain the FWHC 60% as a control, for drought stress maintained 20% of FWHC (soil moisture maintained 1/3 of total FWHC), and for Zn stress once added 400 mg ZnSO 4 × 7 H 2 O kg -1 of soil (dry mass, DM) in each pot.The pots were weighed every second day until the end of the experiment to maintain the desired water field water holding capacity (FWHC), control at 60%, drought at 20% and Zn stress at 60%.After 28 days from the onset of both stresses, all plants were tested for morphological measurements, physiological analysis (photosynthetic performance and relative water content (RWC) determination), and thereafter leaves were harvested in liquid nitrogen for further biochemical analysis.The harvested plant material was stored at −20 • C up to all analyses.

Growth and Biomass Characterization
Plant growth measurements were evaluated for all the hybrids and clones at the end of the treatment (28 days after treatment, DAT, and 58 days after sprouting).For each plant, plant height was measured by using a graduated ruler from the soil surface to the end of the ligule's youngest fully expanded leaves.Similarly, the number of leaves was manually counted.At the end of the experiment, plants were harvested with leaves and stems (except the oldest expanded leaves for biochemical analysis) to determine aboveground shoots (with leaves) dry biomass.Biomass was dried at 60 • C in an oven for 72 h and subsequently, shoots of dry biomass were recorded.

Chlorophyll Fluorescence Measurements
Fluorescence measurements were taken on the last two fully expanded leaves of control and treated plants after 1-h dark adaptation.For each plant, measurements were performed with at least 4 technical replications (on a different portion of the same leaf) by using a Handy PEA Chlorophyll Fluorimeter (Hansatech Instruments Ltd., King's Lynn, UK), with a one-second light pulse of 3500 µmol m −2 s −1 by three LEDs emitting at 650 nm.The initial and maximal fluorescence were determined to measure maximum photosystem II (PSII) photochemical efficiency Fv/Fm (ratio of variable fluorescence to maximum fluorescence).Additionally, the fast fluorescence transient [39] was measured for the determination of the performance index (PI).

Determination of Relative Water Content (RWC)%
The relative water content (RWC) of leaves with the same developmental stage was measured at harvesting.RWC was calculated using the following formula: RWC% = [(FW − DW/TW − DW)] × 100 (where; FW = fresh weight, DW = dry weight, TW = turgid weight).Fresh weight was taken immediately after cutting the leaves from plants, turgid weight after leaving the leaves 24 h in distilled water (in an aluminum tray at room temperature) and dry weight was measured after 72 h drying in an oven at 65 • C.
2.5.Biochemical Assays 2.5.1.Analysis of Proline, Phenol and Malondialdehyde (MDA) After 28 days of stress treatments (drought and Zn), the contents of proline and phenol were measured from leaf samples of both crops.In brief, free proline extraction was performed from frozen leaf tissues by grinding with a mortar and pestle [40].Two hundred milligrams of ground samples were added to 5 mL of a 3% aqueous sulfosalicylic acid solution and vigorously vortexed for 1 min.Thereafter, the extract was centrifuged for 10 min at 4 • C at 10,000 rpm, and then the supernatant was stored at −20 • C in 2 mL Eppendorf tubes.For the determination of proline, 100 µL of the extraction was added with 1 mL of 1% ninhydrin solution which contains a 60:40 ratio of glacial acetic acid: water, and boiled at 95 • C in a water bath for 20 min.The reaction was stopped by submerging the samples in an ice bath.Thereafter, 3 mL of toluene was added and vigorously mixed by vortex and samples were left under dark conditions for 1 h.At the same time, the blank was prepared with 100 µL of a 3% sulfosalicylic acid instead of plant extraction.The light absorbance of the toluene phase was read at 520 nm with a microplate reader (Biotek Synergy 2, Winooski, VT, USA), and then proline concentration was determined by using a standard curve of proline.Results were expressed in µmol g −1 FW.
Total phenol contents in leaf tissues were determined through the Folin-Ciocalteu's method [41].One hundred milligrams of ground frozen plant material was added with 1.5 mL of 70% ethanol (v/v), vortexed, and centrifuged at 10,000 rpm for 5 min.Then 40 µL of the extract was mixed with 200 µL of Folin-Ciocalteu reagent and the solution was diluted by adding 1000 µL distilled water.Afterward, 600 µL of 20% sodium carbonate (Na 2 CO 3 , w/v) was added, samples were heated in a water bath at 85 • C for 1 min, then samples were allowed to stand at room temperature for 1 h in darkness.The absorbance of the samples read at 725 nm.The standard curve of gallic acid (GA) from the range 20, 40, 60, 80, 100, and 120 µg/mL was also prepared at the same time and finally, the results were expressed as total phenol contents mg GA g −1 FW.
The level of lipid peroxidation was quantified by measuring the production of malondialdehyde (MDA) in leaves after 28 days of stress treatments (drought and Zn) following the method described by [42].200 mg of ground fresh leave samples were mixed with 2 mL of reaction solution containing 0.5% (v/v) thiobarbituric acid (TBA) and 20% (v/v) trichloroacetic acid (TCA) and then samples were vortexed for 1 min.After incubation in a water bath at 95 • C for 30 min, the mixture was allowed to cool in an ice bath for 10 min, thereafter at room temperature, and then centrifuged at 10,000 rpm for 10 min.The absorbance of the supernatant was determined spectrophotometrically at 532 and 600 nm.The concentration of MDA was calculated using the formula: MDA (nmol g FW −1 ) = [(OD532 − OD600)]/(ε × FW), where FW is the fresh weight and ε the extinction coefficient (155 mM −1 cm −1 ).Data were expressed as µmol g FW −1 (fresh weight).

Determination of Total Soluble Protein and Antioxidant Enzymes Activity
The total soluble protein was determined by using the kit of bicinchoninic acid (BCA) assay and standard of bovine serum albumin (BSA) (Thermo Fisher Scientific, Illinois, USA), as earlier described [43].Briefly, plant material was ground with liquid nitrogen by mortar and pestle and added 0.1 M Na-phosphate buffer pH 7.0, containing 250 mM sucrose, 1 mM MgCl 2 , 1.0 mM EDTA, 0.1 mM dithiothreitol (DTT) and 1% (w/v) polyvinylpolypyrrolidone (PVPP) in a 1:10 proportion (plant material to buffer vol.).After this,the mixture was vortexed for 1 min before centrifugation at 12,000 rpm for 12 min.The supernatant was then used with BCA reagents in the development of intense purple color and read the absorbance at 562 nm with a microplate reader (Synergy HT Microplate Reader, BioTek Instruments, Inc., Winooski, VT, USA).
For the determination of POD (peroxidase) and PPO (polyphenol oxidases) enzyme activity, the method in ref. [44] method was followed with slight modification.PPO activity was measured as a catechol substrate, and the reaction was with 100 mM potassium phosphate buffer (pH 6.8), pyrogallol (50 µM) and 10 µL of enzyme solution in a volume of 200 µL.For POD activity the assay mixture contained 100 mM potassium phosphate buffer (pH 6.8), pyrogallol (50 µM), 10 µL of enzyme extract, and H 2 O 2 (50 µM).For both activities, the absorbance was read at 420 nm and defined as an increase of 0.1 absorbance units.For the determination of SOD (superoxide dismutase) activity, 20 µL of plant extract was added with 0.1 mM EDTA, 50 mM NaHCO 3 (pH 9.8), and at the end, 0.6 mM of epinephrine [45] and we waited four minutes to confirm the adrenochrome absorbance at 475 nm.To determine APX (ascorbate peroxidase) activity, 20 µL of plant extract was added with 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbic acid, and lastly 0.1 mM H 2 O 2 .Afterward, the decrease in absorbance was measured from 30 s to 1 min at 290 nm, according to [46].The APX activity was calculated based on the extinction coefficient (2.8 mM −1 cm −1 ).

Drought and Zn Tolerance Evaluation
To assess the drought and Zn stress tolerance of different genotypes, the stress tolerance index (STI) was used.STI was calculated using the following [47] formula: where Yp = value of each trait under control conditions, Ys = value of each trait under stress conditions, and Ŷp = mean value of all hybrids/clones under control conditions.

Statistical Analysis and Clustering
SPSS package (Version 26 SPSS Inc., Chicago, IL, USA) was used to analyze the data using a two-way-ANOVA (for genotypes, abiotic stress treatments, and their interactions) between control and treatments (drought and Zn) for all morphological (growth traits), physiological and biochemical analyses.Data were expressed as the mean ± standard deviation (S.D.).The significant differences between treatments mean were evaluated with Tukey's HSD post hoc test at p < 0.05.R statistical software (Version 4.1.1)was used for principal component analysis (PCA) and the ClustVis online tool (http://biit.cs.ut.ee/clustvis/, accessed on 10 September 2021) was used for Hierarchical clustering analysis HCA) based on STI values for each morphological (growth), physiological and physiological parameter.The drought and Zn stress tolerance of different hybrids of Miscanthus and clones of Arundo were assessed using PCA ranking value as earlier stated by [48] using the following formula: In this formula, two major components, PC1 and PC2, were obtained from PCA analysis and visually represented as percentages in the accompanying Figures.Both PC1 and PC2 are the PCA loading of morphological, physiological, and biochemical parameters for seven Miscanthus hybrids, and seven Arundo clones after 28 days of drought and Zn stress, separately.Finally, the numeric rank was calculated from the mean ranking values under drought and Zn treatments to evaluate and compare stress tolerance among the hybrids and clones.

Growth Attributes and Biomass Accumulation
The effect of drought and Zn stress on the growth parameters and shoot dry biomass of seven hybrids of Miscanthus and Arundo clones are presented in Table 2. Based on the results, a significant reduction occurred in plant height and number of leaves per plant after 28 days of exposure to drought and Zn stress in the Miscanthus hybrids and Arundo clones (Table 2).At the same time, the effect of drought and Zn treatments and hybrids and clones, as well as the interactions between hybrids or clones and treatments, were significant (p < 0.05) for all morphological and physiological and biochemical parameters, except shoot dry weight (SDW) and photosynthesis performance index (PI) traits of Miscanthus hybrids and Arundo clones, respectively.The Miscanthus hybrid GRC10, and Arundo clone PC1 showed higher plant height than the other six hybrids and clones, under drought and Zn stress conditions compared with control plants.On the other hand, the most significant reduction in plant height and number of leaves in both treatments was observed in Miscanthus hybrids GRC3 and GRC6, and Arundo clones CT2 and PI1.Under well-watered (control) conditions, Miscanthus GRC10 followed by GRC14 and Arundo PC1, followed by PC7 and PC6, showed the highest levels of shoot-dry biomass accumulation.The highest decrease of shoot-dry weight (SDW) biomass was observed in Miscanthus GRC6, GRC9 (54.0 and 54.5%) and GRC3 (48.2%), and Arundo CT2 (54%), PI1 (53%) under drought stress while a similar trend of decreasing was observed under Zn stress.The smallest decrease in SDW, in comparison to the control plants was measured on the hybrid GRC10 (8% drought and 4% Zn) and Arundo clone PC1 (19% drought and 16% Zn) under drought and Zn stress.

Chlorophyll Fluorescence under Drought and Zn Stress
The dark-adapted maximum quantum yield of PSII, as Fv/Fm and performance index (PI) sharply declined under drought and Zn toxicity.The decline of PSII (Fv/Fm), PI and their significant interactions under treatments compared to control conditions for all hybrids and clones are shown in Table 3. GRC6, and GRC3 Miscanthus hybrids showed the highest reduction in Fv/Fm (62 and 52%) while Arundo PI1 and PC7 clones showed 38 and 31% respectively, under drought conditions.A less significant decline was observed in Miscanthus GRC10 (5% decrease) while no significant difference was measured in Arundo clone PC1 (2% decrease) in drought stress compared with control.On the other hand, under Zn stress, Miscanthus GRC9 (62.5%),GRC15 (60.7%) and GRC3 (52%), and Arundo PI1 (50%) showed the highest reduction in Fv/Fm, whereas the maximum quantum efficiency of hybrids GRC10 and PC1, PC6 clones were maintained under such condition.The PI was significantly reduced for all hybrids of Miscanthus after 28 days of stress (drought and Zn), while no significant difference was observed in Arundo PC1, PC6, and PC7 clones (Table 3).Among the Miscanthus hybrids, GRC10 decreased less (9 and 10%) compared with the more sensitive GRC3 (63 and 52%), and GRC6 (68 and 48%) under both drought and Zn stress, respectively.Arundo clone PC1 showed a higher PI compared to all clones under both stress conditions (Table 3).The highest reduction of PI occurred in Arundo CT2 (42.6 and 34.4%, drought and Zn) and PI1 (22.7 and 31%, drought and Zn) clones under drought and Zn stress (Table 3).Table 4 represented all morpho-physio-biochemical and biomass attributes interactions in treatments (Zn and drought), hybrids and clones.Table 4. Summary of analysis of variance (two-factor ANOVA) for the effects of treatments (drought and Zn), hybrids of Miscanthus (top) and clones of Arundo (below) and the interactions on plant height (PH), number of leaves (NOL), shoot dry weight (SDW), the maximum quantum efficiency of the PSII (Fv/Fm), performance index (PI-ABS), leaf relative water content (RWC), protein, enzymes including polyphenol oxidase (PPO), peroxidase (POD), superoxide dismutase (SOD) and ascorbate peroxidase (APX), lipid peroxidation (MDA), phenol and proline with data after 28 days of stress.Relative water content (RWC) was measured to assess plant water status either under control or stress conditions.All Miscanthus hybrids and Arundo clones showed a high level of leaf RWC (Miscanthus values between 71 to 95% and Arundo 87 to 93%, respectively) under control (well-watered) conditions.Significant effects (p < 0.05) on RWC were observed for all hybrids and clones under drought and Zn stress conditions in comparison to control plants.The highest reduction of RWC occurred in Miscanthus GRC3 in both drought and Zn stress (25 and 23%, drought and Zn) and GRC1 (11 and 22%) and under Zn stressed GRC15 (22.4%).Miscanthus GRC10 showed a lower reduction of RWC under drought and Zn stress (8 and 6%, drought and Zn) than control plants.On the other hand, in Arundo, most of the clones displayed a reduction of RWC under drought and Zn stress.The highest RWC reduction was observed in Arundo CT2, PI1 (drought 28 and 29% and Zn 33 and 23%, respectively) and moderate reduction was recorded in clones A1, PC6, and PC7 (between 16 to 23%) under both stresses.

Effect of Drought and Zn Stress on Proline and Phenol Accumulation
Proline content (PC) and total phenol content (TPC) were significantly affected under drought and Zn stress (Figure 1A-D).In Miscanthus GRC10 and Arundo PC1 showed the highest PC and TPC, while the smallest PC and TPC contents in Miscanthus GRC3, Arundo ASR, and CT2, respectively (Figure 1A,B).PC content considerably increased under drought and Zn stress conditions in all Miscanthus hybrids (except GRC3) by a fold increase ratio from 2.22 to 3.36, similarly, all clones of Arundo (except CT2) also increased by a fold change ratio from 1.44 to 2.54 compared with the control condition (Figure 1A,B).A similar trend in TPC accumulation was observed in all Miscanthus hybrids by fold change ratio from 1.47 to 2.17 while in Arundo clones PC1, PI1, ASR, and PC6, there was a fold change ratio of 1.16 to 1.58 under both stresses compared with control groups (Figure 1C,D).However, the highest accumulation of both TPC and PC was measured in Miscanthus GRC10 (TPC fold-change ratio 2.17, 1.98; PC ratio 2.45, 2.75 droughts and Zn, respectively) (Figure 1A,C) and in Arundo PC1 (TPC fold-change ratio 1.41, 1.37; PC ratio 2.54, 2.42 drought and Zn, respectively) under both drought and Zn stress (Figure 1B,D).

Lipid Peroxidation under Drought and Zn Stress
Malondialdehyde (MDA) content is an important indicator regarding plant oxidative stress and redox signaling, and long exposure to drought or Zn stress in certain Miscanthus hybrids and Arundo clones increased MDA in leaves (Figure 2).Particularly, compared with the control conditions, the highest increase in MDA was recorded in the Miscanthus hybrids GRC3 (229 and 378%, drought and Zn, respectively), GRC6 (64 and 67%) (Figure 2, and in the Arundo clones PI1 (292 and 170%), PC7 (329% and 62%) and A1 (216 and 174%) under drought and Zn stress, respectively (Figure 2).A significant increase in MDA occurred under Zn stress in hybrid GRC14 (111%) and Arundo ASR (184%).Under such conditions (drought or Zn stress) the lowest accumulation was recorded for Miscanthus GRC10 (16 and 25%, drought and Zn, respectively) and Arundo PC1 (22 and 15%, drought and Zn, respectively).

Effect of Drought and Zn Stress on Soluble Protein Accumulation and Activities of Antioxidant Enzymes
Soluble protein is an essential component for cellular osmotic regulations.The total content of soluble protein increased with the prolongation of drought and Zn stress in Miscanthus hybrids (except GRC9) and Arundo clones (except PC7), as reported in Figure 3.Under drought stress compared to control conditions, the total soluble protein content highly increased in Miscanthus GRC1 (70%), GRC3 (70%) and GRC10 (20%) (Figure 3A), and Arundo A1 (82%), PC1 (20%) and PC7 (15%) (Figure 3), but significant reduction occurred in GRC9 (18%) and PC7 (15%), respectively.On the contrary, the accumulation of total soluble protein showed a similar trend under Zn stress for Miscanthus GRC1, GRC3 and GRC10 hybrids and Arundo A1, PC1 and PC7 clones (Figure 3).The activities of all antioxidant enzymes including PPO, POD, SOD and APX in response to drought and Zn stress on Miscanthus hybrids and Arundo clones are shown in Figure 4A-H.However, the increasing and decreasing effect of antioxidant enzymes depended on both Miscanthus hybrids and Arundo clones.The analysis of PPO activity showed a gradual increase in leaves of Miscanthus GRC10, GRC9 and GRC1 and all the clones of Arundo under both drought and Zn stress (Figure 4A,B).Compared with the control condition, under drought and Zn stress, PPO activity revealed the highest increase in GRC10 (32 and 48%, drought and Zn stress, respectively), GRC9 (31 and 45%) (Figure 4A) and in Arundo PC1 (37 and 42%), PC7 (30 and 40%) and PI1 (27% in both stresses) (Figure 4B).On the other hand, such enzyme activity in Miscanthus GRC3, GRC14 and GRC15 and Arundo ASR, CT2, and PC6 remained unchanged under both drought and Zn stress (Figure 4A,B).In the case of POD, Miscanthus GRC9 (35 and 40%, drought and Zn, respectively), GRC10 (23 and 15%) and Arundo PC1 (103 and 52%) and PC7 (63 and 61%) showed the highest activity under such drought and Zn stress (Figure 4C,D).In contrast, Miscanthus GRC1 under drought stress and Arundo A1 under both drought and Zn stress showed a significant (p < 0.05) reduction in POD activity.Miscanthus GRC3, GRC14 and GRC15 and Arundo ASR, CT2 and PC6 remained unchanged for such enzyme activity (Figure 4C,D).SOD activity increased in all Miscanthus hybrids, except GRC1, GRC15, and all giant clones due to drought and Zn stress (Figure 4E,F).The maximum increase in SOD activity was found in Miscanthus GRC10 (30 and 38% drought and Zn stress, respectively), GRC9 (21 and 35%) and GRC14 (11 and 25%), followed by Arundo A1 (196 and 191%), PC1 (75 and 97%) and ASR (32 and 20%) under drought and Zn treatment, respectively.Regarding the SOD activity, Miscanthus GRC3, GR6 and GRC15 remain unchanged like POD in response to drought and Zn stress (Figure 4E,F).
In the analysis of APX enzyme activity, all Miscanthus hybrids, except GRC3 and all Arundo clones, except PC6 and PC7 showed higher peak increase under drought and Zn stress, and among Arundo PC7 decreased significantly under such stresses (Figure 4G,H).Compared with the control, APX significantly increased under drought stress in Miscanthus GRC10, GR6 and GRC14 and Arundo A1 and PC1 by 40%, 31%, 28%, 98% and 41%, respectively.A similar increasing trend of APX activity was found for these hybrids and clones under Zn stress compared with control.However, APX activity was unchanged in Miscanthus GRC3 and followed by GRC9 and Arundo ASR and PI1 (Figure 4G,H).

Ranking among the Hybrids and Clones to Drought and Zn Stress Tolerance
The loading plots of principal components 1 and 2 for the seven Miscanthus hybrids and seven Arundo clones under drought and zinc stress conditions are displayed in Figure 5A-D.These plots are based on an examination of the growth, physiological, and biochemical characteristics.The results from principal components analysis (PCA) of Miscanthus hybrids under drought and Zn stress showed that principal component 1 (PC1) explained approximately 53.4% and 61.2% of the total variations (Figure 5A,B), and the second principal (PC2) 18.2% and 16.2%, respectively (Figure 5A,B) (Supplementary Materials, Figure S1A,B).On the other hand, concerning PCA of Arundo under drought stress and Zn treatment, PC1 explained 62.1% and 61.1% (Figure 5C,D) of the total variance, while the second principal component (PC2) explained 15.7% and 16.7%, respectively (Figure 5C,D) (Supplementary Materials, Figure S1C,D).Regarding Miscanthus hybrids under drought stress, the first component (PC1) was characterized by a high positive score with SDW, phenol, PI, PH and proline (Figure 5A) and under Zn stress, PPO, SDW, PI, Fv/Fm, and APX which were negatively correlated with MDA (Figure 5B).Under drought stress in Miscanthus hybrids, the second component (PC2) was identified with a high score with RWC and APX, while for Zn stress, it was phenol and RWC (Figure 5B).On the other hand, in Arundo clones, under drought stress PC1 was characterized by high positive score proline, NOL, POD, PPO, SDW phenol, and protein which were negatively correlated with MDA, and under Zn stress were PPO, NOL, SDW, proline and POD (Figure 5C).However, under Zn stress, MDA showed a negative correlation with SOD and a positively correlated with Fv/Fm (Figure 5D) in Arundo clones.The second component (PC2) in Arundo clones was identified with high score protein and SOD which were negatively correlated with MDA.Therefore, the Miscanthus hybrids and Arundo clones with high PC1 and PC2 scores had better performance in either drought or Zn stress compared to other hybrids or clones.The heatmap from hierarchical clustering analysis (HCA) showed morphological, physiological, and biochemical parameters under drought and Zn stress could be clustered into four distinct groups (Figure 6A-D).According to the color scale, the dark red color represents the highest values, while the yellow represents the lower STI values of the parameters under drought and Zn treatments.In agreement with PCA observations, 14parameters were grouped into different clusters under drought and Zn stress of Miscanthus hybrids and Arundo clones (Figure 6A-D).The heatmap categorized the seven hybrids and seven clones into four distinct clusters A, B, C, and D based on the results of STI from drought and Zn treatment (Figure 6A-D).Cluster A indicated GRC10 was the most drought and Zn tolerant (highlighted by dark red color), and cluster B including GRC14 and GRC15 indicated moderate drought-tolerant hybrids (Figure 6A,B).Miscanthus GRC1, GRC3, GRC6 and GRC9 indicated the most sensitive hybrids of both drought and Zn stress in clusters C and D, respectively (Figure 6A,B).On the other hand, Arundo clones were also indicated in four clusters, whereas cluster A including PC1 and then ASR indicated the most drought tolerant (Figure 6C) while PC1 was only the most Zn stress-tolerant.For cluster B, PC6 and A1 indicated moderate drought and Zn stress-tolerant (Figure 6C,D).The clones, including CT2 and PI1, were clustered in group C, which indicated the most sensitive clones for drought and Zn stress tolerance (Figure 6C,D).In addition to the PCA and HCA results, a mean ranking value was calculated to represent drought and Zn tolerance for Miscanthus hybrids and Arundo clones.Miscanthus GRC10 then GRC14 and GRC15 displayed higher mean ranking values under drought stress whereas under Zn stress GRC10 and GRC 14 showed higher mean ranking values (Table 5).In contrast, Miscanthus GRC3 and GRC9 showed comparatively lower mean ranking values under both drought and Zn stress, suggesting these hybrids were more sensitive to drought and Zn stress.Additionally, among the Arundo clones, PC1 and ASR showed higher mean ranking values compared to other clones under drought stress, while under Zn stress PC1 and PC6 demonstrated higher mean ranking values (Table 5).Arundo CT2 and PC7 revealed lower mean ranking values among the seven clones, suggesting that these two clones were more sensitive to both drought and Zn stress (Table 5).

Discussion
The plant exhibits several responses to drought and HM stress conditions, and one of the most critical responses is reducing the growth rate.However, plant development and biomass production under drought or toxic metal conditions are associated with the better adjustment of water relations to sustain physiological and biochemical activities [34,49].In the current study, all Miscanthus hybrids and Arundo clones exhibited severe responses under both drought and Zn stress treatments.
In bioenergy crops, biomass yield is a key factor for determining economic viability [50].In previous studies, growth, biomass, and physiological response under drought stress [34,51,52], and under Zn stress [24,36] in Miscanthus spp.were hybrid-specific, while in Arundo were clone or ecotype-specific [36,53,54].Both drought and Zn stress negatively affected the growth of the Miscanthus hybrids and Arundo clones in the present study.In agreement with the previous studies, the growth response was hybrid-specific in Miscanthus and clone-specific in Arundo under both drought and Zn stress.The decrease in total dry weight including stem and number of leaves per shoot under drought or Zn stress might be related to accelerated leaf senescence, reduced stomatal conductance, decreased photosynthesis and increased suppression of cell growth under low turgor pressure [52,55,56].
Chlorophyll fluorescence is known as an effective technique to monitor the physiological status of plants under several abiotic stress [57].The maximum quantum (Fv/Fm) of photosystem II and performance index (PI) are efficient parameter for discriminating among tolerant or sensitive Miscanthus hybrids and Arundo clones.In contrast, Miscanthus GRC10 andGCR15 and Arundo PC1 and ASR showed higher Fv/Fm and PI under both drought and Zn stress.Similar findings were reported in several edible crops and non-food crops tolerant varieties under such stress [34,52,58].
The major consequence of drought is related to water status in plants which defines a plant's potentiality to survive under the water-deficient condition to maintain growth, and photosynthesis activity.Meanwhile, heavy metals limit the water uptake [24].As stated earlier by [59], relative water content (RWC) is one of the common indexes for assessing plant water status.However, increased water retention during dehydration is a crucial static for developing drought resistance [60].Generally, it has been shown that exposure to drought stress changed RWC in several crop species including wheat [61,62], tomato [63], and sorghum [64], for both drought and Zn stress in Miscanthus spp.[24,34].Previous studies confirmed the relationship between RWC and plant genotypes' ability to tolerate drought, with the maintenance of a relatively constant RWC widely regarded as one of the best criteria for identifying and selecting tolerant and sensitive genotypes [34,63].In this study, a high RWC was recorded in Miscanthus hybrids of M. sinensis × M. sacchariflorus GRC10 and then GRC14, while Arundo PC1, PC6 and ASR clones that were the most tolerant hybrids and clones to drought and Zn stress in our study.
Plants have evolved a wide array of different pathways to respond to different stresses, specifically through secondary metabolites production [65,66].Among these, phenolic compounds are important secondary metabolites, whose accumulation in plants increases abiotic stress tolerance [67].Our results revealed that most of the Miscanthus hybrids and Arundo clones increase total phenol contents (TPC) under both drought and Zn stress, and such accumulation was highest in hybrids M. sinensis × M. sacchariflorus GRC10, then GRC14, and GRC15 and in Arundo clones PC1, ASR and PC6.The highest accumulation was observed in Miscanthus spp.under drought conditions [68] in Arundo under nickel (Ni) and copper (Cu) stress [49].Malčovská et al. [69] proposed that plants increase TPC in cells when plants are exposed to heavy metal stress as phenol are reactive oxygen species scavengers along with metal chelators.On the other hand, soluble protein and proline are considered compatible solutes and osmoregulators and they play an adaptative function in stress tolerance in higher plants [70].In our results, soluble protein contents increased highly in Miscanthus hybrids of M. sinensis × M. sacchariflorus mostly GRC10 and GRC14 and Arundo clones PC1 and ASR under both drought and Zn stress.Similar results were found in rice drought stress-tolerant varieties [71] and in Miscanthus cadmium (Cd) stress-tolerant species [72].Proline has been shown to be an energy supplier in membrane and subcellular structures, protect the plant's photosynthetic apparatus by a radical oxygen scavenger and maintain the redox potential that enables plant's growth, development, and survival under stress conditions [60,73,74].The level of accumulation of proline under stress conditions was used to identify stress-tolerant genotypes, as reported by several studies [60,73], of Miscanthus species [72].However, contrary results have also been reported in a correlation between the degree of stress tolerance and proline accumulation [73,75].In our study, Miscanthus hybrids of M. sinensis × M. sacchariflorus GRC10 and thereafter in GRC15 and Arundo clone PC1 showed the highest accumulation of proline under both drought and Zn stress that were the most tolerant hybrids and clones.Considering that the highest dry biomass (Table 2) was also observed in the hybrids of M. sinensis × M. sacchariflorus largely GRC10, and then GRC15 and Arundo PC1 and A1 clones, this might indicate that proline, being involved in cytoplasmic osmotic adjustment, could enhance drought and Zn stress tolerance in Miscanthus GRC10, and GRC15 and Arundo PC1 and A1 clones.
Drought and heavy metals, like other abiotic stresses, increase the formation of reactive oxygen species (ROS) that damage plants.As a general adaptation strategy, plants utilize a few enzymatic and non-enzymatic detoxification systems to protect themselves from oxidative damage caused by ROS [63,76].Among the antioxidant's enzymes, it includes CAT, POD, PPO, SOD, APX and non-enzymatic systems, such as phenol and proline which works together to support plants to survive under stress conditions [77].Nevertheless, such antioxidant enzyme mechanisms are complicated and genetically controlled [78].In our study, Miscanthus hybrids of M. sinensis × M. sacchariflorus, specifically GRC10 and then GRC14 and GRC15, and Arundo clones PC1, ASR and PC6 showed higher activity of PPO, SOD and APX enzymes under both drought and Zn stress which is linked with the lower accumulation of MDA and higher accumulation of proline, phenol and better photosynthesis performance.SOD and POD could play a great role in catalyzing H 2 O 2 into H 2 O and oxygen produced by ROS H 2 O 2 , meanwhile PPO produces rich phenolic compounds [71].In agreement with past studies, Miscanthus tolerant species highly increased PPO, SOD, and APX activity under drought [36] and heavy metal (Cd, Ni, and Zn) stress [33] and Arundo drought and Cd stress [79].Thus, our results support that drought and Zn stress tolerance capability among the different hybrids and clones are positively correlated with PPO, SOD and APX antioxidants activity.
In the present study, PCA and HCA were combined used to cluster seven hybrids of Miscanthus and seven Arundo clones with varying drought and Zn tolerance into four major groups based on their growth and physiological parameters.PCA analysis showed that the variation among the seven hybrids of Miscanthus and seven Arundo clones was largely due to their alternations in growth and physiological parameters (Figure 5A-D).According to the heatmaps, clusters A and B showed better growth, lower MDA, and a higher accumulation of proline under both drought and Zn stress.These clusters had higher mean ranking values among the Miscanthus hybrids and Arundo clones which comprised Miscanthus GRC10 and GRC15 hybrids and Arundo PC1 and ASR clones, suggesting these hybrids and clones have good tolerance to drought and Zn stress.However, the most drought and Zn-tolerant Miscanthus hybrid GRC10 and Arundo clone PC1 were placed in cluster A, especially due to relatively higher levels of proline, phenol and higher growth and lower MDA.Cluster C and D indicated lower growth and lower proline, phenol and higher MDA under both drought and Zn stress for Miscanthus hybrids and Arundo clones.Based on HCA data, Miscanthus GRC3 and Arundo CT2 found the most drought and Znsensitive hybrids and clones which were placed in cluster D and recognized lower mean ranking values.The results suggested that Miscanthus hybrids and Arundo clones might act differentially under drought and Zn stress.

Conclusions
Significant differences in response to drought and Zn tolerance among seven Miscanthus hybrid lines and giant reed clones, based on their growth, physiological and biochemical responses were found in climate chamber pot trials.Based on STI values of each morpho-physiological parameter, hierarchical clustering analysis (HCA) and PCA ranking value, our results showed that Miscanthus hybrid lines M. sinensis × M. sacchariflorus GRC10 was the most drought and Zn stress tolerant.Thereafter, M. sinensis × M. sacchariflorus line GRC15 was identified as drought tolerant, while GRC14 was Zn stress tolerant.Arundo clone PC1 was the most drought and Zn stress tolerant.We also found that M. sinensis × M. sinensis hybrid line GRC3 was the most sensitive hybrid for both drought and Zn stress, while CT2 was the most sensitive clone for both stresses.The physiological and biochemical measurements along with growth parameters measured in this study were effective for discerning differences between Miscanthus and Arundo and a range of variants within these species.The findings of this study provide opportunities for further reductionist experiments needed to investigate specific metabolic and their underlying molecular mechanisms providing tolerance to drought and HM stresses in these two species.
On the other hand, marginal land presents a large opportunity to produce non-food biomass from perennial grasses with low risks of Indirect Land Use Change (low ILUC).It needs to be tested if these trait-based resilience rankings will translate to biomass yield and quality performance in crops growing in real Zn contaminated and marginal soils over multiple years with weather patterns that are becoming more erratic in this era of extreme weather caused by climate change.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biology12121525/s1,Table S1.Estimated drought and Zn stress tolerance indices (stress tolerance indexes, STI) among the seven Miscanthus hybrids lines based on growth including; plant height (PH), number of leaves (NOL), shoot dry weight (SDW), physiological; the maximum quantum efficiency of the PSII (Fv/Fm), performance index (PI-ABS), leaf relative water content (RWC),and biochemical; protein, enzymes including polyphenol oxidase (PPO), peroxidase (POD), superoxide dismutase (SOD) and ascorbate peroxidase (APX), lipid peroxidation (MDA), parameters; Table S2.Estimated drought and Zn stress tolerance indices (stress tolerance indexes, STI) among the seven giant reed clones based on growth including; plant height (PH), number of leaves (NOL), shoot dry weight (SDW), physiological; the maximum quantum efficiency of the PSII (Fv/Fm), performance index (PI-ABS), leaf relative water content (RWC),and biochemical; protein, enzymes including polyphenol oxidase (PPO), peroxidase (POD), superoxide dismutase (SOD) and ascorbate peroxidase (APX), lipid peroxidation (MDA), parameters; Table S3.Contributions and correlations of each growth and physiological parameter to the three main components of the PCA, used in the PCA ranking procedure based on the STI (drought and Zn) of Miscanthus hybrid lines and giant reed clones; Figure S1.The proportion of variance for principal component analysis (PCA) based on the STI of growth and physiological traits of Miscanthus hybrids under drought (A) and under Zn (B) stress conditions and giant reed clones (C) and (D), respectively.

Figure 1 .
Figure 1.Total proline (A,B) and phenol (total phenol) (C,D) contents in leaves of Miscanthus hybrids and Arundo clones, respectively.Data are expressed as mean ± SD (n = 4), and different letters (a, b and c) indicate significant difference and ns = non-significant differences between control and treatments by Tukey's HSD post hoc test at p < 0.05.

Figure 2 .
Figure 2. Drought and Zn both stress-induced the content of Malondialdehyde (MDA) in leaves of Miscanthus hybrids (above) and Arundo clones (below).Data are presented with mean ± SD (n = 4).Different letters (a, b and c) indicate significant difference and ns = non-significant differences between control and treatments by Tukey's HSD post hoc test at p < 0.05.

Figure 3 .
Figure 3. Indicate total protein content in leaves of Miscanthus hybrids (A) and Arundo clones (B), respectively.Data are presented with mean ± SD (n = 4).Different letters (a, b and c) indicate significant differences and ns = non-significant differences between control and treatments by Tukey's HSD post hoc test at p < 0.05.

Figure 4 .
Figure 4. Activities of antioxidant enzymes as (A) Polyphenol oxidases (PPO), (B) peroxidase (POD), (C) Superoxide dismutase (SOD), (D) ascorbate peroxidase (APX) in the leaves of Miscanthus hybrids and (E) PPO, (F) POD, (G) SOD, (H) APX in Arundo leaves under drought and Zn stress.Data are expressed with mean ± SD (n = 4), and different letters (a, b and c) indicate significant differences and ns = non-significant difference between control and treatments by Tukey's HSD post hoc test at p < 0.05.

Figure 5 .
Figure 5. Principal components analysis (PCA) biplot of the growth and physiological parameters of seven Miscanthus hybrids and seven Arundo clones.Here PCA plots (A,B) are based on data under drought and Zn stress of Miscanthus hybrids and (C,D) obtained under drought and Zn stress of Arundo clones, respectively.

Figure 6 .
Figure 6.Heatmap and hierarchical clustering analysis (HCA) for growth and physiological parameters under drought and Zn stress conditions of seven Miscanthus hybrids and seven Arundo clones.HCA, (A,B) based on data under drought and Zn stress of Miscanthus hybrids and (C,D) obtained under drought and Zn of Arundo clones, respectively.The color scale is based on STI values of each trait.The clustering under drought and Zn stress for hybrids and clones was determined four groups as indicated A, B, C & D.

Table 1 .
List of the seven Miscanthus hybrids (left), source, genotypes and seven Arundo clones (right) and their origin considered for this study.

Table 2 .
The growth parameters on plant height (PH) (cm), number of leaves (NOL) and shoot dry weight (SDW) (gm) of Miscanthus hybrids and Arundo clones under control, drought and Zn stress conditions.Data are presented Mean ± SD (mean ± standard deviation, n = 4) and different letters (a, b, and c) indicate a significant difference between control and treatments by Tukey's post hoc test at p < 0.05.

Table 3 .
Dark-adapted chlorophyll fluorescence, the maximum quantum efficiency of the photosystem II (Fv/Fm), performance index (PI-ABS) and relative water content (RWC%) of Miscanthus hybrids and Arundo clones after 28 days of drought and Zn stress.Data are mean ± SD (n = 4) and values followed by different letters indicate significant differences and the same letter indicates no statistically significant difference by Tukey's post hoc test at p < 0.05.

Table 5 .
PCA ranking values are based on stress tolerance index (STI) with three major principal components (PC1, PC2, and PC3) and numeric ranking of Miscanthus hybrids and Arundo clones under drought and Zn stress.