Gibberellin Is Involved in Inhibition of Cucumber Growth and Nitrogen Uptake at Suboptimal Root-Zone Temperatures

<div><p>Suboptimal temperature stress often causes heavy yield losses of vegetables by suppressing plant growth during winter and early spring. Gibberellin acid (GA) has been reported to be involved in plant growth and acquisition of mineral nutrients. However, no studies have evaluated the role of GA in the regulation of growth and nutrient acquisition by vegetables under conditions of suboptimal temperatures in greenhouse. Here, we investigated the roles of GA in the regulation of growth and nitrate acquisition of cucumber (<i>Cucumis sativus</i> L.) plants under conditions of short-term suboptimal root-zone temperatures (T_r). Exposure of cucumber seedlings to a T_r of 16°C led to a significant reduction in root growth, and this inhibitory effect was reversed by exogenous application of GA. Expression patterns of several genes encoding key enzymes in GA metabolism were altered by suboptimal T_r treatment, and endogenous GA concentrations in cucumber roots were significantly reduced by exposure of cucumber plants to 16°C T_r, suggesting that inhibition of root growth by suboptimal T_r may result from disruption of endogenous GA homeostasis. To further explore the mechanism underlying the GA-dependent cucumber growth under suboptimal T_r, we studied the effect of suboptimal T_r and GA on nitrate uptake, and found that exposure of cucumber seedlings to 16°C T_r led to a significant reduction in nitrate uptake rate, and exogenous application GA can alleviate the down-regulation by up regulating the expression of genes associated with nitrate uptake. Finally, we demonstrated that N accumulation in cucumber seedlings under suboptimal T_r conditions was improved by exogenous application of GA due probably to both enhanced root growth and nitrate absorption activity. These results indicate that a reduction in endogenous GA concentrations in roots due to down-regulation of GA biosynthesis at transcriptional level may be a key event to underpin the suboptimal T_r-induced inhibition of root growth and nitrate uptake. These findings may have important practical implications in effective mitigation of suboptimal temperature-induced vegetable loss under greenhouse conditions.</p></div

Effects of suboptimal T_r and GA on root morphological parameters of cucumber seedlings.

<p>(A) Total root length of cucumber seedlings. (B) Average diameter of roots of cucumber seedlings. (C) Number of root tips of cucumber seedlings. (D) Root surface area of cucumber seedlings. 10-day-old seedlings were transferred to 16°C T_r conditions in the presence or absence of exogenous 5 μM GA for 5 d. Data are means±se. Different letters on the top of column indicate significant differences (<i>P <0</i>.<i>05</i>, n = 6).</p

Effect of suboptimal T_r and GA on ¹⁵NO₃^- influx of cucumber.

<p>15-day-old cucumber seedlings were transferred to 22°C T_r and 16°C T_r conditions in the presence or absence of GA 5 μM GA for 8d. Data are means±SE. Different letters on the top of column indicate significant differences (<i>P <0</i>.<i>05</i>, n = 3).</p

Effects of inhibitors of key enzymes in N assimilation on ¹⁵NO₃^- influx of cucumber seedlings.

<p>20-day-old cucumber seedlings were exposed for 6 h to 16°C T_r (16°C), 16°C T_r in the presence of 5 μM GA (16°C+GA), 5 μM GA plus 0.5 mM tungstate (W), 5 μM GA plus 0.25 mM L-methionine sulphoximine (MSX), 5 μM GA plus 0.5 mM azaserine (AZA), and 5 μM GA plus 1 mM aminooxyacetate (AOA). Data are means±SE. Different letters on the top of column indicate significant differences (<i>P <0</i>.<i>05</i>, n = 3).</p

Effect of suboptimal T_r and GA on the growth of cucumber seedlings.

<p>(A) Phenotypes of cucumber seedlings. (B) Root dry mass (DM) of cucumber seedlings. (C) Shoot DM of cucumber seedlings. (D) Leaf area of cucumber seedlings. (E) Root to shoot ratio of cucumber seedlings. 15-day-old cucumber seedlings were transferred to 22°C T_r and 16°C T_r conditions in the presence or absence of GA 5 μM GA for 8d. Data are means±SE. Different letters on the top of column indicate significant differences (<i>P <0</i>.<i>05</i>, n = 6). Bar = 10 cm.</p

Suboptimal T_r regulates the transcript levels of GA biosynthesis genes.

<p>(A) Expression profiles of GA biosynthesis <i>GA 20-oxidase</i> genes. (B) Expression profiles of <i>GA 3-oxidase</i> genes. (C) Expression profiles of <i>GA 2-oxidase</i> genes. (D) Determination of GA₄ concentration in cucumber roots. 10-day-old seedlings were treated with 22°C T_r or 16°C T_r for 5 d. Data are means±SE. Different letters on the top of column indicate significant differences (<i>P <0</i>.<i>05</i>, n = 3).</p

Suboptimal T_r and GA regulate the transcript levels of <i>CsNRT1</i> family genes.

<p>15-day-old cucumber seedlings were transferred to 22°C T_r and 16°C T_r conditions in the presence or absence of GA 5 μM GA for 8 d. The relative expression levels were analyzed by qPCR using <i>Actin</i> as internal control. Data are means±SE. Different letters on the top of column indicate significant differences (<i>P <0</i>.<i>05</i>, n = 3).</p

A Glutamine-Rich Carrier Efficiently Delivers Anti-CD47 siRNA Driven by a “Glutamine Trap” To Inhibit Lung Cancer Cell Growth

It is not efficient enough using the current approaches for tumor-selective drug delivery based on the EPR effect and ligand–receptor interactions, and they have largely failed to translate into the clinic. Therefore, it is urgent to explore an enhanced strategy for effective delivery of anticancer agents. Clinically, many cancers require large amounts of glutamine for their continued growth and survival, resulting in circulating glutamine extraction by the tumor being much greater than that for any organs, behaving as a “glutamine trap”. In the present study, we sought to elucidate whether the glutamine-trap effect could be exploited to deliver therapeutic agents to selectively kill cancer cells. Here, a macromolecular glutamine analogue, glutamine-functionalized branched polyethylenimine (GPI), was constructed as the carrier to deliver anti-CD47 siRNA for the blockage of CD47 “don’t eat me” signals on cancer cells. The GPI/siRNA glutamine-rich polyplexes exhibited remarkably high levels of cellular uptake by glutamine-dependent lung cancer cells, wild-type A549 cells (A549^WT), and its cisplatin-resistant cells (A549^DDP), specifically under glutamine-depleted conditions. It was noted that the glutamine transporter ASCT2 was highly expressed both on A549^WT and A549^DDP but with almost no expression in normal human lung fibroblasts cells. Inhibition of ASCT2 significantly prevented the internalization of GPI polyplexes. These findings raised the intriguing possibility that the glutamine-rich GPI polyplexes utilize the ASCT2 pathway to selectively facilitate their cellular uptake by cancer cells. GPI further delivered anti-CD47 siRNA efficiently both in vitro and in vivo to downregulate the intratumoral mRNA and protein expression levels of CD47. CD47 functions as a “don’t eat me” signal and binds to the immunoreceptor SIRPα inducing evasion of phagocytic clearance. GPI/anti-CD47 siRNA polyplexes achieved significant antitumor activities both on A549^WT and A549^DDP tumor-bearing nude mice. Notably, it had no adverse effect on CD47-expressing red blood cells and platelets, likely because of selective delivery. Therefore, the glutamine-rich carrier GPI driven by the glutamine-trap effect provides a promising new strategy for designing anticancer drug delivery systems