Article Male and Female of Perform Similarly to Flooding in Morphology, Anatomy, and Physiology

1, 1, 2 3 3 Fei-fei Zhai †, Hai-dong Li †, Shao-wei Zhang , Zhen-jian Li , Jun-xiang Liu , Yong-qiang Qian 3, Guan-sheng Ju 3, Yun-xing Zhang 1, Long Liu 1, Lei Han 3 and Zhen-yuan Sun 3,*

1 School of Architectural and Artistic Design, Henan Polytechnic University, Century Avenue, Jiaozuo 454000, China; lkyzff@163.com (F.-f.Z.); [email protected] (H.-d.L.); [email protected] (Y.-x.Z.); [email protected] (L.L.) 2 College of Horticulture and Landscape, Henan Vocational College of Agriculture, Zhengzhou 451450, China; [email protected] 3 State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Key Laboratory of Tree Breeding and Cultivation, State Forestry Administration, Beijing 10091, China; [email protected] (Z.-j.L.); [email protected] (J.-x.L.); [email protected] (Y.-q.Q.); [email protected] (G.-s.J.); [email protected] (L.H.) * Correspondence: [email protected]; Tel.: +86-010-6288-9626 These authors have contributed equally to this work. †  Received: 7 February 2020; Accepted: 11 March 2020; Published: 14 March 2020 

Abstract: Salix viminalis L., a dioecious species, is widely distributed in riparian zones, and flooding is one of the most common abiotic stresses that this species suffers. In this study, we investigated the morphological, anatomical, and physiological responses of male vs. female plants of S. viminalis to flooding. The results showed that the height and root collar diameter were stimulated by flooding treatment, which corresponded with higher dry weight of the stem and . However, the dry weight of the underground part decreased, which might be due to the primary root having stopped growing. The little-influenced net photosynthesis rate (Pn) under flooding treatment could guarantee rapid growth of the aboveground part, while the unaffected leaf anatomical structure and photosynthetic pigment contents could ensure the normal operation of photosynthetic apparatus. Under a flooding - environment, the production ratio of superoxide free radical (O2· ) and malondialdehyde (MDA) contents increased, indicating that the cell membrane was damaged and oxidative stress was induced. At the same time, the antioxidant enzyme system, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and osmotic adjustment substances, involving proline (Pro) and solute protein (SP), began to play a positive role in resisting flooding stress. Different from our expectation, the male and female plants of S. viminalis performed similarly under flooding, and no significant differences were discovered. The results indicate that both male and female plants of S. viminalis are tolerant to flooding. Thus, both male and female plants of S. viminalis could be planted in frequent flooding zones.

Keywords: ; dioecious plant; gender difference; anatomical structure; gas exchange; antioxidant enzyme; osmotic adjustment

1. Introduction Flooding is one of the primary abiotic stresses encountered by many plants, and it develops when the water content in the soil surpasses the field moisture capacity [1]. Soil flooding is usually caused by

Forests 2020, 11, 321; doi:10.3390/f11030321 www.mdpi.com/journal/forests Forests 2020, 11, 321 2 of 16 heavy rainfall, poor soil drainage, and some irrigation practices [2]. The water layer above the soil can be shallow or deep, so partial or complete submergence can be induced [3]. The Intergovernmental Panel on Climate Change (IPCC) (http://www.ipcc.ch) reported that man-induced climate change will increase the frequency of heavy precipitations and tropical cyclone activity, and it is likely that the flood plains (i.e., lowlands), riparian zones, and cultivated lands will suffer from more frequent flooding events [4]. Soil flooding always restricts O2 diffusion to plants, and thereby inhibits their aerobic respiration [5]. This is often accompanied by a decrease in soil pH and an accumulation of toxic soil substances [6,7]. Under low-oxygen conditions, plants usually have typical alternations in morphology, anatomy, and physiology to adapt to the anaerobic environment. At the morphological level, some adaptable plants under flooding conditions are often taller than their non-flooded comparisons, such as marsh dock (Rumex palustris Sm.) [8], rhodes grass (Chloris gayana Kunth), kleingrass (Panicum coloratum L.) [9], and Sentang (Azadirachta excelsa (Jack)Jacobs) [10]. On the contrary, there are also some plants that exhibit shorter stems as a result of the negative impact of the anaerobic environment on growth [11,12]. In addition, an obvious response of plants tolerant to flooding is the formation of adventitious roots, which could help to maintain normal function of water and nutrient uptake and thus relieve the harmful effect of flooding [12,13]. Besides, the generation of aerenchyma in tissues is the most common anatomical response, which could facilitate the transport of oxygen from shoots to roots [3]. Leaf gas exchange parameters are one of the most frequently studied physiological processes of plants under flooding. The photosynthetic rate of many plants has been shown to present a significant declining trend, especially for flooding-sensitive plant species [13–15]. However, flooding-tolerant plants could maintain a high photosynthetic rate or photosynthesis is basically not influenced [14]. The causes for inhibited photosynthesis can be complex, including declined stomatal closure, decreased chlorophyll content, destruction of chloroplast membrane structure, reduction of enzyme activity associated with photosynthesis, and so on [15,16]. Under a flooding environment, the production of reactive oxygen species (ROS), such as superoxide - free radical (O2· ), increases, which causes oxidative damage, lipid peroxidation of cell membranes, and irreversible metabolic dysfunctions, leading to cell death [17–19]. Malondialdehyde (MDA) is the final product of membrane lipid peroxidation, and the content of this substance is often used to assess the degree of oxidative damage of the cell plasma membrane [20,21]. However, the cellular ROS level can be regulated by a variety of antioxidant enzymes and nonenzymatic antioxidants [22]. Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) activities are often activated when plants are subjected to flooding, indicating the efficient role of antioxidant enzymes in scavenging ROS [23,24].In addition, plants can also accumulate osmotic regulators, such as proline (Pro), to adjust the osmotic potential of cells, so that the stress pressure can be relieved [25]. Sexuality was precisely defined by Sachs for the first time, and since then, the study of sex in plants has been an interesting topic for researches [26]. Today, it is well established that dioecious plants account for 5.1% to 6.0% of angiosperm species (15,600 of 304,419 to 261,750) distributed in 987 genera and 175 families [27]. They play an important role in sustaining the stability and continuity of the structure and function of terrestrial ecosystems [28]. Because of different reproductive costs between male and female plants [29], gender (denoting the development of male and female individuals of plants) differences may be manifested under environmental stresses [30–32]. Previous studies reveal that gender differences vary greatly in plant species. It is shown that male plants of cathay poplar (Populus cathayana Rehd.) and yuannan poplar (Populus yunnanensis Dode.) are more resistant to stresses such as drought, salinity, and heavy metal [33–35]. However, the female plants of sea buckthorn (Hippophae rhamnoides L.) and dark-leaved willow (Salix myrsinifolia Salisb.) exhibit stronger tolerance to drought and UV-B stress, respectively [36,37]. So far, limited studies have been carried out to discuss gender-specific responses to flooding. River systems are dominated by plenty of dioecious plants, which are frequently flooded [15,38]. Previous studies indicate that females of narrowleaf cottonwood (Populus angustifolia Forests 2020, 11, 321 3 of 16

James) are more tolerant to flooding than males, which could be related to the greater frequency of females in flood-prone sites [39,40]. Salix viminalis L., a species of Salix, Salicaceae, is predominant in riparian zones, and the female plants are more prevalent in wetter zones that are prone to suffer from flooding. However, whether the male and female plants of S. viminalis perform differently (i.e., the females are more tolerant to flooding than males) under flooding has not been documented. Although there has been research to examine the responses of S. viminalis to soil flooding [41], no studies have been conducted to discuss possible gender differentiation of S. viminalis under flooding. In this study, we investigated the gender-specific responses of S. viminalis in morphology, anatomy, growth, gas exchange, chlorophyll pigment, oxidative stress degree, osmotic adjustment, and enzymatic antioxidants to flooding stress. The objectives of this study were to: (1) explore how flooding affects the morphological, anatomical, and physiological processes of dioecious S. viminalis, and (2) assess whether differences exist between male and female plants of S. viminalis under flooding stress.

2. Materials and Methods

2.1. Plant Materials and Experiment Design A male parent of S. viminalis from Heilihe nature reserve and a female parent from Saihanwula nature reserve were used for controlled intraspecific hybridization. In the F1 progeny containing 570 seedlings, 10 male and 10 female individuals were selected randomly. One-year-old branches were harvested to propagate cuttings from each individual on March 29th, 2018. The cuttings with a length of about 10 cm were grown in nutritive bowls filled with peat soil. Then, five male and five female clones were replanted in 16 cm diameter 15 cm deep plastic pots on May 12th and the substrate × was a 2:1 mixture of garden soil to peat soil. The plants were placed in a greenhouse of the Chinese Academy of Forestry Sciences in Beijing and were watered regularly until flooding application. On June 10th, plants of similar height were selected for flooding treatment. The experimental layout was a completely randomized block design with two main factors (gender and watering regime). Two watering regimes were applied: well-watered treatment as control and flooding treatment. In the control, the plants were watered every day to maintain soil moisture at field capacity. In the flooding treatment, the pots were placed into a larger plastic bucket filled with tap water to 4 cm above the soil surface and watered every day to maintain the water level. During the flooding treatment, the morphological, photosynthetic, and physiological parameters were investigated at 7, 14, 21, 30, and 45 days. For the physiological parameters, the were cut into pieces and mixed, put into the liquid nitrogen, and then stored at –80 ◦C. There were five blocks in this research, and the experiment was conducted during the gowning season from June 10th to July 25th, 2018. During this period, the precipitation is large and flooding events are more prone to occur.

2.2. Growth Characteristics (Height, Diameter, and Biomass) The height and root collar diameter of each individual plant were measured at 0, 7, 14, 21, 30, and 45 days. The plant height was measured by a measuring tape with a precision of 0.1 cm, and the root collar diameter was evaluated by Vernier caliper with an accuracy of 0.01 cm. At the end of the experiment, the root, stem, and leaf were harvested and each component was dried to a constant weight and weighed, respectively.

2.3. Leaf Gas Exchange Parameters Gas exchange parameters were measured in the 3rd~5th fully expanded and mature leaf from the top of the stem between 9:00 and 12:00 in the morning using the Li-Cor 6400XT photosynthesis measuring system (LI-COR, Lincoln, NE, USA) equipped with a red-blue light-emitting diode light source. The parameters included net photosynthesis (Pn), stomatal conductance (Gs), internal CO2 concentrations (Ci), and transpiration rate (Tr). The optimal parameters were set as follows: Forests 2020, 11, 321 4 of 16

2 1 photosynthetic photon flux density of 1200 µmol m− s− and an approximately ambient CO2 concentration 1 of 400 µmol mol− .

2.4. Chlorophyll Pigment About 0.20 g of leaf tissue, 0.2 0.2 cm in size, was immersed into 80% acetone (v:v) for 24 h, × approximately under room temperature until all of the tissue turned white. Meanwhile, the tube was shaken at intervals. Then, the absorbance of the extracting solution was measured at 470, 646, and 663 nm. The content of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid (Caro) were calculated by the equation described by Lichtenthaler [42].

2.5. Proline Content Pro was extracted and measured according to the procedure by Bates et al. [43]. About 0.30 g of leaf tissue was immersed into 5 ml of 3% aqueous sulfosalicylic acid solution. After being extracted in a boiling water bath and filtered, 2.0 ml of glacial acetic acid and 2 ml of 2.5% acid ninhydrin were added into 2 ml of the supernatant. Then, the mixed solution was incubated in a boiling water bath. After the mixture was cooled, 5 ml of toluene was added and the absorbance was measured at 520 nm.

2.6. Production Ratio of Superoxide Free Radical, Malonaldehyde Content, Solute Proteins Content, and Autioxidant Enzyme Activities Determination The frozen leaves were used to test the physiological indicators, including the production ratio of - O2· , the MDA content, the solute proteins (SP) content, the SOD activity, the POD activity, the CAT activity, and the APX activity. All of these indicators were analyzed by applying the respective assay kit (Comin Biotechnology Co. Ltd., Suzhou, China).

2.7. Anatomical Structure Observation The 5th fully expanded and mature leaf from the top of the stem was used for anatomical analysis. The same parts of the leaf were cut into 2 mm strips perpendicular to the main vein and immediately fixed in formalin, acetic acid, and alcohol solution (FAA) solution (70% alcohol/glacial acetic acid/40% formalin = 90:5:5, v/v) and stored at 4 ◦C. Then, the materials were, respectively, dehydrated with gradient ethanol of 75% (4 h), 85% (2 h), 90% (2 h), 95% (1 h), and 100% concentrations (two changes, 30 min). Next, they were immersed in mixed liquor with ethanol and xylene (10 min) and xylene (two changes, 10 min). Thereafter, the samples were embedded in paraffin by the Embedding Center (JB-P5, Wuhan Junjie Electronic Co., Ltd.), and subsequently crosscut into 4-µm-thick slices by a microtome (RM2016, Shanghai Leica Instrument Co. Ltd.). After dyeing in saffron for 2 h and solid green for 40 s, the cross-sections were observed and photographed using a NIKON ECLIPSE E100 microscope (Nikon Corporation, Japan).

2.8. Statistical Analysis All data were analyzed by SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). Before the ANOVAs, the data were checked for normality, as well as the homogeneity of variances using Shapiro-Wilk test and Levene’s test, respectively. Individual differences among the means for each investigated time and each part of plant were identified by Duncan tests of one-way ANOVAs at a significance level of p < 0.05. Two-way ANOVAs were used to test the effects of gender, flooding, and their interaction, which was carried out using the multivariate General Linear Model procedure with Type III sum of squares.

3. Results

3.1. Gender-Specific Responses of Morphology to Flooding All of the plants grew vigorously and no withering, defoliation, or death occurred during the study. At the end of experiment, about 3~5 leaves at the bottom of the plant under the flooding Forests 2020, 11, 321 5 of 16 treatment turned yellow. White hypertrophied lenticels and adventitious roots developed at the base of the submerged shoot after 3 and 5 days of flooding treatment, respectively. However, there were no obvious differences between male and female plants.

3.2. Gender-Specific Responses of Plant Growth to Flooding S. viminalis plants continued to grow for both the control and flooding treatment groups during Forests 2020, 11, x FOR PEER REVIEW 5 of 16 the experiment (Figure1). Flooding did not significantly a ffect the plant height or root collar diameter Forests 2020, 11, x FOR PEER REVIEW 5 of 16 beforeof thethe firstsubmerged 21 days, shoot but theafter two 3 and parameters 5 days of forflood floodinging treatment, treatment respectively. were significantly However, higher there were than theof the controlno submerged obvious at 30 differences and shoot 45 after days. between 3 and However, 5male days and of the floodfemale planting plants. heighttreatment, and respectively. root collar diameterHowever, betweenthere were male andno obvious female plantsdifferences showed between no obviousmale and di femalefferences plants. throughout the experiment (Figure1). Besides, the growth3.2. Gender-Specific parameters Responses were not of significantly Plant Growth a fftoected Flooding by gender flooding interaction. × 3.2. Gender-Specific Responses of Plant Growth to Flooding

140 8 PG 0.8104 0.8794 0.8677 0.9508 0.7771 0.5438 (a) PG 0.5698 0.9050 0.9527 0.7314 0.8989 0.7049 (b) PF 0.2400 0.7250 0.2983 0.0387 0.0024 0.0000 PF 0.3850 0.3134 0.1274 0.0518 0.0012 0.0002 140 120 PG×F 0.8505 0.7850 0.7989 0.9300 0.8975 0.8578 8 7 PG×F 0.7122 0.8608 0.9905 0.7944 0.5479 0.5432 PG 0.8104 0.8794 0.8677 0.9508 0.7771 0.5438 (a) a PG 0.5698 0.9050 0.9527 0.7314 0.8989 0.7049 (b) a PF 0.2400 0.7250 0.2983 0.0387 0.0024 0.0000 a PF 0.3850 0.3134 0.1274 0.0518 0.0012 0.0002 a P 60.7122 0.8608 0.9905 0.7944 0.5479 0.5432 b 120 PG×F 0.8505 0.7850 0.7989 0.9300 0.8975 0.8578a bb 7 G×F aa 100 a a b a b a a b b aa a aa a b a a a a 5 aa a a a a bb 6 aa a aa aa b 100 80 a a a aa a a b b a aa a a a a b b a aa a b aa a a a a a CK-M 5 a 4 a a aa a CK-M a CK-F aa a a CK-F 80 60 aaa a a a FT-M 3 FT-M aa a CK-M FT-F 4 CK-M FT-F CK-F CK-F 60 height/cm Plant 40 FT-M 3 2 FT-M FT-F FT-F Root-collar diameter/cm Root-collar

Plant height/cm Plant 40 20 2 1 Root-collar diameter/cm Root-collar 20 0 1 0 0 7 14 21 30 45 0 7 14 21 30 45 0 Days of flooding treatment/d 0 Days of flooding treatment/d 0 7 14 21 30 45 0 7 14 21 30 45 Days of flooding treatment/d Days of flooding treatment/d FigureFigure 1. The 1. plantThe plant height height (a) and(a) and root root collar collar diameter diameter (b )(b) of of male male vs. vs. female femaleSalix Salix viminalis viminalis plantsplants atat differentdifferent times times of flooding of flooding treatment. treatment. Note: Note: The dataThe da areta the are mean the meanstandard ± standard deviation deviation (SD) (SD) from from five Figure 1. The plant height (a) and root collar diameter (b) of male vs.± female Salix viminalis plants at replicates.differentfive replicates.times Different of flooding Different small letterstreatment. small indicate letters Note: indicate significant The da signta diareificantff erencesthe differencesmean between ± standard between treatments deviation treatments at the(SD) at same fromthe same time pointfive replicates.time according point Differentaccording to Duncan small to testsDuncan letters (p < tests 0.05).indicate (p < CK, 0.05). sign control; ificantCK, control; FT,differences flooding FT, flooding between treatment; treatment; treatments M, male; M, at male; F,the female; sameF, female; PG, gendertimeP pointG, e genderffect; according P Feffect;, flooding to P DuncanF, flooding effect; tests P effect;G (pF ,< gender 0.05).PG×F, genderCK, and control; flooding and flooding FT, interactionflooding interaction treatment; effect. effect. M, male; F, female; × PG, gender effect; PF, flooding effect; PG×F, gender and flooding interaction effect. At theS. endviminalis of the plants flooding continued treatment, to grow we foundfor both that the the cont undergroundrol and flooding biomass treatment of male groups and during female plantstheS. was viminalisexperiment significantly plants (Figure continued decreased, 1). Flooding to grow including did for not both significantly primary the cont rootrol affect and theflooding total plant root, heighttreatment in spite or root groups of thecollar emergenceduring diameter ofthe adventitious experimentbefore the first (Figure root, 21 days, while 1). Flooding but the the aboveground twodid notparameters significantly biomass for flooding affect (stem the treatment and plant leaf) height were was or significantly significantlyroot collar diameter higher increased than (Figurebeforethe2 the ).control However, first 21at days,30 theand but total 45 thedays. dry two weightHowever, parameters presented the for plant flooding no height significant treatment and root di ffcollarwereerences significantly diameter between between thehigher control male than and thethe flooding controlfemale atplants treatment. 30 and showed 45 days. Besides, no However, obvious the dry differencesthe weight plant height of thro eachughout and constituent root the collar experiment anddiameter sapling (Figure between had 1). no male Besides, significant and the difemalefferencesgrowth plants between parameters showed male nowere and obvious not female significantly differences plants. Inaf throfected addition,ughout by gender the the dry experiment × weightflooding was interaction.(Figure not significantly 1). Besides, atheffected bygrowth gender parametersflooding were interaction. not significantly affected by gender × flooding interaction. × 15 PG 0.5659 0.9200 0.5674 0.6831 0.9134 0.9572 PF 0.0000 -- 0.0006 0.0005 0.0006 0.0807 15 P 0.2333 -- 0.2821 0.7108 0.9663 0.5584 PG 0.5659G×F 0.9200 0.5674 0.6831 0.9134 0.9572 a PF12 0.0000 -- 0.0006 0.0005 0.0006 0.0807 a a PG×F 0.2333 -- 0.2821 0.7108 0.9663 0.5584a 12 a a a 9 a 9 CK-M a CK-F 6 a CK-M FT-M a CK-F FT-F 6 weight/g Dry a bb aa FT-M FT-F 3 bb Dry weight/g Dry a aa a bb aa bb b 3 bb a a b aa a abb 0bb a a PR AR TR Stem Leaf Total 0 Plant organ PR AR TR Stem Leaf Total Plant organ

Figure 2. The dry weight of primary root, adventitious root, total root, stem, leaf, and sapling (total) Figure 2. The dry weight of primary root, adventitious root, total root, stem, leaf, and sapling (total) Figureof 2.male The vs. dry female weight Salix of primary viminalis root, plants adventitious at the end root, of totalflooding root, treatment.stem, leaf, PR,and primarysapling (total) root; AR, of male vs. female Salix viminalis plants at the end of flooding treatment. PR, primary root; AR, of maleadventitious vs. female root; Salix TR, viminalis total root. plants at the end of flooding treatment. PR, primary root; AR, adventitious root; TR, total root. adventitious root; TR, total root. At the end of the flooding treatment, we found that the underground biomass of male and femaleAt the plantsend of was the significantlyflooding treatment, decreased, we foundincluding that primarythe underground root and totalbiomass root, of in male spite and of the femaleemergence plants was of adventitious significantly root, decreased, while the including aboveground primary biomass root and (stem total and root, leaf) in was spite significantly of the emergenceincreased of (Figureadventitious 2). However, root, while the totalthe aboveground dry weight presented biomass no(stem significant and leaf) differences was significantly between the increasedcontrol (Figure and the 2). flooding However, treatment. the total dryBesides, weight the presented dry weight no of significant each constituent differences and between sapling thehad no controlsignificant and the differencesflooding treatment. between Besides, male and the femaledry weight plants. of each In addition,constituent the and dry sapling weight had was no not significantsignificantly differences affected between by gender male × flooding and female interaction. plants. In addition, the dry weight was not significantly affected by gender × flooding interaction.

Forests 2020, 11, 321 6 of 16

3.3. Gender-Specific Responses of Leaf Anatomical Structure to Flooding The male and female plants displayed similar cross-section structures, including one-layer upper and lower epidermis, bi-layered compact palisade parenchyma, 2–3-cell-thick loose spongy parenchyma and transversal vascular bundles, etc. (Figure3). After flooding treatment for 45 days, the anatomical Forests 2020, 11, x FOR PEER REVIEW 6 of 16 structure of males and females were not affected significantly and palisade cells were still arranged regularly (Figure3). 3.3. Gender-Specific Responses of Leaf Anatomical Structure to Flooding

CK-M FT-M

CK-F FT-F

Figure 3. LeafFigure cross-sections 3. Leaf cross-sections of male vs. of femalemale vs.Salix female viminalis Salix viminalisplants atplants 45 daysat 45 days of flooding of flooding treatment. treatment. Observations were done using th NIKON ECLIPSE E100 microscope at 20 Magnification. Observations were done using th NIKON ECLIPSE E100 microscope ×at 20 × Magnification. Scale bars Scale bars = 50 µm=. 50 PP, μ palisadem. PP, palisade parenchyma; parenchyma; SP, spongy SP, parenchyma; spongy parenc TVB,hyma; transcurrent TVB, transcurrent vascular bundle vascular. bundle. 3.4. Gender-Specific Responses of Gas Exchange Parameters to Flooding The male and female plants displayed similar cross-section structures, including one-layer The gas exchangeupper and parameters, lower epidermis, including bi-layered Pn, gs,C compacti, and E,palisade were not parenchyma, significantly 2–3-cell-thick affected by loose spongy flooding throughoutparenchyma the experiment, and transversal although vascular they bundles, fluctuated etc. over (Figure time 3). (Figure After4 flooding). The male treatment and for 45 days, female plants oftheS. anatomical viminalis alsostructure presented of males no and obvious females diff erences.were not affected In addition, significantly the gas exchangeand palisade cells were parameters werestill not arranged significantly regularly affected (Figure by gender3). flooding interaction. × 3.4. Gender-Specific Responses of Gas Exchange Parameters to Flooding

24 0.48 PG 0.9826 0.4141 0.2356 0.5409 0.9683 (b) PG 0.9503 0.5719 0.9274 0.1862 0.5788 (a) P 0.4654 0.2862 0.0878 0.3254 0.7873 ) PF 0.9992 0.3013 0.2646 0.9789 0.9993 F -1 )

s P 0.6118 0.9091 0.3769 0.4459 0.8877 -1 PG×F 0.5387 0.9599 0.4568 0.6223 0.8179 G×F

-2 a a a s 20 a 0.40 a a a aa -2 a a a aa a a a a a a a O)m a a 2 a aa a a 16 a aa 0.32 a a a a a aa a a 12 CK-M 0.24 CK-F CK-M FT-M CK-F 8 FT-F 0.16 FT-M FT-F

4 0.08 Net photosynthetic rate/(μmol m rate/(μmol Net photosynthetic

0 conductance/(mol(H Stomatal 0.00 7 14213045 7 14213045 Days of flooding treatment/d Days of flooding treatment/d

Forests 2020, 11, x FOR PEER REVIEW 6 of 16

3.3. Gender-Specific Responses of Leaf Anatomical Structure to Flooding

CK-M FT-M

CK-F FT-F

Figure 3. Leaf cross-sections of male vs. female Salix viminalis plants at 45 days of flooding treatment. Observations were done using th NIKON ECLIPSE E100 microscope at 20 × Magnification. Scale bars = 50 μm. PP, palisade parenchyma; SP, spongy parenchyma; TVB, transcurrent vascular bundle.

The male and female plants displayed similar cross-section structures, including one-layer upper and lower epidermis, bi-layered compact palisade parenchyma, 2–3-cell-thick loose spongy parenchyma and transversal vascular bundles, etc. (Figure 3). After flooding treatment for 45 days, the anatomical structure of males and females were not affected significantly and palisade cells were still arranged regularly (Figure 3). Forests 2020, 11, 321 7 of 16 3.4. Gender-Specific Responses of Gas Exchange Parameters to Flooding

24 0.48 PG 0.9826 0.4141 0.2356 0.5409 0.9683 (b) PG 0.9503 0.5719 0.9274 0.1862 0.5788 (a) P 0.4654 0.2862 0.0878 0.3254 0.7873 ) PF 0.9992 0.3013 0.2646 0.9789 0.9993 F -1 )

s P 0.6118 0.9091 0.3769 0.4459 0.8877 -1 PG×F 0.5387 0.9599 0.4568 0.6223 0.8179 G×F

-2 a a a s 20 a 0.40 a a a aa -2 a a a aa a a a a a a a O)m a a 2 a aa a a 16 a aa 0.32 a a a a a aa a a 12 CK-M 0.24 CK-F CK-M FT-M CK-F 8 FT-F 0.16 FT-M FT-F

4 0.08 Net photosynthetic rate/(μmol m rate/(μmol Net photosynthetic

0 conductance/(mol(H Stomatal 0.00 Forests 2020, 711, x FOR 14213045 PEER REVIEW 7 14213045 7 of 16 Days of flooding treatment/d Days of flooding treatment/d Forests 2020, 11, x FOR PEER REVIEW 7 of 16 ) -1

420 18 PG 0.7124 0.5481 0.2190 0.8402 0.7754 PG 0.7692 0.6254 0.7032 0.7743 0.4979 (c) (d)

) PF 0.8301 0.2484 0.3288 0.8458 0.6794

) mol P 0.5143 0.8760 0.4913 0.0631 0.7231

-1 F 2 PG×F 0.8203 0.9249 0.5453 0.7437 0.7564 420PG×F 0.6459 0.7762 0.2300 0.8274 0.6288 18 PG 0.7124 0.5481 0.2190 0.8402 0.7754 360 PG 0.7692 0.6254 0.7032 0.7743 0.4979 (c) (d)

a a -1) a aaa a aa 15PF 0.8301 0.2484 0.3288 0.8458 0.6794 ) mol PF 0.5143 0.8760aa 0.4913a 0.0631 0.7231 2 aa s a PG×F 0.8203a 0.9249 0.5453 0.7437 0.7564 P 0.6459 0.7762 0.2300 0.8274 0.6288 a -2 360 aG×Fa a a a

a a -1) 300 a aa aaaa a aa 15 a a aa a s 12 a O)m a -2 a

a a 2 300 a a a a 240 12 a O)m

2 a 9 a aa a 240 CK-M a a a CK-M 180 CK-F a a a a a a CK-F CK-M FT-M 9 a a aa aa a a CK-M FT-M 180 CK-F FT-F 6 a a a CK-F FT-F concentration/(μmol(CO FT-M a a FT-M 2 120 a a a FT-F 6 a FT-F concentration/(μmol(CO 2 120 60 3 60 3 0 0 Transpiration rate/(mmol(H Transpiration 0 7 14213045 0 7 14213045 Transpiration rate/(mmol(H Transpiration Intracellular CO Intracellular 7 14213045 7 14213045

Intracellular CO Intracellular Days of flooding treatment/d Days of flooding treatment/d Days of flooding treatment/d Days of flooding treatment/d

FigureFigure 4. Net photosynthesis4. Net photosynthesis rate (Pn)( ratea), stomatal (Pn) (a), conductance stomatal conductance (gs)(b), intracellular (gs) (b), CO intracellular2 concentration CO2 Figure 4. Net photosynthesis rate (Pn) (a), stomatal conductance (gs) (b), intracellular CO2 (Ci)(c),concentration and transpiration (Ci) (c), and rate transpiration (E) (d) of male rate vs. (E) (d) female of maleSalix vs.viminalis female Salixplants viminalis at di plantsfferent at times different of concentration (Ci) (c), and transpiration rate (E) (d) of male vs. female Salix viminalis plants at different flooding treatment. timestimes of of flooding flooding treatment. treatment. 3.5. Gender-Specific Responses of Chlorophyll Pigments to Flooding TheThe gas gas exchange exchange parameters,parameters, including PPn,n ,g gs,s ,C Ci, i,and and E, E, were were not not significantly significantly affected affected by by Thefloodingflooding contents throughout throughout of Chl a, thethe Chl experiment,experiment, b, and Car althoughalthough of S. viminalis they they fluctuated fluctuatedplants were over over nottime time significantly (Figure (Figure 4). 4).The influenced The male male and and by floodingfemalefemale treatment, plants plants andof of S.S. the viminalisviminalis three pigments alsoalso presented also presented nono obviousobvious no differences. differences. significant In diIn addition,ff addition,erence the between the gas gas exchange male exchange and femaleparameters plantsparameters (Figure were were5 ).not not In signi signi addition,fificantlycantly chlorophyll affected byby pigments gender gender × × flooding wereflooding not interaction. interaction. significantly a ffected by gender × flooding interaction. 3.5.3.5. Gender-Specific Gender-Specific Responses Responses of Chlorophyll PigmentsPigments to to Flooding Flooding

1.0 0.28 P 0.5620 0.8730 0.4810 0.9470 0.9800 1.0 PG 0.2067 0.5009 0.8176 0.4490 0.7218 (a) 0.28 G P 0.5620 0.8730 0.4810 0.9470 0.9(b)800 PG 0.2067 0.5009 0.8176 0.4490 0.7218 (a) G (b) PF 0.9370 0.8308 0.6392 0.6485 0.8934 PF 0.8550 0.5930 0.9900 0.3500 0.3200 PF 0.9370 0.8308 0.6392 0.6485 0.8934 PF 0.8550 0.5930 0.9900 0.3500 0.3200 P 0.3733 0.4194 0.8478 0.8555 0.7582 PG×F 0.9800 0.5340 0.6720 0.8630 0.6050 G×F 0.24 P 0.9800 0.5340 0.6720 0.8630 0.6050 PG×F 0.3733 0.4194 0.8478 0.8555 0.7582 0.24 G×F ) 0.8 ) a -1 -1 ) 0.8 a ) a a a a -1 a a a -1 a a a a a aaa aaa a a 0.20 aaa a a a a a a aa a a aaa 0.20 a a a aa a a a a a aa a aa a aa a a a a a 0.6 a aa aa a aaa a aa aa a 0.6 a a a 0.16 a 0.16 CK-M CK-M CK-F CK-M 0.12 CK-F CK-M 0.4 FT-M CK-F 0.12 FT-M CK-F 0.4 FT-F FT-M FT-F FT-M FT-F 0.08 FT-F 0.2 0.08 0.2 0.04 Chlorophyll a content/(mg g a content/(mg Chlorophyll g b content/(mg Chlorophyll 0.04 Chlorophyll a content/(mg g a content/(mg Chlorophyll g b content/(mg Chlorophyll 0.0 0.00 0.0 7 14213045 0.00 7 14213045 7 14213045Days of flooding treatment/d 7Days 14213045 of flooding treatment/d Days of flooding treatment/d Days of flooding treatment/d

0.24 PG 0.1507 0.6434 0.7218 0.3317 0.6193 (c) P 0.8270 0.9249 0.9526 0.5900 0.7369 0.24 F 0.21 PPG×FG 0.38510.1507 0.4518 0.6434 0.9146 0.7218 0. 83270.3317 0.9670 0.6193 (c) PF 0.8270 0.9249 0.9526 0.5900 0.7369 )

-1 a 0.210.18 PG×F 0.3851aa 0.4518 0.9146 0.8327 0.9670 a aaa a aa ) a aaa -1 a a a aa a a a 0.180.15 a aa a a a a a aa a aa a aa a a 0.150.12 CK-M CK-F 0.120.09 FT-M FT-F CK-M CK-F 0.090.06 FT-M FT-F

Carotenoids content/(mg g content/(mg Carotenoids 0.060.03 0.00 Carotenoids content/(mg g content/(mg Carotenoids 0.03 7 14213045 0.00 Days of flooding treatment/d 7 14213045 Days of flooding treatment/d Figure 5. Chlorophyll a (Chl a) (a), Chlorophyll b (Chl b) (b), and Carotenoids (Car) (c) contents of Figure 5. Chlorophyll a (Chl a) (a), Chlorophyll b (Chl b) (b), and Carotenoids (Car) (c) contents of male vs. female Salix viminalis plants at different times of flooding treatment. male vs. female Salix viminalis plants at different times of flooding treatment. Figure 5. Chlorophyll a (Chl a) (a), Chlorophyll b (Chl b) (b), and Carotenoids (Car) (c) contents of male vs. female Salix viminalis plants at different times of flooding treatment. The contents of Chl a, Chl b, and Car of S. viminalis plants were not significantly influenced by flooding treatment, and the three pigments also presented no significant difference between male The contents of Chl a, Chl b, and Car of S. viminalis plants were not significantly influenced by and female plants (Figure 5). In addition, chlorophyll pigments were not significantly affected by flooding treatment, and the three pigments also presented no significant difference between male gender × flooding interaction. and female plants (Figure 5). In addition, chlorophyll pigments were not significantly affected by

gender × flooding interaction.

Forests 2020, 11, 321 8 of 16

3.6. Gender-Specific Responses of Superoxide Free Radical and Lipid Peroxidation to Flooding

- Flooding treatment led to increases in the production ratio of O2· and MDA contents of S. viminalis - (Figure6). The production ratio of O 2· under flooding treatment was significantly higher than that of theForests control 2020,after 11, x FOR 14 days,PEER REVIEW while MDA contents increased significantly after 30 days of flooding8 of 16 - treatment. However, both the production ratios of O2· and MDA contents between male and female 3.6.Forests Gender-Specific 2020, 11, x FOR PEERResponses REVIEW of Superoxide Free Radical and Lipid Peroxidation to Flooding 8 of 16 plants showed no significant differences. In addition, the two parameters were not significantly affected by gender3.6. Gender-Specificflooding interaction. Responses of Superoxide Free Radical and Lipid Peroxidation to Flooding 300 54 × PG 0.6475 0.4053 0.7917 0.7981 0.2001 (a) PG 0.1694 0.7194 0.7799 0.1554 0.5877 (b) PF 0.0060 0.0001 0.0000 0.0000 0.0002 PF 0.1071 0.1559 0.2105 0.0012 0.0026 ) -1 PG×F 0.4054 0.6122 0.2664 0.6052 0.4578a ) PG×F 0.0601 0.4329 0.8985 0.1751 0.9104 250 a a -1 45 300 a 54 a a PG 0.6475 0.4053a 0.7917a 0.7981 0.2001 PG 0.1694 0.7194 0.7799 0.1554 0.5877a a (a) a a b (b) PF 0.0060 0.0001 0.0000 0.0000 0.0002 PF 0.1071 0.1559a 0.2105a 0.0012 0.0026 ) a b a a a c b -1 a ) aa a b 200 PG×F 0.4054a a 0.6122 0.2664 0.6052 0.4578b a 36 PG×F 0.0601 0.4329 0.8985 bc 0.1751 0.9104 a -1 a 250 ab bb a a 45 a a b a a a a a a b a aa a b 150 b a b b CK-M 27 a a a c b CK-M 200 a a b b 36 a a bc b ab bb CK-F a CK-F b FT-M FT-M b FT-F FT-F 100 b b 18 150 b CK-M 27 CK-M CK-F CK-F 50 FT-M 9 FT-M 100 FT-F 18 FT-F Malondialdehyde content/(nmol g content/(nmol Malondialdehyde

Oxygen free radical content/(nmol g content/(nmol radical free Oxygen 0 0 50 7 14213045 9 7 14213045

Days of flooding treatment/d g content/(nmol Malondialdehyde Days of flooding treatment/d Oxygen free radical content/(nmol g content/(nmol radical free Oxygen 0 0 7 14213045 7 14213045 Days of flooding treatment/d ∙- Days of flooding treatment/d Figure 6. Production ratio of superoxide free radical (O2 ) (a) and malondialdehyde (MDA) (b) contents of male vs. female Salix viminalis plants at different- times of flooding treatment. FigureFigure 6. Production 6. Production ratio of ratio superoxide of superoxide free radical free (O radical2· )(a) (O and2∙-) malondialdehyde (a) and malondialdehyde (MDA) (b (MDA)) contents (b) of malecontents vs. female of maleSalix vs. viminalis female Salixplants viminalis at diff erentplants times at different of flooding times treatment.of flooding treatment. Flooding treatment led to increases in the production ratio of O2∙- and MDA contents of S. 3.7. Gender-Specificviminalis (Figure Responses 6). The ofproduction Enzyme Activities ratio of O to2 Flooding∙- under flooding treatment was significantly higher Flooding treatment led to increases in the production ratio of O2∙- and MDA contents of S. than that of the control after 14 days, while MDA contents increased significantly after 30 days of Floodingviminalis (Figure treatment 6). causedThe production significant ratio increases of O2∙- under of SOD, flooding POD, treatment CAT, and was APX significantly activities in higher both flooding treatment. However, both the production ratios of O2∙- and MDA contents between male and than that of the control after 14 days, while MDA contents increased significantly after 30 days of malefemale and female plants plants showed after no 14 days,significant and thedifference four enzymes. In addition, activities werethe two on theparameters rise alongside were thenot flooding treatment. However, both the production ratios of O2∙- and MDA contents between male and floodingsigni treatmentficantly affected time (Figure by gender7). However, × flooding there interaction. were no significant di fferences between males and femalesfemale of S. viminalis,plants showedneither no in thesignificant control nordifference the floodings. In treatment.addition, the Besides, two theparameters enzyme activitieswere not were not3.7.signi significantlyGender-Specificficantly affected a ffResponsesected by gender by of gender Enzyme × flooding Activitiesflooding interaction. to interaction. Flooding × 3.7. Gender-Specific Responses of Enzyme Activities to Flooding 700 40000 PG 0.3933 0.6818 0.8189 0.5932 0.8960 (a) PG 0.0032 0.4132 0.0796 0.9255 0.4590 (b) PF 0.0379 0.0098 0.0248 0.0080 0.0039 PF 0.0004 0.0038 0.0047 0.0086 0.0029 P 0.0194 0.8851 0.8021 0.5127 0.5336 P 0.4798 0.6626 0.9178 0.7161 0.2212 600 G×F a G×F 700 a 3200040000 a PG 0.3933 0.6818 0.8189 0.5932 0.8960a PG 0.0032 0.4132 0.0796 0.9255 0.4590 (b) a a (a) a aa a 500 PF 0.0379 0.0098 0.0248a 0.0080a 0.0039 PF 0.0004 0.0038a 0.0047 0.0086 0.0029

) a ) P 0.4798 0.6626 0.9178 0.7161 0.2212 -1 600 PG×F 0.0194a 0.8851 0.8021 0.5127 0.b5336 -1 G×F a ab b bb a bb b a a a b b aa 2400032000 a bc 400 a b a bb bb c aa a 500 a a a a a ) a CK-M ) ab CK-M -1 a -1 bc a 300 b b CK-F c ab bb bb CK-F a b b FT-M 1600024000 FT-M 400 a bb a bc a bb FT-F bb c FT-F 200 CK-M bc ab CK-M CK-F CK-F SOD activity/(U g g SOD activity/(U 300 g POD activity/(U c FT-M 160008000 FT-M 100 FT-F FT-F 200 SOD activity/(U g g SOD activity/(U 0 g POD activity/(U 80000 100 7 14213045 7 14213045 Days of flooding treatment/d Days of flooding treatment/d 0 0 7 14213045 7 14213045 Days of flooding treatment/d Days of flooding treatment/d 300 240 PG 0.3308 0.1282 0.6687 0.9880 0.5226 (c) PG 0.4113 0.2133 0.1988 0.8561 0.3750 (d) PF 0.0361 0.0003 0.0000 0.0000 0.0000 PF 0.0117 0.0004 0.0001 0.0001 0.0000 PG×F 0.5158 0.8217 0.3280 0.2542 0.0880 PG×F 0.7353 0.9402 0.6301 0.9475 0.5626 250 a a 200 a ) ) 300 a 240 a -1 -1 PG 0.3308 0.1282 0.6687 0.9880a 0.5226 (c) PG 0.4113 0.2133 0.1988 0.8561 a 0.3750 (d) PF 0.0361 0.0003 0.0000a 0.0000 0.0000 PF 0.0117 0.0004 0.0001 0.0001 0.0000 g g a a a

-1 a -1 a 200 PG×F 0.5158 0.8217 0.3280 0.2542 0.0880 160 PG×F 0.7353 0.9402 0.6301 0.9475 0.5626 250 a a a 200 a ) ) aa a a a -1 -1 b a ab a a ab b a b b CK-M g g 150 b a b CK-M 120 a a a

-1 a -1 b a b b CK-F 200 b CK-F 160 b a b FT-M FT-M ab b b bb aa a a b FT-F b FT-F b 100 ab b b b 80 ab CK-M 150 b CK-M 120 a b b b b CK-F b CK-F b b FT-M FT-M ab b b bb 50 40 b FT-F

FT-F min APXactivity/(umol CAT activity/(nmol min activity/(nmol CAT 100 80 b

0 0 50 40 APX activity/(umol min APXactivity/(umol CAT activity/(nmol min activity/(nmol CAT 7 14213045 7 14213045 Days of flooding treatment/d Days of flooding treatment/d 0 0 7 14213045 7 14213045 Figure 7. SuperoxideDays of dismutaseflooding treatment/d (SOD) (a), peroxidase (POD) (b),Days catalase of flooding (CAT)treatment/d (c), and ascorbate Figure 7. Superoxide dismutase (SOD) (a), peroxidase (POD) (b), catalase (CAT) (c), and ascorbate peroxidaseperoxidase (APX) (APX) (d) (d) activities activities of of male male vs.vs. female female SalixSalix viminalis viminalis plantsplants at different at diff timeserent of times flooding of Figure 7. Superoxide dismutase (SOD) (a), peroxidase (POD) (b), catalase (CAT) (c), and ascorbate floodingtreatment. treatment. peroxidase (APX) (d) activities of male vs. female Salix viminalis plants at different times of flooding Floodingtreatment. treatment caused significant increases of SOD, POD, CAT, and APX activities in both male and female plants after 14 days, and the four enzyme activities were on the rise alongside the Flooding treatment caused significant increases of SOD, POD, CAT, and APX activities in both flooding treatment time (Figure 7). However, there were no significant differences between males male and female plants after 14 days, and the four enzyme activities were on the rise alongside the flooding treatment time (Figure 7). However, there were no significant differences between males

Forests 2020, 11, 321 9 of 16

3.8. Gender-Specific Responses of Osmotic Regulation to Flooding Forests 2020, 11, x FOR PEER REVIEW 9 of 16 Compared with the control, the flooding treatment increased Pro and SP contents significantly in S. viminalisand females, but there of S. were viminalis, no obvious neither di ffinerences the control between nor malethe flooding and female treatment. plants Besides, (Figure8 the). The enzyme Pro contentsactivities increased were continually not significantly as time affected went on, by whilegender the × flooding SP contents interaction. presented a fluctuating variation throughout the experiment. Furthermore, the Pro and SP contents were not significantly affected by gender3.8. Gender-Specificflooding interaction. Responses of Osmotic Regulation to Flooding ×

28 8400 P 0.6040 0.5554 0.1842 0.9589 0.5973 PG 0.5160 0.9793 0.3518 0.5864 0.3469 (a) G (b) PF 0.2364 0.1506 0.0006 0.0016 0.0015 PF 0.0025 0.0003 0.0119 0.0001 0.0027 PG×F 0.7893 0.8396 0.5576 0.6135 0.9232 PG×F 0.7602 0.3042 0.5837 0.7795 0.9692 24 ) 7000 aa -1 a ) 20 ab a

-1 a 5600 a bc a a a aa c a ab a 16 a a b b a b b b a a a b CK-M 4200 b b CK-M a a b bb b 12 CK-F b b CK-F a FT-M FT-M FT-F 2800 FT-F 8

Proline content/(ug g 1400 4 g content/(ug protein Solute

0 0 7 14213045 7 14213045 Days of flooding treatment/d Days of flooding treatment/d

FigureFigure 8. Proline 8. Proline (Pro) (Pro) (a) and (a) soluteand solute protein protein (SP) (SP) (b) contents(b) contents of maleof male vs. vs. female femaleSalix Salix viminalis viminalisplants plants at differentat different times times of flooding of flooding treatment. treatment.

4. DiscussionCompared with the control, the flooding treatment increased Pro and SP contents significantly in S. viminalis, but there were no obvious differences between male and female plants (Figure 8). The 4.1. Flooding Treatment Effects Pro contents increased continually as time went on, while the SP contents presented a fluctuating 4.1.1.variation Morphology, throughout Growth, the and experiment. Anatomical Furthermore, Structure the Pro and SP contents were not significantly affected by gender × flooding interaction. S. viminalis is a species of obligated riparian trees that are often distributed along streams and rivers and other4. Discussion wet areas [44]. In this research, we studied the response of males vs. females of S. viminalis in an artificial flooding treatment with water up to 4 cm above the soil surface. Hypertrophied lenticels and adventitious4.1. Flooding rootsTreatment were Effects induced after 3 days of flooding treatment, which are specific changes of flood-tolerant species [1,3]. The hypertrophied lenticels can contribute to air exchange between the 4.1.1. Morphology, Growth, and Anatomical Structure stem and root of a plant and the atmosphere [1,45], while the adventitious roots can replace some functions ofS. viminalis older damaged is a species roots of andobligated help plantsriparian to trees uptake that waterare often and distributed nutrients along normally streams [24, 46and]. Symptomsrivers ofand chlorosis other wet and areas defoliation [44]. In this of leavesresearch, can we also studied be induced the response by flooding of males treatment vs. females [1 ,of47 S.]. However,viminalis only in yellow an artificial leaves of flooding the flooded treatment plants werewith observedwater up in to our 4 study,cm above which the might soil indicatesurface. that S.Hypertrophied viminalis is slightly lenticels damaged and adventitious by long-time roots flooding were induced treatment. after 3 days of flooding treatment, Floodingwhich are can specific bring changes about a of reduction flood-tolerant of plant species height [1,3]. and The root hypertrophied collar diameter, lenticels and the can biomass contribute of the leaf,to air stem, exchange and root between can also the stem decrease and root [13,39 of, a48 plant,49]. and In our the study,atmosphere flooding [1,45], irritated while the the adventitious growth of the abovegroundroots can replace part, accompaniedsome functions by tallerof older plant damage heightd androots thicker and help root plants collar diameter,to uptake which water wasand nutrients normally [24,46]. Symptoms of chlorosis and defoliation of leaves can also be induced by consistent with the larger dry weight of the leaf and stem. However, the growth of the underground flooding treatment [1,47]. However, only yellow leaves of the flooded plants were observed in our part was suppressed, along with a lighter dry weight of the root. Our results concerning morphology study, which might indicate that S. viminalis is slightly damaged by long-time flooding treatment. and biomass are consistent with previous findings of purpul osier (Salix integra Thunb.), Salix variegate Flooding can bring about a reduction of plant height and root collar diameter, and the biomass Franch, and marsh bluegrass (Poa leptocoma Trin.) [38,50,51], which indicate that flooding has a negative of the leaf, stem, and root can also decrease [13,39,48,49]. In our study, flooding irritated the growth effectof on the the aboveground root system, part, while accompanied it promotes by the taller growth plant of height the aerial and part.thicker The root elongation collar diameter, of the which stem mightwas be an consistent effective strategywith the forlargerS. viminalis dry weightunder of our the experimental leaf and stem. conditions However, with the a shallowgrowth waterof the level,underground which might part contribute was suppressed, to escaping along from with severe a lighte floodingr dry weight [52–54 of]. the In root. this study,Our results morphological concerning changes,morphology including and the biomass emergence are consistent of hypertrophied with previous lenticels findings and adventitiousof purpul osier roots (Salix and integra accelerated Thunb.), abovegroundSalix variegate growth, Franch were, conduciveand marsh to bluegrass adapting ( riparianPoa leptocoma zones Trin.) for S. [38,50,51], viminalis. which indicate that Anatomicalflooding has characteristics a negative effect are on also the importantroot system, in wh improvingile it promotes the flooding the growth tolerance of the aerial of plants part. [ 55The]. A previouselongation study of found the stem that flooding-susceptiblemight be an effective poplar strategy displays for S. an viminalis unstable under anatomical our experimental structure in whichconditions the shape ofwith palisade a shallow cells water turns fromlevel, long which columns might intocontribute circles to [47 escaping]. However, from the severe flooding-tolerant flooding [52– species54]. still In ownthis study, regular morphological palisade cells changes, and are including characterized the emergence by the presenceof hypertrophied of aerenchymatous lenticels and adventitious roots and accelerated aboveground growth, were conducive to adapting riparian zones for S. viminalis.

Forests 2020, 11, 321 10 of 16 tissue [47,56]. In our study, the anatomical structure of both male and female S. viminalis was not influenced by flooding, which could ensure the stability of the photosynthetic structure. However, aerenchymatous tissue was not observed in the leaves of S. viminalis, which might be due to the fact that CO2 could diffuse normally and so the formation aerenchymatous tissue was not initiated.

4.1.2. Photosynthesis The photosynthetic rate generally decreases for non-flooding-tolerant species during the flooding period, and the earliest response is stomatal closure [15,49]. However, flooding-tolerant species can maintain a high level or even an unaffected photosynthetic rate [14,57]. Although the Pn of S. viminalis fluctuated over time, it was not significantly affected by flooding. The photosynthetic response of S. viminalis was in accordance with species of the same genus, such as S. integra cv. qingpi [1]. Usually, gs has a positive correlation with Pn [58], which also applied to this study. The high gs may increase gas exchange rates, as well as the total volumes, and hence plants assimilate more photosynthates for growth [59]. Also, the appearance of hypertrophied lenticels and adventitious roots in a short time is conducive to keeping the stoma open and maintaining an unaltered photosynthetic rate [60]. The pigment concentrations in the leaves of plants are closely associated with photosynthesis. Many previous studies have found that the Chl a, Chl b, and Car contents usually decline under flooding conditions, along with a reduction of photosynthesis [15,49,61]. However, the Chl a, Chl b, and Car contents in this study were unchanged, which could ensure the normal operation of photosynthesis. The observation of photosynthesis and chlorophyll indicate that S. viminalis were tolerant to flooding.

4.1.3. Oxidative Stress

- Plants exposed to flooding conditions usually accumulate ROS, such as O2· [62]. The ROS react with unsaturated fatty acids, which results in destruction of the membranes, while MDA is the final - production of membrane lipid peroxidation [62,63]. We observed an enhanced production ratio of O2· and MDA contents for both male and female S. viminalis plants under flooding treatment; the former and the later showed significant increases after 14 days and 30 days of treatment, respectively. Similar - results have been shown in previous studies [19,64], and usually, the production ratios of O2· and MDA contents are positively correlated with flooding treatment time [64,65]. The increased production ratios - of O2· and MDA contents can damage the membrane lipids and plants cells, break the cytomembrane structure, and cause oxidative stress [62,64,66]. Our results indicate that the flooding could induce oxidative stress and cause damage to the leaf cells of S. viminalis.

4.1.4. Antioxidant System Plants have developed an antioxidant system, including a series of enzymes (SOD, POD, CAT, etc.) and substances to defend or alleviate the detrimental effect of ROS by metabolism under abiotic stresses [64]. Usually, the antioxidant enzyme activities are positively correlated with the self-protective - ability of plants [67]. SOD is the first line of defense and it catalyzes the dismutation of O2· to O2 and H2O2 timely and effectively in the cytosol, chloroplasts, and mitochondria [34]. SOD activity significantly increased after 14 days of flooding treatment in this study, suggesting that the detoxification - of O2· in S. viminalis is effective. POD, CAT, and APX can catalyze the decomposition of H2O2 to water and oxygen, eventually clearing up H2O2 in cells [23,34,64]. The three enzymes increased significantly in our study, which contributed to neutralizing the product H2O2 from the previous SOD. Similar result has been obtained in açai berry (Euterpe oleracea Mart.) [62] treated with flooding. The enhanced activities of antioxidant enzymes could increase the antioxidant ability of S. viminalis, and thus were helpful to reduce oxidative stress.

4.1.5. Osmotic Substances Osmotic adjustment has been regarded as one of the most crucial protective mechanisms for plants to adapt to stressful environments, while Pro and SP are major osmotic substances [16,34]. Pro can act as Forests 2020, 11, 321 11 of 16 a cell protector and reducer of osmotic potential, which is involved in many cellular processes to keep a balance of ROS [19]. SP is also an important osmotic protective substance that plays a role in regulating osmotic potential [68]. Previous studies have found that flooding significantly increases Pro content in the leaves of pigeonpea (Cajanus cajan L. Millsp.) [69] and bambara groundnut (Vigna subterranea L. Verdc) [70], while accumulated SP has been discovered in Acorus tatarinowii Schott under flooding conditions [71]. Accumulated Pro and SP contents were observed in our study, indicating that osmotic adjustment was irritated and acted as an effective way for S. viminalis to respond to flooding. However, there is some dispute about the accumulation of Pro. An et al. [21] found that Pro accumulation does not contribute to maintaining water balance and it may be an indicator of injury for fig leaves under flooding condition.

4.2. Gender-Specific Responses It is generally assumed that differences exist in males and females of dioecious plants due to different resource allocation, i.e., that females apply more resources in defense while males invest more resources in growth [72,73]. Gender differences usually appear or are greater under adverse conditions [74], and such differences exist in many plants, such as Populus cathayana [35], European aspen (Populus tremula L.) [75], and grey willow (Salix glauca L.) [76]. Gender-specific differences in flooding resistance vary in plant species, even when the plants are in the same genus. A previous study showed that males of eastern cottonwood (Populus deltoides Marsh) have better cellular defense mechanisms against damage caused by waterlogging stress, whereas females are more responsive to waterlogging stress [48]. In S. variegate, the males can adjust more flexibly their resource allocation, and so they are more tolerant to flooding [77]. However, the female plants of P. angustifolia are more tolerant to flooding, which is strengthened by the increased occurrence of female cottonwoods in streamside zones [39,40]. The favored occurrence of female plants in wetter sites is also apparent in some , and it is observed that female willows grow more vigorously than males [78,79]. In our study, flooding treatment caused morphological and physiological changes in S. viminalis, but male and female plants performed similarly and no significant gender-specific differences were discovered. This result is different from previous studies on gender differences of S. viminalis, which found that female plants may be more severely affected by fungal pathogens and high temperatures than males [80,81]. However, gender differences of S. myrsinifolia are not much affected by simulated climatic changes (enhanced CO2 and temperature or their combination) at the pre-reproductive stage [73], and no specific difference was found in P. angustifolia in response to seasonal changes in water availability [82]. In our study, no obvious differences were found between the two genders, suggesting that similar patterns of morphology, anatomy, and physiology may be adopted by the two genders to co-exist in riparian zones, which is consistent with two dioecious riparian species, namely, Salix myrsinifolia and Salix lapponum [83]. Besides, all plants survived 45 days of flooding with no leaf abscission, fast growth aboveground, unaffected photosynthesis, and strong antioxidant capacity and osmotic adjustment ability, which suggests that both male and female plants of S. viminalis are flood-tolerant. Thus, it is concluded that the greater frequency of females in riparian zones is not influenced by flooding, and other factors, such as demographic parameters, could also influence the spatial distribution of genders [84].To better understand the response of male and female S. viminalis to flooding, further studies at the level of molecular biology are needed.

5. Conclusions The combination of morphological, anatomical, and physiological parameters can help us to better understand the gender-specific responses of S. viminalis to flooding. The present study suggests that the growth of plant height and root collar diameter is stimulated by flooding treatment, accompanied by increased dry weight of the aboveground part. The little-affected photosynthesis could guarantee the rapid growth of plants, while the unaffected leaf anatomical structure and photosynthetic pigment contents could ensure the normal operation of photosynthetic apparatus. However, the dry weight of Forests 2020, 11, 321 12 of 16 the underground part decreased at the end of the experiment, which might be owing to the fact that the primary root stopped growing. The flooding treatment caused oxidative stress in S. viminalis, but they could resist the stress by enhancing antioxidant enzyme activities and osmotic adjustment ability. Different from our expectation, gender-specific differences in S. viminalis were not obviously detected, and the male and female plants performed similarly to flooding. Comprehensive indicators indicate that both male and female plants of S. viminalis are tolerant to flooding, which would guarantee a sustainable population and maintain a stable riparian ecosystem. Thus, both male and female plants of S. viminalis could be planted in frequent flooding zones.

Author Contributions: F.-f.Z. and Z.-y.S. designed the study. F.-f.Z., H.-d.L., S.-w.Z., and Z.-j.L. performed the experiments. J.-x.L., Y.-q.Q., and G.-s.J. analyzed the data. F.-f.Z. and H.-d.L. wrote the original draft. Y.-x.Z., L.L., L.H., and Z.-y.S. revised and edited the article. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (grant number 31700533), the Central Public-interest Scientific Institution Basal Research Fund (grant number CAFYBB2018ZB002), the Key Technologies R & D Program of Henan Province (grant numbers 182102310052 and 192102310307), and the Doctoral Scientific Fund Project of Henan Polytechnic University (grant number B2017-40). Acknowledgments: We would like to express our sincere gratitude to Xia Li, Zhi-cheng Chen, Ping Li and Jun-chao Xing (Chinese Academy of Forestry) for performing the research. Conflicts of Interest: The authors declare no conflict of interest.

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