Phragmites australis root secreted phytotoxin undergoes photo-degradation to execute severe phytotoxicity

Abstract

Our study organism, Phragmites australis (common reed), is a unique invader in that both native and introduced lineages are found coexisting in North America. This allows one to make direct assessments of physiological differences between these different subspecies and examine how this relates to invasiveness. Recent efforts to understand plant invasive behavior show that some invasive plants secrete a phytotoxin to ward-off encroachment by neighboring plants (allelopathy) and thus provide the invaders with a competitive edge in a given habitat. Here we show that a varying climatic factor like ultraviolet (UV) light leads to photo-degradation of secreted phytotoxin (gallic acid) in P. australis rhizosphere inducing higher mortality of susceptible seedlings. The photo-degraded product of gallic acid (hereafter GA), identified as mesoxalic acid (hereafter MOA), triggered a similar cell death cascade in susceptible seedlings as observed previously with GA. Further, we detected the biological concentrations of MOA in the natural stands of exotic and native P. australis. Our studies also show that the UV degradation of GA is facilitated at an alkaline pH, suggesting that the natural habitat of P. australis may facilitate the photo-degradation of GA. The study highlights the persistence of the photo-degraded phytotoxin in the P. australis's rhizosphere and its inhibitory effects against the native plants.

Figure 1

The collage shows the effect of UV irradiated GA on photo-oxidation of GA (A). GA-C refers to unirradiated GA; GA-UV refers to GA irradiated under UV lamp and GA-SL exhibits UV irradiated GA under sunlight. Browning in the UV lamp and sunlight treated GA shows possible photo-oxidation. (B) shows the 4-day-old A. thaliana roots treated with different forms of GA (100 µM) post 72 hrs of treatment and stained for root viability using FDA. Green fluorescence in (B) shows cell viability and loss of fluorescence shows cell death. (C) shows the ROS generation in A. thaliana roots post 10 minutes of GA treatment. ROS is represented by green fluorescence in (C), ROS staining was performed using ROS sensitive dye DCF. (B and C) n = 40 roots. (D) shows the biomass difference in A. thaliana plants treated with different forms of GA. (Values are mean ± S.D, n = 6).

Figure 2

Mesoxalic (MOA) contents in the rhizospheric samples of P. australis collected from different locations in Delaware. Roman numbers on x axis refers to different locations from where samples were collected for MOA analysis. Location I: 75° 44 1365W; 39° 40 7205N. Location II: 75° 9 7783W; 38° 47 2854N. Location III: 75° 9 7796W; 38° 47 2643N. Location IV: 75° 9 8204W; 38° 47 2859N. Location V: 75° 9 9748W; 38° 47 3693N. Location VI: 75° 98 8776W; 38° 46 796N. Location VII: 75° 21 628W; 38° 52 714N. Location VIII: 75° 32 0044W; 39° 11 8686N. Location IX: 75° 33 4075W; 39° 13 0043N. Location X: 75° 42 4394W; 39° 33 1825N. Location XI: 75° 46 2108W; 39° 36 7932N. Values are mean ± S.D., n = 18. Different letters on the bars are used to indicate means that differ significantly (F_(1,87) ≤ 221.5, p < 0.005).

Figure 3

Mesoxalic (MOA) contents in the rhizospheric samples of P. australis competing with Spartina patens. Roman numbers on x axis refers to different locations from where samples were collected for GA and MOA analysis. Location I: 75° 44 1445W; 39° 40 7205N. Location II: 75° 9 6683W; 38° 47 2854N. Location III: 75° 9 8816W; 38° 47 2643N. Values are mean ± S.D., n = 12. Different letters on the bars are used to indicate means that differ significantly (F_(1,33) ≤ 106.2, p < 0.005).

Figure 4

Effect of pHs 5.8 (A) and 8.0 (B) on UV degradation of GA to MOA. Known concentrations of GA were exposed to UV light for 3 hrs and incubated at room temperature. Aliquots for the HPLC analysis were drawn at different time points shown in x axis. Values are mean ± S.D., n = 6.

Figure 5

Effect of Mesoxalic (MOA) on the growth and development of Spartina patens. The x axis refers different concentrations of MOA applied to S. patens plants. Values are mean ± S.D., n = 20. Different letters on the bars are used to indicate means that differ significantly (F_(1,75) ≤ 212.4, p < 0.001).

Figure 6

Effect of UV degraded product MOA, on MT disruption in A. thaliana. Eight-day old A. thaliana transgenic line carrying a GFP fusion with a microtubule specific protein (CTD-PAPK1-GFP) was used for the assay. MOA was administered to the roots at different concentrations (10–50 µM). Roots were imaged for 40 min time course and a representative image at 40 min is shown in the panels. The images are confocal scanning laser micrographs and each image is representative of the roots of at least six independent plants analyzed and imaged. (Scale bars = 20 µm). See the “Materials and Methods” section for the details on microscopy and staining methodology. n = 20 roots.

Figure 7

Effect of UV degraded product of MOA on actin disruption in A. thaliana. Eight-day old A. thaliana seedlings expressing a GFP fusion to the second actin-binding domain (ABD) of Arabidopsis fimbrin 1 (ABD2-GFP) subjected for MOA (10 and 50 µM) treatment for 30 mins and imaged. The arrows in the panel represent the collapsed actin fibers post-MOA treatments. The images are confocal scanning laser micrographs and each image is representative of the roots of at least six independent plants analyzed and imaged. (Scale bars = 5 µm). See the “Materials and Methods” section for the details on microscopy and staining methodology. n = 40 roots.