The Role of MPK6 as Mediator of Ethylene/Jasmonic Acid Signaling in <Emphasis Type="Italic">Serendipita indica</Emphasis>-Colonized Arabidopsis Roots

Serendipita indica is an axenically cultivable fungus, which colonizes a broad range of plant species including the model plant Arabidopsis thaliana. Root colonization by this endophyte leads to enhanced plant fitness and performance and promotes resistance against different biotic and abiotic stresses. The involvement of MPK6 in this mutualistic interaction had been previously shown with an mpk6 A. thaliana mutant, which failed to respond to S. indica colonization. Here, we demonstrate that mpk6 roots are significantly less colonized by S. indica compared to wild-type roots and the foliar application of plant hormones, ethylene, or jasmonic acid, restores the colonization rate at least to the wild-type level. Further, hormone-treated mpk6 plants show typical S. indica-induced growth promotion effects. Moreover, expression levels of several genes related to plant defense and hormone signaling are significantly changed at different colonization phases. Our results demonstrate that the successful root colonization by S. indica depends on efficient suppression of plant immune responses. In A. thaliana, this process relies on intact hormone signaling in which MPK6 seems to play a pivotal role.     

Characteristics of Chlorophyll Fluorescence and Antioxidant-Oxidant Balance in <Emphasis Type="Italic">PEPC</Emphasis> and <Emphasis Type="Italic">PPDK</Emphasis> Transgenic Rice under Aluminum Stress

Aluminum is one of the most important heavy metals inducing stress during plant growth and development. In this study, transgenic rice (Oryza sativa L., cv. Kitaake) plants expressing the maize C₄PEPC and PPDK genes were evaluated for aluminum tolerance. A 4.3 and 19.1 folds increase of PPDK and PEPC activities in transgenic rice produced increases in root exudation of oxalate, malate, and citrate (1.20, 1.41, and 1.65 times, respectively) compared to untransformed (WT) plants. Transgenic rice had enhanced aluminum tolerance compared to WT based on chlorophyll fluorescence and chlorophyll levels. Transgenic plants under aluminum stress also had decreased lipid membrane oxidative damage and higher levels of ROS-scavenging enzyme activity. The PEPC and PPDK genes play an important role in aluminum stress tolerance by increasing the effluxes of organic acids.     

Physiological and biochemical changes in different drought-tolerant alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.) varieties under PEG-induced drought stress

Medicago sativa L. cv. Longzhong is a nutritious forage plant in dryland regions of the Loess Plateau with strong drought tolerance and broad adaptability. To understand the adaptation mechanism of alfalfa (M. sativa L. cv. Longzhong) to drought stress, growth, and physiological parameters including levels of chlorophyll content, osmotic adjustment, reactive oxygen species (ROS), and antioxidant enzymes and antioxidants were measured under simulated levels of drought (? 0.40, ? 0.80, ? 1.20, ? 1.60, and ? 2.00 MPa). The changes in M. sativa L. cv. Longzhong were compared with those of plants of M. sativa L. cv. Longdong control (Variety I) suited to moderate rainfall areas and M. sativa L. cv. Gannong No. 3 (Variety II) suited to irrigated areas. The results showed that root–shoot ratio, the chlorophyll (a + b) and osmolytes contents, the degree of lipid peroxidation and ROS production, and the levels of antioxidative enzymes and antioxidants increased significantly with increasing drought stress, whereas plant height, aboveground biomass, chlorophyll a/b ratio, leaf water potential (Ψ₁), and relative water content (RWC) decreased in response to drought. The Longzhong variety responded early to beginning drought stress (between 0 and ? 0.4 MPa) compared with the controls. Under drought stress (between ? 0.4 and ? 2.0 MPa), the Longzhong variety had significantly higher belowground biomass, root–shoot ratio, Ψ₁, RWC, catalase (CAT) activity and reduced glutathione content than those of Varieties I and II, but hydrogen peroxide and hydroxyl free radical (OH·) contents were significantly lower. Step regression analysis showed that OH·, CAT, malondialdehyde, superoxide anion-free radical (O ₂ ^·? ), and superoxide dismutase of Longzhong had the most marked response to drought stress. In conclusion, the stronger drought tolerance of the Longzhong variety might be due to its higher water-holding capacity, root–shoot ratio, and ability to coordinate enzymatic and non-enzymatic antioxidant systems, which coordinate the peroxidation and oxidative systems.     

Mechanisms Underlying the Regulation of Root Formation in <Emphasis Type="Italic">Malus hupehensis</Emphasis> Stem Cuttings by Using Exogenous Hormones

Malus hupehensis is one of the most important Malus ornamental and rootstock species in the south China Yellow River Basin. In the present study, we treated the stem cuttings of M. hupehensis with one of three exogenous hormones, indole acetic acid (IAA), naphthalene acetic acid (NAA), or a compound plant growth regulator (GGR) to investigate the mechanisms underlying root formation in stem cuttings and to optimize stem cutting propagation techniques. The results showed that immersing the stem cuttings in 100 mg/L of IAA for 2 h before planting was most effective, which reduced the time to root formation by 21 days and increased rooting percentage by 129.4 %, compared to that in the control group. In addition, the levels of endogenous substances (endogenous hormones, soluble proteins, and carbohydrates) dynamically changed, with the time to peak value or time to valley value of each parameter synchronized well with the initiation of adventitious roots. The synchronized change suggested that root formation was coordinated with physiological metabolism. However, exogenous hormone treatment significantly accelerated the catabolism of the root inhibiting hormone, abscisic acid. On the other hand, exogenous hormone treatment significantly enhanced the accumulation of root promoting hormones [IAA, gibberellic acid (GA₃), and zeatin riboside (ZR)] and soluble proteins. Moreover, exogenous hormone treatments accelerated the consumption of starch and soluble sugars. Overall, the results indicated that exogenous hormone treatment (IAA) accelerated the synthesis of endogenous hormones (IAA, GA₃, and ZR), therefore, sped up the metabolism of carbohydrates and soluble proteins, and consequently quickened the root formation process.     

Metabolic alteration of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell suspension cultures overexpressing <Emphasis Type="Italic">geraniol synthase</Emphasis> in the plastids or cytosol

Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus’s geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering.     

Quorum sensing activity of the plant growth-promoting rhizobacterium <Emphasis Type="Italic">Serratia glossinae</Emphasis> GS2 isolated from the sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) rhizosphere

Plant growth-promoting rhizobacteria (PGPR) affect plant growth through various mechanisms, such as indole-3-acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and biofilm formation. The aim of the study reported here was to isolate and characterize rhizobacteria that produce quorum-sensing signal molecules and other PGPR-related molecules. A biofilm-forming bacterium, GS2, was isolated from the rhizosphere of a sesame plant and subsequently found to produce two quorum-sensing signal molecules that were identified as N-hexanoyl-L-homoserine lactone (m/z 200) and N-octanoyl-L-homoserine lactone (m/z 228) by liquid chromatography–tandem mass spectrometry analysis. The strain was also found to produce IAA (17.2 μg mL^?1), gibberellins (113.7 μg mL^?1), and ACC deaminase (9.7 μM α-ketobutyrate mg^?1 protein h^?1). The strain was identified as Serratia glossinae based on a comparison of 16S rRNA gene sequences. Inoculation of the strain promoted growth of a gibberellin-deficient rice dwarf mutant (Waito-C). Different growth attributes, including shoot and root elongation, chlorophyll content, and plant weight could be attributed to the PGPR characteristics of strain GS2. These results suggest that S. glossinae strain GS2 can serve as a microbial agent that improves plant growth.     

Ethylene-dependent adjustment of metabolite profiles in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seedlings during gravitropic response

Metabolite profile adjustments under impact of ethylene synthesis inhibitors were studied in Arabidopsis thaliana (L.) Heynh. seedlings during reorientation of plants relative to the gravity vector (gravistimulation). Metabolite profiles were compared with Principal Component Analysis. We have shown that significant changes in metabolite profiles developed within 60 min of gravistimulation and were most pronounced in 2 mm root tips including the root cap, apical meristem and elongation zone. Gravistimulation resulted in the increased levels of valine, leucine, serine, γ-aminobutyric acid, nicotinic acid, and decreased levels of several monosaccharides, malate and oxalate. Treatment with ethylene synthesis inhibitor, aminoethoxyvinylglycine (10 μM), escaped the effect of gravistimulation on root tip metabolite profile. Metabolite profile adjustments revealed in this study suggest that ethylene may be involved into the regulation of Arabidopsis metabolome during the gravitropic response.     

Influence of different host associations on glutamine synthetase activity and ammonium transporter in <Emphasis Type="Italic">Santalum</Emphasis><Emphasis Type="Italic">album</Emphasis> L.

The present study was aimed at understanding the role of different hosts in ammonium transporter1;2 expressions and glutamine synthetase(GS) activity and their effects on the growth parameters in the sandal. Sandal plant associated with leguminous host expressed better growth parameters. GS activity of leguminous hosts alone and in host associated sandals was analyzed using GS transferase assay. Highest GS activity was expressed in Mimosa pudica—sandal association compared to other leguminous and non-leguminous host associations. The association of N₂ fixing host with sandal enhanced C and N levels in order to maintain the C/N value. The role of ammonium transporters in N nutrition of sandal-host association was elucidated by cloning AMT1;2 from the leaves, haustoria and roots of host associated sandal and quantifying the relative expression by the \( 2^{{ - \Delta \Delta {\text{C}}_{\text{T}} }} \) method. SaAMT1;2 was strongly up-regulated in leaves, roots and haustoria of leguminous host associated sandal compared to non-leguminous host associations. The relative increase in SaAMT1;2 expressions and up-regulated GS activity positively affected the growth parameters in sandal when associated with leguminous hosts.     

<Emphasis Type="Italic">OsNOX3</Emphasis>, encoding a NADPH oxidase,regulates root hair initiation and elongation in rice

Root hairs play important roles in plant nutrient and water acquisition. To better understand the genetic mechanism controlling root hair development in rice (Oryza sativa L.), a rice mutant with root hair defects was isolated and characterized. Cryo-scanning electron microscope showed that the density and length of root hairs in the mutant were significantly reduced compared to the wild type (WT). Map-based cloning and complementation test revealed that the mutation occurred in a NADPH oxidase gene OsNOX3 (LOC_Os01g61880). The OsNOX3 displays high sequence similarity with the previously characterized NOX genes RTH5 in maize and RHD2 in Arabidopsis, which play critical roles in root hair development. Expression pattern analysis indicated that OsNOX3 was expressed in various tissues throughout the plant with high expression in roots and root hairs. Subcellular localization analysis confirmed that OsNOX3 was located on the plasma membrane. Staining assays showed that the content of superoxide and hydrogen peroxide were significantly reduced in root hair tips of Osnox3 when compared to WT. Our results showed critical roles of OsNOX3 in regulating both root hair initiation and elongation in rice, which is similar to RTH5 but different from RHD2, confirming the difference of genetic mechanisms regulating root hair morphogenesis in monocot and dicot plants.     

Media derived from brown seaweeds <Emphasis Type="Italic">Cystoseira myriophylloides</Emphasis> and <Emphasis Type="Italic">Fucus spiralis</Emphasis> for in vitro plant tissue culture

In the present study, the effect of seaweed extract (SE) from Fucus spiralis (Fs), Cystoseira myriophylloides (Cm) and Laminaria digitata (Ld) on in vitro plant tissue culture was examined. Combination of 25?% of SE from Cm with 25?% of MS medium increased adventitious shoot regeneration from Nicotiana benthamiana leaf discs explants by 620?%, when compared to the conventional regeneration medium. Similarly SE from Fs and Ld enhanced regeneration by about 500?%. However, when increasing SE to 50?%, only Cm significantly enhanced shoot regeneration. The effect of SE was also evaluated on in vitro micropropagation of N. benthamiana, grape, plum and apricot by assessing shoot length, number of leaves and internodes. When used alone but at lower concentrations (2.5 and 12.5?%), SE from Fs and Cm resulted in at least the same efficacy as MS alone for micropropagation of N. benthamiana shoots. However, for micropropagation of grapevine, plum and apricot woody plants, a combination of 50?% of SE from Cm or Fs with 50?% of their conventional micropropagation media was necessary. Rooting was also enhanced in N. benthamiana and grapevine, and was correlated with their higher concentrations of indole acetic acid when compared to SE from Ld. This finding, in addition to mineral analysis data, suggests that SE of Fs and Cm contain necessary nutrients and growth regulators to allow their use as medium for in vitro plant culture.     

Characterization of As(III) oxidizing <Emphasis Type="Italic">Achromobacter</Emphasis> sp. strain N2: effects on arsenic toxicity and translocation in rice

Achromobacter sp. strain N2 was isolated from a pyrite-cinder-contaminated soil and presented plant growth promoting traits (ACC deaminase activity, production of indole-3-acetic and jasmonic acids, siderophores secretion, and phosphate solubilization) and arsenic transformation abilities. Achromobacter sp. strain N2 was resistant to different metals and metalloids, including arsenate (100 mM) and arsenite (5 mM). The strain was resistant to ionic stressors (i.e., arsenate and NaCl), whereas bacterial growth was impaired by osmotic stress. Strain N2 was able to oxidize 1.0 mmol L^?1 of arsenite to arsenate in 72 h. This evidence was supported by the retrieval of an arsenite oxidase AioA gene highly homologous to arsenite oxidases of Achromobacter and Alcaligenes species. Rice seeds of Oryza sativa (var. Loto) were bio-primed with ACCD-induced and non-induced cells in order to evaluate the effect of inoculation on rice seedlings growth and arsenic uptake. The bacterization with ACCD-induced cells significantly improved seed germination and seedling heights if compared with the seeds inoculated with non-induced cells and non-primed seeds. Enhanced arsenic uptake was evidenced in the presence of ACCD-induced cells, suggesting a role of ACCD activity on the mitigation of the toxicity of arsenic accumulated by the plant. This kind of responses should be taken into account when proposing PGP strains for improving plant growth in arsenic-rich soils.     

Wound-induced increases in the glucosinolate content of oilseed rape and their effect on subsequent herbivory by a crucifer specialist

Damage to the oilseed rape plant (Brassica napus L.) by the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) induces systemic changes to the glucosinolate profile, most noticeably an increase in the concentration of indole glucosinolates. When jasmonic acid was applied to the cotyledons of the plant, a similar effect was observed. Feeding tests with artificial substrates compared a glucosinolate fraction from jasmonic acid-treated plants with a similar fraction from untreated plants. In these tests, alterations to the glucosinolate profile increased the feeding of a crucifer-specialist feeder (P. chrysocephala). However, in whole plant tests, P. chrysocephala did not feed more on the jasmonic acid treated plants than on the controls. This implies that other aspects of the damage response are being induced by the jasmonic acid treatment and having a negative effect on subsequent herbivory.     

Influence of triacontanol and jasmonic acid on metabolomics during early stages of root induction in cultured tissue of tomato (<Emphasis Type="Italic">Lycopersicon esculentum)</Emphasis>

Influence of n-triacontanol (TRIA) and jasmonic acid (JA) on metabolic profiling during root morphogenesis was studied in Lycopersicon esculentum (cv. PKM-1). Proton nuclear magnetic resonance (¹H NMR) based metabolomics was employed to investigate the variations in metabolic profile. Chenomx NMR suite v.8.1 was used to identify and quantify metabolites based on their respective signature spectra. The levels of 47 metabolites were monitored for 72 h at specific time intervals (0, 3, 6, 9, 12, 24, 36, 48 and 72 h). Principal component analysis was performed to determine the variations in the metabolic profile between control and treatments during in vitro rhizogenesis. TRIA was observed to promote early root emergence (24 h) and also influence the metabolic variation during rhizogenesis between 9 and 24 h post exposure. Compounds such as IAA, ATP, NADPH, UDP-N-acetylglucosamine and gallate predominated at 9 h. Unlike TRIA, JA was unable to promote an early root induction. However, it influenced the synthesis of a relatively higher concentration of IAA at 6 h when compared to ATP, NADPH and trigonelline at 9 h. In the presence of both TRIA and JA (TRIA?+?JA), significant changes in the metabolic profiles were observed 24 h post exposure and the rooting was observed only after 72 h. The study suggests that TRIA may accelerate in vitro rhizogenesis of cultured tomato tissues by mainly increasing the synthesis of other growth promoting metabolites. But in the presence of JA, TRIA’s effect appears to be reduced.     

Bacteria endemic to saline coastal belt and their ability to mitigate the effects of salt stress on rice growth and yields

Increase in soil salinity adversely affects the metabolism and lowers the yield of rice (Oryza sativa L). Application of plant growth-promoting rhizobacteria (PGPR) to ameliorate the effects of salt stress on sensitive rice can be both effective and sustainable. In this study, 20 bacterial strains were isolated from the soil of saline-prone regions of Satkhira, north of the Sundarbans in coastal Bangladesh. Three bacteria among these grew well in the presence of 3 M salt (NaCl) and were Gram positive and non-motile. Their 16S rRNA sequence revealed that they belong to the Halobacillus genus. Two of them were identified as Halobacillus dabanensis strain SB-26 and the other one as Halobacillus sp. GSP 34. A couple of mechanisms by which these microbes could play beneficial role if associated with plants, such as nitrogen fixation and indole acetic acid (IAA) production, were identified. The two bacterial strains showed positive results for nitrogen fixation and indole acetic acid (IAA) activity under salt stress. Their effect on the physiology and yield of a farmer popular but sensitive BRRI dhan 28 rice variety was investigated under both control and salt stress. At the seedling stage, inoculated plants had significantly greater root length, shoot height, total weight, chlorophyll content, but lower electrolyte leakage both in control and salt stress (0, 40, and 80 mM). Performance of the plants was even better when both bacteria were used in combination. At the reproductive stage, the plants also showed better phenology in presence of the inoculated bacteria. Under stress (50 mM NaCl), these plants showed significantly greater plant height, lower spikelet damage, and yield reduction compared to untreated plants. The identified Halobacillus strains can therefore be used to improve the yield of rice by exploiting their plant growth promotion activities in coastal areas affected by moderate salinity, such as those with an ionic conductivity of up to 5 dS m^?1.