X Tutup
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 May 26;9(21):eadg7448.
doi: 10.1126/sciadv.adg7448. Epub 2023 May 26.

LIKE EARLY STARVATION 1 and EARLY STARVATION 1 promote and stabilize amylopectin phase transition in starch biosynthesis

Chun Liu  1 , Barbara Pfister  1 , Rayan Osman  2 , Maximilian Ritter  3 , Arvid Heutinck  1 , Mayank Sharma  1 , Simona Eicke  1 , Michaela Fischer-Stettler  1 , David Seung  1 , Coralie Bompard  2 , Melanie R Abt  1 , Samuel C Zeeman  1
Affiliations

LIKE EARLY STARVATION 1 and EARLY STARVATION 1 promote and stabilize amylopectin phase transition in starch biosynthesis

Chun Liu et al. Sci Adv. .

Abstract

Starch, the most abundant carbohydrate reserve in plants, primarily consists of the branched glucan amylopectin, which forms semi-crystalline granules. Phase transition from a soluble to an insoluble form depends on amylopectin architecture, requiring a compatible distribution of glucan chain lengths and a branch-point distribution. Here, we show that two starch-bound proteins, LIKE EARLY STARVATION 1 (LESV) and EARLY STARVATION 1 (ESV1), which have unusual carbohydrate-binding surfaces, promote the phase transition of amylopectin-like glucans, both in a heterologous yeast system expressing the starch biosynthetic machinery and in Arabidopsis plants. We propose a model wherein LESV serves as a nucleating role, with its carbohydrate-binding surfaces helping align glucan double helices to promote their phase transition into semi-crystalline lamellae, which are then stabilized by ESV1. Because both proteins are widely conserved, we suggest that protein-facilitated glucan crystallization may be a general and previously unrecognized feature of starch biosynthesis.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.. Structures and conservation of ESV1 and LESV proteins.
(A) Conservation of homologous ESV1 and LESV protein sequences. Gap frequencies are based on ≥25 orthologous ESV1 and LESV sequences from (14) (for alignments, see data S1, A and B). Indicated in colored boxes above the Arabidopsis sequences are the chloroplast transit peptide (cTP), Trp-rich region, and Pro-rich region (ESV1 only) of the respective proteins. Colored boxes below represent the AlphaFold secondary structure predictions [see (B)]. Note that the alignment position does not correspond to the Arabidopsis protein residue numbering because of gaps. (B) AlphaFold predictions of ESV1 and LESV. Cartoon representations of the full-length proteins; the side chains of aromatic (Trp, Tyr, and Phe) and acidic residues (Asp and Glu) are shown in pink and yellow, respectively, and the protein backbone is colored according to confidence of the model (pLDDT value; see color key). (C) Isolated β sheet plane regions of ESV1 and LESV, reoriented to reveal the view along the length of the planes.
Fig. 2.
Fig. 2.. Expression of ESV1 and LESV in different yeast genetic backgrounds.
(A) Immunoblots of total protein extracts from yeast strains expressing ESV1 (45 kDa), LESV (72 kDa), or both proteins in the 28 and 29 genetic backgrounds (expression sets indicated above the strain number) (13). ESV1 and LESV were detected using protein-specific antibodies in each case (white arrowheads). (B) Quantification of insoluble and soluble glucans of the yeast strains shown in (A), grown in liquid culture for 5.75 hours under inducing conditions. Values are means ± SE (n = 4); independent replicate cultures arose from different precultures. WW, wet weight. Statistical comparisons were performed using two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test; see data S2 (A and B). Note that only comparisons for insoluble glucans to the respective parental strain are indicated in the graph. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. ns, not significant. (C) Light micrographs (LMs) of iodine-stained yeast cells grown as in (B). For strain specifications, refer to (A) and (B). Scale bar, 10 μm.
Fig. 3.
Fig. 3.. Appearance of soluble and insoluble glucans produced by yeast strains expressing ESV1 and LESV.
(A) TEMs of the indicated yeast strains (for strain specifications, refer to Fig. 2). White arrowheads indicate presumably insoluble glucan structures. (B) SEMs of insoluble particles purified from the indicated yeast strains. Scale bars, 2 μm (A) and (B).
Fig. 4.
Fig. 4.. The lesv single mutant has a conditional mutant phenotype.
(A) Glucan quantification of plants grown in 12-hour light/12-hour dark cycles, harvested as indicated. FW, fresh weight. Values are means ± SE (n = 6). Statistical comparisons were performed using two-way ANOVA with Dunnett’s multiple comparisons test; see data S3 (A and B). Only comparisons to the wild type for soluble glucans are shown. WT, wild type. (B) TEMs of selected lesv chloroplast sections, obtained from plants grown and sampled as in (A). Most lesv plastids appear wild-type like (i), but a few contain unusual glucans (ii). Scale bar, 2 μm. Pie charts show quantitative data of plastid section classifications; black, sections containing regular starch granules; red, sections containing starch and apparent phytoglycogen; blue, sections containing phytoglycogen-like inclusions only. (C) Glucan quantification of plants grown as in (A), subjected to a single prolonged night (16 hours), and harvested as indicated. Values are means ± SE (n = 4). Statistical comparisons were performed as in (A); see data S4 (A and B). Only comparisons to the wild type for soluble glucans are shown. ***P ≤ 0.001. (D) TEMs of lesv plants grown and harvested as in (C). Most chloroplast sections contain either a mixture of starch and phytoglycogen (i) or phytoglycogen-like inclusions only (ii). Scale bar, 2 μm. Pie charts indicate respective quantifications, as in (B). (E and F) Quantification of maltose in the plants in (A) (E) and (C) (F). Note that maltose is not included in the soluble glucans as measured in (A) and (C). Values are means ± SE (n = 4). Statistical comparisons were performed using one-way ANOVA with Dunnett’s multiple comparisons test; see data S5 (A and B).
Fig. 5.
Fig. 5.. Glucan accumulation and turnover in plants overexpressing ESV1 and LESV in the isa1isa2 background.
(A) Endogenous ESV1 and LESV and overexpressed ESV1-YFP and LESV-YFP, as assessed by immunoblotting of total leaf protein extracts. Actin (in red) served as a loading control (omitted in the anti-ESV1 immunoblot for clarity). (B) Arabidopsis rosettes harvested at the EOD and 2 h-EON stained with Lugol’s solution. Scale bar, 1 cm. (C) Glucan quantification of plants harvested at the EOD and 2h-EON. Values are means ± SE (n = 4 biological replicates). Statistical comparisons were performed using two-way ANOVAs with Dunnett’s multiple comparisons test. Comparisons of total summed glucans are indicated in blue, those of soluble glucans (phytoglycogen) in gray, and those of insoluble glucans (starch) in black. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. For clarity, only selected comparisons are shown. See data S6 (A and B).
Fig. 6.
Fig. 6.. Appearance of glucans forming in mesophyll chloroplasts of plants overexpressing ESV1 or LESV in the isa1isa2 background.
Leaf tissue was harvested at the EOD, and the chloroplasts and starch granules therein visualized using TEM. White and red arrowheads indicate starch granules and phytoglycogen, respectively. Scale bar, 2 μm.
Fig. 7.
Fig. 7.. Glucan content of isa1isa2, esv1, lesv, and higher-order mutants thereof.
(A) Entire rosettes were harvested at the EOD and 2h-EON and stained with Lugol’s solution. Scale bar, 1 cm. (B) Glucan quantification in plants, harvested at the EOD and 2h-EON. Values are means ± SE (n = 4 biological replicates). Statistical comparisons were performed using two-way ANOVA with Dunnett’s multiple comparisons test. Comparisons of total summed glucan contents are indicated in blue, soluble glucans (phytoglycogen) in gray, and insoluble glucans (starch) in black. For clarity, only selected comparisons are shown. *P ≤ 0.05 and ***P ≤ 0.001. See data S7 (A and B).
Fig. 8.
Fig. 8.. Glucans in mesophyll and epidermal cell plastids of isa1isa2, esv1, lesv, and higher-order mutants.
Leaf tissue was harvested at the EOD, and plastids and starch granules observed using TEM. Arrowheads indicate starch granules (white), phytoglycogen (red), and plastid sections lacking glucans entirely (yellow). Scale bars, 2 μm.
Fig. 9.
Fig. 9.. SAXS of purified yeast and plant glucans.
(A) Stacked SAXS plots obtained from glucans purified from yeast strains expressing ESV1 and LESV. The local intensity maxima of individual samples were manually selected and are highlighted by colored dots. Vertical lines indicate the respective maxima’s q values. The respective calculated repeat distances (d) are indicated below the strains’ genetic descriptions. As expected, no maximum could be detected, and thus, no d was calculated, for yeast strain 28, due to the absence of insoluble glucans. NA, not applicable. (B) Stacked SAXS plots obtained from insoluble glucans purified from different plant genotypes. Displayed data are as in (A). In both (A) and (B), only one replicate measurement is shown; refer to table S2 for a summary of replicate analyses. a.u., arbitrary unit.
Fig. 10.
Fig. 10.. Model of the proposed ESV1 and LESV functions.
(A) LESV interacts with glucans that have adopted a helical secondary structure via its Trp-rich domain and possibly aided by its N-terminal domain (not shown) and thereby facilitates their arrangement into compact, ordered tertiary structures. Once seeded, regular glucan arrangements can self-propagate and spread. (B) Regions of ordered glucan helices, seeded by LESV, form adjacent blocks of crystalline lamellae. ESV1 binds to and stabilizes exposed helices at the margins of these regions, restricting access to hydrolytic activities. In both (A) and (B), ESV1 and LESV proteins are represented as simplified versions of their predicted AlphaFold structures (only Trp-rich region is shown; aromatic residues are highlighted by color; pLDDT values are disregarded). See Fig. 1B as comparison.
See this image and copyright information in PMC

References

    1. D. Santelia, S. C. Zeeman, Progress in Arabidopsis starch research and potential biotechnological applications. Curr. Opin. Biotechnol. 22, 271–280 (2011). - PubMed
    1. M. Stitt, S. C. Zeeman, Starch turnover: Pathways, regulation and role in growth. Curr. Opin. Plant Biol. 15, 282–292 (2012). - PubMed
    1. S. Pérez, E. Bertoft, The molecular structures of starch components and their contribution to the architecture of starch granules: A comprehensive review. Starch 62, 389–420 (2010).
    1. P. J. Jenkins, R. E. Cameron, A. M. Donald, A universal feature in the structure of starch granules from different botanical sources. Starch 45, 417–420 (1993).
    1. T. Sawada, Y. Nakamura, T. Ohdan, A. Saitoh, P. B. Francisco Jr., E. Suzuki, N. Fujita, T. Shimonaga, S. Fujiwara, M. Tsuzuki, Diversity of reaction characteristics of glucan branching enzymes and the fine structure of α-glucan from various sources. Arch. Biochem. Biophys. 562, 9–21 (2014). - PubMed
X Tutup