Shotgun label-free proteomic and biochemical study of somatic embryos (cotyledonary and maturation stage) in Catharanthus roseus (L.) G. Don

Abstract

Somatic embryogenesis is an important and wonderful biotechnological tool used to develop whole plant from a single or a group of somatic cells. The differentiated somatic cells become totipotent stem cells by drastic reprogramming of a wide range of cellular activities, leading to the acquisition of embryogenic competence. After acquiring competence, the cells pass through globular, heart, torpedo and cotyledonary stages of embryo; however, all advanced embryos do not convert into full plant, produce adventive embryos or callus instead, thus reverses the programming. This is a big limitation in propagation of many plants. Understanding and unraveling the proteins at this 'embryo to plantlet' transition stage will help to get more numbers of plants. Thus, our study was aimed at an identification of differentially abundant proteins between two important advanced stages, i.e. cotyledonary-(T1) and maturation stage (T2) of somatic embryos in Catharanthus roseus. A total of 2949 and 3030 proteins were identified in cotyledonary and maturation stage, respectively. Of these, 1129 proteins were common to both. Several proteins were found to be differentially accumulated in two different embryo stages in which over 60 proteins were most accumulated during somatic embryo maturation time. More chlorophyll accumulation was noted at this time under the influence of gibberellic acid (GA₃). Proteins like Mg-protoporphyrin IX chelatase, chlorophyll a-b-binding protein, photosystem I iron-sulfur center, photosystem II Psb, photosystem II subunit P-1, P-II domain-containing protein, RuBisCO large chain, RuBisCO small chain, RuBisCO activase, RuBisCO large subunit-binding proteins were synthesized. Some of the identified proteins are linked to chlorophyll synthesis, carbohydrate metabolism and stress. The identified proteins are categorized into different groups on the basis of their cellular location, role and other metabolic processes. Biochemical attributes like protein, sugar, proline, antioxidant enzyme (APX, SOD and CAT) activities were high in T2 as compared to T1. The proteins like peroxidases, pathogenesis-related proteins, the late-embryogenesis abundant proteins, argonaute, germin and others have been discussed in C. roseus somatic embryo maturation process.

Keywords: Cotyledonary stage; Embryogenesis abundant proteins; Gel-free proteomic method; Gibberellic acid; Pathogenesis-related proteins; Shotgun; Somatic embryos; Stress protein.

Fig. 1

Cotyledonary and maturation stages of SE, a cotyledonary embryos in medium containing 2 mg/l BAP and 1.5 mg/l NAA; b, c cotyledonary embryos in magnified view d: mature green embryos in mediun containing 2.0 mg/l GA₃, e, f mature green embryos in magnified view (Bars a 0.5 cm; b, c 1 cm; d 0.5 cm; e, f 1 cm)

Fig. 2

Different categorization of proteins found exclusively in T1 (a–c) and T2 (d–f). Some of the proteins have more than one molecular function, cellular location or may be involved in more than one biological process

Fig. 3

Different categorization of proteins found common but in low abundance in T1 (a–c) and T2 (d–f) with respect to each other. Some of the proteins have more than one molecular function, cellular location or may be involved in more than one biological process and several were unknown, hence the sum may not be equal to total number of proteins

Fig. 4

Biochemical attributes of T1 and T2: a–c protein, carbohydrate and proline content, respectively; d–f catalase, ascorbate peroxidase, and superoxide dismutase, respectively. Carbohydrate = glucose. Values are expressed as means ± standard errors of three replicates of two experiments. Means followed by a different letters, are significantly different at p ≤ 0.05 according Duncan’s multiple range test (DMRT)

Fig. 5

a–c Categorization of most accumulated proteins in T2 (T2/T1 ≥ 2.6). Some of the proteins have more than one molecular function, cellular location or may be involved in more than one biological process

Fig. 6

Heat map of total co-expressed proteins showing differential abundance in T1 and T2

Fig. 7

The schematic representation of some of the most accumulated common proteins (proteins with yellow tags) and the proteins present exclusively (proteins with green tags) in maturation stage (T2). ETC electron transport chain, PI and PII photosystem I and photosystem II, direction of arrowheads points out the process or step where a particular protein has some association