Involvement of Ca2+ in Vacuole Degradation Caused by a Rapid Temperature Decrease in Saintpaulia Palisade Cells: A Case of Gene Expression Analysis in a Specialized Small Tissue.

Saintpaulia (African violet) leaves are known to be damaged by a rapid temperature decrease when cold water is applied to the leaf surface; the injury is ascribed to the chloroplast damage caused by the cytosolic pH decrease following the degradation of the vacuolar membrane in the palisade cells. In this report, we present evidence for the involvement of Ca(2+) in facilitating the collapse of the vacuolar membrane and in turn in the temperature sensitivity of Saintpaulia leaves. In the presence of a Ca(2+) chelator (EGTA) or certain Ca(2+) channel inhibitors (Gd(3+) or La(3+)) but not others (verapamil or nifedipine), the pH of the vacuole, monitored through BCECF (2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein) fluorescence, did not increase in response to a rapid temperature drop. These pharmacological observations are consistent with the involvement of mechanosensitive Ca(2+) channels in the collapse of the vacuolar membrane. The high level of expression of an MCA- (Arabidopsis mechanosensitive Ca(2+) channel) like gene, a likely candidate for a mechanosensitive Ca(2+) channel(s) in plant cells, was confirmed in the palisade tissue in Saintpaulia leaves by using a newly developed method of gene expression analysis for the specialized small tissues.

Flower visitors of Streptocarpus teitensis: implications for conservation of a critically endangered African violet species in Kenya.

BACKGROUND: The African violets are endangered plant species restricted mainly to the Eastern Arc Mountains biodiversity hotspots in Kenya and Tanzania. These plants grow well in shaded environments with high humidity. Given their restricted geographical range and published evidence of dependance on insect vectors to facilitate sexual reproduction, understanding their pollination biology is vital for their survival. METHODS: We conducted an empirical study using flower visitor observations, pan trapping and bagging experiments to establish the role of flower visitors in the fruit set of a locally endemic and critically endangered species of African violet in Taita Hills, Kenya, Streptocarpus teitensis. RESULTS: The study found that fruit set is increased by 47.8% in S. teitensis when flowers are visited by insects. However, it is important to note the presence of putative autogamy suggesting S. teitensis could have a mixed breeding system involving self-pollination and cross-pollination since bagged flowers produced 26.9% fruit set. CONCLUSIONS: Insects appear to be essential flower visitors necessary for increased fruit set in S. teitensis. However, there is evidence of a mixed breeding system involving putative self-pollination and cross-pollination suggesting that S. teitensis is somewhat shielded from the negative effects of pollinator losses. Consequently, S. teitensis appears to be protected to a degree from the risks such as reproduction failure associated with pollinator losses by the presence of a safety net in putative self-pollination.

The First Glimpse of Streptocarpus ionanthus (Gesneriaceae) Phylogenomics: Analysis of Five Subspecies' Chloroplast Genomes.

Streptocarpus ionanthus (Gesneriaceae) comprise nine herbaceous subspecies, endemic to Kenya and Tanzania. The evolution of Str. ionanthus is perceived as complex due to morphological heterogeneity and unresolved phylogenetic relationships. Our study seeks to understand the molecular variation within Str. ionanthus using a phylogenomic approach. We sequence the chloroplast genomes of five subspecies of Str. ionanthus, compare their structural features and identify divergent regions. The five genomes are identical, with a conserved structure, a narrow size range (170 base pairs (bp)) and 115 unique genes (80 protein-coding, 31 tRNAs and 4 rRNAs). Genome alignment exhibits high synteny while the number of Simple Sequence Repeats (SSRs) are observed to be low (varying from 37 to 41), indicating high similarity. We identify ten divergent regions, including five variable regions (psbM, rps3, atpF-atpH, psbC-psbZ and psaA-ycf3) and five genes with a high number of polymorphic sites (rps16, rpoC2, rpoB, ycf1 and ndhA) which could be investigated further for phylogenetic utility in Str. ionanthus. Phylogenomic analyses here exhibit low polymorphism within Str. ionanthus and poor phylogenetic separation, which might be attributed to recent divergence. The complete chloroplast genome sequence data concerning the five subspecies provides genomic resources which can be expanded for future elucidation of Str. ionanthus phylogenetic relationships.

Genetic Analysis of Floral Symmetry Transition in African Violet Suggests the Involvement of Trans-acting Factor for CYCLOIDEA Expression Shifts.

With the growing demand for its ornamental uses, the African violet (Saintpaulia ionantha) has been popular owing to its variations in color, shape and its rapid responses to artificial selection. Wild type African violet (WT) is characterized by flowers with bilateral symmetry yet reversals showing radially symmetrical flowers such as dorsalized actinomorphic (DA) and ventralized actinomorphic (VA) peloria are common. Genetic crosses among WT, DA, and VA revealed that these floral symmetry transitions are likely to be controlled by three alleles at a single locus in which the levels of dominance are in a hierarchical fashion. To investigate whether the floral symmetry gene was responsible for these reversals, orthologs of CYCLOIDEA (CYC) were isolated and their expressions correlated to floral symmetry transitions. Quantitative RT-PCR and in situ results indicated that dorsal-specific CYCs expression in WT S. ionantha (SiCYC and SiCYC1B) shifted in DA with a heterotopically extended expression to all petals, but in VA, SiCYC1s' dorsally specific expressions were greatly reduced. Selection signature analysis revealed that the major high-expressed copy of SiCYC had been constrained under purifying selection, whereas the low-expressed helper SiCYC1B appeared to be relaxed under purifying selection after the duplication into SiCYC and SiCYC1B. Heterologous expression of SiCYC in Arabdiopsis showed petal growth retardation which was attributed to limited cell proliferation. While expression shifts of SiCYC and SiCYC1B correlate perfectly to the resulting symmetry phenotype transitions in F1s of WT and DA, there is no certain allelic combination of inherited SiCYC1s associated with specific symmetry phenotypes. This floral transition indicates that although the expression shifts of SiCYC/1B are responsible for the two contrasting actinomorphic reversals in African violet, they are likely to be controlled by upstream trans-acting factors or epigenetic regulations.

Erratum: Genetic Analysis of Floral Symmetry Transition in African Violet Suggests the Involvement of Trans-acting Factor for CYCLOIDEA Expression Shifts.

[This corrects the article DOI: 10.3389/fpls.2018.01008.].

Histogen Layers Contributing to Adventitious Bud Formation Are Determined by their Cell Division Activities.

Saintpaulia ionantha is propagated by adventitious buds in horticulture, and periclinal chimeral cultivars are usually difficult to propagate. However, some periclinal chimeral cultivars can be propagated with adventitious buds, and the mechanism of which has been unknown. Striped flower cultivars "Kaname," "Concord," and "Monique" were used to investigate what causes flower color separation in adventitious shoot-derived plants by tissue culture. These cultivars were revealed to have mutated flavonoid 3', 5' hydroxylase (SiF3'5'H), WDR1 (SiWDR1), or flavonoid 3 hydroxylase (SiF3H), respectively, in their L1 layer. From our previous study using "Kaname," all flowers from adventitious shoots were colored pink, which was the epidermal color of mother plants' flowers. We used "Concrd" and "Monique" from which we obtained not only monochromatic-colored plants the same as the epidermal color of mother plants, but also plants with a monochromatic colored plants, same as the subepidermal color, and a striped flower color the same as mother plants. Histological observations revealed that epidermal cells divided actively at 14 d after culture and they were involved in the formation of adventitious shoots in the cultured leaf segments of "Kaname." On the other hand, in "Concord" and "Monique," the number of divided cells in the subepidermis was rather higher than that of epidermal cells, and subepidermal cells were sometimes involved in shoot formation. In addition, the plant and leaf size of L1-derived plants from "Concord" and "Monique" were non-vigorous and smaller than those derived from the subepidermal layer. In conclusion, periclinal chimeral cultivars of Saintpaulia can be divided into two types. One type has a high cell division activity in the L1 layer, from which only single flower-colored plants derived from L1 can be obtained as adventitious shoots. Another type has a low cell division activity in the L1 layer, from which striped flower-colored plants the same as mother plants derived from several layers including L1 can be obtained as adventitious shoots. In the periclinal chimeral cultivar capable of propagation with adventitious shoots, the possibility was shown that cells in the L2 layer could form shoots by involving cells of the L1 layer with a low division activity.

Stable Transformation of the Saintpaulia ionantha by Particle Bombardment.

BACKGROUND: A highly efficient genetic transformation system is essential for a successful genetic manipulation of the African violet (Saintpaulia ionantha Wendl.). OBJECTIVES: Developing a particle bombardment-based genetic transformation system for the African violet. MATERIALS AND METHODS: A local cultivar of the African violet from Guilan province was used for transformation experiments. The pFF19G and pBin61-Ech42 vectors were used for transient and stable transformation experiments, respectively. The PCR and RT-PCR techniques were used to verify transgene presence and transcript levels in candidate transgenic lines, respectively. RESULTS: Using leaf explants as target tissues, we transferred an endochitinase gene cDNA into African violet. Transgenic plants were regenerated on selection medium at a reasonable frequency (in average, one stable transgenic line per shot). Molecular analysis of transgenic plants by PCR and RT-PCR techniques confirmed successful integration and expression of transgene in several independent transgenic lines. CONCLUSIONS: Our results provide an efficient stable transformation system for genetic transformation of African violet.

Expression and occurrence of uracil-DNA glycosylase in higher plants.

Uracil-DNA glycosylase (UDG) is the first enzyme in the base excision repair pathway for removal of uracil in DNA. DNA repair capacity is likely to be a critical factor in mutagenesis and thereby in the capacity to prevent genetic damage and unwanted variation. We have studied expression of UDG in 9 higher plant species. The highest expression of UDG was measured in Solanum tuberosum. A comparison of 6 Solanum tuberosum cultivars showed that the specific activity ranged from 30 pmol mg1 protein min-1 in the cultivar Laila to 80 pmol mg-1 protein min-1 in the cultivar Ostara. Measurement of UDG in Begonia X cheimantha gave no indications of enzyme activity. The possible effects of no or low UDG activity is discussed. In vitro cultures of Solanum tuberosum and Thymus vulgaris were used to examine the effect of auxin and cytokinin on the UDG activity. Axillary shoots of Solanum tuberosum were cultured on medium including 20 variations in hormone concentration. Auxin (1-naphtaleneacetic acid) increased the expression of UDG. Plants cultured on medium supplemented with 3 mg 1-1 1-naphtaleneacetic acid showed a specific UDG activity which was approximately 3-fold higher than the activity in controls. The cytokinin benzyladenine reduced the specific UDG activity at concentrations in the range 0.25-10 mg 1-1 . In vitro cultured Saintpaulia ionantha was used to examine UDG activity during initiation, conditioning and multiplication cycles. In general, highest expression of UDG was measured in the conditioning cycle on hormone free medium. Measurement of UDG expression during single subculture periods, clearly showed that UDG expression may vary over one culture period. Expression of UDG was in general highest three weeks after transfer to fresh medium. Of different seedling organs from 0- to 15-day-old Brassica napus L., roots and hypocotyls showed the highest UDG activities. In cotyledons a very low and nearly constant specific activity was observed. In 12-day-old seedlings the activity in roots was approximately 20 times higher than the activity in cotyledons.

Histological changes associated with acquisition of competence for shoot regeneration in leaf discs ofSaintpaufla ionantha xconfusa hybrid (African violet) cultured in vitro.

In leaf discs ofSaintpaulia ionantha xconfusa hybrid (cv. Virginia) cultured on shoot-inducing medium, periclinal divisions were initiated in epidermal cells 3-5 days after explant isolation. This timing coincided with the time for competence acquisition determined in tissue-transfer experiments. Some of the daughter cells from periclinal divisions formed the target cells which divided both anticlinally and periclinally to form cell division centers (meristemoids), precursors of adventitious shoots. The target cells were not morphologically distinct from other epidermal cells at the light microscope level. It is suggested that the periclinal divisions in epidermal cells represent the dedifferentiation phase during which target (competent) cells are formed. Once the cells have acquired the ability to divide periclinally, both dedifferentiation and shoot induction occur in the presence of exogenous plant hormones.

Acquisition of competence for shoot regeneration in leaf discs ofSaintpaufla ionantha xconfusa hybrids (African violet) cultured in vitro.

Leaf discs fromSaintpaulia ionantha xconfusa hybrids (African violet) were transferred between basal medium (BM) containing no hormones and shoot-inducing medium (SIM) containing 2.0 mg 1-1 indole acetic acid and 0.08 mg l-1 6-benzylaminopurine to determine whether there is a "window" of competence for shoot regeneration. Leaf discs precultured on BM prior to transfer to SIM formed buds 3 days earlier than the controls (leaf discs not precultured) regardless of whether the discs were placed upside down or right side up on the medium. This suggests that cultured leaf cells were not competent for shoot induction during the first 3 days of culture. Leaf discs cultured right side up (abaxial surface to the medium) did not form buds on BM alone, unlike discs cultured upside down. Leaf disc survival was affected by a delay in hormonal exposure, but surviving leaf discs produced as many shoots as control leaf discs. This suggests that in the absence of exogenous plant hormones, cellular competence to regenerate shoots is not lost in excised leaf discs of African violet.

Micropropagação de violeta-africana (Saintpaulia ionantha Wendl): efeito da Benzilaminopurina na multiplicação / Micropropagation of African-Violet (Saintpaulia ionantha Wendl): effect of Benzylaminopurine on multiplication

A violeta-africana (Saintpaulia ionantha Wendl.) é uma espécie cultivada como ornamental pela beleza de suas flores e folhagem. A Benzilaminopurina (BAP) pode ser utilizada na multiplicação in vitro dessa espécie, no entanto, inexistem informações mais detalhadas sobre as respostas obtidas sobre a multiplicação, em uma faixa ampla de concentrações. Objetivou-se com este trabalho determinar o efeito do BAP na multiplicação in vitro de violeta-africana. Foram utilizados, como explantes, tufos de brotações das cultivares Optimara Miki, Optimara Maki e Optimara Akemi, com tamanhos entre 0,8 e 2,0 cm. Empregou-se o meio MS, com concentrações de nutrientes minerais e vitaminas reduzidas à metade, suplementado com mio-inositol (100mg L-1), sacarose (30 g L-1) e ágar (7 g L-1). Os tratamentos utilizados foram: 0,0; 0,44; 1,78; 3,08 e 4,44 mM de BAP. O pH do meio foi ajustado para 5,8 antes da esterilização. O material vegetal permaneceu em temperatura de 24 ± 2 ºC, fotoperíodo de 14 horas e densidade de fluxo luminoso de 40 mmol c4m-2 s-1. As avaliações do número total de brotações e do número e altura de brotações maiores que 3 mm foram realizadas aos 46 dias. A adição de BAP ao meio de cultura foi essencial para a multiplicação das culturas. As respostas aos tratamentos variaram entre os genótipos utilizados. Maiores resultados de número total de brotações e número de brotações superiores que três milímetros foram observados em concentrações de BAP situadas entre 1,78 e 4,44 mM. A altura das brotações decresceu com a utilização de BAP.

African-Violet (Saintpaulia ionantha Wendl.) is an ornamental plant widely cultivated, because of its beautiful foliage and flowers. Benzylaminopurine (BAP) can be used to violet multiplication, but there weren't information about results with several concentrations. The aim of this work was to determine the effect of different concentrations of BAP on the multiplication of violet cultures. One utilized, as explants, shoot clusters of cultivars Optimara Miki, Optimara Maki e Optimara Akemfrom, with height from 0,8 to 2,0 cm. One utilized MS medium, with half concentration of mineral nutrients and vitamins, suplementated with myo-inositol (100 mg L-1), sucrose (30 g L-1) and agar (7 g L-1). The treatments applied were: 0.0, 0.44, 1.78, 3,08 e 4,44 mM of BAP. The pH of the medium was adjusted to 5,8 before sterilization. Cultures remained at 24 ± 2 ºC, photoperiod of 14 hours and 40 mmol cm-2 s-1 of light density flux. Total shoot number and number and height of shoots with more than three millimeters were evaluated at 46 days. Addition of BAP to the culture medium was essential to multiplication of cultures. Responses of treatments varied between cultivars. Best results of total shoot number and number shoots with more than three millimeters were found with concentrations between 1.78 e 4.44 mM. The height of shoots decreased with the addition of BAP.