Genome-Wide Analysis Regulatory Network of Photomorphogenesis Revealed the Complex Brassinosteroid Effects in

Light and brassinosteroids （BRs） have been proved to be crucial in regulating plant growth and development; however, the mechanism of how they synergistically function is still largely unknown. To explore the underlying mechanisms in photomorphogenesis, genome-wide analyses were carried out through examining the gene expressions of the dark-grown WT or BR biosynthesis-defective mutant det2 seedlings in the presence of light stimuli or exogenous Brassinolide （BL）. Results showed that BR deficiency stimulates, while BL treatment suppresses, the expressions of lightresponsive genes and photomorphogenesis, confirming the negative effects of BR in photomorphogenesis. This is consistent with the specific effects of BR on the expression of genes involved in cell wall modification, cellular metabolism and energy utilization during dark-light transition. Further analysis revealed that hormone biosynthesis and signaling-related genes, especially those of auxin, were altered under BL treatment or light stimuli, indicating that BR may modulate photomorphogenesis through synergetic regulation with other hormones. Additionally, suppressed ubiquitin-cycle pathway during light-dark transition hinted the presence of a complicated network among light, hormone, and protein degradation. The study provides the direct evidence of BR effects in photomorphogenesis and identified the genes involved in BR and light signaling pathway, which will help to elucidate the molecular mechanism of plant photomorphogenesis.     

RNA Sequencing of Laser-Capture Microdissected Compartments of the Maize Kernel Identifies Regulatory Modules Associated with Endosperm Cell Differentiation

Endosperm is an absorptive structure that supports embryo development or seedling germination in angiosperms. The endosperm of cereals is a main source of food, feed, and industrial raw materials worldwide. However, the genetic networks that regulate endosperm cell differentiation remain largely unclear. As a first step toward characterizing these networks, we profiled the mRNAs in five major cell types of the differentiating endosperm and in the embryo and four maternal compartments of the maize (Zea mays) kernel. Comparisons of these mRNA populations revealed the diverged gene expression programs between filial and maternal compartments and an unexpected close correlation between embryo and the aleurone layer of endosperm. Gene coexpression network analysis identified coexpression modules associated with single or multiple kernel compartments including modules for the endosperm cell types, some of which showed enrichment of previously identified temporally activated and/or imprinted genes. Detailed analyses of a coexpression module highly correlated with the basal endosperm transfer layer (BETL) identified a regulatory module activated by MRP-1, a regulator of BETL differentiation and function. These results provide a high-resolution atlas of gene activity in the compartments of the maize kernel and help to uncover the regulatory modules associated with the differentiation of the major endosperm cell types.     

应用系统遗传学与细胞发生的基因调控网络序列标记显示分析

基因型-表现型复杂系统自组织化是基因系统到蛋白质系统、代谢酶系统的遗传信息转换过程。一个协同表达的基因群调控一个相对独立性状的功能模块,基因网络的自组织化建构基因组稳态与遗传适应过程。腺垂体干细胞分化成5种不同的内分泌细胞系,受上丘脑和性腺、胰岛细胞等激素的调控,涉及系列转录因子的诱导表达,成为细胞系发生研究的模型。GH基因的表达受上丘脑激素GRF、GHRP-6刺激以及SMS抑制,经不同受体、G蛋白亚单元和PKA、PKC信号传导路径,转录因子调控细胞再生或GH基因表达、激素分泌。基因表达调控决定于基因序列,如启动子、非翻译RNA区、蛋白质的结构域等,系统生物学包括组学、计算与合成生物技术,序列标志片段显示(STFD),可用于细胞系分化、病理变化等基因表达谱、信号传导网络的系统分析与功能基因克隆。     

New Experimental Approaches to Analysis of Complex Forms of Behavior

The interest of neurophysiologists in clarifying the mechanisms of the most complex function of the brain, namely, the rational activity of animals and man, has grown in recent years [1-3, 5, 7, 12-14, 16]. Contemporary physiology of higher nervous activity embraces the most contradictory views concerning this problem, even going so far as to deny that it exists. Clearly, more experimental studies are necessary for us to reach an understanding of the laws of development of rational activity and its specific manifestations.     

Studies of the Kinetic Mechanism of Maize Endosperm ADP-Glucose Pyrophosphorylase Uncovered Complex Regulatory Properties

ADP-glucose pyrophosphorylase catalyzes the synthesis of ADP-glucose (ADP-Glc) from Glc-1-phosphate (G-1-P) and ATP. Kinetic studies were performed to define the nature of the reaction, both in the presence and absence of allosteric effector molecules. When 3-phosphoglycerate (3-PGA), the putative physiological activator, was present at a saturating level, initial velocity studies were consistent with a Theorell-Chance BiBi mechanism and product inhibition data supported sequential binding of ATP and G-1-P, followed by ordered release of pyrophosphate and ADP-Glc. A sequential mechanism was also followed when 3-PGA was absent, but product inhibition patterns changed dramatically. In the presence of 3-PGA, ADP-Glc is a competitive inhibitor with respect to ATP. In the absence of 3-PGA—with or without 5.0 mm inorganic phosphate—ADP-Glc actually stimulated catalytic activity, acting as a feedback product activator. By contrast, the other product, pyrophosphate, is a potent inhibitor in the absence of 3-PGA. In the presence of subsaturating levels of allosteric effectors, G-1-P serves not only as a substrate but also as an activator. Finally, in the absence of 3-PGA, inorganic phosphate, a classic inhibitor or antiactivator of the enzyme, stimulates enzyme activity at low substrate by lowering the K_M values for both substrates.Plant ADP-Glc pyrophosphorylase (AGPase) catalyzes an important, rate-limiting step in starch biosynthesis: the reversible formation of ADP-Glc from ATP and Glc-1-P (G-1-P). Most AGPases are regulated by effector molecules derived from the prevalent carbon metabolism pathway, with inorganic phosphate (P_i) and 3-phosphoglycerate (3-PGA) being the most studied effectors of higher plants. Interestingly, the barley (Hordeum vulgare) endosperm form of AGPase is unique among higher plant homologs in its insensitivity to both 3-PGA and P_i (Kleczkowski et al., 1993a). Heat lability (as often found for endosperm AGPases) and reductive activation (for those AGPases harboring an N-terminal Cys residue in the small subunit) are also important mechanisms by which AGPases are regulated (Fu et al., 1998; Tiessen et al., 2002).Transgenic plant studies emphasize the importance of allosteric effectors in controlling enzyme activity and, in turn, starch yield. For example, expressing an allosterically enhanced Escherichia coli AGPase resulted in a 35% increase in potato (Solanum tuberosum) tuber starch (Stark et al., 1992) and a 22% to 25% increase in maize (Zea mays) seed starch (Wang et al., 2007). Rice (Oryza sativa) seed weight was increased up to 11% by expression of a second E. coli-derived AGPase mutant with altered allosteric properties (Sakulsingharoj et al., 2004). In another example, expressing a maize AGPase variant with less sensitivity to P_i and enhanced heat stability led to a 38% increase in wheat (Triticum aestivum) yield (Smidansky et al., 2002), a 23% increase in rice yield (Smidansky et al., 2003), and up to a 68% increase in maize yield (L.C. Hannah, unpublished data). Increases in these cases were due to enhanced seed number. Finally, transgenic expression of an allosterically altered potato tuber AGPase enhanced Arabidopsis (Arabidopsis thaliana) leaf transitory starch turnover and improved growth characteristics (Obana et al., 2006) and enhanced the fresh weight of aerial parts of lettuce (Lactuca sativa) plants (Lee et al., 2009).In higher plants, AGPase is a heterotetramer, consisting of two large and two small subunits; by contrast, most bacterial AGPases are homotetramers. Crystal structures of a bacterial AGPase and a nonnative, small subunit homotetramer derived from the potato tuber enzyme have been described recently (Jin et al., 2005; Cupp-Vickery et al., 2008). Unfortunately, since both structures were determined in the presence of high sulfate concentrations, both enzymes are in inactive forms.While AGPase allosteric regulation has received a great deal of attention, the kinetic mechanism has been defined completely only for two cases: the homotetrameric form from the bacterium Rhodospirillum rubrum and the plant heterotetrameric enzyme from barley leaf (Paule and Preiss, 1971; Kleczkowski et al., 1993b). The kinetic mechanism is sequential in both cases, with ATP the first substrate bound and ADP-Glc the final product released. Despite this similarity, there are important differences, most notably the existence of an isomerization step following ADP-Glc release, so that this product and ATP bind to different forms of the barley enzyme. This isomerization step is absent from the bacterial enzyme. Interestingly, isoforms of the closely related nucleoside diphospho-Glc family exhibit fundamentally different kinetic mechanisms. Some UDP-Glc pyrophosphorylases catalyze a sequential BiBi mechanism (Elling, 1996), while others, such as dTDP-Glc and CDP-Glc pyrophosphorylases from Salmonella, employ a ping-pong mechanism (Lindqvist et al., 1993, 1994).Because of the mechanistic diversity exhibited by pyrophosphorylases in general and by the two well-characterized AGPases in particular, we investigated the kinetic mechanism of the recombinant maize endosperm AGPase. We were particularly interested in the roles played by allosteric effectors that appear to be critically important in catalytic efficiency and, thus, starch content. Surprisingly, patterns of initial velocity at varying substrate concentrations as well as product inhibition behavior were identical to those observed for the homotetrameric bacterial enzyme (Paule and Preiss, 1971) and differed significantly from the heterotetrameric barley leaf enzyme (Kleczkowski et al., 1993b). Moreover, we found that both G-1-P and ADP-Glc could stimulate AGPase catalytic activity beyond that expected for simple substrate effects. We also found that the classic inhibitor, P_i, actually enhanced AGPase activity at low substrate concentrations but inhibited activity at high substrate levels. A model is presented to account for this observation. Finally, we determined that 3-PGA only stimulates AGPase activity by 2.5-fold if care is taken to saturate with substrates during assays.     

A Quantitative and Dynamic Model of the Arabidopsis Flowering Time Gene Regulatory Network

Various environmental signals integrate into a network of floral regulatory genes leading to the final decision on when to flower. Although a wealth of qualitative knowledge is available on how flowering time genes regulate each other, only a few studies incorporated this knowledge into predictive models. Such models are invaluable as they enable to investigate how various types of inputs are combined to give a quantitative readout. To investigate the effect of gene expression disturbances on flowering time, we developed a dynamic model for the regulation of flowering time in Arabidopsis thaliana. Model parameters were estimated based on expression time-courses for relevant genes, and a consistent set of flowering times for plants of various genetic backgrounds. Validation was performed by predicting changes in expression level in mutant backgrounds and comparing these predictions with independent expression data, and by comparison of predicted and experimental flowering times for several double mutants. Remarkably, the model predicts that a disturbance in a particular gene has not necessarily the largest impact on directly connected genes. For example, the model predicts that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) mutation has a larger impact on APETALA1 (AP1), which is not directly regulated by SOC1, compared to its effect on LEAFY (LFY) which is under direct control of SOC1. This was confirmed by expression data. Another model prediction involves the importance of cooperativity in the regulation of APETALA1 (AP1) by LFY, a prediction supported by experimental evidence. Concluding, our model for flowering time gene regulation enables to address how different quantitative inputs are combined into one quantitative output, flowering time.     

Combining Theoretical and Experimental Approaches to Understand the Circadian Clock

This review is intended as a summary of our work carried out as part of the German Research Association (DFG) Center Program on Circadian Rhythms. Over the last six years, our approach to understanding circadian systems combined theoretical and experimental tools, and Gonyaulax and Neurospora have proven ideal for these efforts. Both of these model organisms demonstrate that even simple circadian systems can have multiple light input pathways and more than one rhythm generator. They have both been used to elaborate basic circadian features in conjunction with formal models. The models introduce the “zeitnehmer,” i.e., a clock‐regulated input pathway, to the conceptual framework of circadian systems, and proposes networks of individual feedbacks as the basis for circadian rhythmicity.     

Quantitative Analysis of Approaches to Measure Cooperative Phosphate Release in Polymerized Actin

We use stochastic simulations that treat several experimental probes of actin dynamics to explore the extent to which phosphate dissociation in filamentous actin may be cooperative. Phosphate time-courses from polymerization and copolymerization experiments of ATP- and ADP-actin are studied, including the effects of variations in filament-number concentration as well as single-filament depolymerization time-courses. We find that highly cooperative models are consistent with the treated experimental data. We also find that some types of experiments that are believed to provide strong constraints on the cooperativity of actin hydrolysis models do not provide such constraints.     

Dynamic Changes in ANGUSTIFOLIA3 Complex Composition Reveal a Growth Regulatory Mechanism in the Maize Leaf

Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growth-regulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated.