Development of a novel noninvasive quantitative method to monitor Siraitia grosvenorii cell growth and browning degree using an integrated computer-aided vision technology and machine learning.

The rapid, accurate and noninvasive detection of biomass and plant cell browning can provide timely feedback on cell growth in plant cell culture. In this study, Siraitia grosvenorii suspension cells were taken as an example, a phenotype analysis platform was successfully developed to predict the biomass and the degree of cell browning based on the color changes of cells in computer-aided vision technology. First, a self-made laboratory system was established to obtain images. Then, matrices were prepared from digital images by a self-developed high-throughput image processing tool. Finally, classification models were used to judge different cell types, and then a semi-supervised classification to predict different degrees of cell browning. Meanwhile, regression models were developed to predict the plant cell mass. All models were verified with a good agreement by biological experiments. Therefore, this method can be applied for low-cost biomass estimation and browning degree quantification in plant cell culture.

[Rapid cryopreservation for Siraitia grosvenorii cells based on cells' capacitance detection].

A rapid quantitative evaluation method for Siraitia grosvenorii cells was successfully developed based on plant cells' capacitance value detected by a viable cell mass monitor and the cryopreservation of S. grosvenorii suspension cells was optimized. The survival rate of S. grosvenorii cells was quantitatively measured by viable cell mass monitor and 2, 3, 5-triphenyltetrazolium chloride (TTC). An optimum cryoprotectant recipe is that the growth medium contained 10% sucrose and 10% DMSO. The experimental results also showed higher cell survival rates and cell viabilities were achieved when suspension cells were treated with pretreatment of 0.2 mol/L sucrose. With the increase of concentration of sucrose, however, the cell survival rate was decreased. And the cell survival rate represented a bell shape with the increase of pretreatment time. The highest cell survival rate and cell viability were obtained with the 9 h' s pretreatment. In addition, there was a good correlation between the cell survival rate measured by cell recovery test and that measured by viable cell mass monitor, while there were no significant differences in the cell morphology and the ability of mogrosides V production by S. grosvenorii cells cultured in suspension after cryopreservation. Therefore, the evaluation method developed based on the viable cell mass monitor has good feasibility and reliability.

Cytogenetic relationships among Citrullus species in comparison with some genera of the tribe Benincaseae (Cucurbitaceae) as inferred from rDNA distribution patterns.

BACKGROUND: Comparative mapping of 5S and 45S rDNA by fluorescent in situ hybridization (FISH) technique is an excellent tool to determine cytogenetic relationships among closely related species. RESULTS: In this study, the number and position of 5S and 45S rDNA loci in all Citrullus species and subspecies were determined. The cultivated watermelon (C. lanatus subsp. vulgaris), C. lanatus subsp. mucosospermus, C. colocynthis and C. naudinianus (or Acanthosicyos naudinianus) had two 45S rDNA loci and one 5S rDNA locus which was located syntenic to one of the 45S rDNA loci. C. ecirrhosus and C. lanatus subsp. lanatus had one 45S rDNA locus and two 5S rDNA loci, each located on a different chromosome. C. rehmii had one 5S and one 45S rDNA locus positioned on different chromosomes. The distribution of 5S and 45S rDNA in several species belonging to other genera in Benincaseae tribe was also investigated. The distribution pattern of rDNAs showed a great difference among these species. CONCLUSIONS: The present study confirmed evolutionary closeness among cultivated watermelon (C. lanatus subsp. vulgaris), C. lanatus subsp. mucosospermus and C. colocynthis. Our result also supported that C. lanatus subsp. lanatus was not a wild form of the cultivated watermelon instead was a separate crop species. In addition, present cytogenetic analysis suggested that A. naudinianus was more closely related to Cucumis than to Citrullus or Acanthosicyos, but with a unique position and may be a link bridge between the Citrullus and the Cucumis.

Melon necrotic spot virus Replication Occurs in Association with Altered Mitochondria.

Melon necrotic spot virus (MNSV) (genus Carmovirus, family Tombusviridae) is a single-stranded, positive-sense RNA virus that has become an experimental model for the analysis of cell-to-cell virus movement and translation of uncapped viral RNAs, whereas little is known about its replication. Analysis of the cytopathology after MNSV infection showed the specific presence of modified organelles that resemble mitochondria. Immunolocalization of the glycine decarboxylase complex (GDC) P protein in these organelles confirmed their mitochondrial origin. In situ hybridization and immunolocalization experiments showed the specific localization of positive-sense viral RNA, capsid protein (CP), and double-stranded (ds)RNA in these organelles meaning that replication of the virus takes place in association with them. The three-dimensional reconstructions of the altered mitochondria showed the presence of large, interconnected, internal dilations which appeared to be linked to the outside cytoplasmic environment through pores and/or complex structures, and with lipid bodies. Transient expression of MNSV p29 revealed that its specific target is mitochondria. Our data document the extensive reorganization of host mitochondria induced by MNSV, which provides a protected environment to viral replication, and show that the MNSV p29 protein is the primary determinant of this effect in the host.

The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense.

The N-terminal region of the Ourmia melon virus (OuMV) coat protein (CP) contains a short lysine/arginine-rich (KR) region. By alanine scanning mutagenesis, we showed that the KR region influences pathogenicity and virulence of OuMV without altering viral particle assembly. A mutant, called OuMV6710, with three basic residue substitutions in the KR region, was impaired in the ability to maintain the initial systemic infection in Nicotiana benthamiana and to infect both cucumber and melon plants systemically. The integrity of this protein region was also crucial for encapsidation of viral genomic RNA; in fact, certain mutations within the KR region partially compromised the RNA encapsidation efficiency of the CP. In Arabidopsis thaliana Col-0, OuMV6710 was impaired in particle accumulation; however, this phenotype was abolished in dcl2/dcl4 and dcl2/dcl3/dcl4 Arabidopsis mutants defective for antiviral silencing. Moreover, in contrast to CPwt, in situ immunolocalization experiments indicated that CP6710 accumulates efficiently in the spongy mesophyll tissue of infected N. benthamiana and A. thaliana leaves but only occasionally infects palisade tissues. These results provided strong evidence of a crucial role for OuMV CP during viral infection and highlighted the relevance of the KR region in determining tissue tropism, host range, pathogenicity, and RNA affinity, which may be all correlated with a possible CP silencing-suppression activity.

Identification and characterization of ten new water gaps in seeds and fruits with physical dormancy and classification of water-gap complexes.

BACKGROUND AND AIMS: Physical dormancy (PY) occurs in seeds or fruits of 18 angiosperm families and is caused by a water-impermeable palisade cell layer(s) in seed or fruit coats. Prior to germination, the seed or fruit coat of species with PY must become permeable in order to imbibe water. Breaking of PY involves formation of a small opening(s) (water gap) in a morpho-anatomically specialized area in seeds or fruits known as the water-gap complex. Twelve different water-gap regions in seven families have previously been characterized. However, the water-gap regions had not been characterized in Cucurbitaceae; clade Cladrastis of Fabaceae; subfamilies Bombacoideae, Brownlowioideae and Bythnerioideae of Malvaceae; Nelumbonaceae; subfamily Sapindoideae of Sapindaceae; Rhamnaceae; or Surianaceae. The primary aims of this study were to identify and describe the water gaps of these taxa and to classify all the known water-gap regions based on their morpho-anatomical features. METHODS: Physical dormancy in 15 species was broken by exposing seeds or fruits to wet or dry heat under laboratory conditions. Water-gap regions of fruits and seeds were identified and characterized by use of microtome sectioning, light microscopy, scanning electron microscopy, dye tracking and blocking experiments. KEY RESULTS: Ten new water-gap regions were identified in seven different families, and two previously hypothesized regions were confirmed. Water-gap complexes consist of (1) an opening that forms after PY is broken; (2) a specialized structure that occludes the gap; and (3) associated specialized tissues. In some species, more than one opening is involved in the initial imbibition of water. CONCLUSIONS: Based on morpho-anatomical features, three basic water-gap complexes (Types-I, -II and -III) were identified in species with PY in 16 families. Depending on the number of openings involved in initial imbibition, the water-gap complexes were sub-divided into simple and compound. The proposed classification system enables understanding of the relationships between the water-gap complexes of taxonomically unrelated species with PY.

Proximate composition, extraction, characterization and comparative assessment of coconut (Cocos nucifera) and melon (Colocynthis citrullus) seeds and seed oils.

Proximate composition, extraction, characterization and comparative assessment of Cocos nucifera and Colocynthis citrullus seeds and seed oils were evaluated in this work using standard analytical techniques. The results showed the percentage (%) moisture, crude fibre, ash, crude protein, lipids and total carbohydrate contents of the seeds as 7.51 and 4.27, 7.70 and 5.51, 1.02 and 2.94, 10.57 and 11.67, 47.80 and 50.42 and 32.84 and 29.47 while the calorific values were 553.99 and 567.32 Kcal/100 g for C. nucifera and C. citrullus, respectively. The two seed oils were odourless and at room temperature (30 degrees C) liquids, with a pale yellow to yellowish colouration. Lipid indices of the seed oils indicated the Acid Values (AV) as 2.06-6.36 mg NaOH g(-1) and 2.99-6.17 mg NaOH g(-1), Free Fatty Acids (FFA) as 1.03-3.18 and 1.49-3.09%, Saponification Values (SV) as 252.44-257.59 and 196.82-201.03 mg KOH g(-1), Iodine Values (IV) as 9.73-10.99 and 110.93-111.46 mg of I2 g(-1) of oil and Peroxide Values (PV) as 0.21-0.21 and 1.53-2.72 mg O2 kg(-1) for soxhlet-mechanical extracted C. nucifera and C. citrullus seed oils, respectively. The studied characteristics of the oil extracts in most cases compared favourably with most conventional vegetable oils sold in the Nigeria markets; however, there were some observed levels of significant differences in the values at p < or = 0.05. These results suggest that the seeds examined may be nutritionally potent and also viable sources of seed oils judging by their oil yield. The data also showed that the seed oils were edible inferring from their low AV and their corresponding low FFA contents. Industrially, the results revealed the seed oils to have great potentials in soap manufacturing industries because of their high SV. They were also shown to be non-drying due to their low IV which also suggested that the oils contain few unsaturated bonds and therefore have low susceptibility to oxidative rancidity and deterioration as confirmed by their low PV which also serves as indicators of the presence or high levels of anti-oxidants in the oils.

Plasmodesmata distribution and sugar partitioning in nitrogen-fixing root nodules of Datisca glomerata.

To understand carbon partitioning in roots and nodules of Datisca glomerata, activities of sucrose-degrading enzymes and sugar transporter expression patterns were analyzed in both organs, and plasmodesmal connections between nodule cortical cells were examined by transmission electron microscopy. The results indicate that in nodules, the contribution of symplastic transport processes is increased in comparison to roots, specifically in infected cells which develop many secondary plasmodesmata. Invertase activities are dramatically reduced in nodules as compared to roots, indicating that here the main enzyme responsible for the cleavage of sucrose is sucrose synthase. A high-affinity, low-specificity monosaccharide transporter whose expression is induced in infected cells prior to the onset of bacterial nitrogen fixation, and which has an unusually low pH optimum and may be involved in turgor control or hexose retrieval during infection thread growth.

High temperatures activate local viral multiplication and cell-to-cell movement of Melon necrotic spot virus but restrict expression of systemic symptoms.

The infection of melon plants by Melon necrotic spot virus (MNSV) and the development of necrotic disease symptoms are a seasonal occurrence in Japan, which take place between winter and early summer, but not during mid-summer. In this paper we investigate the effect of three different temperatures (15, 20, and 25 degrees C) on the local and systemic expression of MNSV in melon plants. Previously, the incidence of plants expressing systemic symptoms caused by MNSV and other viruses was found to be greater at temperatures less than 20 degrees C. In this study, our temperature-shift experiments support previous studies that found the expression of systemic symptoms increases as temperature falls from 25 to 20 degrees C and decreases as temperature rises from 20 to 25 degrees C. However, MNSV replication in melon cells and local viral movement within leaves following the inoculation of melon protoplasts or cotyledons were more frequent at 25 degrees C than at 15 or 20 degrees C.

Distribution and pathway for phloem-dependent movement of Melon necrotic spot virus in melon plants.

The translocation of Melon necrotic spot virus (MNSV) within tissues of inoculated and systemically infected Cucumis melo L. 'Galia' was studied by tissue-printing and in situ hybridization techniques. The results were compatible with the phloem vascular components being used to spread MNSV systemically by the same assimilate transport route that runs from source to sink organs. Virus RNAs were shown to move from the inoculated cotyledon toward the hypocotyl and root system via the external phloem, whereas the upward spread through the stem to the young tissues took place via the internal phloem. Virus infection was absent from non-inoculated source tissues as well as from both shoot and root apical meristems, but active sink tissues such as the young leaves and root system were highly infected. Finally, our results suggest that the MNSV invasion of roots is due to virus replication although a destination-selective process is probably necessary to explain the high levels of virus accumulation in roots. This efficient invasion of the root system is discussed in terms of natural transmission of MNSV by the soil-borne fungal vector.

Comparative histochemical analyses of oxidative burst and cell wall reinforcement in compatible and incompatible melon-powdery mildew (Podosphaera fusca) interactions.

The spatial-temporal expression patterns of oxidative burst and cell wall reinforcement were analyzed in leaves of resistant and susceptible melon (Cucumis melo L.) cultivars in response to Podosphaera fusca (Fr.) Braun & Shishkoff, the main causal agent of powdery mildew in cucurbits. Extensive development of powdery mildew mycelia and a progressive increase in haustorial count were recorded in the susceptible cultivar after 4d, while in the resistant cultivar powdery mildew failed to grow and small brownish and necrotic leaf areas were frequently observed. Rapid generation of the reactive oxygen intermediates hydrogen peroxide and superoxide radicals 4h after pathogen challenge, but before the fungal haustoria formation, stood upstream in the cascade of events induced during these interactions. This oxidative burst was followed by the accumulation of strengthening polymers of callose and lignin at the cell wall of attacked resistant plant cells. Interestingly, the transcriptional levels of phenylalanine ammonia-lyase (PAL), an important enzyme for phenylpropanoid metabolism, did not significantly change throughout the experiments. Although these physiological changes were observed in both cultivars, their faster kinetics and amplitude in the resistant line compared to the susceptible cultivar governed the differential visual response of these cultivars against P. fusca. These findings, along with data obtained in previous studies, have provided the bases for an integrated model in which the spatial-temporal response patterns of these resistance mechanisms have been arranged, which may ultimately lead to successful protection of melon plants against P. fusca.

Programmed cell death of the nucellus during Sechium edule Sw. seed development is associated with activation of caspase-like proteases.

The nucellus is a maternal tissue that embeds and feeds the developing embryo and secondary endosperm. During seed development, the cells of the nucellus suffer a degenerative process soon after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been considered to be a form of developmentally programmed cell death (PCD). It was investigated whether or not this degenerative process is characterized by apoptotic hallmarks. Evidence showed that cell death is mostly localized in the border region of the tissue adjacent to the expanding endosperm. Cell death is accompanied by profound changes in the morphology of the nuclei and by a huge degradation of nuclear DNA. Moreover, an increase of activity of different classes of proteinases is reported, and the induction of caspase-like proteases sensitive to specific inhibitors was detected. Nucellar caspase-like proteases are characterized by an acid pH optimum suggesting a possible localization in the vacuole.

[Comparative analysis of rDNA distribution in metaphase chromosomes of Cucurbitaceae species].

Fluorescence in situ hybridization (FISH) and double FISH experiments were carried out to ascertain the chromosomal distribution patterns of the 45S and 5S ribosomal DNAs in the three species of Cucurbitaceae. Five pairs of 45S rDNA loci and two pairs of 5S rDNA signals were detected on chromosomes of Cucurbita moschata Duch. Luffa cylindrical Roem. contained five pairs of 45S rDNA loci and one pair of 5S rDNA loci. In Benincasa hispida Cogn., two pairs of 45S rDNA sites and one pair of 5S rDNA site were detected. In this species, 5S rDNA and one pair of the 45S loci were collocated closely in chromosome 7S. 45S rDNA chromosomal distribution patterns were highly conserved among the three species, althoufh their number varied markedly. The 5S rDNA sites on chromosomes among the three species were highly polymorphic. We further discussed differentially evolutionary processes of 45S and 5S rDNA in plant genomes.

Callus formation and cucurbitacin B accumulation in Ecballium elaterium callus cultures.

Ecballium elaterium fruit juice is used for the treatment of sinusitis in Turkish folk medicine. The aim of this study was to increase the yield of cucurbitacin B, an anti-inflammatory compound previously isolated in various organs of E. elaterium, through tissue culture techniques. Higher yields of cucurbitacin B (1.126%) were obtained from the first subculture calluses from stem nodes in the presence of benzyl adenine (BA; 1 mg/l) and naphtalene acetic acid (NAA; 0.1 mg/l) in comparison with the yields obtained from plant material (0.01%).

Formation of rhamnogalacturonan II-borate dimer in pectin determines cell wall thickness of pumpkin tissue.

Boron (B) deficiency results in inhibition of pumpkin (Cucurbia moschata Duchesne) growth that is accompanied by swelling of the cell walls. Monomeric rhamnogalacturonan II (mRG-II) accounted for 80% to 90% of the total RG-II in B-deficient walls, whereas the borate ester cross-linked RG-II dimer (dRG-II-B) accounted for more than 80% of the RG-II in control plants. The results of glycosyl residue and glycosyl linkage composition analyses of the RG-II from control and B-deficient plants were similar. Thus, B deficiency does not alter the primary structure of RG-II. The addition of (10)B-enriched boric acid to B-deficient plants resulted within 5 h in the conversion of mRG-II to dRG-II-(10)B. The wall thickness of the (10)B-treated plants and control plants was similar. The formation and possible functions of a borate ester cross-linked RG-II in the cell walls are discussed.

Identification of a Ca(2+)-pectate binding site on an apoplastic peroxidase.

An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca(2)+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca(2)+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 did not bind at all. This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that APRX bears a Ca(2)+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.

Cucurbit protoplast isolation for the study of plant virus replication.

A cucurbit protoplast isolation protocol was established for the study of plant virus replication in vivo. This protocol is applicable to both cucumber and squash leaf tissue with significant increases in yields of viable protoplasts suitable for electroporation, compared to other published methods. A combination of Cellulase RS, Macerozyme R10 and mannitol was used as digestion enzymes and osmoticum. An average of 1.7x10(7) protoplasts per gram of fresh leaf tissue were obtained from cucumber cultivar Bet-alpha. Both cucumber cultivar Shimson and squash cultivar First Taste produced an average yield of 6.0x10(6) protoplasts per g of fresh leaf tissue. Electroporation of 10 microg of Zucchini yellow mosaic potyvirus (ZYMV-S) RNA into the protoplasts resulted in virus replication and synthesis of coat protein (CP). SDS-PAGE and immunoblotting were used to detect the CP 48 h post-electroporation. This protocol is highly reproducible and will assist researchers who require cucurbit protoplasts to study virus replication.

Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells.

Ascorbate oxidase activity and immunoreactivity were evaluated in crude tissue extracts obtained from callus cell cultures induced by green zucchini sarcocarp and grown in the presence of tunicamycin, a powerful N-glycosylation inhibitor. Tunicamycin at 2 or 4 microg ml(-1) blocked cell growth within a couple of weeks, although a sustained cell viability was observed in the same period. A significant inhibition of total protein synthesis was observed at 10 and 15 days of culture time, with a decrease of 30% and 43% respectively when cells were grown in the presence of 2 microg ml(-1) tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 microg ml(-1). After the same culture times ascorbate oxidase specific activity assayed in crude tissue extracts showed increases of about 1.9-fold and 3.5-fold (10 days) and 1.7-fold and 3.1-fold (15 days) at 2 and 4 microg ml(-1) tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the same growing times. Lectin-blot, based on the use of concanavalin A, indicated a marked decrease of glycosylated proteins in tunicamycin-treated cultures. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarcely recognized the enzyme expressed in tunicamycin-treated cells; on the contrary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures.

[Study on the pharmacognosy of traditional Chinese medicine tubeimu].

In this article, we have studied the traditional Chinese medicine Tubeimu on the pharmacognosy. This paper reports the thickening condition on the epidermis cell walls of the bulbuls of Tubeimu [Bolbostemma paniculatum (Maxim) Franquet] for the first time and corrects the wrong record in before literature.