Proteomics Reveals an Increase in the Abundance of Glycolytic and Ethanolic Fermentation Enzymes in Developing Sugarcane Culms During Sucrose Accumulation

Abstract

Sugarcane is an economically important crop contributing to the sugar and ethanol production of the world with 80 and 40%, respectively. Despite its importance as the main crop for sugar production, the mechanisms involved in the regulation of sucrose accumulation in sugarcane culms are still poorly understood. The aim of this work was to compare the quantitative changes of proteins in juvenile and maturing internodes at three stages of plant development. Label-free shotgun proteomics was used for protein profiling and quantification in internodes 5 (I₅) and 9 (I₉) of 4-, 7-, and 10-month-old-plants (4M, 7M, and 10M, respectively). The I₉/I₅ ratio was used to assess the differences in the abundance of common proteins at each stage of internode development. I₉ of 4M plants showed statistically significant increases in the abundance of several enzymes of the glycolytic pathway and proteoforms of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC). The changes in content of the enzymes were followed by major increases of proteins related to O₂ transport like hemoglobin 2, ROS scavenging enzymes, and enzymes involved in the ascorbate/glutatione system. Besides, intermediates from tricarboxylic acid cycle (TCA) were reduced in I₉-4M, indicating that the increase in abundance of several enzymes involved in glycolysis, pentose phosphate cycle, and TCA, might be responsible for higher metabolic flux, reducing its metabolites content. The results observed in I₉-4M indicate that hypoxia might be the main cause of the increased flux of glycolysis and ethanolic fermentation to supply ATP and reducing power for plant growth, mitigating the reduction in mitochondrial respiration due to the low oxygen availability inside the culm. As the plant matured and sucrose accumulated to high levels in the culms, the proteins involved in glycolysis, ethanolic fermentation, and primary carbon metabolism were significantly reduced.

Keywords: Saccharum spp.; ethanolic fermentation; glycolysis; hypoxia; sucrose accumulation.

Figure 1

Principal component analysis considering the fmol of 1,655 proteins identified by the MassPivot Software, including solely the accession numbers with FDR ≤ 1%, for (I₅) and (I₉) analyzed at 4M (I₅-4M, I₉-4M), 7M (I₅-7M, I₉-7M) and after the drought stress at 10M (I₅-10M, I₉-10M).

Figure 2

Venn diagrams relative to the total number of common and unique proteins identified by the PLGS 2.5.1 Software, including all different accession numbers with FDR ≤ 1%, for I₅ and I₉, analyzed at 4M, 7M, and after the drought stress at 10M. The values of p < 0.05 and p > 0.95 were considered as statistically significant for low or high abundance, respectively, for the I₉/I₅ ratio.

Figure 3

Distribution of differential abundance of the proteins, identified with PLGS 2.5.1 Software, for I₅ (left) and I₉ (right), at 4 M, 7M, and 10M. Proteins were classified according to the BLAST₂GO annotation for biological processes. Black and gray bars represent the unique and highly abundant sequences, respectively. The GO enrichment analysis, relative to the entire proteomic data, was performed using the Fisher Exact Test with FDR < 0.05 and p < 0.01 in the BLAST₂GO Software, considering only the most specific GOTerms.

Figure 4

Schematic view of the quantitative changes in enzymes involved in primary carbon metabolism under hypoxia in I₉ relative to I₅ in 4-month-old-plants (data from Table 3). Green triangles indicate enzymes with at least one proteoform with a statistically significant increase in abundance.