Morphological observation and protein expression of fertile and abortive ovules in Castanea mollissima

Chinese chestnuts (Castanea mollissima Blume.) contain 12–18 ovules in one ovary, but only one ovule develops into a seed, indicating a high ovule abortion rate. In this study, the Chinese chestnut ‘Huaihuang’ was used to explore the possible mechanisms of ovule abortion with respect to morphology and proteomics. The morphology and microstructure of abortive ovules were found to be considerably different from those of fertile ovules at 20 days after anthesis (20 DAA). The fertile ovules had completely formed tissues, such as the embryo sac, embryo and endosperm. By contrast, in the abortive ovules, there were no embryo sacs, and wide spaces between the integuments were observed, with few nucelli. Fluorescence labelling of the nuclei and transmission electron microscopy (TEM) observations showed that cells of abortive ovules were abnormally shaped and had thickened cell walls, folded cell membranes, condensed cytoplasm, ruptured nuclear membranes, degraded nucleoli and reduced mitochondria. The iTRAQ (isobaric tag for relative and absolute quantitation) results showed that in the abortive ovules, low levels of soluble protein with small molecular weights were found, and most of differently expressed proteins (DEPs) were related to protein synthesis, accumulation of active oxygen free radical, energy synthesis and so on. These DEPs might be associated with abnormal ovules formation.

Micromelanconis kaihuiae gen. et sp. nov., a new diaporthalean fungus from Chinese chestnut branches in southern China

Melanconis-like fungi are distributed in several families of Diaporthales, mainly Juglanconidaceae, Melanconidaceae, Melanconiellaceae and Pseudomelanconidaceae. A new Melanconis-like genus of Pseudomelanconidaceae was discovered on branches of Chinese chestnut (Castanea mollissima) in southern China, which was confirmed by both morphology and phylogenetic analysis of combined ITS, LSU, tef1a and rpb2 sequences. The new genus Micromelanconis is characterized by two types of conidia from natural substrate and manual media of PDA, respectively. Conidia from Chinese chestnut branches are pale brown, ellipsoid, multiguttulate, aseptate with hyaline sheath. While conidia from PDA plates are pale brown, long dumbbell-shaped, narrow at the middle and wide at both ends, multiguttulate, aseptate, and also with hyaline sheath. All Pseudomelanconidaceae species were only reported on tree branches in China until now. More interesting taxa may be discovered if detailed surveys on tree-inhabiting fungi are carried out in East Asia in the future.

Transcriptome analysis of genes involved in starch biosynthesis in developing Chinese chestnut (Castanea mollissima Blume) seed kernels

Chinese chestnut (Castanea mollissima Blume) seed kernels (CCSK) with high quality and quantity of starch has emerged as a potential raw material for food industry, but the molecular regulatory mechanism of starch accumulation in developing CCSK is still unclear. In this study, we firstly analyzed the fruit development, starch accumulation, and microscopic observation of dynamic accumulation of starch granules of developing CCSK from 10 days after flowering (DAF) to 100 DAF, of which six representative CCSK samples (50–100 DAF) were selected for transcriptome sequencing analysis. Approximately 40 million valid reads were obtained, with an average length of 124.95 bp, which were searched against a reference genome, returning 38,146 unigenes (mean size = 1164.19 bp). Using the DESeq method, 1968, 1573, 1187, 1274, and 1494 differentially expressed unigenes were identified at 60:50, 70:60, 80:70, 90:80 and 100:90 DAF, respectively. The relationship between the unigene transcriptional profiles and starch dynamic patterns in developing CCSK was comparatively analyzed, and the specific unigenes encoding for metabolic enzymes (SUSY2, PGM, PGI, GPT, NTT, AGP3, AGP2, GBSS1, SS1, SBE1, SBE2.1, SBE2.2, ISA1, ISA2, ISA3, and PHO) were characterized to be involved potentially in the biosynthesis of G-1-P, ADPG, and starch. Finally, the temporal transcript profiles of genes encoding key enzymes (susy2, pgi2, gpt1, agp2, agp3, gbss1, ss1, sbe1, sbe2.1, sbe2.2, isa1, isa2, isa3, and pho) were validated by quantitative real-time PCR (qRT-PCR). Our findings could help to reveal the molecular regulatory mechanism of starch accumulation in developing CCSK and may also provide potential candidate genes for increasing starch content in Chinese chestnut or other starchy crops.