Identification and expression analysis of starch branching enzymes involved in starch synthesis during the development of chestnut (Castanea mollissima Blume) cotyledons

Abstract

Chinese chestnut (Castanea mollissima Blume) is native to China and distributes widely in arid and semi-arid mountain area with barren soil. As a perennial crop, chestnut is an alternative food source and acts as an important commercial nut tree in China. Starch is the major metabolite in nuts, accounting for 46 ~ 64% of the chestnut dry weight. The accumulation of total starch and amylopectin showed a similar increasing trend during the development of nut. Amylopectin contributed up to 76% of the total starch content at 80 days after pollination (DAP). The increase of total starch mainly results from amylopectin synthesis. Among genes associated with starch biosynthesis, CmSBEs (starch branching enzyme) showed significant increase during nut development. Two starch branching enzyme isoforms, CmSBE I and CmSBE II, were identified from chestnut cotyledon using zymogram analysis. CmSBE I and CmSBE II showed similar patterns of expression during nut development. The accumulations of CmSBE transcripts and proteins in developing cotyledons were characterized. The expressions of two CmSBE genes increased from 64 DAP and reached the highest levels at 77 DAP, and SBE activity reached its peak at 74 DAP. These results suggested that the CmSBE enzymes mainly contributed to amylopectin synthesis and influenced the amylopectin content in the developing cotyledon, which would be beneficial to chestnut germplasm selection and breeding.

Fig 1. Starch content, pasting temperature and granule size in developing chestnut cotyledons.

A. Total starch, amylose and amylopectin contents during chestnut development. B. The pasting temperature of chestnut starch at different time points. C. Average starch granule areas at different developmental stages. D. The proportion of different-size starch granules. E. Scanning electron micrographs of starch granules. The number of 60, 64, 68, 71, 74, 77, 80, and 83 represents days after pollination. Scale bars, 5 μm. Data are means ± SE (n = 3).

Fig 2. The expression patterns of genes associated with starch synthesis in developing cotyledon, and CmSBE expression pattern in different organs.

A. Heat map showing gene expression of AGP, GBSS, SSS, PUL and SBE during chestnut development. B and C. The relative expressions of CmSBE I and CmSBE II genes in chestnut cotyledon at different days after pollination (DAP) measured by qRT-PCR and semi-quantitative PCR, respectively. The expression of chestnut β-Actin was used as a control. The number of 60, 64, 68, 71, 74, 77, 80, and 83 in A, B and C represents days of cotyledon development after pollination. D. The relative expression levels of CmSBE I and CmSBE II in cotyledon (74 DAP), leaf, bud and root. Values represent the means ± SE (n = 3).

Fig 3. Identification and conserved domain analysis of CmSBE I and CmSBE II.

A. The identification of CmSBE I and CmSBE II by native-PAGE. The number of 60, 64, 68, 71, 74, 77, 80, and 83 in A and B represents days of cotyledon development after pollination. B and C. Peptides of CmSBE I and CmSBE II were detected by LC-MS/MS and their conserved domains were predicted by CD-Search tool in the NCBI database. Bold fonts in the sequences indicate peptide fragments detected by LC-MS/MS. Blue regions associated with amino acids 143–239 of CmSBE I (B) and 230–324 of CmSBE II (C) indicate the conserved domain of E_set_GBE_euk_N. Yellow regions associated with amino acids 243–651 CmSBE I (B) and 328–743 of CmSBE II (C) indicate the conserved domain of AmyAc_bac_euk_BE. Green regions associated with amino acids 670–772 of CmSBE I (B) and 761–856 of CmSBE II (C) indicate the conserved domain of Alpha-amylase_C.

Fig 4. The protein expression patterns and enzyme activity of CmSBE.

A. Immunoblot analysis of cotyledons showing the accumulation of CmSBE I, CmSBE II and Actin (control). B. CmSBE activity in chestnut cotyledons in development stages. The number of 60, 64, 68, 71, 74, 77, 80, and 83 represents days of cotyledon development after pollination.