Role of the molecular structure of humified organic matter in rice plant response to environmental lead pollution.

Humified organic matter has been shown to decrease Pb toxicity in plants. However, there are still gaps in our understanding of the mechanism by which this phenomenon occurs. In this study, we aimed to assess the ability of humic substances (HSs), humic acids (HAs), and fulvic acids (FAs) to enhance defense mechanisms in rice plants under lead (Pb)-stressed conditions. HS fractions were isolated from vermicompost using the chemical fractionation methodology established by the International Humic Substances Society. These fractions were characterized by solid-state NMR and FTIR. Chemometric analysis was used to compare humic structures and correlate them with bioactivity. Three treatments were tested to evaluate the protective effect of humic fractions on rice plants. The first experiment involved the application of humic fractions along with Pb. The second comprised pretreatment with humic fractions followed by subsequent exposure to Pb stress. The third experiment involved Pb stress and subsequent treatment with humic fractions. The root morphology and components of the antioxidative defense system were evaluated and quantified. The results showed that HS + Pb, HA + Pb, and FA + Pb treatment preserved root growth and reduced the levels of O2- and malondialdehyde (MDA) in the roots by up to 5% and 2%, respectively. Pretreatment of the plants with humic fractions promoted the maintenance of root growth and reduced the contents of O2-, H2O2, and MDA by up to 48%, 22%, and 20%, respectively. Combined application of humic fractions and Pb reduced the Pb content in plant tissues by up to 60%, while pretreatment reduced it by up to 80%. The protective capacity of humic fractions is related to the presence of peptides, lignin, and carbohydrate fragments in their molecular structures. These results suggest that products could be developed that can mitigate the adverse effects of heavy metals on agricultural crops.

Knockdown of OsNRT2.4 modulates root morphology and alters nitrogen metabolism in response to low nitrate availability in rice.

The expression patterns of the NRT2 genes have been well described; however, the role of OsNRT2.4 in root growth is not well known. In this study, we thus aimed at investigating the role of high-affinity NO3- transport OsNRT2.4 in root growth modulation. Through the amiRNA-mediated gene silencing technique, we successfully obtained osnrt2.4 knockdown lines to study the role of OsNRT2.4 on root growth under low nitrate conditions. We performed real-time PCR analysis to investigate the relative gene expression level in root and shoot, soluble metabolites, and measurement of root system. Knockdown of OsNRT2.4 decreased rice growth. The comparison with wild-type (WT) plants showed that (i) knockdown of OsNRT2.4 inhibited root formation under low NO3- supply; (ii) we demonstrated that the mutant lines had significantly increased NO3- uptake than WT plants when grown in different nitrate supplies; (iii) osnrt2.4 knockdown lines showed an alteration in nitrogen metabolism, and this affected the root growth; and (iv) the downregulation of OsNRT2.4 enhanced the expression of gene response of low external NO3- concentrations. Herein we provide new insights in OsNRT2.4 functions. Our data demonstrated that OsNRT2.4 plays a role in root growth, nitrogen metabolic pathway and probably have functions in nitrate transport from root to shoot under low nitrate availability in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01273-6.

Silencing the Oryza sativa plasma membrane H+-ATPase isoform OsA2 affects grain yield and shoot growth and decreases nitrogen concentration.

The plasma membrane (PM) H+-ATPase (EC 3.6.1.3.) is a key component involved in nutrient uptake. There are 10 PM H+-ATPase isoforms in the rice genome (OsA1-OsA10), and OsA2 is highly responsive to nitrate (NO3-). We investigated the role that the OsA2 isoform plays in the total N and growth of rice (Oryza sativa). By the use of artificial microRNA, mutant osa2 rice lines presented ∼70 % downregulated levels of OsA2. Three osa2 lines and control plants (transformed with an empty IRS154 vector and named IRS) were cultivated in the greenhouse to evaluate grain and shoot production. For hydroponic experiments, the same lines were grown in Hoagland solution under two different NO3- levels for 30 days - 0.2 mM NO3--N (low N) or 2.0 mM NO3--N (sufficient N) - or were grown for three days without NO3- (starvation) after 27 days under 2.0 mM NO3--N. In the greenhouse experiments, compared with the IRS plants, the osa2 lines had lower shoot fresh weights, grain yields and SPAD values. Moreover, compared with the IRS plants, the three osa2 lines grown hydroponically under low NO3- levels had lower N concentration and net flux of NO3-. PM H+-ATPase activity was lower in the osa2 mutants than in the IRS plants. The relatively low N concentration in the osa2 lines was not due to lower expression of OsNRT2.1, OsNRT2.2, or OsNAR2.1. These results indicate that the specific PM H+-ATPase isoform OsA2 affects the net flux of NO3-, N concentration, and grain yield.

Dark septate endophytic fungi increase the activity of proton pumps, efficiency of 15N recovery from ammonium sulphate, N content, and micronutrient levels in rice plants.

Plants colonised by dark septate endophytic (DSE) fungi show increased uptake of nutrients available in the environment. The objective of the present study was to evaluate the impact of DSE fungi on the activity of proton pumps, nitrogen (N) recovery from ammonium sulphate, and nutrient accumulation in rice plants. Treatments consisted of non-inoculated plants and plants inoculated with two isolates of DSE fungi, A101 and A103. To determine N recovery from the soil, ammonium sulphate enriched with 15N was added to a non-sterile substrate while parameters associated with the activity of proton pumps and with NO3- uptake were determined in a sterile environment. The A101 and A103 fungal isolates colonised the roots of rice plants, promoting 15N uptake, growth, and accumulation of nutrients as compared with the mock control. A103 induced the expression of the plasma membrane H+-ATPase (PM H+-ATPase) isoforms OsA5 and OsA8, the activity of the PM H+-ATPase and H+-pyrophosphatase. Our results suggest that the inoculation of rice plants with DSE fungi represents a strategy to improve the N recovery from ammonium sulphate and rice plant growth through the induction of OsA5 and OsA8 isoforms and stimulation of the PM H+-ATPase and H+-pyrophosphatase.

Contribution of dark septate fungi to the nutrient uptake and growth of rice plants

ABSTRACT The use of dark septate fungi (DSE) to promote plant growth can be beneficial to agriculture, and these organisms are important allies in the search for sustainable agriculture practices. This study investigates the contribution of dark septate fungi to the absorption of nutrients by rice plants and their ensuing growth. Four dark septate fungi isolates that were identified by Internal transcribed spacer phylogeny were inoculated in rice seeds (Cv. Piauí). The resulting root colonization was estimated and the kinetic parameters Vmax and Km were calculated from the nitrate contents of the nutrient solution. The macronutrient levels in the shoots, and the NO3--N, NH4+-N, free amino-N and soluble sugars in the roots, sheathes and leaves were measured. The rice roots were significantly colonized by all of the fungi, but in particular, isolate A103 increased the fresh and dry biomass of the shoots and the number of tillers per plant, amino-N, and soluble sugars as well as the N, P, K, Mg and S contents in comparison with the control treatment. When inoculated with isolates A103 and A101, the plants presented lower Km values, indicating affinity increases for NO3--N absorption. Therefore, the A103 Pleosporales fungus presented the highest potential for the promotion of rice plant growth, increasing the tillering and nutrients uptake, especially N (due to an enhanced affinity for N uptake) and P.

Contribution of dark septate fungi to the nutrient uptake and growth of rice plants.

The use of dark septate fungi (DSE) to promote plant growth can be beneficial to agriculture, and these organisms are important allies in the search for sustainable agriculture practices. This study investigates the contribution of dark septate fungi to the absorption of nutrients by rice plants and their ensuing growth. Four dark septate fungi isolates that were identified by Internal transcribed spacer phylogeny were inoculated in rice seeds (Cv. Piauí). The resulting root colonization was estimated and the kinetic parameters Vmax and Km were calculated from the nitrate contents of the nutrient solution. The macronutrient levels in the shoots, and the NO3--N, NH4+-N, free amino-N and soluble sugars in the roots, sheathes and leaves were measured. The rice roots were significantly colonized by all of the fungi, but in particular, isolate A103 increased the fresh and dry biomass of the shoots and the number of tillers per plant, amino-N, and soluble sugars as well as the N, P, K, Mg and S contents in comparison with the control treatment. When inoculated with isolates A103 and A101, the plants presented lower Km values, indicating affinity increases for NO3--N absorption. Therefore, the A103 Pleosporales fungus presented the highest potential for the promotion of rice plant growth, increasing the tillering and nutrients uptake, especially N (due to an enhanced affinity for N uptake) and P.

Development and nitrate reductase activity of sugarcane inoculated with five diazotrophic strains.

Diazotrophs are able to stimulate plant growth. This study aimed at evaluating the effect of inoculation of five diazotrophic strains on growth promotion and nitrate reductase (NR, EC 1.7.1.1) activity in sugarcane. An experiment was carried out from three stages of cultivation: sprouting, tubes, and in hydroponics. On the first two stages, seven treatments were adopted: uninoculated control; mixed inoculation with five strains; and individual inoculation with Gluconacetobacter diazotrophicus (Gd), Herbaspirillum rubrisubalbicans (Hr), Herbaspirillum seropedicae (Hs), Nitrospirillum amazonense (Na), and Paraburkholderia tropica (Pt). The four treatments showing the best performance were transferred to the hydroponic system for analysis of NR activity. Hs, Pt, and the mixture of all strains led to the highest seedling biomass in tubes, followed by Hr. In hydroponics, the mixture and the strain Hr had the highest growth-promoting effect. NR activity was influenced by inoculation only under low N supply conditions, with positive effect of Hr, Pt, and the mixture.

Involvement of Hormone- and ROS-Signaling Pathways in the Beneficial Action of Humic Substances on Plants Growing under Normal and Stressing Conditions.

The importance of soil humus in soil fertility has been well established many years ago. However, the knowledge about the whole mechanisms by which humic molecules in the rhizosphere improve plant growth remains partial and rather fragmentary. In this review we discuss the relationships between two main signaling pathway families that are affected by humic substances within the plant: one directly related to hormonal action and the other related to reactive oxygen species (ROS). In this sense, our aims are to try the integration of all these events in a more comprehensive model and underline some points in the model that remain unclear and deserve further research.

Root-Shoot Signaling crosstalk involved in the shoot growth promoting action of rhizospheric humic acids.

Numerous studies have shown the ability of humic substances to improve plant development. This action is normally reflected in an enhancement of crop yields and quality. However, the mechanisms responsible for this action of humic substances remain rather unknown. Our studies have shown that the shoot promoting action of sedimentary humic acids is dependent of its ability to increase root hydraulic conductivity through signaling pathways related to ABA, which in turn is affected in roots by humic acids in an IAA-NO dependent way. Furthermore, these studies also indicate that the primary action of humic acids in roots might also be physical, resulting from a transient mild stress caused by humic acids associated with a fouling-cleaning cycle of wall cell pores. Finally the role of alternative signal molecules, such as ROS, and corresponding signaling pathways are also discussed and modeled in the context of the above-mentioned framework.

Vermicompost humic acids modulate the accumulation and metabolism of ROS in rice plants.

This work aims to determine the reactive oxygen species (ROS) accumulation, gene expression, anti-oxidant enzyme activity, and derived effects on membrane lipid peroxidation and certain stress markers (proline and malondialdehyde-MDA) in the roots of unstressed and PEG-stressed rice plants associated with vermicompost humic acid (VCHA) application. The results show that the application of VCHA to the roots of unstressed rice plants caused a slight but significant increase in root ROS accumulation and the gene expression and activity of the major anti-oxidant enzymes (superoxide dismutase and peroxidase). This action did not have negative effects on root development, and an increase in both root growth and root proliferation occurred. However, the root proline and MDA concentrations and the root permeability results indicate the development of a type of mild stress associated with VCHA application. When VCHA was applied to PEG-stressed plants, a clear alleviation of the inhibition in root development linked to PEG-mediated osmotic stress was observed. This was associated with a reduction in root ROS production and anti-oxidant enzymatic activity caused by osmotic stress. This alleviation of stress caused by VCHA was also reflected as a reduction in the PEG-mediated concentration of MDA in the root as well as root permeability. In summary, the beneficial action of VCHA on the root development of unstressed or PEG-stressed rice plants clearly involves the modulation of ROS accumulation in roots.

Isoforms of plasma membrane H(+)-ATPase in rice root and shoot are differentially induced by starvation and resupply of NO3⁻ or NH4+.

The goal of this study was to evaluate the effects of nitrogen starvation and resupply in 10 PM H+-ATPase isoforms and the expression of NO3⁻ and NH4+ transporters in rice. The net uptake of both forms of NO3⁻-N or NH4+-N was increased with its resupply. Resupply of NO3⁻ resulted in induction of the following PM H+-ATPase isoforms, OsA1, OsA2, OsA5 and OsA7 in the shoots and OsA2, OsA5, OsA7 and OsA8 in the roots. Resupply of NH4+ resulted in the induction of the following OsA1, OsA3 and OsA7 isoforms in the roots while OsA1 was induced in the shoots. It was observed that increased PM H+-ATPase activity also resulted in increased net uptake of NO3⁻ and NH4+. In the roots, OsNRT2.1 and OsNRT2.2 were induced by NO3⁻ resupply, while OsAMT1.1 and OsAMT1.2 were induced by NH4+ deficiency. The results showed that the expression of PM H+-ATPase isoforms is related to NO3⁻ and NH4+ transporters as well as in which section of the plant it takes place. PM H+-ATPase isoforms OsA2 and OsA7 displayed the strongest induction in response to N resupply, therefore indicating that these genes could be involved in N uptake in rice.