Lettuce Protoplasts Research Articles

Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation.

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.

In vitro Bioassay of Allelopathic Activities of Soybean, and Three Isoflavones, Using Protoplast Co-culture Method with Digital Image Analysis

Inhibitory allelopathic activities of dried leaves of potted plants and protoplasts of Glycine max (soybean) cv. Okuhara-wase were studied using two in vitro bioassay methods. Strong inhibition (90%) of growth of recipient lettuce root was obtained with 50 mg soybean leaves using the lettuce seedling growth test (sandwich method), and moderate inhibition (65%) of lettuce protoplast division was obtained with 100 × 103 mL-1 protoplasts of etiolated soybean seedlings using the protoplast co-culture method with digital image analysis (DIA-PP method). Three isoflavones, genistein, genistin (genistein-7-O-glucoside), and daidzin (daidzein-7-O-glucoside) were investigated as putative allelochemicals using the DIA-PP method. The three isoflavones at 100 µM showed early crystal accumulation in lettuce protoplasts and inhibited lettuce protoplast division by 100%. Genistein showed the strongest inhibition. The results were compared and discussed with the previous reports on the invader leguminous plant, Kudzu, and two isoflavones, daidzein and Kudzu-specific puerarin (daidzein-6-C-glucoside), and other allelopathic plants and their putative allelochemicals.

TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts.

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 μM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.

In vitro Bioassay of Allelopathic Activities of a Mangrove Tree, Kandelia obovata, and Fast-growing Trees, Betula platyphylla and Populus alba, Using Protoplast Co-culture Method

Allelopathic activities of a salt-tolerant and low-temperature tolerant mangrove tree, Kandelia obovata, which grows in brackish water regions of sub-tropical areas, and two fast-growing trees, Betula platyphylla and Populus alba, which grow in the temperate area, were examined by two in vitro bioassay methods, the sandwich method using dried leaves and the protoplast co-culture method using leaf protoplasts. Lettuce root growth examined by the sandwich method, was inhibited 50% by 50 mg dried mature leaves of K. obovata. In the protoplast co-culture method, inhibition rates of cell division of lettuce protoplasts were 31% and 69% by leaf protoplasts of K. obovata at densities of 1 × 104 mL-1 and 5 × 104 mL-1, respectively. These results were compared with the inverse relationship between allelopathic activities and salt tolerance of mangrove plants of different families. B. platyphylla showed 37% inhibition by the sandwich method using dried young leaves, but only 10% inhibition at 5 × 104 mL-1 by the protoplast co-culture method using leaf protoplasts of B. platyphylla. Dried young leaves of P. alba showed 66% inhibition, but the leaf protoplasts at the density of 5 × 104 mL-1 showed highly stimulatory activity. Abscisic acid, of which contents in leaf protoplasts of three tree species varies from high to low in relation to salt tolerance and recalcitrance of tissue culture, was discussed as a putative allelochemical.

Allelopathic activities of three carotenoids, neoxanthin, crocin and β-carotene, assayed using protoplast co-culture method with digital image analysis.

Allelopathic activities of three carotenoids of a natural pigment group, neoxanthin, crocin and β-carotene, were assayed by the protoplast co-culture method with digital image analysis (DIA-PP method). Effects on three different growth stages of lettuce protoplasts, i.e., cell wall formation, cell division, and yellow pigment accumulation, were investigated using 96-well culture plates. Cell division was inhibited 65-95% by all three carotenoids at 33-100 µM. Inhibition of cell division stage was stronger than at the cell wall formation stage in neoxanthin, and the water-soluble carotenoid, crocin, whose yellow pigment was incorporated into the vacuole of lettuce protoplasts. Neoxanthin at 33 µM and crocin at higher than 100 µM inhibited more than 100% of the yellow pigment accumulation. By contrast, at low concentrations (0.01-1 µM) β-carotene stimulated growth at the cell division stage. At high concentrations of β-carotene (100-500 µM), inhibition was prominent at all three stages, and also in neighboring wells of zero control, which suggested emission of a volatile compound by β-carotene. They were compared with the report of the volatile compound, tulipalin A. Differences in patterns of inhibition of carotenoids on lettuce protoplast growth were compared with reports of another natural pigment, anthocyanin, and anthocyanin-containing red callus cultured in the light, and with that of neoxanthin-containing yellow callus cultured in the dark.

Evaluation of Isoflavones as Allelochemicals with Strong Allelopathic Activities of Kudzu Using Protoplast Co-Culture Method with Digital Image Analysis

The inhibitory allelopathic activity of Pueraria montana (Kudzu), and activities of two putative allelochemical isoflavones, puerarin and daidzein, were evaluated using the protoplast co-culture method with digital image analysis using lettuce as a recipient (DIA-PP method). Cotyledon protoplasts of Kudzu were isolated using Cellulase R10 and Driselase 20 in 0.6 M mannitol solution. Optimal hormonal condition and density for growth of Kudzu protoplasts were surveyed. Medium for co-culture of Kudzu or isoflavones with lettuce protoplasts was 50 μl liquid MS basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose, and 0.4 M or 0.6 M mannitol. Protoplast division of lettuce was strongly inhibited by Kudzu at a low density (104/ml). Slightly less inhibition by Kudzu on cell wall formation and yellow pigment accumulation stages of lettuce growth was also observed. Puerarin did not inhibit the growth of lettuce protoplasts at three growth stages but slightly stimulated growth at high concentrations. By contrast, daidzein, aglycon of puerarin, inhibited growth at three stages of lettuce protoplast growth and strongly inhibited cell division at 100 μM. Daidzein might be one cause of the strong inhibitory allelopathic activity of Kudzu. Grade of inhibitory activities was compared with that of other allelopathic plants including an invader plant and their allelochemicals studied using the DIA-PP method.

Establishment of Pluripotent Cell Cultures to Explore Allelopathic Activity of Coffee Cells by Protoplast Co-Culture Bioassay Method.

We focused on the demonstration of a new pluripotent coffee cell culture system to control the growth and metabolic functions. Somatic cells in the epidermal layer of in vitro somatic embryos (SEs) of Coffea canephora expressed higher pluripotency to produce secondary SEs than primary or secondary meristematic tissue. SEs were ideal explants to selectively induce functionally-differentiated cell lines, both non-embryogenic callus (nEC) and embryogenic callus (EC). The protoplast co-culture bioassay method was used to explore allelopathic activity of these cultured coffee cells. Cell wall formation of lettuce protoplasts varied after five days of co-culture. A strong stimulative reaction was observed at lower nEC protoplast densities, whereas growth was inhibited at higher densities. The reaction of lettuce protoplasts after 12 days of co-culture was recognized as an inhibitory reaction of colony formation.

Application of the protoplast co-culture method for evaluation of allelopathic activities of volatile compounds, safranal and tulipalin A

The effects of two plant volatile compounds (VOCs), safranal and tulipalin A were investigated using the protoplast method with digital image analysis, which was developed to investigate allelopathic activities of plants at the cellular level in a 50 μL liquid medium. Both VOCs showed inhibition on lettuce protoplasts growth at all three stages, i.e., the cell wall formation stage, the cell division stage, and the yellow colour accumulation stage. Among the three stages of protoplast growth, differences of inhibition patterns were observed. Inhibitions at the cell wall formation stage were stronger than those at the latter two stages in both VOCs. Tulipalin A showed higher inhibitory activity (total inhibition at 100 μM) than that of safranal (44% inhibition at 1 mM) at cell division stage. They were also compared with the direct exposure method in an enclosed vial using germinated lettuce seedlings, which was developed to investigate allelopathic activities of VOCs at plant level. Tulipalin A showed less inhibitory activity on radicle growth of lettuce, than that of safranal reported. Inhibition on hypocotyl growth was stronger than that on radicle in both VOCs. The causes of the differences between two VOCs and two bioassay methods, and the application of the protoplast method, were discussed for clarifying the contribution of VOCs as allelochemicals at cellular level.

High Allelopathic Activity of Carotenoid-accumulating Callus of a Halophilic Mangrove Plant, Avicennia alba: Protoplast Co-culture Method with Digital Image Analysis

A yellow callus strain was established from hypocotyls of a halophilic mangrove plant, Avicennia alba, by subculture in the dark. Allelopathic activities of yellow A. alba callus were assayed using recipient lettuce protoplasts at three growth stages by the protoplast co-culture method with digital image analysis. The protoplast cultures of yellow A. alba callus were halophilic to NaCl, KCl, and MgCl2 (up to 200 mM) but not to CaCl2. By contrast, NaCl and KCl inhibited the growth of non-salt-tolerant lettuce protoplasts, while CaCl2 and MgCl2 stimulated their growth at low concentrations. Highly salt-tolerant or halophilic mangrove plant cells were expected to have low allelopathic activity, but the protoplasts of yellow A. alba callus had very strong allelopathic activity. The inhibition was strongest at the cell division stage with growth being inhibited to 50% and 9% of the control by 104 mL-1 and 5 × 104 mL-1 of A. alba, respectively. There was less inhibition at the yellow pigment accumulation stage of lettuce. Stimulation was observed at the early cell wall formation stage with up to 105 mL-1 of A. alba. The yellow pigment of yellow A. alba callus was extracted with hexane and its absorption spectrum showed the wavelength peaks of a carotenoid, neoxanthin. Using transmission electron microscopy, specific electron-dense structures were found in yellow A. alba callus, which were similar to the undeveloped ultrastructure of a carotenoid. A carotenoid was strongly suggested to be the putative allelochemical in yellow callus of A. alba.

Evaluation of canavanine as an allelochemical in etiolated seedlings of Vicia villosa Roth: protoplast co-culture method with digital image analysis

Strong allelopathic activities of etiolated seedlings of a leguminous plant, Vicia villosa Roth (hairy vetch), were confirmed by a protoplast co-culture method. The combination of 1% Cellulase RS and 1% Driselase 20 in 0.4 M mannitol solution was used for protoplast isolation. The inhibitory effects of V. villosa protoplasts on the growth of recipient Lactuca sativa L. (lettuce) protoplasts were determined using 96-well culture plates with 50 μL of liquid medium consisting of Murashige and Skoog’s basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM 6-benzyladenine, 3% (w/v) sucrose, and 0.4 M mannitol in each well. The allelopathic activity of V. villosa epicotyl protoplasts (2 × 102 to 105 mL−1), on cell division of lettuce protoplasts (6 × 103 to 5 × 104 mL−1), was stronger than that of roots and was stronger than on cell wall formation. Digital image analysis of co-cultured protoplasts (DIA-PP method) showed the effect of V. villosa protoplasts on the accumulation of a yellow color in lettuce protoplasts. Weaker inhibition was seen than at the cell wall formation and cell division stages. Effects of putative allelochemical, canavanine at 10 μM strongly inhibited division of lettuce protoplasts, whereas putative allelochemical, cyanamide was stimulatory at 10 μM, but inhibitory at 1 mM. A high level of canavanine was found in epicotyl protoplasts using gas chromatography-mass spectrometry. Canavanine was suggested to be the allelochemical in V. villosa epicotyl protoplasts of etiolated seedlings, although its effect differed with the growth stages of lettuce protoplasts.

Evaluation of an Anthocyanin, Cyanidin 3,5-di-O-glucoside, as an Allelochemical in Red Callus of a Mangrove Sonneratia ovata, Using Protoplast Co-Culture Bioassay Method with Digital Image Analysis

protoplasts to examine the allelopathic activities. Protoplasts were isolated with Cellulase R10 and Driselase 20 in 0.6 M mannitol solution and purified by density gradient centrifugation on 0.6 M sucrose. Protoplasts were co-cultured in 50 μL of liquid Murashige and Skoog’s (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid and 0.1 μM benzyladenine and 0.6 M mannitol solution in a 96-well culture plate. Protoplast density ranged from 5 × 103/mL to 105/mL. Cell division of lettuce protoplasts was strongly inhibited by addition of S. ovata protoplasts, and non-spherical cell enlargement was slightly inhibited. By contrast, digital image analysis of scanned 96-well culture platesrevealed no inhibition in accumulation of yellow color in lettuce protoplasts. An anthocyanin, cyanidin 3,5-di-O-glucoside (cyanin), was identified and its content in the red callus was ca. 1 mM of fresh weight. The effects of cyanin on the growth of lettuce protoplasts at three stages were similar to those of red S. ovata protoplasts. From these results, cyanin was most likely the allelochemical contained in red callus of S. ovata. The allelopathic activity of cyanin was compared with that of other putative allelochemicals in several plant materials, using the protoplast co-culture method with digital image analysis.

Evaluation of Allelochemicals, Abscisic Acid and Coumarin, in Leaf-Origin Suspension Cultured Cells of <i>Prunus yedoensis</i> Using Protoplast Co-Culture Bioassay Method

Dried leaves of Prunus yedoensis and P. lannesiana (50 mg) showed strong inhibitory allelopathic activities, e.g., more than 97% growth inhibition of lettuce seedling using the sandwich method. Similarly, among suspension cultures induced from leaves and peduncles of two Prunus species, we found the strongest inhibitory allelopathic activities of protoplasts of leaf-origin suspension cells of P. yedoensis, when the protoplast co-culture method for bioassay of allelopathy was applied with lettuce as a recipient plant. Effects of two putative allelochemicals, abscisic acid and coumarin, on both protoplast cultures of lettuce and P. yedoensis were investigated. Coumarin inhibited the growth of lettuce protoplasts from low concentrations, while abscisic acid stimulated. Abscisic acid inhibited the protoplast growth of P. yedoensis from low concentrations, while coumarin did not, but inhibited only at a high concentration (1 mM). Contents of abscisic acid in protoplasts were measured using small scale purification and Enzyme Linked Immno Sorbent Assay, and contents of coumarin in leaf-origin susepension cells of P. yedoensis were measured using Gas Chromatography-Mass Spectrometry. Coumarin was more likely the allelochemical causing the strong inhibitory allelopathic activities of P. yedoensis in the protoplast co-culture bioassay. Effectiveness of the protoplast co-culture bioassay method of allelopathy was discussed.

Morphological characterisation of lettuce plasma membrane ultrastructure and vesicle formation caused by nonylphenol: A scanning electron microscopy study

Fresh produce can come into contact with nonylphenol (NP), a widely used surfactant and industrial endocrine disrupting chemical, through various routes of exposure in the agricultural environment. Nonylphenol has been linked to immunological and behavioural abnormalities as well as infertility and cancer in mammals. One of its modes of action is believed to be through damaging the plasma membrane of cells. While it has been demonstrated that NP can be phytotoxic, its effect on plant cell membrane integrity has not yet been examined. In this study, the plasma membranes of lettuce protoplasts were analysed with a high- resolution scanning electron microscope (SEM). The protoplasts were isolated from plants that had been exposed to 800, 1600, 3200, 6400 and 12,800μg/l NP, respectively, for three weeks. The plasma membrane of the protoplasts that were exposed to 6400 and 12,800μg/l NP showed vesicle-like protrusions, accompanied by flattening of the membrane in its direct vicinity. This study showed that NP caused changes in the ultrastructure of lettuce plasma membranes, thereby compromising its functionality. To the best of our knowledge, this paper describes for the first time the morphology of the plasma membrane using high-resolution SEM.

In vitro bioassay of allelopathy of Arabidopsis thaliana by sandwich method and protoplast co-culture method with digital image analysis.

We examined the allelopathic activities of Arabidopsis thaliana, ecotype Columbia by two in vitro methods. The effect of dried leaves on the growth of recipient lettuce seedlings was examined by the sandwich method. The allelopathic activity on protoplast growth was examined by co-culture with recipient lettuce leaf protoplasts in 50 µl liquid medium using a 96-well culture plate. Non-spherically enlarged or dividing protoplasts of lettuce were counted under an inverted microscope. Inhibition of yellow accumulation during lettuce protoplast growth was quantitated by image analysis of scanned digital images of 96-well culture plates. The results were described as the percentages of control without A. thaliana. The results were compared with those obtained using several plants which had strong allelopathic activities on recipient lettuce using the same methods. The growth of lettuce protoplasts was inhibited at both 4 and 8 days of culture with protoplasts of A. thaliana. The results suggested the usefulness of the protoplast co-culture method for future studies on allelopathy.

<i>In Vitro</i> Bioassay of Allelopathy in Four Bamboo Species; <i>Bambusa multiplex, Phyllostachys bambusoides, P. nigra, Sasa kurilensis</i>, Using Sandwich Method and Protoplast Co-Culture Method with Digital Image Analysis

Moderately strong allelopathic activities were found in four bamboo species, Bambusa multiplex cv. Houraichiku; Phyllostachys bambusoides cv. Madake; P. nigra cv. Hachiku; Sasa kurilensis cv. Chishimazasa, which are of different classification or of different ecological distributions, using the “Sandwich Method”, which assays the dried leaves on growth of lettuce seedlings. Only small difference of activity was found among the four bamboo species. In addition, “Protoplast Co-culture Method” for assay of allelopathy in a 50 μL liquid medium using a 96 well culture plate, was applied to the suspension cultures of the four bamboo species. Protoplasts were isolated from two-week cultured suspension cells of four bamboo species using Cellulase RS and Pectolyase Y-23 in 0.6 M mannitol. At low protoplast densities of bamboo, B. multiplex and P. bambusoides stimulated the recipient lettuce growth, i.e., non-spherically cell enlargement and cell divisions observed under an inverted microscope, while protoplasts of P. nigra and S. kurilensis were less stimulatory or inhibitory. Inhibitory effect of S. kurilensis was the strongest among four bamboo species. Furthermore, highly inhibitory effects of S. kurilensis protoplasts on yellow color accumulation of lettuce protoplasts were clearly observed by analysis of a scanned digital image of a 96-well culture plate. Differences and causes of the allelopathic activities were discussed comparing with other plant species studied using the same assay methods.

Allelopathy in a Leguminous Mangrove Plant, Derris indica: Protoplast Co-culture Bioassay and Rotenone Effect

To investigate allelopathic activity of a leguminous mangrove plant, Derris indica, the 'Protoplasts Co-culture Method' for bioassay of allelopathy was developed using suspension culture. A suspension culture was induced from immature seed and sub-cultured in Murashige and Skoog's (MS) basal medium containing 10 μM each of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). The protoplasts were isolated using the separate wells method with 2% each of Cellulase RS, Driselase 20 and Macerozyme R10 in 0.4 M mannitol solution. Protoplast cultures of D. indica revealed that high concentrations of cytokinins, BA and thidiazuron, were effective for cell divisions. The co-cultures of D. indica protoplasts with recipient lettuce protoplasts using 96 multi-well culture plates were performed in MS basal medium containing 0.4 M mannitol solution and 1 μM 2,4-D and 0.1 μM BA. The protoplast density of D. indica used in co-culturing varied from 6 x 10(3) - 10(5) / mL. Very strong inhibitory allelopathic effects of D. indica protoplasts on lettuce protoplast growth were found. A similar strong inhibitory allelopathic activity of dried young leaves on lettuce seedling growth was also observed by using the sandwich method. Rotenone, which is a component of Derris root, dissolved in DMSO, was highly inhibitory on the growth of lettuce protoplasts in culture and this could be one of the causes of the strong allelopathic activity of D. indica.

Protoplast Co-culture Bioassay for Allelopathy in Leguminous Plants, Leucaena leucocephala and Mucuna gigantea, Containing Allelochemical Amino Acids, Mimosine and L-DOPA

Protoplasts isolated from calluses of two woody leguminous plants, Leucaena leucocephala and Mucuna gigantea, which contain high levels of the allelochemical amino acids, mimosine and l-DOPA, respectively, were co-cultured to examine their allelopathic activities. The young leaves were first confirmed to have strong inhibitory activity by the sandwich method. Callus cultures were induced and sub-cultured from seeds of L. leucocephala and from leaves of M. gigantea in Murashige and Skoog’s (MS) basal media containing high concentrations of auxin and cytokinins. Protoplasts of both calluses were isolated using the separate well method under strong enzymatic conditions, in high osmotic, 0.8 M mannitol solution. Protoplasts of both species had strong inhibitory effects on the recipient protoplasts of cotyledons of Lactuca sativa (lettuce) co-cultured using 96 multi-well culture plates in the MS basal medium, containing 1 ?M 2,4-dichlorophenoxyacetic acid, 0.1 ?M benzyladenine and 0.8 M mannitol solution. Mimosine was less inhibitory than l-DOPA on the growth of lettuce protoplasts at the stage of colony formation. Amino acid profiles of calluses and protoplasts of the above two species and Mucuna pruriens showed very low contents of mimosine and l-DOPA, respectively. The cause of the high inhibitory allelopathic activities found in Leucaena and Mucuna protoplasts on the growth of lettuce protoplasts was discussed.

An inverse relationship between allelopathic activity and salt tolerance in suspension cultures of three mangrove species, Sonneratia alba, S. caseolaris and S. ovata: development of a bioassay method for allelopathy, the protoplast co-culture method.

A bioassay method for allelopathy, the 'protoplast co-culture method' was developed to study the relationship between salt tolerance and allelopathy of three mangrove species, Sonneratia alba, S. caseolaris, and S. ovata. Plants of S. alba grow in the seaward-side high salinity region and plants of the latter two species grow in upstream-side regions of a mangrove forest, respectively. Effects of five sea salts (NaCl, KCl, MgCl2, MgSO4 and CaCl2) on the growth of the suspension cells of the latter two species were first investigated by a small-scale method using 24-well culture plates. S. ovata cells showed higher tolerance than S. caseolaris cells to NaCl and other salts, but were not as halophilic as S. alba cells. Protoplasts isolated from suspension cells were co-cultured with lettuce protoplasts in Murashige and Skoog's (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose and 0.6-0.8 M osmoticum. S. caseolaris protoplasts had a higher inhibitory effect on lettuce protoplast cell divisions than S. alba protoplasts at any lettuce protoplast density, and the effect of S. ovata was intermediate between the two. These results were similar to those obtained from a different in vitro bioassay method for allelopathy, the 'sandwich method' with dried leaves. The inverse relationship between allelopathic activity and salt tolerance in suspension cells of Sonneratia mangroves is discussed.

Functional characterization and expression profiling of a DREB2-type gene from lettuce (Lactuca sativa L.)

Dehydration responsive element binding factors (DREBs) play important roles in plant responses to abiotic stress. In this study, a DREB2-type gene, LsDREB2A, was isolated from lettuce (Lactuca sativa L.) and characterized. LsDREB2A encoded a protein of 322 amino acid residues with the conserved apetala 2 domain and was classified into the A2 subgroup of DREBs. LsDREB2A has no sequence rich in proline, glutamic acid, serine, and threonine to trigger protein degradation. Southern blot analysis indicated that there could be more than ten DREB homologs in lettuce. A gel shift assay indicated that LsDREB2A could specifically recognize the dehydration responsive element (DRE) sequence in vitro. In a yeast one-hybrid assay, LsDREB2A bound specifically to the DRE sequence and activated the expression of both His3 and LacZ reporter genes. A transient expression experiment in lettuce protoplasts showed that LsDREB2A was localized to the nucleus. These results suggest that LsDREB2A might function as a transcription factor. Quantitative real-time polymerase chain reaction revealed that the expression of LsDREB2A was significantly increased by hyperosmotic and high salinity treatment, but not cold, heat, nor abscisic acid (ABA) treatment. Overexpression of LsDREB2A increased the tolerance of salt stress in transgenic plants. These results suggested that LsDREB2A plays an important role during high salinity stress in an ABA-independent pathway.

A strategy for building an amplified transcriptional switch to detect bacterial contamination of plants.

We have designed and tested a transcriptional autofeedback loop that could be used to engineer plants to sense the presence of bacteria. The signal amplification circuit was built based on the biological switch responsive to the presence of bacterial flagellin. Several flagellin- and E. coli-inducible Arabidopsis promoters were cloned and tested in transient expression assays in Arabidopsis and lettuce protoplasts using a flagellin-based peptide. These were investigated either as direct drivers of a reporter gene, or as a component of a transcriptional autofeedback loop. Arabidopsis promoters from the xyloglucan endotransglucosylase/hydrolase 18 (ATXTH18) and cytochrome P450 family CYP82C3 monooxygenase worked well as biological switches. These promoters were incorporated into our feedback loop system for signal amplification. The inclusion of a transcriptional repressor reduced basal expression, thereby increasing fold-amplification of signal detection and fine-tuning the positive autofeedback loop regulation.