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 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

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

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

Table_1_Heterotrimeric G-Protein γ Subunit CsGG3.2 Positively Regulates the Expression of CBF Genes and Chilling Tolerance in Cucumber.DOCX

<p>Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of alpha (Gα), beta (Gβ), and gamma (Gγ) subunits are central signal transducers mediating the cellular response to multiple stimuli, such as cold, in eukaryotes. Plant Gγ subunits, divided into A, B, and C three structurally distinct types, provide proper cellular localization and functional specificity to the heterotrimer complex. Here, we demonstrate that a type C Gγ subunit CsGG3.2 is involved in the regulation of the CBF regulon and plant tolerance to cold stresses in cucumber (Cucumis sativus L.). We showed that CsGG3.2 transcript abundance was positively induced by cold treatments. Transgenic cucumber plants (T1) constitutively over-expressing CsGG3.2 exhibits tolerance to chilling conditions and increased expression of CBF genes and their regulon. Antioxidative enzymes, i.e., superoxide dismutase, catalase, peroxidase, and glutathione reductase activities increased in cold-stressed transgenic plants. The reactive oxygen species, oxygen free radical and H₂O₂, production, as well as membrane lipid peroxidation (MDA) production decreased in transgenic plants, suggesting a better antioxidant system to cope the oxidative-damages caused by cold stress. These findings provide evidence for a critical role of CsGG3.2 in mediating cold signal transduction in plant cells.</p