植物叶片暂时淀粉分解途径研究进展

植物叶片暂时淀粉主要分解途径包括如下过程:叶绿体中半结晶状淀粉粒在葡聚糖-水双激酶(GWD)和磷酸葡聚糖-水双激酶(PWD)作用下磷酸化,使淀粉粒结构松散;异淀粉酶(ISA3)作用于松散淀粉粒而释放出磷酸葡聚糖,再经磷酸葡聚糖磷酸酶(SEX4)水解去除磷酸而生成可溶性线性葡聚糖;葡聚糖在β-淀粉酶(BAM3)催化下水解生成麦芽糖后,再通过麦芽糖载体(MEX1)转运至细胞质.该文主要综述了以上转化过程中涉及的底物、生成物和催化酶类的研究进展情况,同时简述了植物叶片暂时淀粉分解的次要途径和抗逆性相关途径,并提出了该领域目前存在的问题和今后研究方向.     

淀粉含量不同的玉米自交系籽粒淀粉积累及其关键酶活性

以2个高淀粉和2个低淀粉玉米自交系为材料,分析了玉米籽粒淀粉的动态积累规律,同时对高低淀粉玉米籽粒灌浆过程中淀粉生物合成关键酶活性的动态变化及其与淀粉积累动态的相关性进行讨论分析。研究结果表明:灌浆过程中4个自交系淀粉含量变化趋势均呈sigmoid型曲线。灌浆过程中ADPG-PPase(腺苷二磷酸葡萄糖焦磷酸化酶)、SSS(可溶性淀粉合成酶)、GBSS(颗粒结合淀粉合成酶)活性均呈单峰曲线变化,峰值都出现在20～30DAP(授粉后天数)。2个高淀粉自交系的Q酶(淀粉分支酶)活性也呈单峰曲线变化,峰值也出现在20DAP,而2个低淀粉自交系的Q酶活性则呈双峰曲线变化,2个峰值分别出现在15～20DAP和30～35DAP。4个自交系籽粒淀粉的积累速率与各自交系ADPG-PPase、SSS和GBSS的活性变化呈极显著正相关。各自交系关键酶活性之间,ADPG-PPase、SSS和GBSS三者间活性变化呈极显著正相关,这3种酶活性变化与Q酶活性变化也呈不同程度的正相关。     

Lactase Persistence and Lipid Pathway Selection in the Maasai

The Maasai are a pastoral people in Kenya and Tanzania, whose traditional diet of milk, blood and meat is rich in lactose, fat and cholesterol. In spite of this, they have low levels of blood cholesterol, and seldom suffer from gallstones or cardiac diseases. Field studies in the 1970s suggested that the Maasai have a genetic adaptation for cholesterol homeostasis. Analysis of HapMap 3 data using Fixation Index (Fst) and two metrics of haplotype diversity: the integrated Haplotype Score (iHS) and the Cross Population Extended Haplotype Homozygosity (XP-EHH), identified genomic regions and single nucleotide polymorphisms (SNPs) as strong candidates for recent selection for lactase persistence and cholesterol regulation in 143–156 founder individuals from the Maasai population in Kinyawa, Kenya (MKK). The non-synonmous SNP with the highest genome-wide Fst was the TC polymorphism at rs2241883 in Fatty Acid Binding Protein 1(FABP1), known to reduce low density lipoprotein and tri-glyceride levels in Europeans. The strongest signal identified by all three metrics was a 1.7 Mb region on Chr2q21. This region contains the genes LCT (Lactase) and MCM6 (Minichromosome Maintenance Complex Component) involved in lactase persistence, and the gene Rab3GAP1 (Rab3 GTPase-activating Protein Catalytic Subunit), which contains polymorphisms associated with total cholesterol levels in a genome-wide association study of >100,000 individuals of European ancestry. Sanger sequencing of DNA from six MKK samples showed that the GC-14010 polymorphism in the MCM6 gene, known to be associated with lactase persistence in Africans, is segregating in MKK at high frequency (∼58%). The Cytochrome P450 Family 3 Subfamily A (CYP3A) cluster of genes, involved in cholesterol metabolism, was identified by Fst and iHS as candidate loci under selection. Overall, our study identified several specific genomic regions under selection in the Maasai which contain polymorphisms in genes associated with lactase persistence and cholesterol regulation.     

Identification of the Soluble Starch Synthase Activities of Maize Endosperm

This study identified the complement of soluble starch synthases (SSs) present in developing maize (Zea mays) endosperm. The product of the du1 gene, DU1, was shown to be one of the two major soluble SSs. The C-terminal 450 residues of DU1 comprise eight sequence blocks conserved in 28 known or predicted glucan synthases. This region of DU1 was expressed in Escherichia coli and shown to possess SS activity. DU1-specific antisera detected a soluble endosperm protein of more than 200 kD that was lacking in du1- mutants. These antisera eliminated 20% to 30% of the soluble SS activity from kernel extracts. Antiserum against the isozyme zSSI eliminated approximately 60% of the total soluble SS, and immunodepletion of du1- mutant extracts with this antiserum nearly eliminated SS activity. Two soluble SS activities were identified by electrophoretic fractionation, each of which correlated specifically with zSSI or DU1. Thus, DU1 and zSSI accounted for the great majority of soluble SS activity present in developing endosperm. The relative activity of the two isozymes did not change significantly during the starch biosynthetic period. DU1 and zSSI may be interdependent, because mutant extracts lacking DU1 exhibited a significant stimulation of the remaining SS activity.     

Starch and the Control of Kernel Number in Maize at Low Water Potentials

After reproduction is initiated in plants, subsequent reproductive development is sometimes interrupted, which decreases the final number of seeds and fruits. We subjected maize (Zea mays L.) to low water potentials (psi(w)) that frequently cause this kind of failure. We observed metabolite pools and enzyme activities in the developing ovaries while we manipulated the sugar stream by feeding sucrose (Suc) to the stems. Low psi(w) imposed for 5 d around pollination allowed embryos to form, but abortion occurred and kernel number decreased markedly. The ovary contained starch that nearly disappeared during this abortion. Analyses showed that all of the intermediates in starch synthesis were depleted. However, when labeled Suc was fed to the stems, label arrived at the ovaries. Solute accumulated and caused osmotic adjustment. Suc accumulated, but other intermediates did not, showing that a partial block in starch synthesis occurred at the first step in Suc utilization. This step was mediated by invertase, which had low activity. Because of the block, Suc feeding only partially prevented starch disappearance and abortion. These results indicate that young embryos abort when the sugar stream is interrupted sufficiently to deplete starch during early ovary development, and this abortion results in a loss of mature seeds and fruits. At low psi(w), maintaining the sugar stream partially prevented the abortion, but invertase regulated the synthesis of ovary starch and partially prevented full recovery.     

玉米籽粒胚乳细胞增殖及其与淀粉充实的关系

用纤维素酶解离胚乳、滤膜法统计玉米胚乳细胞的数目,进一步借助Logistic方程模拟胚乳细胞增殖动态的结果表明,整个灌浆期间胚乳细胞增殖呈现“慢-快-慢”的变化趋势。授粉15d后,不同类型胚乳的细胞数目依序为普通玉米〉糯玉米〉甜玉米〉爆裂玉米;胚乳细胞数目主要取决于细胞的增殖速率,并与淀粉充实和粒重关系密切。胚乳发育前期以胚乳细胞增殖为主,后期以淀粉积累为主。     

Selection and the Cell Cycle: Positive Darwinian Selection in a Well-Known DNA Damage Response Pathway

Cancer is a common occurrence in multi-cellular organisms and is not strictly limited to the elderly in a population. It is therefore possible that individuals with genotypes that protect against early onset cancers have a selective advantage. In this study the patterns of mutation in the proteins of a well-studied DNA damage response pathway have been examined for evidence of adaptive evolutionary change. Using a maximum likelihood framework and the mammalian species phylogeny, together with codon models of evolution, selective pressure variation across the interacting network of proteins has been detected. The presence of signatures of adaptive evolution in BRCA1 and BRCA2 has already been documented but the effect on the entire network of interacting proteins in this damage response pathway has, until now, been unknown. Positive selection is evident throughout the network with a total of 11 proteins out of 15 examined displaying patterns of substitution characteristic of positive selection. It is also shown here that modern human populations display evidence of an ongoing selective sweep in 9 of these DNA damage repair proteins. The results presented here provide the community with new residues that may be relevant to cancer susceptibility while also highlighting those proteins where human and mouse have undergone lineage-specific functional shift. An understanding of this damage response pathway from an evolutionary perspective will undoubtedly contribute to future cancer treatment approaches.     

The Mac1 Gene: Controlling the Commitment to the Meiotic Pathway in Maize

The switch from the vegetative to the reproductive pathway of development in flowering plants requires the commitment of the subepidermal cells of the ovules and anthers to enter the meiotic pathway. These cells, the hypodermal cells, either directly or indirectly form the archesporial cells that, in turn, differentiate into the megasporocytes and microsporocytes. We have isolated a recessive pleiotropic mutation that we have termed multiple archesporial cells1 (mac1) and located it to the short arm of chromosome 10. Its cytological phenotype suggests that this locus plays an important role in the switch of the hypodermal cells from the vegetative to the meiotic (sporogenous) pathway in maize ovules. During normal ovule development in maize, only a single hypodermal cell develops into an archesporial cell and this differentiates into the single megasporocyte. In mac1 mutant ovules several hypodermal cells develop into archesporial cells, and the resulting megasporocytes undergo a normal meiosis. More than one megaspore survives in the tetrad and more than one embryo sac is formed in each ovule. Ears on mutant plants show partial sterility resulting from abnormalities in megaspore differentiation and embryo sac formation. The sporophytic expression of this gene is therefore also important for normal female gametophyte development.     

多胞质玉米胚乳淀粉粒性状的扫描电镜观察

１１种多胞质系玉米胚乳淀粉粒的扫描电镜观察表明：不同的细胞质对细胞核有不同程度的互作,３种甜质胞质玉米的胚乳淀粉粒多呈球形,排列紧密,存在一定的共性;４种雄性不育胞质玉米的胚乳淀粉粒多呈不规则形,除（Ｔ）Ｍｏ１７外,排列疏松。这１１种玉米胚乳淀粉粒的平均直径为９．７８μｍ￣１４．６９μｍ,通过玉米胚乳淀粉粒形态特征的观察,在玉米淀粉性状和玉米籽粒的商品价值关系上进行一定程度探索,为玉米的进一步发展     

High-throughput Screening of EMS Mutagenized Maize for Altered Starch Digestibility

Carbohydrate research increasingly is focused on changing the biochemical nature of starch to create more efficient substrates for biofuel production; parallel work is aimed at healthier foods for human consumption. A key factor in both of these efforts is the rate at which starch is digested by amylases. Starch digestibility is influenced heavily by genetically controlled factors including starch granular and molecular structure and composition. Maize mutant varieties with increased starch digestibility would help to make more cost-efficient biofuels. To identify such mutants among segregating families of ethyl methane sulfonate-mutagenized maize, we developed a miniaturized, high-throughput single kernel preparation and starch digestion assay that can process over 500 samples per week. In a preliminary screen of 480 families, we have identified 62 mutants with faster rates of digestion as compared to wild type and, in the same screen, an additional 53 lines with slower rates of digestion, thus tremendous potential health benefits. These mutants can be used for detailed structural analysis of starch and flour physical and chemical properties, factors that interact with starch in the cell and analysis of an apparently large number of genes that can impact rates of starch digestion.     

Pathway Evolution by Horizontal Transfer and Positive Selection Is Accommodated by Relaxed Negative Selection upon Upstream Pathway Genes in Purple Bacterial Carotenoid Biosynthesis

Horizontal gene transfer and selection are major forces driving microbial evolution. However, interactions between them are rarely studied. Phylogenetic analyses of purple bacterial carotenoid biosynthesis genes suggest two lineages: one producing spheroidenone and the other producing spirilloxanthin. Of the latter lineage, Rubrivivax gelatinosus S1 and Hoeflea phototrophica DFL-43 also or instead produce spheroidenone. Evolution of the spheroidenone pathway from that producing spirilloxanthin theoretically requires changes in the substrate specificity of upstream pathway enzymes and acquisition of a terminal ketolase (CrtA). In R. gelatinosus and likely also in H. phototrophica, CrtA was acquired from the Bacteroidetes, in which it functions as a hydroxylase. Estimation of nonsynonymous and synonymous mutations using several pairwise methods indicated positive selection upon both genes, consistent with their functional changes from hydroxylases to ketolases. Relaxed negative selection upon all other carotenoid biosynthetic genes in these organisms was also apparent, likely facilitating changes in their substrate specificities. Furthermore, all genes responsible for terminal carotenoid biosynthetic pathway steps were under reduced negative selection compared to those known to govern biosynthetic pathway specificity. Horizontal transfer of crtA into R. gelatinosus and H. phototrophica has therefore likely been promoted by (i) the apparent selective advantage of spheroidenone production relative to spirilloxanthin production, (ii) reduced negative selection upon other carotenoid biosynthetic genes, facilitating changes in their substrate specificities, and (iii) preexisting low enzyme substrate specificities due to relaxed negative selection. These results highlight the importance and complexity of selection acting upon both a horizontally transferred gene and the biochemical network into which it is integrating.Biochemical pathway evolution has been examined extensively, particularly regarding mechanisms by which novel functions can be generated, diversified, and maintained (9, 12). Best studied in this regard is the role of gene duplication followed by divergence, resulting in paralog families that, despite sharing a common evolutionary ancestor, possess different functions (10). In clonally reproducing organisms, such as bacteria and archaea, this type of diversification is further compounded by horizontal gene transfer (18), whereby a divergent ortholog from one organism is introduced into the metabolic network of another, thereby becoming a “xenolog” (29). Horizontal gene transfer between distantly related organisms is especially diversifying due to the likelihood of altering the genome structure or biochemical and regulatory networks of the recipient, in contrast to recombination between close relatives, which may promote genetic cohesion (33).Selection controls phenotypic diversity as a function of evolutionary fitness. Three scenarios can be detected from patterns of nucleotide substitutions (23): (i) positive selection, by which advantageous functionally divergent mutants are further optimized by increased mutational sampling of phenotypic space; (ii) negative (purifying) selection, by which deleterious mutations are purged; and (iii) neutral mutation, in which mutations that do not affect the selected phenotype accumulate, resulting in genetic drift. Considering horizontal transfer, selection will favor fixation of a horizontally transferred gene if its phenotype is advantageous and will disfavor it when either the gene product or the alterations that it causes in the host network are deleterious. Successful horizontal gene transfer resulting in gene fixation is the result of net evolutionary benefit for the host, due both to the horizontally transferred gene itself and to minimal suboptimal alteration of the host metabolic and genetic networks into which it is integrating. Genetic parasites such as plasmids, transposons, and integrated phages are exceptional in directly promoting their own retention.Carotenoids are isoprenoid pigments produced by many bacteria and fungi and all photosynthetic eukaryotes (7, 8). They are typically colored red, orange, and yellow due to their extensively conjugated polyene chains (7). In all photosynthetic organisms, carotenoids facilitate the assembly of the photosynthetic reaction center and interact with it as auxiliary light-harvesting pigments and antioxidant molecules (13-15). Indeed, the niche in which a particular phototroph lives is defined, at least in part, by the absorption spectrum of its pigments, including light-harvesting carotenoids (55). Carotenoids also modulate membrane fluidity and permeability (19); these functions remain poorly understood.In this study, the evolution of carotenoid biosynthesis by horizontal gene transfer and selection is evaluated in the purple bacteria, anoxygenic phototrophic Proteobacteria that can use reduced sulfur compounds as electron sources. Whereas phylogenetically most purple bacteria belong to the Alphaproteobacteria, some belonging to the Betaproteobacteria and Gammaproteobacteria have evolved by horizontal transfer of alphaproteobacterial photosynthetic superoperons, which include carotenoid biosynthetic genes (24, 37). Carotenoid biosynthesis in the purple bacteria (Fig. ​(Fig.1)1) begins with the condensation of two molecules of geranylgeranyl pyrophosphate by the phytoene synthase CrtB, forming phytoene. The phytoene desaturase CrtI then desaturates phytoene either three or four times, producing neurosporene or lycopene, respectively. Both of these intermediates are subsequently hydroxylated at the 1 position by the hydroxylase CrtC, desaturated at the 3 and 4 positions by the CrtI homolog CrtD, methylated at the 1-hydroxyl group by the methyltransferase CrtF, and, in spheroidenone- and 2,2′-diketospirilloxanthin-producing organisms, ketolated at the 2 position by the ketolase CrtA (Fig. ​(Fig.1).1). Considerable subpathway diversity also exists because of the potential for asymmetry between carotenoid ends.Open in a separate windowFIG. 1.Carotenoid biosynthetic pathway in purple bacteria. For simplicity, not all subpathways are shown. Carbon numbers for the ψ end group are shown for phytoene.Carotenoid biosynthesis has been well studied biochemically and genetically in the spheroidene-producing organisms Rhodobacter capsulatus (3, 4, 16, 45) and Rhodobacter sphaeroides (1, 31, 32) and the spirilloxanthin-producing organisms Bradyrhizobium sp. strain ORS278 (17), Thiocapsa roseopersicina (30), and Rubrivivax gelatinosus (16, 21, 22, 40, 41, 43, 52-54). The latter organism produces 2,2′-diketospirilloxanthin (lycopene derived), spheroidenone (neurosporene derived), and their precursors using the same enzymes. Pathway utilization in R. gelatinosus is determined primarily by the substrate specificities of CrtC, CrtD, and CrtI and by the rate of metabolic flux (52-54). These extensive biochemical and genetic studies provide a solid framework for sequence-based evolutionary analyses, making this pathway a valuable model with which to study biochemical pathway evolution.     

玉米淀粉分支酶基因启动子的克隆与功能分析

以玉米品种“吉糯1号”的基因组DNA为模板,通过PCR扩增得到玉米淀粉分支酶基因的启动子序列,克隆到pMD18-TVector上,经测序,该启动子大小为934bp。与已报道的序列比较仅有14个核苷酸发生改变,同源性为98.5%。用该启动子取代植物表达载体pBI121的35S启动子,与GUS基因编码区连接,构建成融合质粒pSBE-GUS。经农杆菌介导法转化烟草,获得了转基因植株。GUS活性检测结果表明,由该启动子序列引导的GUS基因能在种子中表达,而在其他组织中表达微弱或未表达,证实该启动子具有种子特异性表达的功能。     

The Impact of Viral RNA on Assembly Pathway Selection

Many single-stranded RNA viruses self-assemble their protein containers around their genomes. The roles that the RNA plays in this assembly process have mostly been ignored, resulting in a protein-centric view of assembly that is unable to explain adequately the fidelity and speed of assembly in such viruses. Using bacteriophage MS2, we demonstrate here via a combination of mass spectrometry and kinetic modelling how viral RNA can bias assembly towards only a small number of the many possible assembly pathways, thus increasing assembly efficiency. Assembly reactions have been studied in vitro using phage coat protein dimers, the known building block of the T = 3 shell, and short RNA stem-loops based on the translational operator of the replicase cistron, a 19 nt fragment (TR). Mass spectrometry has unambiguously identified two on-pathway intermediates in such reactions that have stoichiometry consistent with formation of either a particle 3-fold or 5-fold axis. These imply that there are at least two sub-pathways to the final capsid. The flux through each pathway is controlled by the length of the RNA stem-loop triggering the assembly reaction and this effect can be understood in structural terms. The kinetics of intermediate formation have been studied and show steady-state concentrations for intermediates between starting materials and the T = 3 shell, consistent with an assembly process in which all the steps are in equilibrium. These data have been used to derive a kinetic model of the assembly reaction that in turn allows us to determine the dominant assembly pathways explicitly, and to estimate the effect of the RNA on the free energy of association between the assembling protein subunits. The results reveal that there are only a small number of dominant assembly pathways, which vary depending on the relative ratios of RNA and protein. These results suggest that the genomic RNA plays significant roles in defining the precise assembly sub-pathway followed to create the final capsid.     

Starch Biosynthesis in Developing Wheat Grain : Evidence against the Direct Involvement of Triose Phosphates in the Metabolic Pathway

We have used ¹³C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of ¹³C between carbons atoms 1 and 6 of [1-¹³C] or [6-¹³C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the ¹³C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the ¹³C sugars were supplied in vivo to the intact plant. The ¹³C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of ¹³C in all glucosyl carbons was quantified by ¹³C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background ¹³C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data and with the current information on the intracellular distribution of enzymes in plant storage tissues.     

Starch Mobilization in Ultradried Seed of Maize (Zea mays L.) During Germination

The effects of ultradry storage on the starch mobilization in maize (Zea mays L.) seed after aging were investigated. The results indicated that there were no significant differences in the content of ATP,starch, and soluble sugar, as well as the activity of amylase, between ultradried seeds and seeds stored at -20 ℃ during germination. These results were consistent with the higher level of vigor of the ultradried seed. Sieve tube introduction of a fluorescence dye (carboxyl fluoresceindiacetate) and laser confocal microscopy were used to study the development of plasmodesmata in the ultradried seeds. The results indicated that plasmodesmata developed well in ultradried seeds. Fluorescence analysis also showed that the fluorescence intensity in the radicle of ultradried seeds was stronger than that in seeds with a higher moisture content. This suggests that ultradry treatment has no adverse effects on the seeds. After seed imbibition, cell orgaelles could be resumed. It is concluded that ultradry seed storage is beneficial for maintaining seed vigor and that starchy mobilization proceeds regularly during germination.     

Modulation of Sucrose and Starch Metabolism by Salicylic Acid Induces Thermotolerance in Spring Maize

Russian Journal of Plant Physiology - Two inbred lines of spring maize (Zea mays L.), CML 32 (stress tolerant) and LM 11 (stress susceptible) were taken to study the effect of salicylic acid (SA)...