Light-Independent and Tissue-Specific Accumulation of Light-Harvesting Chlorophyll a/b Binding Protein and Ribulose Bisphosphate Carboxylase in Dark-Grown Pine Seedlings

The presence of light-harvesting chlorophyll a/b binding proteinsand the small and large subunits of ribulose bisphosphate carboxylasewas revealed in dark-grown pine seedlings. The light-independentaccumulation of the proteins was observed in photosyntheticand/or green tissue, such as cotyledons and hypocotyls, butnot in non-photosynthetic roots. ¹ This work was supported by Grants-in-Aid from the Ministryof Agriculture, Forestry and Fisheries of Japan (IntegratedResearch Program for the Use of Biotechnological Proceduresfor Plant Breeding), and from the Science and Technology Agencyof Japan (Encouragement of Basic Research). (Received May 2, 1991; Accepted August 13, 1991)     

Chlorophyll Synthesis in Dark-Grown Pine Primary Needles

The pigment content of dark-grown primary needles of Pinus jeffreyi L. and Pinus sylvestris L. was determined by high-performance liquid chromatography. The state of protochlorophyllide a and of chlorophylls during dark growth were analyzed by in situ 77 K fluorescence spectroscopy. Both measurements unambiguously demonstrated that pine primary needles are able to synthesize chlorophyll in the dark. Norflurazon strongly inhibited both carotenoid and chlorophyll synthesis. Needles of plants treated with this inhibitor had low chlorophyll content, contained only traces of xanthophylls, and accumulated carotenoid precursors. The first form of chlorophyll detected in young pine needles grown in darkness had an emission maximum at 678 nm. Chlorophyll-protein complexes with in situ spectroscopic properties similar to those of fully green needles (685, 695, and 735 nm) later accumulated in untreated plants, whereas in norflurazon-treated plants the photosystem I emission at 735 nm was completely lacking. To better characterize the light-dependent chlorophyll biosynthetic pathway in pine needles, the 77 K fluorescence properties of in situ protochlorophyllide a spectral forms were studied. Photoactive and nonphotoactive protochlorophyllide a forms with emission properties similar to those reported for dark-grown angiosperms were found, but excitation spectra were substantially red shifted. Because of their lower chlorophyll content, norflurazon-treated plants were used to study the protochlorophyllide a photoreduction process triggered by one light flash. The first stable chlorophyllide photoproduct was a chlorophyllide a form emitting at 688 nm as in angiosperms. Further chlorophyllide a shifts usually observed in angiosperms were not detected. The rapid regeneration of photoactive protochlorophyllide a from nonphotoactive protochlorophyllide after one flash was demonstrated.     

Expression of cab Genes in Douglas-Fir Is Not Strongly Regulated by Light

Dark-grown Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) seedlings had approximately 30% of the major polypeptide of the light-harvesting chlorophyll a/b binding protein, 30% of cab mRNA, 54% of psbA mRNA, and 14% of total chlorophyll, in comparison with amounts in light-grown seedlings. Seedlings entrained under a 24-hour photoperiod of light and dark showed small diurnal fluctuations in cab and psbA mRNA levels and, when transferred to continuous conditions, no circadian rhythms in mRNA levels were apparent. These results suggest that regulation of cab gene expression in Douglas-fir differs from regulation in angiosperms, because in the latter, both light and circadian factors strongly influence the expression of cab genes.     

Purification of Mitochondrial Glutamate Dehydrogenase from Dark-Grown Soybean Seedlings

Proteins in extracts from cotyledons, hypocotyls, and roots of 5-d-old, dark-grown soybean (Glycine max L. Merr. cv Williams) seedlings were separated by polyacrylamide gel electrophoresis. Three isoforms of glutamate dehydrogenase (GDH) were resolved and visualized in gels stained for GDH activity. Two isoforms with high electrophoretic mobility, GDH1 and GDH2, were in protein extracts from cotyledons and a third isoform with the lowest electrophoretic mobility, GDH3, was identified in protein extracts from root and hypocotyls. Subcellular fractionation of dark-grown soybean tissues demonstrated that GDH3 was associated with intact mitochondria. GDH3 was purified to homogeneity, as determined by native and sodium dodecyl sulfate-polyacrylamide gels. The isoenzyme was composed of a single 42-kD subunit. The pH optima for the reductive amination and the oxidative deamination reactions were 8.0 and 9.3, respectively. At any given pH, GDH activity was 12- to 50-fold higher in the direction of reductive amination than in the direction of the oxidative deamination reaction. GDH3 had a cofactor preference for NAD(H) over NADP(H). The apparent Michaelis constant values for [alpha]-ketoglutarate, ammonium, and NADH at pH 8.0 were 3.6, 35.5, and 0.07 mM, respectively. The apparent Michaelis constant values for glutamate and NAD were 15.8 and 0.10 mM at pH 9.3, respectively. To our knowledge, this is the first biochemical and physical characterization of a purified mitochondrial NAD(H)-dependent GDH isoenzyme from soybean.     

Development of Chloroplast in Dark-Grown Wheat Seedlings Irradiated at Elevated Temperature

Elevated temperature inhibited the accumulation of chlorophyll and photosynthetic proteins, and the development of photochemical activity, however, carotenoids continued to accumulate. Signal transduction pathway involved in protochlorophyllide oxidoreductase was unaffected by elevated temperature of 38°C. Two-dimensional gel electrophoresis of stroma proteins showed similar patterns in the dark-grown seedlings and seedlings irradiated at elevated temperature, although some low molecular mass proteins accumulated at 38°C. In contrast, seedlings irradiated at 25°C showed complex pattern of proteins. Hence the development of chloroplast and its associated functions during irradiation of etiolated seedlings are inhibited by elevated temperature.     

氮磷亏缺对小麦TaIPS基因表达的影响

为了解小麦高效利用土壤磷的分子机理和实现对小麦缺磷的分子诊断,以普通小麦(Triticum aestivum L.)小偃54为材料,克隆了5个受缺磷诱导的IPS基因,同源比较结果显示,小麦IPS基因属于典型的受缺磷条件特异诱导的TPSI1/MT4小基因家族.对小麦根系和地上部的半定量RT-PCR研究结果表明,与全营养处理对照相比,3叶期小麦幼苗经过缺氮、缺磷和氮磷同时缺乏处理8d后,缺磷显著增加了根系中3个TaIPS1(TaIPS1.1、TaIPS1.2和TaIPS1.3)基因和地上部TaIPS1.1基因的表达,中度上调了根系中2个TaIPS2基因(TaIPS2.1和TaIPS2.2)的表达,轻度上调了地上部TaIPS1.2和2个TaIPS2基因的表达.通过比较5个基因在根系和地上部对缺磷的响应,认为TaIPS1.1是一个较理想的用于诊断小麦植株磷素丰缺的基因.缺氮不仅降低了3个TaIPS1基因在根系中的表达,并抑制了IPS基因对缺磷的响应.这一研究结果预示了TaIPS基因对低磷胁迫的响应依赖于植株体内的氮素营养状况.     

Control of Mitochondrial Activities by Phytochrome in Cotyledons of Dark-Grown Cucumber Seedlings

Mitochondria isolated from cotyledons of dark-grown cucumberseedlings after illumination with white light showed an increasedcapacity for oxidation of malate. This increase is regulatedby phytochrome, since the rate of oxidation was increased bybrief pulses of red light, the effects of which were reversedby brief pulses of far-red light. Similarly, increases in therates of oxidation of citrate and -ketoglutarate were observedin mitochondria isolated from illuminated cotyledons. By contrast,rates of succinate-, exogenous NADH-, durohydroquinone- andascor-bate-dependent consumption of O₂ were little affectedby the light treatment. Exogenous NAD⁺stimulated the oxidationof malate and -ketoglutarate by mitochondria from dark-grown,control cotyledons, while it did not enhance the rates of oxidationof these substrates by mitochondria isolated from light-treatedcotyledons. The activities of mitochondrial enzymes (malateand succinate dehydrogenase, NAD⁺-malic enzyme and cytochromeoxidase) were not different between illuminated and dark-growncotyledons. Neither cycloheximide nor chloramphenicol inhibitedthe increase in the rate of oxidation of malate. (Received October 24, 1988; Accepted May 24, 1989)     

Stimulation of Cell Elongation by Tetraploidy in Hypocotyls of Dark-Grown Arabidopsis Seedlings

Plant size is largely determined by the size of individual cells. A number of studies showed a link between ploidy and cell size in land plants, but this link remains controversial. In this study, post-germination growth, which occurs entirely by cell elongation, was examined in diploid and autotetraploid hypocotyls of Arabidopsis thaliana (L.) Heynh. Final hypocotyl length was longer in tetraploid plants than in diploid plants, particularly when seedlings were grown in the dark. The longer hypocotyl in the tetraploid seedlings developed as a result of enhanced cell elongation rather than by an increase in cell number. DNA microarray analysis showed that genes involved in the transport of cuticle precursors were downregulated in a defined region of the tetraploid hypocotyl when compared to the diploid hypocotyl. Cuticle permeability, as assessed by toluidine-blue staining, and cuticular structure, as visualized by electron microscopy, were altered in tetraploid plants. Taken together, these data indicate that promotion of cell elongation is responsible for ploidy-dependent size determination in the Arabidopsis hypocotyl, and that this process is directly or indirectly related to cuticular function.     

Dry-matter Production and Photosynthesis in Pine Seedlings

Growth analysis and photosynthesis measurements were carriedout on first-year seedlings of Pinus radiata, P. contorta, andP. nigra. P. sylvestris was also included in two growth-analysisexperiments. There appeared to be no difference in relativegrowth-rate (RGR) between the species mentioned, except betweenP. radiata and P. nigra. It was found that when the level ofmineral nutrition was high, P. radiata had a higher RGR thanP. nigra and that the reverse was true at low mineral nutrientlevels. There was no difference in net assimilation rate (NAR)between the species, but NAR appeared to increase and decreasewith mineral nutrient supply. Net photosynthesis per unit of leaf was somewhat higher in P.radiata than in P. contorta; P. nigra had a lower photosyntheticrate than either P. radiata or P. contorta.     

Hormonal Regulation of Expression of Two Cysteine Endopeptidase Genes in Rice Seedlings

Two cDNA probes (pRP60 and pRP80) were used to examine the hormonalregulation of expression of two cysteine endopeptidase genesin germinated whole seeds and de-embryonated seeds of rice.The pRP60 protein is a major rice cysteine endopeptidase namedREP-1 that digests rice glutelin, the main seed storage protein.The pRP80 protein has features of a cysteine endopeptidase buthas not yet been confirmed to be one. Neither mRNA was detectablein dry seeds, and the levels per seed increased sharply uponimbibition, reached peaks at d 6 to d 9, and then decreased.Both mRNAs were expressed in a seed-specific manner, but theaccumulation of pRP60 mRNA was about ten times greater thanthe other. When seeds were de-embryonated and incubated, theamounts of both mRNAs were reduced to very low levels, but inthe presence of 0.01 to 1 µM. gibberellic acid (GA₃) bothagain reached high levels. Addition of abscisic acid (ABA) oruniconazole, an inhibitor of gibberellin biosynthesis, partlyeliminated the effect of GA₃. Protein immunoblot analysis showedthat the accumulation of the REP-1 protein in seeds was regulatedby GA₃ and ABA similarly to that of pRP60 mRNA, but the changein pRP60 mRNA with time after the onset of imbibition precededthat of the REP-1 protein. (Received May 28, 1997; Accepted September 8, 1997)     

紫萍P143品系植物rbcS基因的cDNA克隆与表达

紫萍P14 3品系离体半叶状体在黑暗下培养 4d以后 ,rbcS基因的表达比完整植株显著降低 ;半叶状体在黑暗下培养 10d ,后再转移到长日照条件下培养 ,rbcS基因的表达能恢复到较高水平     

The Promoter of a Pine Photosynthetic Gene Allows Expression of a {beta}-Glucuronidase Reporter Gene in Transgenic Rice Plants in a Light-Independent but Tissue-Specific Manner

In angiosperms, the expression of the cab gene that encodesthe chlorophyll a/b-binding protein of PSII is light-regulated.However, the pine cab gene is expressed in a light-independentbut cell-type-specific manner. In the present study, the cab-6promoter (1.7 kbp) from pine was fused to a -glucuronidase (GUS)reporter gene and the chimeric gene was introduced into riceprotoplasts by electroporation. The GUS expression was studiedin the resultant transgenic rice plants. Expression of GUS ata substantial level was confirmed in primary leaves of dark-germinatedrice seedlings, and no obvious effect of light on the GUS activitywas observed. The expression of GUS was restricted to photosynthetictissues. The pine cab-6 promoter is, thus, sufficient for inductionof light-independent but cell-type-specific expression in cellsof a monocot, as is the case in the original pine cells. (Received December 17, 1993; Accepted April 22, 1994)     

Transport Gradient of Indoleacetic Acid in Pine Seedlings

Movement of indoleacetic acid (IAA-1-¹⁴C) through 5-min segments from stems and roots of Pinus taeda L., P. jeffreyi Gtrev. and Balf., and P. ponderosa Laws, seedlings was measured. All three species showed the same general pattern of IAA-1-¹⁴C transport. Predominantly downward movement occurred at all points along the stem and root; least IAA-1-¹⁴C transport occurred in segments obtained near the shoot apex; and most occurred in segments obtained near or just below the cotyledons. This pattern of transport gradient is different from that reported in other plants. These results could be interpreted in either of two ways: (1) that transport limitations may control movement of the growth-regulator, or (2) that the transport gradient may be an indication of endogenous growth-regulator levels.     

Shoot--root Plasticity and Episodic Growth in Red Pine Seedlings

Red pine seedlings of a half-sib seed source were grown in growthchambers under thermoperiodic regimes of 30/20 °C, 25/15°C and 20/10 °C day/night temperatures. Classical growthanalyses based on weekly harvests of leaves, stem and rootswere employed to study the first 3 to 15 weeks of seedling development.Leaf and root growth were inversely related and episodic. Significantshort term surges in growth of either organ were effective inreversing periodic imbalances that occurred, thus maintaininga long term dry weight equilibrium between above and below groundseedling parts. Adaptive plasticity in the leaf-root balanceat different temperatures gave plants grown at 25/15 °Ca larger proportion of leaves relative to roots and a greatersize compared to seedlings grown under other regimes. Episodicfluctuations in leaf and root growth occurred simultaneouslywith depressions in net assimilation rate. Apparently, balancedgrowth is maintained at an assimilatory cost to the plant, periodic‘corrections’ of shoot—root imbalances requiringcarbohydrate conversion and energy expenditure. Pinus resinosa Ait., red pine, episodic growth, shoot—root balance, plasticity, net assimilation rate, growth analysis