Root adaptive responses of tall fescue (Festuca arundinacea) growing in sand treated with petroleum hydrocarbon contamination

Phytoremediation is a green technique used to restore polluted sites through plant-initiated biochemical processes. Its effectiveness, however, depends on the successful establishment of plants in the contaminated soil. Soils that are contaminated with polycyclic aromatic hydrocarbons (PAHs), especially low molecular weight, mobile PAHs such as naphthalene pose a significant challenge to this. Plant roots growing in these soils exhibit changes to their structure, physiology and growth patterns. Tall fescue (Festuca arundinacea) roots grown in sand contaminated with either petroleum crude oil (10.8g total extractable hydrocarbons kg-1 sand dw) or naphthalene (0.8g kg-1 sand dw) exhibited a temporary inhibition in elongation with accelerated lateral growth (p<0.01), whilst also showing a deviation from the normal root orientation responses to gravity. Scanning electron micrographs (SEM) revealed that the stele in the contaminated roots was located much further away from the root epidermis, because the cortex was larger (p<0.001) due to the cells being more isodiametric in shape. Once past the initial acclimatisation period of 2.5-3.0 months, no visual differences were observed between control and treated plants, but the root ultrastructural modifications persisted. The fluorescent hydrophobic probe „Nile red‟ was applied to the epidermis of a living root to mimic and visualise the uptake of naphthalene into the root through the transpiration stream. The root sections were also stained with 0.1% (w/v) berberine hemisulphate in order to stain Casparian bands. Overlaying images obtained with the use of Texas red HYQ filter (wavelength 589-615nm) and UV illumination (wavelength 345-458nm) revealed the presence of passage cells in the endodermis and uptake of Nile red into protoxylem vessels beyond the endodermis of control roots. On the other hand, the path of Nile red was blocked at the endodermis of naphthalene- treated roots. The cell walls in the endodermis of naphthalene-treated roots were prominently thickened (p<0.001) and lacked passage cells. The treated roots also possessed a well-formed exodermis (p<0.01). The results suggest that the well-formed endodermis lacking passage cells, the well-formed exodermis as well as the increased cortex zone provided an effective barrier to the flux of hydrophobic xenobiotics towards the inner core of the roots, if previously exposed to the contaminants. The SEM images of naphthalene-treated as well as crude oil-treated roots showed partial collapse in the cortex zone, presumably due to water stress, but the treated plants withstood drought stress better than the control plants. The underlying physiological changes responsible for the adaptive responses of tall fescue to the exposure to naphthalene contamination were studied through metabolic profiling of plant roots and shoots. The results indicated synergistic interactions between sugars or sugar- like compounds and phenolic compounds may assist to create an integrated redox system and contribute to stress tolerance in naphthalene-treated tall fescue. The signal for a compound speculated to be indole acetic acid (IAA) was either subdued or absent in the tissues of naphthalene-treated tall fescue, suggesting the existence of a detoxification mechanism/ defence pathway in the treated plants. The ultra-structural and molecular modifications, resulting from PAH stress enabled tall fescue to resist tougher challenges

Scanning electron microscopic investigations of root structural modifications arising from growth in crude oil-contaminated sand

The choice of plant for phytoremediation success requires knowledge of how plants respond to contaminant exposure, especially their roots which are instrumental in supporting rhizosphere activity. In this study, we investigated the responses of plants with different architectures represented by beetroot (Beta vulgaris), a eudicot with a central taproot and many narrower lateral roots, and tall fescue (Festuca arundinacea), a monocot possessing a mass of threadlike fibrous roots to grow in crude oil-treated sand. In this paper, scanning electron microscopy was used to investigate modifications to plant root structure caused by growth in crude oil-contaminated sand. Root structural disorders were evident and included enhanced thickening in the endodermis, increased width of the root cortical zone and smaller diameter of xylem vessels. Inhibition in the rate of root elongation correlated with the increase in cell wall thickening and was dramatically pronounced in beetroot compared to the roots of treated fescue. The latter possessed significantly fewer (p < 0.001) and significantly shorter (p < 0.001) root hairs compared to control plants. Possibly, root hairs that absorb the hydrophobic contaminants may prevent contaminant absorption into the main root and concomitant axile root thickening by being sloughed off from roots. Tall fescue exhibited greater root morphological adaptability to growth in crude oil-treated sand than beetroot and, thus, a potential for long-term phytoremediation

Responses of tall fescue (Festuca arundinacea) to growth in naphthalene-contaminated sand: xenobiotic stress versus water stress

The adaptations of tall fescue (Festuca arundinacea) arising from growth in naphthalene-contaminated sand (0.8 g kg−1 sand dry weight (dw)) were investigated in the contexts of xenobiotic stress and water stress. The transfer of polycyclic aromatic hydrocarbons (PAHs) across the root endodermis was investigated using the hydrophobic Nile red stain as a PAH homologue. Nile red was applied to the epidermis of a living root to visualise uptake into the root through the transpiration stream, and the distance travelled by the stain into the root tissues was investigated using epi-fluorescence microscopy (Nikon Eclipse 90i). The results showed that the Nile red applied to the roots grown in naphthalene-contaminated sand was unable to penetrate the roots beyond the endodermis, whereas those grown in ‘clean’ sand showed evidence of uptake into the xylem vessels beyond the endodermis. Furthermore, partial collapse was observed in the cortex of naphthalene-treated roots, suggesting drought stress. Interestingly, the treated plants showed visual resilience to drought stress whilst the leaves of the control plants showed signs of wilting

Changes in the abundance of sugars and sugar-like compounds in tall fescue (Festuca arundinacea) due to growth in naphthalene-treated sand

The hydrophilic metabolome of tall fescue (Festuca arundinacea) adapted to grow in naphthalene-treated sand (0.8 g kg−1 sand dw) was analysed using gas chromatography-mass spectrometry, and peaks corresponding to the more abundant compounds were tentatively identified from analysis of their mass spectral features and reference to the NIST Mass Spectral Database. Particular attention was paid to sugars as they are known to play important roles as stress regulators in plants. The results showed that the abundance of sugars was greater in the roots but lesser in the shoots of treated plants when compared to their control counterparts. The results for indole acetic acid (IAA) were notable: IAA was prominently less in the treated roots compared to shoots, and in treated shoots, IAA was particularly subdued compared to untreated shoots consistent with IAA degradation in treated plant tissues. The differences in the molecular phenotype between control and treated plants were expressed in root structural differences. The treated roots were modified to have greater suberisation, enhanced thickening in the endodermis and distortions in the cortical zone as demonstrated through scanning electron and epi-fluorescence microscopy