Cytotoxic and molecular impacts of allelopathic effects of leaf residues of Eucalyptus globulus on soybean (Glycine max)

Abstract

Eucalyptus trees litter plays a crucial role in structuring plant populations and regulating crop quality. To help characterize the allelopathic impact of Eucalyptus plantations and understand the interactions between tree litter and understorey plant populations, we performed two different genomic approaches to determine soybean (Glycine max) crop plant response to biotic stress induced by leaf residues of Eucalyptus globulus trees. For assessing cell death, a qualitative method of DNA fragmentation test (comet assay) was employed to detect cleavage of the genomic DNA into oligonucleosomal fragments and help to characterize the apoptotic event among the experimental samples. In addition, quantitative method of genome analysis at the transcriptional level also was conducted to investigate the expression responses of soybean genome to allelochemicals. Expression of specific genes, which are responsible for the breakdown of proteins during programmed cell death PCD (cysteine proteases and their inhibitors), was examined using semi-quantitative RT-PCR (sqPCR). Results of both conducted analyses proved significant genetic effects of Eucalyptus leaf residues on soybean crop genome, revealed by steady increase in DNA damage as well as variation in the transcript levels of cysteine proteases and inhibitors. Further detailed studies using more sensitive methods are necessary for a comprehensive understanding of the allelopathic effects of Eucalyptus plantations on crops.

Keywords: CC, cystatins; CPE, papain–like cysteine proteases; Comet assay; Cysteine proteases; EUGL, Eucalyptus ground leaves; Eucalyptus globulus; Glycine max; PCD, programmed cell death; VPE, vacuolar processing enzyme (legumain-like cysteine proteases); sqRT-PCR, semiquantitative reverse transcription polymerase chain reaction.

Fig. 1

Damage in nuclei isolated from leaves of Glycine max Exposed in the comet Assay to various Concentrations of EUGL 1: 0 (Control); 2: 10%; 3: 20%; 4: 30%, 5: 40%; 6: 50%. A: Photomicrographs of EtBr-stained DNA from protoplasts of Glycine max cultivated with EUGL. B: analysis of DNA damage using different parameters; TL: Tail length; T. DNA: % tailed DNA; TM: Tail moment, Mean values with the same letter are not significantly different at P < 0.05 level by one-way ANOVA.

Fig. 2

Expression pattern of 5 genes of papain–like cysteine proteases (CP1-5) 3 genes of legumain-like proteases (VEP1-3) and 4 genes of cystatins (CC1-4) in leaves of Glycine max in response to EUGL (1: control; 2: 10%; 3: 20%; 4: 30%; 5: 40% and 6: 50%). A: Transcripts accumulation; B: cysteine proteases genes and their inhibitors / tubulin expression ratio.