Multiple N-Glycans Cooperate in the Subcellular Targeting and Functioning of Arabidopsis KORRIGAN1

Arabidopsis thaliana KORRIGAN1 (KOR1) is an integral membrane endo-β1,4-glucanase in the trans-Golgi network and plasma membrane that is essential for cellulose biosynthesis. The extracellular domain of KOR1 contains eight N-glycosylation sites, N1 to N8, of which only N3 to N7 are highly conserved. Genetic evidence indicated that cellular defects in attachment and maturation of these N-glycans affect KOR1 function in vivo, whereas the manner by which N-glycans modulate KOR1 function remained obscure. Site-directed mutagenesis analysis of green fluorescent protein (GFP)-KOR1 expressed from its native regulatory sequences established that all eight N-glycosylation sites (N1 to N8) are used in the wild type, whereas stt3a-2 cells could only inefficiently add N-glycans to less conserved sites. GFP-KOR1 variants with a single N-glycan at nonconserved sites were less effective than those with one at a highly conserved site in rescuing the root growth phenotype of rsw2-1 (kor1 allele). When functionally compromised, GFP-KOR1 tended to accumulate at the tonoplast. GFP-KOR1Δall (without any N-glycan) exhibited partial complementation of rsw2-1; however, root growth of this line was still negatively affected by the absence of complex-type N-glycan modifications in the host plants. These results suggest that one or several additional factor(s) carrying complex N-glycans cooperate(s) with KOR1 in trans to grant proper targeting/functioning in plant cells.     

Metabolic Profiling and Physiological Analysis of a Novel Rice Introgression Line with Broad Leaf Size

A rice introgression line, NIL-SS1, and its recurrent parent, Teqing, were used to investigate the influence of the introgression segment on plant growth. The current research showed NIL-SS1 had an increased flag leaf width, total leaf area, spikelet number per panicle and grain yield, but a decreased photosynthetic rate. The metabolite differences in NIL-SS1 and Teqing at different developmental stages were assessed using gas chromatography—mass spectrometry technology. Significant metabolite differences were observed across the different stages. NIL-SS1 increased the plant leaf nitrogen content, and the greatest differences between NIL-SS1 and Teqing occurred at the booting stage. Compared to Teqing, the metabolic phenotype of NIL-SS1 at the booting stage has closer association with those at the flowering stage. The introgression segment induced more active competition for sugars and organic acids (OAs) from leaves to the growing young spikes, which resulted in more spikelet number per plant (SNP). The results indicated the introgression segment could improve rice grain yield by increasing the SNP and total leaf area per plant, which resulted from the higher plant nitrogen content across growth stages and stronger competition for sugars and OAs of young spikes at the booting stage.     

拟南芥AtrbohD和AtrbohF缺失对叶蛋白质组的影响

拟南芥NADPH氧化酶AtrbohD和AtrbohF在脱落酸（abscisic acid,ABA）抑制主根伸长、ABA诱导气孔关闭以及植物应答干旱、盐及病菌侵染等逆境胁迫反应中发挥重要作用,但这2个蛋白亚基缺失对拟南芥（Arabidopsis thaliana）蛋白质组的影响还未见报道。我们以营养土中生长16 d的野生型及AtrbohD和AtrbohF双基因突变体atrbohD1/F1叶片为材料进行蛋白组学分析,在双向电泳图谱上可分辨出约1 000个蛋白点,且蛋白表达谱存在差异。选取42个显著差异蛋白点进行MALDI-TOF/TOF质谱鉴定,成功鉴定出20个差异蛋白,这些蛋白主要与氧化还原、能量代谢、蛋白代谢、转录和信号传导等相关,还有一些蛋白功能未知。     

Actin-Dependent and -Independent Functions of Cortical Microtubules in the Differentiation of Arabidopsis Leaf Trichomes

Arabidopsis thaliana tortifolía2 carries a point mutation in α-tubulin 4 and shows aberrant cortical microtubule dynamics. The microtubule defect of tortifolia2 leads to overbranching and right-handed helical growth in the single-celled leaf trichomes. Here, we use tortifolia2 to further our understanding of microtubules in plant cell differentiation. Trichomes at the branching stage show an apical ring of cortical microtubules, and our analyses support that this ring is involved in marking the prospective branch site. tortifolia2 showed ectopic microtubule bundles at this stage, consistent with a function for microtubules in selecting new branch sites. Overbranching of tortifolia2 required the C-terminal binding protein/brefeldin A-ADP ribosylated substrate protein ANGUSTIFOLIA1, and our results indicate that the angustifolia1 mutant is hypersensitive to alterations in microtubule dynamics. To analyze whether actin and microtubules cooperate in the trichome cell expansion process, we generated double mutants of tortifolia2 with distorted1, a mutant that is defective in the actin-related ARP2/3 complex. The double mutant trichomes showed a complete loss of growth anisotropy, suggesting a genetic interaction of actin and microtubules. Green fluorescent protein labeling of F-actin or microtubules in tortifolia2 distorted1 double mutants indicated that F-actin enhances microtubule dynamics and enables reorientation. Together, our results suggest actin-dependent and -independent functions of cortical microtubules in trichome differentiation.     

拟南芥过氧化物酶序列与功能的生物信息学分析

植物过氧化物酶(POD)属于多基因家族,不仅是植物体内清除活性氧自由基的重要酶类之一,而且参与多种生理生化过程,在维系植物生长发育过程中发挥重要的作用。采用生物信息学方法对拟南芥过氧化物酶家族的73个基因编码的蛋白质序列的结构和功能进行了分析,其中包含氨基酸的数量、等电点、跨膜结构域、信号肽、二级结构组成及磷酸化位点等,并用Mega4.0软件构建了去除信号肽前后的系统进化树,旨在了解其结构特征。对已经进行功能研究的成员进行结构分析,以此来揭示结构与功能之间的联系,为植物抵御氧化胁迫方面研究提供理论基础。     

老年女性类风湿关节炎合并骨质疏松症的代谢特点分析

目的:探讨老年女性类风湿关节炎(RA)合并骨质疏松症的代谢特点。方法:选择老年绝经后女性RA患者共59例,检测患者血生化代谢指标如血糖、血脂、CRP等和骨代谢指标如骨钙素(OC)、β-胶原特殊序列(β-Crosslaps)甲状旁腺素(iPTH)等,并进行统计分析。结果:骨质疏松患者的绝经时间、病程长度、OC、β-Crosslaps、iPTH显著高于骨量正常和骨量减少的患者,25羟基维生素D显著低于骨量正常和骨量减少的患者(P均0.05)。RA患者的骨密度水平与是否使用激素和X线分期情况无关。结论:老年女性RA患者易发生骨质疏松,出现骨质疏松的RA女性患者绝经时间更长,可出现脂代谢紊乱及明显的维生素D缺乏,并具有高转换型骨代谢特点。     

拟南芥叶片直接分化雌蕊的诱导及其RAPD分析

以拟南芥为材料，通过培养诱导首次获得了叶片上直接分化雌蕊忱一自然界罕见的拟南芥变异体，雌蕊结构典型，RAPD分析结果表明变异体DNA分子上发生了突变。     

In Vivo Bioluminescent Imaging of Irradiated Orthotopic Pancreatic Cancer Xenografts in Nonobese Diabetic-Severe Combined Immunodeficient Mice: A Novel Method for Targeting and Assaying Efficacy of Ionizing Radiation

Adenocarcinoma of the pancreas is a lethal malignancy, and better models to study tumor behavior in vivo are needed for the development ofmore effective therapeutics. Ionizing radiation is a treatment modality that is commonly used in the clinical setting, in particular, for locally confined disease; however, good model systems to study the effect of ionizing radiation in orthotopic tumors have not been established. In an attempt to create clinically relevant models for studying treatments directed against pancreatic cancer, we have defined a methodology to measure the effect of varying doses of radiation in established human pancreatic cancer orthotopic xenografts using two different pancreatic cancer cell lines (Panc-1 and BXPC3) infected with a lentiviral vector expressing CMV promoter-driven luciferase to allow bioluminescence imaging of live animals in real time. Quantifiable photon emission from luciferase signaling in vivo correlated well with actual tumor growth. Bioluminescence imaging of the established pancreatic xenografts was used to direct delivery of radiation to the orthotopic tumors and minimize off-target adverse effects. Growth delay was observed with schedules in the range of 7.5 Gy in five fractions to 10 Gy in four fractions, whereas doses 3 Gy or higher produced toxic adverse effects. In conclusion, we describe a model in which the effects of ionizing radiation, alone or in combination with other therapeutics, in orthotopic xenografts, can be studied.     

Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

Metabolic labeling of proteins with a stable isotope (¹⁵N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome.     

水稻与拟南芥跨膜蛋白14家族的分子进化特征与功能分析

脂类既是植物生命活动重要的能量来源,也是细胞膜系统不可或缺的结构成分,在植物生长发育和逆境反应等生命活动过程中都起到至关重要的作用。随着脂类代谢研究的不断深入,植物脂类合成通路已渐渐明晰,其中连通不同细胞器间脂类合成中间物质运送的膜蛋白也正被不断发现,但对质体脂类转运蛋白还鲜有报道。跨膜蛋白14家族(Transmembrane 14 family, Tmemb14 family)是一个新发现的跨膜蛋白家族,目前只有拟南芥FAX1 (Fatty Acid Export 1)和斑马鱼TMEM14已被克隆鉴定,该家族其他成员的生物学功能还未见报道。AtFAX1参与植物质体长链脂肪酸的跨膜外运,其功能丧失显著降低植物生物量并影响花粉发育和育性。本研究通过生物信息手段对拟南芥和水稻中的跨膜蛋白14家族成员的进化关系、蛋白理化性质、结构域功能和编码基因的表达模式进行了分析,揭示了Tmemb14家族成员在单、双子叶植物进化中的功能分化,为进一步研究跨膜蛋白14家族成员的生理功能提供了理论依据。     

Diurnal Changes in Maize Leaf Photosynthesis : II. Levels of Metabolic Intermediates of Sucrose Synthesis and the Regulatory Metabolite Fructose 2,6-Bisphosphate

Diurnal changes in the regulatory metabolite, fructose-2,6-bisphosphate (F26BP), and key metabolic intermediates of sucrose biosynthesis were studied in maize (Zea mays L. cv Pioneer 3184) during a day-night cycle. Whole leaf concentrations of dihydroxyacetonephosphate (DHAP) and fructose 1,6-bisphosphate changed markedly during the photoperiod. DHAP concentration was correlated positively with the rate of sucrose formation in vivo (assimilate export plus sucrose accumulation) and extractable activity of sucrose phosphate synthase (SPS). The changes closely followed net photosynthetic rate, which tracked irradiance. The other metabolic intermediates measured (glucose 6-phosphate, fructose 6-phosphate, and UDP-glucose) were either relatively constant over the 24 hour period or changed in a different pattern. Diurnal changes in leaf F26BP concentrations were pronounced, and fundamentally different than the pattern reported with other species. F26BP concentration decreased at the beginning of the day and remained low and constant; a 3- to 4-fold increase occurred with darkness, and slowly declined thereafter. In general, leaf F26BP concentration was negatively correlated with net photosynthetic rate, and also leaf DHAP concentration. Consequently, co-ordination of the regulation of cytosolic fructose 1,6-bisphosphatase and SPS was apparent. The results support the postulate that in maize leaves the activation state of SPS may be dependent on availability of DHAP and possibly other metabolites.     

Functional Analysis and RNA Sequencing Indicate the Regulatory Role of Argonaute1 in Tomato Compound Leaf Development

Regardless of whether a leaf is simple or compound, the mechanism underlying its development will give rise to a full comprehension of plant morphogenesis. The role of Argonaute1 (AGO1) in the development of simple leaves has been established, but its role in the development of compound leaves remains to be characterized. In this paper, a virus-induced gene silencing (VIGS) strategy was used to dramatically down-regulate the expression of AGO1 ortholog in tomatoes, a model plant for research into compound leaves. AGO1-silenced tomato compound leaves exhibited morphological defects of leaf adaxial-abaxial and trichome development. Analysis of global gene expression profiles indicated that the silencing of AGO1 in tomato compound leaf caused significant changes in the expression of several critical genes, including Auxin Response Factor 4 (ARF4) and Non-expressor of PR5 (NPR5), which were involved in adaxial-abaxial formation and IAA15 that was found to contribute to growth of trichomes as well as Gibberellic Acid Insensitive (GAI) which participated in hormone regulation. Collectively, these results shed light on the complicated mechanism by which AGO1 regulates compound leaf development.     

In Vivo Analysis of Lrig Genes Reveals Redundant and Independent Functions in the Inner Ear

Lrig proteins are conserved transmembrane proteins that modulate a variety of signaling pathways from worm to humans. In mammals, there are three family members – Lrig1, Lrig2, and Lrig3 – that are defined by closely related extracellular domains with a similar arrangement of leucine rich repeats and immunoglobulin domains. However, the intracellular domains show little homology. Lrig1 inhibits EGF signaling through internalization and degradation of ErbB receptors. Although Lrig3 can also bind ErbB receptors in vitro, it is unclear whether Lrig2 and Lrig3 exhibit similar functions to Lrig1. To gain insights into Lrig gene functions in vivo, we compared the expression and function of the Lrigs in the inner ear, which offers a sensitive system for detecting effects on morphogenesis and function. We find that all three family members are expressed in the inner ear throughout development, with Lrig1 and Lrig3 restricted to subsets of cells and Lrig2 expressed more broadly. Lrig1 and Lrig3 overlap prominently in the developing vestibular apparatus and simultaneous removal of both genes disrupts inner ear morphogenesis. This suggests that these two family members act redundantly in the otic epithelium. In contrast, although Lrig1 and Lrig2 are frequently co-expressed, Lrig1^−/−;Lrig2^−/− double mutant ears show no enhanced structural abnormalities. At later stages, Lrig1 expression is sustained in non-sensory tissues, whereas Lrig2 levels are enhanced in neurons and sensory epithelia. Consistent with these distinct expression patterns, Lrig1 and Lrig2 mutant mice exhibit different forms of impaired auditory responsiveness. Notably, Lrig1^−/−;Lrig2^−/− double mutant mice display vestibular deficits and suffer from a more severe auditory defect that is accompanied by a cochlear innervation phenotype not present in single mutants. Thus, Lrig genes appear to act both redundantly and independently, with Lrig2 emerging as the most functionally distinct family member.