Polyamines alleviate the inhibitory effect of the DNA cross-linking agent mitomycin C on root growth

ABSTRACT

Polyamines (putrescine, spermidine and spermine) are ubiquitously present in various types of cells of living organisms. They are involved in a variety of cellular processes, including cell proliferation and cell differentiation, and are required for abiotic stress tolerances in plants. However, it is still not understood whether polyamines are involved in the plant growth inhibition caused by DNA-damaging agents. In this study, we examined the effects of polyamines on the inhibition of plant root growth and gene expression in Arabidopsis thaliana treated with mitomycin C (MMC), a genotoxic agent that induces DNA interstrand crosslinks. We found that polyamines alleviated the inhibitory effect caused by MMC on root growth. In addition, we also found that polyamines alleviated the increased expression of AtBRCA1 and AtRAD51 genes induced by MMC treatment. Our study provides the first evidence that polyamines contribute to tolerance against plant-growth inhibition caused by a DNA-damaging chemical.

KEYWORDS:

• Polyamine
• mitomycin C
• DNA damage
• root growth
• Arabidopsis thaliana

Introduction

Polyamines are small polycationic molecules widely distributed in almost all living organisms.¹ Three major polyamines are often extremely abundant in eukaryotes: putrescine, spermidine and spermine (Figure 1). Putrescine (a diamine) is converted to spermidine (a triamine) and then to spermine (a tetraamine) by sequential addition of aminopropyl groups donated by decarboxylated S-adenosylmethionine.² Numerous studies have shown that polyamines are involved in a variety of cellular processes, such as cell proliferation, cell differentiation, apoptosis and cellular senescence.^3,4 Furthermore, it is well known that polyamines in plants increase a plant’s tolerance to abiotic stresses, including salinity, drought and chilling stress.^5,6 In Arabidopsis thaliana (L.) Heynh., the lack of ACL5, a protein that catalyzes the biosynthesis of thermospermine (another tetraamine), strongly reduces stem elongation associated with an over-accumulation of lignin in vascular bundles.^7,8 Thus, polyamines play important roles in various physiological processes involved in both reducing stress tolerance and in morphogenetic processes in plants.

Figure 1. Chemical structures of three polyamines, the diamine putrescine, the triamine spermidine and the tetraamine spermine.

Polyamines mainly bind to negatively charged macromolecules in cells, such as nucleic acids, phospholipids and particular acidic proteins.^9,10 Polyamine/DNA interactions result in the duplex and triplex DNA stabilisations and induce DNA condensation.^11,12 It has also been shown that polyamine depletion in HeLa cells is responsible for inhibiting DNA repair following X-ray-induced DNA strand breaks and production of UV light-induced pyrimidine dimers, ^13,14 suggesting that polyamines are involved in DNA-repair processes in cancer cells.

Mitomycin C (MMC), a DNA-damaging agent, interacts with DNA to induce interstrand crosslink (ICL), a covalent linkage formed between two DNA strands that are highly cytotoxic. ICL can be induced by several classes of carcinogens, such as MMC and cisplatin, and can be induced naturally by endogenous agents, such as formaldehyde and acetaldehyde.^15,16 Williams et al. (1994) found that lung fibroblast V79 cells of Chinese hamsters in which polyamine is depleted by DFMO (an inhibitor of polyamine biosynthesis) are more sensitive to PUVA than control cells (without DFMO).¹⁷ PUVA is an effective therapy for skin disorders because it induces ICL formation when patients are treated with psoralen in combination with UV-A irradiation therapy.¹⁸ Therefore, polyamines likely provide a higher tolerance to cytotoxicity caused by ICL formation in cultured mammalian cells. However, it is still not understood how polyamines are involved in plant-growth inhibition caused by ICL-inducing agents. In this study, we focused on growth inhibition of primary roots caused by treating seedlings with MMC, examine the contribution of polyamines in counteracting the inhibition and investigate changes in gene expression in response to DNA damage.

Results and discussion

We first determined whether the growth of primary roots was affected by any of the polyamine treatments in the growth media. As shown in Figure 2(a), we detected no significant difference (P > .05) in primary root growths between roots in media containing 100 µM putrescine, spermidine or spermine and the control (without a polyamine). This suggests that these polyamines exert no discernible effect on the growth of primary roots, at least under the experimental conditions we imposed.

Figure 2. Extent to which polyamines alleviate the root-growth inhibition caused by MMC treatment. Panels: (a) the growth of primary roots after seedlings were transferred onto growth media with 100 µM of putrescine (Put), spermidine (Spd) or spermine (Spm) additions and the control condition (no polyamines), (b) the growth of primary roots after seedlings were transferred onto growth media with the different concentration of MMC (1, 3 and 5 µg/mL) and onto that without MMC, (c) the growth of primary roots after seedlings were transferred onto the media supplemented with 3 µg/mL MMC (MMC[+]) and polyamine hydrochloride (30 µM or 100 µM) media (Put, Spd or Spm) and a control medium without MMC [termed MMC[–]]. Error bars indicate standard deviation (n > 16). Significant differences between MMC[-] or polyamine-treated roots and their untreated roots were determined with the two-sample t-test at the P < .05 (*) level of significance. The n.s. represents no significant difference (P > .05).

Next, we tested the effect of different concentrations of MMC on the growth of primary roots. Our results showed that MMC inhibits the growth of primary roots depending on its concentration (Figure 2(b)). Under our experimental conditions, it was often observed that the roots of seedlings cultivated on the 5 µg/mL MMC medium were grown to be exposed to air without touching on the agar medium, so we chose the concentration of 3 µg/mL MMC for further experiments to evaluate accurately the effect of MMC treatment.

To reveal the effect of polyamines on the growth of primary roots subjected to treatment with MMC, we compared the lengths of primary roots of seedlings grown on the medium containing MMC (termed MMC[+]) with added polyamines (putrescine, spermidine or spermine) and grown on a medium without any polyamines but with MMC[+]. Figure 2(c) shows that the polyamine supplements slightly alleviated the inhibition of growth associated with the addition of MMC to the growth media. For example, root lengths of seedlings treated with MMC[+] and 100 µM putrescine were 13.6% longer than without putrescine. Similar results were revealed for the two other polyamines: root lengths were 11.5% (30 µM spermidine) and 12.4% (100 µM spermidine) higher than controls, whereas they were 18.2% (30 µM spermine) and 26.9% (100 µM spermine) higher than the controls. Thus, all three polyamines we examined alleviated the inhibitory effect (due to MMC) on primary roots of A. thaliana seedlings.

To investigate whether the polyamines are involved in transcriptional regulation of DNA damage response (DDR) genes in A. thaliana, we examined the change of transcript abundance of the genes AtBRCA1 (At4g21070) and AtRAD51 (At5g20850). The BRCA1 is a human tumor suppressor that plays a critical role in DNA repair and cell-cycle checkpoints, whereas the RAD51 protein is a multifunctional protein implicated in DNA replication and homologous recombination repair.^19,20 AtBRCA1 and AtRAD51 genes were isolated as A. thaliana orthologous genes of BRCA1 and RAD51.^21,22 It has been previously reported that transcript levels of AtBRCA1 and AtRAD51 are markedly increased in A. thaliana by treating it with zeocin, a double strand break (DSB)-inducing agent.^23,24

When we examined the MMC-dependent transcriptional changes of the AtBRCA1 and AtRAD51 genes with quantitative real-time PCR, AtBRCA1 and AtRAD51 gene expressions increased by approximately 8.8-fold and 7.2-fold, respectively, by 24 h after transferring seedlings to the MMC[+] medium (Figure 3(a,b)). Next, we assessed the polyamine-dependent transcriptional changes of seedlings grown in the medium without MMC (termed MMC[–]) medium. Our results showed that putrescine, spermidine and spermine treatments were associated with lower expression of the AtBRCA1 gene than control (by 81.7%, 55.9% and 66.2% compared to the control on average, respectively), whereas expression of AtRAD51 declined by 71.4%, 56.0% and 70.9% as well (respectively). However, we found no significant difference (P > .05) in these gene expression with the addition of polyamines in the MMC[–] medium as determined by two-sample t-test.

Figure 3. Polyamine-dependent changes in AtBRCA1 and AtRAD51 gene expressions. Panels: (a) Relative gene expression level of AtBRCA1, (b) Relative gene expression level of AtRAD51. Expressions of AtBRCA1 and AtRAD51 were normalized using the ACT8 gene as an internal control. The values of control (Ctrl; no polyamine) with MMC[–] were set to 1.0 and other values were calculated relative to those. Error bars indicate standard deviation of biological triplicates. Significant differences between polyamine-treated seedlings and the untreated control for each series were determined with the two-sample t- test at P < .05 (*) level of significance.

We similarly assessed polyamine-dependent changes on the expression of AtBRCA1 and AtRAD51 genes of seedlings grown in MMC[+] medium. We found that treatment of all of three polyamines suppressed the increased expression of AtBRCA1 and AtRAD51 caused by MMC (Figure 3(a,b)). AtBRCA1 gene expression in the MMC[+] medium with putrescine, spermidine and spermine declined by 47.0%, 39.0% and 43.9% compared to the control (without polyamines), respectively, whereas AtRAD51 gene expression declined by 50.8%, 44.8% and 48.5% as well. These lower gene expression levels (relative to controls) were statistically significant (two-sample t-test, P < .05).

We showed that polyamines alleviated the inhibitory effect caused by MMC on the primary-root growth. Although Davis et al. (2016) already showed that several types of genotoxic agents (including MMC) inhibit the growth of primary roots and reduce lateral root density, ²³ our study is the first to provide evidence that polyamines contribute to tolerance against plant growth inhibitions caused by a DNA-damaging chemical. In this study, we have no data to prove that the MMC treatments used in our experiments are enough to induce DNA cross-linking in the plant genome. However, it was shown that cross-linking was detected by a comet assay when the A. thaliana seedlings were treated with 3.3 µg/mL MMC after the application of an alkylating agent N-methyl-N-nitrosourea in a previous study.²⁵ Although our experimental procedures are not identical to the method in the precious study, there seems to be a possibility that the growth inhibition of primary roots induced by MMC treatment is due to a DNA cross-linking.

How are polyamines involved in the root-growth inhibition caused by MMC? It has been shown that polyamines interact genomic DNA to form nuclear aggregation.²⁶ Takata et al. (2013) reported that condensed chromatin of genomic DNA is better protected from DNA damages induced by DSB, heavy ions or genotoxic chemicals than when chromatin is not in a condensed condition.²⁷ One possibility is that polyamines may alleviate the inhibition of root growth (induced by MMC) by interacting with DNA to condense chromatin to prevent DNA from becoming damaged. Another possibility is that polyamines may play a role in facilitating DNA-repair activities, which in turn, consequently affects the expression of AtBRCA1 and AtRAD51 genes. A recent study showed that polyamines facilitate DSB repair by promoting homologous-recombination mediated by increased activity of the Rad51 protein.²⁸ Although MMC induces ICL, it also causes DNA strands to break by producing free radicals.²⁹ Further studies using DSB-inducing agents and reactive oxygen species in DNA-repair defective mutants are needed to determine whether molecular functions of polyamines on DNA damage is specific to a certain type of DNA damage or repair mechanism in plant cells.

Materials and methods

To evaluate changes in root growth following application of MMC and polyamines, the length of primary roots of wild-type seedlings of A. thaliana (Col-0 ecotype) were measured. First, seeds were sterilized with 70% ethanol for 5 min and then applied a sterilization solution [0.5% sodium hypochlorite, 0.05% Tween-20] for 10 min. After completing the sterilization process, the seeds were rinsed three times in sterile water and then placed on 0.8% agar medium [Murashige-Skoog (MS) medium with 2% sucrose (pH 5.8)]. Four-day-old seedlings were transferred to 1% agar MS medium with 2% sucrose (pH 5.8) containing various concentrations of MMC and polyamine salt (putrescine dihydrochloride, spermidine trihydrochloride or spermine tetrahydrochloride). Then the agar plates were set vertically in a growth chamber and subjected to continuous white light at 22°C. After the seedlings were grown for seven more days under these conditions, lengths of primary roots were measured.

For gene expression analysis, 2-week-old seedlings grown on the 0.8% MS agar plate were transferred to an MS liquid medium in a culture flask containing 100 µM polyamine-hydrochloride salt. The culture/seedling mixture was cultivated in the flask and by shaking it for 24 hours. Then, MMC was added to the culture flask to reach a final concentration of 3 µg/mL. After culturing the MMC/seedling mixture for 24 h, the seedlings were immediately frozen in liquid nitrogen and stored at −80 °C. Total RNAs were extracted from the frozen tissues using RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s protocol, and reverse transcribed the RNA with iScript^TM Reverse Transcription Supermix (Bio-Rad) to conduct a quantitative reverse transcription polymerase chain reaction (PCR). Quantitative real-time PCR was performed on the CFX96 Real-Time PCR Detection System (Bio-Rad). The iQSYBR Green Supermix (Bio-Rad) intercalation dye was used as a fluorescent reporter and the following primers for the PCR analysis were used: BRCA1-F (5ʹ-AACCA CTGCA CTCTC AGCCT A-3ʹ), BRCA1-R (5ʹ-AGCCC TGAGC AAGAT AAGAC C-3ʹ), RAD51-F (5ʹ-CGCAA GTAGA TGGTT CAGCT C-3ʹ), RAD51-R (5ʹ-TCCTC TGCTC TTCCT TTCCT C-3ʹ), ACT8-F (5ʹ-CTCGT CGTCT TCAGC TTCAT C-3ʹ) and ACT-R (5ʹ-CGATG AAGAT CTGGC TCACT C-3ʹ). Relative expression levels for each sample were calculated on three biological replicates from standard curves. The expression levels of AtBRCA1 and AtRAD51 genes were normalized relative to the transcript levels of the actin gene ACT8 (At1g49240).

Declaration of interest statement

The authors declare no potential conflicts of interest.

Acknowledgments

The authors would like to thank Enago (www.enago.jp) for the English language review.