The mechanism of Arabidopsis immutans variegation

The Arabidopsis immutans (im) is variegated with green sectors and white sectors containing defective plastids due to a nuclear gene mutation. IM is a plastid terminal oxidase (PTOX) sharing similarity with mitochondrial alternative oxidase (AOX). In order to better understand IM\u27s biological roles and variegation mechanism of im, I characterized im plants at different levels;A structural model of IM, in which the diiron reaction center is composed of two conserved histidine and four glutamate residues, was tested by mutagenesis in vitro and in planta. It that these six conserved residues were found essential for IM activity and do not tolerate changes. Mutagenesis screening of 14 other conserved residues showed that two residues are essential, and four are important but not essential for IM activity. A 16 aa sequence uniquely present in PTOX was also found to be required for PTOX activity and stability. Overexpression of AOX in chloroplasts functionally rescues the variegation phenotype of im. And AOX deleted of the dimerization domain is more efficient to compensate im variegation phenotype, while addition of IM exon-8 sequence costs AOX function in chloroplasts;A proteomic analysis suggested plastids in im white sectors were alive even without photosynthetic capacity. Defective plastids caused by photodamage induced a higher respiratory activity and various upregulated cytosolic proteins. Downregulated FtsZ1 level and microscopy analysis suggested that the division of photooxidized plastids were suppressed. Downregulation of IM to ~3% of wild type levels did not compromise plant growth suggests that IM is normally in excess. Phenotypical observations indicated that IM does not significantly affect proplastids or differentiated plastids, but plays an essential role in early plastid differentiation stages;A hypothesis was proposed to interpret im variegation mechanism. Proplastids develop into defective plastids due to photodamage without carotenoid protection; while some differentiating plastids escape photodamage and form chloroplasts due to shade effects. In a differentiating heteroplastidic cell, division of defective plastids was suppressed while chloroplasts maintain post-mitotic division. Consequently, homoplastidic and heteroplastidic differentiating cells with normal chloroplasts lead to green sector formation, while homoplastidic cells with defective plastids lead to white sector formation

Molecular Characterization of Magnesium Chelatase in Soybean [Glycine max (L.) Merr.]

Soybean (Glycine max) seed yields rely on the efficiency of photosynthesis, which is poorly understood in soybean. Chlorophyll, the major light harvesting pigment, is crucial for chloroplast biogenesis and photosynthesis. Magnesium chelatase catalyzes the insertion of Mg2+ into protoporphyrin IX in the first committed and key regulatory step of chlorophyll biosynthesis. It consists of three types of subunits, ChlI, ChlD, and ChlH. To gain a better knowledge of chlorophyll biosynthesis in soybean, we analyzed soybean Mg-chelatase subunits and their encoding genes. Soybean genome harbors 4 GmChlI genes, 2 GmChlD genes, and 3 GmChlH genes, likely evolved from two rounds of gene duplication events. The qRT-PCR analysis revealed that GmChlI, GmChlD, and GmChlH genes predominantly expressed in photosynthetic tissues, but the expression levels among paralogs are different. In silicon promoter analyses revealed these genes harbor different cis-regulatory elements in their promoter regions, suggesting they could differentially respond to various environmental and developmental signals. Subcellular localization analyses illustrated that GmChlI, GmChlD, and GmChlH isoforms are all localized in chloroplast, consistent with their functions. Yeast two hybrid and bimolecular fluorescence complementation (BiFC) assays showed each isoform has a potential to be assembled into the Mg-chelatase holocomplex. We expressed each GmChlI, GmChlD, and GmChlH isoform in Arabidopsis corresponding mutants, and results showed that 4 GmChlI and 2 GmChlD isoforms and GmChlH1 could rescue the severe phenotype of Arabidopsis mutants, indicating that they maintain normal biochemical functions in vivo. However, GmChlH2 and GmChlH3 could not completely rescue the chlorotic phenotype of Arabidopsis gun5-2 mutant, suggesting that the functions of these two proteins could be different from GmChlH1. Considering the differences shown on primary sequences, biochemical functions, and gene expression profiles, we conclude that the paralogs of each soybean Mg-chelatase subunit have diverged more or less during evolution. Soybean could have developed a complex regulatory mechanism to control chlorophyll content to adapt to different developmental and environmental situations

The mechanism of Arabidopsis immutans variegation

The Arabidopsis immutans (im) is variegated with green sectors and white sectors containing defective plastids due to a nuclear gene mutation. IM is a plastid terminal oxidase (PTOX) sharing similarity with mitochondrial alternative oxidase (AOX). In order to better understand IM's biological roles and variegation mechanism of im, I characterized im plants at different levels;A structural model of IM, in which the diiron reaction center is composed of two conserved histidine and four glutamate residues, was tested by mutagenesis in vitro and in planta. It that these six conserved residues were found essential for IM activity and do not tolerate changes. Mutagenesis screening of 14 other conserved residues showed that two residues are essential, and four are important but not essential for IM activity. A 16 aa sequence uniquely present in PTOX was also found to be required for PTOX activity and stability. Overexpression of AOX in chloroplasts functionally rescues the variegation phenotype of im. And AOX deleted of the dimerization domain is more efficient to compensate im variegation phenotype, while addition of IM exon-8 sequence costs AOX function in chloroplasts;A proteomic analysis suggested plastids in im white sectors were alive even without photosynthetic capacity. Defective plastids caused by photodamage induced a higher respiratory activity and various upregulated cytosolic proteins. Downregulated FtsZ1 level and microscopy analysis suggested that the division of photooxidized plastids were suppressed. Downregulation of IM to ~3% of wild type levels did not compromise plant growth suggests that IM is normally in excess. Phenotypical observations indicated that IM does not significantly affect proplastids or differentiated plastids, but plays an essential role in early plastid differentiation stages;A hypothesis was proposed to interpret im variegation mechanism. Proplastids develop into defective plastids due to photodamage without carotenoid protection; while some differentiating plastids escape photodamage and form chloroplasts due to shade effects. In a differentiating heteroplastidic cell, division of defective plastids was suppressed while chloroplasts maintain post-mitotic division. Consequently, homoplastidic and heteroplastidic differentiating cells with normal chloroplasts lead to green sector formation, while homoplastidic cells with defective plastids lead to white sector formation.</p

Basic Helix-Loop-Helix (bHLH) Transcription Factors Regulate a Wide Range of Functions in Arabidopsis

The basic helix-loop-helix (bHLH) transcription factor family is one of the largest transcription factor gene families in Arabidopsis thaliana, and contains a bHLH motif that is highly conserved throughout eukaryotic organisms. Members of this family have two conserved motifs, a basic DNA binding region and a helix-loop-helix (HLH) region. These proteins containing bHLH domain usually act as homo- or heterodimers to regulate the expression of their target genes, which are involved in many physiological processes and have a broad range of functions in biosynthesis, metabolism and transduction of plant hormones. Although there are a number of articles on different aspects to provide detailed information on this family in plants, an overall summary is not available. In this review, we summarize various aspects of related studies that provide an overview of insights into the pleiotropic regulatory roles of these transcription factors in plant growth and development, stress response, biochemical functions and the web of signaling networks. We then provide an overview of the functional profile of the bHLH family and the regulatory mechanisms of other proteins

PSB27: A thylakoid protein enabling Arabidopsis to adapt to changing light intensity

In earlier studies we have identified FKBP20-2 and CYP38 as soluble proteins of the chloroplast thylakoid lumen that are required for the formation of photosystem II supercomplexes (PSII SCs). Subsequent work has identified another potential candidate functional in SC formation (PSB27). We have followed up on this possibility and isolated mutants defective in the PSB27 gene. In addition to lack of PSII SCs, mutant plants were severely stunted when cultivated with light of variable intensity. The stunted growth was associated with lower PSII efficiency and defective starch accumulation. In response to high light exposure, the mutant plants also displayed enhanced ROS production, leading to decreased biosynthesis of anthocyanin. Unexpectedly, we detected a second defect in the mutant, namely in CP26, an antenna protein known to be required for the formation of PSII SCs that has been linked to state transitions. Lack of PSII SCs was found to be independent of PSB27, but was due to a mutation in the previously described cp26 gene that we found had no effect on light adaptation. The present results suggest that PSII SCs, despite being required for state transitions, are not associated with acclimation to changing light intensity. Our results are consistent with the conclusion that PSB27 plays an essential role in enabling plants to adapt to fluctuating light intensity through a mechanism distinct from photosystem II supercomplexes and state transitions

Impacts of Cropping Systems on Aggregates Associated Organic Carbon and Nitrogen in a Semiarid Highland Agroecosystem.

The effect of cropping system on the distribution of organic carbon (OC) and nitrogen (N) in soil aggregates has not been well addressed, which is important for understanding the sequestration of OC and N in agricultural soils. We analyzed the distribution of OC and N associated with soil aggregates in three unfertilized cropping systems in a 27-year field experiment: continuously cropped alfalfa, continuously cropped wheat and a legume-grain rotation. The objectives were to understand the effect of cropping system on the distribution of OC and N in aggregates and to examine the relationships between the changes in OC and N stocks in total soils and in aggregates. The cropping systems increased the stocks of OC and N in total soils (0-40 cm) at mean rates of 15.6 g OC m-2 yr-1 and 1.2 g N m-2 yr-1 relative to a fallow control. The continuous cropping of alfalfa produced the largest increases at the 0-20 cm depth. The OC and N stocks in total soils were significantly correlated with the changes in the >0.053 mm aggregates. 27-year of cropping increased OC stocks in the >0.053 mm size class of aggregates and N stocks in the >0.25 mm size class but decreased OC stocks in the 0.25 mm aggregate size class accounted for more than 97% of the total increases in the continuous wheat and the legume-grain rotation systems. These results suggested that long-term cropping has the potential to sequester OC and N in soils and that the increases in soil OC and N stocks were mainly due to increases associated with aggregates >0.053 mm

A Proteomic Analysis of Maize Chloroplast Biogenesis

Proteomics studies to explore global patterns of protein expression in plant and green algal systems have proliferated within the past few years. Although most of these studies have involved mapping of the proteomes of various organs, tissues, cells, or organelles, comparative proteomics experiments have also led to the identification of proteins that change in abundance in various developmental or physiological contexts. Despite the growing use of proteomics in plant studies, questions of reproducibility have not generally been addressed, nor have quantitative methods been widely used, for example, to identify protein expression classes. In this report, we use the de-etiolation (“greening”) of maize (Zea mays) chloroplasts as a model system to explore these questions, and we outline a reproducible protocol to identify changes in the plastid proteome that occur during the greening process using techniques of two-dimensional gel electrophoresis and mass spectrometry. We also evaluate hierarchical and nonhierarchical statistical methods to analyze the patterns of expression of 526 “high-quality,” unique spots on the two-dimensional gels. We conclude that Adaptive Resonance Theory 2—a nonhierarchical, neural clustering technique that has not been previously applied to gene expression data—is a powerful technique for discriminating protein expression classes during greening. Our experiments provide a foundation for the use of proteomics in the design of experiments to address fundamental questions in plant physiology and molecular biology

The effects of cropping systems on soil bulk density at the 0–20 and 20–40 cm depths.

<p>FA: Fallow treatment, CA: Continuously cropped alfalfa system; CW: continuously cropped winter wheat system; LG: Legume-grain rotation system.</p