Hydrolysis of lipid and protein reserves in loblolly pine seeds in relation to protein electrophoretic patterns following imbibition

Somatic embryogenesis (SE) of Pinus strobus L. has been greatly improved over the last few years with respect to both the initiation frequencies from a number of seed families and production of mature somatic embryos that readily convert to plants. However, there are no data on biochemical characterization of somatic embryos in relation to zygotic embryos of eastern white pine and on the optimal duration of the maturation stage. It is believed that somatic embryos closely resembling zygotic embryos not only morphologically but biochemically would display more vigorous growth. Hence, in this study the accumulation pattern of the most abundant seed storage proteins in zygotic and somatic embryos were characterized by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and identified by amino acid sequencing and tandem mass spectrometry (MS/MS). This showed that somatic embryos accumulated storage proteins in a similar manner to zygotic embryos and that the most abundant were the buffer‐insoluble 11S‐ globulins MW 59.6 kDa, which dissociated under reduced conditions to 38.2–40.0 and 22.5–23.5 kDa range polypeptides, and buffer‐soluble 7S vicilin‐like proteins MW 46.0–49.0 kDa, which did not separate under reduced conditions. Other relatively abundant soluble proteins were in the ranges of 25–27 and 27–29 kDa. The only group of proteins that showed different migration profiles in the presence of β‐mercaptoethanol (ME) were the low molecular mass proteins of 14.6–16.5 kDa. Somatic embryos that matured for 9 weeks on medium with 6% sucrose accumulated more storage proteins than those matured on medium with 3% sucrose and the extension of the maturation period to 12 weeks resulted in significant reduction of the storage proteins on both media. As expected, somatic embryos matured on medium with 6% sucrose had lower water potential (Ψ) than those from medium with 3% sucrose. Nonetheless, the somatic embryos matured under the best of tested conditions (6% sucrose for 9 weeks) had slightly higher water content; 1.35 ± 0.28 g H2O g−1 DM (mean ± sd) than the mature non‐dried zygotic embryos; (1.16 ± 0.09 g H2O g−1 DM), and accumulated less storage proteins, whose amounts were either similar to (7S‐vicilins) or below (11S‐globulins) those found in the immature zygotic embryos collected 2 weeks prior to the usual cone collection. The implications of these results for further research and development of viable artificial seed is discussed.

Section: Resultsmentioning

confidence: 99%

Accumulation pattern and identification of seed storage proteins in zygotic embryos of Pinus strobus and in somatic embryos from different maturation treatments

Klimaszewska

Morency

Jones‐Overton

et al. 2004

“…1991, Misra and Green 1991, Hakman 1993, Stone and Gifford 1997, Chatthai and Misra 1998). Other studies have reported the identification of proteins that are believed to play important functional roles during specific developmental stages of embryogenesis such as late‐embryogenesis‐abundant (LEA) proteins, germination proteins and proteins involved in reserve hydrolysis (Stabel et al. 1990, Gifford et al.…”

Section: Introductionmentioning

confidence: 99%

“…ization during germination and early seedling growth , Hakman 1993, Stone and Gifford 1997, Chatthai and Misra 1998. Other studies have reported the identification of proteins that are believed to play important functional roles during specific developmental stages of embryogenesis such as late-embryogenesis-abundant (LEA) proteins, germination proteins and proteins involved in reserve hydrolysis , Groome et al 1991, Schneider and Gifford 1994. In contrast, limited data have been reported on events, which regulate early development of conifer zygotic embryos, mainly due to the relative inaccessibility of the embryos within the developing seeds.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the developmental stages of cypress zygotic embryos by two‐dimensional electrophoresis and by cytochemistry

Sallandrouze

Faurobert

Maâtaoui

2002

Biochemical changes that characterize megagametophyte and zygotic embryo development in the conifer Cupressus sempervirens L. (Cupressaceae) were studied by complementary methods of cytochemistry and two-dimensional electrophoresis (2-DE). These analyses revealed that early in their development megagametophytes and embryos were characterized by the predominant elaboration of starch in association with a low protein content. As their development proceeded, starch content gradually decreased while protein body synthesis progressively intensified, both in the megagametophyte and the embryo. In parallel, an increase in protein level as well as an accumulation of specific polypeptides could be observed in the two tissues. During maturation, protein bodies accumulated to high levels both in megagametophyte and embryo cells, whereas starch could no longer be detected. Protein levels were high in mature seeds and reached 12% and 8% of the megagametophyte and embryo DW, respectively. Some sets of polypeptides accumulated more specifically at this time in both megagametophyte and embryo. Some of these began to first accumulate in the megagametophyte during embryo development before their concentration rose in the embryo at cotyledonary stage. Others displayed a more specific-embryo accumulation pattern.

“…In lobloily pine and other conifer seeds, stratification, important species produce seeds that are dormant, pos-germination and post-gertninative seedling growth are sess low germinability and/or have diminished seedling characterized by the accumulation and disappearance of vigor. To the forest industry, the recalcitrant nature of distinct de novo synthesized protein sets (Stabel et al conifer seed germination poses a significant problem to 1990, Gifford et al 1991, Groome et al 1991. Schneider the success and efficiency of reforestation, nursery prac-and Gifford 1994).…”

mentioning

confidence: 99%

“…A better understanding of seeds, electrophoretic analysis of proteins synthesized in vivo indicated that unique protein sets were synthesized in a temporal fashion. Gifford et al (1991) categorized these proteins into three groups; the first one is synthesized coostitutively throughout germination and postgerminative growth, the second one is present in the mature seed and is only synthesized for up to 1 day following imbibition at 28°C, and finally, a third one whose initial synthesis coincides with the completion of germination, and increases thereafter. In Norway spruce embryos, Stabel et al (1990) reported similar differential synthesis of protein sets during germination and postgerminative growth.…”

mentioning

confidence: 99%

Changes in mRNA populations during loblolly pine (Pinus taeda) seed stratification, germination and post‐germinative growth

Mullen

King

Gifford

1996

Self Cite

Changes in the patterns of gene expression were examined during loblolly pine (Pinus taeda L.) seed stratification, germination, and post‐germinative growth. In both the megagametophyte and the embryo, DNA contents remained relatively constant at all stages examined. RNA contents, however, increased in both tissues following seed germination, particularly in the embryo where a 7‐fold increase in the RNA content was observed 5 days after germination. Poly(A)+ RNA, extracted from megagametophytes and embryos, was translated in vitro in a rabbit reticulocyte lysate cell‐free system. Analysis of [35S]‐methionine‐labelled translation products by two‐dimensional electrophoresis/fluorography indicated that there were changes in the populations of mRNAs during all developmental stages examined. In both the megagametophyte and the embryo several distinct mRNA populations, including one constitutively present at all stages examined, were identified. One mRNA population, present in the mature seed, decreased during seed stratification. Another population, not present in the mature seed, rose during the period of stratification that coincided with an increase in seed germinability. A third population, which appeared during seed germination, increased steadily during post‐germinative growth. Besides these similarities, specific differences between megagametophyte and embryo were noted. For example, one mRNA population, which was present in the megagametophyte of the mature seed and remained constant during the stratification period, disappeared immediately following seed germination. In the embryo, one set of messages was germination specific. In total, these results show that mRNA populations change in a temporal fashion that is consistent with the patterns of de novo protein synthesis known to occur in loblolly pine during the same developmental periods.