SEARCH

SEARCH BY CITATION

Keywords:

  • Arabidopsis thaliana ;
  • light intensity;
  • caloric restriction;
  • autophagy;
  • longevity

Summary

  1. Top of page
  2. Summary
  3. Acknowledgments
  4. References
  5. Supporting Information

Caloric restriction (CR) extends lifespan in various heterotrophic organisms ranging from yeasts to mammals, but whether a similar phenomenon occurs in plants remains unknown. Plants are autotrophs and use their photosynthetic machinery to convert light energy into the chemical energy of glucose and other organic compounds. As the rate of photosynthesis is proportional to the level of photosynthetically active radiation, the CR in plants can be modeled by lowering light intensity. Here, we report that low light intensity extends the lifespan in Arabidopsis through the mechanisms triggering autophagy, the major catabolic process that recycles damaged and potentially harmful cellular material. Knockout of autophagy-related genes results in the short lifespan and suppression of the lifespan-extending effect of the CR. Our data demonstrate that the autophagy-dependent mechanism of CR-induced lifespan extension is conserved between autotrophs and heterotrophs.

Arabidopsis is a monocarpic plant with a short life cycle that can be divided into four major stages: seedling development, vegetative development (or leaf rosette development), and partly overlapping flowering and rosette senescence (Boyes et al., 2001). The lifespan of Arabidopsis extends from radicle emergence until complete senescence of the rosette and cessation of flowering. We found that decreasing the light intensity from 150 μE m−2 s−1 [standard conditions recommended for Arabidopsis (Hennig, 2010); hereafter referred to as normal light (NL)] to 100 μE m−2 s−1 (low light; LL) under constant photoperiod (16 h) delayed most of the developmental transitions in wild-type Columbia (WT) plants (Table S1) resulting in extension of the individual stages of life cycle (Fig. 1A, Table S2) and cumulative increase in the mean lifespan up to ~25% (Fig. 1A). To confirm that LL treatment mimics caloric restriction (CR), we measured photosynthetic capacity (SPAD values proportional to chlorophyll content) and glucose content in the rosette leaves at the onset of flowering. As expected, lowering light intensity was accompanied by decrease in both SPAD values and glucose content (Fig. S1).

image

Figure 1. The effect of light intensity and genetic inhibition of autophagy on Arabidopsis lifespan. Full legend is provided in supporting information online. (A) Life cycle of WT, atg and complementation lines grown under NL and LL. X-axis indicates days after radicle emergence. Numbers on the horizontal bars indicate mean length (days) of the developmental stage. Left and right graphs depict results of two representative independent trials, including atg5 and atg7 plants, respectively. P values indicate probability of lifespan extension by LL (Student's t-test). Detailed statistical analysis is provided in Tables S1 and S2. (B) Kaplan–Meier survival curves for the lifespan data from the same trials, as shown in A. atg5 compl, complementation lines atg5 pATG5::ATG5. atg7 compl, complementation lines atg7 pATG7::ATG7.

Download figure to PowerPoint

It has recently been shown in yeast and animal models that CR promotes longevity via activation of autophagy (Madeo et al., 2010; Rubinsztein et al., 2011). To address possible roles of autophagy in LL-induced lifespan extension in Arabidopsis, we analyzed two autophagy markers, the degradation of the autophagic adapter protein neighbor of BRCA1 gene product (NBR1) by Western blots (Svenning et al., 2011; Klionsky et al., 2012) and the expression of ATG8a by real-time quantitative PCR (Thompson et al., 2005) in the leaves of NL- and LL-grown WT plants at the onset of flowering and 10 days later. Under LL, degradation of NBR1 and upregulation of ATG8a over a 10-day period were, respectively, ~3.5 and ~2 times higher, as compared with NL-grown plants (Fig. 2), indicating that LL stimulates autophagy.

image

Figure 2. Low light stimulates autophagy in Arabidopsis. Full legend is provided in supporting information online. (A) Western blot analysis of the total protein extracts from rosette leaves using anti-NBR1. The leaves were sampled at the onset of flowering (0 days after flowering, DAF) and after 10 days (10 DAF). Coomassie blue staining of the total protein (lower panel) was used as a loading control. Percentages shown below the upper panel correspond to the mean relative amount of NBR1 protein. Note dramatic decrease of NBR1 in LL-grown WT plants at 10 DAF, indicative of induction of autophagy. In contrast, atg plants accumulate NBR1 under corresponding conditions reflecting impaired autophagic flux. (B) Real-time quantitative PCR analysis of ATG8a expression in rosette leaves. The data represent mean relative level of ATG8a transcript at 10 DAF compared with 0 DAF. Error bars denote SD (n = 3). Low light induced significant upregulation of expression (P < 0.0001; Student's t-test) in all genotypes. atg5 compl, complementation lines atg5 pATG5::ATG5. atg7 compl, complementation lines atg7 pATG7::ATG7.

Download figure to PowerPoint

Next, we investigated whether autophagy is required for lifespan extension in Arabidopsis using knockout lines atg5-1 (Thompson et al., 2005) and atg7-2 (Hofius et al., 2009). It has previously been shown that autophagy-deficient mutants of Arabidopsis have enhanced expression of ATG8 genes, especially under conditions inducing autophagy (Thompson et al., 2005). Consistent with these data, knockout of either ATG5 or ATG7 led to upregulation of ATG8a in response to the LL conditions to the levels higher than in WT and complementation lines (Fig. 2B). At the same time, both atg5 and atg7 plants grown under LL revealed strong accumulation of NBR1, indicative of impaired autophagic flux (Fig. 2A). ATG knockout plants have previously been shown to exhibit an early-senescence phenotype even under favorable growth conditions (Liu & Bassham, 2012). Apart from the early onset and completion of rosette senescence, faster completion of flowering and accelerated silique shattering (Table S1), we found that the lengths of all major stages of the life cycle in atg5 and atg7 plants were reduced regardless of the light intensity (Fig. 1A, Table S2). As a result, the mean lifespan of atg5 and atg7 mutants was decreased by ~22% and ~12%, respectively, under NL, and by ~32% and ~22%, respectively, under LL. Collectively, these data demonstrate that autophagy attenuates transition through the successive stages of Arabidopsis life cycle and therefore is the prerequisite for longevity.

We have noticed that both atg5 and atg7 plants retained the ability to initiate CR-like response (i.e., decrease in both photosynthetic capacity and glucose content) to LL (Fig. S1). Therefore, we assessed whether these plants were able to sense CR and extend their lifespan similar to WT. The survival data show that the LL-induced increase in plant longevity was dramatically reduced (atg5) or completely abrogated (atg7) in the autophagic mutants, as compared with WT and complementation lines (Fig. 1). Thus, our findings reveal that intact autophagy machinery confers lifespan extension in Arabidopsis under the CR conditions.

Restricting uptake of calories has long been known as a simple and potent method for increasing longevity in heterotrophs (Mair & Dillin, 2008), and has recently been linked to the activation of autophagy (Madeo et al., 2010; Rubinsztein et al., 2011). Our work provides the first experimental setting for studying mechanisms increasing plant longevity under the CR and establishes autophagy as one of the crucial components. The plant autophagic machinery, and especially induction phase of plant autophagy, remains poorly understood, hampering our mechanistic insight into how autophagy is activated under the LL conditions. Once activated, autophagy can act in a number of pathways to confer cytoprotection (e.g., elimination of oxidized proteins and defective/old organelles) and sustain plant development (e.g., remobilization of nutrients) (Liu & Bassham, 2012), which collectively should lead to the lifespan extension.

Being an ephemeral monocarpic plant, Arabidopsis literally reproduces itself to death providing a tractable model to investigate lifespan regulation. Considering a central role of autophagy in the longevity of such evolutionary distant organisms as yeast, Arabidopsis, and mammals, autophagy is likely to play similarly important role in the longevity of polycarpic plants. Therefore, genetic or pharmacological manipulation of autophagy might open new venues for crop and tree production.

Acknowledgments

  1. Top of page
  2. Summary
  3. Acknowledgments
  4. References
  5. Supporting Information

This work was supported by the Swedish Research Council, Pehrssons Fund, the Swedish Foundation for Strategic Research, Olle Engkvist Foundation and the Spanish Ministry of Science and Innovation. V.S-V was a recipient of a FPI fellowship from the Spanish Ministry of Science and Innovation (BES-2008-003592). We thank Richard Vierstra for atg5-1 seeds, Daniel Hofius for atg7-2 seeds, Terje Johansen for anti-NBR1 and Andrei Smertenko, David Clapham, Daniel Hofius, and Hans Ronne for valuable comments on the manuscript.

References

  1. Top of page
  2. Summary
  3. Acknowledgments
  4. References
  5. Supporting Information
  • Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davies KR, Görlach J. (2001) Growth stage-based phenotypic analysis of Arabidopsis: A model for high throughput functional genomics in plants. Plant Cell 13, 14991510.
  • Hennig L (2010) Growth protocols for model plants in developmental biology. Methods Mol. Biol. 655, 110.
  • Hofius D, Schultz-Larsen T, Joensen J, Tsitsigiannis DI, Petersen NH, Mattsson O, Jorgensen LB, Jones JD, Mundy J, Petersen M. (2009) Autophagic components contribute to hypersensitive cell death in Arabidopsis. Cell 137, 773783.
  • Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, Agholme L, Agnello M, Agostinis P, Aguirre-Ghiso JA, et al. (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445544.
  • Liu Y, Bassham DC (2012) Autophagy: pathways for self-eating in plant cells. Annu. Rev. Plant Biol. 63, 215237.
  • Madeo F, Tavernarakis N, Kroemer G (2010) Can autophagy promote longevity? Nat. Cell Biol. 12, 842846.
  • Mair W, Dillin A (2008) Aging and survival: the genetics of life span extension by dietary restriction. Annu. Rev. Biochem. 77, 727754.
  • Rubinsztein DC, Marino G, Kroemer G (2011) Autophagy and aging. Cell 146, 682695.
  • Svenning S, Lamark T, Krause K, Johansen T (2011) Plant NBR1 is a selective autophagy substrate and a functional hybrid of mammalian autophagic adapters NBR1 and p62/SQSTM1. Autophagy 7, 9931010.
  • Thompson AR, Doelling JH, Suttangkakul A, Vierstra RD (2005) Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol. 138, 20972110.

Supporting Information

  1. Top of page
  2. Summary
  3. Acknowledgments
  4. References
  5. Supporting Information
FilenameFormatSizeDescription
acel12048-sup-0001-FigS1.tifimage/tif305KFig. S1 Low light induces caloric restriction in Arabidopsis
acel12048-sup-0002-DataS1.docxWord document40KData S1 Supporting Information containing full legends for Fig. 1 and Fig. 2, the legend for Fig. S1, experimental procedures with the list of primers and additional references.
acel12048-sup-0003-TableS1.pdfapplication/PDF88KTable S1 Timing of developmental transitions in wild-type, atg mutants and complementation lines grown under normal and low light conditions.
acel12048-sup-0004-TableS2.pdfapplication/PDF86KTable S2 Length of major stages of life cycle in wild-type, atg mutants and complementation lines grown under normal and low light conditions.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.