Waterlogging tolerance evaluation of fifteen poplar clones cultivated in the Jianghan Plain of China

To provide references for poplar cultivation in waterlogged prone area of Jianghan Plain of China, the waterlogging tolerance of 15 poplar clones widely cultivated in these areas were evaluated based on their responses to 45-day waterlogging stress followed by 15-day drainage recovery in morphology, growth, biomass accumulation, leaf gas exchange and chlorophyll fluorescence parameters. The results showed that the normal watered seedlings (CK) of the 15 clones grew vigorously during the experiment, and no defoliation and death occurred. For the seedlings under waterlogging treatment (water 10 cm above the soil surface), its morphology changed markedly, including slowing growth, chlorosis and abscission of leaves, development of hypertrophied lenticels and adventitious roots etc. Waterlogging stress significantly inhibited the seedling growth of height and ground diameter, biomass accumulation, as well as leaf gas exchange and chlorophyll fluorescence parameters of the 15 clones with varying degrees. The net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), intercellular CO2 concentration/ environmental CO2 concentration (Ci/Ca), variable fluorescence (Fv), variable fluorescence/ initial fluorescence (Fv/Fo) and PS Ⅱ primary light energy conversion efficiency (Fv/Fm) decreased gradually with the prolonged waterlogging, and reached their bottom on day 45. During the terminal recovery stage, the leaf gas exchange and chlorophyll fluorescence parameters of the most clones increased, but their recovery abilities were significantly different. At the end of the experiment, the highest survival rates (100%) were observed in DHY, HS-1, HS-2, I-72, I-69, I-63 and NL-895, and the lowest (zero) occurred in XYY. Survival rates of the other clones ranged from 33.33% to 83.33%. Both results of cluster analysis and membership function analysis showed that HS-1, I-69, DHY, NL-895 and HS-2 had the strongest waterlogging tolerance, XYY and HBY were the worst, and the other clones were moderate. These results would provide guidance not only for the selection of cultivated varieties in Jianghan Plain, but also for the selection of hybrid parents for waterlogging resistance breeding.


Introduction Introduction Introduction Introduction
Jianghan Plain is located in the central and southern part of Hubei Province of China. It is an important part of the plain in the middle and lower reaches of the Yangtze River, and is one of the lowest plains in China with average altitude of around 27 m. Jianghan Plain has a huge area land prone to waterlogging. Due to the low-lying terrain and excessive seasonal rainfall, waterlogging has become one of the major natural disasters in these areas. Poplar (Populus L.) is one of the most important tree species for afforestation in these areas due to its characteristics of strong waterlogging resistance, rapid growth and broad applications. However, long-term waterlogging stress still adversely affects the growth and survival of poplar seriously (Peng et al., 2018). Under waterlogging stress, due to the lack of oxygen supply to roots, plants often present the phenomenon of leaf chlorosis and abscission, root rotting, photosynthetic capacity decline and so on, which leads to slow growth and even death, coupled with a huge loss of wood yield and ecological function (He et al., 2018;Zhao et al., 2019;Yan et al., 2019).
Previous studies have shown that different poplar clones always possessed significantly different waterlogging tolerance (Gong et al., 2007;Azizi et al., 2017;Sun et al., 2020;Rodriguez et al., 2020). For instance, P. deltoides 'Lux' is waterlogging-tolerant, while P. simonii is waterlogging-susceptible (Du et al., 2010;Liu et al., 2014;Chen et al., 2015). Male poplar plants tend to have stronger waterlogging tolerance than female ones owing to its lower reproductive costs and energy requirements (Correia and Barradas, 2000;Jiang et al., 2009;Yang et al., 2012). At present, there are more than 15 poplar clones cultivated in Jianghan Plain with different growth performance, which might be related to their different waterlogging tolerance. Therefore, it is of great significance to evaluate the waterlogging tolerance of these clones and select waterlogging-tolerant clones for afforestation and waterlogging resistance breeding in Jianghan Plain. In this study, waterlogging tolerance of 15 poplar clones widely planted in Jianghan Plain were evaluated by investigating their responses to waterlogging stress in morphology, growth, biomass accumulation, together with physiological and ecological characteristics, and waterlogging-tolerant clones were selected as well.

Plant materials and experiments
The experiment was performed in greenhouse of Huazhong Agricultural University located in Wuhan, Hubei Province (30°28' N, 114°21' E). The region belongs to Jianghan Plain, and has a warm, temperate climate, with an annual average of 240 frost-free days, 1,269 mm of rainfall, and a mean yearly temperature of 16.3°C. Most rainfall occurs between June and August, accounting for about 40% of the annual rainfall. In a year, the lowest average temperature occurs in January (around 3.0 °C) and the highest average temperature occurs in July (around 29.3 °C) (Du et al., 2008).
In mid-March, one-year-old shoots of the 15 clones were selected and cut into around 15 cm cuttings with three to four buds. The cuttings were planted in 15 cm× 20 cm× 15 cm pots (one cutting per pot) containing mixed soil (natural light loam: sand: peat = 6:1:1, pH= 6.2) after soaking in tap water for 24 h, and grown in greenhouse. The soil consisted of 2-5% N, P2O5 and K2O, with more than 20% organic matter (dry weight). After the plants survived, tap water and 1/2 Hoagland nutrient solution were irrigated once a week, 3 respectively. The plants with a mean height of 30-40 cm were randomly assigned to one of two treatments for a 60-day study: (1) watered (CK); (2) partial submergence (PS). In both treatment groups, there were 12 cuttings of each clone (four blocks, three cuttings arranged randomly per block). The pots of the watered treatment group had drainage holes in the bottom, and the plants were watered using tap water every day as needed to maintain soil moisture at around 75% of the maximum field water capacity. The plants of the waterlogging treatment group were flooded to a depth of 10 cm above the soil surface in an artificial pond with a depth of 70 cm. To reduce the influence of sunlight on water temperature and to replace the water lost due to evaporation and transpiration, water was continuously supplied to maintain water temperature and waterlogging depth. After 45 days, the waterlogged plants were removed from the partial submergence treatment and allowed to recover for 15 days to simulate natural environmental conditions.

Morphology and growth parameters
During the waterlogging period, morphological changes of the plants were observed everyday including leaf chlorosis and abscission, development of hypertrophied lenticels and adventitious roots etc. At the end of the experiment, the survival rate of each clone was recorded.
On day zero, 45 and 60, the seedling height and ground diameter of all clones were measured to calculate its growth parameters during the experiment. At the end of recovery stage (60 d), all of the plants were harvested. Root, stem, and leaf components were dried and weighed. In addition, the stem and its leaves were combined as a total shoot value, that is shoot weight = leaf weight + stem weight, and total biomass = shoot weight + root weight (Rodriguez et al., 2020).

Measurement of leaf gas exchange and chlorophyll fluorescence
On the 0, 15 th , 30 th , 45 th and 60 th day of waterlogging treatment, the 5 th fully expanded and mature leaf from the top of the stem of each clone was chosen to measure leaf gas exchange and chlorophyll fluorescence (Peng et al., 2018). For leaf gas exchange measurement, four plants per clone per treatment were measured between 9:00 am. and 11:30 am. using a LI-6400 photosynthesis system (LI-COR Inc., Lincoln, NE, USA) with a standard LI-COR gas exchange chamber (2 × 3 cm). A 1500 μmol·m -2 ·s -1 light intensity of illumination was provided by red diodes (6400-02 LED Source), and the gas flow rate was set as 500 μmol·s -1 . The gas exchange measurements included net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), intercellular CO2 concentration/ environmental CO2 concentration (Ci/Ca), and the corresponding ambient environmental conditions, such as leaf surface temperature, photosynthetically active radiation, relative atmospheric temperature and relative humidity. Chlorophyll fluorescence of leaves of four plants per treatment per clone was measured using a LI-6400 fluorescence system (LI-COR Inc., USA) after a 20-min dark adaptation under natural conditions, including variable fluorescence (Fv), PSII primary light energy conversion efficiency (Fv/Fm), variable fluorescence to initial fluorescence ratio (Fv/Fo) and so on.

Statistical analysis
The data were analyzed by analysis of variance (ANOVA) and multiple comparisons (Duncan's) with SAS statistical software package version 9.0 (SAS Institute Inc., USA). The waterlogging tolerance of each clone was evaluated by the waterlogging tolerance indexes and membership function values. The waterlogging tolerance index (%) = average value of waterlogging treatment/ average value of watered treatment × 100% (Rodriguez et al, 2020). The calculation formula of membership function value is U(xi)=(x-xmin)/(xmax-xmin) or U(xi)=1-(x-xmin)/(xmax-xmin) for the index which has a positive or negative correlation with waterlogging tolerance, respectively. U(xi) represents the membership function value of an index of clone i; x represents the mean value of an index of clone i; xmax and xmin are, respectively, the maximum and minimum values of an index in all clones (Yan et al., 2020). The cluster analysis of waterlogging tolerance of the 15 clones was carried out based on multivariate analysis (minimum distance method) of all data obtained during the study (Du et al., 2008).

Plant morphology
All watered plants (CK) grew vigorously, and no defoliation occurred. At the end of the experiment, the survival rates of all CK plants were 100%. In terms of waterlogged plants, obvious morphological changes were observed, including chlorosis and abscission of leaves, as well as development of hypertrophied lenticels and adventitious roots. Most clones firstly appeared hypertrophied lenticels on the 5 th -6 th day of waterlogging treatment, together with chlorosis leaves on the 10 th -13 th day, and defoliation on the 19 th -20 th day (Table 1). At the end of drainage recovery, DHY, HS-1, HS-2, I-72, I-69, I-63 and NL-895 displayed the highest survival rates (100%); XYY owned the lowest survival rate (0%), and that of the other clones ranged from 33.33% to 83.33%. Among the 15 clones, all plants of XYY and some plants of HBY and I-214 died during the waterlogging treatment, and XYY died earliest on day 32. Some plants of JBY, JNDY, LNY, I-45/51 and ZL-2025 died during drainage recovery.

Plant growth
Growth of height and ground diameter Height growth of the 15 clones was significantly inhibited by waterlogging treatment to varying degrees (p < 0.05; Figure 1). Among them, DHY, HS-1, HS-2 and NL-895 had better waterlogging tolerance indexes, which were 85.63%, 87.32%, 87.14% and 84.59%, individually; HBY and XYY showed the lowest waterlogging tolerance indexes, which were 42.06% and 37.56% respectively; while that of the other clones ranged from 60% to 80% (Supplementary Table 1). According to the waterlogging tolerance indexes, the slightest inhibition occurred in HS-1, and the most severe inhibition happened in XYY.
Waterlogging treatment significantly reduced ground diameter growth of the 15 clones with different degrees (p < 0.05; Figure 1). Among them, waterlogging tolerance indexes in XYY and HBY were lower than 60%, and the lowest value occurred in XYY (-9.63%); DHY, LNY, HS-1, HS-2 and I-69 had higher waterlogging tolerance indexes (>80%), and that of the other clones ranged from 60% to 80% (Supplementary  Table 1). These waterlogging tolerance indexes indicated that the ground diameter growth of XYY was most affected by waterlogging stress, while that of DHY, LNY, HS-1, HS-2 and I-69 were least affected. Note: Bars labeled with * and ** indicated significant difference between watered and waterlogged seedlings of the same clone at p＜0.05 and p＜0.01, respectively (t test; Mean ± SE, n=4; the same below)

Membership function analysis
According to the parameters used in cluster analysis of waterlogging tolerance, membership function values of the 15 poplar clones were calculated (Table 3). The results showed that HS-1 owned the highest average membership value (0.637), followed by NL-895 (0.620), and XYY had the lowest one (0.436). Identical to the results of cluster analysis, membership function analysis showed that waterlogging tolerance of HS-1, NL-895, I-69, HS-2 and DHY were the best, while XYY and HBY were the worst. Table 3  Table 3  Table 3  Table 3 Lots of previous literatures reported that waterlogging stress would adversely affect morphology, anatomy, nutrient metabolism, hormone balance and photosynthetic performance in many plant species (Loreti et al., 2016). Soil hypoxia is the main cause of waterlogging injury (Voesenek and Bailey-Serres, 2013).
Survival and growth of plants under waterlogging stress are closely related to the function of leaves and roots (Kreuzwieser and Rennenberg, 2014;Striker and Colmer, 2017). Under waterlogging stress, roots of plants were firstly damaged, followed by inhibited growth of aboveground parts, such as retarded leaf initiation and development, decreased growth of height and ground diameter, chlorosis and abscission of leaves, as well as reduction of biomass accumulation (Chen et al., 2010;Štícha et al., 2016). Waterlogging-tolerant plants always can relieve injury by changing their morphology and physiological metabolism (Bailey Serres and Voesenek, 2008;Bejaoui et al., 2012). It is generally believed that the characteristics of development of hypertrophied lenticels and adventitious roots, together with root aerenchyma formation are closely related to waterlogging tolerance of poplar (Kreuzwieser and Rennenberg, 2014;Peng et al., 2017). In the present study, the waterlogging-tolerant clones generally developed hypertrophied lenticels and adventitious roots earlier than waterlogging-susceptible clones. For instance, DHY, HS-1, HS-2, I-69, NL-895, I-72 and I-63 hypertrophied lenticels on day 5-6 of waterlogging treatment, and induced their adventitious roots on day 10-11. While, the corresponding time in XYY, HBY, LNY, I-45/51 and I-214 were day 6-7 and day 16-17, respectively. In hypertrophied lenticels and adventitious roots, abundant aerenchyma is helpful for plants to relieve injury by enhancing absorption of O2, water and nutrient elements from water, accompanied with release of CO2, C2H2, ethanol and other harmful substances (Pedersen et al., 2020). At the end of the experiment, all waterlogged plants of DHY, HS-1, HS-2, I-72, I-69, I-63 and NL-895 survived, and all waterlogged plants of XYY died, and survival rates of the other clones ranged from 33.33% to 83.33%. That means these seven clones could endure long-term waterlogging stress, and XYY was the most waterlogging-susceptible.
In addition to survival rate, growth and biomass accumulation are important indicators of plant waterlogging tolerance as well. Under waterlogging stress, waterlogging-tolerant plants can maintain superior growth and biomass accumulation to susceptible ones (Cao et al., 1999;Gong et al., 2007;Peng et al., 2017).
In our study, DHY, HS-1, HS-2, I-69 and NL-895 exhibited markedly higher waterlogging tolerance indexes compared to the other clones in growth and biomass accumulation, which indicated their superior vitality under waterlogging stress. The negative waterlogging tolerance index of ground diameter growth in XYY might due to the decay of stem base during waterlogging treatment, accompanied with the shrink during the recovery stage. A lower root/shoot ratio reflected the greater reduction in root than shoot growth, which mainly resulted from the decay of the original root system and the inhibition of new root growth (Liu and Dickmann, 1992;Zhou et al., 2019). In our study, waterlogging-tolerant clones generally owned higher root/shoot ratios than waterlogging-sensitive clones, in accordance with those reported in the literatures (Rodriguez et al., 2020).
Gas exchange reflects metabolism activities of plants (Chen et al., 2005). Stomatal closure is one of the earliest responses of plants to waterlogging stress, which can reduce the rate of water loss and relieve waterlogging injury (Blanke and Cooke, 2004;Rood et al., 2010). Waterlogging induced Gs decrease is usually associated with variation of hormone levels in plants, especially the increased concentration of abscisic acid (ABA) (Jackson et al., 2003). Accompanied with the decrease of Gs, values of Pn, Tr and Ci/ Ca changed correspondingly as well (Mielke et al., 2005). In the present study, the leaf gas exchange parameters of all clones decreased in varying levels with the prolonged waterlogging, which was not only related to the waterlogging induced damage of photosynthetic system, but also related to the reduced chlorophyll contents and premature leaf senescence (Sena and Kozlowski, 1980). Waterlogging-tolerant plants generally have higher leaf gas exchange parameters under waterlogging stress compared to waterlogging-sensitive plants (Du et al., 2010;Liu et al., 2011). In our study, the leaf gas exchange parameters of DHY, HS-1, HS-2, I-69 and NL-895 were remarkably less inhibited by waterlogging stress than those of XYY, HBY and the other clones. Therefore, they can survive after the long-term waterlogging stress, and maintain higher growth rate and biomass accumulation.

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Chlorophyll fluorescence is the remission of energy absorbed by photosynthetic pigments, which was widely used as a sensitive measure of stress induced damage to PSII (Kozlowski et al., 1984). Fv, Fv/Fm and Fv/Fo are three important parameters to reveal the photochemical reactions of plants (Bilger et al., 1995). Among them, Fv reflects the reduction of QA (primary quinone-type electron acceptor of PSⅡ); Fv/Fo reflects the potential activity of PS Ⅱ in leaves; Fv/Fm represents the maximum photochemical efficiency of PS Ⅱ in leaves. It has been reported previously that Fv, Fv/Fm and Fv/Fo decrease when plants are suffering from waterlogging stress (Smethurst et al., 2005). The greater reduction levels usually reflect more severe damage to PS Ⅱ (Du et al., 2010;Peng et al., 2017). In this study, Fv, Fv/Fm and Fv/Fo values in all of the 15 clones pronouncedly decreased with the prolonged waterlogging. While, DHY, HS-1, HS-2, I-69 and NL-895 exhibited less decreased values and better recovery abilities than the other clones. That means DHY, HS-1, HS-2, I-69 and NL-895 suffered slighter waterlogging injury among the 15 clones, which was in accordance with their leaf gas exchange parameters.

Conclusions Conclusions Conclusions Conclusions
The waterlogging tolerance of plants is a complicated quantitative trait, which involved in characteristics of morphology, anatomy and physiology. Therefore, multi-indicator evaluation is necessary to accurately reflect waterlogging tolerance of plants (Zhou et al., 2019). In this study, we evaluated waterlogging tolerance of the 15 clones by cluster analysis and membership function analysis, respectively, based on the parameters of their survival rate, growth of height and ground diameter, biomass accumulation, leaf gas exchange and chlorophyll fluorescence. Results of the both analysis methods showed that HS-1, I-69, DHY, NL-895 and HS-2 had the strongest waterlogging tolerance, XYY and HBY were the worst, and the other clones were moderate. Therefore, these waterlogging-tolerant clones should be selected preferentially for afforestation and breeding for waterlogging resistance in Jianghan Plain.