Proteomic identification of differentially expressed proteins between male and female plants in Pistacia chinensis

Abstract

Pistacia chinensis is a strict dioecious plant with male and female flowers in individuals. In China, P. chinensis is widely planted for biodiesel oil due to high oil content in seeds. In practice it requires to grow more female plants for biodiesel production. At present, there are still no reliable methods for sex determination during the long juvenile stage of this species. In order to develop protein molecular markers for sex determination in P. chinensis, proteomic approach was used to identify differentially expressed proteins between male and female plants. Vegetative organs (leaf and stem) rather than reproductive organs/tissues were used for protein extraction so as to develop protein markers which can be used in siblings before flowering. Protein was extracted using a phenol-based protocol. By using two-dimensional electrophoresis, a total of 10 protein spots were found to be differentially expressed in leaf and stem between both sexes, of which 7 were successfully identified by mass spectrometry and matched to 6 functional proteins such as NB-ARC domain containing protein, light harvesting chlorophyll a/b binding protein, asorbate peroxidase (APX), eukaryotic translation initiation factor 5A2, temperature-induced lipocalin (TIL) and phosphoglycerate kinase (PGK). The sex-related difference displayed in a tissue-specific way, especially in stem. PGK existed in high abundance in stem phloem in the female, but was almost not detected in the male; APX and two TIL species were highly abundant in the stem of male plants, while their abundance was much lower in female plants. Moreover, these abundance differences were further confirmed in individual plants. Hence, it is assumed that APX, PGK and TIL might be promising candidates to serve as protein molecular markers for sex determination in P. chinensis. Our results form the basis for a further understanding of the biochemical mechanisms of sex determination in P. chinensis.

Figure 1. 2-DE identification of differentially expressed proteins in leaves between male and female plants (10-year-old) in Pistacia chinensis.

A mixed tissue powder from three different male or female individuals was used for protein extraction. A, 2-DE profile of leaf proteins from female plants as reference. B, magnified gel regions containing spots L1 and L2, accompanied by column configuration of relative abundance (generated by PDQUEST). f = female; m = male. OEE2 = oxygen-evolving enhancer protein 2. Arrow indicates the prominent spot Rubisco.

Figure 2. 2-DE identification of differentially expressed proteins in leaves between male and female plants (40-year-old) in Pistacia chinensis.

A mixed tissue powder from three different male or female individuals was used for protein extraction. A, 2-DE profile of leaf proteins from female plants as reference. B, magnified gel regions containing spots L1 and L2, accompanied by column configuration of relative abundance (generated by PDQUEST). f = female; m = male. OEE2 = oxygen-evolving enhancer protein 2. Arrow indicates the prominent spot Rubisco.

Figure 3. 2-DE identification of differentially expressed proteins in leaves between male and female individuals (10-year-old) in Pistacia chinensis.

Two representative 2-DE gels from three biological replicas are shown. f = female; m = male. OEE2 = oxygen-evolving enhancer protein 2. Arrow indicates the prominent spot Rubisco.

Figure 4. 2-DE analysis of differentially expressed proteins in stem phloems between male and female plants (10-year-old) in Pistacia chinensis.

Stem was sampled in winter. A mixed tissue powder from three different male or female individuals was used for protein extraction. A, 2-DE profile of phloem proteins from female plants as reference. B, magnified gel regions containing spots P1, P2, P4 and P5, accompanied by column configuration of relative abundance (generated by PDQUEST). Spot P3 failed to be identified by MS/MS. f = female; m = male.

Figure 5. 2-DE identification of differentially expressed proteins in stem phloem between male and female individuals (10-year-old) in Pistacia chinensis.

Two representative 2-DE gels from three biological replicas are shown. Stem was sampled in winter. f = female; m = male.

Figure 6. 2-DE identification of differentially expressed proteins in stem xylems between male and female plants (10-year-old) in Pistacia chinensis.

Stem was sampled in winter. A mixed tissue powder from three different male or female individuals was used for protein extraction. A, 2-DE profile of xylem proteins from female plants as reference. B, magnified gel regions containing spots X1-X3, accompanied by column configuration of relative abundance (generated by PDQUEST). Spot X2 failed to be identified by MS/MS. f = female; m = male.

Figure 7. 2-DE identification of differentially expressed proteins in stem xylem between male and female individuals (10-year-old) in Pistacia chinensis.

Up panel, two representative CBB-stained gels, xylem was sampled in winter; down panel, magnified gel regions containing spots X1 (APX) out of a total of 8 individuals, of which 1–4 were sampled in winter and 5–8 sampled in autumn. f = female; m = male.