A RhABF2/Ferritin module affects rose (Rosa hybrida) petal dehydration tolerance and senescence by modulating iron levels.

Plants often develop the capacity to tolerate moderate and reversible environmental stresses, such as drought, and to re-establish normal development once the stress has been removed. An example of this phenomenon is provided by cut rose (Rosa hybrida) flowers, which experience typical reversible dehydration stresses during post-harvest handling after harvesting at the bud stages. The molecular mechanisms involved in rose flower dehydration tolerance are not known, however. Here, we characterized a dehydration- and abscisic acid (ABA)-induced ferritin gene (RhFer1). Dehydration-induced free ferrous iron (Fe2+ ) is preferentially sequestered by RhFer1 and not transported outside of the petal cells, to restrict oxidative stresses during dehydration. Free Fe2+ accumulation resulted in more serious oxidative stresses and the induction of genes encoding antioxidant enzyme in RhFer1-silenced petals, and poorer dehydration tolerance was observed compared with tobacco rattle virus (TRV) controls. We also determined that RhABF2, an AREB/ABF transcription factor involved in the ABA signaling pathway, can activate RhFer1 expression by directly binding to its promoter. The silencing of RhABF2 decreased dehydration tolerance and disrupted Fe homeostasis in rose petals during dehydration, as did the silencing of RhFer1. Although both RhFer1 and Fe transporter genes are induced during flower natural senescence in plants, the silencing of RhABF2 or RhFer1 accelerates the petal senescence processes. These results suggest that the regulatory module RhABF2/RhFer1 contributes to the maintenance of Fe levels and enhances dehydration tolerance through the action of RhFer1 locally sequestering free Fe2+ under dehydration conditions, and plays synergistic roles with transporter genes during flower senescence.

Assessing the influence of structural disorder on the plant epidermal cells' optical properties: a numerical analysis.

Many plant surfaces, such as rose petals, display lens-like epidermal cells that are known to assist the collection and focusing of the sunlight. Those cells form an array with a high degree of structural irregularities including disorder in the height and orientation of the cells, and in their arrangement. In this study, we numerically analyze the influence of structural disorder on the optical properties of a 3D modeled epidermal cell array using ray tracing simulations. We conclude that the anti-reflection properties of such structures are almost unperturbed by disorder effects, although the latter can notably broaden the propagation angle distribution of the collected light. Those results also have a direct implication on the design of plant-inspired light management structures. This aspect is illustrated by introducing the example of a thin-film solar cell covered by a light harvesting epidermal cells replica and simulated for each of the three disorder types considered.

Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening.

Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes' biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme-cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors - especially compounds that generate singlet oxygen ((1)O2) e.g., riboflavin (IC50 29 µM), retinoic acid, eosin (IC50 27 µM) and erythrosin (IC50 36 µM). The riboflavin effect was light-dependent, supporting (1)O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (-)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 µM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 µM mycophenolic acid and (-)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads.

A Rosa canina WUSCHEL-related homeobox gene, RcWOX1, is involved in auxin-induced rhizoid formation.

Homeobox (HB) proteins are important transcription factors that regulate the developmental decisions of eukaryotes. WUSCHEL-related homeobox (WOX) transcription factors, known as a plant-specific HB family, play a key role in plant developmental processes. Our previous work has indicated that rhizoids are induced by auxin in rose (Rosa spp.), which acts as critical part of an efficient plant regeneration system. However, the function of WOX genes in auxin-induced rhizoid formation remains unclear. Here, we isolated and characterized a WUSCHEL-related homeobox gene from Rosa canina, RcWOX1, containing a typical homeodomain with 65 amino acid residues. Real-time reverse transcription PCR (qRT-PCR) analysis revealed that RcWOX1 was expressed in the whole process of callus formation and in the early stage of rhizoid formation. Moreover, its expression was induced by auxin treatment. In Arabidopsis transgenic lines expressing the RcWOX1pro::GUS and 35S::GFP-RcWOX1, RcWOX1 was specifically expressed in roots and localized to the nucleus. Overexpression of RcWOX1 in Arabidopsis increased lateral root density and induced upregulation of PIN1 and PIN7 genes. Therefore, we postulated that RcWOX1 is a functional transcription factor that plays an essential role in auxin-induced rhizoid formation.

An NAC transcription factor controls ethylene-regulated cell expansion in flower petals.

Cell expansion is crucial for plant growth. It is well known that the phytohormone ethylene functions in plant development as a key modulator of cell expansion. However, the role of ethylene in the regulation of this process remains unclear. In this study, 2,189 ethylene-responsive transcripts were identified in rose (Rosa hybrida) petals using transcriptome sequencing and microarray analysis. Among these transcripts, an NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor gene, RhNAC100, was rapidly and dramatically induced by ethylene in the petals. Interestingly, accumulation of the RhNAC100 transcript was modulated by ethylene via microRNA164-dependent posttranscriptional regulation. Overexpression of RhNAC100 in Arabidopsis (Arabidopsis thaliana) substantially reduced the petal size by repressing petal cell expansion. By contrast, silencing of RhNAC100 in rose petals using virus-induced gene silencing significantly increased petal size and promoted cell expansion in the petal abaxial subepidermis (P < 0.05). Expression analysis showed that 22 out of the 29 cell expansion-related genes tested exhibited changes in expression in RhNAC100-silenced rose petals. Moreover, of those genes, one cellulose synthase and two aquaporin genes (Rosa hybrida Cellulose Synthase2 and R. hybrida Plasma Membrane Intrinsic Protein1;1/2;1) were identified as targets of RhNAC100. Our results suggest that ethylene regulates cell expansion by fine-tuning the microRNA164/RhNAC100 module and also provide new insights into the function of NAC transcription factors.

Alternative pathways of dehydroascorbic acid degradation in vitro and in plant cell cultures: novel insights into vitamin C catabolism.

L-Ascorbate catabolism involves reversible oxidation to DHA (dehydroascorbic acid), then irreversible oxidation or hydrolysis. The precursor-product relationships and the identity of several major DHA breakdown products remained unclear. In the presence of added H2O2, DHA underwent little hydrolysis to DKG (2,3-dioxo-L-gulonate). Instead, it yielded OxT (oxalyl L-threonate), cOxT (cyclic oxalyl L-threonate) and free oxalate (~6:1:1), essentially simultaneously, suggesting that all three product classes independently arose from one reactive intermediate, proposed to be cyclic-2,3-O-oxalyl-L-threonolactone. Only with plant apoplastic esterases present were the esters significant precursors of free oxalate. Without added H2O2, DHA was slowly hydrolysed to DKG. Downstream of DKG was a singly ionized dicarboxy compound (suggested to be 2-carboxy-L-xylonolactone plus 2-carboxy-L-lyxonolactone), which reversibly de-lactonized to a dianionic carboxypentonate. Formation of these lactones and acid was minimized by the presence of residual unreacted ascorbate. In vivo, the putative 2-carboxy-L-pentonolactones were relatively stable. We propose that DHA is a branch-point in ascorbate catabolism, being either oxidized to oxalate and its esters or hydrolysed to DKG and downstream carboxypentonates. The oxidation/hydrolysis ratio is governed by reactive oxygen species status. In vivo, oxalyl esters are enzymatically hydrolysed, but the carboxypentonates are stable. The biological roles of these ascorbate metabolites invite future exploration.

Reactive oxygen species-induced release of intracellular ascorbate in plant cell-suspension cultures and evidence for pulsing of net release rate.

SUMMARY: *Apoplastic ascorbate has been proposed to confer resistance to oxidative stresses, e.g. ozone. We investigated reactive oxygen species (ROS)-induced secretion and catabolism of ascorbate. *Late-growth-phase cultured cells of rose and Arabidopsis were preloaded with [(14)C]ascorbate. Radiolabelled metabolites and secretion products were analysed by high-voltage electrophoresis. *In both species, exogenous 1 mM hydrogen peroxide (H(2)O(2)) rapidly stimulated [(14)C]ascorbate and [(14)C]dehydroascorbate accumulation in the medium (apoplast). Net (14)C export was most rapid within 100 s of washing, and often showed superimposed pulses, of c. 10-s duration, whose amplitude was greater after H(2)O(2) treatment. Oxidative stress did not cause indiscriminate metabolite leakage from the cells. H(2)O(2) caused c. 20-40% of the intracellular [(14)C]ascorbate to be irreversibly catabolized to [(14)C]oxalyl-threonate and related products; however, the great majority of the extracellular radioactivity remained as [(14)C]ascorbate and [(14)C]dehydroascorbate. Much of the apoplastic dehydroascorbate was probably reabsorbed by the cells and reduced back to ascorbate. *The data show that exported ascorbate can serve an apoplastic antioxidant role in these late-growth-phase cells without being irreversibly lost, whereas in early-growth-phase cells most extracellular ascorbate is irreversibly degraded. In conclusion, cultured plant cells can respond actively to oxidative stress by reversibly exporting ascorbate into the apoplast.

Determination of subcellular concentrations of soluble carbohydrates in rose petals during opening by nonaqueous fractionation method combined with infiltration-centrifugation method.

Petal growth associated with flower opening depends on cell expansion. To understand the role of soluble carbohydrates in petal cell expansion during flower opening, changes in soluble carbohydrate concentrations in vacuole, cytoplasm and apoplast of petal cells during flower opening in rose (Rosa hybrida L.) were investigated. We determined the subcellular distribution of soluble carbohydrates by combining nonaqueous fractionation method and infiltration-centrifugation method. During petal growth, fructose and glucose rapidly accumulated in the vacuole, reaching a maximum when petals almost reflected. Transmission electron microscopy showed that the volume of vacuole and air space drastically increased with petal growth. Carbohydrate concentration was calculated for each compartment of the petal cells and in petals that almost reflected, glucose and fructose concentrations increased to higher than 100 mM in the vacuole. Osmotic pressure increased in apoplast and symplast during flower opening, and this increase was mainly attributed to increases in fructose and glucose concentrations. No large difference in osmotic pressure due to soluble carbohydrates was observed between the apoplast and symplast before flower opening, but total osmotic pressure was much higher in the symplast than in the apoplast, a difference that was partially attributed to inorganic ions. An increase in osmotic pressure due to the continued accumulation of glucose and fructose in the symplast may facilitate water influx into cells, contributing to cell expansion associated with flower opening under conditions where osmotic pressure is higher in the symplast than in the apoplast.

Rose protoplast isolation and culture and heterokaryon selection by immobilization in extra thin alginate film.

Somatic hybridization has been identified as one method for the genetic improvement of roses. The success of somatic hybridization programmes relies to a great extent upon efficient protoplast isolation and culture and selection of heterokaryons. This paper reports the isolation of rose cell suspension protoplasts by direct sucrose flotation and demonstrates their culture using extra thin alginate film. A comparative assessment of the efficiency of conventional culture techniques versus those with extra thin alginate film or thin alginate layer is also presented. A very high plating efficiency (80%) was obtained using thin alginate layer or extra thin alginate film techniques with improved media formulations. Protoplasts of Rosa damascena and R. bourboniana were fused by using polyethylene glycol as fusogen and later immobilized in the thin layer of alginate. The fused protoplasts were tracked on the basis of differential fluorescent staining, and the hybridity of heterokaryons following their development to callus was confirmed by molecular characterization. This novel selection strategy has general applicability and is faster and simpler to perform during somatic hybridization experiments.

Genomic organization, rapid evolution and meiotic instability of nucleotide-binding-site-encoding genes in a new fruit crop, "chestnut rose".

From chestnut rose, a promising fruit crop of the Rosa genus, powdery mildew disease-resistant and susceptible genotypes and their F(1) progeny were used to isolate nucleotide-binding-site (NBS)-encoding genes using 19 degenerate primer pairs and an additional cloning method called overlapping extension amplification. A total of 126 genes were harvested; of these, 38 were from a resistant parent, 37 from a susceptible parent, and 51 from F(1) progeny. A phylogenetic tree was constructed, which revealed that NBS sequences from parents and F(1) progeny tend to form a mixture and are well distributed among the branches of the tree. Mapping of these NBS genes suggested that their organization in the genome is a "tandem duplicated cluster" and, to a lesser extent, a "heterogeneous cluster." Intraspecific polymorphisms and interspecific divergence were detected by Southern blotting with NBS-encoding genes as probes. Sequencing on the nucleotide level revealed even more intraspecific variation: for the R4 gene, 9.81% of the nucleotides are polymorphic. Amino acid sites under positive selection were detected in the NBS region. Some NBS-encoding genes were meiotically unstable, which may due to recombination and deletion events. Moreover, a transposon-like element was isolated in the flanking region of NBS genes, implying a possible role for transposon in the evolutionary history of resistance genes.

In vitro propagation of rose--a review.

In vitro propagation of rose has played a very important role in rapid multiplication of cultivars with desirable traits and production of healthy and disease-free plants. During the last several years, different approaches have been made for in vitro propagation of rose. Micropropagation using apical buds or nodal segments and understanding the specific requirements at different stages has been comprehensively covered in literature. New challenges for refinements of protocols for high rate of shoot multiplication and development of cost effective methods has gained importance in the recent past. Importance of liquid static culture for shoot proliferation and root induction for rose is also discussed in the present review. Further, the development of protocol for in vitro plant regeneration which is considered as most important step for successful implementation of various biotechnological techniques used for plant improvement programmes has been adequately addressed in literature. In rose, there are several reports which indicate rapid regeneration and multiplication through organogenesis or somatic embryogenesis. On the whole, the present review gives a consolidated account of in vitro propagation in rose.

The novel herbicide oxaziclomefone inhibits cell expansion in maize cell cultures without affecting turgor pressure or wall acidification.

Oxaziclomefone [OAC; IUPAC name 3-(1-(3,5-dichlorophenyl)-1-methylethyl)-3,4-dihydro-6-methyl-5-phenyl-2H-1,3-oxazin-4-one] is a new herbicide that inhibits cell expansion in grass roots. Its effects on cell cultures and mode of action were unknown. In principle, cell expansion could be inhibited by a decrease in either turgor pressure or wall extensibility. Cell expansion was estimated as settled cell volume; cell division was estimated by cell counting. Membrane permeability to water was measured by a novel method involving simultaneous assay of the efflux of (3)H(2)O and [(14)C]mannitol from a 'bed' of cultured cells. Osmotic potential was measured by depression of freezing point. OAC inhibited cell expansion in cultures of maize (Zea mays), spinach (Spinacia oleracea) and rose (Rosa sp.), with an ID(50) of 5, 30 and 250 nm, respectively. In maize cultures, OAC did not affect cell division for the first 40 h. It did not affect the osmotic potential of cell sap or culture medium, nor did it impede water transport across cell membranes. It did not affect cells' ability to acidify the apoplast (medium), which may be necessary for 'acid growth'. As OAC did not diminish turgor pressure, its ability to inhibit cell expansion must depend on changes in wall extensibility. It could be a valuable tool for studies on cell expansion.

Do polyamines contribute to plant cell wall assembly by forming amide bonds with pectins?

Two new reducing glycoconjugates [N-D-galacturonoyl-putrescinamide (GalA-Put) and N,N'-di-D-galacturonoyl-putrescinamide (GalA-Put-GalA)] and homogalacturonan-putrescine (GalAn-Put) conjugates were synthesised as model compounds representing possible amide (isopeptide) linkage points between a polyamine and either one or two pectic galacturonate residues. The amide bond(s) were stable to cold acid and alkali (2M TFA and 0.1M NaOH at 25 degrees C) but rapidly hydrolysed by these agents at 100 degrees C. The amide bond(s) were resistant to Driselase and to all proteinases tested, although Driselase digested GalAn-Put, releasing fragments such as GalA3-Put-GalA3. To trace the possible formation of GalA-polyamine amide bonds in vivo, we fed Arabidopsis and rose cell-cultures and chickpea internodes with [14C]Put. About 20% of the 14C taken up was released as 14CO2, indicating some catabolism. An additional approximately 73% of the 14C taken up (in Arabidopsis), or approximately 21% (in rose), became ethanol-insoluble, superficially suggestive of polysaccharide-Put covalent bonding. However, much of the ethanol-inextractable 14C was subsequently extractable by acidified phenol or by cold 1M TFA. The small proportion of radioactive material that stayed insoluble in both phenol and TFA was hydrolysable by Driselase or hot 6M HCl, yielding 14C-oligopeptides and/or amino acids (including Asp, Glu, Gly, Ala and Val); no free 14C-polyamines were released by hot HCl. We conclude that if pectin-polyamine amide bonds are present, they are a very minor component of the cell walls of cultured rose and Arabidopsis cells and chickpea internodes.

Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-L-threonate.

Increasing the L-ascorbate (vitamin C) content of crops could in principle involve promoting its biosynthesis or inhibiting its degradation. Recent progress has revealed biosynthetic pathways for ascorbate, but the degradative pathways remain unclear. The elucidation of such pathways could promote an understanding of the roles of ascorbate in plants, and especially of the intriguing positive correlation between growth rate and ascorbate oxidase (or its products). In some plants (Vitaceae), ascorbate is degraded via L-idonate to L-threarate (L-tartrate), with the latter arising from carbons 1-4 of ascorbate. In most plants, however (including Vitaceae), ascorbate degradation can occur via dehydroascorbate, yielding oxalate plus L-threonate, with the latter from carbons 3-6 of ascorbate. The metabolic steps between ascorbate and oxalate/L-threonate, and their subcellular location, were unknown. Here we show that this pathway operates extracellularly in cultured Rosa cells, proceeds via several novel intermediates including 4-O-oxalyl-L-threonate, and involves at least one new enzyme activity. The pathway can also operate non-enzymatically, potentially accounting for vitamin losses during cooking. Several steps in the pathway may generate peroxide; this may contribute to the role of ascorbate as a pro-oxidant that is potentially capable of loosening the plant cell wall and/or triggering an oxidative burst.

Evolution by reticulation: European dogroses originated by multiple hybridization across the genus rosa.

The European dogroses (Rosa sect. Caninae (DC.) Ser.) are characterized by a unique meiosis system ("canina-meiosis"), which controls the heterogamous development of tetraploid egg cells and haploid pollen grains resulting in a pentaploid somatic status. This permanent anorthoploidy is supposed to have originated by a hybridization event in the postglacial period. In this study we present molecular evidence by an analysis of nuclear ribosomal DNA data that dogroses are complex allopolyploids resulting from multiple hybridization events. As previously described, the nrITS-1 region does not undergo concerted evolution in dogroses. Thus, different ITS-1 sequences persist within single individuals. Secondary structure predictions do not point to the existence of pseudogenes within these ITS-1 types. Our data suggest that the pentaploid Caninae genome originated from different members of nondogroses and the now extinct Protocaninae.

Density-labelling of cell wall polysaccharides in cultured rose cells: comparison of incorporation of 2H and 13C from exogenous glucose.

Labelling with stable isotopes has under-exploited potential for studies of polysaccharide endotransglycosylation in vivo. Ideally, the labelled polysaccharides should have the highest possible buoyant density. Although [13C6]glucose has previously been used as a precursor, it was unclear whether 2H would be efficiently incorporated from [2H]glucose or lost as D2O. Rose (Rosa sp.) cell-suspension cultures efficiently incorporated 13C from D-[13C6,2H7]glucose into wall polysaccharides with negligible dilution from atmospheric 12CO2. Also, approximately 70% of the 2H atoms in D-[13C6,2H7]glucose were retained during polysaccharide biosynthesis. This shows that relatively few cycles of intermediary metabolism leading to the release of D2O occurred before sugar residues were incorporated into wall polysaccharides. In agreement with these observations, isopycnic centrifugation in caesium trifluoroacetate gradients showed that the hydrated buoyant density of xyloglucan synthesised by rose cells growing on [13C6,2H7]glucose and [13C6]glucose was 3.7 and 2.6% higher, respectively, than in isotopically non-labelled cultures. Thus, [13C,2H]glucose-feeding enabled a 42% better resolution of 'heavy' from 'light' xyloglucan than [13C]glucose-feeding.