David J. Gifford

Amino Acid Utilization in Seeds of Loblolly Pine during Germination and Early Seedling Growth: I. Arginine and Arginase Activity

The mobilization and utilization of the major storage proteins in loblolly pine (Pinus taeda L.) seeds following imbibition were investigated. Most of the seed protein reserves were contained within the megagametophyte. Breakdown of these proteins occurred primarily following radicle emergence and correlated with a substantial increase in the free amino acid pool in the seedling; the majority of this increase appeared to be the result of export from the megagametophyte. The megagametophyte was able to break down storage proteins and export free amino acids in the absence of the seedling. Arginine (Arg) was the most abundant amino acid among the principal storage proteins of the megagametophyte and was a major component of the free amino acid pools in both the seedling and the megagametophyte. The increase in free Arg coincided with a marked increase in arginase activity, mainly localized within the cotyledons and epicotyl of the seedling. Arginase activity was negligible in isolated seedlings. Experiments with phenylphosphorodiamidate, a urease inhibitor, supported the hypothesis that arginase participates in Arg metabolism in the seedling. The results of this study indicate that Arg could play an important role in the nutrition of loblolly pine during early seedling growth.

Regulation of loblolly pine (Pinus taeda L.) arginase in developing seedling tissue during germination and post-germinative growth

After seed germination, hydrolysis of storage proteins provides a nitrogen source for the developing seedling. In conifers the majority of these reserves are located in the living haploid megagametophyte tissue. In the developing loblolly pine (Pinus taeda L.) seedling an influx of free amino acids from the megagametophyte accompanies germination and early seedling growth. The major component of this amino acid pool is arginine, which is transported rapidly and efficiently to the seedling without prior conversion. This arginine accounts for nearly half of the total nitrogen entering the cotyledons and is likely a defining factor in early seedling nitrogen metabolism. In the seedling, the enzyme arginase is responsible for liberating nitrogen, in the form of ornithine and urea, from free arginine supplied by the megagametophyte. In this report we investigate how the seedling uses arginase to cope with the large arginine influx. As part of this work we have cloned an arginase cDNA from a loblolly pine expression library. Analysis of enzyme activity data, accumulation of arginase protein and mRNA abundance indicates that increased arginase activity after seed germination is due to de novo synthesis of the enzyme. Our results suggest that arginase is primarily regulated at the RNA level during loblolly pine seed germination and post-germinative growth.

Purification and Characterization of Catalase from Loblolly Pine (Pinus taeda L.) Megagametophytes.

Catalase (EC 1.11.1.6) was purified to near homogeneity from isolated megagametophytes of germinated loblolly pine (Pinus taeda L.) seeds, and monospecific antibodies were elicited in rabbits. Following a procedure that involved acetone extraction, (NH4)2SO4 fractionation, and four chromatographic steps (i.e. DE-52 cellulose, Superdex-200, hydroxylapatite, and phenyl-Sepharose CL-4B), catalase was purified about 140-fold to a final specific activity of 2215 mmol min-1 mg-1 of protein. Cotton isocitrate lyase antibodies were used, and protein immunoblots revealed that the resolution on hydroxylapatite and phenyl-Sepharose allowed for the complete separation of catalase from contaminating isocitrate lyase. The molecular masses of the native enzyme and its subunit are 235 and 59 kD, respectively, indicating that the pine holoenzyme is a homotetramer. Loblolly pine catalase exists as multiple isoforms. When megagametophytes taken 7 d after imbibition at 30[deg]C were extracted, subjected to nondenaturing isoelectric focusing, and stained for catalase activity, at least four catalase isoforms were observed, including one dominant form with an isoelectric point of 6.87. Purified pine catalase is not a glycoprotein and has a ratio of absorbance at 208 nm to absorbance at 405 nm of 1.5. When probed with loblolly pine catalase antibodies, protein blots of cell-free extracts from megagametophytes of mature, stratified, and germinated loblolly pine seeds, the megagametophyte glyoxysomal fraction, and purified loblolly pine catalase all revealed one immunoreactive 59-kD polypeptide. This indicates that no detectable change in the enzymes monomeric molecular mass occurs during seed stratification and germination, early seedling growth, and purification.

Structural and Biochemical Changes in Loblolly Pine (Pinus taeda L.) Seeds during Germination and Early Seedling Growth. II. Storage Triacylglycerols and Carbohydrates

Triacylglycerols (TAGs) comprised 59% of the total storage reserve in mature loblolly pine (Pinus taeda L.) seeds; 80% of these TAGs were stored in the megagametophyte. The TAG breakdown in the seedling was initiated before radicle emergence (during germination), while in the megagametophyte breakdown occurred after radicle emergence (during early seedling growth). In both seed tissues, the majority of TAG breakdown took place during early seedling growth. Within the seedling, the most rapid rate of TAG breakdown occurred in the radicle and hypocotyl. Unlike TAGs, there was very little carbohydrate stored in loblolly pine seeds at maturity. Levels of 80% ethanol‐soluble carbohydrate in the megagametophyte and seedling decreased during germination and then increased during early seedling growth. This increase coincided with the period of rapid TAG depletion. Accompanying the increase in 80% ethanol‐soluble carbohydrate level was a corresponding increase in 80% ethanol‐insoluble carbohydrates, such as starch, in the seedling during early seedling growth. Accumulation of starch occurred in both the cotyledons and hypocotyl. Starch accumulation in the megagametophyte was more transient, occurring around germination. The megagametophyte was important for the growth and nutrition of the seedling. In the presence of the megagametophyte, the seedling accumulated sucrose during early seedling growth. However, in the absence of the megagametophyte, loblolly pine seedlings failed to accumulate carbohydrates to any great extent.

Structural and Biochemical Changes in Loblolly Pine (Pinus taeda L.) Seeds During Germination and Early-Seedling Growth. I. Storage Protein Reserves

Quantitative and qualitative changes in the storage proteins of loblolly pine (Pinus taeda L.) seeds were followed during germination and early-seedling growth and were correlated with light-microscopic observations. In both the megagametophyte and the embryo, the cells of tissues from fully stratified seeds appeared very similar to the cells of tissues from mature desiccated seeds. A change in the appearance of protein vacuoles, resulting from the hydrolysis of storage proteins, occurred in the seedling prior to the completion of germination (denoted by radicle emergence from the seed coat) and continued during early-seedling growth. Within the seedling, storage proteins were mobilized more rapidly in the root pole, including the hypocotyl and radicle, than in the shoot pole, including the cotyledonary whorl, and shoot apex or epicotyl. In both parts of the seedling, protein hydrolysis was first observed in the procambial and epidermal tissue. In contrast to the seedling, changes in the appearance of protein vacuoles were not evident in the megagametophyte until after germination was completed. Changes in protein vacuoles in the megagametophyte occurred in two directional waves, relative to corrosion cavity proximity.

Patterns of storage protein and triacylglycerol accumulation during loblolly pine somatic embryo maturation

Conifer somatic embryo germination and early seedling growth are fundamentally different than in their zygotic counterparts in that the living maternal megagametophyte tissue surrounding the embryo is absent. The megagametophyte contains the majority of the seed storage reserves in loblolly pine and the lack of the megagametophyte tissue poses a significant challenge to somatic embryo germination and growth. We investigated the differences in seed storage reserves between loblolly pine mature zygotic embryos and somatic embryos that were capable of germination and early seedling growth. Somatic embryos utilized in this study contained significantly lower levels of triacylglycerol and higher levels of storage proteins relative to zygotic embryos. A shift in the ratio of soluble to insoluble protein present was also observed. Mature zygotic embryos had roughly a 3:2 ratio of soluble to insoluble protein whereas the somatic embryos contained over 5-fold more soluble protein compared to insoluble protein. This indicates that the somatic embryos are not only producing more protein overall, but that this protein is biased more heavily towards soluble protein, indicating possible differences in metabolic activity at the time of desiccation.

Dehydration, Damage to the Plasma Membrane and Thylakoids, and Heat-shock Proteins in Lines of Maize Differing in Endogenous Levels of Abscisic Acid and Drought Resistance

Summary We investigated dehydration avoidance, and damage to the plasma and thylakoid membranes, in the high-level abscisic acid drought-resistant line of Zea mays L., Polj 17, and the low-level abscisic acid drought-sensitive line, F-2, under water shortage, and water shortage and high-temperature stress conditions. The synthesis of heat-shock proteins in Polj 17 and F-2 under water shortage and high-temperature stress conditions was also examined. Under water shortage conditions both lines were mildly dehydrated and suffered damage to the plasma and thylakoid membranes to a similar extent. Exposure to water shortage and high-temperature stress revealed that the high-ABA line Polj 17 had greater capability of withstanding stress conditions than the low-ABA line F-2. Polj 17 showed greater ability to avoid dehydration than F-2. Lines Polj 17 and F-2 showed similar damage to the plasma and thylakoid membranes. However, Polj-17 showed much greater ability to tolerate damage to the cell membranes than F-2. Polj 17 and F-2 did not differ in the pattern of synthesis of heat-shock proteins. Both lines synthesized bands of heat-shock proteins of 43-45 kDa, which appear to be uncommon in maize.

Regulation of two loblolly pine (Pinus taeda L.) isocitrate lyase genes in megagametophytes of mature and stratified seeds and during postgerminative growth

Two full-length cDNAs encoding the glyoxysomal enzyme isocitrate lyase(ICL) were isolated from a λZAP cDNA library prepared frommegagametophyte mRNAs extracted from seeds imbibed at 30 °C for 8days. The cDNAs, designated Ptbs ICL 8 and Ptbs ICL 12, have openreading frames of 1740 and 1719 bp, with deduced amino acid sequences of580 and 573 residues, respectively. The predicted amino acid sequencesof Ptbs ICL 8 and Ptbs ICL 12 exhibit a 79% identity with each other,and have a greater than 75% identity with ICLs from variousangiosperm species. The C-termini of Ptbs ICL 8 and Ptbs ICL 12terminate with the tripeptide Ser-Arg-Met and Ala-Arg-Met, respectively,both being conserved variants of the type 1 peroxisomal targetingsignal. RNA blot and slot analysis revealed that Ptbs ICL 8 and PtbsICL 12 mRNAs were present at low levels in the megagametophyte of themature and stratified seeds, and that the level of both transcriptsincreased markedly upon seed germination. Protein blot analysisindicated that the steady-state level of ICL was low in the mature andstratified seed, then increased rapidly upon seed germination, peakingat around 8-10 days after imbibition (DAI). Changes in the level ofICL activity in cell-free extracts was similar to the steady-stateprotein content with the exception that ICL activity was not detected inmegagametophyte extracts of mature or stratified seeds. From 10-12 DAIwhen the megagametophyte tissue senesced, ICL activity decreased rapidlyto near undetectable levels. In contrast, steady-state levels of ICLprotein and mRNA remained relatively constant during megagametophytesenescence. In vivo synthesis of ICL protein was measured to shedlight on these differences. ICL immunoselected from[35S]-methionine labelled proteins indicated that ICL wassynthesized at very low levels during megagametophyte senescence.Together, the results show that loblolly pine ICL gene expression iscomplex. While temporal regulation appears to be primarilytranscriptional, it also involves a number of post-transcriptionalprocesses including at least one translational and/or post-translationalmechanism.

Relationships between glycollate and formate metabolism in greening barley leaves

Abstract The activities of enzymes catalysing glycollate oxidation, formate production and folate-dependent formate utilization were examined in the primary leaves of Hordeum vulgare cv Galt. Seedlings were grown for 6 days in darkness and then transferred to continuous light (500 μinsteins/m 2 per sec) for up to 5 days. Cell-free extracts of the primary leaves contained glycollate oxidase (EC 1.1.3.1), 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5, 10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) and ability to enzymically decarboxylate glyoxylate. These activities increased during greening and at the end of the light treatment were 70–450% higher than etiolated controls. Greened primary leaves also incorporated [ 14 C]formate at rates that were three- to four-fold higher than shown by etiolated leaves. The specific activity of 10-formyltetrahydrofolate synthetase was decreased by 20–35% when the leaves were greened in the presence of 10 mM hydroxysulphonate. This inhibitor also reduced the incorporation of [ 14 C]formate by up to 45%. A potential flow of carbon from glycollate to 10-formyltetrahydrofolate via glyoxylate and formate was suggested by the data.

Loblolly pine seed dormancy: constraints to germination.

Stratification by moist chilling is often used to break seed dormancy in loblolly pine (Pinus taeda L.). The role that moist chilling plays at the cellular level in preparing the embryo for germination and early seedling growth was investigated. Moist chilling did not affect the embryos ability to mobilize seed storage proteins, which is a biochemical marker of early seedling growth. Analysis of in vivo protein synthesis profiles indicated that moist chilling has only a subtle effect on gene expression in the embryo or germinant. The means by which moist chilling affects interactions between the seed coat and the living tissues of the seed also was studied. Although the seed coat is mainly a mechanical barrier to germination, seed coat replacement studies showed that moist chilling alleviates some factor(s) in the seed coat that significantly inhibit germination.