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Endophytism within Vitis represents a topic of critical relevance due to the multiple standpoints from which it can be approached and considered. From the biological and botanical perspectives, the interaction between microorganisms and perennial woody plants falls within the category of stable relationships from which the plants can benefit in multiple ways. The life cycle of the host ensures persistence in all seasons, repeated chances of contact, and consequent microbiota accumulation over time, leading to potentially high diversity compared with that of herbaceous short-lived plants. Furthermore, grapevines are agriculturally exploited, highly selected germplasms where a profound man-driven footprint has indirectly and unconsciously shaped the inner microbiota through centuries of cultivation and breeding. Moreover, since endophyte metabolism can contribute to that of the plant host and its fruits' biochemical composition, the nature of grapevine endophytic taxa identities, ecological attitudes, potential toxicity, and clinical relevance are aspects worthy of a thorough investigation. Can endophytic taxa efficiently defend grapevines by acting against pests or confer enough fitness to the plants to endure attacks? What are the underlying mechanisms that translate into this or other advantages in the hosting plant? Can endophytes partially redirect plant metabolism, and to what extent do they act by releasing active products? Is the inner microbial colonization necessary priming for a cascade of actions? Are there defined environmental conditions that can trigger the unleashing of key microbial phenotypes? What is the environmental role in providing the ground biodiversity by which the plant can recruit microsymbionts? How much and by what practices and strategies can these symbioses be managed, applied, and directed to achieve the goal of a better sustainable viticulture? By thoroughly reviewing the available literature in the field and critically examining the data and perspectives, the above issues are discussed.

Among cereal crops, salinity tolerance is rare and complex. Multiple genes control numerous pathways, which constitute plant's response to salinity. Cell cultures act as model system and are useful to investigate the salinity response which can possibly mimic a plant's response to stress. In the present study two indica rice varieties, KS-282 and Super Basmati which exhibited contrasting sodium chloride (NaCl) stress response were used to establish cell cultures. The cell cultures showed a contrasting response to salt stress at 100 mM NaCl. High level of intracellular hydrogen peroxide (H2O2) and nitric oxide (NO) were observed in sensitive cell culture for prolonged period as compared to the tolerant cells in which an extracellular H2O2 burst along with controlled intracellular H2O2 and NO signal was seen. To evaluate the role of NO in inducing cell death under salt stress, cell death percentage (CDP) was measured after 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) pre-treatment. CDP was reduced significantly in both tolerant and sensitive cell cultures emphasizing NO's possible role in programmed cell death. Expression analysis of apoplastic NADPH oxidase, i.e. OsRbohA and recently characterised OSCA family members i.e. OsOSCA 1.2 and OsOSCA 3.1 was done. Intracellular H2O2/NO levels displayed an interplay between Ca2+ influx and ROS/RNS signal. Detoxifying enzyme (i.e. ascorbate peroxidase and catalase) activity was considerably higher in tolerant KS-282 while the activity of superoxide dismutase was significantly prominent in the sensitive cells triggering greater oxidative damage owing to the prolonged presence of intracellular H2O2. Salt stress and ROS responsive TFs i.e. OsSERF1 and OsDREB2A were expressed exclusively in the tolerant cells. Similarly, the expression of genes involved in maintaining high [K+]/[Na+] ratio was considerably higher and earlier in the tolerant variety. Overall, we suggest that a control over ROS production, and an increase in the expression of genes important for potassium homeostasis play a dynamic role in salinity tolerance in rice cell cultures.

Salinity tolerance has been extensively investigated in recent years due to its agricultural importance. Several features, such as the regulation of ionic transporters and metabolic adjustments, have been identified as salt tolerance hallmarks. Nevertheless, due to the complexity of the trait, the results achieved to date have met with limited success in improving the salt tolerance of rice plants when tested in the field, thus suggesting that a better understanding of the tolerance mechanisms is still required. In this work, differences between two varieties of rice with contrasting salt sensitivities were revealed by the imaging of photosynthetic parameters, ion content analysis and a transcriptomic approach. The transcriptomic analysis conducted on tolerant plants supported the setting up of an adaptive program consisting of sodium distribution preferentially limited to the roots and older leaves, and in the activation of regulatory mechanisms of photosynthesis in the new leaves. As a result, plants resumed grow even under prolonged saline stress. In contrast, in the sensitive variety, RNA-seq analysis revealed a misleading response, ending in senescence and cell death. The physiological response at the cellular level was investigated by measuring the intracellular profile of H2O2 in the roots, using a fluorescent probe. In the roots of tolerant plants, a quick response was observed with an increase in H2O2 production within 5 min after salt treatment. The expression analysis of some of the genes involved in perception, signal transduction and salt stress response confirmed their early induction in the roots of tolerant plants compared to sensitive ones. By inhibiting the synthesis of apoplastic H2O2, a reduction in the expression of these genes was detected. Our results indicate that quick H2O2 signaling in the roots is part of a coordinated response that leads to adaptation instead of senescence in salt-treated rice plants.

Clear evidence has highlighted a role for hormones in the plant stress response, including salt stress. Interplay and cross-talk among different hormonal pathways are of vital importance in abiotic stress tolerance. A genome-wide transcriptional analysis was performed on leaves and roots of three-day salt treated and untreated plants of two Italian rice varieties, Baldo and Vialone Nano, which differ in salt sensitivity. Genes correlated with hormonal pathways were identified and analyzed. The contents of abscisic acid, indoleacetic acid, cytokinins, and gibberellins were measured in roots, stems, and leaves of seedlings exposed for one and three days to salt stress. From the transcriptomic analysis, a huge number of genes emerged as being involved in hormone regulation in response to salt stress. The expression profile of genes involved in biosynthesis, signaling, response, catabolism, and conjugation of phytohormones was analyzed and integrated with the measurements of hormones in roots, stems, and leaves of seedlings. Significant changes in the hormone levels, along with differences in morphological responses, emerged between the two varieties. These results support the faster regulation of hormones metabolism in the tolerant variety that allows a prompt growth reprogramming and the setting up of an acclimation program, leading to specific morpho-physiological responses and growth recovery.

The study of programmed cell death (PCD) activated in a certain group of cells is complex when analyzed in the whole plant. Plant cell suspension cultures are useful when investigating PCD triggered by environmental and developmental stimuli. Due to their homogeneity and the possibility to synchronize their responses induced by external stimuli, these cultures are used for studying the signaling pathways leading to PCD. The first problem in the analysis of PCD in cell cultures is the quantification of cell viability/death over time. Cultured cells from different plant species may have specific mitotic patterns leading to calli or cell chains mixed to single cell suspensions. For this reason, not all cell cultures allow morphological parameters to be investigated using microscopy analysis, and adapted or ad hoc methods are needed to test cell viability.Here we report on some accurate methods to establish and propagate cell cultures from different plant species, including crops, as well as to determine cell viability and PCD morphological and genetic markers. In particular, we describe a protocol for extracting nucleic acids required for real-time PCR analysis which has been optimized for those cell cultures that do not allow the use of commercial kits.

Here, for the first time, a comprehensive transcriptomics study is presented of leaf senescence in the legume model Medicago truncatula, providing a broad overview of differentially expressed transcripts involved in this process. The cDNA-amplification fragment length polymorphism (AFLP) technique was used to identify > 500 genes, which were cloned and sorted into functional categories according to their gene ontology annotation. Comparison between the datasets of Arabidopsis and M. truncatula leaf senescence reveals common physiological events but differences in the nitrogen metabolism and in transcriptional regulation. In addition, it was observed that a minority of the genes regulated during leaf senescence were equally involved in other processes leading to programmed cell death, such as nodule senescence and nitric oxide signalling. This study provides a wide transcriptional profile for the comprehension of key events of leaf senescence in M. truncatula and highlights a possible regulative role for MADS box transcription factors in the terminal phases of the process.

Summary • Here mitochondrial morphology and dynamics were investigated in Medicago truncatula cell-suspension cultures during growth and senescence. • Cell biology techniques were used to measure cell growth and death in culture. Mitochondrial morphology was investigated in vivo using a membrane potential sensor probe coupled with confocal microscopy. • Expression of a senescence-associated gene (MtSAG) was evaluated in different cell-growth phases. Mitochondria appeared as numerous, punctuate organelles in cells at the beginning of the subculture cycle, while interconnected networks were observed in actively growing cells. In senescent cells, giant mitochondria were associated with dying cells. The release of cytochrome c from mitochondria was detected in different growth phases of cultured cells. • Studies on plant cell cultures allowed us to identify physiological and molecular markers of senescence and cell death, and to associate distinct mitochondrial morphology with cells under different physiological conditions.

An in vitro protocol has been developed for callus induction, somatic embryogenesis, and plant regeneration from stigma–style culture of grapevine. Four different grapevine cultivars (Vitis vinifera L.; cvs. ‘Bombino Nero,’ ‘Greco di Tufo,’ ‘Merlot,’ and ‘Sangiovese’) were tested. Explants were cultured on Nitsch and Nitsch medium (NN) supplemented with various combinations of 6-benzylaminopurine (BA; 4.5 and 9.0 mM) and b-naphthoxyacetic acid (NOA; 5.0 and 9.9 mM). Sucrose (88mM) was used as the carbon source. Somatic embryogenesis was induced within 3–7 mo. After culture initiation. Even though explants of different origin (unfertilized ovules and anthers) regenerated somatic embryos, the higher embryogenic potential was observed in stigma and style explants, with the exception of ‘Merlot,’ which regenerated somatic embryos only from unfertilized ovules. The percentages of stigma–style explants producing somatic embryos was 7% in ‘Bombino Nero’ (cultured on NN medium supplemented 9.0 mM BA and 9.9 mM NOA), 14% in ‘Greco di Tufo’ (4.5mM BA and 9.9 mM NOA), and 8% in ‘Sangiovese’ (9.0 mM BA and 9.9 mM NOA). The presence of growth regulators (BA and NOA) in the medium was essential for induction of somatic embryogenesis. Plants were regenerated on hormone-free NN medium containing 88mM sucrose.

Leaf senescence is the last stage of development of an organ and is aimed to its ordered disassembly and nutrient reallocation. Whereas chlorophyll gradually degrades during senescence in leaves, mitochondria need to maintain active to sustain the energy demands of senescing cells. Here we analysed the motility and morphology of mitochondria in different stages of senescence in leaves of grapevine (Vitis vinifera), by stably expressing a GFP (green fluorescent protein) reporter targeted to these organelles. Results show that mitochondria were less dynamic and markedly changed morphology during senescence, passing from the elongated, branched structures found in mature leaves to enlarged and sparse organelles in senescent leaves. Progression of senescence in leaves was not synchronous, since changes in mitochondria from stomata were delayed. Mitochondrial morphology was also analysed in grapevine cell cultures. Mitochondria from cells at the end of their growth curve resembled those from senescing leaves, suggesting that cell cultures might represent a useful model system for senescence. Additionally, senescence-associated mitochondrial changes were observed in plants treated with high concentrations of cytokinins. Overall, morphology and dynamics of mitochondria might represent a reliable senescence marker for plant cells.

Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (²::GFP). Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct, stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably transformed cell line. Electronic supplementary material The online version of this article (doi:10.1007/s00299-008-0510-4) contains supplementary material, which is available to authorized users.

The expression pattern of the KDC1 gene, coding for an inwardly-rectifying K(+) channel of Daucus carota , is described in several embryo stages and seedling tissues. Relative quantitative RT-PCR experiments indicated that, during (somatic) embryonic development, the KDC1 transcript appears as early as the globular stage and that the transcript level remains constant throughout the successive heart and torpedo stages. Thereafter, the KDC1 transcript is preferentially expressed in plant roots, but is also present in other tissues, and in particular, in the shoot apical meristem. In situ hybridisation experiments showed that in embryos KDC1 mRNA is detectable preferentially in protoderm cells with a stage dependent expression pattern. At later times, the hybridisation signal is particularly evident in root hairs, root epidermis and endodermis, but is also observed in single cell layers corresponding to L1 of the shoot apical meristem and leaf primordia. Promoter studies with the beta -glucuronidase reporter gene confirm preferential expression of KDC1 in embryo protoderm cells and in plant root epidermis and root hairs. Western blot analysis of embryonic proteins and immunolocalisation experiments on somatic embryos sections revealed the presence of KDC1 during embryo development. Consistent with these observations, patch-clamp experiments performed on protoplasts isolated from embryos at the torpedo stage demonstrated the presence of functional inward rectifying K(+) channels. This is the first report on the expression of a plant ion channel during embryo development.

Protein tyrosine phosphorylation plays a central role in a variety of signal transduction pathways regulating animal cell growth and differentiation, but its relevance and role in plants are controversial and still largely unknown. We report here that a large number of proteins from all plant subcellular fractions are recognized by recombinant, highly specific, anti-phosphotyrosine antibodies. Protein tyrosine phosphorylation patterns vary among different adult plant tissues or somatic embryo stages and somatic embryogenesis is blocked in vivo by a cell-permeable tyrosyl-phosphorylation inhibitor, demonstrating the involvement of protein tyrosine phosphorylation in control of specific steps in plant development.