Genetic Basis of Carnivorous Leaf Development

Plant carnivory is often manifested as dramatic changes in the structure and morphology of the leaf. These changes appear to begin early in leaf development. For example, the development of the Sarracenia purpurea leaf primordium is associated with the formation of an adaxial ridge, whose growth along with that of the leaf margin resulted in a hollow structure that later developed into a pitcher. In Nepenthes khasiana, pitcher formation occurs during the initial stages of leaf development, although this has not been shown at the primordial stage. The formation of the Utricularia gibba trap resulted from the growth of the dome-shaped primordium in both the longitudinal and transverse directions. Recent research has begun to unfold the genetic basis of the development of the carnivorous leaf. We review these findings and discuss them in relation to the flat-shaped leaves of the model plant Arabidopsis.

Plant-derived insulator-like sequences for control of transgene expression

Stable and consistent transgene expression is necessary to advance plant biotechnology. Stable expression can be achieved by incorporating enhancer-blocking insulators, which are cisregulatory elements that reduce enhancer interference in gene expression, into transgene constructs. Sufficient insulators for plant use are not available, and their discovery has remained elusive. In this work, we computationally mined the compact genome of Utricularia gibba for insulator sequences and identified short (<1 kb) sequences with potential insulator activity. Based on in vivo tests, three of these effectively mitigate the ectopic transgene expression caused by the Cauliflower Mosaic Virus 35S promoter and do so better than previously reported plant insulators. However, all sequences with apparent insulator activity also decrease the effectiveness of the CaMV 35S promoter, and thus may be more accurately classified as silencers. However, since the insulator effect is proportionately much higher than the silencing effect, these sequences are still useful for plant transformation.

New records of flowering plants collected from the Phou Khao Khouay National Biodiversity Conservation Area for the flora of Laos

We report 14 species of flowering plants as new additions to the flora of Laos. These are Illex viridis (Aquifoliaceae), Capparis erycibe (Capparaceae), Euphorbia bokorensis (Euphorbiaceae), Exacum darae (Gentianaceae), Aeschynanthus cambodiensis (Gesneriaceae), Tetraphyllum roseum (Gesneriaceae), Utricularia gibba (Lentibulariaceae), Macrosolen brandisianus (Loranthaceae), Decaschistia siamensis (Malvaceae), Nyssa yunnanensis (Nyssaceae), Adenia penangiana var. penangiana (Passifloraceae), Pentaphylax euryoides (Pentaphylacaceae), Wikstroemia bokorensis (Thymelaeaceae), and Debregeasia wallichiana (Urticaceae). We discovered the species during a botanical survey of the Phou Khao Khouay National Biodiversity Conservation Area (PKKNBCA) of Lao PDR in 2015-2019. In addition, nine rarely collected flowering plant species in Laos are newly reported for the PKKNBCA.

Atypical DNA methylation, sRNA-size distribution, and female gametogenesis in Utricularia gibba

The most studied DNA methylation pathway in plants is the RNA Directed DNA Methylation (RdDM), a conserved mechanism that involves the role of noncoding RNAs to control the expansion of the noncoding genome. Genome-wide DNA methylation levels have been reported to correlate with genome size. However, little is known about the catalog of noncoding RNAs and the impact on DNA methylation in small plant genomes with reduced noncoding regions. Because of the small length of intergenic regions in the compact genome of the carnivorous plant Utricularia gibba, we investigated its repertoire of noncoding RNA and DNA methylation landscape. Here, we report that, compared to other angiosperms, U. gibba has an unusual distribution of small RNAs and reduced global DNA methylation levels. DNA methylation was determined using a novel strategy based on long-read DNA sequencing with the Pacific Bioscience platform and confirmed by whole-genome bisulfite sequencing. Moreover, some key genes involved in the RdDM pathway may not represented by compensatory paralogs or comprise truncated proteins, for example, U. gibba DICER-LIKE 3 (DCL3), encoding a DICER endonuclease that produces 24-nt small-interfering RNAs, has lost key domains required for complete function. Our results unveil that a truncated DCL3 correlates with a decreased proportion of 24-nt small-interfering RNAs, low DNA methylation levels, and developmental abnormalities during female gametogenesis in U. gibba. Alterations in female gametogenesis are reminiscent of RdDM mutant phenotypes in Arabidopsis thaliana. It would be interesting to further study the biological implications of the DCL3 truncation in U. gibba, as it could represent an initial step in the evolution of RdDM pathway in compact genomes.

Atypical DNA methylation, sRNA size distribution and female gametogenesis correlate with genome compaction in Utricularia gibba

The most studied DNA methylation pathway in plants is the RNA Directed DNA Methylation (RdDM), a conserved mechanism that involves the role of noncoding RNAs to control the expansion of the noncoding genome. Genome-wide methylation levels have been reported to correlate with genome size. However, little is known about the catalog of noncoding RNAs and the impact on DNA methylation in compact plant genomes. Because the small genome size of the carnivorous plant Utricularia gibba we investigate the noncoding RNA landscape and global DNA methylation in a compact genome. Here, we report that, compared to other angiosperms, U. gibba has an unusual distribution of noncoding RNAs and reduced global DNA methylation levels, as determined by a novel strategy based on long-read DNA sequencing with the Pacific Bioscience platform and confirmed by whole-genome bisulfite sequencing. Moreover, reduced DNA methylation correlates with lack of a functional RdDM pathway, as U. gibba DICER-LIKE 3 (DCL3), encoding a DICER endonuclease that produces 24-nt small-interfering RNAs lost key domains required for complete function. Our findings unveil that lack of a functional DCL3 in U. gibba correlates with a decreased proportion of 24-nt small-interfering RNAs, low genome methylation levels, and developmental abnormalities during female gametogenesis that are reminiscent of RdDM mutant phenotypes in Arabidopsis thaliana. It would be interesting to further study the biological implications of the DCL3 truncation in U. gibba, as it could represent an initial step in the evolution of apomixis in compact genomes.

Atypical DNA methylation, sRNA size distribution and female gametogenesis correlate with genome compaction in Utricularia gibba

SummaryThe most studied DNA methylation pathway in plants is the RNA Directed DNA Methylation (RdDM), which is a conserved mechanism that involves noncoding-RNAs to control the expansion of intergenic regions. However, little is known about relationship between plant genome size reductions and DNA methylation.Because the compact genome size of the carnivorous plant Utricularia gibba, we investigate in this plant the noncoding-RNA landscape and DNA methylation through a combination of cytological, evolutionary, and genome-wide transcriptomic and methylation approaches.We report an unusual distribution of noncoding RNAs in U. gibba in comparison with other characterized angiosperms, which correlated with a lower level of global genome methylation, as determined by a novel strategy based on long-read DNA sequencing and corroborated by whole-genome bisulfite analysis. Moreover, found that genes involved in the RdDM pathway may not be functionally active in U. gibba, including a truncated DICER-LIKE 3 (DCL3), involved in the production of 24-nt small-RNAs.Our findings suggest that selective pressure to conserve a fully functional RdDM pathway might be reduced in compact genomes and a defective DCL3 correlate with a decreased proportion of 24-nt small-RNAs and developmental alterations in U. gibba, which could represent an initial step in the evolution of apomixis.

Agrobacterium tumefaciens mediated transformation of the aquatic carnivorous plant Utricularia gibba

Background The genus Utricularia belongs to Lentibulariaceae, the largest family of carnivorous plants, which includes terrestrial, epiphytic and aquatic species. The development of specialized structures that evolved for carnivory is a feature of this genus that has been of great interest to biologists since Darwin‘s early studies. Utricularia gibba is itself an aquatic plant with sophisticated bladder traps having one of the most complex suction mechanisms for trapping prey. However, the molecular characterization of the mechanisms that regulate trap development and the biophysical processes involved in prey trapping are still largely unknown due to the lack of a simple and reproducible gene transfer system. Results Here, we report the establishment of a simple, fast and reproducible protocol for genetic transformation of U. gibba based on the T-DNA of Agrobacterium tumefaciens . An in vitro selection system using Phosphinotricin as a selective agent was established for U. gibba . Plant transformation was confirmed by histochemical GUS assays and PCR and qRT-PCR analyses. We report on the expression pattern of the 35S promoter and of the promoter of a trap-specific ribonuclease gene in transgenic U. gibba plants. Conclusions The genetic transformation protocol reported here is an effective method for studying developmental biology and functional genomics of this genus of carnivorous plants and advances the utility of U. gibba as a model system to study developmental processes involved in trap formation.

Production and Characterization of Crude Protease from RS1 Isolate from silage of Floating Bladderwort (Utricularia gibba)

The purpose of this research was to produce and characterizing crude protease from RS1 isolate of swamp plant silage. The optimum production time of RS1 isolate was 40 h. The optimum pH and temperature of protease from RS1 isolate were 10 and 45℃, respectively. Ion Mg3+ increased RS1 protease whereas ion of Na+, K+, Fe2+, and Zn2+ inhibited protease from RS1 isolate. Study on the effect of metals ion indicated that protease from RS1 isolate was metaloenzyme. Based analysis on SDS-PAGE, the molecular weight of RS1 protease had 12 bands with molecular weights ranging from 34.75 kDa to 263.53 kDa.

Agrobacterium tumefaciens mediated transformation of the aquatic carnivorous plant Utricularia gibba

Background The genus Utricularia belongs to Lentibulariaceae, the largest family of carnivorous plants, which includes terrestrial, epiphytic and aquatic species. The development of specialized structures that evolved for carnivory is a feature of this genus that has been of great interest to biologists since Darwin‘s early studies. Utricularia gibba is itself an aquatic plant with sophisticated bladder traps having one of the most complex suction mechanisms for trapping prey. However, the molecular characterization of the mechanisms that regulate trap development and the biophysical processes involved in prey trapping are still largely unknown due to the lack of a simple and reproducible gene transfer system. Results Here, we report the establishment of a simple, fast and reproducible protocol for genetic transformation of U. gibba based on the T-DNA of Agrobacterium tumefaciens . An in vitro selection system using Phosphinotricin as a selective agent was established for U. gibba . Plant transformation was confirmed by histochemical GUS assays and PCR and qRT-PCR analyses. We report on the expression pattern of the 35S promoter and of the promoter of a trap-specific ribonuclease gene in transgenic U. gibba plants. Conclusions The genetic transformation protocol reported here is an effective method for studying developmental biology and functional genomics of this genus of carnivorous plants and advances the utility of U. gibba as a model system to study developmental processes involved in trap formation.