Structure and expression of the gene encoding cystatin D, a novel human cysteine proteinase inhibitor

A new member of the human cystatin multigene family has been cloned from a genomic library using a cystatin C cDNA probe. The complete nucleotide sequence of a 4.3-kilobase DNA segment, containing a complete gene with structure very similar to those of known Family 2 cystatin genes, was determined. The novel gene, called CST4, is composed of three exons and two introns. It contains the coding information for a protein of 142 amino acid residues, which has been tentatively called cystatin D. The deduced amino acid sequence includes a putative signal peptide and presents 51-55% identical residues with the sequences of either cystatin C or the secretory gland cystatins S, SN, or SA. The cystatin D sequence contains all regions of relevance for cysteine proteinase inhibitory activity and also the 4 cysteine residues that form disulfide bridges in the other members of cystatin Family 2. Northern blot analysis revealed that the cystatin D gene is expressed in parotid gland but not in seminal vesicle, prostate, epididymis, testis, ovary, placenta, thyroid, gastric corpus, small intestine, liver, or gall-bladder tissue. This tissue-restricted expression is in marked contrast with the wider distribution of all the other Family 2 cystatins, since cystatin C is expressed in all these tissues and the secretory gland cystatins are present in saliva, seminal plasma, and tears. Cystatin D, being the first described member of a third subfamily within the cystatin Family 2, thus appears to have a distinct function in the body in contrast to other cystatins.     

脱落酸对UV-C胁迫下小麦幼苗光合特性及抗氧化酶活性的影响

研究了UV-C辐射下短期和长期脱落酸（ABA）处理对小麦幼苗CO₂同化作用、羧化效率、光合CO₂响应以及抗氧化酶活性等的影响.结果表明,在无UV-C辐射情况下,短期和长期ABA处理能提高光合速率,比对照增加14.69%和20.46%,降低气孔导度,比对照降低14.74%和17.31%,但对胞间CO₂浓度和羧化效率影响不大.当受到UV-C辐射时,光合速率、羧化效率、气孔导度和胞间CO₂浓度逐渐降低.长期ABA处理变化最小,其次为ABA短期处理,对照降低最大.ABA处理能够提高小麦光合对CO₂的响应,UV-C辐射抑制光合对CO₂的响应.ABA处理能够提高小麦抗氧化酶（CAT、SOD、POD）活性而降低MDA含量.在UV-C辐射下,CAT活性先升高随后降低,在辐射处理1 h时活性达最大值,ABA处理的SOD和POD活性先升高后降低,且ABA长期处理比短期处理增加明显,对照则逐渐降低.ABA处理可能通过提高小麦CO₂同化作用和抗氧化酶活性增强对UV-C胁迫的抗性,且ABA长期处理比短期处理效果更明显.     

Effects of abscisic acid analogues on abscisic acid-induced gene expression in barley aleurone protoplasts: Relationship between structure and function of the abscisic acid molecule

The plant hormone abscisic acid (ABA) mediates gene expression in barley aleurone protoplasts. In order to elucidate the essential functional groups of the ABA molecule, the specificity of a number of ABA analogues for inducing ABA-regulated gene (e.g., RAB, BASI) expression in barley aleurone protoplasts was studied. These analogues have modifications at three different positions of the ABA molecule: (a) the 1-hydroxyl group (1-deoxy ABA), (b) the carboxyl group (ABA-methyl ester or ABA-glucose ester), and (c) both the 1-hydroxyl and 4-carbonyl groups (-ionylidene acetic acid). The importance of the different putative functional groups was analyzed. The dose-response analysis of ABA analogues upon the induction gene expression showed the following order: ABA > ABA methyl ester > 1-deoxy ABA > ABA glucose ester > -ionylidene acetic acid > --ionone.     

Heterologous expression of the cysteine proteinase gene VaCP17 from the cold-adapted grapevine Vitis amurensis increases cold tolerance in Arabidopsis

Plant Cell, Tissue and Organ Culture (PCTOC) - Cysteine proteinases (thiol) carry out diverse and critical functions in plants through their ability to hydrolyze peptide bonds in target proteins....     

Osmotic stress and abscisic acid induce expression of the wheat Em genes

The early-methionine-labelled (Em) polypeptide is the single most abundant cytosolic protein of dry wheat embryos. It is encoded by messenger RNA which accumulates during the later (maturation) stages of embryogenesis. The accumulation of Em mRNA can be induced in isolated developing embryos, in culture, by the application of the plant growth regulator, abscisic acid, which prevents precocious germination. Precocious germination is also inhibited by the culture of embryos under conditions of osmotic stress when accumulation of Em mRNA is induced. This induction occurs in the absence of any significant increase in the endogenous levels of embryonic abscisic acid although there is a requirement for the continued presence of the growth regulator. Additionally, expression of Em genes can be repeated during early germination, if imbibing embryos are subjected to osmotic stress. Induction of Em-gene expression by osmotic stress is consistent with the proposed role of the Em polypeptide in mediating the remarkable tolerance of cereal embryos to the programmed desiccation undergone during their maturation.     

Differential effects of cold, osmotic stress and abscisic acid on polyamine accumulation in wheat

The effects of cold, osmotic stress and abscisic acid (ABA) on polyamine accumulation were compared in the moderately freezing-sensitive wheat (Triticum aestivum L.) variety Chinese Spring (CS) and in two derived chromosome 5A substitution lines, CS(T. spelta 5A) and CS(Cheyenne 5A), exhibiting lower and higher levels of freezing tolerance, respectively. When compared with the other treatments, putrescine (Put) and spermidine (Spd) levels were much greater after cold treatment, spermine (Spm) following polyethylene glycol-induced (PEG) osmotic stress and Spm and cadaverine (Cad) after ABA treatment. During 3-week cold stress, the Put concentration, first exhibited a transient increase and decrease, and then gradually increased. These alterations may be due to changes in the expression of genes encoding the enzymes of Put synthesis. The Put content was higher in the freezing-tolerant chromosome 5A substitution line than in the sensitive one after 3 weeks of cold. In contrast to cold, ABA and PEG induced a continuous decrease in the Spd level when applied for a period of 3 weeks. The Spm content, which increased after PEG and ABA addition, was twice as high as that of Put during ABA treatment at most sampling points, but this difference was lower in the case of PEG. The Cad level, induced to a great extent by ABA, was much lower when compared with that of the other polyamines. The present experiments indicate that cold, osmotic stress and ABA have different effects on polyamines, and that some of these changes are affected by chromosome 5A and correlate with the level of stress tolerance.     

The effect of abscisic acid on cold tolerance and chloroplasts ultrastructure in wheat under optimal and cold stress conditions

The effect of abscisic acid (0.1 mM) on cold tolerance of leaf cells and ultrastructure of chloroplasts in wheat (Triticum aestivum L.) under optimal (22 °C) and cold stress conditions (4 °C) was studied. Results indicated that exogenous abscisic acid induces a rise in the cold tolerance of wheat along with a number of significant ultrastructural changes in chloroplasts both at 22 and at 4 °C. Some of them (increase in density of chloroplasts stroma, formation of “distorted” and “dilated” thylakoids, appearance of invaginations, changes in the shape of chloroplasts and increase of their dimension owing to the stroma area) were common to the two types of treatments. At the same time, the character of changes in the membrane system of plastids was temperature specific, i.e. if at 22 °C the hormone caused a considerable increase in the length of photosynthetic membranes in chloroplast owing the length of both appressed and non-appressed membranes of thylakoids, then in cold stress conditions observed an increase in the number of grana and the length of appressed membranes of thylakoids. These results suggested that the rise in the cold tolerance of abscisic acid-treated plants is associated with the ultrastructural reorganization of chloroplasts aimed to defense plant cells against chilling injury and to maintain the activity of the photosynthetic system.     

Multiple mode regulation of a cysteine proteinase gene expression in rice

In many plants, cysteine proteinases play essential roles in a variety of developmental and physiological processes. In rice (Oryza sativa), REP-1 is a primary cysteine proteinase responsible for the digestion of seed storage proteins to provide nutrients to support the growth of young seedlings. In the present study, the gene encoding REP-1 was isolated, characterized, and designated as OsEP3A. An OsEP3A-specific DNA probe was used to study the effect of various factors on the expression of OsEP3A in germinating seeds and vegetative tissues of rice. The expression of OsEP3A is hormonally regulated in germinating seeds, spatially and temporally regulated in vegetative tissues, and nitrogen-regulated in suspension-cultured cells. The OsEP3A promoter was linked to the coding sequence of the reporter gene, gusA, which encodes beta-glucuronidase (GUS), and the chimeric gene was introduced into the rice genome. The OsEP3A promoter is sufficient to confer nitrogen regulation of GUS expression in suspension-cultured cells. Histochemical studies also indicate that the OsEP3A promoter is sufficient to confer the hormonal regulation of GUS expression in germinating seeds. These studies demonstrate that in rice the REP-1 protease encoded by OsEP3A may play a role in various physiological responses and processes, and that multiple mechanisms regulate the expression of OsEP3A.     

Gene expression of serine and cysteine proteinase inhibitors during cereal leaf beetle larvae feeding on wheat: the role of insect-associated microorganisms

Plants and insects have been coexisting for more than 350 million years. During this time, both have evolved many strategies to successfully exploit or respond to reciprocal adaptation and defense reactions. Plants tend to minimize the damage caused by pest feeding, while pests tend to manipulate plant response by suppressing plant defense mechanisms or developing strategies to overcome plant defense systems. Plants recognize insect pests by the wounding that they cause and elicitors present in pest oral secretions (saliva and/or regurgitant). These elicitors or insect-associated microorganisms can modulate plant response to the benefit of their insect hosts. In this article, we have undertaken an analysis of gene expression in serine and cysteine proteinase inhibitors (SerPI and CysPI, respectively) in wheat (Triticum aestivum) plants exposed to cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae) larvae feeding, and the impact of microbes associated with CLB on the expression levels of these genes. Using three wheat varieties and antibiotic-treated and untreated CLB larvae, we found that SerPI plays a more important role than CysPIs in plant defense against CLB larvae. Additionally, higher levels of SerPI gene expression were observed in systemic leaves in comparison to the wounded leaves (local response). Each of the tested wheat varieties reacted in a specific way to the particular treatment. Moreover, the presence of microbial components associated with insects influenced plant response to CLB larvae feeding.     

Endogenous abscisic acid and wheat germ agglutinin content in wheat grains during development

Abscisic acid (ABA) and wheat germ agglutinin content of immature wheat grains and embryos was determined by immunoassay throughout the development of a field-grown wheat crop ( Triticum aestivum cv. Timmo). Wheat germ agglutinin accumulation in the embryo was not preceded by an increase in endogenous abscisic acid amount or concentration in either embryos or grains. At a later stage in development the endogenous concentration of abscisic acid in both embryos and grains was found to be two orders of magnitude lower than the endogenous levels required to inhibit precocious germination and promote wheat germ agglutinin accumulation in excised embryos cultured in vitro. These findings are discussed in the context of the control of embryo development in vivo by both ABA and the water status of the grain and embryo.     

Potato cold hardiness development and abscisic acid. I. Conjugated abscisic acid is not the source of the increase in free abscisic acid during potato (Solanum commersonii) cold acclimation

Free and conjugated abscisic acid (ABA) levels in stem-cultured plantlets of potato ( Solanum commersonii Dun, PI 458317) during cold acclimation were measured. The levels of free and conjugated ABA were measured by an enzyme immunoassay (EIA) with rabbit anti-ABA-serum. The use of immunoglobulin G fraction purified from rabbit antiserum and the methylated form of ABA resulted in an improved measuring range (0.01 to 10 pmol ABA) and precision (slope of logit-log plot, −1.35) of EIA, compared to the use of antiserum and free ABA. Estimates of the EIA were consistent with those resulting from a commercial EIA. Under a 4/2°C (day/night) temperature regime, the potato plantlets increased cold hardiness from −5°C (warm-grown control) to −10°C by the 7th day. During the same period, there were two transitory increases in free ABA, the first one three-fold from 1.5 to 5.3 nmol (g dry weight)⁻¹ on the 2nd day and the second one five-fold from 1.5 to 7.6 nmol (g dry weight)⁻¹ on the 6th day. Each increase in ABA concentration was followed by an increase in cold hardiness. There was no significant change in conjugated ABA content (4.2±0.6 nmol [g dry weight]⁻¹) throughout the cold acclimation period. The lack of an interrelationship between levels of free and conjugated ABA suggested that the transitory increase in free ABA during cold acclimation was not a result of the conversion of conjugated ABA. The increase in free ABA due to biosynthesis of ABA during potato cold acclimation is discussed.