Fallopia japonica and Fallopia × bohemica extracts cause ultrastructural and biochemical changes in root tips of radish seedlings.

Japanese knotweed (Fallopia japonica) and Bohemian knotweed (Fallopia × bohemica) are invasive plants that use allelopathy as an additional mechanism for colonization of the new habitat. Allelochemicals affect the growth of roots of neighboring plants. In the present study, we analyze the early changes associated with the inhibited root growth of radish seedlings exposed to aqueous extracts of knotweed rhizomes for 3 days. Here, we show that cells in the root cap treated with the knotweed extracts exhibited reduced cell length and displayed several ultrastructural changes, including the increased abundance of dilated ER cisternae filled with electron-dense material (ER bodies) and the accumulation of dense inclusions. Moreover, mitochondrial damage was exhibited in the root cap and the meristem zone compared to the non-treated radish seedlings. Furthermore, malfunction of the intracellular redox balance system was detected as the increased total antioxidative capacity. We also detected increased metacaspase-like proteolytic activities and, in the case of 10% extract of F. japonica, increased caspase-like proteolytic activities. These ultrastructural and biochemical effects could be the reason for the more than 60% shorter root length of treated radish seedlings compared to controls.

Determination of Caspase-Like Activities in Roots by the Use of Fluorogenic Substrates.

Activity of proteases in tissues can be influenced by various intrinsic and extrinsic factors. One of the activities that is regularly monitored in organisms ranging from prokaryotes to metazoans is the -aspase-like activity: activity of proteases, which cleave their substrates after the negatively charged amino acid residues, especially the aspartic acid. This activity is also known as the caspase-like activity, since the caspases, metazoan cysteine proteases, are one of the best characterized proteases with Asp-directed activities. Plants do not contain caspases; however, various plant proteases have been shown to exhibit caspase-like activity including saspases, phytaspases, and legumains (VPEs). The activity of these proteases can change in plants in response to stress. Here we present a simple method for monitoring of the caspase-like protease activity in roots, which have been treated with allelopathic extracts, using a set of commercially available caspase substrates. We show that activity towards some, but not all, caspase substrates is upregulated in treated but not control samples. The protocol can be used also for other plant tissues as well as for other stressors.

Oxidative Stress in Roots: Detection of Lipid Peroxidation and Total Antioxidative Capacity.

Various abiotic and biotic agents disturb the fine balance between cellular oxidants and antioxidants. The resulting oxidative stress occurs either due to the increasing levels of reactive oxygen species (ROS) or weak antioxidative system that cannot scavenge ROS burst. In addition to their harmful role, ROS can also act as signaling molecules, and oxidative stress is often the initial step in the programmed cell death. Here we describe two parameters of oxidative stress that can be measured spectrophotometrically: lipid peroxidation via the content of the by-product malondialdehyde, and the amount of all non-enzymatic antioxidants named as total antioxidative capacity. Both methods are presented using young radish (Raphanus sativus) seedlings after treatment with extract from the invasive plant species Japanese knotweed (Fallopia japonica).

Plant cell responses to allelopathy: from oxidative stress to programmed cell death.

Allelopathy is a plant-plant interaction in which one plant releases biologically active compounds that have negative effects on the fitness of the target plant. The most pronounced effects are inhibition of seed germination and growth of neighboring plants. The roots of these plants are in contact with the allelochemicals released into the soil, as the primary target of the allelopathic action. To date, the best documented allelopathic activities relate to some weeds and invasive alien plants that show rapid spread and successful growth. A better understanding of the mechanisms of allelopathy will help to improve crop production and to manage and prevent plant invasions. At the cellular level, allelochemicals induce a burst of reactive oxygen species in the target plants, which leads to oxidative stress, and can promote programmed cell death. Lipid peroxidation and cell membrane changes, protein modifications, and increased protease activities are the early signs of cell damage. When enzymatic and nonenzymatic antioxidants cannot scavenge reactive oxidants, this can result in hydrolytic or necrotic degradation of the protoplast. Cell organelles then lose their integrity and function. In roots, the structure and activity of the apical meristem are changed, which affects root growth and water absorption. Such allelopathically active compounds might thus be applied to control and manage weeds and invasive plants in a more sustainable way, to reduce chemical pollution.

Root growth inhibition and ultrastructural changes in radish root tips after treatment with aqueous extracts of Fallopia japonica and F. ×bohemica rhizomes.

Allelopathic compounds released by invasive alien plants can suppress the growth of plants in their vicinity. The aim of this study was to investigate changes in tissue and cell structure in roots of radish seedlings treated with 10% aqueous extracts of rhizomes from the invasive knotweeds Fallopia japonica and F. ×bohemica. After 7 days of growth without and with aqueous extracts from these rhizomes, the anatomical and ultrastructural changes in the radish seedling roots were analyzed with light and transmission electron microscopy, and hydrogen peroxide was localized with diaminobenzidine, to define oxidative stress. The roots of radish seedlings treated with the knotweed extracts were shorter and thicker, due to the shorter and wider shapes of their cortex cells, which were organized in more columns than the control roots. There were signs of cell damage and oxidative stress in the root cap cells, and to a lesser extent in the meristematic zone. As well as the irregularly shaped nuclei and plasma membrane detached from the cell wall, the most prominent ultrastructural effects in the root cap cells of these aqueous rhizome extracts were the ring-shaped form of the mitochondria and large endoplasmic reticulum bodies. Excessive vacuolization was seen for the cells of the root apical meristem.