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Using Proteomic Analysis to Investigate Uniconazole-Induced Phytohormone Variation and Starch Accumulation in Duckweed (Landoltia Punctata)

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Using Proteomic Analysis to Investigate Uniconazole-Induced Phytohormone Variation and Starch Accumulation in Duckweed (Landoltia Punctata)

Mengjun Huang et al. BMC Biotechnol.

Abstract

Background: Duckweed (Landoltia punctata) has the potential to remediate wastewater and accumulate enormous amounts of starch for bioethanol production. Using systematical screening, we determined that the highest biomass and starch percentage of duckweed was obtained after uniconazole application. Uniconazole contributes to starch accumulation of duckweed, but the molecular mechanism is still unclear.

Results: To elucidate the mechanisms of high starch accumulation, in the study, the responses of L. punctata to uniconazole were investigated using a quantitative proteomic approach combined with physiological and biochemical analysis. A total of 3327 proteins were identified. Among these identified proteins, a large number of enzymes involved in endogenous hormone synthetic and starch metabolic pathways were affected. Notably, most of the enzymes involved in abscisic acid (ABA) biosynthesis showed up-regulated expression, which was consistent with the content variation. The increased endogenous ABA may up-regulate expression of ADP-glucose pyrophosphorylase to promote starch biosynthesis. Importantly, the expression levels of several key enzymes in the starch biosynthetic pathway were up-regulated, which supported the enzymatic assay results and may explain why there is increased starch accumulation.

Conclusions: These generated data linked uniconazole with changes in expression of enzymes involved in hormone biosynthesis and starch metabolic pathways and elucidated the effect of hormones on starch accumulation. Thus, this study not only provided insights into the molecular mechanisms of uniconazole-induced hormone variation and starch accumulation but also highlighted the potential for duckweed to be feedstock for biofuel as well as for sewage treatment.

Figures

Fig. 1
Fig. 1
Distribution, coverage, and functional category of proteins identified in the study. a Distribution of identified proteins among different molecular weights; (b) Coverage of proteins by the identified peptides; (c) Functional category of identified proteins
Fig. 2
Fig. 2
Effect of uniconazole on endogenous hormone levels of duckweed. The ABA content corresponds to the main Y-axis, the contents of ZR, GA1 + 3 and GA1 + 4 correspond to the minor Y-axis
Fig. 3
Fig. 3
Expression patterns of some enzymes involved in hormones biosynthesis. Red boxes indicate the up-regulated enzymes in response to uniconazole, gray means no significant difference was observed, and white means this enzyme was not found in this study. The numbers in the upper half of the boxes correspond to EC numbers, and the numbers in the lower half correspond to the ratios of expression levels of these enzymes at 2, 5, 72, and 240 h compared with the control levels. 5.5.1.13: ent-copalyl diphosphate synthase; 4.2.3.19: ent-kaurene synthase; 1.14.13.78: ent-kaurene oxidase; 1.14.13.79: ent-kaurenoic acid oxidase; 1.14.11.12: gibberellin-44 dioxygenase; 1.14.11.15: gibberellin 3beta-dioxygenase; 1.14.11.13: gibberellin 2beta-dioxygenase; 1.14.13.90: zeaxanthin epoxidase; 5.3.99.9: neoxanthin synthase; 1.13.11.51: 9-cis-epoxycarotenoid dioxygenase; 1.1.1.288: xanthoxin dehydrogenase; 1.2.3.14: abscisic-aldehyde oxidase; 2.4.1.263: abscisate beta-glucosyltransferase; 3.2.1.21: beta-glucosidase; 2.5.1.27: adenylate dimethylallyltransferase; 2.5.1.75: tRNA dimethylallyltransferase
Fig. 4
Fig. 4
Dry weight, starch content, and activities of AGP, SSS, α- and β-AMY in duckweed. The starch content corresponds to the left of main Y-axis, activities of AGP and SSS correspond to the right of main Y-axis, activities of α- and β-AMY correspond to the left of minor Y-axis, and dry weight corresponds to the right of minor Y-axis
Fig. 5
Fig. 5
Expression patterns of some enzymes involved in carbon metabolism. Red boxes indicate the up-regulated enzymes in response to uniconazole, green for down-regulated, gray means no significant difference was observed, and white means this enzyme was not found in this study. The numbers in the upper half of the boxes correspond to EC numbers, and the numbers in the lower half correspond to the ratios of expression levels of these enzymes at 2, 5, 72, and 240 h compared with the control levels. 2.7.7.27: ADP-glucose pyrophosphorylase; 2.4.1.11: granule bound starch synthase; 2.4.1.21: soluble starch synthase; 2.4.1.1: glucan phosphorylase; 3.6.1.21: ADP-sugar diphosphatase; 2.4.1.18: starch branching enzyme; 3.2.1.1: alpha-amylase; 3.2.1.2: beta-amylase; 2.7.7.9: UDP-glucose pyrophosphorylase; 2.4.1.12: cellulose synthase; 2.4.1.13: sucrose synthase; 2.4.1.14: sucrose phosphate synthase; 3.1.3.24: sucrose-6-phosphate phosphatase; 2.4.1.25: 4-alpha-glucanotransferase; 2.4.1.15: trehalose-6-phosphate synthase; 3.1.3.12: trehalose 6-phosphate phosphatase; 3.2.1.28: trehalase; 3.2.1.26: beta-fructofuranosidase
Fig. 6
Fig. 6
A proposed model in which ABA and other endogenous hormones modulate starch accumulation. Red upward arrow indicates up-regulated protein in response to uniconazole, green downward arrow for down-regulated, gray arrow means no significant difference was observed in this research. The model was constructed to help understanding uniconazole-induced hormone variation and starch accumulation in L. punctata. Uniconazole induces in expression of hormone-associated enzymes that result in changes in endogenous hormone contents. ABA is bound by PYRs and then initiates ABA signaling pathway [49, 50]. The significantly increased ABA and decreased GA modulate the expression of some enzymes involved in starch metabolism, and these enzymes finally result in starch accumulation. There may be an unknown regulation mechanism of starch accumulation, which is indicated by a question mark. See text for detailed description of the model. PYR: pyrabactin resistance; AGP: ADP-glucose pyrophosphorylase; α-AMY: alpha-amylase

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