Photoperiod control of poplar bark storage protein accumulation

Bark storage proteins (BSPs) accumulate in the inner bark parenchyma of many woody plants during autumn and winter. We investigated the effect of a short-day (SD) photoperiod on the accumulation of the 32-kilodalton bark storage protein of poplar (Populus deltoides Bart. ex Marsh.) under controlled environmental and natural growing conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein gel blot analysis revealed that 10 days of SD exposure (8 hours of light) resulted in a 20% increase in the relative abundance of the 32-kilodalton bark storage protein of poplar. After 17 days of SD exposure, the 32-kilodalton bark storage protein accounted for nearly one-half of the soluble bark proteins. In natural field conditions, accumulation of the 32-kilodalton bark storage protein was observed to start by August 18 (daylength 14.1 hours). Immunoprecipitation of in vitro translation products with anti-BSP serum revealed that the SD protein accumulation was correlated with changes in the pool of translatable mRNA. A survey of poplar clones from different geographic origins revealed the presence of the 32-kilodalton BSP in the dormant bark of all the clones tested. These results demonstrate that a SD photoperiod induces, whether directly or indirectly, rapid changes in woody plant gene expression, leading to the accumulation of BSP.     

Purification and characterization of the 22-kilodalton potato tuber proteins

Three abundant proteins of approximate molecular masses of 22, 23, and 24 kilodaltons were purified from potato (Solanum tuberosum L.) tubers by DEAE cellulose and CM-52 cellulose ion exchange column chromatography, electroelution, and high-pressure liquid chromatography (HPLC). Antibodies specific to the gel-purified 22-kilodalton protein were prepared. Immunoblot analysis showed that the 22-, 23-, and 24-kilodalton proteins are immunologically related and that these proteins are present in tubers and as higher molecular mass forms in leaves, but not in stems, roots, and stolons. The ratios of amino acid composition were compared among the three purified proteins, and the aminoterminal amino acid sequences were determined for these three proteins. All three proteins have identical amino-terminal sequences that match the deduced amino acid sequence of an abundant tuber protein cDNA.     

Developmental Biochemistry of Cottonseed Embryogenesis and Germination : XIII. REGULATION OF BIOSYNTHESIS OF PRINCIPAL STORAGE PROTEINS

The synthesis of the principal cottonseed storage proteins during embryogenesis has been followed by analyses of stained protein gels and of fluorographs of protein synthesized in vivo and from purified RNA in vitro in the wheat germ system. The kinetics of in vivo labeling as well as immunochemical cross-reactivity indicate that the 52- and 48-kilodalton mature storage protein sets are derived from 70- and 67-kilodalton precursor protein sets that are abundant proteins in embryonic cotyledons and disappear in late embryogenesis. Identification of the initial translation products of the storage protein mRNA has not been clearly established although products of apparent molecular weights of 69,000 and 60,000 are the likely storage protein precursors.     

Characterization of a soybean leaf protein that is related to the seed lectin and is increased with pod removal

Levels of several polypeptides in addition to the vegetative storage protein (VSP) increase in soybean leaves following depodding. Two of these polypeptides interact specifically with antibodies raised against the seed lectins of Phaseolus vulgaris and soybean. The two polypeptides, which had apparent molecular masses of 29,000 daltons and 33,000 daltons, were present in the sink-deprived plants but not in control podded plants and were the subunit polypeptides of a glycoprotein designated lectin-related protein (LRP). Soybean LRP was purified to near homogeneity by a combination of ammonium sulfate precipitation and gel filtration. Dialysis of the resuspended ammonium sulfate precipitate caused LRP to reprecipitate, and LRP was soluble only in the presence of molar NaCl. The native relative molecular mass of LRP was 119,000 daltons, a size consistent with a tetrameric organization of the two polypeptides. LRP precipitated during dialysis in association with a 28,000 dalton polypeptide. The protein coprecipitating with LRP was identified as the dimer of the 28,000 dalton subunit of VSP, one of three native isomeric forms of VSP occurring in leaves of depodded plants. Although the specific association between LRP and VSP was intriguing, an in vivo interaction between LRP and VSP was doubtful. LRP was shown to be immunologically similar to soybean agglutinin but did not have detectable hemagglutinating activity. LRP also was shown to be made up of polypeptides distinct from soybean agglutinin.     

Complementary DNA cloning of poplar bark storage protein and control of its expression by photoperiod

Bark storage proteins accumulate in the bark of many woody plants during autumn and winter. In poplar (Populus deltoides Bartr. ex Marsh), the accumulation of the 32-kilodalton bark storage protein is controlled by photoperiod. We have isolated a full-length cDNA encoding for the poplar 32-kilodalton bark storage protein and determined its nucleotide sequence. The derived amino acid sequence shows that poplar bark storage protein is rich in serine, leucine, phenylalanine, and lysine. Poplar bark storage protein is similar to the poplar wound-induced cDNA clone 4 and clone 16 (TJ Parsons, HD Bradshaw, MP Gordon [1989] Proc Natl Acad Sci USA 86: 7895-7899). DNA gel blot analysis suggests that poplar bark storage protein is encoded by a multigene family of about five genes. Poplar plants grown in long days contained low levels of mRNA for the bark storage protein. Exposure to short days resulted in an increase in bark storage protein mRNA within 7 days. After 21 days of short day exposure, high levels of mRNA were detected. The accumulation of bark storage protein mRNA in response to short days was also observed in plants exposed to natural shortening daylengths. Our results indicate that the accumulation of poplar bark storage protein mRNA is controlled by photoperiod. This finding will provide a useful system for investigating photoperiodism in woody plants.     

Poplar Bark Storage Protein and a Related Wound-Induced Gene Are Differentially Induced by Nitrogen

Poplars (Populus deltoides Bartr. ex Marsh) accumulate a 32-kD bark storage protein (BSP) in phloem parenchyma and xylem ray cells during autumn and winter. Accumulation of poplar BSP is associated with short-day (SD) photoperiods. Poplar BSP shares sequence similarity with the product of the wound-inducible poplar gene win4. The influence of nitrogen availability and photoperiod on the levels of BSP, BSP mRNA, and win4 mRNA was investigated. In long-day (LD) plants BSP, BSP mRNA, and win4 mRNA levels were correlated with the amount of NH4NO3 provided to the plant. BSP mRNA and BSP were detected only in bark, whereas win4 mRNA was detected only in leaves. In LD plants treated with NH4NO3, BSP mRNA levels were significantly greater than those of win4. In nitrogen-deficient plants exposed to SD conditions, the accumulation of BSP mRNA and BSP was delayed for 2 weeks. This delay was eliminated by further SD exposure, and after 6 weeks of SD treatment similar levels of BSP and BSP mRNA were detected in the bark of SD plants regardless of the level of NH4NO3 treatment. win4 mRNA levels declined to undetectable levels in young leaves of SD plants but increased in mature leaves. These results indicate that BSP accumulation in both LD and SD plants is influenced by nitrogen availability. Although both BSP and win4 appear to be involved in nitrogen storage, our data suggest that BSP is probably the primary protein involved in both seasonal and short-term nitrogen storage in poplar. These results also suggest that nitrogen cycling and storage in poplar could involve a two-component system. In this system the win4 gene product may modulate accumulation and mobilization of leaf nitrogen, whereas BSP is involved in seasonal and short-term nitrogen storage during periods of excess nitrogen availability.     

Expression of bottom component RNA of cowpea mosaic virus in cowpea protoplasts

Upon inoculation of cowpea protoplasts with the bottom component of cowpea mosaic virus, at least six virus-induced proteins (with sizes of 170, 110, 87, 84, 60, and 32 kilodaltons) are synthesized, but not the capsid proteins (37 and 23 kilodaltons). These bottom-component-induced proteins were studied with respect to their genetic origin and mode of synthesis. The analyses were based on their electrophoretic peptide patterns resulting from partial digestion with Staphylococcus aureus protease V8. Comparison of the peptide patterns of the virus-induced proteins with those of the cowpea mosaic virus RNA-coded polypeptides produced in rabbit reticulocyte lysate showed that the 170- and 32-kilodalton polypeptides, which are the first viral products in cowpea mosaic virus-infected cells, were actually coded by the bottom component RNA of the virus. The 110-, 87-, and 84-kilodalton polypeptides, and possibly the 60-kilodalton polypeptide, appeared to have amino acid sequences in common with the 170-kilodalton polypeptide, demonstrating that they were virus coded as well. The results indicated that cowpea mosaic virus bottom component RNA was translated in vivo into a single 200-kilodalton polyprotein from which probably all bottom-component-specific proteins arose by three successive cleavages.     

Degradation of tobacco pathogenesis-related proteins : evidence for conserved mechanisms of degradation of pathogenesis-related proteins in plants

Tobacco (Nicotiana tabacum L.) leaves were found to contain an extracellular proteinase that endoproteolytically cleaves tobacco pathogenesis-related (PR) proteins. This proteinase was partially purified from tobacco leaves and characterized as an aspartyl proteinase with a pH optimum around pH 3 and a molecular mass of 36,000 to 40,000 daltons. In vitro, the enzyme cleaved purified tobacco and tomato PR proteins into discrete fragments. The characteristics of this proteinase were similar to pepsin and identical to those displayed by a previously described tomato 37-kilodalton aspartyl proteinase active against tomato PR proteins (I Rodrigo, P Vera, V Conejero [1989] Eur J Biochem 184: 663-669), suggesting that these extracellular proteases could play a role in a conserved mechanism for PR protein turnover in plants.     

Biosynthesis of Storage Proteins in Ripening Agrostemma githago L. Seeds

The synthesis of storage proteins in ripening Agrostemma githago seeds was studied by in vivo pulse and pulse-chase experiments with labeled amino acids and labeled glucosamine. It was found that storage proteins were not synthesized directly, but via cleavage of several large precursor proteins. Two disulfide-linked proteins of 38 and 25 kilodaltons were synthesized via a single large precursor protein. This precursor protein contained internal disulfide bridges, at least one of which is involved in holding the subunit structure together following cleavage of the precursor. A similar mode of biosynthesis was noted for two other disulfide-linked proteins of 36 and 22 kilodaltons. The half-life of the precursors was about 2 hours. This mode of processing is analogous to the synthesis of legumin in legumes and globulin in oats. A third pair of disulfide-bonded proteins (41 and 23 kilodaltons) was synthesized from a precursor protein in several steps. These included a legumin-like cleavage, whereafter the subunits remained disulfide-bonded. Then, from the largest subunit, a part was cleaved off, probably a storage protein of 17 kilodaltons. This 17-kilodalton protein was not disulfide-bonded to the 41 and 23-kilodalton complex. The first processing step was fast, the second slow: The half-lives of the precursors were about 3 and 10 hours, respectively. Finally, a group of 16- and 17-kilodalton proteins was synthesized by cleavage of large precursor proteins, likely in two steps. After cleavage, the proteins were not disulfide-bonded. The half-life of the precursors was short, less than 1 hour. In addition, for the 38-, 23-, and one of the 17-kilodalton proteins, a small decrease of relative molecular weight was observed as a last processing step. This was likely due to deglycosylation.     

Purification of leucoplast pyruvate kinase from developing castor bean endosperm

Leucoplast pyruvate kinase from endosperm of developing castor oil seeds (Ricinus communis L.; cv Baker) has been purified 1370-fold to a specific activity of 41.1 micromoles pyruvate produced per minute per milligram protein. Nondenaturing polyacrylamide gel electrophoresis of the purified enzyme resulted in a single protein staining band that co-migrated with pyruvate kinase activity. However, following sodium dodecyl sulfate polyacrylamide electrophoresis, two major protein staining bands of 57.5 and 44 kilodaltons, which occurred in an approximate 2:1 ratio, respectively, were observed. The native molecular mass was approximately 305 kilodaltons. Rabbit antiserum raised against the final enzyme preparation effectively immunoprecipitated leucoplast pyruvate kinase. The 57.5- and 44-kilodalton polypeptides are immunologically related as both proteins cross-reacted strongly on Western blots probed with the rabbit anti-(developing castor seed endosperm leucoplast pyruvate kinase) immunoglobulin that had been affinity-purified against the 57.5-kilodalton polypeptide. In contrast, pyruvate kinases from the following sources showed no immunological cross-reactivity with the same immunoglobulin: the cytosolic enzyme from developing or germinating castor bean endosperm; chloroplastic pyruvate kinase from expanding leaves of the castor oil plant; chloroplastic or cytosolic pyruvate kinase from the green alga, Selenastrum minutum; and mammalian or bacterial pyruvate kinases.     

Photosynthetic apparatus in chilling-sensitive plants

Proteins of fresh, cold and dark-stored and illuminated tomato leaves were fractionated by SDS electrophoresis. The total soluble proteins extracted from fresh leaves were separated into 5 main fractions with MWs of 54,000, 45,000, 32,000, 23,000 and 14,000. The cold and dark storage of the leaves causes a marked reduction mainly in the fraction with MW of 45,000 which increased with the illumination of the cold and dark-storaged leaves. The polypeptides with MWs of 54,000 and 14,000 (probably large and small subunits of ribulose, bisphosphate carboxylase) were stable under these conditions. In contrast, the polypeptides with MWs of 54,000 and 14,000 are decreased following the storage of tomato leaves in the dark at room temperature. Chloroplast soluble proteins were seperated by SDS electrophoresis into fractions with MWs of 64,000, 54,000, 20,000 and 14,000. The same fractions in similar proportions were observed in soluble-chloroplast proteins from fresh as well as coold and dark-stored and illuminated leaves. No drastic changes in structural polypeptides were observed following cold and dark-storage and illumination of the leaves. The results indicated that the main protein fraction, which degradated following cold and dark storage of tomato leaves and synthetized during illumination, is the fraction of cytoplasmic protein which in SDS electrophoresis gives polypeptides of about 45,000 MW. The fractions of chloroplast proteins were stable under such conditions.Abbreviations DCIP 2,6-dichlorophenolindophenol - FFA free fatty acid - MW molecular weight - RuBP carboxylase ribulose 1,5-bisphosphate carboxylase - SDS sodium dodecyl sulphate - TCA trichloroacetic acid     

Clonal variation in apical growth and content in vegetative storage proteins in Populus

Summary Apical shoot growth and storage protein content in various poplar species and clones were followed in trees growing in the field and in micropropagated plants cultivated in the growth chamber under a controlled environment. In autumn a 32 kD and a 36 kD vegetative storage protein accumulate in wood, bark and roots of poplar and comprise together about 25% of the soluble proteins. In spring, at the time of dormancy break, the storage proteins are degraded and 3 weeks after budburst these proteins are no longer immunologically detectable. As in autumn, short day exposure of black cottonwood plants (Populus trichocarpa Torr. and Gray) induces cessation of apical growth and accumulation of the 32 kD and 36 kD vegetative storage proteins in all clones studied. In order to simulate spring conditions, short day induced plants were transferred back to long days. Like the situation in spring, budburst and storage protein degradation occurred considerably earlier in clone 9/60 than in clone Muhle Larsen. The latter clone accumulates both in winter and after short day exposure more storage proteins than the former. Furthermore two P. trichocarpa clones differ qualitatively in storage protein content: they possess an additional 34 kD polypeptide which cross-reacts with the anti-32 kD antibody. In conclusion, apical shoot growth and the capacity to synthesize storage proteins can be easily followed in micropropagated poplar cultivated in the growth chamber under inducing photoperiods. This offers the major advantage of independence from the annual growth cycle. Within one species considerable clonal variance in storage protein content and in the induction times needed for dormancy and dormancy break were observed. The suitability of storage protein content and apical growth as early selection traits in breeding programs focusing on nitrogen efficient poplar and clones adapted to specific latitudes will be discussed.     

Protein compositions of mesophyll and paraveinal mesophyll of soybean leaves at various developmental stages

Mesophyll and paraveinal mesophyll protoplasts (PVMP) were isolated from leaves of soybean (Glycine max) at various stages of physiological development, and protein compositions of the two protoplast types were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Polypeptides of 27, 29 (previously shown to be storage proteins), and 94 kilodaltons were found to be PVMP-specific proteins and were present in both nodulated and nonnodulated plants. The 27 and 94 kilodalton polypeptides were major PVMP constituents. All three polypeptides accumulate as early as one-quarter leaf expansion. Immunoblotting and immunocytochemical studies using antibodies against the 27/29 kilodalton proteins confirmed that they are specific to the paraveinal mesophyll (PVM) and that they are localized in the PVM vacuole. The 27 kilodalton polypeptide increased significantly by two weeks depodding, and this accumulation was restricted to the PVM vacuole. Radiolabeling experiments showed that the difference in relative amounts of the 27 and 29 kilodalton polypeptides was due to a greater rate of synthesis of the 27 kilodalton polypeptide. The 94 kilodalton polypeptide accumulated to a maximum at anthesis, but was absent at 2 weeks postanthesis in both depodded and podded nodulated plants, probably because they were nitrogen limited. In nonnodulated plants, it was present through 2 weeks postanthesis. The results confirm that the 27 and 29 kilodalton proteins of soybean leaf are stored in the PVM vacuole and show that they are accumulated early during leaf development while they are still strong sinks for nitrogen. The 94 kilodalton protein, previously found to accumulate in leaves after depodding, is also a PVM protein and is likely a third vegetative storage protein, although its accumulation appears to be more dependent on excess nitrogen availability. The results further support the hypothesis that the PVM is a specialized leaf tissue that functions in synthesis and compartmentation of storage proteins.     

Effect of Nitrogen Starvation on Polypeptide Composition, Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase, and Thylakoid Carotenoprotein Content of Synechocystis sp. Strain PCC6308

Synechocystis sp. strain PCC6308 cells were starved for nitrogen for 5 days. The polypeptide compositions of whole cell extracts and washed membranes of nitrogen-replete and nitrogen-starved cells were compared by one- and two-dimensional electrophoresis. Immunoblotting of one-dimensional gels indicated that pelletable ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was depleted in cells starved for nitrogen, while levels of soluble Rubisco were comparable in nitrogen-starved and nitrogen-replete cells. This is consistent with the hypothesis that pelletable Rubisco may serve as a nitrogen reserve in Synechocystis 6308. Other polypeptides were differentially enriched in the membrane or soluble fractions of nitrogen-replete cells or nitrogen-starved cells, suggesting nitrogen starvation may alter partitioning of polypeptides into soluble and membrane fractions. Degradation of abundant polypeptides during nitrogen starvation appeared to cause an effective magnification of less abundant polypeptides in the molecular mass range of 20 to 40 kilodaltons, as shown by two-dimensional electrophoresis. A 42-kilodalton thylakoid carotenoid protein identified by immunoblotting was conserved in membranes from nitrogen-starved cells. This may be functional for cells depleted of pigment and thus exposed to higher light levels because of decreased self-shading.     

In vivo endoproteolytically cleaved phaseolin is stable and accumulates in developing Phaseolus lunatus L. seeds

Phaseolin is the most abundant storage protein of bean seeds. To modify its amino-acidic composition by protein engineering, for the improvement of its nutritional value, regions which could be modified without detrimental effects on structural features of the protein must be identified. Data presented here, on the characterisation of the major storage protein of lima bean (Phaseolus lunatus L.) seeds, a phaseolin-like glycoprotein, provide good indications on one of such region. Phaseolus lunatus phaseolin consists of four major oligomers containing two subunit classes. Polypeptides of one class show a molecular mass ranging from 38.5 kDa to 32 kDa, while the molecular mass of polypeptides belonging to the other class ranges from 27 kDa to 21 kDa. The subunits originate from the cleavage of precursor forms, with molecular masses of 58 kDa and 54 kDa, which are still present — in residual amounts — in the mature protein. Comparison of their N-terminal sequences with those of the subunits demonstrate that cleavage occurs in a region of the molecule that instead remains uncleaved in phaseolins of the other species. Since this region can accommodate such a drastic modification, we suggest it could be a good candidate for in vitro manipulation.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.Abbreviations kDa kilodalton - LHCP-2 light-harvesting chlorophyll a/b-binding protein - M_r relative molecular mass - PSII photosystem II - SDS sodium dodecyl sulphate     

Comparative proteomic analysis of leaves between photoperiod-sensitive and photoperiod-insensitive maize inbred seedlings under long day treatments

Day length is an important environmental factor affecting the growth and development of maize (Zea mays), a short day (SD) plant grown in different latitudes. Leaf has been recognized as the light perceiving and signal producing organ. Under long day (LD) conditions, photoperiod-sensitive induction phase in maize begins at the fourth fully expanded leaf stage. However, the changes of maize leaf proteome in response to LD are largely unknown. To reveal maize proteome response to LD, proteins extracted from newly expanded fifth, sixth and seventh leaves from maize inbred line 496-10 (photoperiod sensitive) and Huangzao4 (HZ4, photoperiod insensitive) under LD treatments were compared via gel-based proteomic approach. As a result, eleven differentially expressed proteins were identified between 496-10 and HZ4 by mass spectrometry. This difference in protein accumulation was highly reproducible during the fifth to seventh leaf stages and most obvious at the seventh leaf stage. The identified proteins are mainly involved in circadian clock or iron metabolism, light harvesting and photosynthesis, nucleic acid metabolism and carbon fixation or energy metabolism. This study provides new insight into the influences of LD treatment on SD plants, such as maize, at proteome level.     

Differential regulation of trichome formation on the adaxial and abaxial leaf surfaces by gibberellins and photoperiod in Arabidopsis thaliana (L.) Heynh.

In wild-type (WT) Columbia and Landsberg erecta ecotypes of Arabidopsis thaliana (L.) Heynh., trichomes are present on the adaxial surfaces of all rosette leaves but are absent from the abaxial surfaces of the first-formed leaves. We have determined that both long-day (LD) photoperiod and gibberellin (GA) stimulate trichome formation. WT plants grown in LD conditions produce the first abaxial trichome on earlier leaves than plants grown in short-day (SD) conditions. Photoperiod sensitivity of abaxial trichome formation on WT plants develops gradually over time, reaching the maximum sensitivity about 24 d after germination. Application of gibberellic acid to WT plants growing in SD conditions accelerates the onset of abaxial trichomes. Conversely, application of 20 to 80 mg L-1 paclobutrazol, a GA biosynthesis inhibitor, to wild-type plants suppresses trichome initiation on the abaxial epidermis. The GA-deficient mutants ga1-5 and ga4-1 and the GA-insensitive mutant gai-1 exhibit delayed onset of abaxial trichomes when grown in LD conditions. The null mutant ga1-3 produces completely glabrous leaves when grown in SD conditions. Application of gibberellic acid to glabrous ga1-3 plants consistently induces earlier formation of trichomes on the adaxial epidermis than on the abaxial epidermis, demonstrating a difference between the adaxial and abaxial surfaces in their response to GA with regard to trichome formation.