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

Abstract

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.

Keywords: Abortive ovules; Castanea mollissima Blume; Fertile ovules; iTRAQ.

Figure 1. Morphologies of Chinese chestnut burrs, ovaries and ovules.

(A) The morphological observation of Chinese chestnut shell of 20 DAA, the chestnut spines had hardened during this period; (B) ovary of 20 DAA; (C, D) ovules of 20 DAA, the fertile ovules volume greater than the abortive ovules; the arrow points to fertile ovule, DAA, Days after anthesis, Bar = 500 μm in B–D.

Figure 2. The microstructure observation of fertile and abortive ovules at 20 days after anthesis.

(A, B) the cross section of ovary, the ovary consisted of nine locules, and each locule had two inverted ovules; (C, D) The longitudinal section of fertile ovule, the black arrow points to embryo, and embryo developed into a sphere; (E, F) the longitudinal section of abortive ovule, the white arrow points to nucellar tissue; Bar = 100 μm.

Figure 3. Fluorescent labeling of the nuclei of fertile and abortive ovules.

(A, D) Fluorescent labeling of cell nucleus of the outer integument of fertile ovules, the cells showed large nucleus with obvious nucleoli; (B, E) fluorescent labeling of the inner integument of fertile ovules, the nucleolus disappeared; (C, F) fluorescent labeling of the outer integument of abortive ovules, the nucleus shows irregular appearance, “oi” represents outer integument and “ii” represents inner integument; Bar = 20 μm.

Figure 4. The ultrastructure observation of fertile and abortive ovules.

(A–C) The ultrastructural observation of the outer integument of fertile ovules, cells had regular shapes and complete organelles; (D–F) the ultrastructural observation of the inner integument of fertile ovules, cell organelles and nucleus were degraded; (G–I) the ultrastructure observation of abortive ovules integument, a series of changes occurred in cells, such as the grave aggregation of cytoplasm and disintegration of cellular organelle; cn, cell nucleus; ne, nucleolus; mc, mitochondria; Ga, Golgi apparatus; sg, starch grain; ii, inner integument; oi, outer integument; The inner integument and the outer integument were distinguished by black line.

Figure 5. The clustering analysis of the 33 DEPs and detection of GAPDH expression in fertile ovules and abortive ovules.

(A) These DEPs played out in protein synthesis, amino acid metabolism, energy synthesis and so on. “A” represents abortive ovules and “F” represents fertile ovules. (B) The protein gel blot probed with GAPDH antiserum detected a 37 kD band corresponding to the predicted size of GAPDH in the fertile ovules and abortive ovules. “A” represents abortive ovules and “F” represents fertile ovules.