Silencing of OsXDH reveals the role of purine metabolism in dark tolerance in rice seedlings

HAN Rui-cai, Adnan Rasheed, WANG Yu-peng, WU Zhi-feng, TANG Shuang-qin, PAN xiao-hua, SHI Qing-hua, WU Zi-ming

Key Laboratory of Crop Physiology, Ecology and Genetic Breeding, Ministry of Education/Collaborative Innovation Center for the Modernization Production of Double Cropping Rice/Key Laboratory of Crop Physiology, Ecology and Genetic Breeding of Jiangxi Province/College of Agronomy, Jiangxi Agricultural University, Nanchang 330045, P.R.China

Abstract Xanthine dehydrogenase (XDH) is a crucial enzyme involved in purine metabolism. To evaluate the effect of XDH deficiency on rice growth during dark treatment, wild type (WT) Nipponbare (Oryza sativa L.) and two independent transgenic lines with severe RNAi suppression (xdh3 and xdh4) were used in the present experiment. Under normal growth conditions, chlorophyll levels and biomass were indistinguishable between WT and the two RNAi transgenic lines, but XDH enzyme activity and ureide levels were suppressed in XDH RNAi transgenic lines. When XDH RNAi transgenic lines were subjected to dark treatment, chlorophyll content and biomass were significantly decreased, while· production rate and malonaldehyde(MDA) were significantly increased compared to WT. The spraying test of exogenous allantoin raised chlorophyll content and biomass and reduced · production rate and MDA in WT and both transgenic lines, and it also simultaneously reduced differences between RNAi and WT plants caused by XDH deficiency in growth potential and anti-oxidative capacity under dark treatment. These results suggested that fully functional purine metabolism plays an important role in reducing the sensitivity of rice seedlings to dark stress.

Keywords: xanthine dehydrogenase, rice seedlings, dark tolerance, allantoin, purine metabolism

1. Introduction

Rice (Oryza sativaL.) feeds 50% of the world’s population and is a very important crop in many regions (Leeet al.2007). Rice is a heliophyte and light intensity is one of the most significant environmental factors that determine the basic characteristics of rice development (Vazet al.2011). Weak light can lead to decreases in photosynthetic productivity, which seriously affects the growth and development of rice (Wilsonet al. 1992). Reduced light levels or an uneven distribution of light can be caused by covering materials, dust or shading in the greenhouses, or adverse weather conditions during the period of rice fertility(Yanget al.2011; Maet al. 2015). Low-light levels in ricegrowing reasons around the world can cause dark stress in rice and are often due to severe meteorological disasters(Liuet al. 2014). In order to reduce the effect of weak light on rice, it is very important to study molecular mechanisms of rice response to weak light and to use molecular breeding to grow rice cultivars with high physiological adaptability to wide range of light intensity.

Xanthine dehydrogenase (XDH) is a key enzyme in purine metabolism, catalyzing the conversion of xanthine and hypoxanthine to ureides (Andrea and Claus-Peter 2001). Biosynthesis of XDH is also influenced by the external environment, in addition to genetic factors. An experiment onArabidopsisAtXDH1found that salt stress did not significantly affect enzyme activity, cold stress reduced enzyme activity and drought stress increased enzyme activity (Hesberget al. 2004). XDH plays an important role in response to adverse environmentals and in regulating senescence (Montalbini 2000; Zdunek-zastocka and Lips 2003; Zrenneret al. 2006; Brychkovaet al. 2008; Youet al.2017). Variation in the expression level ofXDHleads to corresponding changes in the physiological and biochemical indexes and phenotypes of plants. Earlier studies showed that in transgenic plants created by genetic interference,purine metabolic pathways were blocked and the oxidationreduction environment was changed, leading to decreased tolerance of drought and dark stress and accelerating senescence of plant leaves (Zarepouret al. 2010; Hofmann 2016; Maet al. 2016). Other studies have found that downstream metabolites of purine metabolism such as ureides can improve the adaptive capacity ofArabidopsisto adverse conditions (Lescanoet al.2016). Therefore,this study was conducted to investigate the possible role of XDH in defense mechanisms in rice under dark stress.We evaluated the effect of defects inXDHtranscription on growth and physiology in dark tolerance in rice seedlings,with the goal of determining the physiological mechanism of how XDH alleviates dark stress in the rice seedling stage.

2. Materials and methods

2.1. Plant materials and treatments

We used Nipponbare (Oryza sativaL.) as wild type (WT)and two independent transgenic lines with severe RNAi suppression (xdh3andxdh4in the T3generation). Vector construction and transformation of transgenic rice were completed in the laboratory of Prof. Wan Jianmin, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences.

The experiment was carried out at the Key Laboratory of Crop Physiology, Ecology, and Genetics Breeding of Jiangxi Agricultural University, China. All seeds were soaked in 0.2% KMnO4(w/v) for 24 h, rinsed three times with sterile water and kept in sterile water until germination. Surfacesterilized seeds were sown on silt loamy soil in artificially illuminated incubators. All plants were grown in incubators under 12 h/12 h (day/night), 28°C/25°C (day/night), 22 000 lx/0 lx (day/night), and 75% relative humidity. Plants used in the experiment were approximately 15-day-old with three fully expanded leaves. For the dark treatment, the incubator parameters were set to a full day of darkness for 6 days and then adjusted back to normal light conditions for 6 days. To test the effect of metabolite supplementation, an allantoin solution (0.6%) was sprayed on leaves 6 days prior to the onset of dark treatment and was stopped after 6 days of dark stress treatment. Leaves on the top of the main stem were harvested before dark treatment and after a 6-day dark stress treatment. Same to the time of dry and fresh weight records and whole-plant fresh and dry weights (FW and DW,respectively) were scored before dark treatment and after a 6-day dark stress treatment. Three replicates were used for statistical evaluation of the results. All of the collected samples were frozen immediately with liquid nitrogen and stored at –80°C.

2.2. Analysis of OsXDH expression levels

Total RNA was isolated from seedling leaves using TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, China). About 0.5 μg total RNA was reverse-transcribed to the first-strand cDNAs using PrimeScriptTMRT Master Mix (TaKaRa, China).Real-time quantitative PCR (qRT-PCR) detection was performed on a CFX96 Real-Time PCR Detection System using SYBR?Premix ExTaqTMII (TaKaRa, China).Ubi2was used as the reference gene. During reverse transcription PCR (RT-PCR),OsXDHtranscripts were co-amplified withOsACTINmRNA as an internal control. All primers were designed using NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).

2.3. XDH enzyme activity analysis

Total soluble protein was isolated from leaves. Enzyme separation was carried out using polyacrylamide gels loaded with a comparable amount of soluble proteins. Enzyme activity was detectedin situafter native polyacrylamide gel electrophoresis (PAGE) using hypoxanthine as a substrate and nitroblue tetrazolium as the chromogenic agent (Sagiet al.1998). For a more intuitive understanding of the relative activity of XDH enzymes shown in the paper,quantitative analyses were made by scanning the formazan bands in the gel with a computing laser densitometer(Molecular Dynamic) using Image J2x software (Image J2x Software, USA).

2.4. Measurement of chlorophyll content

Chlorophyll content was determined spectrophotometrically after extraction of shoots with 80% (v/v) acetone, as described by Lichtenthaler and Wellbuen (1983).

2.5. Measurement of allantoin and allantoate

Allantoin and allantoate were extracted from leaves with 80% ethanol and their content was determined according to the method described in Sagiet al. (1998).

2.6. Measurement of · production rate and MDAcontent

2.7. Statistical analysis

Fig. 1 Generation of xanthine dehydrogenase (XDH) RNAi transgenic rice plants. A, schematic representation of the mRNA organization of OsXDH and the T-DNA of the hairpin-RNA expression vector LH-FAD2-1390RNAi-OsXDH. The target sequence(615 bp, between nucleotides 1 and 615 relative to the translation start site) is indicated by the box below the mRNA. The hairpin structure consisting of an antisense target sequence, the Arabidopsis FAD2 intron and the sense target sequence was inserted between the ubiquitin promoter and RB (right border). LB, left border. B and C, transcript levels of OsXDH in wild type (WT) rice plants and two independent transgenic lines (xdh3 and xdh4) of the T3 generation. Total RNA from 30-day-old plants was analyzed by qRT-PCR and RT-PCR. Values represent the mean±SE of three replicates. Significant differences (P＜0.05) are denoted by different lowercase letters. D, XDH enzyme activity levels in WT and T3 transgenic plants. Total soluble protein was extracted from leaves of 30-day-old plants. Each lane in the gel was loaded with 50 μg soluble protein. XDH activity was detected in the gel with hypoxanthine as a substrate.

Individual averages and standard errors of the mean were calculated using the data from independent samples in Excel 2010 (Microsoft, USA). Means of three biological replicates were measured. Analysis of variance (ANOVA)was used to test for significance, significant differences(P＜0.05) between treatments were determined using the multiple comparisons in IBM SPSS Statistics 22 (SPSS 22,SPSS Inc, USA).

3. Results

3.1. Growth phenotype and ureides content in XDH-suppressed plants under normal growth conditions

XDHRNAi transgenic lines (xdh3andxdh4) were generated by infiltration ofAgrobacterium tumefacienscarrying an intron-spliced hairpin RNA construct (LH-FAD2-1390RNAi-OsXDH, Fig. 1-A) that constitutively expresses an inverted repeat of 615 bp spanning exons 1, 2, 3, and 4 ofOsXDHand under the control of aubiquitinpromoter (Fig. 1-A). T0transgenic plants were obtained through tissue culture and genetic transformation. After self-crossing, the resulting T1and T2transgenic plants were screened for homozygosity by PCR. In our subsequent experiments, transgenic plants of the T3generations were used. The qRT-PCR and RT-PCR data indicate thatXDHRNAi transgenic lines had decreased expression ofOsXDH, relative to an internal control gene(Fig. 1-B and C). We also examined the XDH activity inXDHRNAi transgenic lines. Consistent with the results of qRT-PCR and RT-PCR, XDH activity was significantly lower in transgenic plants (Fig. 1-D). Hence, two independent transgenic lines with stable inheritance of XDH activity were established.

In order to determine the influence ofXDHexpression on rice seedlings, we first determined the growth indicators(chlorophyll content and biomass) and ureides content(allantoin and allantoate) produced in purine metabolism of WT andXDHRNAi transgenic lines (xdh3andxdh4) under normal growth conditions (Fig. 2). There were no significant differences in chlorophyll content or biomass between WT and interference lines (P＜0.05). Nevertheless, compared with WT, the content of ureides (allantoin and allantoate)decreased by nearly 50% in both interference lines.

3.2. XDH RNAi transgenic lines were sensitive to dark stress

Because there were no significant differences in the growth phenotype between WT andXDHRNAi transgenic lines under normal growth conditions, we tested WT and interference lines under 24 h of darkness for 6 days. Upon dark stress treatment, compared with WT, the leaves of interference lines were yellower, chlorophyll content decreased significantly (P＜0.05), and biomass was significantly lower (P＜0.05) (Fig. 3-A–D). We also measured enzyme activity and the product of purine metabolism before and after 6 days of dark stress. In-gel staining for XDH activity of transgenic lines revealed that all plants had increased XDH activity after dark stress treatment and that XDH activity was lower inXDHRNAi transgenic lines in comparison to WT (Fig. 3-E), which predictably resulted in a marked increase in ureides (allantoin and allantoate) levels,especially in WT (P＜0.05) (Fig. 3-F and G)

3.3. XDH RNAi transgenic lines had increased ·production rate and MDA accumulation

Stress-induced membrane damage is often mediated by reactive oxygen species (ROS) (Guo and Crawford 2005).Therefore we determined the production rate of· in WT andXDHRNAi transgenic lines (Fig. 4-A). In this study,there were no significant differences among lines before dark stress treatment. Upon dark stress treatment, the· production rate in both lines ofXDHRNAi transgenic lines seedlings were significantly increased compared to WT (P＜0.05).

MDA, as the end product of membrane lipid peroxidation,can partially reflect the degree of damage and cellular resistance to drought. There were no significant differences in MDA content among WT andXDHRNAi transgenic lines before dark treatment (Fig. 4-B). Upon dark stress treatment, seedlings from theXDHRNAi transgenic lines had significantly increased MDA content (P＜0.05), compared with the seedlings of WT (Fig. 4-B).

3.4. Exogenous allantoin alleviated hypersensitivity to dark stress in XDH RNAi transgenic lines

Fig. 2 Growth and ureide levels in xanthine dehydrogenase (XDH) RNAi transgenic rice at the young seedling stage. A and B,chlorophyll and fresh weight (FW) and dry weight (DW) of 30-day-old seedlings from wild type (WT) and two independent RNA interference lines, respectively. C, ureides (allantoin and allantoate) in WT and two XDH RNAi transgenic lines. ALN, allantoin;ALC, allantoate. Values represent the mean±SE of 3, 10, and 3 replicates in A, B, and C, respectively. Significant differences(P＜0.05) are denoted by different lowercase letters.

Fig. 3 Enhanced susceptibility of xanthine dehydrogenase (XDH) RNAi transgenic rice to dark stress. A, leaf phenotypes of wild type (WT) and XDH RNAi transgenic line seedlings after 6 days of dark stress. Chlorophyll content (B), fresh weight (C), and dry weight (D) were measured immediately before stress and after 6 days of dark stress. E, Western blot analysis showing XDH protein levels immediately before stress and after 6 days of dark stress. Each lane in the gel was loaded with equal content soluble protein. XDH activity was detected in the gel with hypoxanthine as a substrate and nitroblue tetrazolium as the chromogenic agent.Numbers above the lanes indicate relative intensity obtained by scanning the formazan bands with a computing laser densitometer using ImageJ2x software. Allantoin (F) and allantoate (G) were measured immediately before stress and after 6 days of dark stress. FW, fresh weight; DW, dry weight. Values represent the mean±SE of at least 3, 10, 10, 3, and 3 replicates in B, C, D, F,and G, respectively. Significant differences (P＜0.05) are denoted by different lowercase letters.

To determine whether purine metabolites actually contribute to the resistance of rice seedlings to dark stress, dark stress treatment was evaluated inXDHRNAi transgenic rice seedlings that had allantoin sprayed on leaves (Fig. 5).We sprayed the maximum possible solubility of allantoin solution (0.6%) on 15-day-old rice seedling leaves, and physiological indexes were not significantiy different compared with treatment without allantoin spaying in the previous experiment (Fig. 3-B–D). Similar results were found in reactive oxygen metabolism in rice seedlings(Fig. 4). Especially noteworthy were increases in biomass and in chlorophyll content, and differences in chlorophyll content, biomass,· production rate, and MDA content were reduced between WT andXDHRNAi transgenic lines upon dark stress treatment with allantoin spraying (Fig. 5).These results indicated that the application of allantoin can promote effective use of nitrogen, increase chlorophyll content and biomass, and reduce the levels of oxidation of leaf blades at the rice seedling stage. In addition, the results showed that the purine metabolite allantoin, and hence the functional metabolism of purines, is involved in the mechanism of dark tolerance of rice seedlings.

Fig. 4 Reduced resistance to oxidation of xanthine dehydrogenase (XDH) RNAi transgenic rice to dark stress. · production rate (A) and malondialdehyde (MDA, B) content in XDH RNAi transgenic rice were measured using the hydroxylamine method before stress and after 6 days of dark stress. FW, fresh weight. Values represent the mean±SE of three replicates. Significant differences (P＜0.05) are denoted by different lowercase letters.

Fig. 5 Restoration of xanthine dehydrogenase (XDH) rice dark-hypersensitive growth by spraying exogenous allantoin. Chlorophyll (A), fresh weight (B), dry weight (C),· production rate (D), and malondialdehyde (MDA) content (E) were measured immediately before stress and after 6 days of dark stress with foliar exogenous allantoin application. FW, fresh weight;DW, dry weight. Values represent the mean±SE of three, 10, 10, three, and three replicates in A, B, C, D, and E. Significant differences (P＜0.05) are denoted by different lowercase letters.

4. Discussion

4.1. The absence of purine metabolism can reduce the tolerance of rice seeding to dark stress

The purpose of this paper was to explore the physiological mechanism of xanthine dehydrogenase in mitigating negative effects of dark stress at the rice seedling stage. We have proven that the interference ofOsXDHseverely impaired stress tolerance in rice seedlings, including significant reductions in chlorophyll content and biomass following dark stress treatment (Fig. 2). The results were consistent with that ofArabidopsismutantAtxdh1in response to dark stress(Watanabeet al. 2010). Studies have shown that negative effects on growth or plant phenotypes are the result ofXDHRNAi transgenic lines causing changes in purine metabolic activity and resulting in a change in purine metabolism,not the accumulation of purines themselves (Nakagawaet al.2007). The contents of allantoin and allantoate were significantly lower than those in the WT (Fig. 3-F and G).Exogenous allantoin can relieve the damage caused by dark stress, and differences in chlorophyll content and biomass are decreased betweenXDHRNAi transgenic lines and WT(Fig. 5-A–C). Our results support the idea that the absence of purine metabolites enhances sensitivity to dark stress.

4.2. Allantoin is involved in regulating active oxygen metabolism

According to the biological radical injury theory (Mchdy 1994), damage to plants is caused by the imbalance between producing and eliminating free radicals in cells under stress, excessive reactive oxygen species (ROS)can lead to membrane lipid peroxidation, which causes the accumulation of MDA, and affects the normal growth of plants (Gill and Tuteja 2010). In the present study, the production rate of· was increased in WT andXDHRNAi transgenic upon dark stress treatment, and the levels of membrane lipid peroxidation and the production rate of· inXDHRNAi transgenic lines were higher than those in WT (Fig. 4). According to previous studies, ureides are effective scavengers of ROS (Pastori and del Rio 1997; Yobiet al. 2013), and increases in the content of allantoin and allantoate can compensate for ROS damage and reduce the death of seedlings (Brychkovaet al.2008). The production rate of· and the degree of membrane lipid peroxidation decreased after leaves were sprayed with exogenous allantoin, and the differences in physiological indexes were reduced between wild type andXDHRNAi transgenic lines(Fig. 5-D and E). The same experimental results were obtained in the study of the critical role for ureides in dark and senescence-induced purine remobilization in theAtxdh1 Arabidopsismutant (Brychkovaet al. 2008).

Although there were significant differences in XDH activity and downstream metabolites between WT andXDHRNAi transgenic lines, there were no significant differences in physiological indicators such as chlorophyll content and biomass (Fig. 2). In contrast, significant differences in these physiological indicators can arise when rice seedlings are grown in a dark environment (Fig. 3-A–D). Plants have a reactive oxygen scavenging system that reduces and eliminates ROS and free radicals, protecting plants from membrane lipid damage and maintaining normal cellular metabolic activity (Wanget al.2007). In the event of severe stress, the free radical content in the plant may increase,and the activity of the reactive oxygen elimination system may be decreased, causing an imbalance in “generationelimination” of free radicals, resulting in damage to cells and tissues (Yang and Gao 2001; Hung and Kao 2003).It is interesting to see that rice seedlings can alleviate the damage caused by dark stress by improving their XDH enzyme activity and the content of downstream metabolites(Fig. 3-E–G). According to our results, we can speculate that, by regulating the activity of XDH, rice can improve the level of purine metabolism, compensate for antioxidant capacity in rice seedlings, and enhance the ability of seedlings to tolerate dark stress.

This study indicated that the use of exogenous allantoin did not completely eliminate the difference between wild type andXDHRNAi transgenic lines under dark stress (Fig. 5).The physiological mechanism for tolerating dark stress was only analyzed from the angle of antioxidation and purine metabolism. At this moment, we have no definitive explanation for the importance of purine metabolism in dark tolerance of rice seedlings. XDH is also involved in nitrogen metabolism and hormone metabolism to deal with adverse environmental factors (Aguey-Zinsouet al.2003; Taylor and Cowan 2004; Sun and Hu 2005). Purine metabolism is an integral part of nitrogen metabolism, and control of purine metabolism can relieve the intrusion of stress factors (Werner and Witte 2011). XDH can relieve the damage caused by dark stress by regulating nitrogen metabolism and reactive oxygen metabolism inArabidopsis(Brychkovaet al. 2008; Watanabeet al. 2010). Moreover,XDH is related to the metabolism of plant hormones such as cytokinin (CTK), abscisic acid (ABA), and indol-3-ylacetic acid (IAA), and participates in the regulation of adverse environmental conditions (Leydeckeret al.1995;Bittneret al. 2001; Sagiet al. 2002; Smith and Atkins 2004;Watanabeet al. 2014). To solve the above problems, other possible mechanisms of plant protection from adverse environmental factors mediated by purine metabolism need to be identified, and we need to evaluate the quantitative contribution of purine metabolites to stress protection in future studies.

5. Conclusion

The study revealed the adverse effects ofOsXDHdownregulation on rice seedlings under dark stress and the important role of purine metabolite allantoin in coping with dark stress. The results showed that there was no significant difference in the growth phenotype betweenXDHRNAi trangenic lines and wild type under normal growth condition.After dark stress, the absence of purine metabolites enhanced sensitivity to dark stress and reactive oxygen species level. The application of exogenous allantoin improved the tolerance and antioxidant capacity of rice seedlings to dark stress. Therefore, it indicated that rice can regulate the activity of XDH enzyme and increase the accumulation of purine metabolites, and compensate for antioxidant capacity and enhance the ability of seedlings to tolerate dark stress.

Acknowledgements

The research was supported by the National Natural Science Foundation of China (31560350 and 31760350)and the Science and Technology Program of Jiangxi, China(20171ACF60018).