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.     

Chitinase accumulates systemically in wounded poplar trees

Young leaves of poplar ( Populus spp.) trees accumulate novel messenger RNAs shortly after the mature leaves have been mechanically wounded. These systemically wound‐induced ( win ) mRNAs are thought to encode proteins involved in plant defense. In the present paper, transgenic tobacco plants that ectopically expressed a win6 cDNA contained a novel chitinase activity that was not present in normal tobacco. This demonstrated Win6 was a chitinase. Win6 and a related protein Win8 accumulated in wounded poplars. Win6 and Win8 had low isoelectric points (ca 4) as predicted from their nucleotide sequence. The wound‐inducible increase in Win6 and Win8 was correlated with an increase in chitinase (EC 3.2.1.14) activity in poplar leaf extracts. We conclude that mechanical wounding induces chitinase in poplar trees, and speculate that the induced chitinase activity could act to increase the tolerance of poplars to opportunistic wound pathogens.     

Induction by nitrogen and low temperature of storage-protein synthesis in poplar trees exposed to long days

The synthesis of storage proteins in trees of poplar (Populus x canadensis Moench) could not only be induced by a shift from long-day to short-day conditions but also by either a low-temperature treatment or by nitrogen feeding under continuous long-day conditions. The synthesis of the protein did not depend on the cessation of growth and the formation of a terminal bud. The accumulation of the storage protein was in all cases preceded by a drastic increase in the level of the corresponding mRNA.Abbreviations cDNA copy DNA - kDA kilodalton     

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.     

The primary structure of a plant storage protein: zein.

The protein sequence of a representative of the zeins, the major storage proteins of maize, has been derived from the nucleotide sequence of a zein cDNA clone. This cDNA was sequence both by the Maxam and Gilbert and the M13-dideoxy techniques. The nucleotide sequence encompasses the non-translated 3' terminus of the mRNA, the entire coding sequence specifying both the mature zein protein and a small signal peptide, and a portion of the non-translated 5' region. The deduced amino acid composition and the amino-terminal amino acid sequence closely resemble those derived from chemical analysis of the zein protein fraction. The data presented represent the first complete amino acid sequence of a plant storage protein.     

Structure and expression of mRNA for a pupal cuticle protein of the silkworm,Bombyx mori

Electrophoretic and immunoblot analyses of proteins extracted from the salt-washed integuments of the silkworm Bombyx mori demonstrated that the pupal cuticle contains structural proteins distinct from those present in the larval cuticle. The cDNA clone encoding a pupal cuticle protein was isolated from the cDNA library constructed from epidermal mRNA of pharate pupae. Northern blot hybridization by use of a cDNA probe provided evidence that mRNA for the pupal cuticle protein accumulate in integument during larval-pupal transformation, though temporal rise of the mRNA level was also noticed at the stages of larval molting. Primary structure of the pupal cuticle protein was deduced from the nucleotide sequence of cDNA. The cloned mRNA sequence encodes a 27 kDa protein rich in alanine and proline, containing characteristic repeats of Ala-Pro-Ala-His-Gln-(Asp/Ser)-Trp-Asn sequence in the carboxyl-proximal domain. The sequence (Ile/Val)-(Leu/Ala)-(Asp/Glu)-Thr-Pro-Glu-Val-Ala-(Gln/Ala)-Ala-Arg-Ala-Ala-His-(Leu/Ile)-(Ala/Ser)-Ala-(Leu/His) occurs in three hydrophobic domains of the molecule.     

Proteins homologous to leaf glycoproteins are abundant in stems of dark-grown soybean seedlings. Analysis of proteins and cDNAs

We report here the cloning and sequence analysis of cDNAs for a pair of closely related proteins from soybean (Glycine max [L.] Merr. cv. Williams 82) stems. Both proteins are abundant in soluble extracts of seedling stems but not of roots. One of these proteins (M _r=28 kDa) is also foundd in the cell wall fraction of stems and actumulates there when seedlings are exposed to mild water deficit for 48 h. The mRNA for these proteins is most abundant in the stem region which contains dividing cells, less abundant in elongating and mature stem cells, and rare in roots. Using antiserum against the 28 kDa protein, we isolated cDNA clones encoding it and an antigenically related 31 kDa protein. The two cDNAs are 80% homologous in nucleotide and amino acid coding sequence. The predicted proteins have similar hydropathy profiles, and contain putative NH₂-terminal signal sequences and a single putative N-linked glycosylation site. The two proteins differ significantly in calculated pI (28 kDa=8.6; 31 kDa=5.8), and the charge difference is demonstrated on two-dimensional gels. The proteins described here may function as somatic storage proteins during early seedling development, and are closely related to glycoproteins which accumulate in vacuoles of paraveinal mesophyll cells of fully expanded soybean leaves when plants are depodded.     

Nucleotide sequence and expression of a ripening and water stress-related cDNA from tomato with homology to the MIP class of membrane channel proteins

The nucleotide sequence and derived amino acid sequence were determined for a full-length version of the tomato cDNA clone, pTOM75, the mRNA for which has previously been shown to accumulate in roots, ripening fruit and senescing leaves. Computer analysis of the predicted protein product, which we have named tomato ripening-associated membrane protein (TRAMP) indicates strong homology to known transmembrane channel proteins from other organisms. Northern analysis showed that this gene was induced by waterstress and that this induction was unaffected in an ABA-deficient genetic back-ground.     

Soybean vegetative storage protein structure and gene expression

Depodded soybean (Glycine max [L] Merr. cv Williams) plants accumulate high levels of a glycoprotein in their leaves that has many features of a storage protein. The protein is found in all vegetative tissues which have been examined but not in the seeds. Translation in vitro indicated that elevated mRNA levels were at least partially responsible for the specific increase in vegetative storage protein. cDNA clones were isolated and sequenced, and an amino acid sequence was predicted. Although the amino acid composition is similar to that of seed storage proteins, no sequence similarity could be detected. Northern blot hybridization confirmed a large increase in vegetative storage protein mRNA in leaves of depodded plants. The vegetative storage proteins are represented by about four gene copies in the haploid genome.     

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.     

Erratum

ErratumSeasonal changes in the concentration of the major storage protein and its mRNA in xylem ray cells of poplar trees     

Induction of cambial reactivation by localized heating in a deciduous hardwood hybrid poplar (Populus sieboldii x P. grandidentata)

BACKGROUND AND AIMS: The timing of cambial reactivation plays an important role in the control of both the quantity and the quality of wood. The effect of localized heating on cambial reactivation in the main stem of a deciduous hardwood hybrid poplar (Populus sieboldii x P. grandidentata) was investigated. METHODS: Electric heating tape (20-22 degrees C) was wrapped at one side of the main stem of cloned hybrid poplar trees at breast height in winter. Small blocks were collected from both heated and non-heated control portions of the stem for sequential observations of cambial activity and for studies of the localization of storage starch around the cambium from dormancy to reactivation by light microscopy. KEY RESULTS: Cell division in phloem began earlier than cambial reactivation in locally heated portions of stems. Moreover, the cambial reactivation induced by localized heating occurred earlier than natural cambial reactivation. In heated stems, well-developed secondary xylem was produced that had almost the same structure as the natural xylem. When cambial reactivation was induced by heating, the buds of trees had not yet burst, indicating that there was no close temporal relationship between bud burst and cambial reactivation. In heated stems, the amount of storage starch decreased near the cambium upon reactivation of the cambium. After cambial reactivation, storage starch disappeared completely. Storage starch appeared again, near the cambium, during xylem differentiation in heated stems. CONCLUSIONS: The results suggest that, in deciduous diffuse-porous hardwood poplar growing in a temperate zone, the temperature in the stem is a limiting factor for reactivation of phloem and cambium. An increase in temperature might induce the conversion of storage starch to sucrose for the activation of cambial cell division and secondary xylem. Localized heating in poplar stems provides a useful experimental system for studies of cambial biology.     

Fluctuation of Vegetative Storage Proteins in the Seedlings of Swietenia macrophylla, Analogous to the Seasonal Changes of Those in the Shoot of the Adult Tree

In order to Identify appropriate plant materials for studying the gene expression and biological function of vegetative storage proteins （VSPs） in woody plants, the VSPs in the seedlings of Swietenla rnacrophylla King were investigated by using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western.blotting. The seed of S. macrophylla was rich in storage proteins that accumulated In the vacuoles of cotyledon parenchyma cells in appearance of compact spherical grains. The growth and development of S. macrophylla seedlings were characterized by an obvious growth rhythm. The storage proteins In seeds disappeared during seedling growth while VSPs appeared in the stem 2 weeks after seedling leaves matured. Thereafter, the VSPs In the seedling stem almost exhausted during new shoot growth, and when the leaves of new shoot Just matured, both the stem beneath the new shoot of seedlings and the stem of new shoot started to accumulate VSPs. Nitrogen application dramatically Increased the level of VSPs, but had little influence on the dynamics of VSP consumption and accumulation in seedling stem. Together with these data, the fluctuation of VSPs in seedlings was very similar to that in the branches of the adult trees. In addition, seedlings are easy to be treated due to their small size. Our results suggested that S. rnacrophylla seedlings were suitable for Investigating the biological roles of VSPs and the mechanism of nitrogen storage in trees.