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101.
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Jessica Jüppner Umarah Mubeen Andrea Leisse Camila Caldana Henrike Brust Martin Steup Marion Herrmann Dirk Steinhauser Patrick Giavalisco 《The Plant journal : for cell and molecular biology》2017,92(2):331-343
Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well‐established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi‐omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two‐phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10–15 million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time‐resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in‐depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems. 相似文献
103.
Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings 下载免费PDF全文
Fu‐Yuan Zhu Mo‐Xian Chen Neng‐Hui Ye Lu Shi Kai‐Long Ma Jing‐Fang Yang Yun‐Ying Cao Youjun Zhang Takuya Yoshida Alisdair R. Fernie Guang‐Yi Fan Bo Wen Ruo Zhou Tie‐Yuan Liu Tao Fan Bei Gao Di Zhang Ge‐Fei Hao Shi Xiao Ying‐Gao Liu Jianhua Zhang 《The Plant journal : for cell and molecular biology》2017,91(3):518-533
In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues; however, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of short‐read RNA sequencing, single molecule long‐read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data from the two sequencing methods, approximately 83.4% of intron‐containing genes were alternatively spliced. Two AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR); however, by contrast to AS events detected under normal conditions, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern, indicated by the increasing number of non‐conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding of AS and alternative translation mechanisms under normal conditions, and in response to ABA treatment. 相似文献
104.
An oligogalacturonide‐derived molecular probe demonstrates the dynamics of calcium‐mediated pectin complexation in cell walls of tip‐growing structures 下载免费PDF全文
Jozef Mravec Stjepan K. Kračun Maja G. Rydahl Bjørge Westereng Daniela Pontiggia Giulia De Lorenzo David S. Domozych William G. T. Willats 《The Plant journal : for cell and molecular biology》2017,91(3):534-546
Pectic homogalacturonan (HG) is one of the main constituents of plant cell walls. When processed to low degrees of esterification, HG can form complexes with divalent calcium ions. These macromolecular structures (also called egg boxes) play an important role in determining the biomechanics of cell walls and in mediating cell‐to‐cell adhesion. Current immunological methods enable only steady‐state detection of egg box formation in situ. Here we present a tool for efficient real‐time visualisation of available sites for HG crosslinking within cell wall microdomains. Our approach is based on calcium‐mediated binding of fluorescently tagged long oligogalacturonides (OGs) with endogenous de‐esterified HG. We established that more than seven galacturonic acid residues in the HG chain are required to form a stable complex with endogenous HG through calcium complexation in situ, confirming a recently suggested thermodynamic model. Using defined carbohydrate microarrays, we show that the long OG probe binds exclusively to HG that has a very low degree of esterification and in the presence of divalent ions. We used this probe to study real‐time dynamics of HG during elongation of Arabidopsis pollen tubes and root hairs. Our results suggest a different spatial organisation of incorporation and processing of HG in the cell walls of these two tip‐growing structures. 相似文献
105.
Amplification of ABA biosynthesis and signaling through a positive feedback mechanism in seeds 总被引:1,自引:0,他引:1
Mariko Nonogaki Khadidiatou Sall Eiji Nambara Hiroyuki Nonogaki 《The Plant journal : for cell and molecular biology》2014,78(3):527-539
Abscisic acid is an essential hormone for seed dormancy. Our previous study using the plant gene switch system, a chemically induced gene expression system, demonstrated that induction of 9‐cis‐epoxycarotenoid dioxygenase (NCED), a rate‐limiting ABA biosynthesis gene, was sufficient to suppress germination in imbibed Arabidopsis seeds. Here, we report development of an efficient experimental system that causes amplification of NCED expression during seed maturation. The system was created with a Triticum aestivum promoter containing ABA responsive elements (ABREs) and a Sorghum bicolor NCED to cause ABA‐stimulated ABA biosynthesis and signaling, through a positive feedback mechanism. The chimeric gene pABRE:NCED enhanced NCED and ABF (ABRE‐binding factor) expression in Arabidopsis Columbia‐0 seeds, which caused 9‐ to 73‐fold increases in ABA levels. The pABRE:NCED seeds exhibited unusually deep dormancy which lasted for more than 3 months. Interestingly, the amplified ABA pathways also caused enhanced expression of Arabidopsis NCED5, revealing the presence of positive feedback in the native system. These results demonstrated the robustness of positive feedback mechanisms and the significance of NCED expression, or single metabolic change, during seed maturation. The pABRE:NCED system provides an excellent experimental system producing dormant and non‐dormant seeds of the same maternal origin, which differ only in zygotic ABA. The pABRE:NCED seeds contain a GFP marker which enables seed sorting between transgenic and null segregants and are ideal for comparative analysis. In addition to its utility in basic research, the system can also be applied to prevention of pre‐harvest sprouting during crop production, and therefore contributes to translational biology. 相似文献
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Constable C 《Bioethics》2012,26(3):157-163
In the United States, the decision of whether to withdraw or continue to provide artificial nutrition and hydration (ANH) for patients in a permanent vegetative state (PVS) is placed largely in the hands of surrogate decision-makers, such as spouses and immediate family members. This practice would seem to be consistent with a strong national emphasis on autonomy and patient-centered healthcare. When there is ambiguity as to the patient's advanced wishes, the presumption has been that decisions should weigh in favor of maintaining life, and therefore, that it is the withdrawal rather than the continuation of ANH that requires particular justification. I will argue that this default position should be reversed. Instead, I will argue that the burden of justification lies with those who would continue artificial nutrition and hydration (ANH), and in the absence of knowledge as to the patient's advanced wishes, it is better to discontinue ANH. In particular, I will argue that among patients in PVS, there is not a compelling interest in being kept alive; that in general, we commit a worse violation of autonomy by continuing ANH when the patient's wishes are unknown; and that more likely than not, the maintenance of ANH as a bridge to a theoretical future time of recovery goes against the best interests of the patient. 相似文献
109.
Telomere truncation has been shown to be an efficient technology for the creation of mini-chromosomes that can be used as artificial chromosome platforms for genetic engineering. Artificial chromosome-based genetic engineering is considered to be superior to the existing techniques of randomized gene integration by Agrobacterium or biolistic-mediated genetic transformation. It organizes multiple transgenes as a unique genetic linkage block for subsequent manipulations in breeding. Telomere truncation technology relies on three components: the telomere sequence that mediates chromosomal truncation, a selection marker that allows the selection of transgenic events, and a site-specific recombination system that can be used to accept future genes into the mini-chromosome by gene targeting. These elements are usually pre-assembled before transformation, a process that is both time and labor consuming. We found in this research that the three elements could be mixed to transform plant cells in a biolistic transformation, and produced efficient chromosomal truncations and mini-chromosomes in rice. This system will allow rapid construction of mini-chromosomes with a flexible selection of resistant markers, site-specific recombination systems and other desirable elements. In addition, a rice telotrisomic line was used as the starting material for chromosomal truncations. Mini-chromosomes from the truncations of both the telocentric chromosome and other chromosomes were recovered. The mini-chromosomes remained stable during 2 years of subculture. The construction of mini-chromosomes in rice, an economically important crop, will provide a platform for future artificial chromosome-based genetic engineering of rice for stacking multiple genes. 相似文献
110.