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. 2021 May 6;16(6):1913309. doi: 10.1080/15592324.2021.1913309

Xanthine-derived metabolites enhance chlorophyll degradation in cotyledons and seedling growth during nitrogen deficient condition in Brassica rapa

So Young Yi a,†, Myungjin Lee a,†, Jana Jeevan Rameneni a, Lu Lu b, Chetan Kaur b, Yong Pyo Lim b,✉
PMCID: PMC8143221  PMID: 33955825

ABSTRACT

Nitrogen (N) deficiency is a main environmental factor that induces early senescence. Cotyledons provide an important N source during germination and early seedling development. In this study, we observed that N deficient condition enhanced gene expression involved in purine catabolism in cotyledons of Chinese cabbage (Brassica rapa ssp. Pekinensis). Seedlings grown with added allopurinol, an inhibitor of xanthine dehydrogenase, in the growth medium showed reduced chlorophyll degradation in cotyledons and lower fresh weight, compared with seedlings grown on normal medium. On the basis of these results, we speculated that xanthine-derived metabolites might affect both seedling growth and early senescence in cotyledons. To confirm this, seedlings were grown with exogenous xanthine to analyze the role of xanthine-derived metabolites under N deficient condition. Seedlings with xanthine as the sole N-source grew faster, and more cotyledon chlorophyll was broken down, compared with seedlings grown without xanthine. The expression levels of senescence- and purine metabolism-related genes in cotyledons were higher than those in seedlings grown without xanthine. These results indicate the possibility that xanthine plays a role as an activator in both purine catabolism and chlorophyll degradation in cotyledons under N deficient condition.

KEYWORDS: N-deficiency-induced leaf senescence, purine catabolism, xanthine dehydrogenase, allopurinol, Brassica rapa

1. Introduction

Premature leaf senescence can be induced by nitrogen (N) deficiency1. During this process, chlorophylls and major chloroplast proteins are degraded with an accompanying decline in photosynthetic capacity.2 Depending on the plant species, up to 80% of N is present in the chloroplasts of mesophyll cells.3 Chloroplast dismantling is therefore a major N source for nutrient recycling and remobilization. In Arabidopsis, SENESCENCE-ASSOCIATED GENE 12 (SAG12), BIFUNCTIONAL NUCLEASE1 (BNF1), and NON-YELLOWING1 (NYE1) are regulated by ORESARA1 (ORE1), a central regulator of senescence whose encoding gene also shows high transcript levels during leaf senescence.4 During senescence, SAG12 plays a role in the degradation of RuBisCO,5 BFN1 regulates nucleic acid breakdown,6 and NYE1 promotes chlorophyll degradation in senescing tissues.7

N is an essential mineral element that affects plant growth and development. N availability is often limited in natural ecosystems. Therefore, plants have efficient mechanisms to remobilize N, for example, from purine nucleobases. Purine catabolism leads to the complete disintegration of the purine ring and enables N recycling.8 The first irreversible rate-limiting reaction of purine oxidation, which is a key step in nucleotide degradation, is catalyzed by xanthine dehydrogenase (XDH; EC 1.1.1.204). Xanthine is the key starting compound in purine catabolism and is oxidized into urate by XDH8,9 Urate is then imported into the peroxisomes and oxidized by urate oxidase (UOX; EC 1.7.3.3) leading to the formation of allantoin and allantoate through a series of metabolic reactions. These end-products of purine catabolism are remobilized for the synthesis of organic molecules to sustain plant growth and development.8–10

The mechanisms of XDH involvement in aging and stress resistance regulation have been studied in Arabidopsis,11 pea,12 maize,13 and grape.14 Arabidopsis XDH-knockdown mutants accumulated xanthine and showed reduced growth and premature senescence caused by the absence of remobilized xanthine-derived catabolites.11,15,16 The XDH inhibitor allopurinol is converted to alloxanthine, which remains tightly attached to the substrate-binding pocket in the MoCo domain of XDH, thereby preventing further substrate turnover.17,18 In experiments on legumes, addition of allopurinol to the growth medium led to xanthine accumulation and the blocking of xanthine catabolism.19,20 Similarly, allopurinol treatment of Arabidopsis plants led to reduced growth and accelerated senescence.9,11 In contrast, rice plants overexpressing XDH showed delayed leaf senescence caused by the accumulation of xanthine-derived catabolites.21 Thus, xanthine’s efficient conversion to active catabolites is an important step in N remobilization that allows plants to grow normally.22 Specific transporters, including ureide permeases (UPSs), are required to remobilize purine-derived catabolites during aging in plants.23 In Arabidopsis, only AtUPS1, AtUPS2, and AtUPS5 have been functionally validated; AtUPS1 has a higher affinity for xanthine and uracil, AtUPS5 has a higher affinity for xanthine and allantoin, and AtUPS2 has a higher affinity for uracil and allantoin.24,25 In studies on legumes, RNAi-UPS1 mutants formed smaller and yellowish leaves caused by blockage of the transport of xanthine-derived catabolites.26 In contrast, overexpression of UPS1 in rice improved plant growth by enhancing the transport of xanthine-derived catabolites to younger tissues under N-deficient conditions.27

The availability of N is an important environmental parameter affecting seedling growth and development. Seed germination is a crucial stage in plant development, and seed nutrients are mobilized and catabolized during plant growth.28,29 Under N-deficient conditions, seed germination and seedling growth rely on seed reserves.30 One of the important functions of cotyledons in plants is to provide N to the developing tissues. Therefore, cotyledons are a suitable plant organ to study processes associated with N mobilization during senescence.28 In particular, seedlings are ideal for phenotypic screens as they can be grown under different conditions on agar plates. Little is known about the mechanism by which N deficiency induces early senescence in Chinese cabbage. In this study, we used cotyledons of cv. Kenshin as the experimental material to study the role of purine catabolites in N-recycling processes under N deficient condition.

2. Materials and methods

2.1. Plant material and growth conditions

Seeds of Chinese cabbage (Brassica rapa ssp. Pekinensis) cv. Kenshin were surface-sterilized in 25% NaOCl solution for 2 min, washed three times in sterile water, and sown on Murashige and Skoog (MS) agar. Kenshin seedlings were grown at 23°C with 60% relative humidity in long-day conditions (16 h light/8 h dark) under white light at 140 μmol m−2 s−1 in a growth chamber. Seedlings were grown on either MS agar (N-sufficient conditions) or N-free MS agar (modified MS salts, MB Cell, Seoul, Korea) for 20 d. To investigate the effects of added purine metabolites, seeds were germinated and grown on MS agar containing 1 mM xanthine (Sigma, St Louis, MO, USA) or 1 mM allopurinol (Sigma) for 20 d. The seedlings were sampled at indicated days after treatment, snap-frozen in liquid N, and stored at −80°C until analysis.

2.2. Measurement of leaf chlorophyll content

Chlorophyll content was measured as described by Park et al. [42]. For chlorophyll determination, cotyledons were sampled from 3-week-old seedlings in each treatment. Chlorophyll was extracted with 90% (v/v) ethanol, and the absorbance of the extracted solution was measured at A646.8 and A663.8 with a SmartSpec Plus spectrophotometer (Bio-Rad, Hercules, CA, USA). The total chlorophyll content was calculated using the following formula: (chlorophyll a + b = 17.67A646.8 + 7.12A663.8).

2.3. RNA extraction and gene expression analysis

Cotyledon samples were collected from three biological replicates, and total RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The extracted RNA was treated with RNase-free DNase I (Qiagen) to degrade DNA. Complementary DNA (cDNA) was synthesized using the ReverTra Ace-α kit with oligo (dT) primers (Toyobo, Osaka, Japan) and 1 µg total RNA. Quantitative reverse transcription PCR (qRT-PCR) was performed using TB Green Prefix Ex Taq premix (Takara Bio, Shiga, Japan) and analyzed using the CFX96 Real-Time PCR system (Bio-Rad). All qRT-PCR experiments were analyzed with three biological and three technical replicates. BrEF1α was used as the internal control for normalization of gene transcript levels. The primers used are listed in Supplementary Table S1.

2.4. Statistical analysis

All results are presented as means and standard deviation (SD). The data for transcript abundance, germination rate, fresh weight, and total chlorophyll contents were obtained from at least three independent biological experiments. Data were analyzed using ANOVA (IBM SPSS Statistics Ver. 26). Comparisons among three or more groups were made using one-way ANOVA followed by Tukey’s honestly significant difference (HSD) tests.

3. Results

3.1. N deficient condition enhances the expression of genes encoding enzymes involved in purine metabolism and senescence in cotyledons of B. rapa.

Purines are the most abundant N heterocyclic compounds in nature and are found in nucleic acids (DNA, RNA) and many other cellular components, including ATP, GTP, and NADH.10 To determine whether purine metabolism in cotyledons was involved in N-remobilization for the new growing parts of the seedling, we examined the expression of senescence- and purine metabolism-related genes under N deficient condition, which promotes major remobilization of cellular components. To this end, Kenshin seeds were grown on normal MS agar for 1 week then transferred to N-free MS agar for 2 weeks. The levels of BrXDH1 and BrUOX transcripts in cotyledons grown on N-free MS agar were found to be about twice those of normal MS agar-grown cotyledons (Figure 1). This N deficient-dependent increase of BrXDH1 and BrUOX transcript levels led us to speculate that N deficient conditions might activate purine catabolism in cotyledons. Furthermore, the expression of BrSAG12, a senescence-related marker, was also markedly enhanced during N deficient condition in cotyledons.

Figure 1.

Figure 1.

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Effect of N deficient condition on the expression of genes encoding enzymes involved in purine metabolism and senescence in cotyledons. Relative expression of BrSAG12, BrXDH1, and BrUOX in cotyledons of 3-week-old seedlings grown in normal Murashige and Skoog (MS) agar medium or N-free MS agar. Relative expression was determined by qRT-PCR and normalized to transcript levels of BrEF1α. Error bars represent standard deviation of three replicates. Similar results were obtained in at least two independent experiments. Different letters indicate significant differences among treatments (α = 0.05, one-way ANOVA and Tukey HSD test; SPSS software)

3.2. Active XDH in cotyledons plays a crucial role in seedling growth

During leaf senescence, XDH is involved in multiple processes, such as the metabolism of N,31 reactive oxygen species,32 and hormones.33 To analyze the role of XDH during early seeding growth, Kenshin seeds were germinated and cultured in normal MS agar containing xanthine or allopurinol for 3 weeks. Seedling growth was not affected by the addition of xanthine but was markedly inhibited by allopurinol: the fresh weight of seedlings grown with allopurinol was about 50% of seedlings grown in normal MS agar (Figure 2b). Furthermore, we found that allopurinol led to early senescence in cotyledons (Figures 2c and 2d). When we compared the expression level of BrORE1, the senescence marker gene, between cotyledons and true leaves, cotyledons showed markedly higher BrORE1 gene expression, compared with that in true leaves (Figure 2c). However, allopurinol treatment suppressed the enhancement of BrORE1 expression in the cotyledons: BrORE1 was expressed at similar levels in cotyledons and true leaves (Figure 2c). Consistent with the expression pattern of BrORE1, the chlorophyll content of allopurinol-treated cotyledons was about 14% higher than that of normal MS-grown cotyledons (Figure 2d). These suppression effects of allopurinol on chlorophyll degradation were only observed in cotyledons, not in true leaves. In other words, XDH inactivation by allopurinol inhibited seedling growth even under N-sufficient conditions, accompanied by suppression in chlorophyll degradation of cotyledons. These results strongly suggested that active XDH in cotyledons is important for early seedling growth.

Figure 2.

Figure 2.

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Effect of xanthine metabolites in seedlings grown under N-sufficient conditions. (a) Kenshin seedlings germinated and grown for 3 weeks on normal MS agar in the presence of xanthine (1 mM) or allopurinol (1 mM). Bar = 1 cm. Fresh weight (b) and total chlorophyll content (d). (c) Relative transcripts level of BrORE1 in cotyledons and true leaves. Transcript levels were analyzed by qRT-PCR and normalized to transcript levels of BrEF1α. Error bars represent standard deviations of three replicates. Similar results were obtained in at least two independent experiments. Different letters indicate significant differences among treatments (α = 0.05, one-way ANOVA and Tukey HSD test; SPSS software)

3.3. Xanthine-derived metabolites correlated with the growth of seedlings under N-deficient conditions

Since N is a significant component of the plant cell, N deficiency affects all aspects of plant function, from metabolism to resource allocation, growth, and development.34 We observed that the suppression effect of seedling growth and chlorophyll degradation by allopurinol treatment was pronounced in cotyledons but not in true leaves (Figure 2). To test whether activation of purine metabolism in cotyledons affected seedling growth, we analyzed seedlings grown under various N-deficient conditions. Kenshin seeds were grown for 1 week on normal MS agar, then seedlings were transferred to three different media and grown for an additional 2 weeks: 1. N-free MS agar; 2. N-free MS agar containing xanthine; 3. N-free MS agar containing allopurinol (Figure 3a). Mutation in AtXDH1 or application of allopurinol to wild-type Arabidopsis plants led to xanthine accumulation during dark stress.11 In our system, we expected allopurinol treatment to reduce production of xanthine-derived metabolites by inhibiting XDH enzyme activity during N-deficient stress. We also expected that xanthine added as the only N-source under N deficient condition would positively affect the normal growth of seedlings through XDH activation. When comparing seedlings grown for 3 weeks under three different N-deficient conditions, the fresh weights of seedlings grown with xanthine as sole N-source (condition 2) were about 38% greater than those of seedlings grown without xanthine (condition 1). In contrast, in the case of seedlings grown with the XDH inhibitor, allopurinol (Figure 3a, condition 3), the fresh weight was 32% lower than the control without allopurinol (Figure 3b). To further analyze the effect of xanthine metabolites on seedling growth, we determined the transcript levels of genes involved in purine catabolism and transport in the cotyledons of Kenshin grown with xanthine or allopurinol under N deficient condition. The transcript levels of BrXDH1, BrUOX, BrUPS1, and BrUPS5 were around two-fold higher in the cotyledons of seedlings grown on N-free MS agar with xanthine than in those grown on N-free MS agar without xanthine (Figure 3d). These results showed that exogenous xanthine not only enhanced the growth of seedlings but also activated the expression of purine catabolism-related genes in cotyledons (Figure 3b and d). Furthermore, the inhibition of XDH by allopurinol reduced both the transcript levels of purine catabolism-related genes (BrXDH, BrUOX, BrUPS1, and BrUPS5) and the growth of seedlings (Figure 3b and e).

Figure 3.

Figure 3.

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Effect of xanthine metabolites in seedlings grown under N-deficient conditions.(a) Kenshin seedlings germinated and grown for 3 weeks on N-free MS agar in the presence of xanthine (1 mM) or allopurinol (1 mM). Bar = 1 cm. Fresh weight of whole seedlings (b) and total chlorophyll content of cotyledon (c). Relative transcripts levels of the purine catabolism genes (BrXDH1, BrUOX, BrUPS1, and BrUPS5) and senescence-related genes (BrORE1, BrSAG12, BrBFN1, and BrNYE1) in cotyledons of seedlings supplemented with 1 mM xanthine as the only N source (d, f) or 1 mM allopurinol (e, g). Transcript levels of the marker genes were determined by qRT-PCR and normalized to transcript levels of BrEF1α. Error bars represent standard deviations of three replicates. Similar results were obtained in at least three independent experiments. Different letters indicate significant differences among treatments (α = 0.05, one-way ANOVA and Tukey HSD test; SPSS software)

As shown in Figure 3, under N deficient condition, exogenous xanthine promoted the growth of seedlings. However, it is difficult to distinguish whether xanthine-derived metabolites themselves were used as the source of N in the growing parts or whether they perhaps participated as an activator of purine catabolism and thereby increased N-remobilization. However, these observations showed that activation of purine catabolism at the transcript level under N-deficient conditions in cotyledons was correlated with seedling growth.

3.4. Xanthine-derived metabolites promote chlorophyll degradation in cotyledons under N deficient condition

We observed that the addition of xanthine to N-free MS agar decreased the content of chlorophyll in cotyledons. In contrast, the addition of the XDH inhibitor allopurinol increased the amount of chlorophyll (Figure 3c). Based on these results, we speculated that XDH activity affects chlorophyll breakdown in cotyledons under N deficient conditions (Figure 3a–c). To further explore chlorophyll degradation in cotyledons, we determined the transcript levels of senescence-associated genes (BrORE1, BrSAG12, BrBNF1, and BrNYE1) in cotyledons of seedlings grown under three N-deficient conditions (Figures 3f and 3g). Consistent with their senescence phenotype (Figure 3a and c), seedlings grown on N-free MS agar with xanthine showed higher transcript levels of all tested senescence-associated genes than seedlings grown on N-free MS agar without xanthine (Figure 3f). Furthermore, allopurinol treatment decreased N-deficiency-induced accumulation of senescence-associated gene transcripts, except for BrSAG12, compared with cotyledons of seedlings grown without allopurinol (Figure 3g). These results showed that xanthine or allopurinol treatment under N deficient condition affected both the expression of senescence-related marker genes and chlorophyll degradation in the cotyledons. Our results suggested that increased production of purine catabolites under N-deficient conditions was related to activation of N-recycling through chlorophyll degradation.

4. Discussion

In this study, we compared and evaluated the responses of Chinese cabbage cv. Kenshin seedlings to xanthine and allopurinol under N-deficient conditions and determined the transcript levels of genes related to purine metabolism and senescence. We identified a role of purine metabolites as a positive regulator of premature senescence of cotyledons under N deficient condition.

In N-sufficient conditions, the effect of allopurinol, an inhibitor of xanthine dehydrogenase (XDH), was a relative reduction in total chlorophyll degradation, which was confined to the cotyledons (Figure 2d). Furthermore, the expression of BrORE1 in cotyledons was five times higher than that in true leaves. However, when allopurinol was added, the expression of BrORE1 in cotyledons was reduced to a level similar to that in true leaves (Figure 2c). These results indicated that the reduction of purine catabolites following allopurinol treatment negatively affected chlorophyll degradation in the cotyledons. Furthermore, the inhibition of chlorophyll degradation in cotyledons was associated with seedling growth inhibition (Figure 2b). These results indicated that active purine catabolism in cotyledons is important for early seedling growth. Cotyledons, the first photosynthetic organs of a plant, are important for establishing seedling growth potential.35 In several species, removing cotyledons at early developmental stages results in reduced growth and later flowering.36–38

To observe the role of purine catabolites during the early seedling stage, Kenshin seedlings were grown with added xanthine under N-deficient conditions. Senescence symptoms and elevated expression of the senescence marker genes BrORE1, BrSAG12, BrBFN1, and BrNYE1 were noted in the cotyledons of seedlings grown with added xanthine under N-deficiency (Figure 3f). Under N-deficient conditions, the xanthine-treated cotyledons also showed significantly higher expression of BrXDH1, BrUOX, BrUPS1, and BrUPS5 transcripts than the cotyledons of seedlings grown without xanthine (Figure 3d). The up-regulation of purine catabolic pathway-related genes and associated transporters indicated their participation in the remobilization of organic N.39,40 In Arabidopsis mutants, a higher accumulation of xanthine was evident in the old leaves than the young leaves of Atxdh1. The young leaves of Ataln accumulated significantly higher allantoin.40 These results indicate that xanthine is primarily degraded in the old leaves, and the majority of the generated allantoin is remobilized to the young leaves.

Considering the clear senescence symptoms in cotyledons and the higher fresh weight of seedlings grown with xanthine under N-deficient conditions, we can speculate that the activation of purine catabolism enabled the seedlings to compensate for N shortage by increasing chloroplast protein degradation in the cotyledons (Figure 3).

The absence of remobilized purine-degraded N from older leaves of Arabidopsis mutant Atxdh1 caused senescence symptoms as a result of increased chloroplastic protein degradation in low-nitrate-grown plants.40 Three independent mutants impaired in purine catabolic pathway-related genes exhibited similar senescence symptoms in their older leaves in response to low nitrate supply.40 The mutant Atxdh1 did not accumulate the antioxidants allantoin and allantoate,11 whereas Ataln accumulated allantoin, and Ataah accumulated both allantoin and allantoate.41 Thus, these reports showed that, in low N conditions, xanthine metabolites play a greater role as an N-source in the growing part than as an antioxidant that reduces senescence symptoms. In our report, the role of xanthine metabolites as a positive regulator of early senescence was limited to the cotyledons of seedlings grown under N-deficient conditions. Our report shows evidence for a role of xanthine metabolites in promoting N-recycling for the growing parts by increasing chloroplast and purine degradation. However, further studies are needed on whether xanthine catabolites in cotyledons are directly used as an N-source for the growing part or whether they play a role in promoting early senescence by activating chlorophyll degradation and/or increasing the expression of senescence-associated genes.

Supplementary Material

Supplemental Material
Click here for additional data file. (18KB, docx)

Acknowledgments

This work was supported by the Korea Institute of Planning and Evaluation for Technology (IPET) in Food, Agriculture, and Forestry through the Agriculture, Food, and Rural Affairs Convergence Technologies Program for Educating Creative Global Leader, funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA) (710011-03-3-DH340). This research was also funded by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea (NRF-2016R1A2B4013170 and NRF-2018K1A3A7A03089858).

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website

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