Overexpression of sirtuins (NAD(+)-dependent protein deacetylases) has been reported to increase lifespan in budding yeast (Saccharomyces cerevisiae), Caenorhabditis elegans and Drosophila ...melanogaster. Studies of the effects of genes on ageing are vulnerable to confounding effects of genetic background. Here we re-examined the reported effects of sirtuin overexpression on ageing and found that standardization of genetic background and the use of appropriate controls abolished the apparent effects in both C. elegans and Drosophila. In C. elegans, outcrossing of a line with high-level sir-2.1 overexpression abrogated the longevity increase, but did not abrogate sir-2.1 overexpression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low-copy-number sir-2.1 overexpression also abrogated longevity. A Drosophila strain with ubiquitous overexpression of dSir2 using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported, but was not long-lived relative to the appropriate transgenic controls, and nor was a new line with stronger overexpression of dSir2. These findings underscore the importance of controlling for genetic background and for the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life-extending effect of dietary restriction on ageing in Drosophila has also been reported to be dSir2 dependent. We found that dietary restriction increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects of sirtuins on lifespan in C. elegans and Drosophila.
The insulin/IGF-like signalling (IIS) pathway has diverse functions in all multicellular organisms, including determination of lifespan. The seven insulin-like peptides (DILPs) in Drosophila are ...expressed in a stage- and tissue-specific manner. Partial ablation of the median neurosecretory cells (mNSCs) in the brain, which produce three DILPs, extends lifespan, reduces fecundity, alters lipid and carbohydrate metabolism and increases oxidative stress resistance. To determine if reduced expression of DILPs is causal in these effects, and to investigate possible functional diversification and redundancy between DILPs, we used RNA interference to lower specifically the transcript and protein levels of dilp2, the most highly expressed of the mNSC-derived DILPs. We found that DILP2 was limiting only for the increased whole-body trehalose content associated with mNSC-ablation. We observed a compensatory increase in dilp3 and 5 mRNA upon dilp2 knock down. By manipulation of dfoxo and dInR, we showed that the increase in dilp3 is regulated via autocrine insulin signaling in the mNSCs. Our study demonstrates that, despite the correlation between reduced dilp2 mRNA levels and lifespan-extension often observed, DILP2 reduction is not sufficient to extend lifespan. Nor is the increased trehalose storage associated with reduced IIS sufficient to extend lifespan. To understand the normal regulation of expression of the dilps and any functional diversification between them will require independent control of the expression of different dilps.
Mating induces profound changes in female insect behavior and physiology. In Drosophila melanogaster, mating causes a reduction in sexual receptivity and an elevation in egg production for at least 5 ...days. Injection of the seminal fluid sex peptide (SP) induces both responses in virgin females, but only for 1-2 days. The role of SP in eliciting the responses to mating remains to be elucidated. Functional redundancy between seminal fluid components may occur. In addition, mating with spermless males results in brief (1-to 2-day) post-mating responses, indicating either that there is a "sperm effect" or that sperm act as carriers for SP or other seminal fluid components. Here we used RNA interference to suppress SP expression, to determine whether SP is required to elicit full post-mating responses, the magnitude of responses due to other seminal fluid components, and whether SP accounts for the "sperm effect." Receptivity was higher and egg production lower in females mated to SP knock-down males than in controls. Comparison with virgins showed that the responses were brief. SP is therefore required for normal magnitude and persistence of post-mating responses. Sperm transfer and use were normal in mates of SP knock-down males, yet their post-mating responses were briefer than after normal matings, and similar to those reported in mates of spermless son-of-tudor males. The prolonged "sperm effect" on female receptivity and egg production is therefore entirely attributable to SP, but sperm are necessary for its occurrence.
Summary
The insulin/insulin growth factor (IGF)‐like signaling (IIS) pathway has a conserved role in regulating lifespan in Caenorhabditis elegans, Drosophila and mice. Extension of lifespan by ...reduced IIS has been shown in C. elegans to require the key IIS target, forkhead box class O (FOXO) transcription factor, DAF‐16. dFOXO, the Drosophila DAF‐16 orthologue, is also an IIS target, and its overexpression in adult fat body increases lifespan. In C. elegans, IIS acts exclusively during adulthood to determine adult survival. We show here, using an inducible overexpression system, that in Drosophila continuous dFOXO overexpression in adult fat body reduces mortality rate throughout adulthood. We switched the IIS status of the flies at different adult ages and examined the effects of these switches on dFOXO expression and mortality rates. dFOXO protein levels were switched up or down by the inducible expression system at all ages examined. If IIS status is reversed early in adulthood, similar to the effects of another intervention that reduces adult mortality in Drosophila, dietary restriction (DR), there is a complete switch of subsequent mortality rate to that of flies chronically exposed to the new IIS regime. At this age, IIS thus acts acutely to determine risk of death. Mortality rates continued to respond to a switch in IIS status up to 4 weeks of adult age, but not thereafter. However, unlike DR, as IIS status was altered at progressively later ages, mortality rates showed incomplete switching and responded with progressively smaller changes. These findings indicate that alteration of expression levels of dFOXO may have declining effects on IIS status with age, that there could be some process that prevents or lessens the physiological response to a switch in IIS status or that, unlike DR, this pathway regulates aging‐related damage. The decreased mortality and increased lifespan of dFOXO overexpressing flies was uncoupled from any effect on female fecundity and from expression levels of Drosophila insulin‐like peptides in the brain.
Plants acclimate to changes in light quantity by altering leaf-cell development and the accumulation of chloroplast components, such that light absorption is favoured under limiting illumination, and ...light utilisation under non-limiting conditions. Previous evidence suggests an involvement of a high-light photosynthetic redox signal in the down-regulation of the accumulation of the light-harvesting complexes of photosystem II (Lhcb) and the expression of the Lhcb genes, and of a blue-light signal in the control of leaf development and in the increase in photosynthetic capacity, as affected by the accumulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). We examined the internal anatomy of leaves, the ultrastructure of chloroplasts and accumulation of light-harvesting complexes and Rubisco in wild-type Arabidopsis thaliana (L.) Heynh. and in mutants in each of the three known blue-light photoreceptors, cryptochrome 1, cryptochrome 2 and phototropin, as well as a mutant in both cryptochromes. Our results indicate an extensive capacity of the Arabidopsis mesophyll cells to adapt to high light fluence rate with an increase in palisade elongation. Under high light, chloroplasts showed increased starch accumulation and reductions in the amount of granal thylakoids per chloroplast, in the proportion of chlorophyll b relative to chlorophyll a, and in the accumulation of the major Lhcb polypeptides. The responses were similar for all four mutants, with respect to their wild types. The results are consistent with either a complete redundancy in function between cryptochromes and phototropin, or their absence of involvement in the light-quantity responses tested. We observed minimal effects of light quantity on Rubisco accumulation over the range of fluence rates used, and conclude that elongation of palisade mesophyll cells and accumulation of Rubisco are controlled separately. This indicates that light acclimation must be the result of a number of individual elementary responses. Quantitative differences in the acclimatory responses were observed between the Landsberg erecta and Columbia wild-type ecotypes used.
Summary
Mammals possess multiple insulin‐like growth factor (IGF) binding proteins (IGFBPs), and related proteins, that modulate the activity of insulin/IGF signalling (IIS), a conserved ...neuroendocrine signalling pathway that affects animal lifespan. Here, we examine if increased levels of an IGFBP‐like protein can extend lifespan, using Drosophila as the model organism. We demonstrate that Imaginal morphogenesis protein‐Late 2 (IMP‐L2), a secreted protein and the fly homologue of the human IGFBP7 tumour suppressor, is capable of binding at least two of the seven Drosophila insulin‐like peptides (DILPs), namely native DILP2 and DILP5 as present in the adult fly. Increased expression of Imp‐L2 results in phenotypic changes in the adult consistent with down‐regulation of IIS, including accumulation of eIF‐4E binding protein mRNA, increase in storage lipids, reduced fecundity and enhanced oxidative stress resistance. Increased Imp‐L2 results in up‐regulation of dilp2, dilp3 and dilp5 mRNA, revealing a feedback circuit that is mediated via the fly gut and/or fat body. Importantly, over‐expression of Imp‐L2, ubiquitous or restricted to DILP‐producing cells or gut and fat body, extends lifespan. This enhanced longevity can also be observed upon adult‐onset induction of Imp‐L2, indicating it is not attributable to developmental changes. Our findings point to the possibility that an IGFBP or a related protein, such as IGFBP7, plays a role in mammalian aging.
Plant photoreceptors detect light cues and initiate responses ranging from chloroplast differentiation to the control of morphogenesis and flowering. The photocontrol of photosynthesis-related ...nuclear genes appears closely related to 'retrograde plastid signals' by which the status of the organelle controls the expression of nuclear genes. However, what specific role, if any, plastid-originated signals play in light responses is poorly understood: it has in the past been proposed that plastid signals play a role in all responses to 'high fluence' far-red light perceived by the light-labile phytochrome A, irrespective of whether they involve photosynthesis-related genes. To explore this further, we have re-examined the phenotype of three cue (cab-underexpressed) Arabidopsis mutants, defective in chloroplast development. The mutants have underdeveloped etioplasts, with increasing impairments in cue6, cue8 and cue3. The mutants show only small defects in photocontrol of hypocotyl elongation and cotyledon opening under prolonged far-red or red light, and normal photocontrol under blue. On the other hand, the expression of photosynthesis-associated nuclear genes is much more impaired in the mutants in the dark and following red or far-red light short treatments or continuous light, than that of those phytochrome-dependent genes tested which are not associated with photosynthesis. Furthermore, red/far-red photoreversible responses involving photosynthesis-related genes (induction of Lhcb1-cab promoter activity, and photoreversible extent of greening) mediated by phytochrome B and other photo-stable phytochromes, both show a reduction in the cue mutants, which correlates with the etioplast defect. Our evidence demonstrates that plastid-derived signals need to be operational in order for the phytochrome control of photosynthetic nuclear genes to occur.
Over-expression of sirtuins (NAD
+
-dependent protein deacetylases) has been reported to increase lifespan in budding yeast,
Caenorhabditis elegans
and
Drosophila melanogaster
1
-
3
. Studies of gene ...effects on ageing are vulnerable to confounding effects of genetic background
4
. We re-examined the reported effects of sirtuin over-expression on ageing and found that standardisation of genetic background and use of appropriate controls abolished the apparent effects in both
C. elegans
and
Drosophila
. In
C. elegans
, outcrossing of a line with high level
sir-2.1
over-expression
1
abrogated the longevity increase, but not
sir-2.1
over-expression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low copy number
sir-2.1
over-expression
2
also abrogated longevity. A
Drosophila
strain with ubiquitous over-expression of
dSir2
using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported
3
, but not relative to the appropriate transgenic controls, and nor was a new line with stronger over-expression of
dSir2
. These findings underscore the importance of controlling for genetic background and the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life extending effect of dietary restriction (DR) on ageing in
Drosophila
has also been reported to be
dSir2
dependent
3
. We found that DR increased fly lifespan independently of
dSir2
. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects on lifespan in
C. elegans
and
Drosophila
.
Lhcb and other nuclear genes for chloroplastic proteins are regulated by several signals. Among them, light and retrograde signals from the plastid itself appear to act through closely related ...mechanisms. To investigate this interaction, we analysed an Arabidopsis mutant, hy1, deficient in plastidic heme oxygenase. hy1 is defective in phytochrome chromophore biosynthesis, which has other indirect effects on tetrapyrrole metabolism. We generated double mutants between hy1-6.2, genetically a null mutation, and three known gun (genome uncoupled) mutants, defective in retrograde plastid signalling. Recent molecular evidence shows GUN5 to be involved in tetrapyrrole metabolism (N. Mochizuki and J. Chory, manuscript in preparation). We observed hy1gun4 to be semi-albino plants, and hy1gun5 albino lethal, in a high-light-sensitive manner. Both double mutants showed defective greening and chloroplast development, and expressed Lhcb at reduced levels specifically in high light. Their degree of 'genome uncoupling' (Lhcb expression in the absence of functional chloroplasts) was similar to that observed in single mutants. These results can be interpreted as a metabolic (rather than genetic) interaction between HY1 and GUN4 or GUN5, and this in turn supports the involvement of tetrapyrroles as plastid signals. The tetrapyrrole precursor 5-aminolevulinic acid (ALA) inhibited Lhcb expression in hy1. Surprisingly, ALA also rescued photomorphogenesis of hy1. We speculate that either one tetrapyrrole intermediate, which can accumulate anomalously in hy1, or an altered ratio between two intermediates, plays a role as a repressor of Lhcb expression. gun1 did not exacerbate the plastid or Lhcb expression phenotype of hy1. This can be interpreted as a role for gun1 strictly on the same pathway as hy1 or, more likely, as evidence for the existence of at least one separate, non-tetrapyrrole related plastid signal.
Summary
Lhcb and other nuclear genes for chloroplastic proteins are regulated by several signals. Among them, light and retrograde signals from the plastid itself appear to act through closely ...related mechanisms. To investigate this interaction, we analysed an Arabidopsis mutant, hy1, deficient in plastidic heme oxygenase. hy1 is defective in phytochrome chromophore biosynthesis, which has other indirect effects on tetrapyrrole metabolism. We generated double mutants between hy1‐6.2, genetically a null mutation, and three known gun (genome uncoupled) mutants, defective in retrograde plastid signalling. Recent molecular evidence shows GUN5 to be involved in tetrapyrrole metabolism (N. Mochizuki and J. Chory, manuscript in preparation). We observed hy1gun4 to be semi‐albino plants, and hy1gun5 albino lethal, in a high‐light‐sensitive manner. Both double mutants showed defective greening and chloroplast development, and expressed Lhcb at reduced levels specifically in high light. Their degree of ‘genome uncoupling’ (Lhcb expression in the absence of functional chloroplasts) was similar to that observed in single mutants. These results can be interpreted as a metabolic (rather than genetic) interaction between HY1 and GUN4 or GUN5, and this in turn supports the involvement of tetrapyrroles as plastid signals. The tetrapyrrole precursor 5‐aminolevulinic acid (ALA) inhibited Lhcb expression in hy1. Surprisingly, ALA also rescued photomorphogenesis of hy1. We speculate that either one tetrapyrrole intermediate, which can accumulate anomalously in hy1, or an altered ratio between two intermediates, plays a role as a repressor of Lhcb expression. gun1 did not exacerbate the plastid or Lhcb expression phenotype of hy1. This can be interpreted as a role for gun1 strictly on the same pathway as hy1 or, more likely, as evidence for the existence of at least one separate, non‐tetrapyrrole related plastid signal.
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