Functions of nitric oxide-mediated post-translational modifications under abiotic stress.

Environmental conditions greatly impact plant growth and development. In the current context of both global climate change and land degradation, abiotic stresses usually lead to growth restriction limiting crop production. Plants have evolved to sense and respond to maximize adaptation and survival; therefore, understanding the mechanisms involved in the different converging signaling networks becomes critical for improving plant tolerance. In the last few years, several studies have shown the plant responses against drought and salinity, high and low temperatures, mechanical wounding, heavy metals, hypoxia, UV radiation, or ozone stresses. These threats lead the plant to coordinate a crosstalk among different pathways, highlighting the role of phytohormones and reactive oxygen and nitrogen species (RONS). In particular, plants sense these reactive species through post-translational modification (PTM) of macromolecules such as nucleic acids, proteins, and fatty acids, hence triggering antioxidant responses with molecular implications in the plant welfare. Here, this review compiles the state of the art about how plant systems sense and transduce this crosstalk through PTMs of biological molecules, highlighting the S-nitrosylation of protein targets. These molecular mechanisms finally impact at a physiological level facing the abiotic stressful traits that could lead to establishing molecular patterns underlying stress responses and adaptation strategies.

Physiological roles of Casparian strips and suberin in the transport of water and solutes.

The formation of Casparian strips (CS) and the deposition of suberin at the endodermis of plant roots are thought to limit the apoplastic transport of water and ions. We investigated the specific role of each of these apoplastic barriers in the control of hydro-mineral transport by roots and the consequences on shoot growth. A collection of Arabidopsis thaliana mutants defective in suberin deposition and/or CS development was characterized under standard conditions using a hydroponic system and the Phenopsis platform. Mutants altered in suberin deposition had enhanced root hydraulic conductivity, indicating a restrictive role for this compound in water transport. In contrast, defective CS directly increased solute leakage and indirectly reduced root hydraulic conductivity. Defective CS also led to a reduction in rosette growth, which was partly dependent on the hydro-mineral status of the plant. Ectopic suberin was shown to partially compensate for defective CS phenotypes. Altogether, our work shows that the functionality of the root apoplastic diffusion barriers greatly influences the plant physiology, and that their integrity is tightly surveyed.

Non-invasive hydrodynamic imaging in plant roots at cellular resolution.

A key impediment to studying water-related mechanisms in plants is the inability to non-invasively image water fluxes in cells at high temporal and spatial resolution. Here, we report that Raman microspectroscopy, complemented by hydrodynamic modelling, can achieve this goal - monitoring hydrodynamics within living root tissues at cell- and sub-second-scale resolutions. Raman imaging of water-transporting xylem vessels in Arabidopsis thaliana mutant roots reveals faster xylem water transport in endodermal diffusion barrier mutants. Furthermore, transverse line scans across the root suggest water transported via the root xylem does not re-enter outer root tissues nor the surrounding soil when en-route to shoot tissues if endodermal diffusion barriers are intact, thereby separating 'two water worlds'.

Two chemically distinct root lignin barriers control solute and water balance.

Lignin is a complex polymer deposited in the cell wall of specialised plant cells, where it provides essential cellular functions. Plants coordinate timing, location, abundance and composition of lignin deposition in response to endogenous and exogenous cues. In roots, a fine band of lignin, the Casparian strip encircles endodermal cells. This forms an extracellular barrier to solutes and water and plays a critical role in maintaining nutrient homeostasis. A signalling pathway senses the integrity of this diffusion barrier and can induce over-lignification to compensate for barrier defects. Here, we report that activation of this endodermal sensing mechanism triggers a transcriptional reprogramming strongly inducing the phenylpropanoid pathway and immune signaling. This leads to deposition of compensatory lignin that is chemically distinct from Casparian strip lignin. We also report that a complete loss of endodermal lignification drastically impacts mineral nutrients homeostasis and plant growth.

Local root ABA/cytokinin status and aquaporins regulate poplar responses to mild drought stress independently of the ectomycorrhizal fungus Laccaria bicolor.

The relatively better performance of mycorrhizal plants subjected to drought stress has commonly been linked to improved root water uptake through the fungal regulation of plant aquaporins and hormones. In this study, we examined the role of ectomycorrhizal fungi in plant water relations and plant hormonal balance under mild drought using split-root seedlings of Populus trichocarpa × deltoides either with or without inoculation with Laccaria bicolor. The root compartments where the drought treatment was applied had higher ABA and lower cytokinin tZR contents, and greater expression of the plant aquaporins PtPIP1;1, PtPIP1;2, PtPIP2;5, and PtPIP2;7. On the other hand, the presence of L. bicolor within the roots down-regulated PtPIP1;4, PtPIP2;3, and PtPIP2;10, and reduced the abundance of PIP2 proteins. In addition, expression of the fungal aquaporins JQ585595 and JQ585596 were positively correlated with root ABA content, while tZR content was positively correlated with PtPIP1;4 and negatively correlated with PtPIP2;7. The results demonstrate a coordinated plant-fungal system that regulates the different mechanisms involved in water uptake in ectomycorrhizal poplar plants.

Ectomycorrhizal symbiosis helps plants to challenge salt stress conditions.

Soil salinity is an environmental condition that is currently increasing worldwide. Plant growth under salinity induces osmotic stress and ion toxicity impairing root water and nutrient absorption, but the association with beneficial soil microorganisms has been linked to an improved adaptation to this constraint. The ectomycorrhizal (ECM) symbiosis has been proposed as a key factor for a better tolerance of woody species to salt stress, thanks to the reduction of sodium (Na+) uptake towards photosynthetic organs. Although no precise mechanisms for this enhanced plant salt tolerance have been described yet, in this review, we summarize the knowledge accumulated so far on the role of ECM symbiosis. Moreover, we propose several strategies by which ECM fungi might help plants, including restriction of Na+ entrance into plant tissues and improvement of mineral nutrition and water balances. This positive effect of ECM fungi has been proven in field assays and the results obtained point to a promising application in forestry cultures and reforestation.

Phenotypic and molecular traits determine the tolerance of olive trees to drought stress.

Olive trees are known for their capacity to adapt to drought through several phenotypic and molecular variations, although this can vary according to the different provenances of the same olive cultivar. We confronted the same olive cultivar from two different location in Spain: Freila, in the Granada province, with low annual precipitation, and Grazalema, in the Cadiz province, with high annual precipitation, and subjected them to five weeks of severe drought stress. We found distinctive physiological and developmental adaptations among the two provenances. Thus, trees from Freila subjected to drought stress exhibited increasing root dry weights and decreasing leaf numbers and relative stem heights. On the other hand, the treatment with drought in Grazalema trees reduced their leaf chlorophyll contents, but increased their relative stem diameter and their root hydraulic conductivity. The physiological responses of Freila tree roots to drought were linked to different molecular adaptations that involved the regulation of genes related to transcription factors induced by ABA, auxin and ethylene signaling, as well as, the action of a predicted membrane intrinsic protein (MIP). On the other hand, the responses of Grazalema trees were related with different root genes related to oxidation-reduction, ATP synthesis, transduction and posttranslational regulation, with a special mention to the cytokinins signaling through the transcript predicted as a histidine-containing phosphotransfer protein. Our results show that olive trees adapted to dry environments will adjust their growth and water uptake capacity through transcription factors regulation, and this will influence the different physiological responses to drought stress.

Role of urban ectomycorrhizal fungi in improving the tolerance of lodgepole pine (Pinus contorta) seedlings to salt stress.

With large forested urban areas, the city of Edmonton, Alberta, Canada, faces high annual costs of replacing trees injured by deicing salts that are commonly used for winter road maintenance. Ectomycorrhizal fungi form symbiotic associations with tree roots that allow trees to tolerate the detrimental effects of polluted soils. Here, we examined mycorrhizal colonization of Pinus contorta by germinating seeds in soils collected from different locations: (1) two urban areas within the city of Edmonton, and (2) an intact pine forest just outside Edmonton. We then tested the responses of seedlings to 0-, 60-, and 90-mM NaCl. Our results showed lower abundance and diversity of ectomycorrhizal fungi in seedlings colonized with the urban soils compared to those from the pine forest soil. However, when subsequently exposed to NaCl treatments, only seedlings inoculated with one of the urban soils containing fungi from the genera Tuber, Suillus, and Wilcoxina, showed reduced shoot Na accumulation and higher growth rates. Our results indicate that local ectomycorrhizal fungi that are adapted to challenging urban sites may offer a potential suitable source for inoculum for conifer trees designated for plating in polluted urban environments.

Surveillance of cell wall diffusion barrier integrity modulates water and solute transport in plants.

The endodermis is a key cell layer in plant roots that contributes to the controlled uptake of water and mineral nutrients into plants. In order to provide such functionality the endodermal cell wall has specific chemical modifications consisting of lignin bands (Casparian strips) that encircle each cell, and deposition of a waxy-like substance (suberin) between the wall and the plasma membrane. These two extracellular deposits provide control of diffusion enabling the endodermis to direct the movement of water and solutes into and out of the vascular system in roots. Loss of integrity of the Casparian strip-based apoplastic barrier is sensed by the leakage of a small peptide from the stele into the cortex. Here, we report that such sensing of barrier integrity leads to the rebalancing of water and mineral nutrient uptake, compensating for breakage of Casparian strips. This rebalancing involves both a reduction in root hydraulic conductivity driven by deactivation of aquaporins, and downstream limitation of ion leakage through deposition of suberin. These responses in the root are also coupled to a reduction in water demand in the shoot mediated by ABA-dependent stomatal closure.

Involvement of the def-1 Mutation in the Response of Tomato Plants to Arbuscular Mycorrhizal Symbiosis Under Well-Watered and Drought Conditions.

Jasmonic acid (JA) and arbuscular mycorrhizal (AM) symbioses are known to protect plants against abiotic and biotic stresses, but are also involved in the regulation of root hydraulic conductance (L). The objective of this experiment was to elucidate the role of JA in the water relations and hormonal regulation of AM plants under drought by using tomato plants defective in the synthesis of JA (def-1). Our results showed that JA is involved in the uptake and transport of water through its effect on both physiological parameters (stomatal conductance and L) and molecular parameters, mainly by controlling the expression and abundance of aquaporins. We observed that def-1 plants increased the expression of seven plant aquaporin genes under well-watered conditions in the absence of AM fungus, which partly explain the increment of L by this mutation under well-watered conditions. In addition, the effects of the AM symbiosis on plants were modified by the def-1 mutation, with the expression of some aquaporins and plant hormone concentration being disturbed. On the other hand, methyl salicylate (MeSA) content was increased in non-mycorrhizal def-1 plants, suggesting that MeSA and JA can act together in the regulation of L. In a complementary experiment, it was found that exogenous MeSA increased L, confirming our hypothesis. Likewise, we confirmed that JA, ABA and SA are hormones involved in plant mechanisms to cope with stressful situations, their concentrations being controlled by the AM symbiosis. In conclusion, under well-watered conditions, the def-1 mutation mimics the effects of AM symbiosis, but under drought conditions the def-1 mutation changed the effects of the AM symbiosis on plants.

Ethylene sensitivity and relative air humidity regulate root hydraulic properties in tomato plants.

MAIN CONCLUSION: The effect of ethylene and its precursor ACC on root hydraulic properties, including aquaporin expression and abundance, is modulated by relative air humidity and plant sensitivity to ethylene. Relative air humidity (RH) is a main factor contributing to water balance in plants. Ethylene (ET) is known to be involved in the regulation of root water uptake and stomatal opening although its role on plant water balance under different RH is not very well understood. We studied, at the physiological, hormonal and molecular levels (aquaporins expression, abundance and phosphorylation state), the plant responses to exogenous 1-aminocyclopropane-1-carboxylic acid (ACC; precursor of ET) and 2-aminoisobutyric acid (AIB; inhibitor of ET biosynthesis), after 24 h of application to the roots of tomato wild type (WT) plants and its ET-insensitive never ripe (nr) mutant, at two RH levels: regular (50%) and close to saturation RH. Highest RH induced an increase of root hydraulic conductivity (Lpo) of non-treated WT plants, and the opposite effect in nr mutants. The treatment with ACC reduced Lpo in WT plants at low RH and in nr plants at high RH. The application of AIB increased Lpo only in nr plants at high RH. In untreated plants, the RH treatment changed the abundance and phosphorylation of aquaporins that affected differently both genotypes according to their ET sensitivity. We show that RH is critical in regulating root hydraulic properties, and that Lpo is affected by the plant sensitivity to ET, and possibly to ACC, by regulating aquaporins expression and their phosphorylation status. These results incorporate the relationship between RH and ET in the response of Lpo to environmental changes.

Effects of different arbuscular mycorrhizal fungal backgrounds and soils on olive plants growth and water relation properties under well-watered and drought conditions.

The adaptation capacity of olive trees to different environments is well recognized. However, the presence of microorganisms in the soil is also a key factor in the response of these trees to drought. The objective of the present study was to elucidate the effects of different arbuscular mycorrhizal (AM) fungi coming from diverse soils on olive plant growth and water relations. Olive plants were inoculated with native AM fungal populations from two contrasting environments, that is, semi-arid - Freila (FL) and humid - Grazalema (GZ) regions, and subjected to drought stress. Results showed that plants grew better on GZ soil inoculated with GZ fungi, indicating a preference of AM fungi for their corresponding soil. Furthermore, under these conditions, the highest AM fungal diversity was found. However, the highest root hydraulic conductivity (Lpr ) value was achieved by plants inoculated with GZ fungi and growing in FL soil under drought conditions. So, this AM inoculum also functioned in soils from different origins. Nine novel aquaporin genes were also cloned from olive roots. Diverse correlation and association values were found among different aquaporin expressions and abundances and Lpr , indicating how the interaction of different aquaporins may render diverse Lpr values.

Autochthonous arbuscular mycorrhizal fungi and Bacillus thuringiensis from a degraded Mediterranean area can be used to improve physiological traits and performance of a plant of agronomic interest under drought conditions.

Studies have shown that some microorganisms autochthonous from stressful environments are beneficial when used with autochthonous plants, but these microorganisms rarely have been tested with allochthonous plants of agronomic interest. This study investigates the effectiveness of drought-adapted autochthonous microorganisms [Bacillus thuringiensis (Bt) and a consortium of arbuscular mycorrhizal (AM) fungi] from a degraded Mediterranean area to improve plant growth and physiology in Zea mays under drought stress. Maize plants were inoculated or not with B. thuringiensis, a consortium of AM fungi or a combination of both microorganisms. Plants were cultivated under well-watered conditions or subjected to drought stress. Several physiological parameters were measured, including among others, plant growth, photosynthetic efficiency, nutrients content, oxidative damage to lipids, accumulation of proline and antioxidant compounds, root hydraulic conductivity and the expression of plant aquaporin genes. Under drought conditions, the inoculation of Bt increased significantly the accumulation of nutrients. The combined inoculation of both microorganisms decreased the oxidative damage to lipids and accumulation of proline induced by drought. Several maize aquaporins able to transport water, CO2 and other compounds were regulated by the microbial inoculants. The impact of these microorganisms on plant drought tolerance was complementary, since Bt increased mainly plant nutrition and AM fungi were more active improving stress tolerance/homeostatic mechanisms, including regulation of plant aquaporins with several putative physiological functions. Thus, the use of autochthonous beneficial microorganisms from a degraded Mediterranean area is useful to protect not only native plants against drought, but also an agronomically important plant such as maize.

Mild salt stress conditions induce different responses in root hydraulic conductivity of phaseolus vulgaris over-time.

Plants respond to salinity by altering their physiological parameters in order to maintain their water balance. The reduction in root hydraulic conductivity is one of the first responses of plants to the presence of salt in order to minimize water stress. Although its regulation has been commonly attributed to aquaporins activity, osmotic adjustment and the toxic effect of Na+ and Cl- have also a main role in the whole process. We studied the effects of 30 mM NaCl on Phaseolus vulgaris plants after 9 days and found different responses in root hydraulic conductivity over-time. An initial and final reduction of root hydraulic conductivity, stomatal conductance, and leaf water potential in response to NaCl was attributed to an initial osmotic shock after 1 day of treatment, and to the initial symptoms of salt accumulation within the plant tissues after 9 days of treatment. After 6 days of NaCl treatment, the increase in root hydraulic conductivity to the levels of control plants was accompanied by an increase in root fructose content, and with the intracellular localization of root plasma membrane aquaporins (PIP) to cortex cells close to the epidermis and to cells surrounding xylem vessels. Thus, the different responses of bean plants to mild salt stress over time may be connected with root fructose accumulation, and intracellular localization of PIP aquaporins.

The symbiosis with the arbuscular mycorrhizal fungus Rhizophagus irregularis drives root water transport in flooded tomato plants.

It is known that the presence of arbuscular mycorrhizal fungi within the plant roots enhances the tolerance of the host plant to different environmental stresses, although the positive effect of the fungi in plants under waterlogged conditions has not been well studied. Tolerance of plants to flooding can be achieved through different molecular, physiological and anatomical adaptations, which will affect their water uptake capacity and therefore their root hydraulic properties. Here, we investigated the root hydraulic properties under non-flooded and flooded conditions in non-mycorrhizal tomato plants and plants inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. Only flooded mycorrhizal plants increased their root hydraulic conductivity, and this effect was correlated with a higher expression of the plant aquaporin SlPIP1;7 and the fungal aquaporin GintAQP1. There was also a higher abundance of the PIP2 protein phoshorylated at Ser280 in mycorrhizal flooded plants. The role of plant hormones (ethylene, ABA and IAA) in root hydraulic properties was also taken into consideration, and it was concluded that, in mycorrhizal flooded plants, ethylene has a secondary role regulating root hydraulic conductivity whereas IAA may be the key hormone that allows the enhancement of root hydraulic conductivity in mycorrhizal plants under low oxygen conditions.

Responses of Rat Root ( Raf.) Plants to Salinity and pH Conditions.

Growth and physiological parameters were examined in rat root ( Raf.) plants grown under controlled environment conditions in hydroponics and subjected to different pH and salinity treatments to determine whether these environmental factors may contribute to poor establishment of in oil sands constructed wetlands. When plants were subjected to a root zone pH ranging from 6.0 to 9.5, the plants that were growing at pH 7.0 showed the highest relative growth rates and chlorophyll concentrations compared with lower and higher pH levels. The greatest inhibition of growth occurred at pH ranging from 8.0 to 9.5. High pH also triggered significant reductions in tissue concentrations of N, P, and microelements, whereas the concentrations of Mg increased at pH >8. When NaCl (25, 50, and 100 mmol L) was added to the nutrient solution at pH 7.0 and 8.5, higher mortality and greater tissue concentrations of Na and Cl were measured in plants growing at pH 8.5 compared with pH 7.0. The results show that plants growing at the optimum pH of 7.0 can better tolerate salinity compared with plants exposed to high root zone pH. Both pH and salinity may present important environmental constraints to growth and establishment of plants in oil sands constructed wetlands.

Role of adventitious roots in water relations of tamarack (Larix laricina) seedlings exposed to flooding.

BACKGROUND: Flooding reduces supply of oxygen to the roots affecting plant water uptake. Some flooding-tolerant tree species including tamarack (Larix laricina (Du Roi) K. Koch) produce adventitious roots in response to flooding. These roots were reported to have higher hydraulic conductivity under flooding conditions compared with non-adventitious roots. In the present study, we examined structural and functional modifications in adventitious roots of tamarack seedlings to explain their flooding tolerance. RESULTS: Seedlings were subjected to the flooding treatment for six months, which resulted in an almost complete disintegration of the existing root system and its replacement with adventitious roots. We compared gas exchange parameters and water relations of flooded plants with the plants growing in well-drained soil and examined the root structures and root water transport properties. Although flooded seedlings had lower needle chlorophyll concentrations, their stomatal conductance, net photosynthesis rates and shoot water potentials were similar to non-flooded plants, indicative of flooding tolerance. Flooded adventitious roots had higher activation energy and a higher ratio of apoplastic to cell-to-cell water flow compared with non-flooded control roots as determined with the 1-hydroxypirene 3,6,8-trisulfonic acid apoplastic tracer dye. The adventitious roots in flooded plants also exhibited retarded xylem and endodermal development and accumulated numerous starch grains in the cortex. Microscopic examination of root sections treated with the PIP1 and PIP2 antibodies revealed high immunoreactivity in the cortex of non-flooded roots, as compared with flooded roots. CONCLUSIONS: Structural modifications of adventitious roots suggest increased contribution of apoplastic bypass to water flow. The reduced dependence of roots on the hypoxia-sensitive aquaporin-mediated water transport is likely among the main mechanisms allowing tamarack seedlings to maintain water balance and gas exchange under flooding conditions.

Hebeloma crustuliniforme facilitates ammonium and nitrate assimilation in trembling aspen (Populus tremuloides) seedlings.

This study examined the role of ectomycorrhizal associations in nitrogen assimilation of Populus tremuloides seedlings. Seedlings were inoculated with Hebeloma crustuliniforme and compared with non-inoculated plants. Nitrogen-metabolizing enzymatic properties were also determined in H. crustuliniforme grown in sterile culture. The seedlings and fungal cultures were subjected to nitrogen treatments (including NO3⁻, NH4⁺ and a combination of NO3⁻ + NH4⁺) for 2 months to examine the effects on growth, nitrogen-assimilating enzyme activities and xylem sap concentrations of NH4⁺ and NO3⁻. Seedlings were also provided for 3 days with ¹5N-labeled NH4⁺ and NO3⁻, and leaf and root ¹5N content relative to total nitrogen was measured. Both NO3⁻ and NH4⁺ were effective in supporting seedling growth when either form was provided separately. When NO3⁻ and NH4⁺ were provided together, seedling growth decreased while enzymatic assimilation of NH4⁺ increased. Additionally, nitrogen assimilation in inoculated seedlings was less affected by the form of nitrogen compared with non-inoculated plants. Fungal ability to enzymatically respond to and assimilate NH4⁺ combined with aspen's enzymatic responsiveness to NO3⁻ was likely the reason for efficient assimilation of both nitrogen forms by mycorrhizal plants.

Role of aquaporins in root water transport of ectomycorrhizal jack pine (Pinus banksiana) seedlings exposed to NaCl and fluoride.

Effects of ectomycorrhizal (ECM) fungus Suillus tomentosus on water transport properties were studied in jack pine (Pinus banksiana) seedlings. The hydraulic conductivity of root cortical cells (L(pc)) and of the whole root system (L(pr)) in ECM plants was higher by twofold to fourfold compared with the non-ECM seedlings. HgCl2 had a greater inhibitory effect on L(pc) in ECM compared with non-ECM seedlings, suggesting that the mercury-sensitive, aquaporin (AQP)-mediated water transport was largely responsible for the differences in L(pc) between the two groups of plants. L(pc) was rapidly and drastically reduced by the 50 mM NaCl treatment. However, in ECM plants, the initial decline in L(pc) was followed by a quick recovery to the pre-treatment level, while the reduction of L(pc) in non-ECM seedlings progressed over time. Treatments with fluoride reduced L(pc) by about twofold in non-ECM seedlings and caused smaller reductions of L(pc) in ECM plants. When either 2 mM KF or 2 mM NaF were added to the 50 mM NaCl treatment solution, the inhibitory effect of NaCl on L(pc) was rapidly reversed in both groups of plants. The results suggest that AQP-mediated water transport may be linked to the enhancement of salt stress resistance reported for ECM plants.

Effects of NaCl on responses of ectomycorrhizal black spruce (Picea mariana), white spruce (Picea glauca) and jack pine (Pinus banksiana) to fluoride.

Black spruce (Picea mariana), white spruce (Picea glauca) and jack pine (Pinus banksiana) were inoculated with Suillus tomentosus and subjected to potassium fluoride (1 mM KF and 5 mM KF) in the presence and absence of 60 mM NaCl. The NaCl and KF treatments reduced total dry weights in jack pine and black spruce seedlings, but they did not affect total dry weights in white spruce seedlings. The addition of 60 mM NaCl to KF treatment solutions alleviated fluoride-induced needle injury in ectomycorrhizal (ECM) black spruce and white spruce, but had little effect in jack pine seedlings. Both KF and 60 mM NaCl treatments reduced E values compared with non-treated control seedlings. However, with the exception of small reductions of K(r) by NaCl treatments in black spruce, the applied KF and NaCl treatments had little effect on K(r) in ECM plants. Chloride tissue concentrations in NaCl-treated plants were not affected by the presence of KF in treatment solutions. However, shoot F concentrations in ECM black spruce and white spruce treated with 5 mM KF + 60 mM NaCl were significantly reduced compared with the 5 mM KF treatment. The results point to a possible competitive inhibition of F transport by Cl. We also suggest that the possibility that aquaporins may be involved in the transmembrane transport of F should be further investigated.