Differential expression of ribosome-inactivating protein genes during somatic embryogenesis in spinach (Spinacia oleracea).
Root segments from spinach (Spinacia oleracea L. cv. Jiromaru) seedlings form embryogenic callus (EC) that responded to exogenous GA(3) by accumulating a 31-kDa glycoprotein [BP31 or S. oleracea ribosome-inactivating protein (EC 3.2.2.22) (SoRIP1)] in association with the expression of embryogenic potential. Microsequencing of this protein revealed significant similarity with type 1 RIPs. We identified cDNAs for SoRIP1 and S. oleracea RIP2 (SoRIP2), a novel RIP having a consensus shiga/ricin toxic domain and performed a comparative analysis of the expression of SoRIPs during somatic embryogenesis. Western blotting and quantitative polymerase chain reaction analyses revealed that the expression of SoRIP1 in calli increased remarkably in association with the acquisition of embryogenic potential, although the expression in somatic embryos decreased moderately with their development. However, the expression of SoRIP2 in calli remained low and constant but increased markedly with the development of somatic embryos. Treatment of callus with GA(3) and/or ABA for 24 h, or with ABA for a longer period, failed to stimulate the expression of either gene. Immunohistochemistry showed that SoRIP1 preferentially accumulated in the proembryos and peripheral meristem of somatic embryos early in development. Appreciable expression of SoRIP2 was not detected in the callus, but intense expression was found in the epidermis of somatic embryos. These results suggest that the expression of spinach RIP genes is differentially regulated in a development-dependent fashion during somatic embryogenesis in spinach.
Kawade
Kensuke
K
Laboratory of Plant Functional Biology, Department of Agrobiology, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Ishizaki
Takuma
T
Masuda
Kiyoshi
K
D000040:Abscisic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D015153:Blotting, Western; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005875:Gibberellins / Q000494:pharmacology; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010937:Plant Growth Regulators / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054788:Ribosome Inactivating Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000196:embryology / Q000235:genetics*
D018076:DNA, Complementary; D005875:Gibberellins; D010937:Plant Growth Regulators; D010940:Plant Proteins; D000040:Abscisic Acid; C007842:gibberellic acid; D054788:Ribosome Inactivating Proteins