Salicylic Acid Regulates Root Gravitropic Growth via Clathrin-Independent Endocytic Trafficking of PIN2 Auxin Transporter in Arabidopsis thaliana.

The phytohormone salicylic acid (SA) plays a crucial role in plant growth and development. However, the mechanism of high-concentration SA-affected gravitropic response in plant root growth and root hair development is still largely unclear. In this study, wild-type, pin2 mutant and various transgenic fluorescence marker lines of Arabidopsis thaliana were investigated to understand how root growth is affected by high SA treatment under gravitropic stress conditions. We found that exogenous SA application inhibited gravitropic root growth and root hair development in a dose-dependent manner. Further analyses using DIRECT REPEAT5 (DR5)-GFP, auxin sensor DII-VENUS, auxin efflux transporter PIN2-GFP, trans-Golgi network/early endosome (TGN/EE) clathrin-light-chain 2 (CLC2)-mCherry and prevacuolar compartment (PVC) (Rha1)-mCherry transgenic marker lines demonstrated that high SA treatment severely affected auxin accumulation, root-specific PIN2 distribution and PIN2 gene transcription and promoted the vacuolar degradation of PIN2, possibly independent of clathrin-mediated endocytic protein trafficking. Our findings proposed a new underlying mechanism of SA-affected gravitropic root growth and root hair development via the regulation of PIN2 gene transcription and PIN2 protein endocytosis in plants.

Four plasma membrane-localized MGR transporters mediate xylem Mg2+ loading for root-to-shoot Mg2+ translocation in Arabidopsis.

Magnesium (Mg2+), an essential structural component of chlorophyll, is absorbed from the soil by roots and transported to shoots to support photosynthesis in plants. However, the molecular mechanisms underlying root-to-shoot Mg2+ translocation remain largely unknown. We describe here the identification of four plasma membrane (PM)-localized transporters, named Mg2+ release transporters (MGRs), that are critical for root-to-shoot Mg transport in Arabidopsis. Functional complementation assays in a Mg2+-uptake-deficient bacterial strain confirmed that these MGRs conduct Mg2+ transport. PM-localized MGRs (MGR4, MGR5, MGR6, and MGR7) were expressed primarily in root stellar cells and participated in the xylem loading step of the long-distance Mg2+ transport process. In particular, MGR4 and MGR6 played a major role in shoot Mg homeostasis, as their loss-of-function mutants were hypersensitive to low Mg2+ but tolerant to high Mg2+ conditions. Reciprocal grafting analysis further demonstrated that MGR4 functions in the root to determine shoot Mg2+ accumulation and physiological phenotypes caused by both low- and high-Mg2+ stress. Taken together, our study has identified the long-sought transporters responsible for root-to-shoot Mg2+ translocation in plants.

A metal tolerance protein, MTP10, is required for the calcium and magnesium homeostasis in Arabidopsis.

Nutrient antagonism typically refers to the fact that too high a concentration of one nutrient inhibits the absorption of another nutrient. In plants, Ca2+ (Calcium) and Mg2+ (Magnesium) are the two most abundant divalent cations, which are known to have antagonistic interactions. Hence, maintaining their homeostasis is crucial for plant growth and development. In this study, we showed that MTP10 (Metal Tolerance Protein 10) is an important regulator for maintaining homeostasis of Mg and Ca in Arabidopsis. The mtp10 mutant displayed severe growth retardation in the presence of excess Mg2+, to which the addition of Ca2+ was able to rescue the phenotype of mtp10 mutant. Additionally, the deficiency of Ca2+ in the culture medium accelerated the high-Mg sensitivity of the mtp10 mutant. The yeast complementation assay suggested that AtMTP10 had no Ca2+ transport activity. And the ICP-MS data further confirmed the antagonistic relationship between Ca2+ and Mg2+, with the addition of Ca2+ reducing the excessive accumulation of Mg2+ and high-Mg inhibiting the uptake of Ca2+. We conclude that the Arabidopsis MTP10 is essential for the regulation of Mg and Ca homeostasis.

An Arabidopsis vasculature distributed metal tolerance protein facilitates xylem magnesium diffusion to shoots under high-magnesium environments.

Magnesium (Mg2+ ) is an essential metal for plant growth; however, its over-accumulation in cells can be cytotoxic. The metal tolerance protein family (MTP) belongs to an ubiquitous family of cation diffusion facilitator (CDF) proteins that export divalent metal cations for metal homeostasis and tolerance in all organisms. We describe here the identification of MTP10 to be critical for xylem Mg homeostasis in Arabidopsis under high Mg2+ conditions. The Arabidopsis plant contains 12 MTP genes, and only knockout of MTP10 decreased the tolerance of high-Mg stress. The functional complementation assays in a Mg2+ -uptake-deficient bacterial strain MM281 confirmed that MTP10 conducted Mg2+ transport. MTP10 is localized to the plasma membrane of parenchyma cells around the xylem. Reciprocal grafting analysis further demonstrated that MTP10 functions in the shoot to determine the shoot growth phenotypes under high Mg2+ conditions. Moreover, compared to the wild type, the mtp10 mutant accumulated more Mg2+ in xylem sap under high-Mg stress. This study reveals that MTP10 facilitates Mg2+ diffusion from the xylem to shoots and thus determines Mg homeostasis in shoot vascular tissues during high-Mg stress.

An ICln homolog contributes to osmotic and low-nitrate tolerance by enhancing nitrate accumulation in Arabidopsis.

Nitrate (NO3- ) is a source of plant nutrients and osmolytes, but its delivery machineries under osmotic and low-nutrient stress remain largely unknown. Here, we report that AtICln, an Arabidopsis homolog of the nucleotide-sensitive chloride-conductance regulatory protein family (ICln), is involved in response to osmotic and low-NO3- stress. The gene AtICln, encoding plasma membrane-anchored proteins, was upregulated by various osmotic stresses, and its disruption impaired plant tolerance to osmotic stress. Compared with the wild type, the aticln mutant retained lower anions, particularly NO3- , and its growth retardation was not rescued by NO3- supply under osmotic stress. Interestingly, this mutant also displayed growth defects under low-NO3 stress, which were accompanied by decreases in NO3- accumulation, suggesting that AtICln may facilitate the NO3- accumulation under NO3- deficiency. Moreover, the low-NO3- hypersensitive phenotype of aticln mutant was overridden by the overexpression of NRT1.1, an important NO3- transporter in Arabidopsis low-NO3- responses. Further genetic analysis in the plants with altered activity of AtICln and NRT1.1 indicated that AtICln and NRT1.1 play a compensatory role in maintaining NO3- homeostasis under low-NO3- environments. These results suggest that AtICln is involved in cellular NO3- accumulation and thus determines osmotic adjustment and low-NO3- tolerance in plants.

Overexpression of Pyrabactin Resistance-Like Abscisic Acid Receptors Enhances Drought, Osmotic, and Cold Tolerance in Transgenic Poplars.

Abscisic acid (ABA) has been known participate in a wider range of adaptive responses to diverse environmental abiotic stresses such as drought, osmosis, and low temperatures. ABA signaling is initiated by its receptors PYR/PYL/RCARs, a type of soluble proteins with a conserved START domain which can bind ABA and trigger the downstream pathway. Previously, we discovered that poplar (Populus trichocarpa) genome encodes 14 PYR/PYL/RCAR orthologs (PtPYRLs), and two of them, PtPYRL1 and PtPYRL5 have been functionally characterized to positively regulate drought tolerance. However, the physiological function of these ABA receptors in poplar remains uncharacterized. Here, we generated transgenic poplar plants overexpressing PtPYRL1 and PtPYRL5 and found that they exhibited more vigorous growth and produced greater biomass when exposed to drought stress. The improved drought tolerance was positively correlated with the key physiological responses dictated by the ABA signaling pathway, including increase in stomatal closure and decrease in leaf water loss. Further analyses revealed that overexpression lines showed improved capacity in scavenging reactive oxygen species and enhanced the activation of antioxidant enzymes under drought stress. Moreover, overexpression of PtPYRL1 or PtPYRL5 significantly increased the poplar resistance to osmotic and cold stresses. In summary, our results suggest that constitutive expression of PtPYRL1 and PtPYRL5 significantly enhances the resistance to drought, osmotic and cold stresses by positively regulating ABA signaling in poplar.

Calcium-dependent protein kinase CPK31 interacts with arsenic transporter AtNIP1;1 and regulates arsenite uptake in Arabidopsis thaliana.

Although arsenite [As(III)] is non-essential and toxic for plants, it is effectively absorbed through various transporters into the roots. Here we identified a calcium-dependent protein kinase (CPK31) response for As(III) tolerance in Arabidopsis. We identified CPK31 as an interacting protein of a nodulin 26-like intrinsic protein (NIP1;1), an aquaporin involved in As(III) uptake. Similarly to the nip1;1 mutants, the loss-of-function mutants of CPK31 improved the tolerance against As(III) but not As(V), and accumulated less As(III) in roots than that of the wild-type plants. The promoter-ß-glucuronidase and quantitative Real-Time PCR analysis revealed that CPK31 displayed overlapping expression profiles with NIP1;1 in the roots, suggesting that they might function together in roots. Indeed, the cpk31 nip1;1 double mutants exhibited stronger As(III) tolerance than cpk31 mutants, but similar to nip1;1 mutants, supporting the idea that CPK31 might serve as an upstream regulator of NIP1;1. Furthermore, transient CPK31 overexpression induced by dexamethasone caused the decrease in As(III) tolerance of transgenic Arabidopsis lines. These findings reveal that CPK31 is a key factor in As(III) response in plants.

Overexpression of Poplar Pyrabactin Resistance-Like Abscisic Acid Receptors Promotes Abscisic Acid Sensitivity and Drought Resistance in Transgenic Arabidopsis.

Drought stress is an important environmental factor limiting productivity of plants, especially fast growing species with high water consumption like poplar. Abscisic acid (ABA) is a phytohormone that positively regulates seed dormancy and drought resistance. The PYR1 (Pyrabactin Resistance 1)/ PYRL (PYR-Like)/ RCAR (Regulatory Component of ABA Receptor) (PYR/PYL/RCAR) ABA receptor family has been identified and widely characterized in Arabidopsis thaliana. However, their functions in poplars remain unknown. Here, we report that 2 of 14 PYR/PYL/RCAR orthologues in poplar (Populus trichocarpa) (PtPYRLs) function as a positive regulator of the ABA signal transduction pathway. The Arabidopsis transient expression and yeast two-hybrid assays showed the interaction among PtPYRL1 and PtPYRL5, a clade A protein phosphatase 2C, and a SnRK2, suggesting that a core signalling complex for ABA signaling pathway exists in poplars. Phenotypic analysis of PtPYRL1 and PtPYRL5 transgenic Arabidopsis showed that these two genes positively regulated the ABA responses during the seed germination. More importantly, the overexpression of PtPYRL1 and PtPYRL5 substantially improved ABA sensitivity and drought stress tolerance in transgenic plants. In summary, we comprehensively uncovered the properties of PtPYRL1 and PtPYRL5, which might be good target genes to genetically engineer drought-Resistant plants.