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. 2022 Mar 9:2022:5455593.
doi: 10.1155/2022/5455593. eCollection 2022.

Characterization of Various Subunit Combinations of ADP-Glucose Pyrophosphorylase in Duckweed (Landoltia punctata)

Mingxiu Wang  1   2 , Ya Dai  2 , Xinyu Li  2   3 , Xinrong Ma  2 , Caixia Li  2 , Xiang Tao  1
Affiliations

Characterization of Various Subunit Combinations of ADP-Glucose Pyrophosphorylase in Duckweed (Landoltia punctata)

Mingxiu Wang et al. Biomed Res Int. .

Abstract

Background: Landoltia punctata can be used as renewable and sustainable biofuel feedstock because it can quickly accumulate high starch levels. ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step during starch biosynthesis in higher plants. The heterotetrameric structure of plant AGPases comprises pairs of large subunits (LSs) and small subunits (SSs). Although several studies have reported on the high starch accumulation capacity of duckweed, no study has explored the underlying molecular accumulation mechanisms and their linkage with AGPase. Therefore, this study focused on characterizing the roles of different L. punctate AGPases. Methodology. Expression patterns of LpAGPs were determined through comparative transcriptome analyses, followed by coexpressing their coding sequences in Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana tabacum.

Results: Comparative transcriptome analyses showed that there are five AGPase subunits encoding cDNAs in L. punctata (LpAGPS1, LpAGPS2, LpAGPL1, LpAGPL2, and LpAGPL3). Nutrient starvation (distilled water treatment) significantly upregulated the expression of LpAGPS1, LpAGPL2, and LpAGPL3. Coexpression of LpAGPSs and LpAGPLs in Escherichia coli generated six heterotetramers, but only four (LpAGPS1/LpAGPL3, LpAGPS2/LpAGPL1, LpAGPS2/LpAGPL2, and LpAGPS2/LpAGPL3) exhibited AGPase activities and displayed a brownish coloration upon exposure to iodine staining. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays validated the interactions between LpAGPS1/LpAGPL2, LpAGPS1/LpAGPL3, LpAGPS2/LpAGPL1, LpAGPS2/LpAGPL2, and LpAGPS2/LpAGPL3. All the five LpAGPs were fusion-expressed with hGFP in Arabidopsis protoplasts, and their green fluorescence signals were uniformly localized in the chloroplast, indicating that they are plastid proteins.

Conclusions: This study uncovered the cDNA sequences, structures, subunit interactions, expression patterns, and subcellular localization of AGPase. Collectively, these findings provide new insights into the molecular mechanism of fast starch accumulation in L. punctata.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Expression patterns of LpAGPs during starch accumulation under nutrient starvation. Landoltia punctata 0202 was cultivated in standard Hoagland nutrient solution for 14 days, and then 0.5 g fronds were transferred into 50 mL distilled water in 250 mL culture flask for further cultivation. Frond samples were collected at 0 h (NS_0), 2 h (NS_2), and 24 h (NS_24) for RNA-Seq. NS: nutrient starvation; comp27906_c1_seq1: LpAGPS1; comp27906_c1_seq2: LpAGPS1; Comp19992_c0_seq1: LpAGPS2; comp43482_c0_seq3: LpAGPL1; comp43482_c0_seq23: LpAGPL1; comp43482_c0_seq26: LpAGPL1; comp43464_c0_seq1: LpAGPL2; comp43482_c0_seq2: LpAGPL3.
Figure 2
Figure 2
Phylogenetic analyses of AGPase large and small subunits between L. punctata and other plants. Protein sequences were retrieved from NCBI, and the plastid transit peptides were cut off. Then, protein sequence alignment and calculation of phylogenetic distance were performed by ClustalW. The phylogenetic tree was generated by MEGA 6.0.
Figure 3
Figure 3
Yeast two-hybrid analyses of L. punctata AGPase subunit interactions. The yeast two-gybrid analyses were carried out on a SD/-Leu-Trp-His-Ade medium and confirmed on a SD/-Leu-Trp-His -Ade + X-α-gal medium. CK1: positive control, pGADT7-T/pGBKT7-53; CK2: negative control, pGADT7, and pGBKT7-LpAGPS1; S1: LpAGPS1; S2: LpAGPS2; L1: LpAGPL1; L2: LpAGPL2; L3: LpAGPL3.
Figure 4
Figure 4
The results of bimolecular fluorescence complementation (BIFC) assay. Scale bar, 20 μm. (a) Positive control (pSPYNE-ABI2 and pSPYCE-RCAR1). (b) Negative control (pSPYNE-LpAGPS2). (c) LpAGPS1-LpAGPL1. (d) LpAGPS1-LpAGPL2. (e) LpAGPS1 -LpAGPL3. (f) LpAGPS2-LpAGPL1. (g) LpAGPS2-LpAGPL2. (h) LpAGPS2-LpAGPL3.
Figure 5
Figure 5
Enzyme activity analyses between large and small subunit combinations of LpAGPs. CK: control check, pRSF, and pACYC; S1: pACYC-LpAGPS1, S2: pACYC-LpAGPS2, L1: pRSF-LpAGPL1, L2: pRSF-LpAGPL2, L3: pRSF-LpAGPL3.
Figure 6
Figure 6
Subcellular localization of LpAGPL1. (a) bright, (b) DAPI, (c) acridine orange (AO), (d) GFP, (e) GFP + DAPI merge, and (f) GFP + AO merge. Bar = 10 μm.
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