Modelling transport of inhibiting and activating signals and their combined effects on floral induction: application to apple tree.

Floral induction (FI) in shoot apical meristems (SAM) is assumed to be triggered by antagonistic endogenous signals. In fruit trees, FI occurs in some SAM only and is determined by activating and inhibiting signals originating from leaves and fruit, respectively. We developed a model (SigFlow) to quantify on 3D structures the combined impact of such signals and distances at which they act on SAM. Signal transport was simulated considering a signal 'attenuation' parameter, whereas SAM fate was determined by probability functions depending on signal quantities. Model behaviour was assessed on simple structures before being calibrated and validated on a unique experimental dataset of 3D digitized apple trees with contrasted crop loads and subjected to leaf and fruit removal at different scales of tree organization. Model parameter estimations and comparisons of two signal combination functions led us to formulate new assumptions on the mechanisms involved: (i) the activating signal could be transported at shorter distances than the inhibiting one (roughly 50 cm vs 1 m) (ii) both signals jointly act to determine FI with SAM being more sensitive to inhibiting signal than activating one. Finally, the genericity of the model is promising to further understand the physiological and architectural determinisms of FI in plants.

Fresh, freeze-dried or cell wall samples: Which is the most appropriate to determine chemical, structural and rheological variations during apple processing using ATR-FTIR spectroscopy?

Attenuated total reflectance Fourier transform spectroscopy (ATR-FTIR) was applied on fresh (NF), freeze-dried (FD) and cell wall materials (AIS) of raw and processed apples. These samples prepared from 36 apple sets and the corresponding 72 purees, issued from different varieties, agricultural practices, storage periods and processing conditions, were used to build models including exploratory analysis, supervised classification and multivariate calibration. Fresh and freeze-dried samples presented similar fingerprint spectral variations due to processing. ATR-FTIR directly on fresh purees satisfactorily predicted textural properties such as particle average size and volume (RPD > 3.0), while freeze-drying improved assessment of chemical (RPD > 3.2) and rheological (RPD > 3.1) parameters using partial least-squares regression. The assessment of texture and macrocomponents of purees can be obtained with a limited sample preparation. For research applications because of a need of sample preparation, changes of cell wall composition during fruit processing could be assessed in relationship with pectin degradation.

A Novel Effector Protein of Apple Proliferation Phytoplasma Disrupts Cell Integrity of Nicotiana spp. Protoplasts.

Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by 'Candidatus Phytoplasma mali' (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called "Protein in Malus Expressed 2" (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.

Tailored nanocellulose structure depending on the origin. Example of apple parenchyma and carrot root celluloses.

Cellulose is the major polysaccharide of cell walls in every plant, making it one of the most abundant natural polymers on Earth. However, despite many decades of investigations, the supramolecular structure of cellulose and especially its variation in the cell walls of different plants have still not been fully revealed. In the present study, cellulose from the parenchymatic tissue of apple fruits and carrot roots was isolated, and nanocellulose was further prepared by high-intensity ultrasonication. AFM revealed that the obtained nanocellulose differed in dimension between the two plant species. Compared with carrot cellulose, whose nanocellulose was obtained in the form of whiskers, apple cellulose had longer and thinner nanofibrils. Both nanocellulose types also differed in terms of their crystalline structure. XRD data indicated that, compared with the apple cellulose, the carrot cellulose had a higher degree of crystallinity and larger crystallites. Moreover, FTIR and Raman spectroscopy revealed differences between the cellulose types in terms of their methine environment, hydroxymethyl conformations and skeletal vibrations. Additionally, with respect to their mechanical properties, the less crystalline apple cellulose and nanocellulose films were more elastic than the stiffer carrot cellulose and nanocellulose films. The possible reason for such differences between the two cellulose types is related to differences in plant tissue morphology and function. During development, apple fruit cell walls must withstand increasing turgor, probably higher that in the case of carrot tissue; therefore, the cellulose scaffolding must be elastic and strong. On the other hand, carrot, a root vegetable, also has to be strong enough to penetrate the soil as well as for its own growth; thus, the cell wall and cellulose scaffold have to be stiff and tough. Thus the structure of nanocellulose depends not only on the treatment but also on the cellulose source.

Effect of arabinoxylan and ß-glucan stearic acid ester coatings on post-harvest quality of apple (Royal Delicious).

The effect of wheat straw arabinoxylan (AX) and ß-glucan stearic acid ester (SABG) composite coating on the quality and storage life of apple (Royal Delicious) was studied at 22 °C (±2) with relative humidity of 65% and 85% for 60 days. Fresh fruits were coated with surface coatings of AX-SABG, shellac in the concentration range of 1-4%. Application of both AX-SABG (1-4%) and shellac (1-4%) coatings was found to significantly reduce weight loss, respiration rate, fruit softening process, ripening index, color degradation and polyphenol oxidase activity compared to control during the storage period of more than 30 days. However, an AX-SABG coating was more effective in reducing fruit decay and loss of aroma volatiles followed by shellac coated apples; the un-coated apples being showing maximum quality deterioration. These findings confirmed the potential benefits of applying AX-SABG coating to extend the shelf life and quality of apples especially during transportation and storage.

Inhibition of adventitious root development in apple rootstocks by cytokinin is based on its suppression of adventitious root primordia formation.

Cytokinin (CK) inhibits adventitious root (AR) formation in stem cuttings. Little is known, however, about the mechanism underlying the inhibitory effect. In this study, 2 mg l-1 of exogenous 6-benzyl adenine (6-BA) was administered to 3 and 7-day-old apple rootstocks 'M.26' cuttings (3 and 7 days 6-BA) by transferring them from a rooting medium containing indole-3-butanoic acid to the medium containing 6-BA. Anatomical and morphological observations revealed that the exogenous application of 6-BA inhibited primordia formation in the 3 days 6-BA but not the 7 days 6-BA group. The concentration of auxin (IAA), the ratios of IAA/CK and IAA/abscisic acid were lower in 3 days 6-BA than in 7 days 6-BA. Expression analysis of genes known to be associated with AR formation was also analyzed. In the 3 days 6-BA group, high level of CK inhibited the synthesis and transport of auxin, as a result, low endogenous auxin level suppressed the auxin signaling pathway genes, as were other AR development and cell cycle related genes; all of which had an inhibitory impact on AR primordium formation. On the contrary, low CK level in the 7 days 6-BA, reduced the inhibitory impact on auxin levels, leading to an upregulated expression of genes known to promote AR primordia formation. Collectively, our data indicated that 3-7 days is the time period in which AR primordia formation occurs in cuttings of 'M.26' and that the inhibition of AR development by CK is due to the suppression of AR primordia development over 3-7 days period after culturing in rooting medium.

Effect of trisodium phosphate dipping treatment on the quality and energy metabolism of apples.

The effects of postharvest trisodium phosphate (TSP) dipping (0.5 mg/mL) on the quality and mitochondrial energy metabolism of apple fruit (cv. Golden delicious) were studied. The results indicated that TSP treatment inhibited the respiration intensity, delayed the increase of weight loss, and inhibited the decrease of flesh firmness, ascorbic acid (AsA), titratable acid (TA) and soluble solids content (SSC) of apple fruit. The results also indicated that TSP treatment delayed the decline of the content of ATP, ADP and energy charge of apple fruit, and enhanced the activity of H+-ATPase, Ca2+-ATPase, succinate dehydrogenase and cytochrome C oxidase. These results suggested that TSP could maintain the quality of apple fruit by mediating respiration and mitochondrial energy metabolism.

Molecular cloning and functional analysis of a biphenyl phytoalexin-specific O-methyltransferase from apple cell suspension cultures.

MAIN CONCLUSION: This manuscript describes the cloning and functional characterization of a biphenyl phytoalexin biosynthetic gene, 3,5-dihydroxybiphenyl O-methyltransferase from elicitor-treated cell cultures of scab resistant apple cultivar 'Florina'. Apples belong to the subtribe Malinae of the Rosaceae family. Biphenyls and dibenzofurans are the specialized phytoalexins of Malinae, of which aucuparin is the most widely distributed biphenyl. The precursor of aucuparin, 3,5-dihydroxybiphenyl, is a benzoate-derived polyketide, which is formed by the sequential condensation of three molecules of malonyl-CoA and one molecule of benzoyl-CoA in a reaction catalyzed by biphenyl synthase (BIS). This 3,5-dihydroxybiphenyl then undergoes sequential 5-O-methylation, 4-hydroxylation, and finally 3-O-methylation to form aucuparin. A cDNA encoding O-methyltransferase (OMT) was isolated and functionally characterized from the cell cultures of scab-resistant apple cultivar 'Florina' (Malus domestica cultivar 'Florina'; MdOMT) after treatment with elicitor prepared from the apple scab causing fungus Venturia inaequalis. MdOMT catalyzed the regiospecific O-methylation of 3,5-dihydroxybiphenyl at the 5-position to form 3-hydroxy-5-methoxybiphenyl. The enzyme showed absolute substrate preference for 3,5-dihydroxybiphenyl. The elicitor-treated apple cell cultures showed transient increases in the MdOMT (GenBank ID MF740747) and MdBIS3 (GenBank ID JQ390523) transcript levels followed by the accumulation of biphenyls (aucuparin and noraucuparin) and dibenzofuran (eriobofuran) phytoalexins. MdOMT fused with N- and C-terminal yellow fluorescent protein showed cytoplasmic localization in the epidermis of Nicotiana benthamiana leaves. In scab inoculated greenhouse-grown 'Florina' plants, the expression of MdOMT was transiently induced in the stem followed by the accumulation of biphenyl phytoalexins.

Multilacunarity as a spatial multiscale multi-mass morphometric of change in the meso-architecture of plant parenchyma tissue.

The lacunarity index (monolacunarity) averages the behavior of variable size structures in a binary image. The generalized lacunarity concept (multilacunarity) on the basis of generalized distribution moments is an appealing model that can account for differences in the mass content at different scales. The model was tested previously on natural images [J. Vernon-Carter et al., Physica A 388, 4305 (2009)]. Here, the computational aspects of multilacunarity are validated using synthetic binary images that consist of random maps, spatial stochastic patterns, patterns with circular or polygonal elements, and a plane fractal. Furthermore, monolacunarity and detrended fluctuation analysis were employed to quantify the mesostructural changes in the intercellular air spaces of frozen-thawed parenchymatous tissue of pome fruit [N. A. Valous et al., J. Appl. Phys. 115, 064901 (2014)]. Here, the aim is to further examine the coherence of the multilacunarity model for quantifying the mesostructural changes in the intercellular air spaces of parenchymatous tissue of pome and stone fruit, acquired with X-ray microcomputed tomography, after storage and ripening, respectively. The multilacunarity morphometric is a multiscale multi-mass fingerprint of spatial pattern composition, assisting the exploration of the effects of metabolic and physiological activity on the pore space of plant parenchyma tissue.

Gentian (Gentiana triflora) prevents transmission of apple latent spherical virus (ALSV) vector to progeny seeds.

MAIN CONCLUSION: Gentian plants ( Gentiana triflora ) severely restrict apple latent spherical virus (ALSV) invasion to the gametes (pollens and ovules) and block seed transmission to progeny plants. Early flowering of horticultural plants can be induced by infection of ALSV vector expressing Flowering Locus T (FT) gene. In the present study, flowering of gentian plants was induced by infection with an ALSV vector expressing a gentian FT gene and the patterns of seed transmission of ALSV in gentian were compared with those in apple and Nicotiana benthamiana. Infection of gentian progeny plants with ALSV was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and enzyme-linked immunosorbent assay (ELISA). ALSV was not transmitted to the progeny gentian plants, whereas small proportions of apple and N. benthamiana progeny plants are infected with ALSV. The in situ hybridization analyses indicated that ALSVs are not present in gentian pollen and ovules, but detected in most of gametes in apple and N. benthamiana. Collectively, these results suggest that seed transmission of ALSV is blocked in gentian plants through the unknown barriers present in their gametes. On the other hand, apple and N. benthamiana seem to minimize ALSV seed transmission by inhibiting viral propagation in embryos.

Combining Thermotherapy with Cryotherapy for Efficient Eradication of Apple stem grooving virus from Infected In-vitro-cultured Apple Shoots.

Apple stem grooving virus (ASGV), a difficult-to-eradicate virus from apple propagative materials, causes serious damage to apple production. The use of virus-free plants has been and is an effective strategy for control of plant viral diseases. This study aimed to eradicate ASGV from virus-infected in-vitro-cultured shoots of four apple cultivars and one rootstock by combining thermotherapy with cryotherapy. In vitro stock shoots infected with ASGV were thermo-treated using an alternating temperature of 36°C (day) and 32°C (night). Shoot tips were excised from the treated stock shoots and subjected to cryotherapy. Results showed that, although thermotherapy did not influence shoot survival rates, it reduced shoot growth and proliferation of in vitro shoots. Shoot regrowth rates decreased while virus eradication frequencies increased in cryo-treated shoot tips as time durations of thermotherapy increased from 0 to 6 weeks. Shoot regrowth and frequency of virus eradication were positively and negatively correlated, respectively, with the size of shoot tips. The protocol established here yielded shoot regrowth rates and virus eradication frequencies of 33 to 76% and 30 to 100%, respectively, in the four apple cultivars and one rootstock. Thermotherapy altered virus distribution patterns, subsequently resulting in production of a larger virus-free area in the thermo-treated shoot tips. Many cells in the top layers of apical dome and some cells in the youngest leaf primordia survived in cryo-treated shoot tips; these cells were most likely free of virus infection. Thus, plants regenerated from the procedure of combining thermotherapy with cryotherapy were free of ASGV, as judged by reverse-transcription polymerase chain reaction. To the best of our knowledge, this is the widest-spectrum technique reported thus far for the production of ASGV-free plants and provides a novel biotechnology for the production of virus-free plants in Malus spp.

Callus Induction from Various Organs of Dragon Fruit, Apple and Tomato on some Mediums.

BACKGROUND AND OBJECTIVE: Dragon fruit (Hylocereus spp.), apple (Malus sylvestris Mill.) and tomato (Solanum lycopersicum L.) are high potential sources of antioxidant compounds such as phenolics. The compounds have the capability of protecting cells and tissues against free radicals. Secondary metabolite produced by callus cell culture from plant organs also acts as a source of antioxidants. This study aimed to determine the optimal ratio of sucrose and 2,4-D in Murashige and Skoog (MS) medium for callus induction from different plant organ explants. With all of characteristic, callus can be used further for the development of natural cell regeneration agent. METHODOLOGY: This study was conducted using analytical technique. Suitable explants were obtained. They were developed in various concentrations of combination between MS medium and 2,4-D. Callus growth, including their weight and surface was then measured and analyzed by using one-way analysis of variance (ANOVA). RESULTS: Callus was able to grow from its explants in 5-7 days after induction process. They were clear in color and had friable texture. The highest value of fresh weight of dragon fruit callus was obtained through MS supplemented with 1 µL L-1 2,4-D and 30 g sucrose. However, apple and tomato callus induction and growth maintenance reached optimal medium on MS supplemented with 30 g sucrose and 2 µL L-1 2,4-D. CONCLUSION: Callus of apple, dragon fruit and tomato was maintained upon MS supplemented with 30-40 g sucrose and 1-2 µL L-1 2,4-D for optimum induction and growth. The optimization of growth medium will give advantages for further development of natural cell regeneration agent.

Lipid droplet-associated gene expression and chromatin remodelling in LIPASE 5'-upstream region from beginning- to mid-endodormant bud in 'Fuji' apple.

KEY MESSAGE: We found that lipid accumulation in the meristem region and the expression of MdLIP2A, which appears to be regulated by chromatin remodeling, coincided with endodormancy induction in the 'Fuji' apple. In deciduous trees, including apples (Malus × domestica Borkh.), lipid accumulation in the meristem region towards endodormancy induction has been thought to be an important process for the acquisition of cold tolerance. In this study, we conducted histological staining of crude lipids in the meristem region of 'Fuji' apples and found that lipid accumulation coincided with endodormancy induction. Since a major component of lipid bodies (triacylglycerol) is esterified fatty acids, we analysed fatty acid-derived volatile compounds and genes encoding fatty acid-modifying enzymes (MdLOX1A and MdHPL2A); the reduction of lipid breakdown also coincided with endodormancy induction. We then characterised the expression patterns of lipid body-regulatory genes MdOLE1 and MdLIP2A during endodormancy induction and found that the expression of MdLIP2A correlated well with lipid accumulation towards endodormancy induction. Based on these results, we conducted chromatin remodelling studies and localized the cis-element in the 5'-upstream region of MdLIP2A to clarify its regulatory mechanism. Finally, we revealed that chromatin was concentrated - 764 to - 862 bp of the 5'-upstream region of MdLIP2A, which harbours the GARE [gibberellin responsive MYB transcription factor binding site] and CArG [MADS-box transcription factor binding site] motifs-meristem development-related protein-binding sites.

miR156 switches on vegetative phase change under the regulation of redox signals in apple seedlings.

In higher plants, miR156 regulates the vegetative phase change via the target SBP/SPL genes. The regulation of miR156 during ontogenetic processes is not fully understood. In the apple genome, of 31 putative MdMIR156 genes that encode pre-miR156, seven were dominantly expressed. However, the transcript levels of only MdMIR156a5 and MdMIR156a12 decreased significantly during the vegetative phase change, which was consistent with the mature miR156 level, indicating that miR156 is under transcriptional regulation. Leaf H2O2 content was higher in the adult phase than in the juvenile phase because of excess H2O2 accumulation in chloroplasts. When in vitro shoots were treated with menadione, diphenyleneiodonium, L-2-oxothiazolidine-4-carboxylic acid or buthionine sulphoximine, the expressions of MdMIR156a5, MdMIR156a12, and as well miR156 were coordinated with reduced glutathione (GSH) contents and glutathione/glutathione disulfide ratio but not H2O2 contents. Alteration of miR156 expression level by MdMIR156a6-overexpressing or miR156-mimetic transgenic Nicotiana benthamiana did not cause a corresponding change in reactive oxygen species or GSH status. Collectively, the results indicate that the vegetative phase change in apple is controlled by the MdMIR156a5 and MdMIR156a12 transcriptional regulatory network in response to the plastid-nucleus redox signals, such as GSH.

Raman imaging of changes in the polysaccharides distribution in the cell wall during apple fruit development and senescence.

MAIN CONCLUSION: Du ring on-tree ripening, the pectin distribution changed from polydispersed in cell wall to cumulated in cell wall corners. During apple storage, the pectin distribution returned to evenly dispersed along the cell wall. The plant cell wall influences the texture properties of fruit tissue for example apples become softer during ripening and postharvest storage. This softening process is believed to be mainly connected with changes in the cell wall composition due to polysaccharides undergoing an enzymatic degradation. These changes in polysaccharides are currently mainly investigated via chemical analysis or monoclonal labeling. Here, we propose the application of Raman microscopy for evaluating the changes in the polysaccharide distribution in the cell wall of apples during both ripening and postharvest storage. The apples were harvested 1 month and 2 weeks before optimal harvest date as well as at the optimal harvest date. The apples harvested at optimal harvest date were stored for 3 months. The Raman maps, as well as the chemical analysis were obtained for each harvest date and after 1, 2 and 3 months of storage, respectively. The analysis of the Raman maps showed that the pectins in the middle lamella and primary cell wall undergo a degradation. The changes in cellulose and hemicellulose were less pronounced. These findings were confirmed by the chemical analysis results. During development changes of pectins from a polydispersed form in the cell walls to a cumulated form in cell wall corners could be observed. In contrast after 3 months of apple storage we could observe an substantial pectin decrease. The obtained results demonstrate that Raman chemical imaging might be a very useful tool for a first identification of compositional changes in plant tissue during their development. The great advantage Raman microspectroscopy offers is the simultaneous localization and identification of polysaccharides within the cell wall and plant tissue.

Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet-vitrification and encapsulation-dehydration procedures.

A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration procedure that was previously reported by us. In both procedures, three types of shoot tip recovery were observed following cryopreservation: callus formation without shoot regrowth, leaf formation without shoot regrowth, and shoot regrowth. Three categories of histological observations were also identified in cross-sections of shoot tips recovered after cryopreservation using the two cryogenic procedures. In category 1, almost all of the cells (94-95%) in the apical dome (AD) were damaged or killed and only some cells (30-32%) in the leaf primordia (LPs) survived. In category 2, only a few cells (18-20%) in the AD and some cells (30-31%) in the LPs survived. In category 3, majority of the cells (60-62%) in the AD and some cells (30-33%) in the LPs survived. These data suggest that shoot regrowth is correlated to the presence of a majority of surviving cells in the AD after liquid nitrogen exposure. No polymorphic bands were detected by inter-simple sequence repeats or by random amplified polymorphic DNA assessments, and ploidy levels analyzed by flow cytometry were unchanged when plants recovered after cryoexposure were compared to controls. The droplet-vitrification procedure appears to be robust since seven genotypes representing four Malus species and one hybrid recovered shoots following cryopreservation. Mean shoot regrowth levels of these seven genotypes were 48% in the droplet-vitrification method, which were lower than those (61%) in the encapsulation-dehydration procedure reported in our previous study, suggesting the latter may be preferred for routine cryobanking applications for Malus shoot tips.

Multi-scale simulation of plant stem reinforcement by brachysclereids: A case study in apple fruit peduncles.

Sclereid formation in addition to or in gaps of fragmented fibre rings is common in dicotyledonous plant stems. Whether this sclereid formation is force-triggered remains open so far. In fruit peduncles of several Malus species as modified plant stems, for example, the persistent fibre ring is displaced to the centre by formation of cortex parenchyma during growth. Parenchyma cells subsequently differentiate into an additional layer of brachysclereids, previously interpreted as an adaptation to continuously rising fruit loads. The present study pursues a multi-scale numerical modelling approach, to verify the important effect for different cellular architectures in both sclerenchyma categories on the stiffness of these tissues and the entire peduncle. First, different material properties are simulated analogue to plant tissues on the basis of three cell types. A regular three-dimensional and a random Voronoi microstructure combined with various mechanical cell wall parameters are applied. Using homogenisation simulations based on HILL's principle, numerical calculations predict a lower effective homogenised tissue stiffness of isodiametric brachysclereids compared to those of fibres, confirming experimentally obtained data from Malus fruit peduncles. Furthermore, a curved peduncle model with a complex arrangement of different material layers is generated. Diverse material sets are tested under three representative loadings, using an adaptive diffuse domain approach (AMDiS). The model explains the function of sclereids as considerable contributors to the stiffness against bending and tensile deformations, as well as torsion, especially in consequence of superimposed load conditions in the case of a curved plant stem.

Apple russeting as seen through the RNA-seq lens: strong alterations in the exocarp cell wall.

Russeting, a commercially important defect in the exocarp of apple (Malus × domestica), is mainly characterized by the accumulation of suberin on the inner part of the cell wall of the outer epidermal cell layers. However, knowledge on the underlying genetic components triggering this trait remains sketchy. Bulk transcriptomic profiling was performed on the exocarps of three russeted and three waxy apple varieties. This experimental design was chosen to lower the impact of genotype on the obtained results. Validation by qPCR was carried out on representative genes and additional varieties. Gene ontology enrichment revealed a repression of lignin and cuticle biosynthesis genes in russeted exocarps, concomitantly with an enhanced expression of suberin deposition, stress responsive, primary sensing, NAC and MYB-family transcription factors, and specific triterpene biosynthetic genes. Notably, a strong correlation (R(2) = 0.976) between the expression of a MYB93-like transcription factor and key suberin biosynthetic genes was found. Our results suggest that russeting is induced by a decreased expression of cuticle biosynthetic genes, leading to a stress response which not only affects suberin deposition, but also the entire structure of the cell wall. The large number of candidate genes identified in this study provides a solid foundation for further functional studies.

Differential response of cell-cycle and cell-expansion regulators to heat stress in apple (Malus domestica) fruitlets.

Temperature is one of the most significant factors affecting physiological and biochemical aspects of fruit development. Current and progressing global warming is expected to change climate in the traditional deciduous fruit tree cultivation regions. In this study, 'Golden Delicious' trees, grown in a controlled environment or commercial orchard, were exposed to different periods of heat treatment. Early fruitlet development was documented by evaluating cell number, cell size and fruit diameter for 5-70 days after full bloom. Normal activities of molecular developmental and growth processes in apple fruitlets were disrupted under daytime air temperatures of 29°C and higher as a result of significant temporary declines in cell-production and cell-expansion rates, respectively. Expression screening of selected cell cycle and cell expansion genes revealed the influence of high temperature on genetic regulation of apple fruitlet development. Several core cell-cycle and cell-expansion genes were differentially expressed under high temperatures. While expression levels of B-type cyclin-dependent kinases and A- and B-type cyclins declined moderately in response to elevated temperatures, expression of several cell-cycle inhibitors, such as Mdwee1, Mdrbr and Mdkrps was sharply enhanced as the temperature rose, blocking the cell-cycle cascade at the G1/S and G2/M transition points. Moreover, expression of several expansin genes was associated with high temperatures, making them potentially useful as molecular platforms to enhance cell-expansion processes under high-temperature regimes. Understanding the molecular mechanisms of heat tolerance associated with genes controlling cell cycle and cell expansion may lead to the development of novel strategies for improving apple fruit productivity under global warming.

bcpmr1 encodes a P-type Ca(2+)/Mn(2+)-ATPase mediating cell-wall integrity and virulence in the phytopathogen Botrytis cinerea.

The cell wall of fungi is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. These usually contain both N- and O-linked oligosaccharides, coupled to the proteins by stepwise addition of mannose residues by mannosyltransferases in the endoplasmic reticulum and the Golgi apparatus. In yeast, an essential luminal cofactor for these mannosyltransferases is Mn(2+) provided by the Ca(2+)/Mn(2+)-ATPase known as Pmr1. In this study, we have identified and characterized the Botrytis cinerea pmr1 gene, the closest homolog of yeast PMR1. We hypothesized that bcpmr1 also encodes a Ca(2+)/Mn(2+)-ATPase that plays an important role in the protein glycosylation pathway. Phenotypic analysis showed that bcpmr1 null mutants displayed a significant reduction in conidial production, radial growth and diameter of sclerotia. Significant alterations in hyphal cell wall composition were observed including a 83% decrease of mannan levels and an increase in the amount of chitin and glucan. These changes were accompanied by a hypersensitivity to cell wall-perturbing agents such as Calcofluor white, Congo red and zymolyase. Importantly, the Δbcpmr1 mutant showed reduced virulence in tomato (leafs and fruits) and apple (fruits) and reduced biofilm formation. Together, our results highlight the importance of bcpmr1 for protein glycosylation, cell wall structure and virulence of B. cinerea.