Influence of mixed and single infection of grapevine leafroll-associated viruses and viral load on berry quality.

Grapevine leafroll disease is a viral disease that affects grapevines (Vitis vinifera L.) and has a severe economic impact on viticulture. In this study, the effect of grapevine leafroll-associated viruses (GLRaV) on berry quality was investigated in clones of cultivar cv. Crimson Seedless table grapes infected with GLRaV. RT-PCR confirmed the identity of the clones: clone 3236, infected only with GLRaV-3 (termed single); clone 3215, infected with GLRaV-3, GLRaV-4 strain 9 and grapevine virus A (termed mixed); and a viral free clone of the same genetic background of the infected clones (termed control). The berry quality indices of size, sugar, acidity and anthocyanin content were measured at harvest maturity. RT-qPCR was used to determine the viral load. The study was repeated over 2 year. A two-way, multivariate analysis of variance was applied with clone and year as independent variables and the measured berry quality parameters as a dependent variable. All dependent variables were significantly affected by viral infection (Wilks, λ, (2,33) = 0.033895, P-value <0.001), while only titratable acidity was affected by year. The average berry dry mass decreased (P-value <0.001). The water content of both infected clones was greater than that of the control (P-value <0.001). Both infected clones displayed reduced sugar content as a fraction of the berry dry mass (P-value <0.001). The anthocyanin and the phenol content of the infected clones were significantly reduced compared with the control clone (P < 0.001, P < 0.05, clone 3236 and clone 3215, respectively). Finally, the viral load was highly variable, and no quantitative relationship between viral load and berry composition was found.

Hydrogen Cyanamide Causes Reversible G2/M Cell Cycle Arrest Accompanied by Oxidation of the Nucleus and Cytosol.

Hydrogen cyanamide (HC) has been widely used in horticulture to trigger bud burst following dormancy. Its use has been banned in some countries due to human health concerns, however the search for effective safe alternatives is delayed by lack of knowledge of the mechanism of HC action. Earlier studies demonstrate that HC stimulates the production of reactive oxygen species (ROS) and alters the rate of cell division. However, the relationships between HC effects on ROS, redox (reduction/oxidation) homeostasis and cell division are unknown. This study used Arabidopsis thaliana ((L.) Heynh.) seedlings expressing the redox reporter roGFP2 to measure the oxidation states of the nuclei and cytosol in response to HC treatment. The Cytrap dual cell cycle phase marker system and flow cytometry were used to study associated changes in cell proliferation. HC (1.5 mM) reversibly inhibited root growth during a 24 h treatment. Higher concentrations were not reversible. HC did not synchronise the cell cycle, in contrast to hydroxyurea. Rather, HC caused a gradual accumulation of cells in the G2/M phase and decline of G1/S phase cells, 16 to 24 h post-treatment. This was accompanied by increased oxidation of both the nuclei and cytosol. Taken together, these findings show that HC impairs proliferation of embryonic root meristem cells in a reversible manner through restriction of G2/M transition accompanied by increased cellular oxidation.

An Evaluation of Nuclei Preparation of the Dormant Axillary Bud of Grapevine for Cell Cycle Analysis by Flow Cytometry.

Whether the division of cells of a dormant meristem may be arrested, e.g., in the G1 phase, has proven to be an extremely difficult hypothesis to test. This is particularly so for woody perennial buds, where dormant and quiescent states are diffuse, and the organ may remain visibly unchanged for 6-9 months of the year. Flow cytometry (FCM) has been widely applied in plant studies to determine the genome size and endopolyploidy. In this study, we present the application of FCM to measure the cell cycle status in mature dormant buds of grapevine (Vitis vinifera cv. Cabernet Sauvignon), which represent a technically recalcitrant structure. This protocol illustrates the optimisation and validation of FCM data analysis to calculate the cell cycle status, or mitotic index, of dormant grapevine buds. We have shown how contamination with debris can be experimentally managed and give reference to the more malleable tomato leaves. We have also given a clear illustration of the primary pitfalls of data analysis to avoid artefacts or false results. Data acquisition and analysis strategies are detailed and can be readily applied to analyse FCM data from other recalcitrant plant samples.

The bud dormancy disconnect: latent buds of grapevine are dormant during summer despite a high metabolic rate.

Grapevine (Vitis vinifera L.) displays wide plasticity to climate; however, the physiology of dormancy along a seasonal continuum is poorly understood. Here we investigated the apparent disconnect between dormancy and the underlying respiratory physiology and transcriptome of grapevine buds, from bud set in summer to bud burst in spring. The establishment of dormancy in summer was pronounced and reproducible; however, this was coupled with little or no change in physiology, indicated by respiration, hydration, and tissue oxygen tension. The release of dormancy was biphasic; the depth of dormancy declined substantially by mid-autumn, while the subsequent decline towards spring was moderate. Observed changes in physiology failed to explain the first phase of dormancy decline, in particular. Transcriptome data contrasting development from summer through to spring also indicated that dormancy was poorly reflected by metabolic quiescence during summer and autumn. Gene Ontology and enrichment data revealed the prevailing influence of abscisic acid (ABA)-related gene expression during the transition from summer to autumn, and promoter motif analysis suggested that photoperiod may play an important role in regulating ABA functions during the establishment of dormancy. Transcriptomic data from later transitions reinforced the importance of oxidation and hypoxia as physiological cues to regulate the maintenance of quiescence and resumption of growth. Collectively these data reveal a novel disconnect between growth and metabolic quiescence in grapevine following bud set, which requires further experimentation to explain the phenology and dormancy relationships.

The initiation of bud burst in grapevine features dynamic regulation of the apoplastic pore size.

The physiological constraints on bud burst in woody perennials, including vascular development and oxygenation, remain unresolved. Both light and tissue oxygen status have emerged as important cues for vascular development in other systems; however, grapevine buds have only a facultative light requirement, and data on the tissue oxygen status have been confounded by the spatial variability within the bud. Here, we analysed apoplastic development at early stages of grapevine bud burst and combined molecular modelling with histochemical techniques to determine the pore size of cell walls in grapevine buds. The data demonstrate that quiescent grapevine buds were impermeable to apoplastic dyes (acid fuchsin and eosin Y) until after bud burst was established. The molecular exclusion size was calculated to be 2.1 nm, which would exclude most macromolecules except simple sugars and phytohormones until after bud burst. We used micro-computed tomography to demonstrate that tissue oxygen partial pressure data correlated well with structural heterogeneity of the bud and differences in tissue density, confirming that the primary bud complex becomes rapidly and preferentially oxygenated during bud burst. Taken together, our results reveal that the apoplastic porosity is highly regulated during the early stages of bud burst, suggesting a role for vascular development in the initial, rapid oxygenation of the primary bud complex.

Developmental control of hypoxia during bud burst in grapevine.

Dormant or quiescent buds of woody perennials are often dense and in the case of grapevine (Vitis vinifera L.) have a low tissue oxygen status. The precise timing of the decision to resume growth is difficult to predict, but once committed, the increase in tissue oxygen status is rapid and developmentally regulated. Here, we show that more than a third of the grapevine homologues of widely conserved hypoxia-responsive genes and nearly a fifth of all grapevine genes possessing a plant hypoxia-responsive promoter element were differentially regulated during bud burst, in apparent harmony with resumption of meristem identity and cell-cycle gene regulation. We then investigated the molecular and biochemical properties of the grapevine ERF-VII homologues, which in other species are oxygen labile and function in transcriptional regulation of hypoxia-responsive genes. Each of the 3 VvERF-VIIs were substrates for oxygen-dependent proteolysis in vitro, as a function of the N-terminal cysteine. Collectively, these data support an important developmental function of oxygen-dependent signalling in determining the timing and effective coordination bud burst in grapevine. In addition, novel regulators, including GASA-, TCP-, MYB3R-, PLT-, and WUS-like transcription factors, were identified as hallmarks of the orderly and functional resumption of growth following quiescence in buds.

On the language and physiology of dormancy and quiescence in plants.

The language of dormancy is rich and poetic, as researchers spanning disciplines and decades have attempted to understand the spell that entranced 'Sleeping Beauty', and how she was gently awoken. The misleading use of 'dormancy', applied to annual axillary buds, for example, has confounded progress. Language is increasingly important as genetic and genomic approaches become more accessible to species of agricultural and ecological importance. Here we examine how terminology has been applied to different eco-physiological states in plants, and with pertinent reference to quiescent states described in other domains of life, in order to place plant quiescence and dormancy in a more complete context than previously described. The physiological consensus defines latency or quiescence as opportunistic avoidance states, where growth resumes in favourable conditions. In contrast, the dormant state in higher plants is entrained in the life history of the organism. Competence to resume growth requires quantitative and specific conditioning. This definition applies only to the embryo of seeds and specialized meristems in higher plants; however, mechanistic control of dormancy extends to mobile signals from peripheral tissues and organs, such as the endosperm of seed or subtending leaf of buds. The distinction between dormancy, quiescence, and stress-hardiness remains poorly delineated, most particularly in buds of winter perennials, which comprise multiple meristems of differing organogenic states. Studies in seeds have shown that dormancy is not a monogenic trait, and limited study has thus far failed to canalize dormancy as seen in seeds and buds. We argue that a common language, based on physiology, is central to enable further dissection of the quiescent and dormant states in plants. We direct the topic largely to woody species showing a single cycle of growth and reproduction per year, as these bear the majority of global timber, fruit, and nut production, as well being of great ecological value. However, for context and hypotheses, we draw on knowledge from annuals and other specialized plant conditions, from a perspective of the major physical, metabolic, and molecular cues that regulate cellular activity.

Spatio-temporal relief from hypoxia and production of reactive oxygen species during bud burst in grapevine (Vitis vinifera).

BACKGROUND AND AIMS: Plants regulate cellular oxygen partial pressures (pO2), together with reduction/oxidation (redox) state in order to manage rapid developmental transitions such as bud burst after a period of quiescence. However, our understanding of pO2 regulation in complex meristematic organs such as buds is incomplete and, in particular, lacks spatial resolution. METHODS: The gradients in pO2 from the outer scales to the primary meristem complex were measured in grapevine (Vitis vinifera) buds, together with respiratory CO2 production rates and the accumulation of superoxide and hydrogen peroxide, from ecodormancy through the first 72 h preceding bud burst, triggered by the transition from low to ambient temperatures. KEY RESULTS: Steep internal pO2 gradients were measured in dormant buds with values as low as 2·5 kPa found in the core of the bud prior to bud burst. Respiratory CO2 production rates increased soon after the transition from low to ambient temperatures and the bud tissues gradually became oxygenated in a patterned process. Within 3 h of the transition to ambient temperatures, superoxide accumulation was observed in the cambial meristem, co-localizing with lignified cellulose associated with pro-vascular tissues. Thereafter, superoxide accumulated in other areas subtending the apical meristem complex, in the absence of significant hydrogen peroxide accumulation, except in the cambial meristem. By 72 h, the internal pO2 gradient showed a biphasic profile, where the minimum pO2 was external to the core of the bud complex. CONCLUSIONS: Spatial and temporal control of the tissue oxygen environment occurs within quiescent buds, and the transition from quiescence to bud burst is accompanied by a regulated relaxation of the hypoxic state and accumulation of reactive oxygen species within the developing cambium and vascular tissues of the heterotrophic grapevine buds.