Label-Free Quantitative Proteomics of Embryogenic and Non-Embryogenic Callus during Sugarcane Somatic Embryogenesis

The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somatic embryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E) and non-embryogenic (NE) callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L^-1) of activated charcoal (AC). Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L^-1 AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days) in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project), including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus.     

Correlation Between Appearance of Embryogenic Cells and the IAA Levels in Rice Somatic Cell Culture

Changes of endogenous IAA level and IAA action in cultured rice ( Oryza sativa L. ) somatic cells during the period from 7th to 15th day which was the transition from somatic to embryogenic cells were observed. The study was carded out in three experimental systems viz. mature caryopsis and young panicles (2 ~ 5 mm long) of rice cv. "Guangluai 4" under normal osmosis (3% sucrose), mature caryopses from rice cv. "Yanjing 2" or "Guangluai 4" under normal and higher osmosis (5% sucrose or 2.5 % sorbitol). During this period, endogenous IAA contents were greatly increased in young-panicle calli under normal osmosis and mature-caryoptic calli under higher osmosis but decreased in mature-caryoptic calli under normal osmosis. Exogenous IAA could induce the appearance of embryogenic cell from nonembryogenic callus at a lower frequency. And 2,3,5-tri-iodobenzoic acid could increase the frequency of embryogenic cell induction. From these results it could be concluded that accumulation of higher IAA level in the cultured rice cells was essential for induction of embryogenic cell appearance. Since 2,4-D was involved in all induction medium with the same concentration but exerted different effects on embryogenic cell induction, it was suggested that it might act through mediating the endogenous IAA metabolism.     

Detection of an Embryogenic Cell Antigen in Carrot

Monoclonal antibodies were raised against proteins in a microsomalfraction from carrot embryogenic cells. The presence of a 31-kDaembryogenic cell antigen detected by one antibody (ID 11) wasdemonstrated in embryogenic cells but not in other plant materials,such as non-embryogenic cells, somatic embryos, crown gallsand hairy roots. The antigen was also present in organ segmentsthat carried somatic embryos having been induced by exposureto various stresses. In non-embryogenic cells, the antibodyrecognized a small amount of a 32-kDa antigen. Both the 31-and the 32-kDa antigens accumulated in carrot seeds during theirdevelopment and then disappeared after germination. (Received April 16, 1990; Accepted July 16, 1990)     

Inositol Trisphosphate Metabolism in Carrot (Daucus carota L.) Cells

The metabolism of exogenously added d-myo-[1-³H]inositol 1,4,5-trisphosphate (IP₃) has been examined in microsomal membrane and soluble fractions of carrot (Daucus carota L.) cells grown in suspension culture. When [³H]IP₃ was added to a microsomal membrane fraction, [³H]IP₂ was the primary metabolite consisting of approximately 83% of the total recovered [³H] by paper electrophoresis. [³H]IP was only 6% of the [³H] recovered, and 10% of the [³H]IP₃ was not further metabolized. In contrast, when [³H]IP₃ was added to the soluble fraction, approximately equal amounts of [³H]IP₂ and [³H]IP were recovered. Ca²⁺ (100 micromolar) tended to enhance IP₃ dephosphorylation but inhibited the IP₂ dephosphorylation in the soluble fraction by about 20%. MoO₄²⁻ (1 millimolar) inhibited the dephosphorylation of IP₃ by the microsomal fraction and the dephosphorylation of IP₂ by the soluble fraction. MoO₄²⁻, however, did not inhibit the dephosphorylation of IP₃ by the soluble fraction. Li⁺ (10 and 50 millimolar) had no effect on IP₃ metabolism in either the soluble or membrane fraction; however, Li⁺ (50 millimolar) inhibited IP₂ dephosphorylation in the soluble fraction about 25%.     

Rapid Effects of IAA on Cell Surface Proteins from Intact Carrot Suspension Culture Cells

Suspension cultures of carrot (Daucus carrota L.) which had an absolute requirement for exogenously supplied auxin were grown in medium containing indoleacetic acid (IAA) as the sole auxin source. Putative cell surface proteins were extracted from the intact cells. Resupply of IAA to cultures partially depleted of auxin resulted in rapidly increased activities of three enzyme activities subsequently extracted. Two of the enzyme activities which increased, peroxidase and pectinesterase, have been implicated in the literature as important to cell wall development, structure, and growth. The other enzyme activity which was increased, IAA oxidase, may be involved in the degradation of IAA In vivo. Polypeptides in the extracts were found to increase equally as rapidly as the enzymes in response to IAA as determined with sodium dodecyl sulfate-polyacrylamide electrophoretic gels stained with silver. It is not known whether the changes in enzyme and polypeptide levels in the protein extracts were due to auxin effects on protein synthesis, transport, or extractability.     

Organic Acid Metabolism in Aluminum-Phosphate Utilizing Cells of Carrot (Daucus carota L.)

Carbohydrate metabolism in Al-phosphate utilizing cells of carrot[designated as IPG, Koyama et al. (1992) Plant Cell Physiol.33: 171], which grow normally in Al-phosphate medium accompaniedby citrate excretion, was investigated. The excretion of citratewas strongly related to the availability of sucrose in medium,indicating that citrate excretion was severely limited by sucrosein medium. The ratio of the amount of carbon in the excretedcitrate to the consumed sucrose, was significantly higher inIPG cells than in wild-type cells. When 50% of the sucrose inthe medium was consumed, the ratio was 0.6% for the IPG cellsand 0.2% the wild-type cells. Under these conditions, IPG cellsshowed altered citrate synthesis metabolism, which resultedin increased citrate production. Specific activity of mitochondrialcitrate synthase was higher in IPG cells than in wild-type cells,whereas the activity of cytosolic NADP-specific isocitrate dehydrogenasewas lower in IPG cells than in wild-type cells. (Received August 27, 1998; Accepted February 21, 1999)     

Metabolism of Polyamines in Transgenic Cells of Carrot Expressing a Mouse Ornithine Decarboxylase cDNA

The metabolisms of arginine (Arg), ornithine (Orn), and putrescine were compared in a nontransgenic and a transgenic cell line of carrot (Daucus carota L.) expressing a mouse Orn decarboxylase cDNA. [¹⁴C]Arg, [¹⁴C]Orn, and [¹⁴C]putrescine were fed to cells and their rates of decarboxylation, uptake, metabolism into polyamines, and incorporation into acid-insoluble material were determined. Transgenic cells showed higher decarboxylation rates for labeled Orn than the nontransgenic cells. This was correlated positively with higher amounts of labeled putrescine production from labeled Orn. With labeled Arg, both the transgenic and the nontransgenic cells exhibited similar rates of decarboxylation and conversion into labeled putrescine. When [¹⁴C]putrescine was fed, higher rates of degradation were observed in transgenic cells as compared with the nontransgenic cells. It is concluded that (a) increased production of putrescine via the Orn decarboxylase pathway has no compensatory effects on the Arg decarboxylase pathway, and (b) higher rates of putrescine production in the transgenic cells are accompanied by higher rates of putrescine conversion into spermidine and spermine as well as the catabolism of putrescine.     

Polyamine Metabolism in Embryogenic Cells of Daucus carota: II. Changes in Arginine Decarboxylase Activity

Embryogenic cultured cells of Daucus carota have been shown to synthesize putrescine from exogenously supplied [¹⁴C]arginine at twice the rate of control nonembryogenic cells. In the present paper, the activity of arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19), an important enzyme in the synthesis of putrescine, was assayed and also found to be elevated by as much as 2-fold in embryogenic cells. This difference between embryogenic and nonembryogenic cells was observed as early as 6 hours after the induction of embryogenesis and appeared not to result from the presence of a diffusible inhibitor or activator. It seemed to be dependent upon concomitant RNA and protein synthesis, as judged using 6-methyl-purine and cycloheximide. After cycloheximide addition to the culture medium, arginine decarboxylase activity declined with a half-time of about 30 minutes in both embryogenic and nonembryogenic cells. It is suggested that elevated arginine decarboxylase activity is involved in the mechanism leading to elevated putrescine levels in these cells and hence may play a role in the embryogenic process.     

Somatic Embryogenesis in Cultured Carrot Cells

Somatic embryogenesis in cultured plant cells is an ideal system for investigating the whole process of differentiation and development from single cells to whole plants, and especially the molecular mechanism of expression of totipotency. This review reports recent progress the studies on somatic embryogenesis.     

Factors Affecting Phytoalexin Production in Cultured Carrot Cells

The production of 6-methoxymellein, a phytoalexin, in culturedcarrot cells under various growth conditions was studied usingtwo induction methods; by adding partial hydrolysates obtainedby treating the cells with pectinase or trypsin, or by directlyadding these enzymes to growing cells to cause the release ofcellular components as endogenous elicitor. 6-Methoxymellein production depended greatly on the cell cultureage. Maximal production was found in cells at the early stationaryphase, while actively dividing cells had only negligible amounts.Release of elicitor from the cells by pectinase or trypsin wasalso influenced by the culture stage. Effective elicitor wasobtained only from cells in the late logarithmic and early stationaryphases. 6-Methoxymellein production required the presence of 2,4-D.IAA could not substitute for 2,4-D, though partial hydrolysatesprepared from these cells grown with IAA or without auxin showedsignificant elicitor activity. On the other hand, the productionwas inhibited by actinomycin D or cycloheximide, suggestingthat de novo syntheses of RNA and protein are required for thephytoalexin production. (Received November 17, 1984; Accepted March 2, 1985)     

植物中的吲哚族芥子油苷与生长素代谢途径的关系

文章介绍了植物中的吲哚族芥子油苷代谢与生长素合成途径相互关系的研究进展。     

Carotenoid and Steroid Syntheses by Carrot Cells in Suspension Culture

Biosynthesis of carotenoids and steroids in tissue cultures of carrot cells I Daucus carota L.) was studied by measuring the change in their contents and incorporation of ^14lC-acelate. The rate of synthesis of these plant products varied significantly during the growth cycle in batch culture. Carotenoids were most actively synthesized in the early logarithmic phase and the synthetic rate sharply declined as the culture aged, whereas the rate of accumulation of phytosterols was highest at the end of the growth phase. Newly synthesized carotenes were shown to undergo turnover during the growth. The synthesis of both carotecnoid and steroid was stimulated by 2.- 4-dichlorophenoxyacetic acid.     

Relationship Between Metabolism and the Lateral Transport of IAA in Corn Coleoptiles

The lateral movement of IAA in coleoptiles of Zea mays has been investigated under aerobic and anaerobic conditions. The IAA-1-¹⁴C was supplied asymmetrically to the apical end of the segment. The results were as follows: A) In air more ¹⁴C was found in the lower half of horizontal segments supplied with an upper donor than in the half opposite the donor in vertical segments. The enhanced lateral movement of ¹⁴C in geotropically stimulated segments of corn coleoptiles under aerobic conditions has thus been confirmed. B) This increased lateral movement of ¹⁴C in geotropically stimulated segments is greatly reduced, but is not completely abolished, under anaerobic conditions. C) The lateral movement of ¹⁴C in vertical segments is significantly less under anaerobic conditions than in air. D) Under anaerobic conditions, the lateral movement of ¹⁴C in horizontal segments can be reduced to the level found in vertical segments by pre-soaking the tissue in a 1 mm solution of the metabolic inhibitor sodium fluoride for 2 hours. The inhibitor has no effect on lateral movement of ¹⁴C in vertical anaerobic segments. E) In air, sodium fluoride has no effect on the lateral movement of ¹⁴C in either vertical or horizontal segments.     

Isolation and Characterization of a cDNA Encoded an Embryogenic Cell Protein 63 Related to Embryogenesis from Carrot

cDNA library with about 6.0 x 108 plaques per μg phage from carrot ( Daucus carota L. cv. Hammaki) torpedo-shaped embryos was constructed by a eDNA cloning system (λgtl0). A fulllength cDNA for embryogenic cell protein 63 (ECP63), an embryogenic cell protein from carrot with a relative molecular weight of 63 000, was isolated from a eDNA library using PCR-amplified DNA as a probe. The nucleotide sequence of ECP63 cDNA was 1 989 bp in length. The cDNA encoded a polypeptide of 569 amino acids, and the calculated molecular weight of this pelypeptide was 62 000. The Northern blot analysis using labeled full-length ECP63 cDNA as a probe showed that the gene for ECP63 was expressed in embryogenic cells, globular, heart-, and torpedo-shaped embryos, but not in seedlings and non-embryogenic cells.     

Ethylene-Mediated Phospholipid Catabolic Pathway in Glucose-Starved Carrot Suspension Cells

Glucose (Glc) starvation of suspension-cultured carrot (Daucus carota L.) cells resulted in sequential activation of phospholipid catabolic enzymes. Among the assayed enzymes involved in the degradation, phospholipase D (PLD) and lipolytic acyl hydrolase were activated at the early part of starvation, and these activities were followed by β-oxidation and the glyoxylate cycle enzymes in order. The activity of PLD and lipolytic acyl hydrolase was further confirmed by in vivo-labeling experiments. It was demonstrated that Glc added to a medium containing starving cells inhibited the phospholipid catabolic activities, indicating that phospholipid catabolism is negatively regulated by Glc. There was a burst of ethylene production 6 h after starvation. Ethylene added exogeneously to a Glc-sufficient medium activated PLD, indicating that ethylene acts as an element in the signal transduction pathway leading from Glc depletion to PLD activation. Activation of lipid peroxidation, suggestive of cell death, occurred immediately after the decrease of the phospholipid degradation, suggesting that the observed phospholipid catabolic pathway is part of the metabolic strategies by which cells effectively survive under Glc starvation.     

Intranuclear and Cytoplasmic Inclusions in Embryogenic and Embryoidal Cells of Panax quinque folius

The presence of intranuclear and cytoplasmic inclusions in cells of embryogenic callus and developing embryoids from tissue cultures of Panax quinquefolius L. was described. These cellular structures were not found in non-embryogenic cells. The size of intranuclear and cytoplasmic inclusions seemed to be related to the developmental status of the cell.     

狗牙根颖果胚性愈伤组织的诱导和胚性细胞的超微结构及植株再生

以普通狗牙根[Cynodon dacylon(L.)Pers.cv.'Suncitv']颖果为外植体,以MS为基本培养基,外加浓度在2.0～6.0mg/L的2,4-D,能高频率地诱导出高质量的胚性愈伤组织,其中以4.0 mg/L为最佳.胚性愈伤组织最佳继代及分化的培养方法为:用MS 2,4-D 4.0mg/L继代1～2次,然后转入1/2 MS 2,4-D 2.0 mg/L中继代1～2次,再在无激素的1/2MS中光照培养10 d,最后在MS 6-BA 3.0 mg/L中诱导分化,分化成苗率达31.7%.经电镜观察发现,胚性愈伤组织结构紧密,细胞较小,内容物丰富,而非胚性愈伤组织结构疏松,细胞巨大,内含一大液泡,几无细胞器.