Rhizosphere microbiomes of resurrection plants Ramonda serbica and R. nathaliae: comparative analysis and search for bacteria mitigating drought stress in wheat (Triticum aestivum L.).

Rhizosphere microbial communities play an important role in maintaining the health and productivity of the plant host. The rhizobacteria Pseudomonas putida P2 of Ramonda serbica and Bacillus cereus P5 of R. nathaliae were selected for treatment of the Belija wheat cultivar because of their plant growth-promoting (PGP) properties. Compared to the non-treated drought-stressed plants, the plants treated with rhizobacteria showed increased activity of the two major antioxidant enzymes, superoxide dismutase, and ascorbate peroxidase. Plants treated with the B. cereus P5 strain exhibited higher proline content under drought stress, suggesting that proline accumulation depends on the relative water content (RWC) status of the plants studied. Inoculation of wheat seeds with the P. putida P2 strain improved water status by increasing RWC and alleviating oxidative stress by reducing H2O2 and malondialdehyde concentrations in plants exposed to severe drought, possibly also helping plants to overcome drought through its 1-aminocyclopropane-1-carboxylic acid deaminase activity. Analysis of data from Next Generation sequencing (NGS) revealed that the dominant bacterial taxa in the rhizosphere of resurrection plants R. serbica and R. nathaliae were extremophilic, thermotolerant, Vicinamibacter silvestris, Chthoniobacter flavus, and Gaiella occulta. From the fungi detected Penicillium was the most abundant in both samples, while Fusarium and Mucor were present only in the rhizosphere of R. serbica and the entomopathogenic fungi Metarhizium, and Tolypocladiumu only in the rhizosphere of R. nathaliae. The fungal communities varied among plants, suggesting a stronger environmental influence than plant species. Our study demonstrates the importance of in vivo experiments to confirm the properties of PGP bacteria and indicates that the rhizosphere of resurrection plants is a valuable source of unique microorganisms that can be used to improve the drought stress tolerance of crops.

Physiological and cell ultrastructure disturbances in wheat seedlings generated by Chenopodium murale hairy root exudate.

Chenopodium murale L. is an invasive weed species significantly interfering with wheat crop. However, the complete nature of its allelopathic influence on crops is not yet fully understood. In the present study, the focus is made on establishing the relation between plant morphophysiological changes and oxidative stress, induced by allelopathic extract. Phytotoxic medium of C. murale hairy root clone R5 reduced the germination rate (24% less than control value) of wheat cv. Natasa seeds, as well as seedling growth, diminishing shoot and root length significantly, decreased total chlorophyll content, and induced abnormal root gravitropism. The R5 treatment caused cellular structural abnormalities, reflecting on the root and leaf cell shape and organization. These abnormalities mostly included the increased number of mitochondria and reorganization of the vacuolar compartment, changes in nucleus shape, and chloroplast organization and distribution. The most significant structural changes were observed in cell wall in the form of amoeboid protrusions and folds leading to its irregular shape. These structural alterations were accompanied by an oxidative stress in tissues of treated wheat seedlings, reflected as increased level of H2O2 and other ROS molecules, an increase of radical scavenging capacity and total phenolic content. Accordingly, the retardation of wheat seedling growth by C. murale allelochemicals may represent a consequence of complex activity involving both cell structure alteration and physiological processes.

Water deficit down-regulates miR398 and miR408 in pea (Pisum sativum L.).

MicroRNAs (miRNAs), recently recognized as important regulator of gene expression at posttranscriptional level, have been found to be involved in plant stress responses. The observation that some miRNAs are up- or down regulated by stress implies that they could play vital roles in plant resistance to abiotic and biotic stress. We investigated the effect of water stress treatment during 10 days on expression of conserved miRNAs-miR398a/b and miR408 in pea plants. This time frame reflects the changes as close as possible to the changes where water stress causes visible effects under field condition. It was observed that dehydration strongly down regulates the expression of both miR398a/b and miR408 in pea roots and shoots. The down-regulation of miR398a/b and the up-regulation of potential target genes - copper superoxide dismutase, CSD1, highlight the involvement of this miRNA in pea stress response. To the contrary, the mRNA level of cytochrome c oxidase subunit 5 (COX5b) did not change in roots and shoots of water-stressed plants, compared to control (well) hydrated plants. This suggests that COX5b is not the target of miR398, or that its expression is regulated by some other mechanism. P1B-ATPase expression increased during water deficit only in the shoots of pea; in the roots there were no changes in expression. Our results help to understand the possible role of investigated miRNAs and their contribution to pea capacity to cope with water deficit.

Resurrection plants of the genus Ramonda: prospective survival strategies - unlock further capacity of adaptation, or embark on the path of evolution?

Paleoendemic species of the monophyletic genus Ramonda (R. myconi, R. serbica and R.~nathaliae) are the remnants of the Tertiary tropical and subtropical flora in Europe. They are the rare resurrection plants of Northern Hemisphere temperate zone. Ramonda serbica and R. nathaliae are chorologically differentiated in the Balkan Peninsula and occupy similar habitats in calcareous, northward slopes in canyons and mountainsides. They remain well-hydrated during spring, late autumn and even in winter. In summer and early autumn when plants are subjected to drought and thermal stress, their desiccation tolerance comes into operation and they fall into anabiosis. Investigations revealed the permanent presence of ubiquitine and its conjugates, high amounts of oxalic acid and proline. Both species are homoiochlorophyllous. It enables them to rapidly resume photosynthesis upon rehydration, but also makes them susceptible to reactive oxygen species formation. Dehydration induces activation of antioxidative enzymes (ascorbate peroxidase, glutathione reductase, polyphenol oxidase), increase in amounts of AsA and GSH, phenolic acids, dehydrins, sucrose, and inorganic ions. Plasma membranes, characterized by high amount of cholesterol, are subjected to decrease in membrane fluidity mostly on account of increased level of lipid saturation. Cytogenetic analysis revealed that R. nathaliae is a diploid (2n = 48) and probably evolutionary older species, while R. serbica is a hexaploid (2n = 144). Two species live together in only two localities forming hybrid individuals (2n = 96). Polyploidization is the major evolutionary mechanism in the genus Ramonda that together with hybridization ability indicates that these relict species which have preserved an ancient survival strategy are not the evolutionary "dead end."The species of the genus Ramonda are promising sources of data important for understanding the complex strategy of resurrection plants' survival, appraised through a prism of their evolutionary and adaptive potential for multiple environmental stresses.

Ubiquitous aspartic proteinase as an actor in the stress response in buckwheat.

The aspartic protease (FeAP9) gene from buckwheat resembles the exon-intron structure characteristic for typical aspartic proteinases, including the presence of the leader intron in the 5'-UTR. RT PCR experiments and gel protein blot analysis indicated that FeAP9 was present in all analyzed organs: developing seeds, seedlings, flowers, leaves, roots and stems. Using Real-time PCR, we found that FeAP9 expression is upregulated in buckwheat leaves under the influence of different abiotic stresses, including dark, drought and UV-B light, as well as wounding and salicylic acid.

Two types of aspartic proteinases from buckwheat seed--gene structure and expression analysis.

Two types of aspartic proteinase (AP) genes have been isolated from the cDNA library of developing buckwheat seeds. Analysis of their sequences showed that one of these, FeAP9, resembled the structure and shared high homology with the so-called typical plant APs characterized by the presence of a plant-specific insert (PSI), an element unique among APs. The other cDNA, FeAPL1, encoded an AP-like protein lacking that domain. Different expression profiles were observed for FeAP9 and FeAPL1. FeAPL1 mRNAs were restricted to the seeds only, whereas FeAP9 mRNAs were also present in the other plant tissues - leaves, roots, and flowers. Higher levels of FeAP9 were observed in senescent leaves compared with green leaves. The differential expression pattern of these two unique APs raises the interesting possibility that these proteinases have unique substrate specificity and may have different roles in plant development and other physiological processes.

Antioxidative enzymes in the response of buckwheat (Fagopyrum esculentum moench) to ultraviolet B radiation.

The behavior of the enzymatic antioxidant defense system was studied in buckwheat leaves and seedlings subjected to short-term enhanced UV-B radiation. The effects of UV-B action were monitored immediately after irradiation as well as after recovery. The applied dose induced an increase in lipid peroxidation and total flavonoid content, a decrease in chlorophyll content, and a change in enzymatic digestibility of extracted DNA. The activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase, and soluble peroxidase, as well as the isoelectric focusing (IEF) pattern of peroxidase isoforms, was analyzed. In treated as well as recovered seedlings, soluble and ascorbate peroxidase activities were increased. The activity of SOD was not altered, whereas CAT activity was decreased. In contrast to seedlings, only CAT activity was increased in treated and recovered leaves.

Vicilin-like storage globulin from buckwheat (Fagopyrum esculentum Moench) seeds.

An 8S storage globulin from buckwheat seed, which resembles the structure and features common to the vicilin-like family of seed storage proteins, was analyzed for this paper. It was found that expression of the 8S globulin gene precedes that of the 13S globulin (the main buckwheat storage protein) and starts from an early stage of buckwheat seed development (9-11 days after flowering), continuing to accumulate throughout seed development to contribute approximately 7% of total seed proteins. This protein fraction might be more interesting for biotechnological application than the 13S buckwheat legumin consisting of 23-25 kDa subunits reported to be the major buckwheat allergen. A partial cDNA was also isolated, showing high homology with cDNAs coding for vicilin-like storage proteins from various plant species, and its expression profile throughout seed development as well as in different buckwheat tissues was analyzed.

Characterization of an aspartic proteinase activity in buckwheat (Fagopyrum esculentum Moench) seeds.

The pepstatin A sensitive acidic proteolytic activity of total protein extracts of buckwheat seeds has been analyzed in developing, mature, and germinating seeds by activity measurements as well as by electrophoretic and immunochemical techniques. Immunoblot analysis using cross-reactive antibodies raised against barley phytepsin suggested that specific proteolytic activity could be attributed to a 47 kDa heterodimeric polypeptide, composed of two subunits: 31 and 16 kDa polypeptides. The analysis of time course expression revealed that the 47 kDa heterodimer accumulated during seed maturation starting from 12 days after pollination and was also present at the beginning of germination. Milk-clotting activity of this proteinase was also indicated.