Reduced expression of BjRCE1 gene modulated by nuclear-cytoplasmic incompatibility alters auxin response in cytoplasmic male-sterile Brassica juncea

The signal from organelle to nucleus, namely retrograde regulation of nuclear gene expression, was largely unknown. Due to the nuclear-cytoplasmic incompatibility in cytoplasmic male-sterile (CMS) plants, we employed CMS Brassica juncea to investigate the retrograde regulation of nuclear gene expression in this study. We studied how reduced BjRCE1 gene expression caused by the nuclear-cytoplasmic incompatibility altered the auxin response in CMS of B. juncea. We isolated the BjRCE1 gene that was located in the nucleus from B. juncea. Over-expression of BjRCE1 enhanced auxin response in transgenic Arabidopsis. The expression of BjRCE1 was significantly reduced in CMS compared with its maintainer fertile (MF) line of B. juncea. There were fewer lateral roots in CMS than MF under normal and treatment of indole-3-acetic acid (IAA) conditions. Expression patterns of several auxin-related genes together with their phenotypes indicated a reduced auxin response in CMS compared to MF. The phenotypes of auxin response and auxin-related gene expression pattern could be mimicked by inhibiting mitochondrial function in MF. Taken together, we proposed reduced expression of BjRCE1 gene modulated by nuclear-cytoplasmic incompatibility alters auxin response in CMS B. juncea. This may be an important mechanism of retrograde regulation of nuclear gene expression in plants.     

Relationship between cytoplasmic male sterility and SPL-like gene expression in stem mustard

We studied how mitochondria–nuclear interactions may give rise to cytoplasmic male sterility (CMS) in stem mustard exhibiting abnormal microsporogenesis. In this system, expression of SPL-like , the counterpart of the Arabidopsis nuclear gene SPOROCYTELESS , is specifically lost in buds of CMS plants. When mitochondrial-specific inhibitors were applied to wild-type fertile stem mustard plants, expression of SPL-like was repressed to some extent. As a consequence, the shape and vigor of pollen grains were severely affected, whereas the fertility of pistils remained unaltered. Thereby, we suggest that a probable pathway responsible for CMS in stem mustard involves mitochondrial retrograde regulation, with SPL-like as a target nuclear gene for a mitochondrial signal.     

Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers

To gain new insights into the mechanism underlying cytoplasmic male sterility (CMS), we compared the nuclear gene expression profiles of flowers of a Brassica napus CMS line with that of the fertile B. napus maintainer line using Arabidopsis thaliana flower-specific cDNA microarrays. The CMS line used has a B. napus nuclear genome, but has a rearranged mitochondrial (mt) genome consisting of both B. napus and A. thaliana DNA. Gene expression profiling revealed that a large number of genes differed in expression between the two lines. For example, nuclear genes coding for proteins that are involved in protein import into organelles, genes expressed in stamens and pollen, as well as genes implicated in either cell-wall remodeling or architecture, were repressed in the CMS line compared with B. napus. These results show that the mt genome of the CMS line strongly influences nuclear gene expression, and thus reveal the importance of retrograde signalling between the mitochondria and the nucleus. Furthermore, flowers of the CMS line are characterized by a replacement of stamens with carpelloid organs, and thus partially resemble the APETALA3 (AP3) and PISTILLATA (PI) mutants. In accordance with this phenotype, AP3 expression was downregulated in the stamens, shortly before these organs developed carpelloid characteristics, even though it was initiated correctly. Repression of PI succeeded that of AP3 and might be a consequence of a loss of AP3 activity. These results suggest that AP3 expression in stamens depends on proper mt function and a correct nuclear-mt interaction, and that mt alterations cause the male sterility phenotype of the CMS line.     

Implication of chlorophyll biosynthesis on chloroplast-to-nucleus retrograde signaling

The biogenesis and function of chloroplast are controlled both by anterograde mechanisms involving nuclear-encoded proteins targeted to chloroplast and by retrograde signals from plastid to nucleus contributing to regulation of nuclear gene expression. A number of experimental evidences support the implication of chlorophyll biosynthesis intermediates on the retrograde signaling, albeit an earlier-postulated direct link between accumulation of chlorophyll intermediates and changes in nuclear gene expression has recently been challenged. By characterization of Arabidopsis mutants lacking the chloroplast localized NADPH-thioredoxin reductase (NTRC) we have recently proposed that imbalanced activity of chlorophyll biosynthesis in developing cells modifies the chloroplast signals leading to alterations in nuclear gene expression. These signals appear to initiate from temporal perturbations in the flux through the pathway from protoporphyrin to protochlorophyllide rather than from the accumulation of a single intermediate of the tetrapyr-role pathway.Key words: chloroplast biogenesis, NADPH-thioredoxin reductase, porphyrins, ROS, signaling, tetrapyrrole, thioredoxinOrchestrated regulation of gene expression in the nucleus and plastids is crucial for the proper biogenesis of the organelle during the development and for the acclimation of plants to environmental cues. Multiple potential candidates for initiating plastidial signals have been recognized, including intermediates of the tetrapyrrole biosynthetic pathway, redox state of chloroplast electron transfer components and reactive oxygen species (ROS). These multiple signaling pathways are likely to interact with each others, resulting in a complex signaling network between plastid and nucleus (reviewed in ref. ¹).     

Cytoplasmic male sterility: a window to the world of plant mitochondrial-nuclear interactions

Mitochondrial function depends on the coordinate action of nuclear and mitochondrial genomes. The genetic dissection of these interactions presents special challenges in obligate aerobes, because the viability of these organisms depends on mitochondrial respiration. The plant trait cytoplasmic male sterility (CMS) is determined by the mitochondrial genome and is associated with a pollen sterility phenotype that can be suppressed or counteracted by nuclear genes known as restorer-of-fertility genes. Here, I review the nature and the origin of the genes that determine CMS, together with recent investigations that have exploited CMS to provide new insights into plant mitochondrial-nuclear communication. These studies have implicated mitochondrial signaling pathways, including those involved in regulating cell death and nuclear gene expression, in the elaboration of CMS. The molecular cloning of nuclear genes that restore fertility (i.e. restorer-of-fertility genes) has identified genes encoding pentatricopeptide-repeat proteins as key regulators of plant mitochondrial gene expression.     

DCW11, down-regulated gene 11 in CW-type cytoplasmic male sterile rice, encoding mitochondrial protein phosphatase 2c is related to cytoplasmic male sterility

Causes of cytoplasmic male sterility (CMS) in plants have beenstudied for two decades, and mitochondrial chimeric genes havebeen predicted to induce CMS. However, it is unclear what happensafter CMS-associated proteins accumulate in mitochondria. Inour previous study of microarray analysis, we found that 140genes are aberrantly regulated in anthers of CW-type CMS ofrice (Oryza sativa L.). In the present study, we investigatedDCW11, one of the down-regulated genes in CW-CMS encoding aprotein phosphatase 2C (PP2C). DCW11 mRNA was preferentiallyexpressed in anthers, with the highest expression in maturepollen. As predicted by the N-terminal sequence, DCW11 signalpeptide–green fluorescent protein (GFP) fusion proteinwas localized in mitochondria. Knockdown of DCW11 in wild-typerice by RNA interference caused a major loss of seed-set fertility,without visible defect in pollen development. Since this knockdownphenotype resembled that of CW-CMS, we concluded that the down-regulationof DCW11 is correlated with CW-CMS. This idea was supportedby the up-regulation of alternative oxidase 1a (AOX1a), whichis known to be regulated by mitochondrial retrograde signaling,in DCW11 knockdown lines. Down-regulation of DCW11 and up-regulationof AOX1a were also observed in two other types of rice CMS.Our result indicates that DCW11 could play a role as a mitochondrialsignal transduction mediator in pollen germination.     

Genetic characterization of a pentatricopeptide repeat protein gene,orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish

Cytoplasmic male sterility (CMS) in plants is a maternally inherited inability to produce functional pollen, and is often associated with mitochondrial DNA abnormalities. Specific nuclear loci that suppress CMS, termed as restorers of fertility (Rf), have been identified. Previously, we identified an Rf for the CMS Kosena radish and used genetic analysis to identify the locus and create a contig covering the critical interval. To identify the Rf gene, we introduced each of the lambda and cosmid clones into the CMS Brassica napus and scored for fertility restoration. Fertility restoration was observed when one of the lambda clones was introduced into the CMS B. napus. Furthermore, introduction of a 4.7-kb BamHI/HpaI fragment of the lambda clone is enough to restore male fertility. A cDNA strand isolated from a positive fragment contained a predicted protein (ORF687) of 687 amino acids comprising 16 repeats of the 35-amino acid pentatricopeptide repeat (PPR) motif. Kosena CMS radish plants were found to express an allele of this gene possessing four substituted amino acids in the second and third repeats of the PPR suggesting that the domains formed by these repeats in ORF687 are essential for fertility restoration. Protein levels of the Kosena CMS-associated mitochondrial protein ORF125 were considerably reduced in plants in which fertility was restored, although mRNA expression was normal. Regarding the possible role for PPR-containing proteins in the regulation of the mitochondrial gene, we propose that ORF687 functions either directly or indirectly to lower the levels of ORF125, resulting in the restoration of fertility in CMS plants.     

The fertility restorer gene, Rf2, for Lead Rice-type cytoplasmic male sterility of rice encodes a mitochondrial glycine-rich protein

Cytoplasmic male sterility (CMS) is associated with a mitochondrial mutation that causes an inability to produce fertile pollen. The fertility of CMS plants is restored in the presence of a nuclear-encoded fertility restorer (Rf) gene. In Lead Rice-type CMS, discovered in the indica variety 'Lead Rice', fertility of the CMS plant is restored by the single nuclear-encoded gene Rf2 in a gametophytic manner. We performed map-based cloning of Rf2, and proved that it encodes a protein consisting of 152 amino acids with a glycine-rich domain. Expression of Rf2 mRNA was detected in developing and mature anthers. An RF2-GFP fusion was shown to be targeted to mitochondria. Replacement of isoleucine by threonine at amino acid 78 of the RF2 protein was considered to be the cause of functional loss in the rf2 allele. As Rf2 does not encode a pentatricopeptide repeat protein, unlike a majority of previously identified Rf genes, the data from this study provide new insights into the mechanism for restoring fertility in CMS.     

Mitochondrial ORF79 levels determine pollen abortion in cytoplasmic male sterile rice

Cytoplasmic male sterility (CMS) is an important agricultural trait characterized by lack of functional pollen, and caused by ectopic and defective mitochondrial gene expression. The pollen function in CMS plants is restored by the presence of nuclear‐encoded restorer of fertility (Rf) genes. Previously, we cloned Rf2, which restores the fertility of Lead Rice (LD)‐type CMS rice. However, neither the function of Rf2 nor the identity of the mitochondrial gene causing CMS has been determined in LD–CMS rice. Here, we show that the mitochondrial gene orf79 acts as a CMS‐associated gene in LD–CMS rice, similar to its role in BT–CMS rice originating from Chinsurah Boro II, and Rf2 weakly restores fertility in BT–CMS rice. We also show that RF2 promotes degradation of atp6–orf79 RNA in a different manner from that of RF1, which is the Rf gene product in BT–CMS rice. The amount of ORF79 protein in LD–CMS rice was one‐twentieth of the amount in BT–CMS rice. The difference in ORF79 protein levels probably accounts for the mild and severe pollen defects in LD–CMS and BT–CMS rice, respectively. In the presence of Rf2, accumulation of ORF79 was reduced to almost zero and 25% in LD–CMS and BT–CMS rice, respectively, which probably accounts for the complete and weak fertility restoration abilities of Rf2 in LD–CMS and BT–CMS rice, respectively. These observations indicate that the amount of ORF79 influences the pollen fertility in two strains of rice in which CMS is induced by orf79.     

Role of Reactive Oxygen Species in the Initiation of Plant Retrograde Signaling

The interaction between the nucleus and the different organelles is important in the physiology of the plant. Reactive oxygen species (ROS) are a by-product of the oxidation of organic molecules to obtain energy by the need to carry out the electron transfer between the different enzymatic complexes. However, they also have a role in the generation of what is known as retrograde signaling. This signal comes from the different organelles in which the oxidation of molecules or the electron transference is taking place such as mitochondria and chloroplasts. Furthermore, ROS can also induce the release of signals from the apoplast. It seems that these signals plays a role communicating to the nucleus the current status of the different parts of the plant cell to induce a changes in gene expression. In this review, the molecular mechanism of ROS retrograde signaling is described.

     

Pollen Fertility Restoration by Nuclear Gene Fr in Cms Bean: Nuclear-Directed Alteration of a Mitochondrial Population

Two nuclear genes, Fr and Fr2, have been identified that restore pollen fertility to cytoplasmic male sterile (CMS) common bean (Phaseolus vulgaris L.) by apparently distinct mechanisms. Whereas Fr2 appears to suppress the expression of a male sterility associated mitochondrial sequence (designated pvs), Fr restores pollen fertility by causing the elimination of this unusual mitochondrial DNA segment. To further investigate the mechanism of Fr action, Fr and Fr2 were cointroduced into the nucleus of a bean line containing the sterility inducing cytoplasm. When the effect of pvs was suppressed by Fr2, the presence of Fr no longer directed the elimination of the mitochondrial pvs sequence. This result suggests that the Fr function is dependent on proper expression of the pvs sequence. To evaluate the temporal and spatial patterns of Fr action, we undertook a polymerase chain reaction-based approach to trace the fate of the pvs sequence in different tissues of F(2) and F(3) fertile-restored plants derived from a genetic cross between a cytoplasmic male sterile line of common bean, CMS-Sprite (frfr), and fertility restorer line R351 (FrFr). We demonstrate that the Fr-directed disappearance of pvs sequence occurs during flower development. Elimination of the pvs sequence from developing megaspores results in permanent fertility restoration in the following generations. Genetic analysis demonstrated that permanent fertility restoration, that is, the complete elimination of pvs from reproductive tissues requires two doses of the Fr allele or the absence of fr in F(2) individuals. The effect of Fr was reversible until full fertility was achieved. On the basis of these results, we propose a model for the mechanism of pvs elimination by the Fr gene and discuss the dynamics of pvs-containing mitochondrial transmission in the presence of the Fr gene.     

A light and electron microscopy analysis of the events leading to male sterility in Ogu-INRA CMS of rapeseed (Brassica napus)

Ogura cytoplasmic male sterility (CMS) occurs naturally in radishand has been introduced into rapeseed (Brassica napus) by protoplastfusion. As with all CMS systems, it involves a constitutivelyexpressed mitochondrial gene which induces male sterility tootherwise hermaphroditic plants (so they become females) anda nuclear gene named restorer of fertility that restores pollenproduction in plants carrying a sterility-inducing cytoplasm.A correlative approach using light and electron microscopy wasapplied to define what stages throughout development were affectedand the subcellular events leading to the abortion of the developingpollen grains upon the expression of the mitochondrial protein.Three central stages of development (tetrad, mid-microsporeand vacuolate microspore) were compared between fertile, restored,and sterile plants. At each stage observed, the pollen in fertileand restored plants had similar cellular structures and organization.The deleterious effect of the sterility protein expression startedas early as the tetrad stage. No typical mitochondria were identifiedin the tapetum at any developmental stage and in the vacuolatemicrospores of the sterile plants. In addition, some strikingultrastructural alterations of the cell's organization werealso observed compared with the normal pattern of development.The results showed that Ogu-INRA CMS was due to premature celldeath events of the tapetal cells, presumably by an autolysisprocess rather than a normal PCD, which impairs pollen developmentat the vacuolate microspore stage, in the absence of functionalmitochondria. Key words: Brassica napus, cell death, light and electron microscopy, mitochondria, plastids, pollen development, Ogu-INRA cytoplasmic male sterility, transgenic-restored plants, tapetum Received 30 September 2007; Revised 11 December 2007 Accepted 20 December 2007     

Nerve growth factor signaling regulates motility and docking of axonal mitochondria

Axonal transport is thought to distribute mitochondria to regions of the neuron where their functions are required. In cultured neurons, mitochondrial transport responds to growth cone activity, and this involves both a transition between motile and stationary states of mitochondria and modulation of their anterograde transport activity. Although the exact cellular signals responsible for this regulation remain unknown, we recently showed that mitochondria accumulate in sensory neurons at regions of focal stimulation with NGF and suggested that this involves downstream kinase signaling. Here, we demonstrate that NGF regulation of axonal organelle transport is specific to mitochondria. Quantitative analyses of motility show that the accumulation of axonal mitochondria near a focus of NGF stimulation is due to increased movement into bead regions followed by inhibition of movement out of these regions and that anterograde and retrograde movement are differentially affected. In axons made devoid of F-actin by latrunculin B treatment, bidirectional transport of mitochondria continues, but they can no longer accumulate in the region of NGF stimulation. These results indicate that intracellular signaling can specifically regulate mitochondrial transport in neurons, and they suggest that axonal mitochondria can respond to signals by locally altering their transport behavior and by undergoing docking interactions with the actin cytoskeleton.     

高粱雄性不育系热激前后线粒体的变化与育性的关系

高粱雄性不育系3197A（3A）在花粉母细胞期进行热激处理后由不育变为可育。提取其处理小穗线粒体用流式细胞仪计数进行分析。结果发现经热激处理后,3A不育系的线粒体数目由1.55×106个／mg鲜穗增加至7.1×106／mg。同时对热激处理后3A的花粉育性及同工酶进行了研究,发现热激后3A的花粉粒变的饱满并可被I2朘I 溶液染色,其细胞色素氧化酶和过氧化物酶的电泳酶谱中出现与保持系3B一致的酶带。     

Rtg2 protein: at the nexus of yeast longevity and aging

Firm support for the notion that metabolism and particularly mitochondrial metabolism plays a significant role in aging has been gathered in studies on yeast. As in other organisms, mitochondria contribute to aging through their propensity to generate reactive oxygen species. There is more to the involvement of mitochondria in aging than this, however. Mitochondrial dysfunction, which accumulates during aging, triggers the retrograde response, an intracellular signaling pathway that activates genes that compensate for this dysfunction. A key signaling protein in this pathway is the Rtg2 protein. Recent studies have provided evidence that this protein lies at the nexus of the four major processes that are involved in aging in yeast and in other organisms; namely, metabolism, stress resistance, chromatin-dependent gene regulation, and genome stability. The details of this central role of Rtg2 protein explain the delicate balance between longevity and aging, which ultimately must tip towards the latter. Phenomena that resemble the retrograde response appear to exist in human cells, with both common and cell type-specific gene expression changes as the output.