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. 2023 Jun 26;24(13):10671.
doi: 10.3390/ijms241310671.

The Influence of Lead and Acyrthosiphon pisum (Harris) on Generation of Pisum sativum Defense Signaling Molecules and Expression of Genes Involved in Their Biosynthesis

Agnieszka Woźniak  1 , Jacek Kęsy  2 , Paulina Glazińska  2 , Wojciech Glinkowski  2 , Dorota Narożna  3 , Jan Bocianowski  4 , Renata Rucińska-Sobkowiak  5 , Van Chung Mai  6 , Włodzimierz Krzesiński  7 , Sławomir Samardakiewicz  8 , Beata Borowiak-Sobkowiak  9 , Mateusz Labudda  10 , Philippe Jeandet  11 , Iwona Morkunas  1
Affiliations

The Influence of Lead and Acyrthosiphon pisum (Harris) on Generation of Pisum sativum Defense Signaling Molecules and Expression of Genes Involved in Their Biosynthesis

Agnieszka Woźniak et al. Int J Mol Sci. .

Abstract

The main aim of this study was to understand the regulation of the biosynthesis of phytohormones as signaling molecules in the defense mechanisms of pea seedlings during the application of abiotic and biotic stress factors. It was important to identify this regulation at the molecular level in Pisum sativum L. seedlings under the influence of various concentrations of lead-i.e., a low concentration increasing plant metabolism, causing a hormetic effect, and a high dose causing a sublethal effect-and during feeding of a phytophagous insect with a piercing-sucking mouthpart-i.e., pea aphid (Acyrthosiphon pisum (Harris)). The aim of the study was to determine the expression level of genes encoding enzymes of the biosynthesis of signaling molecules such as phytohormones-i.e., jasmonates (JA/MeJA), ethylene (ET) and abscisic acid (ABA). Real-time qPCR was applied to analyze the expression of genes encoding enzymes involved in the regulation of the biosynthesis of JA/MeJA (lipoxygenase 1 (LOX1), lipoxygenase 2 (LOX2), 12-oxophytodienoate reductase 1 (OPR1) and jasmonic acid-amido synthetase (JAR1)), ET (1-aminocyclopropane-1-carboxylate synthase 3 (ACS3)) and ABA (9-cis-epoxycarotenoid dioxygenase (NCED) and aldehyde oxidase 1 (AO1)). In response to the abovementioned stress factors-i.e., abiotic and biotic stressors acting independently or simultaneously-the expression of the LOX1, LOX2, OPR1, JAR1, ACS3, NCED and AO1 genes at both sublethal and hormetic doses increased. Particularly high levels of the relative expression of the tested genes in pea seedlings growing at sublethal doses of lead and colonized by A. pisum compared to the control were noticeable. A hormetic dose of lead induced high expression levels of the JAR1, OPR1 and ACS3 genes, especially in leaves. Moreover, an increase in the concentration of phytohormones such as jasmonates (JA and MeJA) and aminococyclopropane-1-carboxylic acid (ACC)-ethylene (ET) precursor was observed. The results of this study indicate that the response of pea seedlings to lead and A. pisum aphid infestation differed greatly at both the gene expression and metabolic levels. The intensity of these defense responses depended on the organ, the metal dose and direct contact of the stress factor with the organ.

Keywords: Pisum sativum; ethylene; genes encoding enzymes of phytohormone biosynthesis; jasmonates; lead; pea aphid.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1
Figure 1
Octadecanoid pathway of jasmonic acid (JA) biosynthesis [28]. The initial steps of JA biosynthesis including lipoxygenase (LOX), allene oxide synthase (AOS) and allene oxide cyclase (AOC) lead to the production of 12-oxo-phytodienoic acid (12-OPDA). 12-OPDA is then transported to the peroxisome and reduced to 3-oxo-2-(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0), which undergoes three rounds of β-oxidation to yield JA. Genes encoding enzymes involved in the biosynthesis of JA, whose expression was studied, are highlighted in red in the scheme.
Figure 2
Figure 2
Pathway of ethylene (ET) biosynthesis [29]. Genes encoding enzymes involved in the biosynthesis of ET, whose expression was studied, are highlighted in red in the scheme.
Figure 3
Figure 3
Pathway of abscisic acid (ABA) biosynthesis [30]. Genes encoding enzymes involved in the biosynthesis of ABA, whose expression was studied, are highlighted in red in the scheme.
Figure 5
Figure 5
Effect of lead and A. pisum on the accumulation of 1-aminocyclopropane-1-carboxylate (ACC) (a,b) in the organs of pea seedlings. The data were obtained from three independent experiments and were statistically analyzed using ANOVA (p < 0.001).
Figure 7
Figure 7
Expression of lipoxygenase (LOX2) in roots (a) and leaves (b) of pea seedlings exposed to lead and A. pisum. Gene expression level was assessed by RT-qPCR. Phosphoprotein phosphatase 2A (PP2A) was used as a reference gene. The data were obtained from three independent experiments and statistically analyzed using ANOVA (p < 0.001).
Figure 8
Figure 8
Expression of 12-oxophytodienoate reductase 1 (OPR1) in roots (a) and leaves (b) of pea seedlings exposed to lead and A. pisum. Gene expression level was assessed by RT-qPCR. Phosphoprotein phosphatase 2A (PP2A) was used as the reference gene. The data were obtained from three independent experiments and statistically analyzed using ANOVA (p < 0.001).
Figure 9
Figure 9
Expression of jasmonic acid-amido synthetase (JAR1) in roots (a) and leaves (b) of pea seedlings exposed to lead and A. pisum. Gene expression level was assessed by RT-qPCR. Phosphoprotein phosphatase 2A (PP2A) was used as the reference gene. The data were obtained from three independent experiments and statistically analyzed using ANOVA (p < 0.001).
Figure 10
Figure 10
Expression of 1-aminocyclopropane-1-carboxylate synthase 3 (ACS3) in roots (a) and leaves (b) of pea seedlings exposed to lead and A. pisum. Gene expression level was assessed by RT-qPCR. Phosphoprotein phosphatase 2A (PP2A) was used as the reference gene. The data were obtained from three independent experiments and statistically analyzed using ANOVA (p < 0.001).
Figure 11
Figure 11
The effect of lead and A. pisum on expression levels of 9-cis-epoxycarotenoid dioxygenase (NCED) in the organs of pea seedlings. (a) roots, (b) leaves. The data were obtained from three independent experiments and statistically analyzed using ANOVA (p < 0.001).
Figure 12
Figure 12
Expression of aldehyde oxidase 1 (AO1) in roots (a) and leaves (b) of pea seedlings exposed to lead and A. pisum. Gene expression level was assessed by RT-qPCR. Phosphoprotein phosphatase 2A (PP2A) was used as the reference gene. The data were obtained from three independent experiments and statistically analyzed using ANOVA (p < 0.001).
Figure 13
Figure 13
Heatmaps for Pearson’s correlation coefficients between all pairs of the ten observed traits in roots (a) and in leaves (b). The correlation coefficients ranged from −1 (blue) to 1 (red); rcr;0.05 = 0.19, rcr;0.01 = 0.25, rcr;0.001 = 0.32.
Figure 4
Figure 4
Effect of lead and A. pisum on accumulation of jasmonic acid (JA) (a,b) and methyl jasmonate (MeJA) (c,d) in the organs of pea seedlings. The data were obtained in three independent experiments and were statistically analyzed using ANOVA (p < 0.001).
Figure 6
Figure 6
Expression of lipoxygenase (LOX1) in roots (a) and leaves (b) of pea seedlings exposed to lead and A. pisum. Gene expression level was assessed by RT-qPCR. Phosphoprotein phosphatase 2A (PP2A) was used as the reference gene. The data were obtained from three independent experiments and statistically analyzed using ANOVA (p < 0.001).
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