植物热激蛋白的研究进展

热激蛋白是一类在有机体受到逆境(如高温等)刺激后大量表达的蛋白。主要参与生物体内新生肽的运输、折叠、组装、定位,以及变性蛋白的复性和降解,综述了植物热激蛋白的诱导合成、种类、生物学功能及其基因的表达调控。     

Treatment of maize pollen to reduce nuclease activity

Recently it has been reported that maize (Zea mays L.) pollen can be stored up to several hours in a hypertonic aqueous medium at 0°C without losing its germinability (Broglia and Brunori 1994). We found that both release and activity of pollen nucleases are diminished in a cold hypertonic aqueous medium. Nucleases can be washed off while preserving germination ability and thus preserving the possibility of passive uptake of exogeneous DNA into germinating pollen grains. Alternatively, active DNA transfer into non-germinating pollen grains in the storage medium itself may be facilitated, due to the very reduced nuclease activity in this medium.     

Gene flow in maize fields with different local pollen densities

The development of maize (Zea mays L.) varieties as factories of pharmaceutical and industrial compounds has renewed interest in controlling pollen dispersal. The objective of this study was to compare gene flow into maize fields of different local pollen densities under the same environmental conditions. Two fields of approximately 36 ha were planted with a nontransgenic, white hybrid, in Ankeny, Iowa, USA. In the center of both fields, a 1-ha plot of a yellow-seeded stacked RR/Bt transgenic hybrid was planted as a pollen source. Before flowering, the white receiver maize of one field was detasseled in a 4:1 ratio to reduce the local pollen density (RPD). The percentage of outcross in the field with RPD was 42.2%, 6.3%, and 1.3% at 1, 10, and 35 m from the central plot, respectively. The percentage of outcross in the white maize with normal pollen density (NPD) was 30.1%, 2.7%, and 0.4%, respectively, at these distances. At distances greater than 100 m, the outcross frequency decreased below 0.1 and 0.03% in the field with RPD and NPD, respectively. A statistical model was used to compare pollen dispersal based on observed outcross percentages. The likelihood ratio test confirmed that the models of outcrossing in the two fields were significantly different (P is practically 0). Results indicated that when local pollen is low, the incoming pollen has a competitive advantage and the level of outcross is significantly greater than when the local pollen is abundant. This journal paper of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa, Project No. 3638, was supported by Hatch Act, State of Iowa fund and by a USDA-CREES BRA Grant.     

Heat-shock response in germinating pine pollen

Summary The in vitro culture of pine pollen at various temperatures reveals only a moderate degree of thermotolerance, with considerably reduced levels of growth at and above 35° C. Unlike the pollen of many previously studied species, pine pollen shows some ability to recover from short periods of growth at temperatures as high as 40° C, especially when such exposures occur during the early stages of pollen germination. The pollen of Pinus taeda, unlike that of most other species, shows both quantitative and qualitative changes in the proteins synthesized during germination in vitro following a switch to elevated temperatures (37° C). This response, which can be elicited both during the very early stages of germination as well as during the later stages of pollen tube growth, is reversible following a shift back to the lower temperatures. As previously shown with vegetative tissue of other plant species, the heat-shock response not only involves the induction of high-molecular-weight proteins (most notably 82 kDa and 70 kDa proteins), but also a number of low-molecular-weight (10–20 kDa) species. Two-dimensional gel electrophoretic analysis reveals a small number of qualitative differences in the types of low-molecular-weight heat-shock proteins synthesized in pollen versus vegetative tissue.     

Developmental accumulation of hydroxyproline and hydroxyproline-containing proteins in Zea mays pollen

The hydroxyproline content of developing Zea mays (maize) pollen was determined. The level of hydroxyproline in uninucleate microspores early in pollen development was low (0.004% of dry weight). In contrast, mature pollen is enriched for this amino acid (0.1% of dry weight). In mature pollen, 90% of the hydroxyproline is in the soluble fraction. Upon in vitro pollen germination, hydroxyproline associated with the insoluble fraction increased from 10% to 26% of the total hydroxyproline. Antibodies specific to extensins and arabinogalactan proteins (AGPs), two major classes of hydroxyproline-containing proteins, recognized two distinct groups of proteins in maize pollen by Western analysis. The two types of pollen hydroxyproline-containing proteins could also be distinguished based on their behavior upon anion exchange chromatography.     

Ultraviolet irradiation of maize (Zea Mays L.) pollen grains

Summary Mature pollen grains from two single cross (F₁) hybrids, Wf9 X H55 and K64 X K55, were exposed to eleven levels (0 to 6.80 erg/cm² × 10⁵ at 0.68 intervals) of ultraviolet irradiation and then were used to pollinate their source. Height and kernel characteristics (kernel weight, weight/100 kernels, kernel number) of individual F₂ plants produced by the normal F₂ kernels obtained from these pollinations were measured within each level and population. Highly significant exposure X population interactions were found for all characters, indicating that the effect of irradiation depended on the genetic source of the pollen grains. Increasing exposure increased or did not change the mean of Wf9 X H55 and decreased the mean in K64 X K55 for all characters. For coefficient of variation values, the interaction, exposure X population, was not significant for any character measured, indicating that irradiation-induced variability was unrelated to pollen source. The results indicate that pollen source strongly influenced the effect of ultraviolet irradiation on plant means but had no influence on variability.     

Heat-shock gene expression and cell cycle changes during mammalian embryonic development

Synchronized regulation of cell division during gastrulation is essential for the regional proliferation of cells and pattern formation of the early CNS. The neural plate and neuroectoderm cells are a rapidly dividing and differentiating population of cells with a unique and rapid heat-shock response. Heat shock and the heat-shock genes were studied during neural plate development in a whole rat embryo culture system at 9.5-11.5 days. A lethal heat shock can cause cell death and severe developmental defects to the forebrain and eye during organogenesis. Heat shock can also result in acquired thermotolerance whereby cell progression is delayed at the G1/S and S/G2 boundaries of the cell cycle. This delay in cell cycle progression caused an overall lengthening of the cell cycle time of at least 2 hr. The heat shock genes may therefore function as cell cycle regulators in neuroectoderm induction and differentiation. The kinetics and expression of the hsp genes were examined in neuroectodermal cells by flow cytometry and Northern analysis. The levels of hsp mRNA 27, 71, 73, and 88 were identified following exposure at 42°C (nonlethal), 43deg;C (lethal) and 42deg;/43deg;C (thermotolerant) heat shock. Examination of hsp gene expression in the neural plate showed tight regulation in the cell cycle phases. Hsp 88 expression was enhanced at Go and hsp71 induction at G2 + M of the cell cycle. Cells exposed to a thermotolerant heat shock of 42deg;C induced hsp71 mRNA expression in all phases of the cell cycle with the mRNA levels of hsp27, 73, and 88 increased but relatively constant. Following a lethal heat shock, dramatic changes in hsp expression were seen especially enhanced hsp71 induction in late S phase. The regulated expression of hsps during the cell cycle at various phases could play a unique and important role in the fate and recovery of neuroectoderm cells during early mammalian embryo development. © 1993Wiley-Liss, Inc.     

Quantitative trait loci (QTLs) for pollen thermotolerance detected in maize

Pollen thermotolerance is an important component of the adaptability of crops to high temperature stress. The tolerance level of the different genotypes in a population of 45 maize recombinant inbred lines was determined as the degree of injury caused by high temperature to pollen germinability (IPGG) and pollen tube growth (IPTG) in an in vitro assay. Both traits revealed quantitative variability and high heritability. The traits were genetically dissected by the analysis of molecular markers using 184 mapped restriction fragment length polymorphisms (RFLPs). Significant genetic correlation between the markers and the trait allowed us to identify a minimum number of five quatitative trait loci (QTLs) for IPGG and six QTLs for IPTG. Their chromosomal localization indicated that the two characters are controlled by different sets of genes. In addition, IPGG and IPTG were shown to be basically independent of the pollen germination ability and pollen tube growth rate under non-stress conditions. These results are discussed in relation to their possible utilization in a breeding strategy for the improvement of thermotolerance in maize.     

表达载体pHsh对大肠杆菌热休克系统中负调控机制的影响

pHsh是一种由σ32识别和启动外源基因表达的新型高效的大肠杆菌表达载体。正常E.coli细胞在热激诱导条件下,σ32的浓度在5 min内到达高峰,随后被3个负调控蛋白Dnak、DnaJ、GrpE结合导致失活或降解,整个热休克反应持续约12min。在携带有外源基因的高拷贝pHsh 的E.coli细胞中,外源基因却能持续高效表达4～10 h,这一现象表明了此时细胞中的σ32比没有携带质粒的细胞内σ32的浓度要高。σ32浓度的增高有可能是由于3个负调控蛋白Dnak、DnaJ、GrpE在细胞内的含量比正常情况下降低的结果。为了验证这一推测,从E.coli中克隆了Dnak、DnaJ、GrpE的编码基因,表达并初步纯化了其重组蛋白以作分子标记,采用双向电泳技术,分析携带质粒（pHsh+）和不携带质粒的E.coli(pHsh-)细胞在热休克后胞内蛋白质组的差异。该项实验通过与检索到的标准的E.coli蛋白质组图谱进行比较鉴别出的两个蛋白Dnak、GrpE,并通过对比目标点的大小和深浅发现pHsh+中的Dnak均少于pHsh─中的目标蛋白,所得结果与上述假设一致。     

Expression of alcohol-soluble endosperm proteins in maize single and double mutants

Summary Many maize (Zea mays L.) mutant genes exist. Some affect protein content or composition, while others modify carbohydrates or kernel phenotype. In doublemutant lines, two mutant genes are present. We know little about interactions of such genes, however. We therefore examined a normal maize inbred, B37, 10 near-isogenic single mutants and 46 double mutants to analyze quantitative effects on alcohol-soluble endosperm proteins. Proteins were extracted with 70% ethanol0.5% sodium acetate-5% mercaptoethanol, and fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). Early peaks were alcohol-soluble glutelin (ASG) subunits, while late peaks contained zein. Results were quantified and statistically analyzed. In many double mutants, protein compositions differed significantly from averages of compositions of corresponding single mutants. For example, a high-methionine, water-insoluble ASG is absent when the opaque-2 (o2) gene combines with shrunken-1 (sh1) or surgary-1 (su1). Another water-insoluble ASG nearly doubled when floury-2 (fl2) andsu1 combined. A high-proline, high-histidine, water-soluble ASG nearly doubled in combinations offl2 witho2,su1 and sugary-2 (su2). Zein was about half its expected value wheno2 combined with amylose-extender (ae), floury-1 (fl1), soft-starch (h),sh1 andsu1. Thus, rapid protein extraction and quantitative RP-HPLC showed major new epistatic and synergistic effects of several mutant genes on protein composition. Unexpectedly, these effects often involve genes that primarily affect starch composition or kernel phenotype. Alcohol-soluble proteins often vary in amount, as ino2 lines. They also differ in nutritional value. Thus, RP-HPLC analysis of these proteins can identify nutritionally superior genotypes, and may help explain the basis of such quality.Presented at the XVI International Congress of Genetics, Toronto, Canada, August 20–27, 1988     

Ultrastructural analysis of the sperm cells of mature pollen of maize,Zea mays

Summary Ultrastructural analysis of the mature viable unhydrated pollen of maize,Zea mays from dehiscent anthers shows that the sperm cells are physically distant, each bounded by an envelope comprising their own plasma membrane and the inner plasma membrane of the vegetative cell. The chondriome is unusual in containing one or more filamentous complexes, up to 12m in length appressed to the side of the sperm nucleus. The extensions at each end of the elongate sperm cells contain longitudinally-oriented arrays of endoplasmic lamellae. In a three-dimensional reconstruction of serial thin sections, there is a long J-shaped sperm, c. 35 × 5m and up to 1m in thickness, sited within pointed evaginations of the vegetative nucleus and a second shorter sperm c. 20 × 5m and up to 3.5m in thickness.Abbreviations PA-TCH-SP periodic acid-thiocarbohydrazide-silver proteinate - DAPI 4,6-diamino-2-phenylindole - SC sperm cell - Sn sperm nucleus - Ua-Pb Uranyl acetate-lead citrate staining - ER endoplasmic reticulum     

Pollen-pistil interaction in maize: effects on genetic variation of pollen traits

Various factors (pollen diameter, in vitro germination and tube length, in vivo growth rate in selfed and nonselfed styles) which could possibly contribute to the competitive ability of pollen were investigated on 30 Zea mays L. inbred lines. The only factor with which pollen diameter was positively correlated was in vitro pollen-tube growth. Traits related to the early stages of growth (in vitro germination, in vitro tube length, early in vivo pollen growth rate) were all positively correlated with each other, and these early characteristics were negatively correlated with late in vivo tube growth rate, which is largely influenced by the stylar genotype.     

Genetic analysis of heat shock proteins in maize

A genetic analysis of heat shock protein (HSP) synthesis was performed in seedling leaf tissue of two maize inbred lines, their F₁ hybrid and F₂ progeny. Protein synthesis following a high temperature treatment was visualized by [³⁵S]-methionine in vivo labelling and two-dimensional gel electrophoresis. The parental lines' HSP synthesis patterns revealed both qualitative and quantitative polymorphisms implicative of differences in HSP structural genes and regulatory factors. The F₁ hybrid HSP profile indicated that synthesis of all parental HSPs conformed to dominant inheritance patterns, including complete dominance, over-dominance and co-dominance. Alleles for six low-molecularweight HSPs in F₂ progeny assorted according to typical 31 Mendelian ratios for dominant gene expression. There is evidence for unlinked gene loci of four different HSP gene pairs, but data for three other HSP gene pairs were inconclusive, perhaps reflecting linkage for one pair and complex regulatory factor interactions for the other two pairs of genes. These results clearly indicate the existence of genetic variability in HSP synthesis and emphasize the potential of partitioning their roles in thermal tolerance using genetic and molecular analyses.     

The accumulation of abundant soluble proteins changes early in the development of the primary roots of maize (Zea mays L.)

A reference database of the major soluble proteins of the primary root of the maize inbred line B73 was generated 5 days after germination (DAG) using a combination of 2-DE and MALDI-TOF MS. A total of 302 protein spots were detected with CBB in a pH 4-7 range and 81 proteins representing 74 distinct Genbank accessions were identified. Only 28% of the major proteins identified in 5 DAG primary roots were identified in similarly analyzed 9 DAG primary roots documenting remarkable changes in the accumulation of abundant soluble proteins early in primary root development.     

MAP kinase signaling during pollen development

The stereotypical events of pollen grain maturation and its coordinated development with other flower tissues requires the interplay of different signalling pathways in order to ensure efficient fertilisation and, eventually, seed set. In recent years evidence has accumulated that members of the mitogen-activated protein kinase (MAP kinase) family are expressed in pollen and may function as regulators of both pollen development and germination. In this review we describe what is known about MAP kinases in pollen and discuss their possible function(s) in pollen biology. Received: 14 December 2000 / Accepted: 6 June 2001     

Male gametophytic selection in maize

Summary There is evidence that male gametophyte selection is a widespread phenomenon in higher plants. The pollen tube growth rate is one of the main components of gametophyte selective value; genetic variability for this trait, due to the effect of single genes or to quantitative variation, has been described in maize. However, indication of gametophytic selection has been indirectly obtained; its effect was revealed by the positive relation observed between gametophyte competitive ability and sporophyte metrical traits.This paper considers the results of selection applied to gametophyte populations produced from single plants. The competitive ability of the lines was evaluated in comparison with that of a standard line by means of the pollen mixture technique. Sporophytic traits were measured in the hybrid progeny obtained by crossing selected S₃ and S₄ families with an unrelated single cross and an inbred line. Gametophyte selection produced inbred lines with high gametophyte competitive ability. In view of the selection procedure adopted, this result was interpreted as an indication of haploid expression of genes involved in the control of pollen tube growth. Moreover, this gametophytic trait was positively correlated with sporophytic traits (seedling weight, kernel weight and root tip growth in vitro), indicating that both groups of characters have a common genetic basis.