A tonoplast intrinsic protein (TIP) is present in seeds, roots and somatic embryos of Norway spruce (Picea abies)

Many plant species contain a seed-specific tonoplast intrinsic protein (TIP) in their protein storage vacuoles (PSVs). Although the function of the protein is not known, its structure implies it to act as a transporter protein, possibly during storage nutrient accumulation/breakdown or during desiccation/imbibition of seeds. As mature somatic embryos of Picea abies (L.) Karst. (Norway spruce) contain PSVs, we examined the presence of TIP in them. Both the megagametophyte and seed embryo accumulate storage nutrients, but at different times and we therefore studied the temporal accumulation of TIP during seed development. Antiserum against the seed-specific a-TIP of Phaseolus vulgaris recognized an abundant 27 kDa tonoplast protein in mature seeds of P. abies. By immunogold labeling of sectioned mature megagametophytes we localized the protein to the PSV membrane. We also isolated the membranes of the PSVs from mature seeds and purified an integral membrane protein that reacted heavily with the antiserum. A sequence of 11 amino acid residues [AEEATHPDSIR], that was obtained from a polypeptide after in-gel trypsin digestion of the purified membrane protein, showed high local identity to a-TIP of Arabidopsis thaliana and to a-TIP of P. vulgaris. The greatest accumulation of TIP in the megagametophytes occurred at the time of storage protein accumulation. A lower molecular mass band also stained from about the time of fertilization until early embryo development. The staining of this band disappeared as the higher molecular mass (27 kDa) band accumulated in the megagametophyte during seed development. Total protein was also extracted from developing zygotic embryos and from somatic embryos. In zygotic embryos low-levels of TIP were seen at all stages investigated, but stained most at the time of storage protein accumulation. The protein was also present in mature somatic embryos but not in proliferating embryogenic tissues in culture. In addition to the seed tissue material, the antiserum also reacted with proteins present in extracts from roots and hypocotyls but not cotyledons from 13-day-old seedlings.     

Proteins arising during the late stages of embryogenesis in Pisum sativum L.

Two abscisic acid (ABA)-responsive seed proteins, ABR17 and ABR18 (ABA-responsive 17000-M_r and 18000-M_r, respectively), previously found to be induced in cultured embryos of pea (Pisum sativum L.) are major components synthesised during normal seed desiccation. The ABR17 and ABR18 proteins showed different patterns of accumulation. The ABR18 protein was abundant in the testa during early seed development but in desiccating seed it was synthesised in the embryo, indicating spacial as well as temporal regulation of expression. The ABR18 protein was undetectable soon after germination but reappeared after adding ABA. The ABR17 protein was not detected in the testa but appeared in the embryo just prior to maximum fresh weight. The ABR17 protein continued to be synthesised during germination and was also present in non-stressed leaves. A high level of endogenous ABA or added ABA increased levels of translatable ABR17 mRNA. The ABR17 and ABR18 proteins were further characterised so as to help determine their structure and function. Neither protein appeared to contain a signal peptide but both proteins appeared to be glycosylated. The proteins had similar amino-acid compositions and limited Nterminal analysis showed 56% sequence identity. Neither protein had any significant N-terminal sequence homology to any of the late embryogenesis-abundant (LEA) proteins or dehydrins. Both proteins, however, show striking homology with a pea disease-resistance-response protein and the major birch pollen allergen, indicating that the ABR17 and ABR18 proteins may be members of a distinct group of stress-induced proteins.Abbreviations ABA (±) cis,trans-abscisic acid - ABR17 M_r-17200 ABA-responsive protein - ABR18 M_r-18 100 ABA-responsive protein - FW fresh weight - I_gG immunoglobulin G - LEA late embryogenesis-abundant - M_r apparent molecularmass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid This work was supported by the Agricultural and Food Research Council via grants-in-aid to Long Ashton Research Station.     

Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein,and enhancement of its accumulation by abscisic acid in somatic embryos

ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70–80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227–291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10^–6 M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LEA protein late-embryogenesis-abundant protein To whom correspondence should be addressedThis work was supported in part by a grant-in-aid for Special Research in Priority Areas (Project No. 02242102) from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.     

Developmental and organ-specific expression of an ABA- and stress-induced protein in barley

An mRNA species, HVA1, has been shown to be rapidly induced by abscisic acid (ABA) in barley aleurone layers (Hong, Uknes and Ho, Plant Mol Biol 11: 495–506, 1988). In the current work we have investigated the expression of HVA1 in other organs of barley plants. In developing seeds, HVA1 mRNA is not detected in starchy endosperm cells, yet it accumulates in aleurone layers and embryo starting 25 days after anthesis, and its level remains high in these organs in dry seeds. Although the levels of HVA1 mRNA are equivalent in the dry embryos of dormant and nondormant barley seeds, upon imbibition HVA1 mRNA declines much slower in the dormant than in the nondormant embryos. The HVA1 mRNA and protein levels are highly induced by ABA treatment in all organs of 3-day-old seedlings. However, the induction in the leaf of 7-day-old seedlings is less than one tenth the level observed in the leaf of 3-day-old seedlings. In the leaf, HVA1 mRNA and protein are induced mainly at the base. These observations indicate that the expression of HVA1 is under developmental regulation. Besides the HVA1 protein, a smaller protein (p20) of approximately 20 kDa cross-reacting with anti-HVA1 polyclonal antibodies, is induced by ABA in barley seedlings but not in seeds. HVA1 mRNA is induced by drought, NaCl, cold or heat treatment. Similar to ABA treatment, the drought induction of HVA1 occurs in all the tissues of 3-day-old seedling, but the induction decreases dramatically in the leaf of 7-day-old plants. The significance of organ-specific, developmentally regulated, and stress-induced expression of HVA1 is discussed.     

Expression of a Late Embryogenesis Abundant Protein Gene,HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice

A late embryogenesis abundant (LEA) protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated. Expression of the barley HVA1 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic rice plants. Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. We also found that the extent of increased stress tolerance correlated with the level of the HVA1 protein accumulated in the transgenic rice plants. Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under water-or salt-stress conditions. Thus, LEA genes hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.     

荞麦糊粉层和子叶中贮藏蛋白质积累过程的超微结构

应用透射电镜技术对荞麦（Fagopyrum esculentum)子叶和糊粉层细胞中贮藏蛋白质的积累过程进行了研究。荞麦开花后15天,胚乳最外细胞的液泡中开始积累蛋白质。开花后25天,最外层胚乳细胞中积累较多的糊粉粒（直径1－2μm)形成糊粉层。开花后20天,子叶细胞中蛋白质开始在液泡和细胞质中积累,同时液泡通过膜的向内生长和缢裂两种方式形成体积较小的液泡。开花后25天,成熟的子叶细胞中含有丰富的蛋白质,贮藏蛋白质主要积累在液泡中形成体积较大的蛋白质贮藏液泡（PSVs,protein storage vacuoles,直径1－3μm）。在荞麦子叶积累蛋白质的各个阶段,细胞质中都有一些来源于高尔基体,含蛋白质的电子不透明小泡（直径0.1-0.7μm)存在,观察到有些小泡正进入液泡,推断这种来自高尔基体膜囊的小泡不仅将蛋白质运输到液泡形成PSVs的作用,也可能是荞麦成熟子叶积累贮藏蛋白质的一种结构。     

Endogenous abscisic acid and protein contents during seed development of <Emphasis Type="Italic">Araucaria angustifolia</Emphasis>

This paper describes a proteome analysis and changes in endogenous abscisic acid (ABA) contents during seed development of Araucaria angustifolia (Bert.) O. Ktze. Megagametophytes and embryonic axis tissues exhibited a similar ABA variation pattern during seed development, reaching maximum values at the pre-cotyledonary stage. The embryonic axis protein content increased until the cotyledonary stage with following stabilization at mature seed. The two-dimensional electrophoresis at the torpedo developmental stage showed approximately 230 polypeptides against 340 in the mature stage. Peptide mass fingerprinting analyses identified three polypeptides, corresponding to an AtSAC4, a late embryogenesis abundant (LEA) and a storage protein, respectively.     

The stress- and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms

Abscisic acid (ABA) induces the expression of a battery of genes in mediating plant responses to environmental stresses. Here we report one of the early ABA-inducible genes in barley (Hordeum vulgare L.), HVA22, which shares little homology with other ABA-responsive genes such as LEA (late embryogenesis-abundant) and RAB (responsive to ABA) genes. In grains, the expression of HVA22 gene appears to be correlated with the dormancy status. The level of HVA22 mRNA increases during grain development, and declines to an undetectable level within 12 h after imbibition of non-dormant grains. In contrast, the HVA22 mRNA level remains high in dormant grains even after five days of imbibition. Treatment of dormant grains with gibberellin (GA) effectively breaks dormancy with a concomitant decline of the level of HVA22 mRNA. The expression of HVA22 appears to be tissue-specific with the level of its mRNA readily detectable in aleurone layers and embryos, yet undetectable in the starchy endosperm. The expression of HVA22 in vegetative tissues can be induced by ABA and environmental stresses, such as cold and drought. Apparent homologues of this barley gene are found in phylogenetically divergent eukaryotic organisms, including cereals, Arabidopsis, Caenorhabitis elegans, man, mouse and yeast, but not in any prokaryotes. Interestingly, similar to barley HVA22, the yeast homologue is also stress-inducible. These observations suggest that the HVA22 and its homologues encode a highly conserved stress-inducible protein which may play an important role in protecting cells from damage under stress conditions in many eukaryotic organisms.     

A late embryogenesis abundant protein HVA1 regulated by an inducible promoter enhances root growth and abiotic stress tolerance in rice without yield penalty

Regulation of root architecture is essential for maintaining plant growth under adverse environment. A synthetic abscisic acid (ABA)/stress‐inducible promoter was designed to control the expression of a late embryogenesis abundant protein (HVA1) in transgenic rice. The background of HVA1 is low but highly inducible by ABA, salt, dehydration and cold. HVA1 was highly accumulated in root apical meristem (RAM) and lateral root primordia (LRP) after ABA/stress treatments, leading to enhanced root system expansion. Water‐use efficiency (WUE) and biomass also increased in transgenic rice, likely due to the maintenance of normal cell functions and metabolic activities conferred by HVA1 which is capable of stabilizing proteins, under osmotic stress. HVA1 promotes lateral root (LR) initiation, elongation and emergence and primary root (PR) elongation via an auxin‐dependent process, particularly by intensifying asymmetrical accumulation of auxin in LRP founder cells and RAM, even under ABA/stress‐suppressive conditions. We demonstrate a successful application of an inducible promoter in regulating the spatial and temporal expression of HVA1 for improving root architecture and multiple stress tolerance without yield penalty.     

A stress-induced,developmentally regulated,highly polymorphic protein family in Pisum sativum L.

The expression of members of two closely related abscisic acid (ABA)-responsive pea protein families, ABR17 and ABR18 (ABA-responsive 17200-M_r and 18100-M_r, respectively), is developmentally, tissueand stress-specifically regulated. Two-dimensional polyacrylamide gel electrophoresis revealed a number of ABR polypeptides on fluorographs of immunoprecipitated translation products of mRNAs, depending on the tissue, stage of development or type of stress. High endogenous ABA, or added ABA, enhanced the accumulation of translatable mRNA for specific ABR members under certain conditions, but high endogenous ABA was not a pre-requisite for accumulation of translatable ABR mRNA. The accumulation of ABR polypeptides was examined by Western blot analysis of acetate-buffer-extracted proteins. In fully expanded, young unstressed leaves, the ABR17 polypeptides (ABR18 polypeptides not detectable) accumulated to markedly higher levels in the epidermis than in the mesophyll. Dehydration stress caused an increased (ABR17) and detectable (ABR18) polypeptide accumulation which occurred predominantly in the epidermis. Detached leaves were used further to characterise factors affecting ABR polypeptide accumulation. An enhanced (ABR17) and detectable (ABR18) polypeptide accumulation occurred in the presence of ABA (10^–4 M) but ABR18-polypeptide accumulation required light. The accumulation of both ABR polypeptides was stimulated in the presence of metabolisable and non-metabolisable carbohydrate sources but not in water or glutamine, indicating an osmotic rather than metabolic response. This carbohydrate-stimulated accumulation was markedly enhanced by light but unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthesis, indicating other photoreceptive processes besides photosynthesis were involved. The function of the ABR proteins remains unknown but their accumulation in aging tissues indicates a role in senescence. The results clearly demonstrate highly complex interactions between different environmental and developmental signals leading to the expression of these stressrelated proteins. In light of these results, the induction of protein expression of the newly-termed intracellular pathogenesis-related proteins, to which the ABR proteins are closely related, is discussed.Abbreviations ABA (±)cis, trans-abscisic acid - ABR17 M_r17200 ABA-responsive protein - ABR18 M_r-18100 ABA-responsive protein - 2-D two-dimensional - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FW fresh weight - IgG immunoglobulin G - M_r apparent molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

TIP,an integral membrane protein of the protein-storage vacuoles of the soybean cotyledon undergoes developmentally regulated membrane accumulation and removal

Protein storage vacuoles (PSVs) in soybean (Glycine max (L.) Merr.) cotyledon cells are formed by subdivision of the central vacuole early in seed maturation. They persist until the fifth or sixth day after germination when the central vacuole re-forms. The major integral membrane protein of PSVs, called Tonoplast Integral Protein or TIP, is highly conserved in the seeds of higher plants (K.D. Johnson et al. 1989, Plant Physiol. 91, 1006–1013). The primary sequence of TIP indicates that it may be a pore protein, although of unknown function (K.D. Johnson et al. 1990, Plant Cell 2, 525–532). TIP is apparently seed-specific and is localized in the protein-storage-vacuole membrane of the storageparenchyma cells and the tonoplast of provascular cells. Using correlated immunoblot and electron microscopicimmunocytochemical assays, we have studied TIP accumulation during seed maturation and its disappearance during seed germination. We have determined that the accumulation of TIP in the protein-storage-vacuole membrane is not correlated with the presence or concentration of stored protein in the organelle. Accumulation of TIP occurs primarily after the division of the central vacuole into protein-storage vacuoles is complete and most of the stored protein has been deposited. Transport of TIP to the PSV membrane is apparently mediated by the Golgi apparatus. Quantitative SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)-immunoblots indicate that, after germination is initiated, TIP abundance is unchanged for the first 4d, but that between days 5 and 7 of growth its abundance decreases drastically. TIP is removed from the PSV membrane prior to the completion of storageprotein mobilization and concurrently with re-formation of the central vacuole. The mechanism of TIP removal appears to involve autophagic sequestering of membrane inside the PSV. The developmental regulation of TIP insertion and removal indicates a physiological function of TIP during late seed maturation or early seedling growth.Abbreviations PSV protein storage vacuoles - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TIP Tonoplast Integral Protein The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentionedWe are grateful to Drs. Ken D. Johnson and Maarten J. Chrispeels (University of California/San Diego, La Jolla, USA) for the gift of anti-TIP antiserum and for their continuing interest in this project. We are also grateful to Dr. Robert Yaklich (Plant Germplasm and Quality Enhancement Laboratory, U.S. Department of Agriculture, Beltsville, Md.) for the soybeans used in this study. We thank Dr. Maria L. Ghirardi for her assistance with the laser densitometry.     

Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana

We analyzed the Arabidopsis thaliana genome sequence to detect Late Embryogenesis Abundant (LEA) protein genes, using as reference sequences proteins related to LEAs previously described in cotton or which present similar characteristics. We selected 50 genes representing nine groups. Most of the encoded predicted proteins are small and contain repeated domains that are often specific to a unique LEA group. Comparison of these domains indicates that proteins with classical group 5 motifs are related to group 3 proteins and also gives information on the possible history of these repetitions. Chromosomal gene locations reveal that several LEA genes result from whole genome duplications (WGD) and that 14 are organized in direct tandem repeats. Expression of 45 of these genes was tested in different plant organs, as well as in response to ABA and in mutants (such as abi3, abi5, lec2 and fus3) altered in their response to ABA or in seed maturation. The results demonstrate that several so-called LEA genes are expressed in vegetative tissues in the absence of any abiotic stress, that LEA genes from the same group do not present identical expression profile and, finally, that regulation of LEA genes with apparently similar expression patterns does not systematically involve the same regulatory pathway.