EffEcts of partial dEfoliation on thE growth, ion rElations and photosynthEsis of Lycium chinense Mill. undEr salt strEss

In this study, we investigated the effects of artificial defoliation on the growth and physiological response of Lycium chinense Mill. to salt stress. Our results show that partial defoliation increases the plant relative growth rate, leaf water content and dry weight-based leaf Na+ content, and reduces the fresh weight-based leaf Na+ content under salt stress. In response to defoliation, the leaf Na+/Ca2+ and Na+/Mg2+ ratios were decreased, but the K+ content remained unchanged. The contents of ROS and MDA were decreased in defoliated plants. Net The photosynthetic rate (PN), stomatal conductance (gs), electron transport rate (ETR), actual photochemical quenching (ΦPSII) and photochemical quenching (qp) were enhanced by defoliation. Together, these findings indicate that partial defoliation mitigates the salt-induced growth inhibition and physiological damage in L. chinense.


introduction
Salt stress is one of the major environmental stresses affecting plant growth and productivity (Flowers, et al., 1977).Salinity triggers physiological damage and growth inhibition via various factors, including ion toxicity, changes in water relation, impairment of mineral nutrition and inactivation of photosynthetic machinery (Bartels and Sunkar, 2005;Greenway and Munns, 1980;Radić, et al., 2013).Salt stress accelerates the degradations of chlorophyll, protein and RNA in leaves, along with an imbalance of ethylene and abscisic acid to induce leaf abscission (Parida and Das, 2005).
Leaf abscission is an essential morphological adaptation of plant in response to biotic or abiotic stresses.Some species such as Fouquieria splendens (ocotillo) in the deserts of North America produce and lose leaves as many as six times a year in response to water deficit (Lambers, et al., 2008).When the leaves of cottonwood species are subject to gall aphids, premature defoliation is induced to reduce aphid population, acting as an adaptive plant response to pest attack (Williams and Whitham, 1986).
Artificial defoliation has been extensively used in previous works as a surrogate to simulate biotic-and abiotic-induced leaf abscission (Conrad and Dhileepan, 2007;Eyles, et al., 2013;Griffith and Anderson, 2013;Watt, et al., 2007).Under salt stress, defoliation aggravates growth inhibition in some African grass species (Griffith and Anderson, 2013) and soybean species (Li, et al., 2006).Chen et al. (2002) reported that under salt stress, defoliation enhanced the growth of poplar P×euramericana cv.I-214, whereas there were no significant changes in P. 'popularis 35-44' .Some species possess compensation mechanisms which mitigate the negative effects of defoliation on growth under stress rather than increase physiological damage (Anderson, et al., 2013;Ferraro and Oesterheld, 2002;McNaughton, 1979).Partial artificial defoliation changes the relationship between carbon source and sink at whole plant level (Layne and Flore, 1995;Pinkard, et al., 2011), improves the water status of the remaining foliage per unit area (Lavigne, et al., 2001;Man, et al., 2013), and increases the ratio of root to shoot (Welker and Menke, 1990) and gas exchange in tree plants (Eyles, et al., 2011;Reich, et al., 1993;Turnbull, et al., 2007).It is presumed that partial defoliation may be beneficial for plant salt resistance.
Lycium chinense Mill is one of the most valuable Chinese traditional medicinal plants, which can grow under high saline soil (Wei, et al., 2006).In the coast area of Hebei province of China, the leaves of L. chinense shed during the leaf expansion stage when the soil salt level is high.However, the physiological and morphological mechanisms of L. chinense against high salinity are rarely reported.It was hypothesized that partial defoliation would alter the ecophysiological processes of plant under salinity stress, thereby enhancing plant salt-stress resistance.Here, the growth and physiological responses of L. chinense (cv.HaiQi) were investigated to evaluate the effect of partial defoliation on plant salt tolerance.

Experimental design
There were four treatments with six replications each: no salinity and no defoliation (Control), 300 mM NaCl with no defoliation (salt × 0% D), 300 mM NaCl with 50% defoliation (salt × 50% D), and 300 mM NaCl with 75% defoliation (salt × 75% D).Leaves were removed from the crown apex downwards based on the opposite leaf pattern of L. chinense.All plants were grown in half-strength Hoagland's nutrient solution.The treatments were conducted for 8 days.

Measurement of plant relative growth rate
Plant height was measured every day at 18:00 h.The first data were recorded at the onset of defoliation and salt treatment.The plant relative growth rate (RGR) was calculated as RGR=(lnH 2 -lnH 1 )/(t 2 -t 1 ), where H 1 and H 2 are the heights of the plants at times t 1 and t 2 , respectively (Chen et al., 2002).

Measurement of leaf water content
The leaf fresh weight (FW) was measured after the leaves were collected from the same plant, washed with distilled water and dried with filter paper.The leaf dry weight (DW) was measured after the leaves were dried at 70°C for 48 h.Leaf water content (LWC) was calculated as LWC=(FW-DW)/ FW×100% determination of na + , K + , ca 2+ and Mg 2+ contents Oven-dried leaf samples (0.200 g) were used for Na + , K + , Ca 2+ and Mg 2+ content determination by an atomic absorption spectrophotometer (Hitachi Z-8000).Analysis was performed according to Wei et al. (2006).

Measurement of photosynthesis and chlorophyll fluorescence
Photosynthesis and chlorophyll fluorescence were measured simultaneously using an open gas exchange system with an integrated chlorophyll fluorescence chamber (Portable Photosynthesis System LI-6400, LI-COR, Lincoln, NE, USA).The measurements were conducted under conditions where the photosynthetic active radiation was 600 μmol m -2 s -1 and the flow rate was 300 μmol s -1 .The minimal fluorescence (F 0 ), maximal fluorescence (F m ), maximal efficiency of PSII photochemistry (F v /F m ) and potential photosynthetic capacity (F v /F 0 ) were determined at the dark-adapted status, which was achieved by wholenight darkness adaptation.Light-adapted status was achieved by more than two hours of illumination with a sodium lamp after dark adaptation.Actual photochemical quenching efficiency (ΦPSII) of PSII was calculated as ΦPSII=(F m '−F s )/F m ' .Non-photochemical quenching (NPQ) was calculated from: NPQ=(F m / F m ')−1.The stomatal limitation (LS) was calculated as LS=1−C i /C a , where C i is the internal CO 2 concentration and C a is the atmospheric CO 2 concentration.

determination of malondialdehyde content
Leaf malondialdehyde (MDA) content was determined according to the method of Heath and Packer (1968) with modifications.Approximately 0.5 g (fresh weight) of leaf tissues were homogenized in 5 mL 10% trichloroacetic acid (TCA) (w/v).The homogenates were centrifuged at 3000 g for 10 min and 2 mL of the supernatant was mixed with 2 mL 0.5% 2-thiobarb-tiuric acid (TBA) (w/v).The mixture was incubated in boiling water at 100°C for 15 min (centrifugation was conducted if precipitate was present).Absorbance was measured using a spectrophotometer (UV-1750 spectrophotometer, Shimadzu Corporation, Japan) at 450 nm, 532 nm and 600 nm.MDA content was calculated using the formula: C (μmol/L) = 6.45×(A 532 − A 600 )−0.56×A 450 .

ros detection
Leaves from the top part of the plants were used for the detection of H 2 O 2 .Leaf H 2 O 2 was examined using a DCFH-diacetate (DA) fluorescent probe according to modified protocol (Rodríguez, et al., 2002).DCFH-DA solution was diluted to 1/1000 with double-distilled water (ddH 2 O), and light was blocked with an aluminum foil.The leaf hypodermis was peeled off, placed in PCR tubes and incubated for 10 min at 37°C in 500 μl diluted DCFH-DA.The leaf hypodermis was then rinsed with distilled water three times, followed by fluorescence observation using a fluorescence microscope with 490 nm excitation and 530 nm emission filters (Leica Digital MicroscopeDM5500B, Wetzlar, Germany).Clear and stained stomata were photographed.Data were expressed as fluorescent brightness calculated with Adobe Photoshop CS 5.

data analysis
Statistical analysis was performed using SPSS for Windows (ver.16.0).All data were subjected to oneway ANOVA at p<0.05.Figures were processed using Origin Pro 9.0.

rEsults plant growth and leaf water content
The plant relative growth rate (RGR) was significantly decreased by salinity compared with control plants (Fig. 1).However, defoliated plants had a higher RGR than non-defoliated plants under salt stress, although there was no distinct difference between 50% defoliation and 75% defoliation (Fig. 1A).The plants with 50% defoliation showed the highest LWC, followed by plants with 75% defoliation, non-defoliated plants under salinity stress and control plants.However, no significant differences were observed between plants with 50% and 75% defoliation (Fig. 1B).

ion content in leaves
Salinity significantly increased the Na + content and reduced the uptakes of K + , Ca 2+ and Mg 2+ in leaves.Compared with non-defoliated plants under salt stress, leaf Na + content was increased by 8.7% and 18.7% when calculated with dry weight, while it was decreased by 19.7% and 9.2% when calculated with fresh weight in 50% and 75% defoliated plants under salt stress, respectively (Table 1).Defoliation had little effect on leaf K + content, but increased the leaf Ca 2+ and Mg 2+ contents compared with no defoliation treatment under salt stress.As a result of defoliation, leaf Na + /K + ratio was increased, but both leaf Na + / Ca 2+ and Na + /Mg 2+ ratios were decreased under salt stress (Table 1).g s , E and LS between 50% defoliation and 75% defoliation treatments under salt stress (Fig. 3).

chlorophyll fluorescent parameters
There were no significant differences in minimal fluorescence (F 0 ) and maximal fluorescence (F m ) among the treatments (Fig. 4A and 4B).Salt stress decreased potential photosynthetic capacity (F v /F 0 ) and maximal efficiency of PSII photochemistry (F v /F m ), while there were no significant differences among defoliation treatments and non-defoliation treatment under salinity (Fig. 4C and 4D).Photochemical quenching (qp), electron transport rate (ETR) and actual photochemical quenching (ΦPSII) were notably decreased in response to salinity, but were increased significantly in 50% defoliation compared with no defoliation under salt stress (Fig. 4E, 4G and 4H).Salt stress significantly increased non-photochemical quenching (NPQ), but there was no significant difference between non-defoliation, 50% and 75% defoliation treatments under salt stress (Fig. 4F).

regression analysis of fresh weight na+ content and leaf water content
Regression analysis between the Na+ content based on the fresh weight (FW) and leaf water content (LWC) demonstrated that the decrease in FW based on the leaf Na+ content which triggered by defoliation was mainly the result of the increase in LWC (Fig. 2) (R2=0.950).

photosynthetic parameters
Leaf net photosynthetic rate (P N ), stomatal conductance (g s ) and transpiration rate (E) were significantly decreased by salt stress.However, defoliation increased P N and E and decreased stomatal limitation (LS), which were significant in 50% and 75% defoliated plants under salt stress (Fig. 3).Defoliation had the tendency to increase g s with no significant difference compared with non-defoliation treatment under salt stress.There were no significant difference in P N ,

leaf chlorophyll and carotenoid contents
Salinity remarkably decreased the contents of chla, chlb, carotenoid and total chlorophyll (Table 2).Under salt stress, the contents of chla, chlb, carotenoid and total chlorophyll were higher in defoliation treatments than those in no defoliation treatments, especially in 50% defoliation treatment, where they were higher than in other treatments (Table 2).

Mda and ros accumulation
Salt stress significantly increased the MDA and ROS contents in non-defoliated plants subjected to salinity compared with those in control plants (Fig. 5).However, 75% defoliation decreased the MDA and ROS contents under salt stress, but there was no significant difference between 50% defoliation and non-defoliation treatments.Compared with non-defoliated plants under salt stress, the MDA content was decreased by 30.3% and 18.2%, and ROS content by 25.0% and 27.2% in plants with 50% defoliation and 75% defoliation, respectively.There were no significant differences in MDA and ROS content between 50% defoliation and 75% defoliation treatments (Fig. 5B, Fig. 5C).

regression analysis of g s and ros accumulation
The regression analysis between stomatal conductance (g s ) and H 2 O 2 accumulation showed that the g s was well associated with the H 2 O 2 accumulation in leaf stomata (R 2 =0.781) (Fig. 6).

discussion
Upon exposure to NaCl, defoliated L. chinense had a reduced relative growth rate (RGR).However, the RGR was increased by partial defoliation compared with non-defoliated plants.This is consistent with the result of a previous study (Chen, et al., 2002).Under salt stress, the Na + content was increased in the remaining leaves after defoliation when calculated based on dry weight; but was decreased when calculated based on fresh weight.This was associated with the increase of leaf water content in response to defoliation.Succulence per se, i.e., high water content in leaves or stems, is an adaptive feature of many halophytes (Short and Colmer, 1999).The effect of sodium on increasing succulence is particularly relevant to salt tolerance for vascular plants (Jennings, 1968; Thomas and Bohnert, table 2. Chlorophyll and carotenoid content in leaves of plants with partial defoliation under salt stress.Means (n = 3 per treatment ± SE) followed by the same letters in the same column are not significantly different at p < 0.05.
Under salt stress, intracellular K + and Na + homeostasis is important for the activities of many cytosolic enzymes, and for maintaining membrane potential and an appropriate osmotic pressure for cell volume regulation.Salinity causes Na + to enter cells and accumulate to high levels, becoming toxic to plants (Hasegawa, et al., 2000).Ca 2+ plays an essential role in stress signal transduction (Mahajan, et al., 2008;Zhu, 2001).An elevated cellular Ca 2+ level confers plant salt adaptation (Batistič and Kudla, 2012;Knight, et al., 1997).Mg 2+ is the central atom of the chlorophyll molecule and is essential for the function of many cellular enzymes and for the aggregation of ribosomes.Mg 2+ modulates ionic currents across the chloroplast and the vacuolar membranes, and might thus regulate ion balance in the cell and stomatal opening (Shaul, 2002).Salinity was shown to reduce the accumulation of Mg 2+ in leaves (Essa, 2002).In this study, K + uptake is not affected by defoliation under salt stress.Nevertheless, a higher Na + /K + ratio is induced by defoliation, and may restrict the growth of plant (Greenway and Munns, 1980).However, significant declines in Na + /Ca 2+ and Na + /Mg 2+ ratios were observed in de- foliated plants as a result of increased accumulations of Ca 2+ and Mg 2+ , which may also be involved in the salt tolerance of L. chinense, because lower Na + /Ca 2+ (Grieve and Maas, 1988;Wu and Zou, 2009) and Na + /Mg 2+ ratios (Koyro, 2000) are beneficial to plant growth under salt stress.
ROS are generated during plant metabolism, photosynthesis, photorespiration (Foyer and Noctor, 2000), fatty acid oxidation and senescence (Vitória, et al., 2001), and play an essential role in the regulation of plant development (Apel and Hirt, 2004).However, excess ROS in plant cells leads to oxidative stress (Gill and Tuteja, 2010).ROS have the potential to interact non-specifically with many cellular components, triggering peroxidative reactions and causing significant damage to membranes and other essential macromolecules such as photosynthetic pigments, protein, nucleic acids and lipids (Foyer, et al., 1994;Lin and Kao, 2000;Shalata and Tal, 1998).The increase of ROS is shown to occur as a response to drought (Smirnoff, 1993), salt (Baxter, et al., 2014;Suzuki, et al., 2012), extreme temperatures (Penfield, 2008), nutrient deficiency (Iturbe-Ormaetxe, et al., 1995) and air pollution (Simkhovich, et al., 2008).Consequently, ROS could act as an important indicator in plant stress tolerance.Salinity has been shown to cause an overproduction of ROS, thereby leading to lipid peroxidation and subsequently the generation of MDA (Demiral and Türkan, 2005).Similar results were seen in the current study because non-defoliated plants had a higher H 2 O 2 and MDA content under salt stress than those grown in non-stress conditions.This may have resulted from the disequilibrium between H 2 O 2 production and H 2 O 2 removal induced by salt stress.
Previous studies showed that H 2 O 2 is synthesized in guard cells as a consequence of environmental stress (Allan and Fluhr, 1997;Lee, et al., 1999;Bright, et al., 2006), which in turn triggers stomatal closure (Chen and Gallie, 2004;McAinsh, et al., 1996).The results of our study supported this viewpoint, because g s was strongly associated with the change of H 2 O 2 accumulation in leaf stomata.We also found that both 50% and 75% defoliated plants had reduced content of H 2 O 2 in guard cells.Consequently, 50% and 75% defoliation plants had a higher g s and net photosynthetic rate than non-defoliation plants, thus appropriate foliage loss improved the gas exchange of L. chinense under salt stress.
The decrease in MDA content was observed in 75% defoliated plants, suggesting that partial defoliation alleviated the salt-induced membrane damage.Salt stress leads to the degradation of chlorophyll and carotenoid (Lutts, et al., 1996;Rao, et al., 2007;Sultana, et al., 1999).Similar results were seen in the current study.However, compared with non-defoliation treatments under salinity, 50% and 75% defoliation increased chla, carotenoid and total chlorophyll content, though it was not significant in 75% defoliation, which also provided evidence that partial defoliation alleviated the salt stress in leaves.
Plants resist photo inhibition by thermal dissipation of excessive excitation energy in the PSII antennae (non-photochemical quenching) and the ability of PSII to transfer electrons to various acceptors within the chloroplast (photochemical quenching) (Proctor and Smirnoff, 2011).In this study, partial defoliation had no effect on non-photochemical quenching (NPQ), but significantly increased photochemical quenching (qp) and actual photochemical quenching (ΦPSII).Electron transport rate (ETR) was markedly improved by 50% defoliation but immune to 75% defoliation.Therefore, 50% defoliation improved the transformation efficiency of energy in the photosystem reaction center.This indicated that the protection of photosynthetic machinery in L. chinense was dependent on the ability of PSII to transfer electrons to acceptors, rather than on thermal dissipation.
In conclusion, under salt stress, partial defoliation increased leaf water content, Na + /Ca 2+ and Na + / Mg 2+ ratios and net photosynthetic rate, whereas it decreased MDA and ROS content in leaves, which ultimately mitigated the salt stress-induced growth inhibition of L. chinense.

fig. 1 .
fig. 1.The effect of partial defoliation on plant relative growth rate (RGR) (A) and leaf water content (LWC) (B) under salt stress.Means (n=3 per treatment±SE) with at least one common letter are not significantly different at p<0.05.

fig. 3 .
fig. 3. Effect of partial defoliation under salt stress on photosynthetic parameters.(A) net photosynthetic rate (P N ); (B) transpiration rate (E); (C) stomatal conductance (g s ), and (D) stomatal limitation (LS) measured on fully expanded leaves located in the top part of the plant.Means (n=6 per treatment ± SE) with at least one same letter are not significantly different at p<0.05.

fig. 4 .
fig. 4. The effect of partial defoliation on chlorophyll fluorescence under salt stress.(A) minimal fluorescence (F 0 ), (B) maximal fluorescence (F m ), (C) potential photochemistry efficiency (F v /F 0 ), (D) maximal efficiency of PSII photochemistry (F v /F m ), (E) photochemical quenching (qp), (F) Non-photochemical quenching (NPQ), (G) Electron transport rate (ETR), and (H) efficiency of photosystem II (ΦPSII) measured for fully expanded leaves located in the top part of the plant.Means (n=6 per treatment±SE) with at least one same letter are not significantly different at p<0.05.

fig. 5 .
fig. 5.The effect of partial defoliation on malondialdehyde (MDA) (A) and reactive oxygen species (ROS) accumulation under salt stress (B, C).The four fluorescence images above represent the approximate average fluorescence brightness of each treatment (C).Magnification: 10×.Means (n=3 per treatment±SE) with at least one same letter are not significantly different at p<0.05.

acknowledgments:
This work was supported by the National Key Technologies R&D Program (2013BAC02B01).authors' contributions: Yuan Guo conducted experiments and wrote the paper.Xiaojing Liu designed the experiment, interpreted data and contributed to writing.Qiong Yu, Xiaohui Feng and Zhixia Xie contributed to data analysis and provided methodological assistance during experiments.conflict of interest disclosure: The authors declare that they have no conflict of interest.

table 1 .
Effect of partial defoliation on leaf ion contents under salt stress.Means (n = 3 per treatment ± SE) followed by the same letters in the same row are not significantly different at p < 0.05.
fig. 2.Regression analysis of the fresh weight (FW)-based Na + content and the leaf water content (LWC).The data of FW Na + content and LWC in control plant were excluded in this regression analysis p<0.05.