Arabidopsis tonoplast intrinsic protein and vacuolar H+-adenosinetriphosphatase reflect vacuole dynamics during development of syncytia induced by the beet cyst nematode Heterodera schachtii.

Plant parasitic cyst nematodes induce specific hypermetabolic syncytial nurse cell structures in host roots. A characteristic feature of syncytia is the lack of the central vacuole and the formation of numerous small and larger vesicles. We show that these structures are formed de novo via widening of ER cisternae during the entire development of syncytium, whereas in advanced stages of syncytium development, larger vacuoles are also formed via fusion of vesicles/tubules surrounding organelle-free pre-vacuole regions. Immunogold transmission electron microscopy of syncytia localised the vacuolar markers E subunit of vacuolar H+-adenosinetriphosphatase (V-ATPase) complex and tonoplast intrinsic protein (γ-TIP1;1) mostly in membranes surrounding syncytial vesicles, thus indicating that these structures are vacuoles and that some of them have a lytic character. To study the function of syncytial vacuoles, changes in expression of AtVHA-B1, AtVHA-B2 and AtVHA-B3 (coding for isoforms of subunit B of V-ATPase), and TIP1;1 and TIP1;2 (coding for γ-TIP proteins) genes were analysed. RT-qPCR revealed significant downregulation of AtVHA-B2, TIP1;1 and TIP1;2 at the examined stages of syncytium development compared to uninfected roots. Expression of VHA-B1 and VHA-B3 decreased at 3 dpi but reached the level of control at 7 dpi. These results were confirmed for TIP1;1 by monitoring At-γ-TIP-YFP reporter construct expression. Infection test conducted on tip1;1 mutant plants showed formation of larger syncytia and higher numbers of females in comparison to wild-type plants indicating that reduced levels or lack of TIP1;1 protein promote nematode development.

Localization of calreticulin and calcium ions in mycorrhizal roots of Medicago truncatula in response to aluminum stress.

Aluminum (Al) toxicity limits growth and symbiotic interactions of plants. Calcium plays essential roles in abiotic stresses and legume-Rhizobium symbiosis, but the sites and mechanism of Ca2+ mobilization during mycorrhizae have not been analyzed. In this study, the changes of cytoplasmic Ca2+ and calreticulin (CRT) in Medicago truncatula mycorrhizal (MR) and non-mycorrizal (NM) roots under short Al stress [50 µM AlCl3 pH 4.3 for 3 h] were analyzed. Free Ca2+ ions were detected cytochemically by their reaction with potassium pyroantimonate and anti-CRT antibody was used to locate this protein in Medicago roots by immunocytochemical methods. In MR and NM roots, Al induced accumulation of CRT and free Ca2+. Similar calcium and CRT distribution in the MR were found at the surface of fungal structures (arbuscules and intercellular hyphae), cell wall and in plasmodesmata, and in plant and fungal intracellular compartments. Additionally, degenerated arbuscules were associated with intense Ca2+ and CRT accumulation. In NM roots, Ca2+ and CRT epitopes were observed in the stele, near wall of cortex and endodermis. The present study provides new insight into Ca2+ storage and mobilization in mycorrhizae symbiosis. The colocalization of CRT and Ca2+ suggests that CRT is essential for calcium mobilization for normal mycorrhiza development and response to Al stress.

Heavy metal tolerance in contrasting ecotypes of Alyssum montanum.

The response of metallicolous (M) and nonmetallicolous (NM) Alyssum montanum ecotypes to multi-metal stress was investigated under in vitro condition and compared in this study. Shoot cultures were simultaneously treated with 0.7 mM ZnSO4, 3.0 µM Pb(NO3)2 and 16.4 µM CdCl2 for 8 weeks and evaluated for their morphogenetic and ultrastructural reaction, growth tolerance as well as ability to Zn, Pb, and Cd uptake. Moreover, tissue localization and concentrations of antioxidant compounds were determined in order to elucidate the potential role of ROS-scavenging machinery in plant tolerance to metal toxicity. The results clearly demonstrated that M specimens treated with heavy metals showed less phytotoxic symptoms and low level of lipid peroxidation than reference NM one. The enhanced tolerance of M ecotype resulted from heavy metals detoxification in trichomes and intracellular leaf compartments as well as balanced ROS accumulation. The inactivation of ROS in M plants was based on peroxidase-flavonoid system, while in NM plants such relationship was not detected and amounts of antioxidant enzymes or phenolic compounds was comparable to untreated specimens or decreased significantly. Considering the procumbent growth of such hemicryptophyte which reproduce effectively in the presence of heavy metals but is characterized by low biomass production, it is proposed to exploit M ecotype of A. montanum in revegetation schemes of polluted calamine wastes to provide the prompt stabilization of areas prone to erosion.

Movement of fluorescent dyes Lucifer Yellow (LYCH) and carboxyfluorescein (CF) in Medicago truncatula Gaertn. roots and root nodules.

Lucifer Yellow (LYCH) and carboxyfluorescein (CF) served in Medicago truncatula roots and root nodules as the markers of apoplastic and symplastic transport, respectively. The aim of this study was to understand better the water and photoassimilate translocation pathways to and within nodules. The present study shows that in damaged roots LYCH moves apoplastically through the vascular elements but it was not detected within the nodule vascular bundles. In intact roots, the outer cortex was strongly labeled but the dye was not present in the interior of intact root nodules. The inwards movement of LYCH was halted in the endodermis. When the dye was introduced into a damaged nodule by infiltration, it spread only in the cell walls and the intercellular spaces up to the inner cortex. Our research showed that in addition to the outer cortex, the inner tissue containing bacteroid-infected cells is also an apoplastic domain. Our results are consistent with the hypothesis that nodules do not receive water from the xylem but get it and photoassimilates from phloem. A comparison between using LYCH and LYCH followed by glutaraldehyde fixation indicates that glutaraldehyde is responsible for fluorescence of some organelles within root nodule cells. The influence of the fixation on nodule fluorescence has not been reported before but must be taken into consideration to avoid errors. An attempt was made to follow carboxyfluorescein (6(5) CF) translocation from leaflets into roots and root nodules. In root nodules, CF was present in all or a couple of vascular bundles (VB), vascular endodermis and some adjacent cells. The leakage of CF from the VBs was observed, which suggests symplastic continuity between the VBs and the nodule parenchyma. The lack of CF in inner tissue was observed. Therefore, photoassimilate entry to the infected region of nodule must involve an apoplastic pathway.

Structural changes in Medicago truncatula root nodules caused by short-term aluminum stress.

Aluminum in the form of Al3+ is one of the most toxic heavy metal pollutants in nature and its effects are primarily root-related. Roots of Medicago truncatula exposed to 50 µM of AlCl3 for 2 h and 24 h were examined by light and electron microscopy. Changes in the appearance of the host cells, infection threads and bacteroidal tissue occurred during the first 2 h of Al stress. Microscopic observations showed that aluminum: (1) induced thickening of plant cell and infection threads (ITs) walls, (2) stimulated IT enlargement, (3) caused disturbances in bacterial release from the ITs, (4) modified cell vacuolation and induced synthesis of granular material and its deposition in the cytoplasm, (5) and caused structural alterations of organella and bacteroids.