Soybean (Glycine max L. Merr.) seedlings response to shading: leaf structure, photosynthesis and proteomic analysis

Abstract

Background: Intercropping and close planting are important cultivation methods that increase soybean yield in agricultural production. However, plant shading is a major abiotic stress factor that influences soybean growth and development. Although shade affects leaf morphological parameters and decreases leaf photosynthesis capacity, information on the responses of soybean leaf photosynthesis to shading at proteomic level is still lacking.

Results: Compared with leaves under normal light (CK) treatment, leaves under shading treatment exhibited decreased palisade and spongy tissue thicknesses but significantly increased cell gap. Although shade increased the number of the chloroplast, the thickness of the grana lamella and the photosynthetic pigments per unit mass, but the size of the chloroplast and starch grains and the rate of net photosynthesis decreased compared with those of under CK treatment. A total of 248 differentially expressed proteins, among which 138 were upregulated, and 110 were downregulated, in soybean leaves under shading and CK treatments were detected via isobaric tags for relative and absolute quantification labeling in the three biological repeats. Differentially expressed proteins were classified into 3 large and 20 small groups. Most proteins involved in porphyrin and chlorophyll metabolism, photosynthesis-antenna proteins and carbon fixation in photosynthetic organisms were upregulated. By contrast, proteins involved in photosynthesis were downregulated. The gene family members corresponding to differentially expressed proteins, including protochlorophyllide reductase (Glyma06g247100), geranylgeranyl hydrogenase (Ggh), LHCB1 (Lhcb1) and ferredoxin (N/A) involved in the porphyrin and chlorophyll metabolism, photosynthesis-antenna proteins and photosynthesis pathway were verified with real-time qPCR. The results showed that the expression patterns of the genes were consistent with the expression patterns of the corresponding proteins.

Conclusions: This study combined the variation of the soybean leaf structure and differentially expressed proteins of soybean leaves under shading. These results demonstrated that shade condition increased the light capture efficiency of photosystem II (PSII) in soybean leaves but decreased the capacity from PSII transmitted to photosystem II (PSI). This maybe the major reason that the photosynthetic capacity was decreased in shading.

Keywords: Leaf structure; Photosynthesis; Proteomics; Shading; Soybean; iTRAQ.

Fig. 1

Photosynthetic rate (a) and pigment contents (b) of soybean leaves under shading and CK treatments. P_n: photosynthetic rate, G_s: stomatal conductance, C_i: intercellular CO₂ concentration, Chl a: chlorophyll a content, Chl b: chlorophyll b content, Car: carotene content, Chl a/b: the ratio of Chl a to Chl b. Significant differences between shading and CK treatments are indicated by different small letters (P < 0.05), respectively

Fig. 2

Stomatal density of soybean leaves under shading and CK treatments. Stomatal density on the adaxial side of leaves under CK (a) and shading (b) treatments. Stomatal density on the abaxial side of leaves under CK (c) and shading (d) treatments. Significant differences between shading and CK treatments are indicated by different small letters (P < 0.05), respectively

Fig. 3

Anatomical structure of soybean leaves under CK and shading treatments. Significant differences between shading and CK treatments are indicated by different small letters (P < 0.05), respectively

Fig. 4

Chloroplast ultrastructure of soybean leaves under shading and CK treatments. a, c CK treatment and b, d shading treatment. Ch: chloroplast, CW: cell wall, Gr: grana, and Th: thylakoid. Significant differences between shading and CK treatments are indicated by different small letters (P < 0.05), respectively

Fig. 5

GO annotation of differentially abundant proteins in soybean leaves under shading treatment. GO are classified into three domains, namely, (a) biological process, (b) molecular function, and (c) cellular component

Fig. 6

Subcellular locations of proteins in soybean leaves under shading

Fig. 7

Expression analysis of nine soybean leaf genes in CK and shading treatments using Real-time RT-PCR. Glyma06g247100: Protochlorophyllide reductase (por); Ggh: Geranylgeranyl hydrogenase (Ggh); Glyma12g052500: Tetrapyrrole-binding protein (TBP); Lhcb1: light-harvesting complex II chlorophyll a/b binding protein 1 (LHCB1); Lhcb2: light-harvesting complex II chlorophyll a/b binding protein 2 (LHCB2); Lhcb4: light-harvesting complex II chlorophyll a/b binding protein 4 ((LHCB4); Glyma13g302100: Protein THYLAKOID FORMATION1 (PTF); Glyma11g152400: NADPH:quinone oxidoreductase (NQO); N/A: Ferredoxin (Fd)