Combined physiological, transcriptome, and cis-regulatory element analyses indicate that key aspects of ripening, metabolism, and transcriptional program in grapes (Vitis vinifera L.) are differentially modulated accordingly to fruit size.

BACKGROUND: In wine grape production, management practices have been adopted to optimize grape and wine quality attributes by producing, or screening for, berries of smaller size. Fruit size and composition are influenced by numerous factors that include both internal (e.g. berry hormone metabolism) and external (e.g. environment and cultural practices) factors. Combined physiological, biochemical, and transcriptome analyses were performed to improve our current understanding of metabolic and transcriptional pathways related to berry ripening and composition in berries of different sizes. RESULTS: The comparison of berry physiology between small and large berries throughout development (from 31 to 121 days after anthesis, DAA) revealed significant differences in firmness, the rate of softening, and sugar accumulation at specific developmental stages. Small berries had significantly higher skin to berry weight ratio, lower number of seeds per berry, and higher anthocyanin concentration compared to large berries. RNA-sequencing analyses of berry skins at 47, 74, 103, and 121 DAA revealed a total of 3482 differentially expressed genes between small and large berries. Abscisic acid, auxin, and ethylene hormone pathway genes were differentially modulated between berry sizes. Fatty acid degradation and stilbenoid pathway genes were upregulated at 47 DAA while cell wall degrading and modification genes were downregulated at 74 DAA in small compared to large berries. In the late ripening stage, concerted upregulation of the general phenylpropanoid and stilbenoid pathway genes and downregulation of flavonoid pathway genes were observed in skins of small compared to large berries. Cis-regulatory element analysis of differentially expressed hormone, fruit texture, flavor, and aroma genes revealed an enrichment of specific regulatory motifs related to bZIP, bHLH, AP2/ERF, NAC, MYB, and MADS-box transcription factors. CONCLUSIONS: The study demonstrates that physiological and compositional differences between berries of different sizes parallel transcriptome changes that involve fruit texture, flavor, and aroma pathways. These results suggest that, in addition to direct effects brought about by differences in size, key aspects involved in the regulation of ripening likely contribute to different quality profiles between small and large berries.

A revised and unified pressure-clamp/relaxation theory for studying plant cell water relations with pressure probes: in-situ determination of cell volume for calculation of volumetric elastic modulus and hydraulic conductivity.

The cell-pressure-probe is a unique tool to study plant water relations in-situ. Inaccuracy in the estimation of cell volume (νo) is the major source of error in the calculation of both cell volumetric elastic modulus (ε) and cell hydraulic conductivity (Lp). Estimates of νo and Lp can be obtained with the pressure-clamp (PC) and pressure-relaxation (PR) methods. In theory, both methods should result in comparable νo and Lp estimates, but this has not been the case. In this study, the existing νo-theories for PC and PR methods were reviewed and clarified. A revised νo-theory was developed that is equally valid for the PC and PR methods. The revised theory was used to determine νo for two extreme scenarios of solute mixing between the experimental cell and sap in the pressure probe microcapillary. Using a fully automated cell-pressure-probe (ACPP) on leaf epidermal cells of Tradescantia virginiana, the validity of the revised theory was tested with experimental data. Calculated νo values from both methods were in the range of optically determined νo (=1.1-5.0nL) for T. virginiana. However, the PC method produced a systematically lower (21%) calculated νo compared to the PR method. Effects of solute mixing could only explain a potential error in calculated νo of <3%. For both methods, this discrepancy in νo was almost identical to the discrepancy in the measured ratio of ΔV/ΔP (total change in microcapillary sap volume versus corresponding change in cell turgor) of 19%, which is a fundamental parameter in calculating νo. It followed from the revised theory that the ratio of ΔV/ΔP was inversely related to the solute reflection coefficient. This highlighted that treating the experimental cell as an ideal osmometer in both methods is potentially not correct. Effects of non-ideal osmotic behavior by transmembrane solute movement may be minimized in the PR as compared to the PC method.

The relationship between root hydraulics and scion vigour across Vitis rootstocks: what role do root aquaporins play?

Vitis vinifera scions are commonly grafted onto rootstocks of other grape species to influence scion vigour and provide resistance to soil-borne pests and abiotic stress; however, the mechanisms by which rootstocks affect scion physiology remain unknown. This study characterized the hydraulic physiology of Vitis rootstocks that vary in vigour classification by investigating aquaporin (VvPIP) gene expression, fine-root hydraulic conductivity (Lp(r)), % aquaporin contribution to Lp(r), scion transpiration, and the size of root systems. Expression of several VvPIP genes was consistently greater in higher-vigour rootstocks under favourable growing conditions in a variety of media and in root tips compared to mature fine roots. Similar to VvPIP expression patterns, fine-root Lp(r) and % aquaporin contribution to Lp(r) determined under both osmotic (Lp(r)(Osm)) and hydrostatic (Lp(r)(Hyd)) pressure gradients were consistently greater in high-vigour rootstocks. Interestingly, the % aquaporin contribution was nearly identical for Lp(r)(Osm) and Lp(r)(Hyd) even though a hydrostatic gradient would induce a predominant flow across the apoplastic pathway. In common scion greenhouse experiments, leaf area-specific transpiration (E) and total leaf area increased with rootstock vigour and were positively correlated with fine-root Lp(r). These results suggest that increased canopy water demands for scion grafted onto high-vigour rootstocks are matched by adjustments in root-system hydraulic conductivity through the combination of fine-root Lp(r) and increased root surface area.

Leaf scorch symptoms are not correlated with bacterial populations during Pierce's disease.

Xylella fastidiosa (Xf) is a xylem-limited bacterium that lives as a harmless endophyte in most plant species but is pathogenic in several agriculturally important crops such as coffee, citrus, and grapevine (Vitis vinifera L.). In susceptible cultivars of grapevine, Xf infection results in leaf scorch, premature leaf senescence, and eventually vine death; a suite of symptoms collectively referred to as Pierce's disease. A qPCR assay was developed to determine bacterial concentrations in planta and these concentrations were related to the development of leaf-scorch symptoms. The concentration of Xf in leaves of experimental grapevines grown in the greenhouse was similar to the concentration of Xf in leaves of naturally infected plants in the field. The distribution of Xf was patchy within and among leaves. Some whole leaves exhibited severe leaf-scorch symptoms in the absence of high concentrations of Xf. Despite a highly sensitive assay and a range of Xf concentrations from 10(2) to 10(9) cells g(-1) fresh weight, no clear relationship between bacterial population and symptom development during Pierce's disease was revealed. Thus, high and localized concentrations of Xf are not necessary for the formation of leaf-scorch symptoms. The results are interpreted as being consistent with an atiology that involves a systemic plant response.

Lateral root initiation in Arabidopsis: developmental window, spatial patterning, density and predictability.

BACKGROUND AND AIMS: The basic regulatory mechanisms that control lateral root (LR) initiation are still poorly understood. An attempt is made to characterize the pattern and timing of LR initiation, to define a developmental window in which LR initiation takes place and to address the question of whether LR initiation is predictable. METHODS: The spatial patterning of LRs and LR primordia (LRPs) on cleared root preparations were characterized. New measures of LR and LRP densities (number of LRs and/or LRPs divided by the length of the root portions where they are present) were introduced and illustrate the shortcomings of the more customarily used measure through a comparative analysis of the mutant aux1-7. The enhancer trap line J0121 was used to monitor LR initiation in time-lapse experiments and a plasmolysis-based method was developed to determine the number of pericycle cells between successive LRPs. KEY RESULTS: LRP initiation occurred strictly acropetally and no de novo initiation events were found between already developed LRs or LRPs. However, LRPs did not become LRs in a similar pattern. The longitudinal spacing of lateral organs was variable and the distance between lateral organs was proportional to the number of cells and the time between initiations of successive LRPs. There was a strong tendency towards alternation in LR initiation between the two pericycle cell files adjacent to the protoxylem poles. LR density increased with time due to the emergence of slowly developing LRPs and appears to be unique for individual Arabidopsis accessions. CONCLUSIONS: In Arabidopsis there is a narrow developmental window for LR initiation, and no specific cell-count or distance-measuring mechanisms have been found that determine the site of successive initiation events. Nevertheless, the branching density and lateral organ density (density of LRs and LRPs) are accession-specific, and based on the latter density the average distance between successive LRs can be predicted.

Genetic engineering of phytochrome biosynthesis in bacteria.

The bilin prosthetic groups of the phytochrome photoreceptors and the light-harvesting phycobiliprotein antennae arise from the oxygen-dependent ring opening of heme. Two ferredoxin-dependent enzymes contribute to this conversion: a heme oxygenase and a bilin reductase with discrete double-bond specificity. Using a dual plasmid system, one expressing a truncated cyanobacterial apophytochrome 1, Cph1(N514), and the other expressing a two-gene operon consisting of a heme oxygenase and a bilin reductase, these studies establish the feasibility of producing photoactive phytochromes in any heme-containing cell. Heterologous expression systems for phytochromes not only will facilitate genetic analysis of their assembly, spectrophotometric activity, and biological function, but also might afford the means to regulate gene expression by light in nonplant cells.

Deletion of the trichodiene synthase gene of Fusarium venenatum: two systems for repeated gene deletions.

The trichodiene synthase (tri5) gene of Fusarium venenatum was cloned from a genomic library. Vectors were created in which the tri5 coding sequence was replaced with the Neurospora crassa nitrate reductase (nit3) gene and with the Aspergillus nidulans acetamidase (amdS) gene flanked by direct repeats. The first vector was utilized to transform a nitrate reductase (niaD) mutant of F. venenatum to prototrophy, and the second vector was utilized to confer acetamide utilization to the wild-type strain. Several of the transformants lost the capacity to produce the trichothecene diacetoxyscirpenol and were shown by hybridization analysis to have gene replacements at the tri5 locus. The nit3 gene was removed by retransformation with a tri5 deletion fragment and selection on chlorate. The amdS gene was shown to excise spontaneously via the flanking direct repeats when spores were plated onto fluoroacetamide.

Targeted mutations in a Trametes villosa laccase. Axial perturbations of the T1 copper.

Trametes villosa laccase was mutated on a tetrapeptide segment near the type 1 site. The mutations F463M and F463L were at the position corresponding to the type 1 copper axial methionine (M517) ligand in Zucchini ascorbate oxidase. The mutations E460S and A461E were near the T1 copper site. The mutated Trametes laccases were expressed in an Aspergillus oryzae host and characterized. The E460S mutation failed to produce a transformant with meaningful expression. The F463L and A461E mutations did not significantly alter the molecular and enzymological properties of the laccase. In contrast, the F463M mutation resulted in a type 1 copper site with an EPR signal intermediate between that of the wild type laccase and plastocyanin, an altered UV-visible spectrum, and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation led to a more basic optimal pH as well as an increase in kcat and Km. These effects are attributed to a significant perturbation of the T1 copper center caused by the coordination of the axial methionine (M463) ligand.