New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development

Pollen germination on the surface of compatible stigmatic tissues is an essential step for plant fertilization. Here we report that the Arabidopsis mutant bcll is male sterile as a result of the failure ofpollen germination. We show that the bcll mutant allele cannot be transmitted by male gametophytes and no homozygous bcll mutants were obtained. Analysis of pollen developmental stages indicates that the bcll mutation affects pollen germination but not pollen maturation. Molecular analysis demonstrates that the failure of pollen germination was caused by the disruption of AtBECLIN 1. AtBECLIN 1 is expressed predominantly in mature pollen and encodes a protein with significant homology to Beclin1/Atg6/Vps30 required for the processes of autophagy and vacuolar protein sorting （VPS） in yeast. We also show that AtBECLIN 1 is required for normal plant development, and that genes related to autophagy, VPS and the glycosylphosphatidylinositol anchor system, were affected by the deficiency of AtBECLIN 1.     

Disruption of the novel plant protein NEF1 affects lipid accumulation in the plastids of the tapetum and exine formation of pollen, resulting in male sterility in Arabidopsis thaliana

A novel male-sterile mutant of Arabidopsis thaliana was isolated by means of T-DNA tagging. Pollen abortion of the mutant was evident after microspore release, and pollen grains were completely absent at anthesis. Transmission electron microscope analysis revealed that primexine was coarsely developed, and that although sporopollenin was produced, it was not deposited onto the microspore plasma membrane. The sporopollenin that failed to be deposited aggregated and accumulated within the locule and on the locule wall. Finally, as no exine formation was observed, the mutant was named nef1. The plastoglobuli within the plastids of the tapetum were reduced, and lipid accumulation was considerably decreased. The mutant had a significantly altered leaf chloroplast ultrastructure and showed various growth defects. Lipid analysis revealed that the total lipid content in nef1 was lower than that in the wild type, which indicated that Nef1 was involved in lipid metabolism. Cloning of the full-length Nef1 indicated that the gene encodes a novel plant protein of 1123 amino acids with limited sequence similarities to membrane proteins or transporter-like proteins, and the NEF1 is predicted to be a plastid integral membrane protein. Motif analysis revealed that NEF1 contains prokaryotic membrane lipoprotein lipid attachment sites that are involved in maintaining cell envelope integrity. It is predicted that the Nef1 encodes a membrane protein that maintains the envelope integrity in the plastids.     

Effects of light on cyanide‐resistant respiration and alternative oxidase function in Arabidopsis seedlings

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)‐resistant respiration and plays a role in optimizing photosynthesis. Although it has been demonstrated that leaf AOX is upregulated after illumination, the in vivo mechanism of AOX upregulation by light and its physiological significance are still unknown. In this report, red light and blue light‐induced AOX (especially AOX1a) expressions were characterized. Phytochromes, phototropins and cryptochromes, all these photoreceptors mediate the light‐response of AOX1a gene. When aox1a mutant seedlings were grown under a high‐light (HL) condition, photobleaching was more evident in the mutant than the wild‐type plants. More reactive oxygen species (ROS) accumulation and inefficient dissipation of chloroplast reducing‐equivalents in aox1a mutant may account for its worse adaptation to HL stress. When etiolated seedlings were exposed to illumination for 4 h, chlorophyll accumulation was largely delayed in aox1a plants. We first suggest that more reduction of the photosynthetic electron transport chain and more accumulation of reducing‐equivalents in the mutant during de‐etiolation might be the main reasons.     

SD-RLK28 positively regulates pollen hydration on stigmas as a PCP-Bβ receptor in Arabidopsis thaliana

Pollen hydration on dry stigmas is strictly regulated by pollen–stigma interactions in Brassicaceae. Although several related molecular events have been described, the molecular mechanism underlying pollen hydration remains elusive. Multiple B-class pollen coat proteins(PCP-Bs) are involved in pollen hydration. Here, by analyzing the interactions of two PCP-Bs with three Arabidopsis thaliana stigmas strongly expressing S-domain receptor kinase(SD-RLK), we determined that SD-RLK28 directly intera...     

拟南芥活性氧不敏感型突变体的筛选与特性分析

采用 EMS化学诱变方法与 H2 O2 氧化胁迫选择 ,以根在重力作用下的弯曲生长为指标 ,筛选得到拟南芥活性氧不敏感型突变体。对突变体杂交后代遗传分析表明 ,突变株对活性氧不敏感性状为隐性单基因突变所致 ;生理生化分析表明突变体对 H2 O2 有很强的抗性 ,表现为气孔开度对 H2 O2 不敏感和 H2 O2 胁迫时较低的膜脂过氧化水平。运用 L SCM技术并结合 H2 O2 荧光探针 H2 DCFDA检测外源 ABA诱导保卫细胞内产生 H2 O2 的情况 ,结果显示突变体体内荧光强度比对照低 ,暗示了突变体体内消除 H2 O2 的能力可能有所提高 ,增强了植株对氧化胁迫的抗性。拟南芥活性氧不敏感突变体的筛选 ,不仅为人们深入研究活性氧在细胞内的作用提供良好的实验材料 ,而且还将大大加深人们对信号转导途径的再认识     

基于光子学技术研究弱光胁迫早期拟南芥光合损伤机理

弱光限制植物的光合作用,降低了光合作用效率,造成农业产量下降.本文主要研究了弱光处理早期,拟南芥光合作用相关指标的变化.研究中发现在弱光处理的早期,植株生长表型和最大光化学效率（Fv/Fm）没有明显变化,实际光化学效率Y（Ⅱ）以及光系统电子传递效率（ETR）下降较明显.此外,弱光处理原生质体,利用2 ＇,7＇-二氯二氢荧光素二乙酯（dichlorofluorescin diacetate,H2DCF-DA）染色,共聚焦显微镜观察,发现细胞中有较明显的活性氧（ROS）合成,且定位于叶绿体.该研究结果为植物弱光耐受性的研究提供理论依据.     

Mitochondrial ATP synthase subunit d,a component of the peripheral stalk,is essential for growth and heat stress tolerance in Arabidopsis thaliana

As rapid changes in climate threaten global crop yields, an understanding of plant heat stress tolerance is increasingly relevant. Heat stress tolerance involves the coordinated action of many cellular processes and is particularly energy demanding. We acquired a knockout mutant and generated knockdown lines in Arabidopsis thaliana of the d subunit of mitochondrial ATP synthase (gene name: ATPQ, AT3G52300, referred to hereafter as ATPd), a subunit of the peripheral stalk, and used these to investigate the phenotypic significance of this subunit in normal growth and heat stress tolerance. Homozygous knockout mutants for ATPd could not be obtained due to gametophytic defects, while heterozygotes possess no visible phenotype. Therefore, we used RNA interference to create knockdown plant lines for further studies. Proteomic analysis and blue native gels revealed that ATPd downregulation impairs only subunits of the mitochondrial ATP synthase (complex V). Knockdown plants were more sensitive to heat stress, had abnormal leaf morphology, and were severely slow growing compared to wild type. These results indicate that ATPd plays a crucial role in proper function of the mitochondrial ATP synthase holoenzyme, which, when reduced, leads to wide-ranging defects in energy-demanding cellular processes. In knockdown plants, more hydrogen peroxide accumulated and mitochondrial dysfunction stimulon (MDS) genes were activated. These data establish the essential structural role of ATPd and support the importance of complex V in normal plant growth, and provide new information about its requirement for heat stress tolerance.     

Ethylene response pathway is essential for ARABIDOPSIS A-FIFTEEN function in floral induction and leaf senescence

ARABIDOPSIS A-FIFTEEN (AAF) encodes a plastid protein and was originally identified as a SENESCENCE-ASSOCIATED GENE. Previously, we found that overexpression of AAF (AAF-OX) in Arabidopsis led to accumulated reactive oxygen species and promoted leaf senescence induced by oxidative stress, which was suppressed by a null mutant, ein2-5, in ethylene response pathway. Whether AAF function is involved in ethylene biosynthesis and/or the response pathway remained unknown. Here we show that neither overexpression (AAF-OX) nor a null mutant (aaf-KO) of AAF generates a higher level of ethylene than the wild type and display a typical triple-response phenotype in etiolated seedlings treated with 1-aminocyclopropane-1-carboxylic acid (ACC). Nevertheless, ein2-5 suppresses the phenotypes of early flowering and age-dependent leaf senescence in AAF-OX plants. We reveal that a functional ethylene response is essential for AAF function in leaf senescence and floral induction, but AAF is unlikely a regulatory component integral to the ethylene pathway.     

Identification of superoxide dismutase isoenzymes in tobacco pollen

We investigated the possible existence of superoxide dismutase (SOD; EC 1.15.1.1) isoenzymes in the pollen of Nicotiana tabacum (Petit Havana SR-1 cultivar). To detect SOD activity, crude extracts from tobacco pollen were subjected to native polyacrylamide gel electrophoresis followed by staining with nitroblue tetra-zolium (NBT). The presence of six SOD isoenzymes was detected in tobacco pollen. Treatment with SOD inhibitors indicated the presence of one manganese SOD (Mn SOD), five copper-zinc SOD (Cu/Zn SOD) isoenzymes, and the absence of iron SOD (Fe SOD).     

Alterations in bioenergetic function induced by Parkinson's disease mimetic compounds: lack of correlation with superoxide generation

J. Neurochem. (2012) 122, 941-951. ABSTRACT: In vitro and in vivo models of Parkinson's disease (PD) suggest that increased oxidant production leads to mitochondrial dysfunction in dopaminergic neurons and subsequent cell death. However, it remains unclear if cell death in these models is caused by inhibition of mitochondrial function or oxidant production. The objective of this study was to determine the relationship between mitochondrial dysfunction and oxidant production in response to multiple PD neurotoxicant mimetics. MPP(+) caused a dose-dependent decrease in the basal oxygen consumption rate in dopaminergic N27 cells, indicating a loss of mitochondrial function. In parallel, we found that MPP(+) only modestly increased oxidation of hydroethidine as a diagnostic marker of superoxide production in these cells. Similar results were found using rotenone as a mitochondrial inhibitor, or 6-hydroxydopamine (6-OHDA) as a mechanistically distinct PD neurotoxicant, but not with exposure to paraquat. In addition, the extracellular acidification rate, used as a marker of glycolysis, was stimulated to compensate for oxygen consumption rate inhibition after exposure to MPP(+) , rotenone, or 6-OHDA, but not paraquat. Together these data indicate that MPP(+) , rotenone, and 6-OHDA dramatically shift bioenergetic function away from the mitochondria and towards glycolysis in N27 cells.     

Vacuolar digestion of entire damaged chloroplasts in Arabidopsis thaliana is accomplished by chlorophagy

In yeast and mammals, selective vacuolar delivery and degradation of whole mitochondria, or mitophagy, represents an important quality control system and is achieved by a cargo recognition mechanism enabling selective elimination of dysfunctional mitochondria. As photosynthetic organelles that need light for energy production, plant chloroplasts accumulate sunlight-induced damage. Plants have evolved multiple mechanisms to avoid, relieve, or repair chloroplast photodamage. Our recent study showed that vacuolar degradation of entire chloroplasts, termed chlorophagy, is induced to degrade chloroplasts that are collapsed due to photodamage. Our results underscore the involvement of autophagy in the quality control of endosymbiotic, energy-converting organelles in eukaryotes.     

Non-specific phospholipases C2 and C6 redundantly function in pollen tube growth via triacylglycerol production in Arabidopsis

Non-specific phospholipase Cs (NPCs) are responsible for membrane lipid remodeling that involves hydrolysis of the polar head group of membrane phospholipids. Arabidopsis NPC2 and NPC6 are essential in gametogenesis, but their underlying role in the lipid remodeling remains elusive. Here, we show that these NPCs are required for triacylglycerol (TAG) production in pollen tube growth. NPC2 and NPC6 are highly expressed in developing pollen tubes and are localized at the endoplasmic reticulum. Mutants of NPC2 and NPC6 showed reduced rate of pollen germination, length of pollen tube and amount of lipid droplets (LDs). Overexpression of NPC2 or NPC6 induced LD accumulation, which suggests that these NPCs are involved in LD production. Furthermore, mutants defective in the biosynthesis of TAG, a major component of LDs, showed defective pollen tube growth. These results suggest that NPC2 and NPC6 are essential in gametogenesis for a role in hydrolyzing phospholipids and producing TAG required for pollen tube growth. Thus, lipid remodeling from phospholipids to TAG during pollen tube growth represents an emerging role for the NPC family in plant developmental control.