Biologia plantarum, 2012 (vol. 56), issue 2
Reviews
Sexual incompatibility in Rosaceae fruit tree species: molecular interactions and evolutionary dynamics
A. Hegedûs, J. Lénárt, J. Halász
Biologia plantarum 56:201-209, 2012 | DOI: 10.1007/s10535-012-0077-3 
Fruit crops have a growing economic importance worldwide and molecular genetics might be useful in solving many problems that arise during commercial production. One of the fields that have attracted intense attention is the molecular basis of self-incompatibility that may result in low fruit set. In tree fruits of the Rosaceae family, the incompatibility reactions take place between the pistil S-ribonuclease (S-RNase) and the pollen-expressed S-haplotype specific F-box (SFB) proteins. In most cases, the loss of self-incompatibility was associated with mutations in the S-RNase or SFB genes. A...
Genes involved in the synthesis and signaling pathway of strigolactone, a shoot branching inhibitor
L. Gong, Y. -J. Yang, J. Zhou
Biologia plantarum 56:210-214, 2012 | DOI: 10.1007/s10535-012-0078-2
Branching is an important step in higher plant development, which not only determines the configuration of the plant directly, but also affects its adaptability to the environment. The interactions between hormones, genes, environmental and other factors subtly regulate the process of branching. Strigolactone is a newly recognized phytohormone and its content and distribution might be a key factor affecting branching. This review is focused on the genes related to synthesis and transduction pathway of strigolactone, and summarizes the inhibitory role of strigolactone in plant branching. Discussions about the issues to be clarified and prospects of...
Original Papers
Cellular origin and development of secondary somatic embryos in Oncidium leaf cultures
J. -T. Chen, P. -I. Hong
Biologia plantarum 56:215-220, 2012 | DOI: 10.1007/s10535-012-0079-1
We report here for the first time that fully differentiated stomatal guard cells have the ability to form directly secondary somatic embryos. Histological and scanning electron microscopy studies reveal that the stomata are formed on primary embryos of both Oncidium cultivars Gower Ramsey and Sweet Sugar. Secondary embryogenesis from these guard cells could be induced by several plant growth regulators (PGRs), including N6-benzyladenine, kinetin, thidiazuron, 1-aminocyclopropane-1carboxylic acid (ACC), ancymidol and 2,3,5-triiodobenzoic acid. When compared with other PGRs, ACC at concentration 1 mg dm-3 resulted in highest...
Lipid composition of Silybum marianum cell cultures treated with methyl jasmonate
M. Cacho, R. Peláez, P. Corchete
Biologia plantarum 56:221-226, 2012 | DOI: 10.1007/s10535-012-0080-8
Elicitation of cell cultures of Silybum marianum with methyl jasmonate (MeJA) increases the production and release of the secondary metabolite silymarin into the culture medium and this process seems to be dependent on phospholipase D activity and its product phosphatidic acid (PA). However, MeJA did not alter total membrane lipid content or overall fatty acid composition. A progressive increase in some galactolipids was observed with elicitation time. Phospholipids were mainly represented by phosphatidylcholine (PC) followed by phosphatidylethanolamine (PE) and phosphatidylinositol (PI). MeJA caused losses of PC species that contain two unsaturated...
Nitric oxide promoted rhizome induction in Cymbidium shoot buds under magnesium deficiency
S. Guha, I. Usha Rao
Biologia plantarum 56:227-236, 2012 | DOI: 10.1007/s10535-012-0081-7
Cymbidium shoot buds grown under Mg2+ deficiency without naphthalene acetic acid (NAA) showed knotted appearance. Ultrastructure of the cortical cells showed a progressive disorganization and disintegration of chloroplast membranes. The growth of shoots was resumed with the addition of 10 μM NAA. Specific NO scavenger, cPTIO induced deformation in shoot growth in 80 % of cultures. In longitudinal sections of shoots treated with cPTIO, depositions of densely stained particles in cells were observed. These inhibitory responses of cPTIO were ameliorated by 10 μM NAA. The NO donor, sodium nitroprusside (SNP), treated shoot buds...
Improved salt tolerance and delayed leaf senescence in transgenic cotton expressing the Agrobacterium IPT gene
Y. D. Liu, Z. J. Yin, J. W. Yu, J. LI, H. L. Wei, X. L. Han, F. F. Shen
Biologia plantarum 56:237-246, 2012 | DOI: 10.1007/s10535-012-0082-6
The manipulation of cytokinin contents via Agrobacterium-mediated transformation is an efficient tool for delaying leaf senescence and improving the resistance to environmental stresses. In the present study, cotton transformants harbouring the Agrobacterium tumefaciens isopentenyl transferase (IPT) gene under the control of the promoter of Gossypium hirsutum cysteine proteinase (Ghcysp) were generated. PCR and Southern blot analysis indicated that the foreign DNA fragment was successfully integrated into the cotton genome. The chlorophyll and cytokinin contents, and ROS-scavenging enzymatic activities were significantly...
Identification and characterization of a bacteria-like sequence in the genome of some Silene species
M. Talianová, J. ®lùvová, R. Hobza, B. Vyskot, B. Janou¹ek
Biologia plantarum 56:247-253, 2012 | DOI: 10.1007/s10535-012-0083-5
The aim of this work was to characterize a nucleotide sequence MK14 that originated from a plasmid library obtained via degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) amplification of laser microdissected Y-chromosomes of Silene latifolia. This sequence showed significant similarity to parts of two adjoining genes from bacterial representatives of the genus Ralstonia. MK14 sequence contains a part of a conserved domain, and phylogenetic analysis based on this region confirmed its relationship to Ralstonia-derived sequences. Genomic Southern blot analysis proved the presence of this fragment in the...
Nucleotide polymorphism pattern and multiple maternal origin in Thinopyrum intermedium inferred by trnH-psbA sequences
J. Zeng, X. Fan, L. -N. Sha, H. -Y. Kang, H. -Q. Zhang, J. Liu, X. -L. Wang, R. -W. Yang, Y. -H. Zhou
Biologia plantarum 56:254-260, 2012 | DOI: 10.1007/s10535-012-0084-4
Thinopyrum intermedium is an important species with potential utilization value in breeding of wheat. In this study, the non-coding intergenic region of trnH-psbA was investigated to assess the genetic diversity and infer the maternal origin within T. intermedium accessions. Eleven haplotypes were distinguished among the thirty-five accessions of T. intermedium. They showed a relatively low nucleotide diversity (π) of 0.00473 ± 0.00037 and a moderately high haplotype diversity (Hd) of 0.733 ± 0.061. In the phylogenetic analysis, all accessions of T. intermedium were positioned into...
Isolation and functional analysis of 4-coumarate:coenzyme A ligase gene promoters from Salvia miltiorrhiza
X. Q. Jin, Z. W. Chen, R. H. Tan, S. J. Zhao, Z. B. Hu
Biologia plantarum 56:261-268, 2012 | DOI: 10.1007/s10535-012-0085-3
The enzyme 4-coumarate:coenzyme A ligase (4CL) plays an important role in phenylpropanoid metabolism. The 5'-upstream regions of two Sm4CL genes were isolated from danshen (Salvia miltiorrhiza Bunge) and their functions were characterized by promoter-directed GUS gene expression assay in transgenic Arabidopsis. Seedlings containing pSm4CL1 promoter:GUS fusions showed apparent GUS staining in hypocotyl and those harboring pSm4CL2 promoter:GUS fusions were clearly stained in cotyledon vasculars and roots. Mature Arabidopsis transformed with pSm4CL1 promoter:GUS exhibited GUS expression which was weak in the shoots and...
Analysis of DNA methylation during the germination of wheat seeds
F. -R. Meng, Y. -C. Li, J. Yin, H. Liu, X. -J. Chen, Z. -F. Ni, Q. -X. Sun
Biologia plantarum 56:269-275, 2012 | DOI: 10.1007/s10535-012-0086-2
DNA methylation is known to play a crucial role in regulating plant development and organ or tissue differentiation. Here, we focused on the DNA methylation dynamics during the germination of wheat seeds using the adapted AFLP technique so called methylation-sensitive amplified polymorphism (MSAP). The MSAP profiles of genomic DNA in embryo and endosperm tissues of germinating seeds, as well as dry seeds were characterized and notable changes of cytosine methylation were detected. Comparisons of MSAP profiles in different tissues tested showed that the methylation level in dry seeds is the highest. The alteration analysis of cytosine methylation displayed...
Frequencies and variation in cytosine methylation patterns in diploid and tetraploid cytotypes of Paspalum notatum
M. P. Rodriguez, G. D. L. Cervigni, C. L. Quarin, J. P. A. Ortiz
Biologia plantarum 56:276-282, 2012 | DOI: 10.1007/s10535-012-0087-1
Paspalum notatum Flügge is a grass species organized as an agamic complex. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in diploid and tetraploid genotypes, and to determine the occurrence of methylation changes associated with tetraploidization by using methylation-sensitive amplification polymorphism (MSAP) markers. No differences were found in the average proportions of methylated CCGG sites between cytotypes, but methylation patterns were significantly more variable in tetraploids. In both groups of plants, epigenetic and non-epigenetic variation correlated significantly...
Genetic diversity in white clover and its progenitors as revealed by DNA fingerprinting
A. Badr, H.H. El-Shazly, L. Mekki
Biologia plantarum 56:283-291, 2012 | DOI: 10.1007/s10535-012-0088-0
The genetic diversity and ancestral relationships of a number of Trifolium species was revealed by using the amplified fragment length polymorphism (AFLP) and the random amplified polymorphic DNA (RAPD) markers. Both markers produced few species-specific markers. Using distance and parsimony methods, in NTSYS-pc and PAUP software programs, we clearly differentiated the accessions of white clover from other closely related progenitors. The phylogenetic trees, produced by PAUP, also reinforced the close affinity of T. nigrescens and the allopolyploid white clover in support of former views that this diploid species...
Low genetic diversity as revealed by SPAR methods possibly leads to extinction of two critically-endangered and endemic species of Mantisia
S. K. Sharma, S. S. D. Bhowmik, S. Kumaria, P. Tandon, S. Rama Rao
Biologia plantarum 56:292-300, 2012 | DOI: 10.1007/s10535-012-0089-z
Mantisia spathulata Schult. and M. wengeri Fischer, two critically-endangered, endemic and rare species of the genus Mantisia (Zingiberaceae), have been rediscovered from Lunglei province of Mizoram, India, after two decades. For sustainable conservation and utilization of the Mantisia species, in vitro seed and clonal propagation methods have been developed earlier by our research group and plantlets have been reintroduced to their natural habitat for species recovery. To comprehend the plausible reasons for endemism and endangeredness of both the species at DNA level, they were analyzed to assess natural...
Plastid division and morphology in the genus Peperomia
M. Ahmadabadi, R. Bock
Biologia plantarum 56:301-306, 2012 | DOI: 10.1007/s10535-012-0090-6
We have investigated several factors determining plastid size and number in Peperomia, a genus in the Piperaceae family whose species naturally display great interspecific variation in chloroplast size and number per cell. Using microscopic techniques, we show that chloroplast size and number are differently regulated in the palisade parenchyma and the spongy parenchyma, suggesting that chloroplast division in these cell types is controlled in different ways. Microscopic studies of iodine-stained root cells revealed a correlation between amyloplast size in root cells and chloroplast size in palisade parenchyma cells. However, despite...
Structure-function relationships during metaphloem sieve elements development in Triticum aestivum
C. N. Yang, Z. Q. Zhou, H. Y. Fan, Z. Jiang, F. Z. Mei
Biologia plantarum 56:307-312, 2012 | DOI: 10.1007/s10535-012-0091-5
The differentiation of metaphloem sieve element (MSEs) in the developing caryopsis of wheat (Triticum aestivum L.) was a programmed cell semi-death process. We studied the changes of microtubules and polysaccharide contents during MSEs development. Some significant features are presented in MSEs, such as cell wall non-uniform thickening, chromatin condensation and so on. During the period of MSEs differentiation, numerous microtubules are distributed in the vicinity of the cell wall, but finally they vanished in mature MSEs. Large glycoconjugates in the cell wall and small glycoconjugates in the Golgi apparatus were observed in the developing...
Role of hydrogen peroxide in regulating glucose-6-phosphate dehydrogenase activity under salt stress
Y. Liu, Q. Wan, R. Wu, X. Wang, H. Wang, Z. Wang, C. Shi, Y. Bi
Biologia plantarum 56:313-320, 2012 | DOI: 10.1007/s10535-012-0092-4
The role of hydrogen peroxide in the regulation of glucose-6-phosphate dehydrogenase (G6PDH) activity in the red kidney bean (Phaseolus vulgaris L.) roots under salt stress (100 mM NaCl) was investigated. Salt stress caused the increase of the activities of G6PDH and antioxidative enzymes including ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), as well as H2O2 production. The application of H2O2 (1 mM) also enhanced the activities of G6PDH as well as antioxidative enzymes. In the presence of exogenous CAT, H2O2 content was decreased,...
Hydrogen peroxide and nitric oxide promote reproductive growth in Litchi chinensis
B. Zhou, N. Li, Z. Zhang, X. Huang, H. Chen, Z. Hu, X. Pang, W. Liu, Y. Lu
Biologia plantarum 56:321-329, 2012 | DOI: 10.1007/s10535-012-0093-3
Vegetative growth and reproductive growth strongly competes with each other during panicle development in litchi (Litchi chinensis Sonn.). We herein investigated the roles of hydrogen peroxide and nitric oxide in the competition between growth of rudimentary leaves and panicle development. The results show that the chilling-induced flowering increased H2O2 and NO contents in the mixed buds. Treatments with sodium nitroprusside (SNP), the NO donor, and methyl viologen dichloride hydrate (MV), the superoxide generator, increased NO and H2O2 contents in the mixed buds. MV and SNP treatments promoted abscission...
Exogenous H2O2 increased catalase and peroxidase activities and proline content in Nitraria tangutorum callus
Y. L. Yang, Y. Y. Zhang, J. Lu, H. Zhang, Y. Liu, Y. Jiang, R. X. Shi
Biologia plantarum 56:330-336, 2012 | DOI: 10.1007/s10535-012-0094-2
Antioxidative responses and proline accumulation induced by exogenous H2O2 were investigated in the callus from halophyte Nitraria tangutorum Bobr. H2O2-treated callus exhibited higher H2O2 content than untreated callus. The activities of catalase (CAT) and peroxidase (POD) significantly increased in the callus treated with H2O2, while ascorbate peroxidase (APX) activity decreased. In addition, significantly enhanced proline content was observed in the callus treated by H2O2, which could be alleviated by H2O2 scavenger...
The effect of water deficit and excess copper on proline metabolism in Nicotiana benthamiana
H. -M. Ku, C. -W. Tan, Y. -S. Su, C. -Y. Chiu, C. -T. Chen, F. -J. Jan
Biologia plantarum 56:337-343, 2012 | DOI: 10.1007/s10535-012-0095-1
Fluctuation in proline content is a widespread phenomenon among plants in response to heavy metal stress. To distinguish between the participation of water deficit and copper on changes in proline metabolism, potted plants and floating leaf discs of tobacco were subjected to CuSO4 treatments. The application of copper increased the proline content in the leaves concomitantly with decreased leaf relative water content and increased abscisic acid (ABA) content in the potted plant. Excess copper increased the expression of two proline synthesis genes, pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT) and suppressed...
Root adaptations to cadmium-induced oxidative stress contribute to Cd tolerance in the hyperaccumulator Sedum alfredii
S. K. Tian, L. L. Lu, X. E. Yang, H. G. Huang, K. Wang, P. H. Brown
Biologia plantarum 56:344-350, 2012 | DOI: 10.1007/s10535-012-0096-0
Short-term responses of Sedum alfredii roots to Cd exposure was compared in Cd hyperaccumulator (HE) and nonhyperaccumulating ecotype (NHE). Cadmium exposure significantly inhibited root elongation and induced loss of plasma membrane integrity and lipid peroxidation of roots tips in the NHE, whereas these effects were much less pronounced in the HE plants. A strong accumulation of reactive oxygen species with increasing Cd concentration was noted in the NHE root tips, but not in HE. After Cd exposure, a dose-dependent decrease in oxidized glutathione and marked increase in reduced glutathione and non-protein thiols were observed in root tips...
Do fern gametophytes have the capacity for irradiance acclimation?
B. Fernández-Marín, S. J. Arroyo Alfaro, J. M. Becerril, J. I. García-Plazaola
Biologia plantarum 56:351-356, 2012 | DOI: 10.1007/s10535-012-0097-z
Ferns present two alternant generations: sporophyte and gametophyte. In the present work we address the question of whether fern gametophytes have the potential to acclimate to different irradiances as vascular plants do. We studied the gametophytes of three different fern species belonging to the Aspleniaceae family with different ecological requirements (Asplenium trichomanes, Asplenium scoloprendrium and Ceterach officinarum). Fern spores were germinated and the gametophytes cultivated under photon flux density (PFD) of 10, 50 or 100 μmol m-2 s-1. From the early stages of spore germination (the...
Brief Communications
Changes in antioxidative enzymes activities during Tacitus bellus direct shoot organogenesis
A. Mitroviæ, D. Jano¹eviæ, S. Budimir, J. Bogdanoviæ Pristov
Biologia plantarum 56:357-361, 2012 | DOI: 10.1007/s10535-012-0098-y
Changes in antioxidative enzymes activities during Tacitus bellus direct shoot organogenesis from leaf explants were examined. During the early stages of shoot organogenesis there was a decrease in superoxide dismutase (SOD) and an increase in catalase (CAT) activity, and later during organogenesis there was an increase in peroxidase (POD) and polyphenol oxidase (PPO) activity. Two highly regulated turning points may be distinguished regarding activities and isoforms of antioxidative enzymes: the initiation of shoot organogenesis and the shoot bud formation. Our data suggest the role of specific CAT, POD, SOD and PPO isoforms in separate processes...
Micropropagation of Pinus peuce
D. Stojièiæ, D. Jano¹eviæ, B. Uzelac, V. Èoke¹a, S. Budimir
Biologia plantarum 56:362-364, 2012 | DOI: 10.1007/s10535-012-0099-x
In Pinus peuce zygotic embryo culture grown on Gresshoff and Doy (1972; GD) basal medium, 2.22 μM benzyladenine (BA) was superior in promoting adventitious bud induction during 4 weeks comparing to kinetin or BA + kinetin. Shoot elongation was achieved on half-strength GD medium devoid of plant growth regulators and containing activated charcoal. Pulse treatment with 1 mM indole-3-butyric acid (IBA) for 2 h, followed by transfer to half-strength GD medium, produced the most efficient rooting. Rooted shoots were transplanted to the greenhouse and plantlets continued to grow and developed into phenotypically normal plants. Up to 10 plants per...
Multiple effects of inhibition of mitochondrial alternative oxidase pathway on photosynthetic apparatus in Rumex K-1 leaves
L.T. Zhang, H.Y. Gao, Z.S. Zhang, Z.C. Xue, Q.W. Meng
Biologia plantarum 56:365-368, 2012 | DOI: 10.1007/s10535-012-0100-8
The effects of inhibition of mitochondrial alternative oxidase (AOX) respiratory pathway on photosynthetic apparatus in Rumex K-1 leaves were studied. Under high irradiance, the inhibition of AOX pathway caused over-reduction of photosystem (PS) 2 acceptor side, a decrease in the energy transfer in the PS 2 units, damage of donor side of PS 2 and decrease in pool size of electron acceptors. The inhibition of AOX pathway also decreased photosynthetic performance index (PIABS), actual photochemical efficiency (ΦPS2), photochemical quenching (qP) and photosynthetic O2 evolution rate. The results demonstrate...
Mitochondrial minisatellite polymorphisms in fodder and sugar beets reveal genetic bottlenecks associated with domestication
Y. Yoshida, M. Matsunaga, D. Cheng, D. Xu, Y. Honma, T. Mikami, T. Kubo
Biologia plantarum 56:369-372, 2012 | DOI: 10.1007/s10535-012-0101-7
Historically, sugar beets were selected from fodder beets. We used mitochondrial minisatellite loci to analyze cytoplasmic genetic diversity in fodder beet and sugar beet. Among the 8 sugar beet accessions examined we identified 3 multi-locus haplotypes. These 3 haplotypes were a subset of 5 haplotypes identified among the 29 fodder beet accessions examined. All but one haplotype in fodder beet comprised, in turn, a subset of 12 haplotypes identified previously in leaf beets. Such apparent decreases in cytoplasmic genetic diversity must result from genetic bottlenecks associated with domestication and the ensuing breeding processes. We also detected...
Monitoring of cultivar identity in micropropagated olive plants using RAPD and ISR markers
A. R. Leva, R. Petruccelli
Biologia plantarum 56:373-376, 2012 | DOI: 10.1007/s10535-012-0102-6
Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were applied to assess the genetic stability of micropropagated olive (Olea europaea L. cv. Maurino) plants regenerated by axillary buds. Initial olive explants, isolated from one donor tree, were multiplied on Murashige and Skoog medium for 12 repeated subcultures. A total of 40 RAPD and 10 ISSR markers resulted in 301 distinct and reproducible band classes showing homogeneous RAPD and ISSR patterns. The amplification products revealed genetic stability among the micropropagated plants and between them and the donor plant. The results demonstrate the genetic...
Na+/H+ antiport activity in plasma membrane and tonoplast vesicles isolated from NaCl-treated cucumber roots
K. Kaba³a, M. Janicka-Russak
Biologia plantarum 56:377-382, 2012 | DOI: 10.1007/s10535-012-0103-5
Sodium/proton antiporter activity in the plasma membrane and tonoplast of cucumber seedling roots treated with 200 mM NaCl for 24 h was determined. It was observed that plasma membrane and tonoplast antiporter activity was only present in membranes from salt-treated plants. In addition, the plasma membrane antiporter protein was present in membranes after induction with NaCl, whereas tonoplast antiporter protein was observed in control and at elevated level in NaCl-treated plants. Moreover, based on the affinity of studied antiporter proteins to sodium ions, it could be assumed that excess sodium ions are firstly translocated from the cytosol to the...
Sodium nitroprusside modulates gene expression involved in glutathione synthesis in Zea mays leaves
C. Souza Mello, V. S. Hermes, M. P. Guerra, A. C. M. Arisi
Biologia plantarum 56:383-388, 2012 | DOI: 10.1007/s10535-012-0104-4
To investigate a possible involvement of nitric oxide in gene regulation of glutathione and flavonoid synthesis pathways, maize seedlings were treated with sodium nitroprusside (SNP), a NO donor, and apocynin (APO), an inducer of NO production. After 12-h treatment, the transcripts of γ-glutamylcysteine synthetase (γ-ecs), glutathione synthetase (gsh-s), chalcone synthase (chs), phenylalanine ammonia lyase (pal.1), myb-related protein P (P1) and actin 1 (act) genes were quantified in maize leaves by real time PCR, using α-tubulin as standard transcript. The level of γ-ecs and...
Expression of the BBM gene during somatic embryogenesis of Arabidopsis thaliana
K. Kulinska-Lukaszek, M. Tobojka, A. Adamiok, E. U. Kurczynska
Biologia plantarum 56:389-394, 2012 | DOI: 10.1007/s10535-012-0105-3
The relationship between somatic embryogenesis (SE) and the expression of the BABY BOOM (BBM) gene was studied in cultured immature zygotic embryos (IZEs) using a transgenic line of Arabidopsis thaliana containing a BBMPro::GUS construct. Results showed spatio-temporal differences in BBM expression in explants during culture. BBM promoter activity was observed in freshly isolated IZEs except distal parts of cotyledons. At the beginning of culture, considerable increase of GUS staining intensity was observed in all parts of explants, which maintained at high level over next few days and coincide with cell divisions....
Genetic variability in regenerated plants of Ungernia victoris
O. M. Bublyk, I. O. Andreev, K. V. Spiridonova, V. A. Kunakh
Biologia plantarum 56:395-400, 2012 | DOI: 10.1007/s10535-012-0106-2
To determine the suitability of micropropagation techniques developed for conserving rare medicinal herb Ungernia victoris we estimated the genetic fidelity of plants produced through direct regeneration from the bulb scale segments and organogenesis from long-term callus culture. Average value of the Jaccard's distances between explant-derived regenerants and maternal plants calculated from RAPD data was 0.5 %, while that of estimated between callus-derived regenerants and maternal cell line was 4.2 %; average distances between the objects among the explant-derived and callus-derived regenerants were 0.7 % and 2.5 %, respectively. The data...