Anti-Inflammatory and Antiatopic Effects of Rorippa cantoniensis (Lour.) Ohwi in RAW 264.7 and HaCaT Cells.

This study evaluated the effects of Rorippa cantoniensis (Lour.) ohwi extract (RCE) on factors associated with inflammation-related skin lesions in RAW 264.7 and HaCaT cells. RCE inhibited the levels of proinflammatory mediators and cytokines such as nitric oxide (NO), prostaglandin E2 (PGE2), interleukin (IL)-6, and tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In addition, RCE significantly inhibited the expression of chemokines and cytokines such as MDC/CCL22, TARC/CCL17, RANTES/CCL5, CTSS, IL-6, IL-1ß, and TNF-α in HaCaT cells costimulated by TNF-α and interferon (IFN)-γ in a concentration-dependent manner. These results suggest that RCE attenuated the TNF-α- and IFN-γ-induced release of proinflammatory chemokines and cytokines probably by suppressing the activation of MAPK (JNK and p38), NF-κB, and STAT1 signaling. Moreover, RCE significantly increased the expression of skin components such as hyaluronic acid and aquaporin, which play important roles in the physical and chemical barriers of the skin. These results suggest that RCE has significant anti-inflammatory and antiatopic activities, which may be beneficial for the topical treatment of inflammatory skin disorders.

Rewiring of hormones and light response pathways underlies the inhibition of stomatal development in an amphibious plant Rorippa aquatica underwater.

Land plants have evolved the ability to cope with submergence. Amphibious plants are adapted to both aerial and aquatic environments through phenotypic plasticity in leaf form and function, known as heterophylly. In general, underwater leaves of amphibious plants are devoid of stomata, yet their molecular regulatory mechanisms remain elusive. Using the emerging model of the Brassicaceae amphibious species Rorippa aquatica, we lay the foundation for the molecular physiological basis of the submergence-triggered inhibition of stomatal development. A series of temperature shift experiments showed that submergence-induced inhibition of stomatal development is largely uncoupled from morphological heterophylly and likely regulated by independent pathways. Submergence-responsive transcriptome analysis revealed rapid reprogramming of gene expression, exemplified by the suppression of RaSPEECHLESS and RaMUTE within 1 h and the involvement of light and hormones in the developmental switch from terrestrial to submerged leaves. Further physiological studies place ethylene as a central regulator of the submergence-triggered inhibition of stomatal development. Surprisingly, red and blue light have opposing functions in this process: blue light promotes, whereas red light inhibits stomatal development, through influencing the ethylene pathway. Finally, jasmonic acid counteracts the inhibition of stomatal development, which can be attenuated by the red light. The actions and interactions of light and hormone pathways in regulating stomatal development in R. aquatica are different from those in the terrestrial species, Arabidopsis thaliana. Thus, our work suggests that extensive rewiring events of red light to ethylene signaling might underlie the evolutionary adaption to water environment in Brassicaceae.

Leaf Cell Morphology Alternation in Response to Environmental Signals in Rorippa aquatica.

Heterophylly, the phenomenon by which plants alter leaf forms to adapt to surrounding conditions, is apparent in amphibious plant species. In response to submergence, they emerge leaves with narrower blade areas. The pathway that receives the submergence signals and the mechanism regulating leaf form via cell proliferation and/or expansion systems have not yet been fully identified yet. Our anatomical study of Rorippa aquatica, an amphibious plant that exhibits heterophylly in response to various signals, showed that leaf thickness increased upon submergence; this was caused by the expansion of mesophyll cell size. Additionally, these submergence effects were inhibited under blue-light conditions. The ANGUSTIFOLIA3 (AN3)/GROWTH-REGULATING FACTOR (GRF) pathway regulating cell proliferation and cell expansion was downregulated in response to submergence; and the response was blocked under the blue-light conditions. These results suggest that submergence and light quality determine leaf cell morphology via the AN3/GRF pathway.

Molecular Basis for Natural Vegetative Propagation via Regeneration in North American Lake Cress, Rorippa aquatica (Brassicaceae).

Some plant species have a striking capacity for regeneration in nature, including regeneration of the entire individual from explants. However, due to the lack of suitable experimental models, the regulatory mechanisms of spontaneous whole plant regeneration are mostly unknown. In this study, we established a novel model system to study these mechanisms using an amphibious plant within Brassicaceae, Rorippa aquatica, which naturally undergoes vegetative propagation via regeneration from leaf fragments. Morphological and anatomical observation showed that both de novo root and shoot organogenesis occurred from the proximal side of the cut edge transversely with leaf vascular tissue. Time-series RNA-seq analysis revealed that auxin and cytokinin responses were activated after leaf amputation and that regeneration-related genes were upregulated mainly on the proximal side of the leaf explants. Accordingly, we found that both auxin and cytokinin accumulated on the proximal side. Application of a polar auxin transport inhibitor retarded root and shoot regeneration, suggesting that the enhancement of auxin responses caused by polar auxin transport enhanced de novo organogenesis at the proximal wound site. Exogenous phytohormone and inhibitor applications further demonstrated that, in R. aquatica, both auxin and gibberellin are required for root regeneration, whereas cytokinin is important for shoot regeneration. Our results provide a molecular basis for vegetative propagation via de novo organogenesis.

Hyperaccumulation of Cd by Rorippa globosa (Turcz.) Thell. from soil enriched with different Cd compounds, and impact of soil amendment with glutathione (GSH) on the hyperaccumulation efficiency.

Rorippa globosa (Turcz.) Thell. is known as Cd hyperaccumulator, however neither hyperaccumulation nature, nor affecting factors like the effect of Cd compounds entering soil from different sources, or of specific soil amendments, are not yet satisfactorily clarified. In the pot culture experiment, Cd accumulation by R. globosa from soils spiked with 3 and 9 mg Cd kg-1 in the form of Cd(NO3)2, CdCl2, CdBr2, CdI2, CdSO4, CdF2, Cd(OH)2, CdCO3, Cd3(PO4)2, CdS and effect of soil amendment with glutathione (GSH) were investigated. Accumulation capacity of R. globosa for Cd appeared to reflect its extractability in soils and was about two-fold bigger for high soluble compounds than for low-soluble ones. At that, the differences between the accumulation of Cd originating from high soluble compound group did not exceed 20%, while the differences within the low soluble compound group were insignificant (p < 0.05). The analysis of Cd uptake, uptake factor (UF), enrichment factor (EF) and translocation factor (TF) patterns revealed that Cd hyperaccumulating properties of R. globosa are based on the high water/nutrients demand and strong tolerance to Cd, although weak protection against Cd uptake by root system was also observed. Amendment with GSH enhanced Cd availability to plant and its uptake from soil, but exerted no effect on Cd translocation in plants. In the light of the results, the use of R. globosa for phytoremediation of moderately polluted agricultural lands as forecrop or aftercrop, and the GSH-assisted phytoremediation of highly polluted post-industrial sites seem to be viable options.

Uranium accumulation in aquatic macrophytes in an uraniferous region: Relevance to natural attenuation.

Phytoremediation potential of uranium (U) was investigated by submerged, free-floating and rooted emergent native aquatic macrophytes inhabiting along the streams of Horta da Vilariça, a uraniferous geochemical region of NE Portugal. The work has been undertaken with the following objectives: (i) to relate the U concentrations in water-sediment-plant system; and (ii) to identify the potentialities of aquatic plants to remediate U-contaminated waters based on accumulation pattern. A total of 25 plant species culminating 233 samples was collected from 15 study points along with surface water and contiguous sediments. Concentrations of U showed wide range of variations both in waters (0.61-5.56 µg L(-1), mean value 1.98 µg L(-1)) and sediments (124-23,910 µg kg(-1), mean value 3929 µg kg(-1)) and this is also reflected in plant species examined. The plant species exhibited the ability to accumulate U several orders of magnitude higher than the surrounding water. Maximum U concentrations was recorded in the bryophyte Scorpiurium deflexifolium (49,639 µg kg(-1)) followed by Fontinalis antipyretica (35,771 µg kg(-1)), shoots of Rorippa sylvestris (33,837 µg kg(-1)), roots of Oenanthe crocata (17,807 µg kg(-1)) as well as in Nasturtium officinale (10,995 µg kg(-1)). Scorpiurium deflexifolium displayed a high bioconcentration factor (BF) of ∼2.5 × 10(4) (mean value). The species Fontinalis antipyretica, Nasturtium officinale (roots) and Rorippa sylvestris (shoots) exhibited the mean BFs of 1.7 × 10(4), 5 × 10(3) and 4.8 × 10(3) respectively. Maximum translocation factor (TF) was very much pronounced in the rooted perennial herb Rorippa sylvestris showing extreme ability to transport U for the shoots and seems to be promising candidate to be used as bioindicator species.

Effect of different nitrogenous nutrients on the cadmium hyperaccumulation efficiency of Rorippa globosa (Turcz.) Thell.

This experiment was used to explore whether the 11 nitrogenous nutrients affect the hyperaccumulation of Rorippa globosa (Turcz.) Thell. to Cd. Pot culture experiments using soil spiked with Cd as CdCl2·2.5H2O and 11 nitrogen-containing chemicals were conducted to determine the efficiency of the accumulation of Cd by R. globosa. Application of all 11 nitrogenous nutrients significantly (p < 0.05) enhanced Cd accumulation by R. globosa (Turcz.) Thell. Two major modes of Cd accumulation were observed: (i) through increase of biomass yield without reduction of Cd uptake and (ii) through increase of Cd uptake efficiency in parallel with increase of biomass yield. Bicarbonate > phosphate > chloride compounds of NH4 enhanced the biomass yield to the greatest extent, while oxalate > nitrate > chloride > and bicarbonate caused a significant increase of Cd uptake by R. globosa. Competition between N and Cd translocation caused either significant reduction of Cd translocation factor or decrease of biomass yield. Of studied nutrients, ammonium bicarbonate NH4HCO3 and ammonium chloride NH4Cl exerted the best joint effect of these two processes on the efficiency of R. globosa as a Cd hyperaccumulator. Application of these chemicals caused increase of Cd concentrations in roots of R. globosa by 35.1 and 41.1 %, and in shoots by 13.9 and 56.4 %, while biomasses of roots increased by 5.8- and 3.8-fold and in shoots by 7.4-fold, and 6.4-fold, respectively, compared to the control. As a result, accumulated load (µg pot(-1)) of Cd in roots increased by 8.2- and 5.8-fold and in shoots by 8.6- and 10.6-fold in both pots. Consequently, chemicals (NH4HCO3 and NH4Cl) that enhanced both Cd enrichment and biomass yield had the greatest effect on the bioaccumulation capacity of R. globosa.

Chemical defenses (glucosinolates) of native and invasive populations of the range expanding invasive plant Rorippa austriaca.

Due to global warming, species are expanding their range to higher latitudes. Some range expanding plants have become invasive in their new range. The Evolution of Increased Competitive Ability (EICA) hypothesis and the Shifting Defense Hypothesis (SDH) predict altered selection on plant defenses in the introduced range of invasive plants due to changes in herbivore pressures and communities. Here, we investigated chemical defenses (glucosinolates) of five native and seven invasive populations of the Eurasian invasive range expanding plant, Rorippa austriaca. Further, we studied feeding preferences of a generalist and a specialist herbivore among the populations. We detected eight glucosinolates in the leaves of R. austriaca. 8-Methylsulfinyloctyl glucosinolate was the most abundant glucosinolate in all plants. There were no overall differences between native and invasive plants in concentrations of glucosinolates. However, concentrations among populations within each range differed significantly. Feeding preference between the populations by a generalist herbivore was negatively correlated with glucosinolate concentrations. Feeding by a specialist did not differ between the populations and was not correlated with glucosinolates. Possibly, local differences in herbivore communities within each range may explain the differences in concentrations of glucosinolates among populations. Little support for the predictions of the EICA hypothesis or the SDH was found for the glucosinolate defenses of the studied native and invasive R. austriaca populations.

Extreme flooding tolerance in Rorippa.

Low oxygen stress imposed by floods creates a strong selection force shaping plant ecosystems in flood-prone areas. Plants inhabiting these environments adopt various adaptations and survival strategies to cope with increasing water depths. Two Rorippa species, R. sylvestris and R. amphibia that grow in naturally flooded areas, have high submergence tolerance achieved by the so-called quiescence and escape strategies, respectively. In order to dissect the molecular mechanisms involved in these strategies, we investigated submergence-induced changes in gene expression in flooded roots of Rorippa species. There was a higher induction of glycolysis and fermentation genes and faster carbohydrate reduction in R. amphibia, indicating a higher demand for energy potentially leading to faster mortality by starvation. Moreover, R. sylvestris showed induction of genes improving submergence tolerance, potentially enhancing survival in prolonged floods. Additionally, we compared transcript profiles of these 2 tolerant species to relatively intolerant Arabidopsis and found that only Rorippa species induced various inorganic pyrophosphate dependent genes, alternatives to ATP demanding pathways, thereby conserving energy, and potentially explaining the difference in flooding survival between Rorippa and Arabidopsis.

Root transcript profiling of two Rorippa species reveals gene clusters associated with extreme submergence tolerance.

Complete submergence represses photosynthesis and aerobic respiration, causing rapid mortality in most terrestrial plants. However, some plants have evolved traits allowing them to survive prolonged flooding, such as species of the genus Rorippa, close relatives of Arabidopsis (Arabidopsis thaliana). We studied plant survival, changes in carbohydrate and metabolite concentrations, and transcriptome responses to submergence of two species, Rorippa sylvestris and Rorippa amphibia. We exploited the close relationship between Rorippa species and the model species Arabidopsis by using Arabidopsis GeneChip microarrays for whole-genome transcript profiling of roots of young plants exposed to a 24-h submergence treatment or air. A probe mask was used based on hybridization of genomic DNA of both species to the arrays, so that weak probe signals due to Rorippa species/Arabidopsis mismatches were removed. Furthermore, we compared Rorippa species microarray results with those obtained for roots of submerged Arabidopsis plants. Both Rorippa species could tolerate deep submergence, with R. sylvestris surviving much longer than R. amphibia. Submergence resulted in the induction of genes involved in glycolysis and fermentation and the repression of many energy-consuming pathways, similar to the low-oxygen and submergence response of Arabidopsis and rice (Oryza sativa). The qualitative responses of both Rorippa species to submergence appeared roughly similar but differed quantitatively. Notably, glycolysis and fermentation genes and a gene encoding sucrose synthase were more strongly induced in the less tolerant R. amphibia than in R. sylvestris. A comparison with Arabidopsis microarray studies on submerged roots revealed some interesting differences and potential tolerance-related genes in Rorippa species.

Hyperaccumulative property comparison of 24 weed species to heavy metals using a pot culture experiment.

The screening of hyperaccumulators is still very much needed for phytoremediation. With properties such as strong tolerance to adverse environment, fast growing and highly reproductive rate, weed species may be an ideal plant for phytoremediation. The objectives of this study were to examine the tolerance and hyperaccumulative characteristics of 24 species in 9 families to Cd, Pb, Cu and Zn by using the outdoor pot-culture experiment. In the screening experiment, only Conyza canadensis and Rorippa globosa displayed Cd-hyperaccumulative characteristics. In a further concentration gradient experiment, C. canadensis was affirmed that it is not a Cd hyperaccumulator. Only R. globosa, indicated all Cd hyperaccumulative characteristics, especially Cd concentration in its stems and leaves were higher than 100 mg/kg, the minimum Cd concentration what a Cd-hyperaccumulator should accumulate. Thus, R. globosa was further validated as a Cd-hyperaccumulator.

Different flooding responses in Rorippa amphibia and Rorippa sylvestris, and their modes of expression in F1 hybrids.

The river floodplain species Rorippa amphibia, Rorippa sylvestris, and their hybrid Rorippa x anceps were studied here, with the aim of identifying potential species differences with respect to flooding tolerance, and of assessing their expression in F1 hybrids. Parents and their F1 hybrids were subjected to three flooding treatments mimicking natural conditions, and growth-related and leaf morphological traits were compared. In contrast to R. sylvestris, R. amphibia responded to waterlogging by forming specialized roots, and its growth was not reduced. These traits were dominantly expressed in hybrids. Both species and the hybrids established shoot growth over 2 wk of complete submergence. Only in R. sylvestris was this not at the expense of root biomass, suggesting that R. sylvestris can photosynthesize underwater. Rorippa sylvestris also showed a hyponastic response. Hybrids were intermediate to the parents in this respect. This study shows that phenotypic expression of parental traits in F1 hybrids is mostly additive, but can also be dominant. This suggests that a large overlap in habitat use of parents and hybrids is likely. If such an overlap occurs, the main evolutionary consequences of hybridization in Rorippa will be the introgression of genes, as the hybrids are fully fertile.

[Growth responses of Rorippa globosa and its accumulation characteristics of Cd and as under the Cd-As combined pollution].

Rorippa globosa has been identified as a new Cd-hyperaccumulating plant species. In the present study, growth responses of Rorippa globosa and its accumulation characteristics of Cd and As were examined under the condition of Cd-As combined pollution. The results showed that Cd and As had an antagonistic effect on enhancing the growth of the plants and Cd uptake and accumulation under the low concentration Cd and As treatments. When Cd and As concentrations were 10 mg/kg and 50 mg/kg, the highest height of the plants and the dry weight of shoots were up to 35.9 cm and 2.2 g/pot, respectively; and the accumulation of Cd in the leaves under the combined pollution was higher than that at the same level under single Cd pollution. However, there were synergic adverse effects on plant growth and Cd uptake under the high concentration Cd and As combined pollution. Meanwhile the accumulation of As in the roots was higher than that in the shoots, the translocation factor < or = 0.3 and the bioaccumulation factor < or = 0.6, which showed that Rorippa globosa had an excluding effect on As uptake. These results confirmed that Rorippa globosa had the strong tolerance ability to the Cd-As combined pollution, and the potential applied to phytoremediation of contaminated soil by Cd and As.

Phytoremediation of cadmium-contaminated soils by Rorippa globosa using two-phase planting.

BACKGROUND: Phytoextraction of contaminated soils by heavy metals can provide a great promise of commercial development. Although there are more than 400 species of hyperaccumulators found in the world, phytoremediation technology is rarely applied in field practice for remedying contaminated soils, partially due to low biomass and long growth duration for most of discovered hyperaccumulating plants. In order to enhance the metal-removing efficiency in a year, the two-phase planting countermeasure of phytoextraction by harvesting anthesis biomass was investigated on the basis of the newly found Cd-hyperaccumulator Rorippa globosa (Turcz.) Thell. with 107.0 and 150.1 mg/kg of the Cd accumulation in stems and leaves, respectively, when soil Cd added was concentrated to 25.0 mg/kg. METHODS: The field pot-culture experiment was used to observe the distribution property of R. globosa aboveground biomass and to examine characteristics of accumulating Cd by the plant at different growth stages. The concentration of Cd in plants and soils was determined using atomic absorption spectrophotometry (AAS). RESULTS AND DISCUSSION: The results indicated that the total dry stem and leaf biomass of R. globosa harvested at the flowering phase was up to 92.3% of that at its full maturity and the concentration of Cd in stems and leaves harvested at the flowering phase was up to 73.8% and 87.7% of that at the mature phase, respectively. The Cd-removing ratio by shoots of R. globosa harvested at the flowering phase was up to 71.4% of that at the mature phase. It was also found, by observing the growth duration of R. globosa, that the frostless period at the experiment site was twice as long as the growth time from the seedling-transplanted phase to the flowering phase of the hyperaccumulator. CONCLUSION: R. globosa could be transplanted into contaminated soils twice in one year by harvesting the hyperaccumulator at its flowering phase based on climatic conditions of the site and traits of the plant growth. In this sense, the extraction efficiency of Cd in shoots of R. globosa increased 42.8% compared to that of at its single maturity when the plant was transplanted into contaminated soils after it had been harvested at its flowering phase and the plant accumulated Cd from soil at the same extraction ratio at its second flowering phase. Thus, the method of anthesis biomass regulation by the two-phase planting is very significant to increase the Cd-removing efficiency by phytoremediation used in practice over the course of a year. RECOMMENDATION AND OUTLOOK: As for some hyperaccumulators that the growth duration from the seedling-transplanted phase to the flowering phase are short and the concentrations of heavy metals accumulated in their shoots at the flowering phase are high, the efficiency of phytoremediation can greatly be improved using the method of the two-phase planting.