STOP1 activates NRT1.1-mediated nitrate uptake to create a favorable rhizospheric pH for plant adaptation to acidity.

Protons (H+) in acidic soils arrest plant growth. However, the mechanisms by which plants optimize their biological processes to diminish the unfavorable effects of H+ stress remain largely unclear. Here, we showed that in the roots of Arabidopsis thaliana, the C2H2-type transcription factor STOP1 in the nucleus was enriched by low pH in a nitrate-independent manner, with the spatial expression pattern of NITRATE TRANSPORTER 1.1 (NRT1.1) established by low pH required the action of STOP1. Additionally, the nrt1.1 and stop1 mutants, as well as the nrt1.1 stop1 double mutant, had a similar hypersensitive phenotype to low pH, indicating that STOP1 and NRT1.1 function in the same pathway for H+ tolerance. Molecular assays revealed that STOP1 directly bound to the promoter of NRT1.1 to activate its transcription in response to low pH, thus upregulating its nitrate uptake. This action improved the nitrogen use efficiency (NUE) of plants and created a favorable rhizospheric pH for root growth by enhancing H+ depletion in the rhizosphere. Consequently, the constitutive expression of NRT1.1 in stop1 mutants abolished the hypersensitive phenotype to low pH. These results demonstrate that STOP1-NRT1.1 is a key module for plants to optimize NUE and ensure better plant growth in acidic media.

Palmitoyltransferase ZDHHC3 is essential for the oncogenic activity of PML/RARα in acute promyelocytic leukemia.

The oncogenic fusion protein promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) is critical for acute promyelocytic leukemia (APL). PML/RARα initiates APL by blocking the differentiation and increasing the self-renewal of leukemic cells. The standard clinical therapies all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), which induce PML/RARα proteolysis, have dramatically improved the prognosis of APL patients. However, the emergence of mutations conferring resistance to ATRA and ATO has created challenges in the treatment of APL patients. Exploring pathways that modulate the oncogenic activity of PML/RARα could help develop novel therapeutic strategies for APL, particularly for drug-resistant APL. Herein, we demonstrated for the first time that palmitoylation of PML/RARα was a critical determinant of its oncogenic activity. PML/RARα palmitoylation was found to be catalyzed mainly by the palmitoyltransferase ZDHHC3. Mechanistically, ZDHHC3-mediated palmitoylation regulated the oncogenic transcriptional activity of PML/RARα and APL pathogenesis. The knockdown or overexpression of ZDHHC3 had respective effects on the expression of proliferation- and differentiation-related genes. Consistently, the depletion or inhibition of ZDHHC3 could significantly arrest the malignant progression of APL, particularly drug-resistant APL, whereas ZDHHC3 overexpression appeared to have a promoting effect on the malignant progression of APL. Thus, our study not only reveals palmitoylation as a novel regulatory mechanism that modulates PML/RARα oncogenic activity but also identifies ZDHHC3 as a potential therapeutic target for APL, including drug-resistant APL.

Inhibition of shoot-expressed NRT1.1 improves reutilization of apoplastic iron under iron-deficient conditions.

Iron deficiency is a major constraint for plant growth in calcareous soils. The interplay between NO3 - and Fe nutrition affects plant performance under Fe-deficient conditions. However, how NO3 - negatively regulates Fe nutrition at the molecular level in plants remains elusive. Here, we showed that the key nitrate transporter NRT1.1 in Arabidopsis plants, especially in the shoots, was markedly downregulated at post-translational levels by Fe deficiency. However, loss of NRT1.1 function alleviated Fe deficiency chlorosis, suggesting that downregulation of NRT1.1 by Fe deficiency favors plant tolerance to Fe deficiency. Further analysis showed that although disruption of NRT1.1 did not alter Fe levels in both the shoots and roots, it improved the reutilization of apoplastic Fe in shoots but not in roots. In addition, disruption of NRT1.1 prevented Fe deficiency-induced apoplastic alkalization in shoots by inhibiting apoplastic H+ depletion via NO3 - uptake. In vitro analysis showed that reduced pH facilitates release of cell wall-bound Fe. Thus, foliar spray with an acidic buffer promoted the reutilization of Fe in the leaf apoplast to enhance plant tolerance to Fe deficiency, while the opposite was true for the foliar spray with a neutral buffer. Thus, downregulation of the shoot-part function of NRT1.1 prevents apoplastic alkalization to ensure the reutilization of apoplastic Fe under Fe-deficient conditions. Our findings may provide a basis for elucidating the link between N and Fe nutrition in plants and insight to scrutinize the relevance of shoot-expressed NRT1.1 to the plant response to stress.

The ferroxidases are critical for Fe(II) oxidation in xylem to ensure a healthy Fe allocation in Arabidopsis thaliana.

The long-distance transport of iron (Fe) in the xylem is critical for maintaining systemic Fe homeostasis in plants. The loading form of Fe(II) into the xylem and the long-distance translocation form of Fe(III)-citrate have been identified, but how Fe(II) is oxidized to Fe(III) in the xylem remains unknown. Here, we showed that the cell wall-resided ferroxidases LPR1 and LPR2 (LPRs) were both specifically expressed in the vascular tissues of Arabidopsis thaliana, while disruption of both of them increased Fe(II) in the xylem sap and caused excessive Fe deposition in the xylem vessel wall under Fe-sufficient conditions. As a result, a large amount of Fe accumulated in both roots and shoots, hindering plant growth. Moreover, under low-Fe conditions, LPRs were preferentially induced in old leaves, but the loss of LPRs increased Fe deposition in the vasculature of older leaves and impeded Fe allocation to younger leaves. Therefore, disruption of both LPRs resulted in severer chlorosis in young leaves under Fe-deficient conditions. Taken together, the oxidation of Fe(II) to Fe(III) by LPRs in the cell wall of vasculature plays an important role in xylem Fe allocation, ensuring healthy Fe homeostasis for normal plant growth.

Phloem iron remodels root development in response to ammonium as the major nitrogen source.

Plants use nitrate and ammonium as major nitrogen (N) sources, each affecting root development through different mechanisms. However, the exact signaling pathways involved in root development are poorly understood. Here, we show that, in Arabidopsis thaliana, either disruption of the cell wall-localized ferroxidase LPR2 or a decrease in iron supplementation efficiently alleviates the growth inhibition of primary roots in response to NH4+ as the N source. Further study revealed that, compared with nitrate, ammonium led to excess iron accumulation in the apoplast of phloem in an LPR2-dependent manner. Such an aberrant iron accumulation subsequently causes massive callose deposition in the phloem from a resulting burst of reactive oxygen species, which impairs the function of the phloem. Therefore, ammonium attenuates primary root development by insufficiently allocating sucrose to the growth zone. Our results link phloem iron to root morphology in response to environmental cues.

Improved Plant Nitrate Status Involves in Flowering Induction by Extended Photoperiod.

The floral transition stage is pivotal for sustaining plant populations and is affected by several environmental factors, including photoperiod. However, the mechanisms underlying photoperiodic flowering responses are not fully understood. Herein, we have shown that exposure to an extended photoperiod effectively induced early flowering in Arabidopsis plants, at a range of different nitrate concentrations. However, these photoperiodic flowering responses were attenuated when the nitrate levels were suboptimal for flowering. An extended photoperiod also improved the root nitrate uptake of by NITRATE TRANSPORTER 1.1 (NRT1.1) and NITRATE TRANSPORTER 2.1 (NRT2.1), whereas the loss of function of NRT1.1/NRT2.1 in the nrt1.1-1/2.1-2 mutants suppressed the expression of the key flowering genes CONSTANS (CO) and FLOWERING LOCUS T (FT), and reduced the sensitivity of the photoperiodic flowering responses to elevated levels of nitrate. These results suggest that the upregulation of root nitrate uptake during extended photoperiods, contributed to the observed early flowering. The results also showed that the sensitivity of photoperiodic flowering responses to elevated levels of nitrate, were also reduced by either the replacement of nitrate with its assimilation intermediate product, ammonium, or by the dysfunction of the nitrate assimilation pathway. This indicates that nitrate serves as both a nutrient source for plant growth and as a signaling molecule for floral induction during extended photoperiods.

Knockout of FER decreases cadmium concentration in roots of Arabidopsis thaliana by inhibiting the pathway related to iron uptake.

Identifying the genes that affect cadmium (Cd) accumulation in plants is a prerequisite for minimizing dietary Cd uptake from contaminated edible parts of plants by genetic engineering. This study showed that Cd stress inhibited the expression of FERONIA (FER) gene in the roots of wild-type Arabidopsis. Knockout of FER in fer-4 mutants downregulated the Cd-induced expression of several genes related to iron (Fe) uptake, including IRT1, bHLH38, NRAMP1, NRAMP3, FRO2 andFIT. In addition, the Cd concentration in fer-4 mutant roots reduced to approximately half of that in the wild-type seedlings. As a result, the Cd tolerance of fer-4 was higher. Furthermore, increased Fe supplementation had little effect on the Cd tolerance of fer-4 mutants, but clearly improved the Cd tolerance of wild-type seedlings, showing that the alleviation of Cd toxicity by Fe depends on the action of FER. Taken together, the findings demonstrate that the knockout of FER might provide a strategy to reduce Cd contamination and improve the Cd tolerance in plants by regulating the pathways related to Fe uptake.

Knockdown of BTS may provide a new strategy to improve cadmium-phytoremediation efficiency by improving iron status in plants.

The identification of the key genes related to cadmium (Cd) tolerance and accumulation is a major element in genetically engineering improved plants for Cd phytoremediation. Owing to the similarity between the ionic hydrated radius of Cd2+ and Fe2+, this study investigated how the Cd tolerance and accumulation of Arabidopsis plants was affected by the knockdown of BTS, a gene that negatively regulates Fe nutrition. After exposure to 40 µM Cd, the BTS-knockdown mutant, bts-1, exhibited greater Fe nutrition and better growth than wild-type plants. In addition, the Cd concentration in both roots and shoots was approximately 50% higher in the bts-1 mutant than in wild-type plants. Consequently, the bts-1 mutant accumulated approximately 100% and 150% more Cd in the roots and shoots, respectively, than wild-type plants. Further study showed that Fe removal from the growth medium and inhibition of the Fe transporter gene, IRT1, removed the differences observed in the growth and Cd concentration of the bts-1 and wild-type plants, respectively. These results demonstrated that BTS knockdown improved Cd tolerance and accumulation in plants by improving Fe nutrition; thus, the knockdown of BTS via biotechnological pathways may represent a valuable strategy for the improvement in the efficiency of Cd phytoremediation.

Ammonium aggravates salt stress in plants by entrapping them in a chloride over-accumulation state in an NRT1.1-dependent manner.

Global climate change has exacerbated flooding in coastal areas affected by soil salinization. Ammonium (NH4+) is the predominant form of nitrogen in flooded soils, but the role played by NH4+ in the plant response to salt stress has not been fully clarified. We investigated the responses of Arabidopsis thaliana, Oryza sativa, and Nicotiana benthamiana plants fed with NH4+. All species were hypersensitive to NaCl stress and accumulated more Cl- and less Na+ than those fed with NO3-. Further investigation of A. thaliana indicated that salt hypersensitivity induced by the presence of NH4+ was abolished by removing the Cl- but was not affected by the removal of Na+, suggesting that excess accumulation of Cl- rather than Na+ is involved in NH4+-conferred salt hypersensitivity. The expression of nitrate transporter NRT1.1 protein was also up-regulated by NH4+ treatment, which increased root Cl- uptake due to the Cl- uptake activity of NRT1.1 and the absence of uptake competition from NO3-. Knockout of NRT1.1 in plants decreased their root Cl- uptake and retracted the NH4+-conferred salt hypersensitivity. Our findings revealed that NH4+-aggravated salt stress in plants is associated with Cl- over-accumulation through the up-regulation of NRT1.1-mediated Cl- uptake. These findings suggest the significant impact of Cl- toxicity in flooded coastal areas, an issue of ecological significance.

Analysis of cardiovascular disease-related NF-κB-regulated genes and microRNAs in TNFα-treated primary mouse vascular endothelial cells.

Activated nuclear factor-κB (NF-κB) plays an important role in the development of cardiovascular disease (CVD) through its regulated genes and microRNAs (miRNAs). However, the gene regulation profile remains unclear. In this study, primary mouse vascular endothelial cells (pMVECs) were employed to detect CVD-related NF-κB-regulated genes and miRNAs. Genechip assay identified 77 NF-κB-regulated genes, including 45 upregulated and 32 downregulated genes, in tumor necrosis factor α (TNFα)-treated pMVECs. Ten of these genes were also found to be regulated by NF-κB in TNFα-treated HeLa cells. Quantitative real-time PCR (qRT-PCR) assay confirmed the up-regulation of Egr1, Tnf, and Btg2 by NF-κB in the TNFα-treated pMVECs. The functional annotation revealed that many NF-κB-regulated genes identified in pMVECs were clustered into classical NF-κB-involved biological processes. Genechip assay also identified 26 NF-κB-regulated miRNAs, of which 21 were upregulated and 5 downregulated, in the TNFα-treated pMVECs. Further analysis showed that nine of the identified genes are regulated by seven of these miRNAs. Finally, among the identified NF-κB-regulated genes and miRNAs, 5 genes and 12 miRNAs were associated with CVD by miRWalk and genetic association database analysis. Taken together, these findings show an intricate gene regulation network raised by NF-κB in TNFα-treated pMVECs. The network provides new insights for understanding the molecular mechanism underlying the progression of CVD.