Lack of fructose 2,6‐bisphosphate compromises photosynthesis and growth in Arabidopsis in fluctuating environments

The balance between carbon assimilation, storage and utilisation during photosynthesis is dependent on partitioning of photoassimilate between starch and sucrose, and varies in response to changes in the environment. However, the extent to which the capacity to modulate carbon partitioning rapidly through short‐term allosteric regulation may contribute to plant performance is unknown. Here we examine the physiological role of fructose 2,6‐bisphosphate (Fru‐2,6‐P₂) during photosynthesis, growth and reproduction in Arabidopsis thaliana (L.). In leaves this signal metabolite contributes to coordination of carbon assimilation and partitioning during photosynthesis by allosterically modulating the activity of cytosolic fructose‐1,6‐bisphosphatase. Three independent T‐DNA insertional mutant lines deficient in 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (F2KP), the bifunctional enzyme responsible for both the synthesis and degradation of Fru‐2,6‐P₂, lack Fru‐2,6‐P₂. These plants have normal steady‐state rates of photosynthesis, but exhibit increased partitioning of photoassimilate into sucrose and have delayed photosynthetic induction kinetics. The F2KP‐deficient plants grow normally in constant environments, but show reduced growth and seed yields relative to wildtype plants in fluctuating light and/or temperature. We conclude that Fru‐2,6‐P₂ is required for optimum regulation of photosynthetic carbon metabolism under variable growth conditions. These analyses suggest that the capacity of Fru‐2,6‐P₂ to modulate partitioning of photoassimilate is an important determinant of growth and fitness in natural environments.     

Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue

In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl‐constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas reinhardtii (Chlamydomonas) does not use the ER pathway; however, the Chlamydomonas genome encodes presumed plant orthologues of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER‐to‐chloroplast lipid trafficking in plants. To resolve this conundrum, we identified a mutant of Chlamydomonas deleted in the TGD2 gene and characterized the respective protein, CrTGD2. Notably, the viability of the mutant was reduced, showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldiacylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conversion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impaired in the tgd2 mutant.     

Structures of Arg‐ and Gln‐type bacterial cysteine dioxygenase homologs

In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ～15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg‐type” enzymes) and some having a Gln substituted for this Arg (“Gln‐type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg‐type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln‐type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron‐bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln‐type” CDO enzymes, we conclude that the “Gln‐type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3‐mercaptopropionate dioxygenases.     

编者按: 脂代谢与心血管疾病

心血管病是我国成年人致死的最主要的疾病,而脂代谢异常是心血管疾病的独立危险因素。因此,阐述研究揭示脂代谢异常在心血管疾病发生发展中的作用及其机制,具有重要的理论意义和临床实用价值。本期专题主要一方面综合评述了脂蛋白组分与脂质代谢,以及甘氨酸的心血管疾病保护作用等相关领域的研究新进展,同时并分别展示了国内学者有关于PCSK9/LDLR通路、模拟人apoE结构域的小分子多肽EpK、多不饱和脂肪酸、脂联素、LXRα- ABCA1途径和普罗布考等在脂代谢中的作用及相应分子机制等方面的研究成果,以期让更多的人进一步深入了解脂质代谢以及,了解心血管病发病的复杂机制和及研究现状。     

外源H_2S对NO_3~-胁迫下番茄幼苗生理生化特性的影响

采用营养液水培方法,通过外源施加H2S供体NaHS(100μmol/L),研究了信号分子H2S对100mmol/L NO3-胁迫下番茄幼苗生理生化特性的影响。结果表明:(1)NO3-胁迫下,随着处理时间的延长,番茄幼苗的株高、根长、鲜重和干重显著降低,叶绿素(a、b)含量、净光合速率、气孔导度、蒸腾速率均显著降低,而胞间CO2浓度以及丙二醛(MDA)、H2O2含量增加,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性显著降低,抗坏血酸(AsA)和还原性谷胱甘肽(GSH)含量显著降低。(2)与NO3-胁迫处理相比,外源NaHS处理1、3、5d后,番茄幼苗的株高、根长、鲜重和干重显著增加,叶绿素(a、b)含量、净光合速率、气孔导度、蒸腾速率均显著升高,而胞间CO2浓度显著降低;MDA和H2O2含量降低,SOD、POD、CAT和APX活性显著增强,AsA和GSH含量显著增加,而且幼苗的硝酸还原酶、谷氨酰胺合成酶、谷氨酸合酶的活性显著增强;L-半胱氨酸脱巯基酶活性和内源H2S含量增加。研究认为,外源H2S可能通过提高抗氧化物酶的活性和增加抗氧化物质含量来缓解NO3-对番茄幼苗造成的伤害,从而增强其对NO3-胁迫耐性。     

烟草叶片对不同光质短期应答的代谢轮廓

在烟草上部叶片生理成熟期给以白、紫外A、蓝、绿、黄、红6种LED灯光处理,研究了烟草叶片对不同光质的短期应答.结果表明: 采用非靶标检测方法共检测到68种GC/MS(气相色谱/质谱)稳定的代谢产物,在PLS-DA得分图中,6种光处理的样品被明显分开;有61种在质谱库中得到鉴定,其中45种代谢物质含量在不同光处理间存在显著性差异,主要为有机酸类、糖类、TCA循环中间产物、氨基酸类等初级代谢产物;采用差异性代谢物的热力图及聚类分析将其分为5类,同时也将6个处理明显分开,其中红、蓝光处理差异最为明显,除B类物质外,其他4类均以红光处理高于蓝光处理.采用靶标性检测方法测定了3种生物碱、5种多酚及茄尼醇的含量,4种(加上非靶标测定到的烟碱)生物碱在不同光质处理间的变化趋势一致,红光和黄光明显促进了生物碱积累;莰菲醇基芸香苷和芸香苷在不同处理之间的变化趋势一致,以蓝光处理最低、黄光最高,光质对其他3种多酚的影响各异;黄光明显抑制了茄尼醇的积累,蓝光处理的叶片中茄尼醇含量最高.说明光质变化对烟草脂肪酸代谢、糖代谢、生物碱代谢、氨基酸代谢、TCA循环和莽草酸途径等多条代谢途径均有显著的影响.     

The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase

Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2′/3′-O-(N′-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a K_D = 15.6 μm and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased K_D for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life.     

The Conserved Modular Elements of the Acyl Carrier Proteins of Lipid Synthesis Are Only Partially Interchangeable

Prior work showed that expression of acyl carrier proteins (ACPs) of a diverse set of bacteria replaced the function of Escherichia coli ACP in lipid biosynthesis. However, the AcpAs of Lactococcus lactis and Enterococcus faecalis were inactive. Both failed to support growth of an E. coli acpP mutant strain. This defect seemed likely because of the helix II sequences of the two AcpAs, which differed markedly from those of the proteins that supported growth. To test this premise, chimeric ACPs were constructed in which L. lactis helix II replaced helix II of E. coli AcpP and vice versa. Expression of the AcpP protein L. lactis AcpA helix II allowed weak growth, whereas the L. lactis AcpA-derived protein that contained E. coli AcpP helix II failed to support growth of the E. coli mutant strain. Replacement of the L. lactis AcpA helix II residues in this protein showed that substitution of valine for the phenylalanine residue four residues downstream of the phosphopanthetheine-modified serine gave robust growth and allowed modification by the endogenous AcpS phosphopantetheinyl transferase (rather than the promiscuous Sfp transferase required to modify the L. lactis AcpA and the chimera of L. lactis AcpA helix II in AcpP). Further chimera constructs showed that the lack of function of the L. lactis AcpA-derived protein containing E. coli AcpP helix II was due to incompatibility of L. lactis AcpA helix I with the downstream elements of AcpP. Therefore, the origins of ACP incompatibility can reside in either helix I or in helix II.