Above- and below-ground morpho-physiological traits indicate that biochar is a potential peat substitute for grapevine cuttings nursery production.

The growing demand for grapevine planting materials, due to growing global viticulture, is promoting research studies to improve vineyard sustainability. In greenhouse nurseries, peat is the most common growing medium component used although is an expensive and non-renewable material. Indeed, the reduction of peat exploitation is receiving great attention, and currently, several materials are being investigated as peat substitutes for composing the cultivation substrates. Biochar, a carbon-rich, recalcitrant charred organic co-product of the pyrolysis or gasification process, has emerged as a potentially promising replacement for soilless substrates in nursery plant material propagation. Although several studies carried out at greenhouse nurseries have shown that biochar, can improve plant growth, only a few studies have focused on the production of grapevine plant material. To fulfil this knowledge gap and push forward the sustainability of the nursery sector, we evaluated above and below-ground morpho-physiological traits of one-year-old potted grapevine cuttings growing with 30% volume of four different biochar types (i.e., from pyrolysis and gasification) mixed with commercial peat. The present study shows that biochar can be used in growing media mixes without adverse effects on roots, improves soil water retention and leaf water potential, and improves the effects on soil microbiology.

Long term effect of biochar on soil plant water relation and fine roots: Results after 10 years of vineyard experiment.

Biochar is widely suggested to improve soil physical properties and soil-water-plant interactions. Furthermore, the application of biochar to the soil can alter the dynamics of the roots and, in turn, affect the performance of the plant. Nevertheless, the long-term evolution of these effects is unknown and of critical importance because biochar persists in soil for centuries. The results of this work are part of a long-term study in the vineyard started in 2009 and still ongoing. In this work, the effect of applying biochar to soil on the plant-water relationships of Vitis vinifera, soil properties and fine root traits is evaluated 10 years after application. Even after 10 years, the ecophysiological measurements indicated an increase in soil water content and a significant increase in the water status of the plants in the plots treated with biochar. Independently of the diameter class considered, both doses of biochar led in the entire 40 cm of soil to a general reduction of the fine-root standing biomass and length, which is probably due to the lower need for fine root foraging. Moreover, the SRL did not show differences among different treatments. When fine-root traits were analysed along the soil depth at 10 cm intervals, we noted that both length and biomass were significantly higher in the control plant only in the upper soil layers (20 cm) and SRL was significantly higher only in the upper 10 cm of soil. These findings underscore how control plants plastically respond to the lower content of water in the soil by decreasing the fine-root cost-to-benefit ratio, especially in the topsoil layer. Research on the effect of biochar in viticulture can provide an effective contribution to the mitigation of climate change by increasing the water status of the soil and plants even 10 years after its application.

Transcription factors PRE3 and WOX11 are involved in the formation of new lateral roots from secondary growth taproot in A. thaliana.

The spatial deployment of lateral roots determines the ability of a plant to interact with the surrounding environment for nutrition and anchorage. This paper shows that besides the pericycle, the vascular cambium becomes active in Arabidopsis thaliana taproot at a later stage of development and is also able to form new lateral roots. To demonstrate the above, we implemented a two-step approach in which the first step leads to development of a secondary structure in A. thaliana taproot, and the second applies a mechanical stress on the vascular cambium to initiate formation of a new lateral root primordium. GUS staining showed PRE3, DR5 and WOX11 signals in the cambial zone of the root during new lateral root formation. An advanced level of wood formation, characterized by the presence of medullar rays, was achieved. Preliminary investigations suggest the involvement of auxin and two transcription factors (PRE3/ATBS1/bHLH135/TMO7 and WOX11) in the transition of some vascular cambium initials from a role as producers of xylem/phloem mother cells to founder cells of a new lateral root primordium.

The influence of slope on Spartium junceum root system: morphological, anatomical and biomechanical adaptation.

Root systems have a pivotal role in plant anchorage and their mechanical interactions with the soil may contribute to soil reinforcement and stabilization of slide-prone slopes. In order to understand the responses of root system to mechanical stress induced by slope, samples of Spartium junceum L., growing in slope and in plane natural conditions, were compared in their morphology, biomechanical properties and anatomical features. Soils sampled in slope and plane revealed similar characteristics, with the exception of organic matter content and penetrometer resistance, both higher in slope. Slope significantly influenced root morphology and in particular the distribution of lateral roots along the soil depth. Indeed, first-order lateral roots of plants growing on slope condition showed an asymmetric distribution between up- and down-slope. Contrarily, this asymmetric distribution was not observed in plants growing in plane. The tensile strength was higher in lateral roots growing up-slope and in plane conditions than in those growing down-slope. Anatomical investigations revealed that, while roots grown up-slope had higher area covered by xylem fibers, the ratio of xylem and phloem fibers to root diameter did not differ among the three conditions, as also, no differences were found for xylem fiber cell wall thickness. Roots growing up-slope were the main contributors to anchorage properties, which included higher strength and higher number of fibers in the xylematic tissues. Results suggested that a combination of root-specific morphological, anatomical and biomechanical traits, determines anchorage functions in slope conditions.

Interspecific variation in functional traits of oak seedlings (Quercus ilex, Quercus trojana, Quercus virgiliana) grown under artificial drought and fire conditions.

To face summer drought and wildfire in Mediterranean-type ecosystems, plants adopt different strategies that involve considerable rearrangements of biomass allocation and physiological activity. This paper analyses morphological and physiological traits in seedlings of three oak species (Quercus ilex, Quercus trojana and Quercus virgiliana) co-occurring under natural conditions. The aim of this study was to evaluate species-specific characteristics and the response of these oak seedlings to drought stress and fire treatment. Seedlings were kept in a growth chamber that mimicked natural environmental conditions. All three species showed a good degree of tolerance to drought and fire treatments. Differences in specific biomass allocation patterns and physiological traits resulted in phenotypic differences between species. In Q. ilex, drought tolerance depended upon adjustment of the allocation pattern. Q. trojana seedlings undergoing mild to severe drought presented a higher photosystem II (PSII) efficiency than control seedlings. Moreover, Q. trojana showed a very large root system, which corresponded to higher soil area exploitation, and bigger leaf midrib vascular bundles than the other two species. Morphological and physiological performances indicated Q. trojana as the most tolerant to drought and fire. These characteristics contribute to a high recruitment potential of Q. trojana seedlings, which might be the reason for the dominance of this species under natural conditions. Drought increase as a result of climate change is expected to favour Q. trojana, leading to an increase in its spatial distribution.

Drf1, a novel regulatory subunit for human Cdc7 kinase.

Studies in model organisms have contributed to elucidate multiple levels at which regulation of eukaryotic DNA replication occurs. Cdc7, an evolutionarily conserved serine-threonine kinase, plays a pivotal role in linking cell cycle regulation to genome duplication, being essential for the firing of DNA replication origins. Binding of the cell cycle-regulated subunit Dbf4 to Cdc7 is necessary for in vitro kinase activity. This binding is also thought to be the key regulatory event that controls Cdc7 activity in cells. Here, we describe a novel human protein, Drf1, related to both human and yeast Dbf4. Drf1 is a nuclear cell cycle-regulated protein, it binds to Cdc7 and activates the kinase. Therefore, human Cdc7, like cyclin-dependent kinases, can be activated by alternative regulatory subunits. Since the Drf1 gene is either absent or not yet identified in the genome of model organisms such as yeast and Drosophila, these findings introduce a new level of complexity in the regulation of DNA replication of the human genome.

p27 cytoplasmic localization is regulated by phosphorylation on Ser10 and is not a prerequisite for its proteolysis.

The activity of the cyclin-dependent kinase inhibitor p27 is controlled by its concentration and subcellular localization. However, the mechanisms that regulate its intracellular transport are poorly understood. Here we show that p27 is phosphorylated on Ser10 in vivo and that mutation of Ser10 to Ala inhibits p27 cytoplasmic relocalization in response to mitogenic stimulation. In contrast, a fraction of wild-type p27 and a p27(S10D)-phospho-mimetic mutant translocates to the cytoplasm in the presence of mitogens. G1 nuclear export of p27 and its Ser10 phosphorylation precede cyclin-dependent kinase 2 (Cdk2) activation and degradation of the bulk of p27. Interestingly, leptomycin B-mediated nuclear accumulation accelerates the turnover of endogenous p27; the p27(S10A) mutant, which is trapped in the nucleus, has a shorter half-life than wild-type p27 and the p27(S10D) mutant. In summary, p27 is efficiently degraded in the nucleus and phosphorylation of Ser10 is necessary for the nuclear to cytoplasmic redistribution of a fraction of p27 in response to mitogenic stimulation. This cytoplasmic localization may serve to decrease the abundance of p27 in the nucleus below a certain threshold required for activation of cyclin-Cdk2 complexes.

The de-ubiquitinating enzyme Unp interacts with the retinoblastoma protein.

The ubiquitin pathway is involved in the proteolytic turnover of many short-lived cellular regulatory proteins. Since selective degradation of substrates of this system requires the covalent attachment of a polyubiquitin chain to the substrates, degradation could be counteracted by de-ubiquitinating enzymes (or isopeptidases) which selectively remove the polyubiquitin chain. Unp is a human isopeptidase with still poorly understood biological functions. Here, we show that cellular Unp specifically interacts with the retinoblastoma gene product (pRb).

Increased proteasome degradation of cyclin-dependent kinase inhibitor p27 is associated with a decreased overall survival in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive neoplasm characterized by the deregulated expression of cyclin D1 by t(11;14). The molecular mechanisms responsible for MCL's clinical behavior remain unclear. The authors have investigated the expression of p53, E2F-1, and the CDK inhibitors p27 and p21 in 110 MCLs, relating their expression to proliferative activity (Ki-67). For comparison, they have similarly analyzed low-grade (12 MALT, 16 CLL/SLL) and high-grade (19 DLCL) lymphomas. p53 was detected more frequently in large-cell MCL (l-MCL; 5 of 7) than in classical MCL (s-MCL; 13 of 103) and DLCL (8 of 19). In MCL and DLCL, the percentage of E2F-1+ nuclei was high, correlating with high Ki-67 expression. Most MCLs (91 of 112) and DLCLs (12 of 19) showed a loss of p27; MALT and CLL/SLL, however, were p27 positive. Reverse transcription-polymerase chain reaction and in vitro protein degradation assays demonstrated that MCLs have normal p27 mRNA expression but increased p27 protein degradation activity via the proteasome pathway. Correlation of MCL p53 and p27 expression with clinical data showed an association between reduced overall survival rates and the overexpression of p53 (P =.001), the loss of p27 (P =. 002), or both. Loss of p27 identified patients with a worse clinical outcome among p53 negative cases (P =.002). These findings demonstrated that MCL has a distinct cell cycle protein expression similar to that of high-grade lymphoma. The loss of p27 and the overexpression of p53 in MCL are prognostic markers that identify patients at high risk. The demonstration that low levels of p27 in MCL result from enhanced proteasome-mediated degradation should encourage additional clinical trials. (Blood. 2000;95:619-626) (Blood. 2000;95:619-626)

Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation.

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor.

Overexpression of the nerve growth factor-inducible PC3 immediate early gene is associated with growth inhibition.

PC3 (pheochromocytoma cell-3) is an immediate early gene isolated as sequence induced in the rat PC12 cell line during neuronal differentiation by nerve growth factor (NGF). PC3, which is expressed in vivo in the neuroblast when it ceases proliferating and differentiates into a neuron, has partial homology with two antiproliferative genes, BTG1 and Tob. Here we report that overexpression of PC3 in NIH3T3 and PC12 cells leads to marked inhibition of cell proliferation. In stable NIH3T3 clones expressing PC3, the transition from G1 to S phase was impaired, whereas the retinoblastoma (RB) protein was detected as multiple isoforms of M(r) 105,000-115,000 (indicative of a hyperphosphorylated state) only in low-density cultures. Such findings are consistent with a condition of growth inhibition. Thus, PC3 might be a negative regulator of cell proliferation, possibly acting as a transducer of factors influencing cell growth and/or differentiation, such as NGF, by a RB-dependent pathway. This is the first evidence of a NGF-inducible immediate early gene displaying antiproliferative activity.

Inhibition of differentiation in myoblasts deprived of the interferon-related protein PC4.

PC4 (pheochromocytoma cell-4) is an immediate early gene related to IFN-gamma, the mRNA of which is induced during the course of neuronal differentiation by nerve growth factor in the PC12 cell line. Here we report that PC4 mRNA is also expressed in the myoblast C2C12 cell line and is regulated during differentiation; its expression decreases within 6 h from the onset of differentiation, attains a minimum after 12 h, and returns to basal level within 36 h. This transient down-regulation of PC4 expression in C2C12 myoblasts is prevented by transforming growth factor beta, a molecule which inhibits the differentiation of muscle. Sense and antisense PC4 cDNA transfection strategies in C2C12 cells were then used to clarify the role of PC4 in muscle differentiation. While no effect was seen by over-expression of PC4, stable transfectants underexpressing PC4 exhibited a delay in attaining the differentiated phenotype, with an impairment of myogenin and myosin expression. Myogenin was also inhibited in C2C12 cells microinjected with the anti-PC4 polyclonal antibody A451. We thus postulate a role for PC4 as a positive regulator during muscle differentiation.

Nerve growth factor regulates the subcellular localization of the nerve growth factor-inducible protein PC4 in PC12 cells.

The immediate early gene (IEG) PC4, which encodes a protein related to gamma interferon, is activated at the onset of the neuronal differentiation induced by nerve growth factor (NGF) in PC12 cells. With an antibody raised to a bacterial beta gal-PC4 fusion protein, the PC4 protein is detected as an immunoreactive molecular species of 49 kDa, whose synthesis is rapidly induced by NGF in parallel with the induction of its mRNA. Immunofluorescence, electron microscopy and subfractionation studies indicate that the PC4 immunoreactivity is localized in the cytoplasm of PC12 cells, where it is increased transiently by NGF within 3 hr of treatment. In addition, the PC4 immunoreactivity presents an NGF-dependent pattern of intracellular localization. In fact, within 3 hr after addition of NGF, PC4 is also significantly expressed on the inner face of the plasma membrane, to which it is physically associated. After longer NGF treatment, PC4 disappears from the plasma membrane and appears in the nucleus, with reduced cytoplasmic expression. Localization in the nucleus is reversed by removal of NGF and closely parallels changes in the state of differentiation of the cell. The existence within the PC4 protein of a consensus sequence for the addition of myristic acid and of a putative sequence for the nuclear localization suggests possible mechanisms for the NGF-dependent redistribution. For an NGF-inducible IEG product, such growth factor-dependent localization of PC4 is a novel type of regulation in the pathways from the NGF receptor to the adjacent membrane proteins and to the nucleus.