Wojciech Borucki

Mutation in the pssB-pssA intergenic region of Rhizobium leguminosarum bv. trifolii affects the surface polysaccharides synthesis and nitrogen fixation ability

Summary A Rhizobium leguminosarum bv. trifolii Tn 5 transposon mutant deficient in exopolysaccharide biosynthesis was found to form non-nitrogen fixing nodules on clover. Root nodules induced by the mutant contained aberrant infection threads and few bacteroids. Sequence analysis of the transposon insertion site localized the mutation in the pssB-pssA intergenic region affecting the exopolysaccharide biosynthesis. The mutant also showed decreased sensitivity to SDS and deoxycholate and displayed a changed lipopolysaccharide (LPS) banding pattern compared to the wild-type strain TA1. The alteration in the O-polysaccharide part of LPS was confirmed by Western immunoblotting with polyclonal antibodies. LPS preparations of strain TA1 and the mutant strain only reacted with their homologous sera. The common epitopes in LPS from bacteroids and free-living rhizobia were revealed by immunogold assay. The results of this study indicate that the pssB-pssA region of R. leguminosarum bv. trifolii is important for the polysaccharide synthesis.

Vacuolar organization in the nodule parenchyma is important for the functioning of pea root nodules.

Different models have been proposed to explain the operation of oxygen diffusion barrier in root nodules of leguminous plants. This barrier participates in protection of oxygen-sensitive nitrogenase, the key enzyme in nitrogen fixation, from inactivation. Details concerning structural and biochemical properties of the barrier are still lacking. Here, the properties of pea root nodule cortical cells were examined under normal conditions and after shoot removal. Microscopic observations, including neutral red staining and epifluorescence investigations, showed that the inner and outer nodule parenchyma cells exhibit different patterns of the central vacuole development. In opposition to the inner part, the outer parenchyma cells exhibited vacuolar shrinkage and formed cell wall infoldings. Shoot removal induced vacuolar shrinkage and formation of infoldings in the inner parenchyma and uninfected cells of the symbiotic tissue, as well. It is postulated that cells which possess shrinking vacuoles are sensitive to the external osmotic pressure. The cells can give an additional resistance to oxygen diffusion by release of water to the intercellular spaces.Immunolocalization studies proved higher expression of endo-β-1,4-glucanases within expanding cells of the outer cortex of pea nodules comparing with nodule endodermis or nodule parenchyma, so it is suggested that (1) endo-glucanases are involved in growth related modifications of cell walls and (2) enlarged cells decrease nodule conductance to oxygen.

Localization of hydrogen peroxide accumulation and diamine oxidase activity in pea root nodules under aluminum stress

Aluminum (Al) is one of the environmental stressors that induces formation of reactive oxygen species (ROS) in plants. Hydrogen peroxide (H2O2) and H2O2-generated apoplast diamine oxidase (DAO) activity were detected cytochemically via transmission electron microscopy (TEM), in pea (Pisum sativum L.) root nodules exposed to high (50 μM AlCl3, for 2 and 24h) Al stress. The nodules were shown to respond to Al stress by disturbances in infection thread (IT) growth, bacteria endocytosis, premature degeneration of bacteroidal tissue and generation of H2O2 in nodule apoplast. Large amounts of peroxide were found at the same sites as high DAO activity under Al stress, suggesting that DAO is a major source of Al-induced peroxide accumulation in the nodules. Peroxide distribution and DAO activity in the nodules of both control plants and Al-treated ones were typically found in the plant cell walls, intercellular spaces and infection threads. However, 2 h Al treatment increased DAO activity and peroxide accumulation in the nodule apoplast and bacteria within threads. A prolonged Al treatment (24 h) increased the H2O2 content and DAO activity in the nodule apoplast, especially in the thread walls, matrix and bacteria within infection threads. In addition to ITs, prematurely degenerated bacteroids, which occurred in response to Al, were associated with intense staining for H2O2 and DAO activity. These results suggest the involvement of DAO in the production of a large amount of H2O2 in the nodule apoplast under Al stress. The role of reactive oxygen species in pea-Rhizobium symbiosis under Al stress is discussed.

Pectins esterification in the apoplast of aluminum-treated pea root nodules

Aiming to elucidate the possible involvement of pectins in aluminum-mediated growth inhibition the distribution of pectins in the apoplast of root nodules was investigated. Experiments were performed on the pea (Pisum sativum L.) root nodules treated with aluminum (50 μM AlCl3, for 2 or 24h). For histochemical acidic pectin localization we used ruthenium red staining. Immunolabeling techniques with monoclonal antibodies specific to high methyl-esterified pectin (JIM7), low methyl-esterified pectin (JIM5) and calcium cross-linked pectin (2F4) were used to re-examine the pattern of pectin esterification and distribution. After immunolabeling the samples were observed using a fluorescent and transmission electron microscope. Ruthenium red staining showed that acid pectin content increased in the apoplast of Al-treated nodules and immunolocalization of pectin epitopes revealed that the fraction of de-esterified pectins increased significantly under Al stress. JIM5 and 2F4 epitopes were located on the inner surface of the primary cell wall with higher intensity at cell corners lining the intercellular spaces and at infection threads (ITs) walls. By contrast, JIM 7 labels all walls uniformly throughout the nodule. In the presence of Al, the increase of JIM5 and 2F4 labeling in thick plant and IT walls, together with a decrease of JIM7 labeling was observed. These results indicate a specific role for pectin de-esterification in the process of wall thickening and growth inhibition. In particular, Al-dependent increase in pectin content and their low methyl esterification degree correlate with wall thickness and higher rigidity, and in this way, may affect IT and nodules growth.

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.

Accumulation and localization of extensin protein in apoplast of pea root nodule under aluminum stress

Cell wall components such as hydroxyproline-rich glycoproteins (HRGPs, extensins) have been proposed to be involved in aluminum (Al) resistance mechanisms in plants. We have characterized the distribution of extensin in pea (Pisum sativum L.) root nodules apoplast under short (for 2 and 24h) Al stress. Monoclonal antibodie LM1 have been used to locate extensin protein epitope by immunofluorescence and immunogold labeling. The nodules were shown to respond to Al stress by thickening of plant and infection thread (IT) walls and disturbances in threads growth and bacteria endocytosis. Immunoblot results indicated the presence of a 17-kDa band specific for LM1. Irrespective of the time of Al stress, extensin content increased in root nodules. Further observation utilizing fluorescence and transmission electron microscope showed that LM1 epitope was localized in walls and intercellular spaces of nodule cortex tissues and in the infection threads matrix. Al stress in nodules appears to be associated with higher extensin accumulation in matrix of enlarged thick-walled ITs. In addition to ITs, thickened walls and intercellular spaces of nodule cortex were also associated with intense extensin accumulation. These data suggest that Al-induced extensin accumulation in plant cell walls and ITs matrix may have influence on the process of IT growth and tissue and cell colonization by Rhizobium bacteria.

Visualisation of plastid outgrowths in potato (Solanum tuberosum L.) tubers by carboxyfluorescein diacetate staining

Key messageWe describe two types of plastid outgrowths visualised in potato tubers after carboxyfluorescein diacetate staining. Probable esterase activity of the outgrowths has been demonstrated for the first time ever.AbstractPlastid outgrowths were observed in the phelloderm and storage parenchyma cells of red potato (S. tuberosum L. cv. Rosalinde) tubers after administration of carboxyfluorescein diacetate stain. Endogenous esterases cleaved off acetic groups to release membrane-unpermeable green fluorescing carboxyfluorescein which accumulated differentially in particular cell compartments. The intensive green fluorescence of carboxyfluorescein exhibited highly branched stromules (stroma-filled plastid tubular projections of the plastid envelope) and allowed distinguishing them within cytoplasmic strands of the phelloderm cells. Stromules (1) were directed towards the nucleus or (2) penetrated the whole cells through the cytoplasmic bands of highly vacuolated phelloderm cells. Those directed towards the nucleus were flattened and adhered to the nuclear envelope. Stromule-like interconnections between two parts of the same plastids (isthmuses) were also observed. We also documented the formation of another type of the stroma-filled plastid outgrowths, referred to here as protrusions, which differed from previously defined stromules in both morphology and esterase activity. Unlike stromules, the protrusions were found to be associated with developmental processes leading to starch accumulation in the storage parenchyma cells. These results strongly suggest that stromules and protrusions exhibit esterase activity. This has been demonstrated for the first time. Morphological and biochemical features as well as possible functions of stromules and protrusions are discussed below.

Localization of acid phosphatase activity in the apoplast of pea (Pisum sativum L.) root nodules grown under phosphorus deficiency

ACPase activity was localized in the apoplast of pea root nodules under phosphorus deficiency. Pea plants (Pisum sativum L. cv. Sze ciotygodniowy) where inoculated with Rhizobium leguminosarum bv. viciae 248 and were cultured on nitrogen-free medium with phosphate (−N/+P) or phosphate-deficient (−N/−P) one. In comparison with control nodules, P-deficient nodules showed the increase of ACPase activity in plant cell walls and the infection threads. The increase in bacterial ACPase activity under P-deficiency may reflect higher demand for inorganic phosphorus that is necessary for bacteria multiplication within the infection threads. The increase of ACPase activity in nodule apoplast under P stress may enlarge the availability of phosphate for plant and bacteria.