A Plant biostimulant prepared from Ascophyllum nodosum Induces Flowering by Regulating the MIR156-mediated Age Pathway in Arabidopsis.

Flowering, the change from vegetative development to the reproductive phase, represents a crucial and intricate stage in the life cycle of plants, which is tightly controlled by both internal and external factors. In this study, we investigated the effect of Ascophyllum nodosum extract (ANE) on the flowering time of Arabidopsis. We found that a 0.1% concentration of ANE induced flowering in Arabidopsis, accompanied by the upregulation of key flowering time genes: FT (FLOWERING LOCUS T), SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1), and LFY (LEAFY). Further investigation showed that ANE specifically promotes flowering through the MIR156-mediated age pathway. ANE treatment resulted in the repression of negative regulator genes, MIR156, while simultaneously enhancing the expression of positive regulator genes, including SPLs and MIR172. This, in turn, led to the downregulation of AP2-like genes, which are known as floral repressors. It is worth noting that ANE did not alleviate the late flowering phenotype of MIR156-overexpressing plants and spl mutants. Furthermore, ANE-derived fucoidan mimics the function of sugars in regulating MIR156, closely mirroring the effects induced by ANE treatments. It suppresses the transcript levels of MIR156 and AP2-like genes while inducing those of SPLs and MIR172, thereby reinforcing the involvement of fucoidan in the control of flowering by ANE. In summary, our results demonstrate that ANE induces flowering by modulating the MIR156-SPL module within the age pathway, and this effect is mediated by fucoidan.

CAX3 (cation/proton exchanger) mediates a Cd tolerance by decreasing ROS through Ca elevation in Arabidopsis.

KEY MESSAGE: Over-expression of CAX3 encoding a cation/proton exchanger enhances Cd tolerance by decreasing ROS (Reactive Oxygen Species) through activating anti-oxidative enzymes via elevation of Ca level in Arabidopsis CAXs (cation/proton exchangers) are involved in the sequestration of cations such as Mn, Li, and Cd, as well as Ca, from cytosol into the vacuole using proton gradients. In addition, it has been reported that CAX1, 2 and 4 are involved in Cd tolerance. Interestingly, it has been reported that CAX3 expressions were enhanced by Cd in Cd-tolerant transgenic plants expressing Hb1 (hemoglobin 1) or UBC1 (Ub-conjugating enzyme 1). Therefore, to investigate whether CAX3 plays a role in increasing Cd tolerance, CAX3 of Arabidopsis and tobacco were over-expressed in Arabidopsis thaliana. Compared to control plants, both transgenic plants displayed an increase in Cd tolerance, no change in Cd accumulation, and enhanced Ca levels. In support of these, AtCAX3-Arabidopsis showed no change in expressions of Cd transporters, but reduced expressions of Ca exporters and lower rate of Ca efflux. By contrast, atcax3 knockout Arabidopsis exhibited a reduced Cd tolerance, while the Cd level was not altered. The expression of Δ90-AtCAX3 (deletion of autoinhibitory domain) increased Cd and Ca tolerance in yeast, while AtCAX3 expression did not. Interestingly, less accumulation of ROS (H2O2 and O2-) was observed in CAX3-expressing transgenic plants and was accompanied with higher antioxidant enzyme activities (SOD, CAT, GR). Taken together, CAX3 over-expression may enhance Cd tolerance by decreasing Cd-induced ROS production by activating antioxidant enzymes and by intervening the positive feedback circuit between ROS generation and Cd-induced spikes of cytoplasmic Ca.

Genotypic variation for cadmium tolerance in common bean (Phaseolus vulgaris L.).

Given the limitation of crop production in Cd-polluted areas, the identification and selection of plant genotypes tolerant to Cd stress are of great significance. In the present work, we show the existence of genotypic variation for Cd tolerance in common bean. The laboratory screening of 25 bean genotypes indicated a significant positive correlation of the mean productivity (MP) and the geometric mean productivity (GMP) with plant fresh weight both in control and Cd-treated plants. A principal component analysis further confirmed this variation and, together with other analyses, led to the selection of genotypes G-11867, Taylor, Emerson, and D-81083 as tolerant genotypes. A total of six bean genotypes with different degrees of Cd tolerance were selected, and their long-term physiological responses to Cd (0, 45, and 90 mg/kg soil) were evaluated. Increasing Cd concentrations led to higher Cd accumulation both in roots and shoots, and to significant rises in the levels of the oxidative stress biomarkers malondialdehyde (MDA), dityrosine (D-T), and 8-hydroxy-2'-deoxyguanosine (8-OH-2'-dG). Remarkable reductions in plant hormone levels and chlorophyll contents, as well as in dry and fresh weight, were observed in Cd-treated plants. Among the examined genotypes, Emerson, Taylor, and G-11867 were found to be more tolerant to Cd owing to lower Cd accumulation and lower oxidative stress levels, as well as higher chlorophyll and hormone contents. Our results contribute to the understanding of the physiological and biochemical basis of Cd tolerance in bean plants and may therefore, be useful for breeding programs directed towards obtaining bean varieties showing low Cd accumulation.

Development of bisphenol A (BPA)-sensing indicator Arabidopsis thaliana which synthesizes anthocyanin in response to BPA in leaves.

Bisphenol A (BPA) is an estrogenic endocrine disruptor which disturbs a normal animal development. We generated an indicator plant that senses and provides a clear visual indicator of an estrogen-like compound BPA in the environment. We developed transgenic Arabidopsis lines expressing a construct designed to synthesize anthocyanin (thus showing a red color) in response to BPA. We transformed Arabidopsis with a recombinant vector containing the chimeric estrogen receptor (XVE region), LAP and coding region of PtrMYB119 (transcription factor involved in anthocyanin biosynthesis in poplar and Arabidopsis). Upon binding of the estrogen compound to the ligand-binding domain of E (estrogen receptor) in XVE, the XV domain binds to LAP promoter and triggering the transcription of PtrMYB119 with a subsequent enhancement of anthocyanin biosynthetic gene expression, resulting in anthocyanin synthesis. The leaves of the transgenic Arabidopsis line XVE-PtrMYB119 turned red in the presence of 10 ppm BPA. The transcript level of PtrMYB119 peaked at day 3 of BPA exposure, then decreased to its minimal level at day 5. Similar expression patterns to that of PtrMYB119 were detected for genes encoding the anthocyanin biosynthetic enzymes chalcone synthase, chalcone flavanone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, and UFGT (UGT78D2). The leaves of transgenic plants did not turn red in response to BPA at concentrations below 10 ppm, but PtrMYB119 expression was induced by BPA at concentrations as low as 1 ppt BPA. Since this transgenic plant turns red in the presence of BPA without any experimental procedures, this line can be easily used by non-scientists.

Over-expression of tobacco UBC1 encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (Nicotiana tabacum).

Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca2+/H+ exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.

Tobacco NtUBC1 and NtUBQ2 enhance salt tolerance by reducing sodium accumulation and oxidative stress through proteasome activation in Arabidopsis.

The ubiquitin/proteasome system plays a crucial role in the regulation of plant responses to environmental stress. Here, we studied the involvement of the UBC1 and UBQ2 genes encoding a ubiquitin conjugating enzyme (E2) and ubiquitin extension protein, respectively, in the response to salt stress. Our results showed that the constitutive expression of tobacco NtUBC1 and NtUBQ2 in Arabidopsis thaliana improved salt tolerance, along with the lower Na+ level and higher K+/Na+ ratio compared to control plants. Moreover, the expression levels of sodium transporters, including AtHKT1 (High-Affinity K+ Transporter1) and AtSOS1 (Salt Overly Sensitive 1), were higher in NtUBC1- and NtUBQ2-Arabidopsis. However, the transcript level of AtNHX1 (Na+/H+ Exchanger 1) was similar between control and transgenic plants. After salt exposure, the activity of the 26S proteasome markedly increased in NtUBC1- and NtUBQ2-expressing plants; however, ubiquitinated protein levels decreased compared to control plants. Furthermore, higher activity of antioxidant enzymes and lower ROS production were observed in UBC1- and UBQ2-expressing plants. We further challenged atubc1, atubc2, and atubq2 single mutants and atubc1ubc2 double mutant lines with salt stress; interestingly, the salt sensitivity and sodium levels of the studied mutants were enhanced, while the potassium levels were reduced. However, the atubc1ubc2 double mutant illustrated a more severe phenotype than the single mutants, probably due to the redundant function of UBC1 and UBC2 in Arabidopsis. Taken together, NtUBC1 and NtUBQ2 enhance salt tolerance by enhancing 26S proteasome activity and reducing Na+ accumulation, ROS, and ubiquitinated/salt-denatured proteins.

Ethylene and ROS mediate root growth inhibition induced by the endocrine disruptor bisphenol A (BPA).

Bisphenol A (BPA) functions as a detrimental substance that disrupts the endocrine system in animals while also impeding the growth and development of plants. In our previous study, we demonstrated that BPA hinders the growth of roots in Arabidopsis by diminishing cell division and elongation, which is ascribed to the increased accumulation and redistribution of auxin. Here, we examined the mediation of ROS and ethylene in BPA-induced auxin accumulation and root growth inhibition. BPA enhanced ROS levels, and ROS increased auxin contents but reduced cell division activity and the expression of EXPA8 involved in root elongation. ROS scavenger treatment reversed BPA-triggered root growth retardation, auxin accumulation, and cell division inhibition. In addition, BPA induced ethylene, and ethylene synthesis inhibitor treatment reversed BPA-triggered root growth retardation and auxin accumulation. Taken together, ROS and ethylene are involved in BPA-inhibited cell elongation and cell division by mediating auxin accumulation and redistribution.

A Plant Biostimulant from Ascophyllum nodosum Potentiates Plant Growth Promotion and Stress Protection Activity of Pseudomonas protegens CHA0.

Abiotic stresses, including salinity stress, affect numerous crops, causing yield reduction, and, as a result, important economic losses. Extracts from the brown alga Ascophyllum nodosum (ANE), and compounds secreted by the Pseudomonas protegens strain, CHA0, can mitigate these effects by inducing tolerance against salt stress. However, the influence of ANE on P. protegens CHA0 secretion, and the combined effects of these two biostimulants on plant growth, are not known. Fucoidan, alginate, and mannitol are abundant components of brown algae and of ANE. Reported here are the effects of a commercial formulation of ANE, fucoidan, alginate, and mannitol, on pea (Pisum sativum), and on the plant growth-promoting activity of P. protegens CHA0. In most situations, ANE and fucoidan increased indole-3-acetic acid (IAA) and siderophore production, phosphate solubilization, and hydrogen cyanide (HCN) production by P. protegens CHA0. Colonization of pea roots by P. protegens CHA0 was found to be increased mostly by ANE and fucoidan in normal conditions and under salt stress. Applications of P. protegens CHA0 combined with ANE, or with fucoidan, alginate, and mannitol, generally augmented root and shoot growth in normal and salinity stress conditions. Real-time quantitative PCR analyses of P. protegens revealed that, in many instances, ANE and fucoidan enhanced the expression of several genes involved in chemotaxis (cheW and WspR), pyoverdine production (pvdS), and HCN production (hcnA), but gene expression patterns overlapped only occasionally those of growth-promoting parameters. Overall, the increased colonization and the enhanced activities of P. protegens CHA0 in the presence of ANE and its components mitigated salinity stress in pea. Among treatments, ANE and fucoidan were found responsible for most of the increased activities of P. protegens CHA0 and the improved plant growth.

Decreases in arsenic accumulation by the plasma membrane intrinsic protein PIP2;2 in Arabidopsis and yeast.

Arsenic (As) is a toxic pollutant that mainly enters the human body via plants. Therefore, understanding the strategy for reducing arsenic accumulation in plants is important to human health and the environment. Aquaporins are ubiquitous water channel proteins that bidirectionally transport water across cell membranes and play a role in the transportation of other molecules, such as glycerol, ammonia, boric acid, and arsenic acid. Previously, we observed that Arabidopsis PIP2;2, encoding a plasma membrane intrinsic protein, is highly expressed in NtCyc07-expressing Arabidopsis, which shows a higher tolerance to arsenite (As(III)). In this study, we report that the overexpression of AtPIP2;2 enhanced As(III) tolerance and reduced As(III) levels in yeast. Likewise, AtPIP2;2-overexpressing Arabidopsis exhibited improved As(III) tolerance and lower accumulation of As(III). In contrast, atpip2;2 knockout Arabidopsis showed reduced As(III) tolerance but no significant change in As(III) levels. Interestingly, the AtPIP2;2 transcript and protein levels were increased in roots and shoots of Arabidopsis in response to As(III). Furthermore, As(III) efflux was enhanced and As(III) influx/accumulation was reduced in AtPIP2;2-expressing plants. The expression of AtPIP2;2 rescued the As(III)-sensitive phenotype of acr3 mutant yeast by reducing As levels and slightly reduced the As(III)-tolerant phenotype of fps1 mutant yeast by enhancing As content, suggesting that AtPIP2; 2 functions as a bidirectional channel of As(III), while the As(III) exporter activity is higher than the As(III) importer activity. All these results indicate that AtPIP2;2 expression promotes As(III) tolerance by decreasing As(III) accumulation through enhancing As(III) efflux in Arabidopsis. This finding can be applied to the generation of low arsenic crops for human health.

Mechanism for Higher Tolerance to and Lower Accumulation of Arsenite in NtCyc07-Overexpressing Tobacco.

Arsenite [As(III)] is a highly toxic chemical to all organisms. Previously, we reported that the overexpression of NtCyc07 enhanced As(III) tolerance and reduced As(III) accumulation in yeast (Saccharomyces cerevisiae) and tobacco (Nicotiana tabacum). To understand a mechanism for higher As(III) tolerance and lower As(III) accumulation in NtCyc07-overexpressing tobacco, we examined the expression levels of various putative As(III) transporters (aquaporin). The expressions of putative As(III) exporter NIP1;1, PIP1;1, 1;5, 2;1, 2;2, and 2;7 were enhanced, while the expressions of putative As(III) importer NIP3;1, 4;1, and XIP2;1 were decreased, contributing to the reduced accumulation of As(III) in NtCyc07-overexpressing tobacco. In addition, the levels of oxidative stress indicators (H2O2, superoxide and malondialdehyde) were lower, and the activities of antioxidant enzymes (catalase, superoxide dismutase and glutathione reductase) were higher in NtCyc07-tobacco than in the control tobacco. This suggests that the lower oxidative stress in transgenic tobacco may be attributed to the higher activities of antioxidant enzymes and lower As(III) levels. Taken together, the overexpression of NtCyc07 enhances As(III) tolerance by reducing As(III) accumulation through modulation of expressions of putative As(III) transporters in tobacco.

The mechanism of root growth inhibition by the endocrine disruptor bisphenol A (BPA).

Bisphenol A (BPA) is a harmful environmental contaminant acting as an endocrine disruptor in animals, but it also affects growth and development in plants. Here, we have elucidated the functional mechanism of root growth inhibition by BPA in Arabidopsis thaliana using mutants, reporter lines and a pharmacological approach. In response to 10 ppm BPA, fresh weight and main root length were reduced, while auxin levels increased. BPA inhibited root growth by reducing root cell length in the elongation zone by suppressing expansin expression and by decreasing the length of the meristem zone by repressing cell division. The inhibition of cell elongation and cell division was attributed to the enhanced accumulation/redistribution of auxin in the elongation zone and meristem zone in response to BPA. Correspondingly, the expressions of most auxin biosynthesis and transporter genes were enhanced in roots by BPA. Taken together, it is assumed that the endocrine disruptor BPA inhibits primary root growth by inhibiting cell elongation and division through auxin accumulation/redistribution in Arabidopsis. This study will contribute to understanding how BPA affects growth and development in plants.

Expression of the Tobacco Non-symbiotic Class 1 Hemoglobin Gene Hb1 Reduces Cadmium Levels by Modulating Cd Transporter Expression Through Decreasing Nitric Oxide and ROS Level in Arabidopsis.

Hemoglobin (Hb) proteins are ubiquitous in plants, and non-symbiotic class 1 hemoglobin (Hb1) is involved in various biotic and abiotic stress responses. Here, the expression of the tobacco (Nicotiana tabacum) hemoglobin gene NtHb1 in Arabidopsis (Arabidopsis thaliana) showed higher cadmium (Cd) tolerance and lower accumulations of Cd, nitric oxide (NO), and reactive oxygen species (ROS) like hydrogen peroxide (H2O2). NtHb1-expressing Arabidopsis exhibited a reduced induction of NO levels in response to Cd, suggesting scavenging of NO by Hb1. In addition, transgenic plants had reduced accumulation of ROS and increased activities of antioxidative enzymes (catalase, superoxide dismutase, and glutathione reductase) in response to Cd. While the expression of the Cd exporters ABC transporter (PDR8) and Ca2+/H+ exchangers (CAXs) was increased, that of the Cd importers iron responsive transporter 1 (IRT1) and P-type 2B Ca2+ ATPase (ACA10) was reduced in response to Cd. When Col-0 plants were treated with the NO donor sodium nitroprusside (SNP) and H2O2, the expression pattern of Cd transporters (PDR8, CAX3, IRT1, and ACA10) was reversed, suggesting that NtHb1 expression decreased the Cd level by regulating the expression of Cd transporters via decreased NO and ROS. Correspondingly, NtHb1-expressing Arabidopsis showed increased Cd export. In summary, the expression of NtHb1 reduces Cd levels by regulating Cd transporter expression via decreased NO and ROS levels in Arabidopsis.

A Convenient Plant-Based Detection System to Monitor Androgenic Compound in the Environment.

Environmental androgen analogues act as endocrine disruptors, which inhibit the normal function of androgen in animals. In the present work, through the expression of a chimeric gene specified for the production of the anthocyanin in response to androgen DHT (dihydrotestosterone), we generated an indicator Arabidopsis that displays a red color in leaves in the presence of androgen compounds. This construct consists of a ligand-binding domain of the human androgen receptor gene and the poplar transcription factor gene PtrMYB119, which is involved in anthocyanin biosynthesis in poplar and Arabidopsis. The transgenic Arabidopsis XVA-PtrMYB119 displayed a red color in leaves in response to 10 ppm DHT, whereas it did not react in the presence of other androgenic compounds. The transcript level of PtrMYB119 peaked at day 13 of DHT exposure on agar media and then declined to its normal level at day 15. Expressions of anthocyanin biosynthesis genes including chalcone flavanone isomerase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, UFGT (UGT78D2), and anthocyanidin synthase were similar to that of PtrMYB119. It is assumed that this transgenic plant can be used by nonscientists for the detection of androgen DHT in the environment and samples such as food solution without any experimental procedures.

The Density and Length of Root Hairs Are Enhanced in Response to Cadmium and Arsenic by Modulating Gene Expressions Involved in Fate Determination and Morphogenesis of Root Hairs in Arabidopsis.

Root hairs are tubular outgrowths that originate from epidermal cells. Exposure of Arabidopsis to cadmium (Cd) and arsenic [arsenite, As(III)] increases root hair density and length. To examine the underlying mechanism, we measured the expression of genes involved in fate determination and morphogenesis of root hairs. Cd and As(III) downregulated TTG1 and GL2 (negative regulators of fate determination) and upregulated GEM (positive regulator), suggesting that root hair fate determination is stimulated by Cd and As(III). Cd and As(III) increased the transcript levels of genes involved in root hair initiation (RHD6 and AXR2) and root hair elongation (AUX1, AXR1, ETR1, and EIN2) except CTR1. DR5::GUS transgenic Arabidopsis showed a higher DR5 expression in the root tip, suggesting that Cd and As(III) increased the auxin content in the root tip. Knockdown of TTG1 in Arabidopsis resulted in increased root hair density and decreased root hair length compared with the control (Col-0) on 1/2 MS media. This phenotype may be attributed to the downregulation of GL2 and CTR1 and upregulation of RHD6. By contrast, gem mutant plants displayed a decrease in root hair density and length with reduced expression of RHD6, AXR2, AUX1, AXR1, ETR1, CTR1, and EIN2. Taken together, our results indicate that fate determination, initiation, and elongation of root hairs are stimulated in response to Cd and As(III) through the modulation of the expression of genes involved in these processes in Arabidopsis.