Interruption of Jasmonic Acid Biosynthesis Causes Differential Responses in the Roots and Shoots of Maize Seedlings against Salt Stress

Abstract

Jasmonates (JAs) together with jasmonic acid and its offshoots are lipid-derived endogenous hormones that play key roles in both developmental processes and different defense responses in plants. JAs have been studied intensively in the past decades for their substantial roles in plant defense comebacks against diverse environmental stresses among model plants. However, the role of this phytohormone has been poorly investigated in the monocotyledonous species against abiotic stresses. In this study, a JA biosynthesis mutant opr7opr8 was used for the investigation of JA roles in the salt stress responses of maize seedlings, whose roots were exposed to 0 to 300 mM NaCl. Foliar stomatal observation showed that opr7opr8 had a larger stomatal aperture than wild type (WT) (B73) under salinity stress, indicating that JA positively regulates guard cell movement under salt stress. The results regarding chlorophyll content and leaf senescence showed that opr7opr8 exhibited delayed leaf senescence under salt stress as compared to WT, indicating that JA plays a role in salt-inducing cell death and subsequent leaf senescence. Moreover, the morphological parameters, including the length of the shoots and roots, and the fresh and dry weights of the shoots and roots, showed that after 7 days of salt treatment, opr7opr8 had heavier and longer shoots than WT but slighter and shorter roots than WT. In addition, ion analysis showed that opr7opr8 accumulated less sodium but more potassium in the leaves than WT but more sodium and less potassium in the roots than WT, suggesting that JA deficiency causes higher salt stress to the roots but less stress to the leaves of the seedlings. Reactive oxygen species (ROS) analysis showed that opr7opr8 produced less H₂O₂ than WT in the leaves but more H₂O₂ in the roots under salt treatment, and correspondingly, ROS-scavenging enzymes superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) showed a similar variation, i.e., opr7opr8 has lower enzymatic activities in the shoots but higher activities in the roots than WT under salt treatment. For osmotic adjustment, opr7opr8 produced less proline in the shoots at 100 and 300 mM NaCl treatments but more in the roots than the WT roots under all salt treatments. In addition, the gene expression for abscisic acid (ABA) biosynthesis under salt stress was investigated. Results showed that the expression levels of four key enzymes of ABA biosynthesis, ZEP1, NCED5, AO1, and VP10, were significantly downregulated in the shoots as compared to WT under salt treatment. Putting all the data together, we concluded that JA-deficiency in maize seedlings reduced the salt-stress responses in the shoots but exaggerated the responses in the roots. In addition, endogenous JA acted as a positive regulator for the transportation of sodium ions from the roots to the shoots because the mutant opr7opr8 had a higher level of sodium in the roots but a significantly lower level in the shoots than WT. Furthermore, JA may act as a positive regulator for ABA biosynthesis in the leaves under salt stress.

Keywords: ABA biosynthesis; ROS; Zea mays; jasmonate; proline; salt response.

Figure 1

opr7opr8 displays a higher stomatal aperture than wild type (B73) under salt (NaCl) treatment. (a) Pore aperture index of stomata. (b) Stomatal aperture index (SAI) of B73 and opr7opr8 after 24 h salt treatment with 0 to 300 mM NaCl. (c) Stomata image of B73 and opr7opr8 after 24 h under 200 mM NaCl treatment at 40x magnification. (d) Stomatal density on the third leaf of B73 and opr7opr8. The asterisks denote significant differences between B73 and the opr7opr8 mutant at p < 0.05 (*) or p < 0.01 (**) by analysis of variance.

Figure 2

opr7opr8 undergoes delayed leaf senescence upon salt (NaCl) stress compared to B73. (a) Chlorosis symptom of leaves of B73 and opr7opr8 seedlings whose roots were treated with 0–300 mM NaCl in the hydroponic system. The third leaves of the two genotypes were used to take the pictures at 7 days of salt treatment. (b) Measurement of chlorophyll A content in the leaves of B73 and opr7opr8 seedlings at 2 days of salt stress. (c) Measurement of chlorophyll B content in the leaves of B73 and opr7opr8 seedlings at 2 days of salt stress. The asterisks denote significant differences between wild type (WT) and opr7opr8 at p < 0.01 (**) by analysis of variance.

Figure 3

Salt treatments inhibit the growth of shoots and roots of B73 and opr7opr8 seedlings. The V3-stage plants of B73 and opr7opr8 were treated with 0–300 mM NaCl in the hydroponic system, and the (a) shoot length, (b) root length, (c) shoot fresh weight, (d) root fresh weight, (e) shoot dry weight, and (f) root dry weight were measured after 7 days of salt treatments. The shoot length is the distance from the first node (the coleoptiles node) to the tip of the leaves. The root length was measured from the first node to the far-end of the root system. The asterisks show significant differences for WT and the mutant at p ≤ 0.05 (*) or p ≤ 0.01 (**) by analysis of variance.

Figure 4

The leaves and roots of B73 and opr7opr8 seedlings accumulate sodium and potassium under NaCl treatments. The samples were taken at 7 days after salt treatment. (a) Na⁺ ion content in the leaf of B73 and opr7opr8 plants. (b) Na⁺ ion content in the roots. (c) K⁺ ion content in the leaves. (d) K⁺ ion content in the roots. The asterisks denote significant differences between WT and the mutant at p ≤ 0.05 (*) or p ≤ 0.01 (**) by analysis of variance.

Figure 5

Reactive oxygen species (ROS) production in the meristematic zone of the roots of B73 and opr7opr8 under 0–300 mM NaCl treatments. (a) Visualization detection of ROS production under bright field (BF) and green light (480–550 nm) fluorescence (GLF) with a confocal microscope using 2′,7′-dichlorofluorescin diacetate (H2DCFDA) at 4 h after salt treatments. (b) Relative fluorescence quantification of ROS production in root meristematic zones of B73 and opr7opr8 under salt treatments by software ImageJ scan. The values of B73 and opr7opr8 were given 1 at 0 mM for relative ROS quantification. The asterisks denote significant differences between WT and the mutant at p ≤ 0.05 (*) or p ≤ 0.01 (**) by analysis of variance.

Figure 6

Malondialdehyde (MDA) and H₂O₂ accumulation in opr7opr8 and B73 under salt stress. The MDA levels in the (a) leaves and (b) roots were detected after 48 h of 0–300 mM NaCl treatments. The H₂O₂ content in the (c) leaves and (d) roots was measured after 48 h of salt treatments. The asterisks denote significant differences between WT and the mutant at p ≤ 0.05 (*) or p ≤ 0.01 (**) by analysis of variance.

Figure 7

ROS-scavenging enzyme activities in the shoots and roots of opr7opr8 and B73 seedlings under 0–300 mM NaCl treatments. The samples were taken 2 days after the salt was applied to the hydroponic solution. (a) SOD activity in leaves and roots, (b) POX activity in leaves and roots, (c) CAT activity in leaves and roots, and (d) APX activity in leaves and roots.

Figure 8

Enzymatic activities of glutathione reductase (GR) and glutathione-S-transferase (GST) in B73 and opr7opr8 seedlings under 0–300 mM NaCl treatments. The samples were taken 2 days after the salt treatments. GST activity in (a) the leaves and (b) roots and GR activity in the (c) leaves and (d) roots. The asterisks denote significant differences between WT and the mutant at p ≤ 0.05 (*) or p ≤ 0.01 (**) by analysis of variance.

Figure 9

Proline contents were detected in (a) the leaves and (b) roots at two days after 0–300 mM NaCl was applied to the roots. The asterisks denote significant differences between WT and the mutant at p ≤ 0.05 (*) or p ≤ 0.01 (**) by analysis of variance.

Figure 10

Quantitative real-time polymerase chain reaction (qRT-PCR) expression level analysis of (a) NCED5, (b) AO1, (c) VP10, and (d) ZEP1 genes in the leaves of the B73 and opr7opr8 plants under 200 mM NaCl treatment. The relative expression level was calculated according to the expression of the gene at 0 h of treatment. The asterisks denote significant differences between WT and the mutant opr7opr8 at p ≤ 0.05 (*) or p ≤ 0.01 (**) by analysis of variance.