Title: Transcriptome Analysis of Cell Wall and NAC Domain Transcription Factor Genes during Elaeis guineensis Fruit Ripening: Evidence for Widespread Conservation within Monocot and Eudicot Lineages
The oil palm (Elaeis guineensis), a monocotyledonous
cotyledon: 子叶
species in the family Arecaceae, has an extraordinarily oil rich fleshy mesocarp, and presents an original model to examine the ripening processes and regulation in this particular monocot fruit. Histochemical analysis
Histochemical analysis(组织化学分析) is the use of chemical reactions performed in situ on tissue sections to localize specific molecules — carbohydrates, lipids, proteins, phenolics, nucleic acids, or enzyme activities — while preserving the spatial organization of the tissue. The core idea is that you’re not homogenizing the sample and assaying a bulk extract; you’re asking where in the tissue something is.
Tissue is usually fixed and sectioned via cryosection(冷冻切片), paraffin（石蜡切片）, or resin（树脂切片）, and treated with a reagent that yields a colored or fluorescent product where the target is present. Classic examples are:
- PAS (periodic acid–Schiff) — magenta（品红） for polysaccharides/starch
- Sudan/Nile red（苏丹红） — neutral lipids and oil bodies
- Toluidine(甲苯胺) blue O — metachromatic(changes color when it binds to specific chemicals); distinguishes lignified(木质化) vs. pectinaceous(富含果胶的) walls
- Phloroglucinol-HCl / Mäule — lignin and lignin subtype
- Enzyme histochemistry — a substrate is converted by endogenous enzyme into an insoluble precipitate (e.g., peroxidase, phosphatase activity)
and cell parameter measurements revealed cell wall and middle lamella expansion and degradation during ripening and in response to ethylene.The middle lamella(胞间层/中胞层，主要是果胶 pectin) is a layer that cements together the primary cell walls of two adjoining plant cells.
Cell wall related transcript profiles suggest a transition from synthesis to degradation is under transcriptional control during ripening, in particular a switch from cellulose, hemicellulose, and pectin synthesis to hydrolysis and degradation.Now we need to understand what are “cell wall related transcript profiles”. In the “Mesocarp Transcriptome Data Mining” section in “Materials and Methods”,
Transcriptome data of the developing mesocarp previously clustered (clusters A, B, C, and D) was searched for transcripts with expression profiles that either increase or decrease during the burst of ethylene production observed during oil palm fruit ripening between 100 and 160 DAP (Supplementary Table 1; Tranbarger et al., 2011).So first of all, the transcript needs to responding to ethylene. Then they talked about how the transcripts were annotated:
The BLAST2GO and InterProScan web services with BLASTX using an E-value cutoff of 1e-5 were used to annotate the gene sets (Altschul et al., 1990; Zdobnov and Apweiler, 2001; Götz et al., 2008).Now some filtering:
- “Cell wall sequences were identified by searching the GO annotated sequences for InterPro accessions and key words related to cell wall processes”
- “by searching (TBLASTX) the 454 sequence database with known candidates related to cell wall biosynthesis and degradation.”
This ends up with “A total of 75 transcripts for cell wall related activities were found to be differentially expressed in the mesocarp during ripening, 63% of which have expression peaks at 140 and 160 DAP (including EgPG4) concomitant with the ethylene burst as measured previously (Tranbarger et al., 2011).”
The data provide evidence for the transcriptional activation of expansin, polygalacturonase, mannosidase, beta-galactosidase, and xyloglucan endotransglucosylase/hydrolase proteins in the ripening oil palm mesocarp, suggesting widespread conservation of these activities during ripening for monocotyledonous and eudicotyledonous fruit types.
These are hand-picked genes that are important in the ripening of tomato and banana.
Profiling of the most abundant oil palm polygalacturonase (EgPG4) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) transcripts during development and in response to ethylene demonstrated both are sensitive markers of ethylene production and inducible gene expression during mesocarp ripening, and provide evidence for a conserved regulatory module between ethylene and cell wall pectin degradation."
First of all, what is EgPG4? “Recent studies by our group identified a polygalacturonase (EgPG4) highly induced by ethylene in oil palm fruit abscission zone cells and associated with the cell separation and fruit abscission (Roongsattham et al., 2012, 2016).” While in this 2016 Paper, “Previous studies revealed that oil palm fruit AZ cell walls are rich in unmethylated pectin and that PG activity and the EgPG4 transcript is highly expressed in the AZ in response to ethylene (Henderson and Osborne, 1994; Henderson et al., 2001; Roongsattham et al., 2012).”. Indeed, this (2012 Paper)[https://link.springer.com/article/10.1186/1471-2229-12-150] was all about the PG gene family in oil palm. Also in this paper, EgPG4 was reported to be “the most highly induced in the fruit base, with a 700–5000 fold increase during the ethylene treatment.” among all the PGs.
Secondly, “profiling” here means real-time RT-PCR. This paper did not generate any new RNAseq data.
“A comprehensive analysis of NAC transcription factors confirmed at least 10 transcripts from diverse NAC domain clades are expressed in the mesocarp during ripening, four of which are induced by ethylene treatment, with the two most inducible (EgNAC6 and EgNAC7) phylogenetically similar to the tomato NAC-NOR master-ripening regulator.”
Overall, the results provide evidence that despite the phylogenetic distance of the oil palm within the family Arecaceae from the most extensively studied monocot banana fruit, it appears ripening of divergent monocot and eudicot fruit lineages are regulated by evolutionarily conserved molecular physiological processes.