Water deficit in developing endosperm of maize: cell division and nuclear DMA endoreduplication

Water deficit severely decreases maize (Zea mays L.) kernel growth; the effect is most pronounced in apical regions of ears. The capacity for accumulation of storage material in endosperms is thought to he partially determined by the extent of cell division and endoreduplication (post-mitotic nuclear DNA synthesis). To gain a better understanding of the regulatory mechanisms involved, we have examined the effect of water deficit on cellular development during the post-fertilization period. Greenhouse-grown maize was subjected to water-limited treatments during rapid cell division [from 1 to 10days after pollination (DAP)] or rapid endoreduplication (9 to 15 DAP). The number of nuclei and the nuclear DNA content were determined with flow cytometry. Water deficit from 1 to 10 DAP substantially decreased the rate of endosperm cell division in apical-region kernels, but had little effect on middle-region endosperms. Rewatcring did not allow cell division to recover in apical-region endosperms. Water deficit from 9 to 15 DAP also decreased cell division in apical-region endosperms. Endoreduplication was not affected by the late treatment in either region of the car, but was inhibited by the early treatment in the apical region. In particular, the proportion of nuclei entering higher DN A-content size classes was reduced. We conclude that cell division is highly responsive to water deficit, whereas endoreduplication is less so. We also conclude that the reduced proportion of nuclei entering higher DNA-content size classes during endoreduplication is indicative of multiple control points in the mitotic and endoreduplication cycles.     

DNA endoreduplication in maize endosperm cells: the effect of exposure to short-term high temperature

DNA endoreduplication in Zea mays L. (cv. A619 × W64A) endosperm peaks between 16 and 18 d after pollination (DAP). The physiological function of DNA endoreduplication is not known but it is believed to be important in maize kernel development. In the present study, we investigated how 2, 4 or 6 d of high temperature (35 °C) affected DNA endoreduplication and maize kernel development in comparison with control kernels grown at 25 °C. Data were collected on fresh weight (FW), nuclei number, mitotic index, and DNA endoreduplication. Maize endosperm FW and nuclei number were reduced by exposure to 4 or 6 d of high temperature. At 18 DAP, the 2 d high temperature treatment (HTT) caused a reduction in FW and nuclei number, but had no effect on DNA endoreduplication and average DNA content per endosperm. However, when the exposure to high temperature was increased to 4 or 6 d, FW, nuclei number and the magnitude of DNA endoreduplication were progressively reduced, and the peak mitotic index was delayed compared with the control endosperm. At 18 DAP, the 4 d treatment showed 54·7% of the cells were 3 or 6 C, whereas only 41·2% were 12 C or higher. Six days of high temperature also resulted in a reduction in endosperm FW, nuclei number and a delay in the peak of mitotic index. DNA endoreduplication occurred in the kernels exposed to this treatment, although the magnitude was severely reduced compared with the control kernels. Nuclear DNA content was highly correlated (r = 0·93) with kernel FW, suggesting an important role of DNA endoreduplication in determining endosperm FW. The data suggest that high temperature during endosperm cell division exerted negative effects on DNA endoreduplication by dramatically reducing the nuclei number, leaving fewer nuclei available for DNA endoreduplication. However, the data also suggest that prolonged exposure to high temperature restricts entry of mitotic cells into the endoreduplication phase of the cell cycle.     

Preferential allocation of sulphur into y‐glutamylcysteinyl peptides in wheat plants grown at low sulphur nutrition in the presence of cadmium

Plants respond to Cd by synthesising phytochelatins (PC) and similar S‐rich peptides which are important in alleviating Cd toxicity. The hypothesis that S nutrition influences the sensitivity of plants to Cd was examined by measuring the growth, PC and Cd content of wheat plants ( Triticum aestivum cv. Condor) grown at 10–1000 µ M S with and without 30 µ M Cd. In the absence of Cd, 100 µ M S was marginally S‐limiting. Cd severely inhibited root growth at 100 µ M S but the concentrations of PC, Cd–PC complexes and Cd in the root were similar to those of plants grown at 1000 µ M S which exhibited no evidence of Cd toxicity. Plants grown at low S (10 and 30 µ M ) contained lower concentrations of Cd. However, they produced very low amounts of PC and were very sensitive to Cd. At 10–300 µ M S (but not at 1000 µ M ), Cd enhanced the concentration of S in the root. The data are consistent with a model in which plants preferentially allocate S to PC synthesis. When S was marginally limiting (100 µ M ), the S supply was sufficient for PC synthesis but not root growth, effectively causing Cd‐induced S deficiency. This did not occur at high S (1000 µ M ). Conversely, at low S (10 and 30 µ M ), the synthesis of PC₂–PC₄ was decreased by 87 and 66%, respectively, thereby resulting in decreased Cd uptake while also making the plants especially sensitive to Cd.     

Regulation of elongase activity by abscisic acid and temperature in microspore‐derived embryos of oilseed rape (Brassica napus)

Growth temperature and ABA both affect the level of erucic acid (22:1) in microspore‐derived embryos (MDEs) of oilseed rape. We have previously shown that these stimuli act independently. In the present study we investigated the effects of growth temperature (15 vs 25°C) and ABA (0 vs 5 µ M ) on the characteristics and activity of the elongase complex, the enzymes synthesising 22:1. Due to inhibition by the substrate oleoyl‐CoA at low concentrations (< 10 µ M ) it was not possible to determine values for K_m and V_max. Elongase activities from preparations extracted from MDEs grown under different conditions showed an optimum temperature higher than 30°C, with a Q₁₀ value of about 3. We found considerable effects of temperature and exogenous ABA on total elongase activity in MDEs. Our results suggest that the accumulation of 22:1 is regulated by the amount of elongase enzyme and not by changes in the intrinsic characteristics of the enzyme. Elongase activity correlated closely with the absolute amount of 22:1 (µmol), whereas the correlation between elongase activity and the fraction of 22:1 (% of fatty acids) in oil was poorer. Including the total activity of acyltransferases did not improve the correlation. Acyltransferase activity itself correlated poorly with the total amount of oil formed.     

Analysis of chromatin and DNA during chromosome endoreduplication in the endosperm ofTriticum durum Desf.

Summary Chromatin structure was studied in nuclei of the endosperm of durum wheat (Triticum durum Desf., cv. Creso), where a large number of cells undergo chromosome endoreduplication during caryopsis development. Optical density profiles of interphase nuclei at different ploidy levels after Feulgen staining were determined cytophotometrically. It was observed that, within each development stage, polyploid nuclei (6–12C and 12–24C) show more condensed chromatin than euploid nuclei (3–6C): this should indicate that endoreduplication is accompanied by some reduction of nuclear activity. Within the same ploidy level, 3–6C and 6–12C nuclei become increasingly condensed with development (except for the last stage), while 12-24C nuclei are identical at all stages. DNA methylation at different stages of caryopsis development was then analyzed in genomic DNA, highly repeated sequences and ribosomal DNA, by digestion with cytosine-methylation-sensitive restriction enzymes. We observed that (i), depending on the enzyme, DNA from caryopses may show higher mean length than DNA from shoot apices and variations occur during endosperm development; (ii) highly repeated DNA sequences also show some variation in base methylation between apices and endosperms and among endosperm development stages, even though to a lesser extent than genomic DNA; (iii) rDNA shows variations only between endosperm and apices while no variation was observed among endosperm development stages in relation to chromosome endoreduplication. Our data may be explained by assuming the occurrence, during endosperm development, of processes of chromatin condensation possibly involved in silencing the activity of extra copies of DNA resulting from chromosome endoreduplication. At least in part, DNA methylation is involved in the process of chromatin condensation. rDNA shows no variation during endosperm development: this suggests that rDNA copies are actively transcribed in both triploid and endoreduplicated nuclei.     

Changes in Nuclear DNA Content of Endosperm Cells During Grain Development in Rice (Oryza sativa)

Morphological, cytological and quantitative DNA changes associatedwith endosperm development in rice caryopsis were investigated.Following a brief free-nuclear phase, the endosperm became cellularby the 4th d after anthesis. While the mean length and breadthof grain attained maximum values at about 10, d after anthesis,f. wt of the whole grain, and of the endosperm separately, continuedto increase until about 16 d after anthesis. Cell divisionsin the endosperm continued until 10 d and following stabilizationof the cell number, the nuclei attained irregular shapes. Thesize of the nuclei and nuclei and the amount of nuclear DNAvaried considerably during endosperm development. The endospermnuclei did not retain the expected 3C–6C DNA level afterthe first few rounds of division and nuclei having more than30C DNA were frequent 8 d past anthesis. The highest C valuerecorded was 74C in a 16-d-old endosperm cell. Oryza sativa, rice, caryopsis, endosperm, cell number, nuclear area, nuclear DNA content, endoreduplication     

Potential role of the rice OsCCS52A gene in endoreduplication

In eukaryotes, the cell cycle consists of four distinct phases: G1, S, G2 and M. In certain condition, the cells skip M-phase and undergo endoreduplication. Endoreduplication, occurring during a modified cell cycle, duplicates the entire genome without being followed by M-phase. A cycle of endoreduplication is common in most of the differentiated cells of plant vegetative tissues and it occurs extensively in cereal endosperm cells. Endoreduplication occurs when CDK/Cyclin complex low or inactive caused by ubiquitin-mediated degradation by APC and their activators. In this study, rice cell cycle switch 52 A (OsCCS52A), an APC activator, is functionally characterized using the reverse genetic approach. In rice, OsCCS52A is highly expressed in seedlings, flowers, immature panicles and 15 DAP kernels. Localization studies revealed that OsCCS52A is a nuclear protein. OsCCS52A interacts with OsCdc16 in yeast. In addition, overexpression of OsCCS52A inhibits mitotic cell division and induces endoreduplication and cell elongation in fission yeast. The homozygous mutant exhibits dwarfism and smaller seeds. Further analysis demonstrated that endoreduplication cycles in the endosperm of mutant seeds were disturbed, evidenced by reduced nuclear and cell sizes. Taken together, these results suggest that OsCCS52A is involved in maintaining normal seed size formation by mediating the exit from mitotic cell division to enter the endoreduplication cycles in rice endosperm.     

Do Genetic Make-up and Growth Manipulation Affect Tomato Fruit Size by Cell Number,or Cell Size and DNA Endoreduplication?

This work investigated the link between genetic and developmental controls of fruit size and composition. On two isogenic lines (CF12-C and CF14-L), differing by fruit weight and sugar content quantitative trait loci (QTLs) identified previously, basal and tip fruits were characterized at anthesis and at maturity through their growth, dry matter and sugar content, number and size of cells and nuclei DNA content. The influence of competition was assessed by removing either basal or tip ovaries at anthesis. On an intact inflorescence, CF12-C fruits grew less than CF14-L fruits, with 1.67 fewer cell layers and similar cell size, suggesting that genes controlling cell division may be responsible for this fruit size variation. Truss thinning masked the QTL effect on fruit size, mainly by reducing the difference in cell number between the two lines and by promoting cell expansion in tip fruits, so that fruit growth was similar at both positions and for both lines. Thus, in these lines, cell number exerts a control on final fruit size only when there is competition among fruits. Different responses of basal and tip fruits after flower removal suggested that this treatment induced changes in hormonal relationships within the truss. No fixed relationship between DNA endoreduplication and cell size was found, as while cell size and dry matter and sugar contents differed with tomato lines, fruit position and truss size, endoreduplication patterns were the same. CF12-C fruits had a higher dry matter (+0.3% of fresh weight) and carbohydrates (+8% of dry matter) content than CF14-L fruits. The percentage dry matter was independent of truss size but decreased slightly from basal to tip fruits.     

Transduction Mechanisms Involved in Thrombin Receptor-Induced Nerve Growth Factor Secretion and Cell Division in Primary Cultures of Astrocytes

Abstract: In astrocytes, thrombin and thrombin receptor-activating activating peptide (TRAP-14), a 14-amino-acid agonist of the proteolytic activating receptor for thrombin (PART), significantly increased cell division as assessed by [³H]thymidine incorporation into DNA (EC₅₀ = 1 n M and +650% at 100 n M for thrombin; EC₅₀ = 3 µ M and +600% at 100 µ M for TRAP-14) and nerve growth factor (NGF) secretion (approximately twofold at 100 n M thrombin or 100 µ M TRAP-14). The [³H]thymidine incorporation was prevented by protein kinase C inhibitors (staurosporine and H7) or by down-regulation of this enzyme by chronic exposure of astrocytes to phorbol 12-myristate 13-acetate (PMA). Thrombin-induced NGF secretion was completely inhibited by protein kinase C inhibitors. Treatment with PMA stimulated NGF secretion 19-fold, and this effect was not further enhanced by thrombin. These data suggest an absolute requirement of protein kinase C activity for thrombin-induced NGF secretion and cell division. Pretreatment of astrocytes with pertussis toxin (PTX) reduced thrombin- and TRAP-14-induced DNA synthesis. PART activation caused a decrease in forskolin-stimulated cyclic AMP accumulation. PTX treatment prevented the inhibitory effect of PART activation on cyclic AMP accumulation, suggesting that a PTX-sensitive G protein, such as G_i or G_o, is involved in thrombin-induced cell division. In contrast, thrombin-induced NGF secretion was not inhibited by PTX. Finally, the protein tyrosine kinase inhibitor herbimycin A partially but significantly prevented thrombin- and TRAP-14-induced cell division but was without effect on NGF secretion. Taken together, these results demonstrate that, in astrocytes, PART(s)-triggered cell division or NGF secretion is mediated by distinct transduction mechanisms.     

Endoreduplication of nuclear DNA in the developing maize endosperm

Multiparametric flow cytometry was used to analyze the development of the endosperm in Zea mays L. during the period from 8 to 20 days after pollination (dap). Nuclear size, DNA content per nucleus, and frequencies of nuclei with varying properties were measured in preparations that included all of the endosperm nuclei of single kernels of the inbred strain Al88. Characteristics of nuclear populations from different kernels on the same ear showed minimal variation. The dynamic changes of non-mitotic cells involved in endosperm development consisted of alternating periods of DNA replication with non-replication. Seven rounds of DNA replication had occurred in some nuclei in the later developmental stages with the rate averaging approximately one round per 24-hour period. Analysis of the DNA levels in the nuclei showed an exact doubling pattern indicating an endoreduplication process, that is, replication of the entire genome during each round. The loosely organized polytenization of the chromatin occurred to varying extents among the nuclei within an endosperm. A weak positive correlation existed between DNA content and size of nuclei suggesting that DNA increases and nuclear growth may not be highly coordinated in this tissue. Increased proportions of the larger nuclei occurred in the later stages of endosperm development. Considering the entire endosperm, the average DNA content per nucleus at the 15-dap peak level was approximately 12.8 C constituting a 2.7-fold overall increase from 8 dap.     

Identification of quantitative trait loci that affect endoreduplication in maize endosperm

Endoreduplication in maize endosperm precedes the onset of starch and storage protein synthesis, and it is generally thought to influence grain filling. We created four backcross populations by reciprocally crossing the F₁ progeny of a cross between Sg18 and Mo17 to the parental inbreds, which differ in endoreduplication by two parameters—mean ploidy and percentage of endoreduplicated nuclei. This four-backcross design allowed us to estimate and test the additive and dominant genetic effects of quantitative trait loci (QTLs) affecting endoreduplication. An analysis of endosperm from the four backcross populations at 16 days after pollination using a modified triploid mapping approach identified three endosperm QTLs influencing mean ploidy and two endosperm QTLs affecting the percentage of endoreduplicated nuclei. Some of these QTLs may manifest their effects on endoreduplication via expression in the embryo. The QTLs detected display strong dominance or over-dominance and interacted epistatically with an embryo-expressed QTL. This helps to explain the genetic basis for transgressive segregation in the backcross progeny. Although the favorable alleles that increase mean ploidy and percentage of endoreduplicated nuclei can be contributed by both parents, the Mo17-derived alleles for endoreduplication were often dominant or over-dominant to the Sg18-derived allele. One QTL on chromosome 7 that may be expressed in both the embryo and endosperm exerted a pleiotropic effect on two different parameters of endoreduplication. The results from this study shed light on the regulation of endoreduplication in maize endosperm and provide a marker-assisted selection strategy for potentially improving grain yield. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. C. M. Coelho and S. Wu contributed equally to this work and should be considered as first authors.     

A Comparative Study on the Role of Cytokinins in Caryopsis Development in the Maize miniature1 Seed Mutant and Its Wild Type

We report here on a comparative developmental profile of plant hormone cytokinins in relation to cell size, cell number and endoreduplication in developing maize caryopsis of a cell wall invertase-deficient miniature1 ( mn1 ) seed mutant and its wild type, Mn1 , genotype. Both genotypes showed extremely high levels of total cytokinins during the very early stages of development, followed by a marked and genotype specific reduction. While the decrease of cytokinins in Mn1 was associated with their deactivation by 9-glucosylation, the absolute and the relative part of active cytokinin forms was higher in the mutant. During the exponential growth phase of endosperm between 6 d after pollination and 9 d after pollination, the mean cell doubling time, the absolute growth rate and the level of endoreduplication were similar in the two genotypes. However, the entire duration of growth was longer in Mn1 compared with mn1 , resulting in a significantly higher cell number in the Mn1 endosperm. These data correlate with the previously reported peak levels of the Mn1 -encoded cell wall invertase-2 (INCW2) at 12 d after pollination in the Mn1 endosperm. A model showing possible crosstalk among cytokinins, cell cycle and cell wall invertase as causal to increased cell number and sink strength of the Mn1 developing endosperm is discussed.     

Mechanisms of Al‐induced iron chlorosis in wheat (Triticum aestivum). Al‐inhibited biosynthesis and secretion of phytosiderophore

Although Al‐induced iron chlorosis has been observed in many plants, the mechanisms responsible for this phenomenon are yet to be understood. We investigated the effect of Al on iron acquisition in a Strategy II plant, wheat ( Triticum aestivum L.) using both Al‐tolerant (Atlas 66) and ‐sensitive (Scout 66) cultivars. When iron was supplied as insoluble iron, ferric hydroxide, in the culture solution, both cultivars without Al treatment grew normally, while those with 100 µ M AlCl₃ developed chlorosis of the young leaves after 3 days of the treatment. A 21‐h treatment with 100 µ M AlCl₃ in 0.5 m M CaCl₂ solution (pH 4.5) decreased the amount of 2'‐deoxymugineic acid (DMA) secreted by Fe‐deficient Atlas 66 and Scout 66 plants by 85 and 90%, respectively. The amount of DMA secreted decreased with increasing external Al concentrations. Al treatment during the biosynthesis process caused the inhibition of that of DMA within 3 h. The secretion process was also found to be inhibited by Al, resulting in the biosynthesized DMA remaining in the roots. These results demonstrate the inhibition by Al of both biosynthesis and secretion of DMA attributed to Al‐induced iron chlorosis.     

Antipodal complex development in the embryo sac of wheat

Dynamics of an antipodal complex formation in wheat (Tritiñum aestivum L.) has been observed in detail using a reconstruction of serial semifine sections. Three consecutive crucial stages have been identified in the development of the antipodal complex: (1) proliferation of initial cells, (2) growth and functional differentiation of antipodal cells, and (3) cell apoptosis. Specific features of the mitotic division of antipodal cells have been characterized. It has been shown that the structure of interphase nuclei and mitotic chromosomes of proliferating antipodal cells is similar to that of nucellar cells surrounding the embryo sac. According to the reconstruction of appropriately oriented serial sections, the division of antipodal cells is asynchronous. DNA content in differentiated antipodal cells has been determined by a cytophotometric analysis; in the case of a mature embryo sac, the ploidy of antipodal cells varied from 8 to 32C. Proliferation and DNA endoreduplication processes in the antipodal complex proceed at different time; the second process starts only after the termination of the first one. DNA endoreduplication is accompanied by total chromatin remodeling; as a result, giant chromosomes are formed in the nuclei of antipodal cells. The final stage of the antipodal complex development is programmed cell death or apoptosis. A model for the structural organization of an antipodal complex has been proposed based on the layer arrangement of cells. The secretory activity of antipodal cells directed towards the endosperm syncytium has been detected for the first time. The analysis of “truncated” ovules with an undeveloped endosperm has shown that developing endosperm can be a possible inductor, which stimulates the functional activity of antipodal cells and triggers their terminal differentiation. The obtained results evidence the functional role of antipodal cells in the development of the endosperm and embryo.     

The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth

The size of tomato fruit results from the combination of cell number and cell size, which are respectively determined by the cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of fleshy pericarp tissue is characterized by numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. Although a clear relationship exists between endoreduplication and cell growth in plants, the exact role of endoreduplication has not been clearly elucidated. To decipher the molecular basis of endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the tomato cyclin-dependent kinase inhibitor SlKRP1. We studied the kinetics of pericarp development in tomato fruit at the morphological and cytological levels, and demonstrated that endoreduplication is directly proportional to cell and fruit diameter. We established a mathematical model for tissue growth according to the number of divisions and endocycles. This model was tested in fruits where we managed to decrease the extent of endoreduplication by over-expressing SlKRP1 under the control of a fruit-specific promoter expressed during early development. Despite the fact that endoreduplication was affected, we could not observe any morphological, cytological or metabolic phenotypes, indicating that determination of cell and fruit size can be, at least conditionally, uncoupled from endoreduplication.     

Inheritance of nuclear DNA content in leaf epidermal cells of Zea mays L.

 The nuclear DNA content (ploidy level) of maize leaf-epidermal cells was investigated by Feulgen cytophotometry in two lines, Illinois High Protein (IHP) and Illinois Low Protein (ILP), their reciprocal hybrids, and their F₂s. Epidermal cells have a 2C, 4C or 8C nuclear DNA content. The mean DNA content per nucleus in IHP was significantly higher than in ILP; the mean DNA content per nucleus in hybrids was intermediate between the parental lines, and the same DNA content was measured in reciprocal crosses. In F₂s the same mean DNA content as in F₁s was observed but with larger variability than in the F₁, possibly indicating genetic segregation. It is inferred that the ploidy level in the leaf epidermis is inherited, and incomplete dominance occurs in hybrids. The same behaviour in the different genotypes was observed for epidermal cell-surface area, except that an increase of mean surface area occurred in the F₁, probably due to heterotic effects. The difference in the accumulation of 4C and 8C nuclei in leaf epidermis parallels that reported between two genotypes for the endosperm tissue: to the greater chromosome endoreduplication found in the endosperm there were correspondingly higher frequencies of 4C and 8C nuclei in the leaf epidermis, indicating a higher general tendency to chromosome endoreduplication in IHP than in ILP. It is suggested that the accumulation of 4C nuclei (G₂-block) in the leaf epidermis may be regarded as the initial step of chromosome endoreduplication, the two phenomena being related to the control of the sequence DNA synthesis-mitosis, possibly involving the same genes in both endosperm and leaf. However, the inheritance of DNA content per nucleus in epidermal tissue seems to be different from that observed in endosperm tissue of the same genotypes, suggesting that differences may occur in the regulation of the activity of these genes. Received: 19 November 1996 / Accepted: 29 November 1996     

Endoreduplication in cucumber (Cucumis sativus) seeds during development, after processing and storage, and during germination

Flow cytometry was used to study endoreduplication in developing, stored and germinating seeds of cucumber ( Cucumis sativus ). Fruits growing in a commercial seed production field were collected every 7 days, starting 14 days after pollination (DAP) up to 63 DAP (commercial harvest time). Seeds were isolated and the proportion of nuclei with different DNA contents in the whole seeds and in the embryos was analysed. Germination capacity of fresh and dried seeds at 25°C was established. In addition, the same analyses were performed on the seeds after processing (fermentation, drying and cleaning), following 1 and 2 years of storage, and after imbibition for 3, 6 and 12 h. In the young developing seeds, endoreduplication up to 128C occurred but this decreased to 8C by maturity. The proportion of endosperm nuclei was the highest at 21 DAP (30%) and then decreased to below 14% at harvest and 8% after processing. In the mature processed seeds, the majority of embryo nuclei (about 80%) contained 2C DNA; however, about 2% of endoreduplicated (8C) nuclei were still present. Seeds did not show any germination capacity up to 21 DAP; then it gradually increased to reach 100% as early as 49 DAP, 2 weeks before commercial harvest time. The relationship between seed maturity, germination and cell cycle status is discussed. The mean C-value of the seed cells as well as the (4C + 8C + 16C)/2C ratio are recommended as markers of cucumber seed maturity and the advancement of germination/priming (the stage of germination sensu stricto ).