Genotype-dependent responses to HIPV exposure in citrus: repression of CsPUB21 and activation of SA/JA signaling

Abstract

Herbivore-induced plant volatiles (HIPVs) are known to activate immune signaling in plants; however, their effectiveness can vary depending on the genotype and the signaling pathway involved. In this study, we evaluated the transcriptional response of four citrus rootstocks (Carrizo citrange, Forner-Alcaide 5 (FA5), Forner-Alcaide 74 (FA74), and Microcitrus australasica) to six synthetic HIPVs [(Z)-3-hexen-1-ol, (Z)-3-hexenyl acetate, (Z)-3-hexenyl butyrate, (Z)-3-hexenyl propanoate, methyl jasmonate, and methyl salicylate]. We focused on genes associated with the salicylic acid (SA) and jasmonic acid (JA) pathways, as well as the susceptibility gene CsPUB21. Overall, the SA pathway was more consistently activated than the JA pathway, with upstream and intermediate genes induced across most genotypes and treatments. In contrast, downstream markers showed more variable expression, suggesting that synthetic HIPVs may induce a primed rather than fully activated defense state. Among the volatiles tested, (Z)-3-hexenyl propanoate and (Z)-3-hexen-1-ol were the most effective, activating genes in both pathways. Importantly, these two compounds also consistently repressed CsPUB21 expression, a gene recently associated with huanglongbing (HLB) susceptibility, through coordinated transcriptional and post-translational regulation. Carrizo citrange showed the strongest transcriptional response, while FA74 exhibited more moderate activation, emphasizing the influence of genetic background on HIPV perception and signaling. These findings highlight the potential of selected synthetic HIPVs as sustainable defense priming agents capable of enhancing citrus immunity by simultaneously activating immune pathways and repressing susceptibility genes such as CsPUB21. This dual mode of action offers promising tools for the integrated management of HLB and other citrus diseases.

Keywords: CsPUB21; abiotic stress; biotic stress; citrus rootstocks; defense gene expression; jasmonic acid signaling; salicylic acid pathway; volatile organic compounds.

Figure 1

Expression of defense-related genes involved in the salicylic acid (SA) and jasmonic acid (JA) signaling pathways in Carrizo citrange rootstock exposed six synthetic HIPV treatments: methyl salicylate (MeSA), methyl jasmonate (MeJA), (Z)-3-hexenyl hexanoate (Z3C6OH), (Z)-3-hexenyl propanoate [(Z)-3-HP], (Z)-3-hexenyl butanoate [(Z)-3-HB], and (Z)-3-hexenyl acetate [(Z)-3-HA]. (A) CsICS, (B) CsPAL, (C) CsNPR1, (D) CsPR2, (E) CsLOX2, (F) CsJAR1, (G) CsCOI, (H) CsMYC2. Letters indicate significant differences between treatments based on Tukey’s test (P < 0.05).

Figure 2

Expression of defense-related genes involved in the salicylic acid (SA) and jasmonic acid (JA) signaling pathways in Forner-Alcaide 5 (FA5) rootstock exposed six synthetic HIPV treatments: methyl salicylate (MeSA), methyl jasmonate (MeJA), (Z)-3-hexenyl hexanoate (Z3C6OH), (Z)-3-hexenyl propanoate [(Z)-3-HP], (Z)-3-hexenyl butanoate [(Z)-3-HB], and (Z)-3-hexenyl acetate [(Z)-3-HA]. (A) CsICS, (B) CsPAL, (C) CsNPR1, (D) CsPR2, (E) CsLOX2, (F) CsJAR1, (G) CsCOI, (H) CsMYC2. Letters indicate significant differences between treatments based on Tukey’s test (P < 0.05).

Figure 3

Expression of defense-related genes involved in the salicylic acid (SA) and jasmonic acid (JA) signaling pathways in Forner-Alcaide 74 (FA74) rootstock exposed six synthetic HIPV treatments: methyl salicylate (MeSA), methyl jasmonate (MeJA), (Z)-3-hexenyl hexanoate (Z3C6OH), (Z)-3-hexenyl propanoate [(Z)-3-HP], (Z)-3-hexenyl butanoate [(Z)-3-HB], and (Z)-3-hexenyl acetate [(Z)-3-HA]. (A) CsICS, (B) CsPAL, (C) CsNPR1, (D) CsPR2, (E) CsLOX2, (F) CsJAR1, (G) CsCOI, (H) CsMYC2. Letters indicate significant differences between treatments based on Tukey’s test (P < 0.05).

Figure 4

Expression of defense-related genes involved in the salicylic acid (SA) and jasmonic acid (JA) signaling pathways in Microcitrus rootstock exposed to six synthetic HIPV treatments: methyl salicylate (MeSA), methyl jasmonate (MeJA), (Z)-3-hexenyl hexanoate (Z3C6OH), (Z)-3-hexenyl propanoate [(Z)-3-HP], (Z)-3-hexenyl butanoate [(Z)-3-HB], and (Z)-3-hexenyl acetate [(Z)-3-HA]. (A) CsICS, (B) CsPAL, (C) CsNPR1, (D) CsPR2, (E) CsLOX2, (F) CsJAR1, (G) CsCOI, (H) CsMYC2. Letters indicate significant differences between treatments based on Tukey’s test (P < 0.05).

Figure 5

Relative expression of the susceptibility gene CsPUB21 in citrus rootstocks (Carrizo citrange, FA5, FA74, and Microcitrus) exposed to six synthetic HIPV treatments: methyl salicylate (MeSA), methyl jasmonate (MeJA), (Z)-3-hexenyl hexanoate (Z3C6OH), (Z)-3-hexenyl propanoate [(Z)-3-HP], (Z)-3-hexenyl butanoate [(Z)-3-HB], and (Z)-3-hexenyl acetate [(Z)-3-HA]. (A) Carrizo citrange, (B) Forner-Alcaide 5 (FA5), (C) Forner-Alcaide 74 (FA74), (D) Microcitrus. Letters indicate significant differences between treatments based on Tukey’s test (P < 0.05).

Figure 6

Heatmaps showing hierarchical clustering of fold-change expression levels of defense-related genes in citrus rootstocks exposed to synthetic HIPVs. Log₂ fold-change (FC) values were calculated from qPCR data for eight defense-related genes (CsICS, CsPAL, CsNPR1, CsPR2, CsLOX2, CsJAR1, CsCOI, CsMYC2, and CsPUB21) following exposure to synthetic HIPVs. (A) Gene expression profiles under six synthetic HIPV treatments: methyl salicylate (MeSA), methyl jasmonate (MeJA), (Z)-3-hexenyl hexanoate (Z3C6OH), (Z)-3-hexenyl propanoate [(Z)-3-HP], (Z)-3-hexenyl butanoate [(Z)-3-HB], and (Z)-3-hexenyl acetate [(Z)-3-HA]. (B) Expression patterns across four citrus genotypes: Carrizo citrange, FA5, FA74, and Microcitrus. (C) Combined heatmap showing average gene expression responses across both synthetic HIPV treatments and genotypes. Hierarchical clustering was performed using Euclidean distance and complete linkage. Color scales represent log₂ FC values, with darker shades indicating stronger induction levels.