Analysis of aquaporins in northern grasses reveal functional importance of Puccinellia nuttalliana PIP2;2 in salt tolerance.

To better understand the roles of aquaporins in salt tolerance, we cloned PIP2;1, PIP2;2, PIP2;3, PIP1;1, PIP1;3, and TIP1;1 aquaporins from three northern grasses varying is salt tolerance including the halophytic grass Puccinellia nuttalliana, moderately salt tolerant Poa juncifolia, and relatively salt sensitive Poa pratensis. We analysed aquaporin expression in roots by exposing the plants to 0 and 150 mM for 6 days in hydroponic culture. NaCl treatment upregulated several PIP transcripts in P. nuttalliana while decreasing PnuTIP1;1. The PnuPIP2;2 transcripts increased by about six-fold in P. nuttalliana, two-fold in Poa juncifolia, and did not change in Poa pratensis. The NaCl treatment enhanced the rate of water transport in yeast expressing PnuPIP2;2 by 56% compared with control. PnuPIP2,2 expression also resulted in a higher Na+ uptake in yeast cells compared with an empty vector suggesting that PnuPIP2;2 may have both water and ion transporting functions. Structural analysis revealed that the transport properties of PnuPIP2;2 could be affected by its unique pore characteristics, which include a combination of hourglass, cylindrical, and increasing diameter conical entrance shape with pore hydropathy of -0.22.

Salinity Tolerance of Halophytic Grass Puccinellia nuttalliana Is Associated with Enhancement of Aquaporin-Mediated Water Transport by Sodium.

In salt-sensitive plants, root hydraulic conductivity is severely inhibited by NaCl, rapidly leading to the loss of water balance. However, halophytic plants appear to effectively control plant water flow under salinity conditions. In this study, we tested the hypothesis that Na+ is the principal salt factor responsible for the enhancement of aquaporin-mediated water transport in the roots of halophytic grasses, and this enhancement plays a significant role in the maintenance of water balance, gas exchange, and the growth of halophytic plants exposed to salinity. We examined the effects of treatments with 150 mM of NaCl, KCl, and Na2SO4 to separate the factors that affect water relations and, consequently, physiological and growth responses in three related grass species varying in salt tolerance. The grasses included relatively salt-sensitive Poa pratensis, moderately salt-tolerant Poa juncifolia, and the salt-loving halophytic grass Puccinellia nuttalliana. Our study demonstrated that sustained growth, chlorophyll concentrations, gas exchange, and water transport in Puccinellia nuttalliana were associated with the presence of Na in the applied salt treatments. Contrary to the other examined grasses, the root cell hydraulic conductivity in Puccinellia nuttalliana was enhanced by the 150 mM NaCl and 150 mM Na2SO4 treatments. This enhancement was abolished by the 50 µM HgCl2 treatment, demonstrating that Na was the factor responsible for the increase in mercury-sensitive, aquaporin-mediated water transport. The observed increases in root Ca and K concentrations likely played a role in the transcriptional and (or) posttranslational regulation of aquaporins that enhanced root water transport capacity in Puccinellia nuttalliana. The study demonstrates that Na plays a key role in the aquaporin-mediated root water transport of the halophytic grass Puccinellia nuttalliana, contributing to its salinity tolerance.

Transcriptome and Metabolome Analyses Reveal Potential Salt Tolerance Mechanisms Contributing to Maintenance of Water Balance by the Halophytic Grass Puccinellia nuttalliana.

Elevated soil salinity exacerbated by human activities and global climate change poses serious threats to plant survival. Although halophytes provide many important clues concerning salt tolerance in plants, some unanswered questions remain to be addressed, including the processes of water and solute transport regulation. We performed high-throughput RNA-sequencing in roots and metabolome characterizations in roots and leaves of Puccinellia nuttalliana halophytic grass subjected to 0 (control) and 150 mM NaCl. In RNAseq, a total of 31 Gb clean bases generated were de novo assembled into 941,894 transcripts. The PIP2;2 and HKT1;5 transcript levels increased in response to the NaCl treatment implying their roles in water and ion homeostasis. Several transcription factors, including WRKY39, DEK3, HY5, and ABF2, were also overexpressed in response to NaCl. The metabolomic analysis revealed that proline and dopamine significantly increased due to the upregulation of the pathway genes under salt stress, likely contributing to salt tolerance mechanisms. Several phosphatidylcholines significantly increased in roots suggesting that the alterations of membrane lipid composition may be an important strategy in P. nuttalliana for maintaining cellular homeostasis and membrane integrity under salt stress. In leaves, the TCA cycle was enriched suggesting enhanced energy metabolism to cope with salt stress. Other features contributing to the ability of P. nuttalliana to survive under high salinity conditions include salt secretion by the salt glands and enhanced cell wall lignification of the root cells. While most of the reported transcriptomic, metabolomics, and structural alterations may have consequences to water balance maintenance by plants under salinity stress, the key processes that need to be further addressed include the role of the changes in the aquaporin gene expression profiles in the earlier reported enhancement of the aquaporin-mediated root water transport.

Ericoid mycorrhizal fungi enhance salt tolerance in ericaceous plants.

To examine the effects of ericoid mycorrhizal (ERM) fungi on salt tolerance of ericaceous plants, we inoculated roots of velvetleaf blueberry (Vaccinium myrtilloides), Labrador tea (Rhododendron groenlandicum), and lingonberry (Vaccinium vitis-idaea) with ericoid mycorrhizal fungi Oidiodendron maius and Meliniomyces variabilis. Plants were subjected to 0 (NaCl control) and 30 mM NaCl treatments, and plant dry weights, gas exchange, and leaf chlorophyll concentrations were compared in inoculated and non-inoculated plants. M. variabilis increased root dry weights in all three species of NaCl-treated plants, and O. maius enhanced root dry weights of lingonberry plants treated with NaCl. Both fungal species were especially effective in enhancing root and shoot dry weights in control (0 mM NaCl) and NaCl-treated lingonberry seedlings. Leaf chlorophyll concentrations were enhanced by fungal inoculation in all three plant species, and this effect persisted under salt stress in Labrador tea and lingonberry. Salt treatment drastically reduced transpiration rates (E) and lowered net photosynthesis (Pn) to the negative values in all three species of non-inoculated plants, and this effect was partly or almost completely reversed by the inoculation with O. maius and M. variabilis. Fungal inoculation was especially effective in reducing NaCl effects on Pn in lingonberry. Oidiodendron maius and M. variabilis were also equally effective in reversing NaCl-induced declines of E in velvetleaf blueberry and lingonberry. However, in Labrador tea, O. maius reversed the decline of E in NaCl-treated plants less compared with M. variabilis resulting in high photosynthetic water use efficiency values. The results support the hypothesis that, similarly to arbuscular mycorrhizal and ectomycorrhizal associations, ERM association increases salt tolerance of plants.

Effects of pH and Mineral Nutrition on Growth and Physiological Responses of Trembling Aspen (Populus tremuloides), Jack Pine (Pinus banksiana), and White Spruce (Picea glauca) Seedlings in Sand Culture.

Responses of trembling aspen (Populus tremuloides), jack pine (Pinus banksiana), and white spruce (Picea glauca) seedlings to root zone pH ranging from 5 to 9 were studied in sand culture in the presence of two mineral nutrition levels. After eight weeks of treatments, effects of pH on plant dry weights varied between the plant species and were relatively minor in white spruce. Higher nutrient supply significantly increased dry weights only in trembling aspen subjected to pH 5 treatment. There was little effect of pH and nutrition level on net photosynthesis and transpiration rates in white spruce and jack pine, but net photosynthesis markedly declined in aspen at high pH. Chlorophyll concentrations in young foliage decreased the most in trembling aspen and jack pine. The effects of high pH treatments on the concentrations of Mg, P, Ca, Mn, Zn, and Fe in young foliage varied between the plant species with no significant decreases of Fe and Zn recorded in trembling aspen and white spruce, respectively. This was in contrast to earlier reports from the studies carried out in hydroponic culture. The sand culture system that we developed could be a more suitable alternative to hydroponics to study plant responses to pH in the root zone. Plant responses to high pH appear to involve complex events with a likely contribution of nutritional effects and altered water transport processes.

Water transport properties of root cells contribute to salt tolerance in halophytic grasses Poa juncifolia and Puccinellia nuttalliana.

Plant water uptake and aquaporin-mediated root water transport are among the most salt-sensitive processes in most plants, but even relatively high salt concentrations do not appear to impair water transport processes in halophytes. To develop better understanding of these processes in halophytic plants, we compared the responses to NaCl of the two halophytic grasses varying in salt tolerance, Puccinellia nuttalliana and Poa juncifolia, with the glycophytic grass Poa pratensis. The plants were hydroponically grown and subjected to different NaCl concentrations for up to 10 days. At the lower NaCl concentrations, shoot and root dry weights were drastically reduced in Poa pratensis, but increased in Puccinellia nuttalliana and Poa juncifolia. The examined treatment concentrations of up to 300 mM NaCl had either no effect (Puccinellia nuttalliana) or little effect (Poa juncifolia) on the net photosynthesis and transpiration rates in plants, but severely decreased the gas exchange parameters in Poa pratensis. Similarly, to growth and gas exchange, leaf water content in Puccinellia nuttalliana was not affected even by the highest, 300 mM NaCl concentration, while Poa pratensis showed decreased shoot water content in all examined NaCl treatments and Poa juncifolia in 150 and 300 mM NaCl. Cell hydraulic conductivity in roots of Poa pratensis also showed high sensitivity to NaCl and was drastically reduced in all examined NaCl concentrations. Cell hydraulic conductivity in Poa juncifolia roots was less affected by NaCl compared with Poa pratensis and in Puccinellia nuttalliana, cell hydraulic conductivity increased in response to NaCl treatments. Both Puccinellia nuttalliana and Poa juncifolia accumulated less Na in their shoot tissues compared with Poa pratensis. The concentrations of K in the roots of Poa pratensis sharply decreased with increasing NaCl treatment concentrations while in Puccinellia nuttalliana, K root concentrations remained high in all NaCl treatments and in Poa juncifoila, root K decreased only in the 300 mM NaCl treatment. Since K efflux from the cytoplasm can contribute to the acidification of the cytoplasm, this process could potentially lead to the inhibition of aquaporin function and reduction of root hydraulic conductivity. The, significance of stable K root concentrations in the roots of halophytes should be further investigated as a possible salt tolerance mechanism that could contribute to the maintenance of aquaporin function and root water transport under salt stress conditions.

Plant water transport and aquaporins in oxygen-deprived environments.

Oxygen deprivation commonly affects plants exposed to flooding and soil compaction. The resulting root hypoxia has an immediate effect on plant water relations and upsets water balance. Hypoxia inhibits root water transport and triggers stomatal closure. The processes contributing to the inhibition of root hydraulic conductivity and conductance (hydraulic conductivity of the whole root system) are complex and involve changes in root morphology and the functions of aquaporins. Aquaporins (AQPs) comprise a group of membrane intrinsic proteins that are responsible for the transport of water, as well as some small neutral solutes and ions. They respond to a wide range of environmental stresses including O2 deprivation, but the underlying functional mechanisms are still elusive. The aquaporin-mediated water transport is affected by the acidification of the cytoplasm and depletion of ATP that is required for aquaporin phosphorylation and membrane functions. Cytoplasmic pH, phosphorylation, and intracellular Ca2+ concentration directly control AQP gating, all of which are related to O2 deprivation. This review addresses the structural determinants that are essential for pore conformational changes in AQPs, to highlight the underlying mechanisms triggered by O2 deprivation stress. Gene expression of AQPs is modified in hypoxic plants, which may constitute an important, yet little explored, mechanism of hypoxia tolerance. In addition to water transport, AQPs may contribute to hypoxia tolerance by transporting O2, H2O2, and lactic acid. Responses of plants to O2 deprivation, and especially those that contribute to maintenance of water transport, are highly complex and entail the signals originating in roots and shoots that lead to and follow the stomatal closure. These complex responses may involve ethylene, abscisic acid, and possibly other hormonal factors and signaling molecules in ways that remain to be elucidated.