The bright side of parasitic plants: what are they good for?

Parasitic plants are mostly viewed as pests. This is caused by several species causing serious damage to agriculture and forestry. There is however much more to parasitic plants than presumed weeds. Many parasitic plans exert even positive effects on natural ecosystems and human society, which we review in this paper. Plant parasitism generally reduces the growth and fitness of the hosts. The network created by a parasitic plant attached to multiple host plant individuals may however trigger transferring systemic signals among these. Parasitic plants have repeatedly been documented to play the role of keystone species in the ecosystems. Harmful effects on community dominants, including invasive species, may facilitate species coexistence and thus increase biodiversity. Many parasitic plants enhance nutrient cycling and provide resources to other organisms like herbivores or pollinators, which contributes to facilitation cascades in the ecosystems. There is also a long tradition of human use of parasitic plants for medicinal and cultural purposes worldwide. Few species provide edible fruits. Several parasitic plants are even cultivated by agriculture/forestry for efficient harvesting of their products. Horticultural use of some parasitic plant species has also been considered. While providing multiple benefits, parasitic plants should always be used with care. In particular, parasitic plant species should not be cultivated outside their native geographical range to avoid the risk of their uncontrolled spread and the resulting damage to ecosystems.

Comparative transcriptome analyses reveal core parasitism genes and suggest gene duplication and repurposing as sources of structural novelty.

The origin of novel traits is recognized as an important process underlying many major evolutionary radiations. We studied the genetic basis for the evolution of haustoria, the novel feeding organs of parasitic flowering plants, using comparative transcriptome sequencing in three species of Orobanchaceae. Around 180 genes are upregulated during haustorial development following host attachment in at least two species, and these are enriched in proteases, cell wall modifying enzymes, and extracellular secretion proteins. Additionally, about 100 shared genes are upregulated in response to haustorium inducing factors prior to host attachment. Collectively, we refer to these newly identified genes as putative "parasitism genes." Most of these parasitism genes are derived from gene duplications in a common ancestor of Orobanchaceae and Mimulus guttatus, a related nonparasitic plant. Additionally, the signature of relaxed purifying selection and/or adaptive evolution at specific sites was detected in many haustorial genes, and may play an important role in parasite evolution. Comparative analysis of gene expression patterns in parasitic and nonparasitic angiosperms suggests that parasitism genes are derived primarily from root and floral tissues, but with some genes co-opted from other tissues. Gene duplication, often taking place in a nonparasitic ancestor of Orobanchaceae, followed by regulatory neofunctionalization, was an important process in the origin of parasitic haustoria.

Trans-specific gene silencing of acetyl-CoA carboxylase in a root-parasitic plant.

Parasitic species of the family Orobanchaceae are devastating agricultural pests in many parts of the world. The control of weedy Orobanchaceae spp. is challenging, particularly due to the highly coordinated life cycles of the parasite and host plants. Although host genetic resistance often provides the foundation of plant pathogen management, few genes that confer resistance to root parasites have been identified and incorporated into crop species. Members of the family Orobanchaceae acquire water, nutrients, macromolecules, and oligonucleotides from host plants through haustoria that connect parasite and host plant roots. We are evaluating a resistance strategy based on using interfering RNA (RNAi) that is made in the host but inhibitory in the parasite as a parasite-derived oligonucleotide toxin. Sequences from the cytosolic acetyl-CoA carboxylase (ACCase) gene from Triphysaria versicolor were cloned in hairpin conformation and introduced into Medicago truncatula roots by Agrobacterium rhizogenes transformation. Transgenic roots were recovered for four of five ACCase constructions and infected with T. versicolor against parasitic weeds. In all cases, Triphysaria root viability was reduced up to 80% when parasitizing a host root bearing the hairpin ACCase. Triphysaria root growth was recovered by exogenous application of malonate. Reverse-transcriptase polymerase chain reaction (RT-PCR) showed that ACCase transcript levels were dramatically decreased in Triphysaria spp. parasitizing transgenic Medicago roots. Northern blot analysis identified a 21-nucleotide, ACCase-specific RNA in transgenic M. truncatula and in T. versicolor attached to them. One hairpin ACCase construction was lethal to Medicago spp. unless grown in media supplemented with malonate. Quantitative RT-PCR showed that the Medicago ACCase was inhibited by the Triphysaria ACCase RNAi. This work shows that ACCase is an effective target for inactivation in parasitic plants by trans-specific gene silencing.

The TvPirin gene is necessary for haustorium development in the parasitic plant Triphysaria versicolor.

The rhizosphere is teemed with organisms that coordinate their symbioses using chemical signals traversing between the host root and symbionts. Chemical signals also mediate interactions between roots of different plants, perhaps the most obvious being those between parasitic Orobanchaceae and their plant hosts. Parasitic plants use specific molecules provided by host roots to initiate the development of haustoria, invasive structures critical for plant parasitism. We took a transcriptomics approach to identify parasitic plant genes associated with host factor recognition and haustorium signaling and previously identified a gene, TvPirin, which is transcriptionally up-regulated in roots of the parasitic plant Triphysaria versicolor after being exposed to the haustorium-inducing molecule 2,6-dimethoxybenzoquinone (DMBQ). Because TvPirin shares homology with proteins associated with environmental signaling in some plants, we hypothesized that TvPirin may function in host factor recognition in parasitic plants. We tested the function of TvPirin in T. versicolor roots using hairpin-mediated RNA interference. Reducing TvPirin transcripts in T. versicolor roots resulted in significantly less haustoria development in response to DMBQ exposure. We determined the transcript levels of other root expressed transcripts and found that several had reduced basal levels of gene expression but were similarly regulated by quinone exposure. Phylogenic investigations showed that TvPirin homologs are present in most flowering plants, and we found no evidence of parasite-specific gene duplication or expansion. We propose that TvPirin is a generalized transcription factor associated with the expression of a number of genes, some of which are involved in haustorium development.

A single-electron reducing quinone oxidoreductase is necessary to induce haustorium development in the root parasitic plant Triphysaria.

Parasitic plants in the Orobanchaceae develop haustoria in response to contact with host roots or chemical haustoria-inducing factors. Experiments in this manuscript test the hypothesis that quinolic-inducing factors activate haustorium development via a signal mechanism initiated by redox cycling between quinone and hydroquinone states. Two cDNAs were previously isolated from roots of the parasitic plant Triphysaria versicolor that encode distinct quinone oxidoreductases. QR1 encodes a single-electron reducing NADPH quinone oxidoreductase similar to zeta-crystallin. The QR2 enzyme catalyzes two electron reductions typical of xenobiotic detoxification. QR1 and QR2 transcripts are upregulated in a primary response to chemical-inducing factors, but only QR1 was upregulated in response to host roots. RNA interference technology was used to reduce QR1 and QR2 transcripts in Triphysaria roots that were evaluated for their ability to form haustoria. There was a significant decrease in haustorium development in roots silenced for QR1 but not in roots silenced for QR2. The infrequent QR1 transgenic roots that did develop haustoria had levels of QR1 similar to those of nontransgenic roots. These experiments implicate QR1 as one of the earliest genes on the haustorium signal transduction pathway, encoding a quinone oxidoreductase necessary for the redox bioactivation of haustorial inducing factors.

Macroscopic and microscopic study on floral biology and pollination of Cinnamomum verum Blume (Sri Lankan).

Cinnamomum verum Blume (syn Cinnamomum zeylanicum) commonly known as Ceylon cinnamon, has gained worldwide attention due to its health benefits and its unique quality. Therefore, maintaining the yield quality and quantity is essential, especially for high-end value-added products. Knowledge on floral behaviour and reproductive biology is essential for breeding superior varieties and is critical for commercial cultivation efforts. However, limited literature is available on the floral biology of C. verum. Here in this study, we assessed the seasonal flowering, floral development and pollination of two cultivars of C. verum. Both macroscopic and microscopic data were collected on floral biology, pollination, and male and female floral organs before and after pollination. Cinnamomum verum is morpho-anatomically, structurally, and physiologically adapted for cross-pollination, possible between the two cultivars; type A (Sri Gemunu) and type B (Sri Wijaya) flowers; naturally evolved with Protogynous Dichogamy. However, due to changes in environmental conditions, female and male stages in the same tree overlap for about 45-60 min suggesting possible close-pollination within the same plant. During this event some of the pollens were observed hydrated even during self-pollination. In mean time, 4-8% of the flowers formed fruits after natural close and hand pollination which is between male and female phases of the same tree. Although C. verum is adapted for cross-pollination, natural close-pollination is also possible. The data suggest the complex nature of the sexual reproduction of C. verum. Well-managed breeding attempts with controlled factors like temperature and humidity will help to develop superior C. verum varieties.

Internet of things (IoT) for smart agriculture: Assembling and assessment of a low-cost IoT system for polytunnels.

Internet of things (IoT) applications in smart agricultural systems vary from monitoring climate conditions, automating irrigation systems, greenhouse automation, crop monitoring and management, and crop prediction, up to end-to-end autonomous farm management systems. One of the main challenges to the advancement of IoT systems for the agricultural domain is the lack of training data under operational environmental conditions. Most of the current designs are based on simulations and artificially generated data. Therefore, the essential first step is studying and understanding the finely tuned and highly sensitive mechanism plants have developed to sense, respond, and adapt to changes in their environment, and their behavior under field and controlled systems. Therefore, this study was designed to achieve two specific objectives; to develop low-cost IoT components from basic building blocks, and to study the performance of the developed systems, and generate real-time experimental data, with and without plants. Low-cost IoT devices developed locally were used to convert existing basic polytunnels to semi-controlled and monitoring-only polytunnels. Their performances were analyzed and compared with each other based on several matrices while maintaining the planted tomato variety and agronomic practices similar. The developed system performed as expected suggesting the possibility of commercial applications and research purposes.

Correction: Internet of Things (IoT) for Smart Agriculture: Assembling and assessment of a low-cost IoT system for polytunnels.

[This corrects the article DOI: 10.1371/journal.pone.0278440.].

Chloroplast genome, nuclear ITS regions, mitogenome regions, and Skmer analysis resolved the genetic relationship among Cinnamomum species in Sri Lanka.

Cinnamomum species have gained worldwide attention because of their economic benefits. Among them, C. verum (synonymous with C. zeylanicum Blume), commonly known as Ceylon Cinnamon or True Cinnamon is mainly produced in Sri Lanka. In addition, Sri Lanka is home to seven endemic wild cinnamon species, C. capparu-coronde, C. citriodorum, C. dubium, C. litseifolium, C. ovalifolium, C. rivulorum and C. sinharajaense. Proper identification and genetic characterization are fundamental for the conservation and commercialization of these species. While some species can be identified based on distinct morphological or chemical traits, others cannot be identified easily morphologically or chemically. The DNA barcoding using rbcL, matK, and trnH-psbA regions could not also resolve the identification of Cinnamomum species in Sri Lanka. Therefore, we generated Illumina Hiseq data of about 20x coverage for each identified species and a C. verum sample (India) and assembled the chloroplast genome, nuclear ITS regions, and several mitochondrial genes, and conducted Skmer analysis. Chloroplast genomes of all eight species were assembled using a seed-based method.According to the Bayesian phylogenomic tree constructed with the complete chloroplast genomes, the C. verum (Sri Lanka) is sister to previously sequenced C. verum (NC_035236.1, KY635878.1), C. dubium and C. rivulorum. The C. verum sample from India is sister to C. litseifolium and C. ovalifolium. According to the ITS regions studied, C. verum (Sri Lanka) is sister to C. verum (NC_035236.1), C. dubium and C. rivulorum. Cinnamomum verum (India) shares an identical ITS region with C. ovalifolium, C. litseifolium, C. citriodorum, and C. capparu-coronde. According to the Skmer analysis C. verum (Sri Lanka) is sister to C. dubium and C. rivulorum, whereas C. verum (India) is sister to C. ovalifolium, and C. litseifolium. The chloroplast gene ycf1 was identified as a chloroplast barcode for the identification of Cinnamomum species. We identified an 18 bp indel region in the ycf1 gene, that could differentiate C. verum (India) and C. verum (Sri Lanka) samples tested.

Establishment of pomegranate (Punica granatum) hairy root cultures for genetic interrogation of the hydrolyzable tannin biosynthetic pathway.

In contrast to the numerous reports on the human therapeutic applications of hydrolyzable tannins (HTs), genes involved in their biosynthesis have not been identified at the molecular level from any plant species. Although we have previously identified candidate HT biosynthetic genes in pomegranate using transcriptomic and bioinformatic analyses, characterization of in planta enzyme function remains a critical step in biochemical pathway elucidation. We here report the establishment of a pomegranate (Punica granatum) hairy root culture system that produces HTs. Agrobacterium rhizogenes strains transformed with a binary vector harboring a yellow fluorescent protein (YFP) gene were used for hairy root induction, allowing visual, non-destructive, detection of transgene incorporation. It also demonstrated that the pomegranate hairy root culture system is suitable for expressing heterologous genes (YFP in this case). Expression of 26 putative UDP-glycosyltransferase (UGT) genes, obtained from a pomegranate fruit peel (a tissue highly abundant in HTs) RNA-Seq library, were verified in wild type and hairy roots. In addition, two candidate UGTs for HT biosynthesis were identified based on HPLC and differential gene expression analyses of various pomegranate tissues. Together with in vitro enzyme activity assays, the hairy root culture system holds great promise for revealing the undivulged HT biosynthetic pathway using pomegranate as a model system.

A CTAB protocol for obtaining high-quality total RNA from cinnamon (Cinnamomum zeylanicum Blume).

Cinnamomum zeylanicum Blume is an endemic Sri Lankan species commonly known as Ceylon cinnamon or true cinnamon. It is considered the king of spices in addition to its medicinal benefits. Despite recent scientific evidence on its medicinal properties and the industrial demand, cinnamon breeding and crop improvement are not been improved to the expectation. It is mainly due to the limited availability of the genomic information of cinnamon, linked with technical challenges caused by abundant secondary metabolites in all plant parts. Therefore, obtaining high-quality RNA is the fundamental step of transcriptomic analysis and the gene discovery process of cinnamon. We have optimized a CTAB based protocol for high-quality RNA extraction from different cinnamon tissues at various maturity stages collected from the field. Regular pH around 8 and the presence of Polyvinylpyrrolidone (PVP) in CTAB buffer increased the viscosity of the cinnamon lysate. Adjusting the pH of the lysis buffer to 6-6.5 reduced the viscosity of lysate while chloroform precipitates protein efficiently at the adjusted pH with no phenol. Therefore, this protocol excludes PVP and phenol extraction steps. Nanodrop spectrophotometer, gel electrophoresis, and bioanalyzer readings confirmed the quality of extracted RNA. RNA-seq libraries prepared were sequenced with Illumina Sequencing by synthesis technology and obtained good quality data to be used for transcriptomic analysis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02756-1.

Universal barcoding regions, rbcL, matK and trnH-psbA do not discriminate Cinnamomum species in Sri Lanka.

The genus Cinnamomum consists of about 250 species spread globally. Out of these, C. verum (C. zeylanicum), also known as true cinnamon or Ceylon cinnamon, has gained worldwide attention due to its culinary uses and medicinal values. Sri Lanka is the largest true cinnamon producer in the world and accounts for about 80-90% of global production. Other than the cultivated species, Sri Lankan natural vegetation is home to seven endemic wild species of the genus Cinnamomum. While these are underutilized, proper identification and characterization are essential steps in any sustainable conservation and utilization strategies. Currently, species identification is purely based on morphological traits, and intraspecific diversity has made it more challenging. In this study, all the eight Cinnamomum species found in Sri Lanka, C. capparu-coronde, C. citriodorum C. dubium, C. litseifolium, C. ovalifolium, C. rivulorum, C. sinharajaense, and C. verum were collected in triplicates and identified using typical morphological traits. DNA extracted with the same collection was assessed with universal barcoding regions, rbcL, matK, and trnH-psbA. While no intraspecific sequence differences were observed in C. citriodorum, C. rivulorum, and C. verum, the others had polymorphic sites in one, two, or all regions assessed. Interestingly, two individuals of C. sinharajaense had identical barcodes to the cultivated species C. verum, while the other one had one variable cite in matK region and three cites in trnH-psbA reigon. Further, one C. dubium and one C. capparu-coronde accession each had identical, rbcL, and trnH-psbA sequences while those had only a single nucleotide variation observed in matK region. Overall, the phylogeny of Cinnamomum species found in Sri Lanka could not be completely resolved with DNA barcoding regions studied.

Factors affecting the efficiency of Rhizobium rhizogenes root transformation of the root parasitic plant Triphysaria versicolor and its host Arabidopsis thaliana.

BACKGROUND: Rhizobium rhizogenes transformation is commonly used to generate transgenic roots traditionally called hairy roots, for both investigative and commercial applications. While fertile plants can be regenerated from transgenic roots, the transgenic roots are more typically used directly, either to investigate root biology or to produce valuable secondary metabolites. Hairy roots have been particularly useful for genetic studies of rhizosphere interactions; including the recognition of host plant roots by the roots of parasitic angiosperms. RESULTS: In this manuscript we analyzed various environmental, nutritional and procedural conditions for their effects on transformation of the model hemi-parasitic plant Triphysaria versicolor and Arabidopsis thaliana, one of its hosts. We first examined the effects of media, gelling agents and co-incubation times on Triphysaria root transformation and determined that while all three affected transformation rates, the media were the most significant. Once those primary conditions were fixed, we examined the roles of seedling age, explant type, acetosyringone, temperature and illumination on Triphysaria hairy root transformation rates. Using the optimized procedure approximately 70% of Triphysaria seedlings developed transgenic roots as judged by expression of YFP. These conditions were then used to transform Arabidopsis and similar transformation rates were obtained. CONCLUSIONS: Analyses of root transformation factors provides a method recovering transgenic roots from both parasitic plants and their hosts at high frequency. In addition to providing an effective in vitro approach for genetic investigations of parasitic plant-host plant interactions, these results are applicable to genetic studies of non-parasitic plants as well.

Biochemical Composition and Expression of Anthocyanin Biosynthetic Genes of a Yellow Peeled and Pinkish Ariled Pomegranate ( Punica granatum L.) Cultivar are Differentially Regulated in Response to Agro-Climatic Conditions.

The accumulation of beneficial biochemical compounds in different parts of pomegranate ( Punica granatum L.) fruit determines fruit quality and highly depends on environmental conditions. We investigated the effects of agro-climatic conditions on major biochemical compounds and on the expression of major anthocyanin biosynthetic genes in the peels and arils of a yellow-peeled and pink-ariled pomegranate cultivar in three agro-climatologically different locations in Sri Lanka. Drier and warmer climates promoted the accumulation of the measured biochemical compounds, i.e. total phenolic content (TPC), antioxidant capacity (AOX), and α, ß, and total punicalagin, in both peels and arils compared to wetter and cooler climates. Pomegranate DFR, F3H, and ANS transcripts in both peels and arils showed higher relative expression in hotter and drier regions, compared to those grown in cooler and wetter conditions. Therefore, growing pomegranates in drier and warmer environments maximizes the production of beneficial biochemical compounds and associated gene expression in pomegranate fruit.