Effects of the interaction between shade and drought on physiological characteristics in Calamus viminalis seedlings

Recently, the endangerment of wild rattan population draws attention on the conservation and sustainable utilization of rattan resources. Rattan growing usually faces the light and water stress. Therefore, we aim to explore the combined effects of shade and drought on seedling growth, thus providing a theoretical ground for the conservation and artificial cultivation of the rattan. The combined effects of shade and drought on physiological and biochemical traits were studied in two-years-old Calamus viminalis seedlings. Photosynthetic indices including Pn, Gs, Tr, and Ci and physiological indices including MDA, SOD, POD, CAT, and Pro were measured under four levels of water treatments and four levels of shade. Shade, drought and their interaction have a significant effect on C. viminalis seedlings growth. Generally, moderate shade could alleviate the impact induced by drought. However, mild drought usually enhances the effect caused by shading. The result showed that the shade decreased Pn, Gs, and Tr but increased Ci, MDA content and Pro content. Either with the shading or drought increasing, the activity of SOD, POD, and CAT firstly increase and then declined. Drought reduced Pn, Gs, Tr, and Ci but increased the content of MDA and Pro. Overall, the result suggests that 25-50% shading and 65% RSWC water treatment are most beneficial for the growth of C. viminalis seedlings.


Introduction
Rattans are a group of climbing plants assigned to Family Palmae, Calameae, widely distributed in Indo-China and Chinese tropical and sub-tropical forests (Jiang et al., 2007;Wang, 2015b). Rattans, so-called "green 306 gold", are well known as an important non-timber product in southeastern Asia, most species of which are good material for handicraft and furniture. Moreover, some species' shoots and fruits can be made into food products. Notably, the rattan plantation and trade are of enormous economic value and developmental potential (Li et al., 2002;Jiang et al., 2007;Jiang, 2013;Wang, 2015b). However, the over-exploitation of rattan resources leads to its habitat loss and a dramatic drop of the natural rattan populations, thus largely impeding the development of rattan industry and trade (Jiang et al., 2007;Dong et al., 2015;Chen et al., 2017;Peng, 2017). The research to investigate physiological characteristics and the affecting factors of rattan seedling growth was called for better conservation of rattan resources and sustainable utilization.
Light and water supply are the most significant factors for the rattan seedling growing (Ingram and Bartels, 1996;Jiang et al., 2007;Liu et al., 2011). Many studies demonstrated that the seedlings of most rattan species are supposed to grow well under some extent of shade and different species are grown under a various degree of the irradiance (Mori, 1980;Yin et al., 1988;Dransfield, 1992;Sun et al., 2013). One-year-old Calamus manan seedlings grow well under 10%-15% shading (Mori, 1980). The most beneficial light condition for the growth of C. simplicifolius seedlings is 20%-50% relative light intensity while that of C. teradactylus is 50%-60% (Yin et al., 1988). Contrary to the former species, Daemonorops margaritae and C. rhabdocladus seedlings require very high relative light intensity about 80% (Yin et al., 1988(Yin et al., , 1993. The water deficiency or drought was the main factor resulting in a remarkable reduction of rattan population (Yin et al., 1993;Li et al., 2002). The growth of most rattan species needs sufficient water supply, such as C. caesius (Manocaran, 1981a(Manocaran, ,b, 1982a. Only a few species like Daemonorops jenkinsiana and C. rhabdocladus have relatively high drought tolerance and can survive in less humidity condition (Yin et al., 1993;Li, 2003). The combine effects of shade and drought on woody seedling have often been described and there is growing evidence that the shade and drought have a significant interactive effect on plant growth (Sack and Grubb, 2002;Aranda et al., 2005;Climent et al., 2006). However, the opinions on whether the drought stress has a stronger impact on seedling growth in deep shade or at higher irradiance are divergent (Holmgren, 2000;Sack and Grubb, 2002). One of the popular opinions is that moderate shade can improve plant performance under drought (Rousset and Lepart, 2000). Tree seedling survival under dry conditions was higher in shade than in high light thus providing support for this opinion (Amissah et al., 2015). In addition, the study on Arthraxon hispidus suggests that the interaction of some degree of shading and drought can facilitate the photosynthetic capacity, metabolic rate, and resistance potential . As for rattans, previous studies mostly concentrated on the effect of irradiance or water supply solely on the growth of rattan seedlings (Yin et al., 1988;Li et al., 2002Li et al., , 2003Sun et al., 2013;Dong et al., 2015), however, the interactive effect of the two factors on the growth of rattan seedlings remains unknown.
C. viminalis, widely distributed in southern Asia, is one of the excellent rattan species for the rattan plantation and industry. However, the research about its physiological characteristics and responses to shade and drought stress are still lacking. In the present study, C. viminalis seedlings were subjected to 2×4 factorial (shade, drought) experiment to test the interactive effects of shade and drought on seedling growth and their physiological resistance. We aim to provide a theoretical ground and support for the conservation and artificial cultivation of C. viminalis.

Materials and Methods
Plant growth conditions and experimental site Two-years-old C. viminalis seedlings were collected from Hainan Zhongteng Co., Ltd., Hainan province, China. Each seedling was separately transplanted to a pot (diameter: 21 cm; height: 19 cm) with the same amount of red clay soil obtained from Ganzhaling, Hainan province. The physiological conditions of the soil were measured as follows. PH: 5.6, water content: 17.26%, organic content: 11.4 g·kg -1 , available N content: 127.75 mg·kg -1 , available phosphorus content: 7.13 mg·kg -1 , available potassium contents: 80.05 mg·kg -1 . The seedlings were grown at the greenhouse of Yongmao ecological breeding base, which located at Jiyang, Sanya in Hainan province (109°35′41"E, 18°19′21"N) from 5 April to 21 June 2018.

Experimental design
After one-month establishment phase of the seedlings, the seedlings were subjected to 2×4 factorial (shade, drought) experiment for a total of 16 treatment combinations and each treatment had 15 pots (8 May 2018). The experiment consists of four water regimes: 85% relative soil water content (CK, W0), 65% RSWC (mild drought, W1), 45% RSWC (moderate drought, W2) and 25% RSWC (severer drought, W3) and four shade treatments: full sunlight (CK), 25% shading, 50% shading, and 75% shading. For the water treatment, relative soil water content was measured by the soil moisture tester (TDR300, USA) at 15:00-18:00 every day and watered as needed to maintain soil moisture during the experiment. For the shade treatment, the seedlings are covered by the shade-cloth. Physiological indices were measured after 45 days of treatment.

Physiological traits measurement
Net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO2 concentration (Ci), and stomatal conductance (Gs) were measured by a portable photosynthesis system Li-6400 (Lincoln, USA) at 9:00-11:00 on sunny days. Five random replicates and from each of replicates three functional leaves were selected for measurement.
The content of MDA and Pro were measured by thiobarbital acid colorimetry and acid ninhydrin method respectively. The activity of SOD, POD, and CAT were measured using NBT method, guaiacol method and ultraviolet absorption method respectively. Three replicates were imposed for each index measurement.

Statistical analyses
All data were subjected to an analysis of variance and Duncan comparisons at a threshold p=0.05 to test the differences between the treatments with an SPSS 20.0 (IBM, USA). All the graphs were created and processed by using Origin 8.5.

Effects of the shade and drought on photosynthetic indices of Calamus viminalis seedlings
With the shading degree increasing, the photosynthetic indices including the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and intercellular carbon dioxide concentration (Ci) all experienced an early raise and a subsequent decline (Table 1). For 25% shading, Tr and Ci significantly increased whereas Pn and Gs showed no statistically significant difference (p>0.05). For 50% shading, Pn decreased significantly by 30%, however, Tr and Ci showed a significant increase by 12% and 8% respectively (p<0.05). A significant decrease was found for Pn, Gs, and Tr at 75% shading (p<0.05), where the decrease was 70%, 45%, and 11% respectively. Pn and Gs reached a peak at 25% shading while Tr and Ci experienced the greatest increase at 50% shading. Conclusively, all the photosynthetic indices were found significantly declined at 75% shading.
Drought decreased Tr and Ci. And Pn and Gs increased under W1 but decreased under W2 (Table 1). Under mild drought (W1), Pn and Gs significantly increased by 27% and 47% respectively compared to control group (p<0.05) but Tr and Ci markedly decreased by 9% and 12% respectively. Under moderate drought (W2), Tr and Ci showed a significant decline by 34% and 15% respectively but Pn showed no significant decrease from the control group. When the seedlings submitted to the severe drought (W3), water deficiency induced a prominent decrease for Pn, Gs, Tr, and Ci by 67%, 64%, 65%, and 20% respectively.  The interactive effects of shade and drought were significant for the growth of C. viminalis seedlings. The moderate shade can alleviate the drought influences on seedlings. Twenty-five shading can reduce the decrease of Pn, Gs, Tr, and Ci induced by drought, however, 50% shading enhanced the reduction of all the photosynthetic indices. Meanwhile, mild drought (W1) significantly increased Pn at 25% shading (p<0.05), whereas significantly decreased Pn at 75% shading (p<0.05). Moderate drought (W2) significantly reduced Pn, Tr at 75% shading (p<0.05). A significant reduction of Pn, Tr, Ci, and Gs was observed by 93%, 51%, 55%, and 83% respectively under the severe water stress (W3) and lowest irradiance (75% shading) (p<0.05). In concluding, C. viminalis seedlings exhibited the greatest Pn (4.55 μmol·m -2 ·s -1 ) under 25% shading and mild drought (W1).
The double factor variance analysis suggests the shade and drought both have highly significant effects on photosynthetic indices (p<0.001) ( Table. 2). The examining value of F shows that the influence of shade has the ranking as follows Ci>Pn>Tr>Gs. The influence of drought has the same ranking as shade. A distinct interaction of shade × drought is also shown in Table 2 (p<0.001) with a ranking Tr>Ci>Pn>Gs.
Effects of shade and drought on the content of MDA in C. viminalis leaves Shade led to a reduction of the content of MDA in C. viminalis seedlings under 25% and 50% shade degree by 10% and 8% respectively while a significant increase under 75% shade degree by 17% (P<0.05) (Fig.  1). Drought stress increasingly raised the content of MDA in C. viminalis seedlings. Mild drought didn't induce a significant increase in the content of MDA (p>0.05). Moderate drought and severer drought led to a significant increase in the content of MDA in seedlings by 31% and 42% respectively in response to water deficiency (p<0.05). Table 2. Double factor variance analysis on photosynthetic indices. The photosynthetic rate, stomatal conductance, intercellular CO2 concentration and transpiration rate were measured in (Pn)/(μmol·m-2·s-1), (Gs)/(mmol·m-2·s-1), (Ci)/(μmol·mol-1), and (Tr)/(mmol·m-2·s-1) respectively  Moreover, a significant interactive effect of shade and drought was found on the content of MDA (p<0.05). Twenty-five shading can reduce the growing of MDA induced by drought. By contrast, more than 50% shading would assist in the promotion of MDA under drought stress in seedlings. On the other hand, mild drought stress promoted the increase of MDA significantly at 75% shading. The content of MDA increased significantly by 60% compared with the control group under the combined effect of severe drought and lowest irradiance.
The variance analysis indicates that shade and drought have a highly significant influence on the content of MDA in C. viminalis seedlings. The magnitude of influence ranks as follows Shade>Drought> Shade > Drought (Table 3).

Effects of shade and drought on the activity of SOD, POD, and CAT of C. viminalis leaves
With shading increasing, the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased first and then declined (Fig. 2). The activity of SOD increased significantly by 12% compared with the control group at 25% shading (p<0.05). For 50% shading, the activity of SOD and POD have observed a prominent increase by 39% and 25% respectively (p<0.05). However, the activity of SOD, POD, and CAT show no significant difference (p>0.05) at 75% shading. Mild drought (W1) and moderate drought (W2) increased the activity of SOD significantly by 26% and 23% respectively (p<0.05). On the contrary, the activity of SOD, POD, and CAT declined but not significantly under the severe drought (W3) (p>0.05). The interactive effect of shade and drought was significant for the activities of SOD, POD, and CAT (p<0.05). The greatest activity of SOD was observed when seedlings submitted to 50% shading and moderate drought, with an increase up to 66% of the control group. The activity of POD and CAT reached a peak at 50% shading and moderate drought, increasing by 134% and 97% respectively. The combined effect of deep shading (75% shading) and severer drought (W3) significantly declined the activity of SOD, POD, and CAT.
The variance analysis suggests that shade and drought have highly significant effects on the activity of SOD, POD, and CAT (p<0.01) ( Table 4). The effects of shading on antioxidase rank as follows SOD>CAT>POD and the effects of drought rank SOD>POD>CAT. The interaction of shade and drought was prominent for the activity of SOD and POD, however not significant for that of CAT, where the ranking of effects is SOD>POD>CAT. Table 4. Two ways variance analysis of antioxidase activity. The superoxide dismutase, peroxidase activity and gatalase activity were measured in (SOD)/(U·min -1 ·g -1 ), (POD)/ (U·min -1 ·g -1 ) and (CAT)/(U·min -1 ·g -1 ) respectively

Effects of shade and drought on the content of proline of C. viminalis leaves
The content of proline was observed no significant difference when submitted to 50% shading (p>0.05). However, 75% shading increased proline significantly by 46% (p<0.05). The drought stress significantly increased the content of proline, which increases up to 46%, 77%, and 108% respectively under mild drought (W1), moderate drought (W2) and severe drought (W3) stress.
Shade and drought have a significant interactive effect on the content of proline (p<0.05). The increase of proline was reduced at 25% shading, however, increased at 50% and 75% shading. Drought stress greatly further increased the growing of the proline induced by shading. Mild drought (W1) significantly increased the content of proline at 50% shading. The severe drought (W3) and the lowest irradiances induced a significant increase of proline by 161% compared with the control group (Fig. 3). The variance analysis showed that shade and drought both have a highly significant effect on proline (p<0.01) (Table 5), ranking as Drought > Shade >Shade × Drought.

Effects of shade and drought on photosynthesis
Shading impacts the photosynthesis by reducing the light intensity and duration and regulating the transpiration rate and stomatal closure (Tang et al., 2013). The present study shows that shading has a significant influence on the photosynthesis of C. viminalis seedlings. With the shading degree increasing, Pn, Gs, Tr, and Ci increased first and then declined. Pn and Gs reached a peak at 25% shading while Tr and Ci reached a peak at 50% shading (Table 1). Therefore, the growth of C. viminalis seedlings favors some extent of shading, especially among the range of 25%-50%. This result agrees with the general opinion that moderate shading could facilitate the growth of rattan seedlings but overshading is greatly harmful to its growth (Bøgh, 1996;Yin et al., 1998). However, different species have various shading tolerance. C. manan, C. simplicifolius, and C. teradactylus have similar shading tolerance with C. viminalis, approximately ranging from 20%-65% (Mori, 1980;Yin et al., 1988Yin et al., , 1993Sun et al., 2013). C. gracilis seedlings can grow well under up to 75% shading (Dong et al., 2015). By contrast, C. rhabdocladus seedling growth requires very high light intensity up to 80% full light (Yin et al., 1993).
Water stress leads to the reduction of photosynthetic material transportation, thus directly decreasing the efficiency of photosynthesis (Lawlor, 1995;Han et al., 2014;Yuan et al., 2016). In the present study, with an increasing degree of water stress, Pn and Gs increased first but then declined. The result is not confirmed to the opinion that mild drought decreases photosynthetic capacity (Yang et al., 2007;Dai et al., 2009). Contrarily the mild drought (W1) could promote Pn and Gs to the greatest value in the present study (Table 1) (Deng et al., 2016). And water stress started to decrease Pn and Gs from moderate drought (W2) but not significantly. A significant decline of Pn, Gs, Tr, and Ci was observed only when seedlings submitted to severe drought (W3) ( Table 1). Therefore, the result suggests that C. viminalis seedlings have relatively high drought tolerance.
A significant interactive effect of shading and drought on the growth of C. viminalis seedlings was observed in the present study. Mild drought (W1) could generally improve the photosynthetic capacity. On the other hand, 25% shading could alleviate the decrease of photosynthetic capacity induced by drought. But the interaction of overshading and severer drought could lead to marked reduction of the photosynthetic capacity. The same result was also observed in Arthraxon hispidus  Effects of shade and drought on the content of MDA The content of MDA is an indicator of membrane lipid peroxidation, reflecting the degree of cell membrane damage in response to the light or water stress (Chen, 1989;Yang et al., 2017). The MDA content would dramatically increase in the plant when the plant is at an adverse condition (Wei et al., 2014;Liu et al., 2018). In the paper, the MDA content at 25% shading was even lower than that of the control group (Fig. 1). The MDA content started to increase at 50% shading but still lower than that of the control group (Fig. 1). However, when the seedlings submitted to 75% shading, the MDA content remarkably increased to its maximum value (Fig. 1). Therefore, 25% shading could promote the performance of the seedling, but 50% shading is a threshold that would do harm to the growth of seedlings. On the other hand, the drought stress induces a significant increase in MDA content (Wang et al., 1990;. Moderate drought and severe drought lead to a prominent increase of MDA content, suggesting an enhancement of lipid peroxidation in cells (Sang et al., 2011;Qian et al., 2015;Jia et al., 2015Jia et al., , 2018. The similar result was found in the study of Rhodiola kirilowii seedlings (Yang et al., 2017). The interactive effect of shading and drought is significant on the growth of C. viminalis seedlings. Low irradiance like 25% shading could lower the MDA content, thus alleviating the damage caused by drought on the cell membrane. However, more than 50% shading would augment the MDA content induced by drought.
Effects of shade and drought on the activity of SOD, POD, and CAT The antioxidant enzyme system, including SOD (superoxide dismutase), POD (Peroxidase) and CAT (catalase), is an important and complicated protective mechanism of plants against the adverse condition (Wu et al., 2016). When plants are facing water or light stress, the activity of SOD, POD, and CAT are promoted to decrease the damage induced by the environment stress (Wu et al., 2016;Sun et al., 2018). The activity of SOD, POD, and CAT generally show an increasing trend at the early stage under stress. The activity of SOD, POD, and CAT started to increase significantly at 25% shading and decline at 75% shading but not significantly (Table 4). Different plant species differ in the responding order and magnitude of the antioxidant enzyme system under stress. In Arthraxon hispidus, the activity of POD significantly increased at 10% shading whereas that of C. viminalis significantly increased at 30% shading . Moreover, the activities of SOD, POD, and CAT in Arthraxon hispidus prominently decreased at 70% shading, however, those of C. viminalis declined at 75% shading but not significantly . Probably, the C. viminalis seedlings have a higher resistance than Arthraxon hispidus under low irradiance.
For the drought stress, the activity of SOD was promoted significantly when facing mild drought (W1) and the activity of POD increased significantly when seedlings submitted to moderate drought (W2) ( Table  4). Under severe drought, the activity of SOD, POD, and CAT all experienced a decline but the decreases are not significant (Table 4). Consequently, C. viminalis seedlings may have powerful antioxidant enzyme regulating ability to protect plants from damage induced by adverse conditions. However, when the interactive effect of deep shading and severer drought obviously exceeds the threshold of the regulation of antioxidant enzyme system, the activity of SOD, POD and CAT significantly decline (Jing et al., 2013;Li et al., 2014;Ren et al., 2017). This strong combined effect is also observed in Arthraxon hispidus .

Effects of shade and drought on the content of proline
The content of free proline, an important osmolyte, plays a vital role in regulating the osmotic pressure of the plant cell and alleviating the damage induced by adverse condition (Ren et al., 2000;Mahajan and Tuteja, 2005;Haffani et al., 2014). In this study, Pro had lower content than the control group at 25% and 50% shading but increased significantly by 46% at 75% shading. That suggests the damage induced by full light and deep shading triggers the great accumulation of Pro in the plant (Arora and Saradhi, 2002;Xue et al., 2011;Xie et al., 2013).
The drought has a greater impact on the content of Pro than shading. The water stress significantly increased the content of Pro at mild drought, moderate drought, and severe drought by 46%, 77%, and 108% respectively (compared with the control group). The accumulation of Pro can protect plant cell from peroxidative processes induced by water stress through osmotic regulation (Shao et al., 2006;Liu et al., 2007;Sang et al., 2011;Hou et al., 2014). This result is supported by many studies in terms of maize, sorghum, Arthraxon hispidus and Eucommia ulmoides (Shao et al., 2006;Liu et al., 2007;Sun et al., 2018). The interaction of shading and drought exert great influences on the content of Pro. Twenty-five percent shading could alleviate the impact induced by drought, but more than 50% shading enhances the accumulation of Pro caused by drought. This result is in full accordance with the recent study on Arthraxon hispidus . Conclusions Shade, drought and their interaction have a significant effect on C. viminalis seedlings growth. Generally, moderate shade could alleviate the impact induced by drought. However, mild drought usually enhances the effect caused by shading. The interactive effect of deep shading and severe drought leads to a significant decrease of the photosynthetic capacity, the decrease of the activity of the antioxidant enzyme and the accumulation of MDA and Pro. When facing the light and water stress, the seedling adjusts itself by regulating photosynthetic indices, enhancing the activity of antioxidant enzyme system, and increasing Pro content to reduce the damage and gradually establish a protective mechanism to adapt the adverse conditions. The result shows that C. viminalis seedlings have relatively high shading and drought resistance and 25%-50% shading and 65% relative soil water content is most beneficial for C. viminalis seedlings growth. As a result, C. viminalis seedlings are more favorably planted in well-drained soil and moderate shade condition than poorlydrained soil and full light condition. When rattans are grown in the natural high-density forests, proper weeding and thinning the crown of the trees would facilitate their growth.