Array and distribution of actin filaments in guard cells contribute to the determination of stomatal aperture

Actin filaments in guard cells and their dynamics function in regulating stomatal movement. In this study, the array and distribution of actin filaments in guard cells during stomatal movement were studied with two vital labeling, microinjection of alexa-phalloidin in Vicia faba and expression of GFP-mTn in tobacco. We found that the random array of actin filaments in the most of the closed stomata changed to a ring-like array after stomatal open. And actin filaments, which were throughout the cytoplasm of guard cells of closed stomata (even distribution), were mainly found in the cortical cytoplasm in the case of open stomata (cortical distribution). These results revealed that the random array and even distribution of actin filaments in guard cells may be required for keeping the closed stomata; similarly, the ring-like array and cortical distribution of actin filaments function in sustaining open stomata. Furthermore, we found that actin depolymerization, the trait of moving stomata, facilitates the transformation of actin array and distribution with stomatal movement. So, the depolymerization of actin filaments was favorable for the changes of actin array and distribution in guard cells and thus facilitated stomatal movement.     

NADK2, an <Emphasis Type="Italic">Arabidopsis</Emphasis> Chloroplastic NAD Kinase,Plays a Vital Role in Both Chlorophyll Synthesis and Chloroplast Protection

As one of terminal electron acceptors in photosynthetic electron transport chain, NADP receives electron and H⁺ to synthesize NADPH, an important reducing energy in chlorophyll synthesis and Calvin cycle. NAD kinase (NADK), the catalyzing enzyme for the de novo synthesis of NADP from substrates NAD and ATP, may play an important role in the synthesis of NADPH. NADK activity has been observed in different sub-cellular fractions of mitochondria, chloroplast, and cytoplasm. Recently, two distinct NADK isoforms (NADK1 and NADK2) have been identified in Arabidopsis. However, the physiological roles of NADKs remain unclear. In present study, we investigated the physiological role of Arabidiposis NADK2. Sub-cellular localization of the NADK2–GFP fusion protein indicated that the NADK2 protein was localized in the chloroplast. The NADK2 knock out mutant (nadk2) showed obvious growth inhibition and smaller rosette leaves with a pale yellow color. Parallel to the reduced chlorophyll content, the expression levels of two POR genes, encoding key enzymes in chlorophyll synthesis, were down regulated in the nadk2 plants. The nadk2 plants also displayed hypersensitivity to environmental stresses provoking oxidative stress, such as UVB, drought, heat shock and salinity. These results suggest that NADK2 may be a chloroplast NAD kinase and play a vital role in chlorophyll synthesis and chloroplast protection against oxidative damage.     

水分胁迫下蚕豆（Vicia faba L．）气孔关闭与叶片细胞中ABA区隔化与再分配的关系

应用胶体金免疫电镜定位技术研究水分胁迫下叶片ＡＢＡ的再分配。表明：水分胁迫初期蚕豆叶肉细胞中ＡＢＡ的量与对照相比无明显变化,但水分胁迫可导致表皮细胞质外体ＡＢＡ含量明显增加;保卫细胞在水分胁迫前即含有大量ＡＢＡ;ＡＢＡ主要分布在叶绿体和细胞核,细胞的腹壁及相邻外壁和内壁也有大量ＡＢＡ存在,但背壁ＡＢＡ的量很少;气孔完全开放时背壁ＡＢＡ含量更少,当水分胁迫导致气孔关闭时,保卫细胞背壁ＡＢＡ含量大增,说明气孔运动与保卫细胞中ＡＢＡ的区隔化与再分配密切相关。     

内质网在纳米材料毒性效应形成中的作用及机制

随着纳米材料在食品、药物、生物医学等多领域的应用,其在生产使用过程中对人类健康的影响引起了广泛关注.内质网是蛋白质折叠与加工修饰、脂质合成以及Ca~(2+)储存的主要场所,是维护细胞内稳态的重要细胞器.内质网作为纳米材料的主要靶细胞器之一,在纳米材料引起的毒性效应中起重要作用.本文结合近年来国内外相关研究进展,阐述了纳米银(Ag-NPs)、纳米金(Au-NPs)、纳米二氧化钛(TiO_2-NPs)、纳米氧化锌(ZnO-NPs)、纳米二氧化硅(SiO_2-NPs)、富勒烯(C_(60))、单壁与多壁碳纳米管(SWCNTs/MWCNTs)以及石墨烯与氧化石墨烯(GO)等典型纳米材料对内质网结构与功能的影响,并归纳总结了内质网在不同纳米材料诱导的毒性效应中的作用及其异同点.纳米材料可通过引起内质网应激诱导细胞凋亡、炎症反应以及细胞自噬,还可通过激活IP_3信号通路诱导内质网Ca~(2+)释放激活钙依赖的细胞凋亡.纳米材料可在内质网中积累造成结构损伤及功能障碍,还可诱导内质网自噬.     

AtMTM1, a novel mitochondrial protein,may be involved in activation of the manganese-containing superoxide dismutase in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Mtm1p is essential for the posttranslational activation of manganese-containing superoxide dismutase (SOD2) in Saccharomyces cerevisiae; however, whether the same holds true for Arabidopsis thaliana is unknown. In this study, by using the yeast mtm1 mutant complementation method, we identified a putative MTM gene (AtMTM1, At4g27940) that is necessary for SOD2 activation. Further, analysis of SOD activity revealed that an SOD2 defect is rescued in the yeast mutant Y07288 harboring the AtMTM1 gene. Related mRNA-level analysis showed the AtMTM1 gene is induced by paraquat but not by hydrogen peroxide, which indicates that this gene is related to the superoxide scavenger SOD. In addition, an AtMTM1::GFP fusion construct was transiently expressed in the protoplasts, and it was localized to the mitochondria. Furthermore, sequence deletion analysis of AtMTM1 revealed that the code region (amino acid (aa) 60–198) of Mtm1p plays an important role in localization of the protein to the mitochondria. Regulation of AtMTM1 gene expression was analyzed using a fusion construct of the 1,766 bp AtMTM1 promoter and the GUS (β-glucuronidase) reporter gene. The screen identified GUS reporter gene expression in the developing cotyledons, leaves, roots, stems, and flowers but not in the siliques. Our results suggest that AtMTM1 encodes a mitochondrial protein that may be playing an important role in activation of MnSOD1 in Arabidopsis.     

Extensive Distribution of Acetylcholinesterase in Guard Cells of Vicia faba

Acetylcholinesterase, an enzyme responsible for hydrolyzing of acetylcholine to choline and acetic acid residues, is detected in the guard cell protoplasts. Extensive acetylcholinesterase activity has been found in the guard cell protoplasts as compared with the mesophyll cell protoplasts. Moreover, light could stimulate the enzyme activity. Localization of acetylcholinesterase in the stomata of Vicia faba L. was undertaken using Karnovsky and Roots cytochemical method. It was found that in the stomata of this plant products of acetylcholinesterase enzymatic reaction mainly appeared in the outer side of the guard cell ventral wall and inner wall. When the staining time was prolonged, products of acetylcholinesterase enzymatic reaction could also be found in the ventral and inner wall of the guard cells. In addition, more extensive product of enzymatic reaction was observed in the opened stomata than in the closed stomata. It was assumed that acetylcholineaterase may participate in the regulation of stomatal movement by hydrolyzing acetylcholine around the stomata.     

Confocal Fluorescence Microscopic Observation on the Transcellular Distribution of Actin Filaments in the Epidermis of Garlic Clove Sheath

By means of paraformaldehyde fixation, Triton X-100 extraction and TRITC-phalloidin staining, the presence and distribution patterns of F-actin in the outer epidermal cells of the garlic (Allium sativum L.) sheath were studied with fluorescence probe technique and confocal laser scanning microscopy. There were a lot of actin filaments (AFs) impenetrate the cell wall, but the AFs with red fluorescence were absent when the cells were treated with cytochalasin D before fixation; the same result was obtained when the cells were treated with unlabeled phalloidin. These results indicate the presence of F-actin in the intercellular channels and that it is related to the plasmodesmata and intercellular trafficking of macromolecules.     

玉米根V-ATPase的A亚基磷酸化位点的鉴定

以玉米(Zea mays L)根的高纯度液泡膜为材料进行的磷酸化反应表明,液泡膜蛋白的磷酸化可明显提高v型H -ATPase(V-ATPase)的ATP水解活性和H 转运活性.进一步研究表明,纯化的液泡膜蛋白能被硫代磷酸化,用V-ATPase的A亚基抗体将一条约69 kD的条带鉴定为A亚基.为了测定V-ATPase的A亚基的磷酸化位点,从硫代磷酸化的凝胶中切下A亚基条带并用胰蛋白酶彻底消化.用RP-HPLC分离纯化酶解片断,收集纯化的硫代磷酸化肽段进行质谱分析所测定的分子量为573.83 Da.A亚基胰蛋白酶彻底消化后能产生61个肽段,只有F56肽段的分子量573.66 Da与573.83 Da最接近,而且F56肽段上只有第525位的丝氨酸可以被磷酸化.因此可以确定,玉米根V-AT-Pase A亚基的潜在磷酸化位点为Ser525.就我们所知,这是首次确定植物V-ATPase A亚基的磷酸化位点.