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Comparative Proteomics of Bark and Xylem Provides Insights into Age-Dependent Corticular Photosynthesis in Eucalyptus grandis

ABSTRACT: Eucalyptus species are vital for the global forestry industry, known for their rapid growth, adaptability, and high biomass production. Beyond their economic importance, eucalyptus plantations contribute to carbon sequestration, playing a crucial role in climate change mitigation by capturing atmospheric CO₂ and storing it as biomass. Vascular tissues, such as phloem and xylem, represent the primary bioproducts of interest in woody plants, exhibiting unique metabolic adaptations to meet the energetic demands for growth and maintenance. However, understanding the metabolic mechanisms that sustain growth, particularly under hypoxic conditions within woody tissues, remains unknown. Insights into the differential regulation of proteins across vascular tissues at contrasting developmental stages can facilitate the monitoring of metabolic responses during xylogenesis and support plant breeding programs. This study investigates the role of corticular photosynthesis in sustaining energy metabolism within eucalyptus stems by analyzing bark and xylem tissues from 4- and 12-year-old Eucalyptus grandis clonal plants. To achieve this, we employed fluorescence microscopy to quantify chloroplast abundance across bark developmental stages and performed proteomic profiling of both tissues. The results revealed a higher number of chloroplasts in younger bark, contrasting with their absence in xylem. A total of 3,113 non-redundant proteins were identified, and enrichment analysis demonstrated a consistent hypoxic response across tissues and ages, alongside age-specific metabolic processes. Proteomic analysis further uncovered distinct proteoforms abundance patterns associated with glycolysis, the tricarboxylic acid cycle, and fermentation pathways. Notably, we observed the presence of differential proteoforms abundance of alcohol dehydrogenase and aldehyde dehydrogenase in both xylem tissues and younger bark, indicating a metabolic shift toward fermentative pathways under hypoxic conditions. Additionally, younger bark tissues exhibited higher abundance of proteins linked to the Calvin-Benson cycle, suggesting active corticular photosynthesis that enhances oxygen availability for mitochondrial respiration in the cambial zone. These findings underscore the adaptive metabolic strategies of eucalyptus stems, emphasizing the pivotal role of corticular photosynthesis in supporting energy production, growth, and wood formation.

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ORGANISM(S): Eucalyptus Grandis

TISSUE(S): Xylem, Bark

SUBMITTER: Felipe Silva

LAB HEAD: Carlos Alberto Labate

PROVIDER: PXD067762 | Pride | 2026-02-23

REPOSITORIES: Pride

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Project description:In order to pinpoint the most differentially expressed genes between Eucalyptus grandis leaf blades and vascular (xylem) tissues as well as between E. grandis and Eucalyptus globulus xylem tissues, a total number of nine 50mer-oligoprobes covering the length of each one of 21,432 unique sequences derived from the Genolyptus EST dataset were synthesized “on-chip” in duplicate, randomly distributed in two blocks of each slide. Probes were also synthesized from ten cDNA sequences encoding known human proteins as negative controls, totaling 21,442 sequences. Leaves and xylem samples were taken from two E. grandis clonal trees, i.e., both derived from the same matrix tree and harboring the same genotype. Two additional xylem samples were collected from two other E. grandis clonal trees of a different genotype, as well as from two E. globulus clonal trees. Therefore, ten cDNA samples and ten identical chips were produced at Roche NimbleGen for the microarray assays, with a total number of 385,956 features per slide. Besides the discovery of differentially expressed genes between leaf and xylem, we wanted to test the validity of the assumed “technical” and “biological duplicates” since all trees were field-grown and four years-old in age. A ten chip study using total RNA recovered from mature leaf and vascular (xylem) tissues of Eucalyptus grandis and xylem from Eucalyptus globulus trees. Two clonal trees of E. grandis (E.grandis_Clone A_Ramet 1 and E.grandis_Clone A_Ramet 2), derived from a single matrix tree and therefore genomically identical, were the source of two samples of leaf RNA and two samples of xylem RNA, individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Two other clonal trees of E. grandis (E.grandis_Clone B_Ramet 1 and E.grandis_Clone B_Ramet 2), derived from a different matrix tree, were the source of two additional samples of xylem RNA individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Likewise, two pairs of clonal trees of E. globulus (E.globulus_Clone A_Ramet 1 and E.globulus_Clone A_Ramet 2/ E.globulus_Clone B_Ramet 1 and E.globulus_Clone B_Ramet 2), derived from two distinct matrix trees, were the source of four additional samples of xylem RNA, individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Each chip measures the expression level of 21,432 genes from Eucalyptus sp. and ten human genes (negative controls) with nine 50-mer probe pairs (PM/MM) per gene in two separate blocks per chip (technical duplicate), totalizing 18 hybridization signal values per gene per chip.

Project description:Comparison of transcriptomes from bark, developing xylem and xylem of P. radiata saplings exposed to 0 or 1mg of Ethephon in lanolin for 1 or 8 weeks We developed an oligonucleotide microarray using sequences (mostly from Pinus taeda) from public sequence databases. These sequences were reconstituted into a non-redundant database by CAP3 assembly and used as templates for automated design of 60-mer oligonucleotide probes through eArray, AgilentM-bM-^@M-^Ys online facility. The microarray slides, manufactured by Agilent, were used to monitor gene expression in an Ethephon-induction experiment. Ethephon was dispersed in lanolin paste and applied in a 3 cm band near the base of the stem of 2-year old Pinus radiata saplings. RNA was extracted from bark, cambial region, also known as M-bM-^@M-^\developing xylemM-bM-^@M-^], and xylem tissues exposed for 1 or 8 weeks to Ethephon. The transcriptomes from these extracts were compared by hybridization onto the All-Pinus microarray slides. Statistically significant differentially expressed genes identified by limma (Linear Models for Microarray Data) were subsequently analysed by singular enrichment analysis through the Database for Annotation, Visualization and Integrated Discovery (DAVID) portal. Results revealed that bark, cambial region and xylem generate mostly mutually exclusive cohorts of genes and Gene Ontology (GO) classes. Ethephon induction led to the upregulation of xylem genes related to the metabolism of phenylpropanoids and flavonoids and to defence responses, specifically, fungal/insect attack and oxidative stress. Independent validation of the microarray data for five genes was obtained by quantitative RT-PCR. The results are also interpreted in reference to gross and microscopic morphological changes. These results confirm the utility of the All-Pinus microarray for transcriptomic research in P. radiata. Series of 2-color, 2 condition experiments in 12 180k arrays. Main comparison is within tissues exposed to 0 [control] or 1 mg Ethephon. 2nd level of comparison is between tissues [bark, xylem scraping, xylem]. Third level of comparison is between time [1 or 8 week exposure]. One slide is hybridized with cRNA generated from control and treated tissues with the same duration of exposure to Ethephon. Two biological replicates [each biological rep is a 2 y old cutting propagated clone] for treated plants whilst control consists of RNA pooled, in equal proportions [estimated by UV absorbance], from 2 untreated biological replicates.]. Dyes used for each sample are indicated in sample description.

			Action	DRS
	12A2_BARK.raw.rar	Raw
	12A_BARK.raw.rar	Raw
	12A_XYLEM.raw.rar	Raw
	12B_BARK.raw.rar	Raw
	12B_XYLEM.raw.rar	Raw

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Comparative Proteomics of Bark and Xylem Provides Insights into Age-Dependent Corticular Photosynthesis in Eucalyptus grandis

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