Abstract
Jasmonoyl-L-isoleucine (JA-Ile) is a powerful oxylipin responsible for the genome-wide transcriptional reprogramming in plants that results in major physiological shifts from growth to defense. The double T-DNA insertion Arabidopsis mutant, cyp94b1cyp94b3 (b1b3), defective in cytochrome p450s, CYP94B1 and CYP94B3, which are responsible for oxidizing JA-Ile, accumulates several fold higher levels of JA-Ile yet displays dampened JA-Ile–dependent wound responses—the opposite of what is expected. Transcriptomic and proteomic analyses showed that while the transcriptional response to wounding was largely unchanged in b1b3 compared to wild type (WT), many proteins were found to be significantly reduced in the mutant, which was verified by immunoblot analyses of marker proteins. To understand this protein phenotype and their hypothesized contribution to the b1b3 phenotypes, wounded rosette leaf samples from both WT and b1b3 were subject to a translating ribosome affinity purification RNA sequencing analysis. More than 1,600 genes whose transcripts do not change in abundance by wounding changed their association with the ribosomes after wounding in WT leaves. Consistent with previous observations, the total pool of mRNA transcripts was similar between WT and b1b3; however, the ribosome-associated pool of transcripts was changed significantly. Most notably, fewer transcripts were associated with the ribosome pool in b1b3 than in WT, potentially explaining the reduction of many proteins in the mutant. Among those genes with fewer ribosome-associated transcripts in b1b3 were genes relating to stress response, specialized metabolism, protein metabolism, ribosomal subunits, and transcription factors, consistent with the biochemical phenotypes of the mutant. These results show previously unrecognized regulations at the translational level that are affected by misregulation of JA homeostasis during the wound response in plants.
Highlights
Much progress has been made in understanding the genomewide transcriptional reprogramming after wounding
At the center of this transcriptional reprogramming is the biosynthesis of jasmonoyl-L-Ile (JA-Ile), a bioactive metabolite form of JA, which can activate transcriptional changes through physical binding to a nuclear-located hormone receptor cocomplex consisting of CORONATINE INSENSITIVE1 (COI1) and JASMONATE ZIM-domain (JAZ) proteins (Chini et al, 2007; Thines et al, 2007; Yan et al, 2007; Fonseca et al, 2009; Sheard et al, 2010; Howe et al, 2018)
It has previously been observed through multiple biochemical and physiological studies that much of the downstream JA-dependent wound responses including anthocyanin accumulation (Figure 1A) are down-regulated in the cyp94b1cyp94b3 (b1b3) mutant when compared to wild type (WT) despite containing threefold to fourfold higher levels of JA-Ile in the mutant (Koo et al, 2014; Poudel et al, 2016)
Summary
Much progress has been made in understanding the genomewide transcriptional reprogramming after wounding. As part of our investigation to understand how this pathway is inactivated, two genes belonging to the CYP94 clade of Arabidopsis cytochrome P450 enzymes, CYP94B1 and CYP94B3, were identified (Kitaoka et al, 2011; Koo et al, 2011, 2014; Heitz et al, 2012). These enzymes act as JA-Ile-12-hydroxylases in the so-called ω-oxidation pathway that oxidizes JA-Ile in a sequential manner to eventually catabolize the hormone (Koo and Howe, 2012; Koo, 2018). In maize, it was discovered that the genetic lesion responsible for the feminized tassels of the classical Tasselseed mutant results from overexpression of ZmCYP94B1 that depletes the bioactive pool of JA-Ile that is required to abort silks in the tassel (Lunde et al, 2019), consistent with the role of CYP94s in JA-Ile turnover
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