Trifurcate Feed-Forward Regulation of Age-Dependent Cell Death Involving miR164 in Arabidopsis

See allHide authors and affiliations

Science  20 Feb 2009:
Vol. 323, Issue 5917, pp. 1053-1057
DOI: 10.1126/science.1166386


Aging induces gradual yet massive cell death in higher organisms, including annual plants. Even so, the underlying regulatory mechanisms are barely known, despite the long-standing interest in this topic. Here, we demonstrate that ORE1, which is a NAC (NAM, ATAF, and CUC) transcription factor, positively regulates aging-induced cell death in Arabidopsis leaves. ORE1 expression is up-regulated concurrently with leaf aging by EIN2 but is negatively regulated by miR164. miR164 expression gradually decreases with aging through negative regulation by EIN2, which leads to the elaborate up-regulation of ORE1 expression. However, EIN2 still contributes to aging-induced cell death in the absence of ORE1. The trifurcate feed-forward pathway involving ORE1, miR164, and EIN2 provides a highly robust regulation to ensure that aging induces cell death in Arabidopsis leaves.

In most organisms, aging leads to organ- and organism-level senescence that eventually causes death and limits longevity. In plants, age-associated senescence and cell death are most dramatically observed in the leaves of annual plants and deciduous trees. Leaf senescence and the associated cell death are developmentally programmed processes that occur in an age-dependent manner, integrating multiple developmental and environmental signals (1, 2). Age-associated cell death in plant leaves is a type of programmed cell death (PCD), but it occurs more slowly and massively than other acute PCD observed during tissue wounding or viral infection, for example. Although leaf senescence and the associated cell death are widely observed in nature and are regarded as a developmental strategy for plant fitness (3), the underlying molecular mechanisms remain elusive.

The Arabidopsis oresara1-1 (ore1-1, oresara means “long-living” in Korean) mutant was initially identified as a delayed leaf senescence mutant (4). We isolated and genetically analyzed the ore1-2 allele in this study (table S1) (5). Leaf yellowing due to the loss of chlorophyll is a typical characteristic of leaf senescence. The phenotype of the ore1-2 mutant plants, with delayed loss of chlorophyll at a later age, is shown in Fig. 1A. We then examined age-associated characteristics in single leaves along their ages. Wild-type leaves showed a reproducible aging pattern; the third and fourth foliar leaves showed a life span of ∼36 days from their emergence (Fig. 1, B and C). Delayed loss of chlorophyll content and of photochemical efficiency (Fv/Fm) (6) with leaf aging was observed in ore1 mutants (Fig. 1, B and C) (4). The expression of the chlorophyll a/b-binding protein 2 (CAB2) gene and the cystein protease-encoding senescence-associated gene 12 (SAG12) exhibited delayed reduction and induction, respectively, with aging (Fig. 1D). We also found that aging-induced cell death was delayed in the ore1 mutants, as shown by slower increases in membrane ion leakage (7) of the leaves with aging (Fig. 1E) and by the absence of trypan blue–stained cells (7) in aged (28-day-old) leaves (Fig. 1F). Thus, ORE1 is a positive regulator of aging-induced cell death and senescence in Arabidopsis leaves.

Fig. 1.

Age-dependent cell death is delayed in the ore1 mutants. (A) Whole plant phenotype of the wild type (WT) and ore1-2 at 45 days after germination (DAG). WT, Col; scale bar, 1 cm. (B) Chlorophyll loss in WT, ore1-1, and ore1-2 with leaf aging. The third rosette leaves at the indicated days after emergence (leaf age) are shown. Scale bar, 0.5 cm. (C to F) Photochemical efficiency (Fv/Fm) of photosystem II (C), expression of CAB2 and SAG12 (D), membrane ion leakage (E), and trypan blue staining (F) of WT and ore1 leaves at the indicated leaf age. In (C) and (E), values are means ± 95% confidence intervals (± 95 CI; n = 12 to 14 leaves). In (F), WT leaves developed blue-colored patches of cells (arrows), indicating areas of dead or dying cells.

Map-based cloning of the ORE1 locus with the use of ore1-2 (fig. S1) revealed that the mutation was caused by a 5–base pair deletion in At5g39610, which encodes a NAC (NAM, ATAF, and CUC) transcription factor, AtNAC2 (fig. S2, A and B) (8). The nuclear localization of ORE1/AtNAC2 (fig. S2C) is consistent with its role as a transcription factor. A search of microarray data (9) revealed that ORE1 expression increases in senescing leaves, and time-course analysis (Fig. 2, A and E) confirmed that ORE1 expression increases with leaf aging. This result implies that ORE1 regulates aging-induced cell death and senescence via its increased expression with leaf aging, which would, in turn, induce senescence-associated downstream genes as a transcription factor.

Fig. 2.

ORE1 mRNA is a target of miR164. (A) Inverse expression pattern of ORE1 and miR164 with leaf aging. rRNA and tRNA and 5S rRNA, loading controls; EtBr, ethidium bromide stain; SYBR, SYBR gold stain. (B) Expression of ORE1 in control vector (Vec) and miR164B-overexpressing (miR164B-ox) lines. The arrow indicates a smaller fragment derived from the ORE1 transcript. (C) Alignment of the miR164 sequence with wild-type ORE1 (ORE1), mutant ORE1 (mORE1), and a truncated EST (RAFL07-50-B15). (D) Effect of miR164-target sequence on the level of ORE1 mRNA. In 22-day-old leaves, ORE1 mRNA levels were measured in the F1 progenies of transgenic lines overexpressing wild-type (ORE1-31xVec, ORE1-31xmiR164B-ox, ORE1-52xVec, and ORE1-52xmiR164B-ox) or mutant ORE1 (mORE1-20xVec, mORE1-20xmiR164B-ox, mORE1-75xVec, and mORE1-75xmiR164B-ox). (E) Level of ORE1 mRNA in WT and the mir164abc mutant leaves with leaf age. (Inset) Smaller view of the graph at earlier ages. (F) Age-dependent decline of the ratio of ORE1 level in the mir164abc mutant relative to that in WT. Error bars indicate SE of the mean in (D), (E), and (F).

The ORE1/AtNAC2 mRNA possesses a potential miR164-binding sequence in its third exon (fig. S2A) (10). The miR164 family in Arabidopsis includes three isoforms [miR164A, miR164B, and miR164C (11)] and represses the expression of a group of NAC family genes by cleaving the target mRNAs (1215). We found that the amount of miR164 declined with leaf aging (Fig. 2A). The inverse correlation between the expression of miR164 and ORE1 with leaf aging led to the hypothesis that miR164 negatively regulates ORE1 mRNA, thereby antagonizing age-dependent senescence. We first tested if ORE1 mRNA is a target of miR164-mediated cleavage. The level of ORE1 mRNA increased and reduced, respectively, in the mir164abc triple mutant (Fig. 2E and fig. S3) (14) and in the miR164B-overexpressing lines (Fig. 2B and fig. S4). In the miR164B-overexpressing lines, a smaller transcript was found, which is probably a cleavage product of the full-length ORE1 transcript (Fig. 2B). We found that several EST sequences of ORE1 are truncated at the potential miR164-target sequence (Fig. 2C). Furthermore, in transgenic lines expressing a mutant version of ORE1 (mORE1) where six mismatches were introduced into the potential miR164-binding sequence (Fig. 2C), the level of the mutant ORE1 transcript was less affected by overexpression of miR164B (Fig. 2D). The minor reduction observed in the transgenic plants overexpressing mutant ORE1 and miR164B is probably due to the cleavage of the endogenous wild-type ORE1 mRNA present in the transgenic lines. These results confirmed that ORE1 mRNA is a target of miR164-guided cleavage.

We then tested whether miR164 controls the level of the ORE1 transcript during leaf aging by examining its level in the mir164abc triple mutant. The differences in ORE1 transcript levels between the wild type and the mir164abc mutant were greater in younger leaves and became negligible when the level of miR164 was barely detectable in older leaves (Fig. 2, E and F). This result suggests that ORE1 expression is negatively regulated by miR164 at earlier stages, which is relieved at later stages because of the age-dependent down-regulation of miR164 expression. This observation led us to test whether miR164 functions as a negative regulator of aging-induced leaf cell death. Aging-induced cell death was accelerated in the mir164abc mutant, as indicated by a faster decline in chlorophyll content (fig. S5A) and photochemical efficiency and a faster increase in membrane ion leakage and SAG12 expression with leaf aging (Fig. 3A). The opposite trends were observed in the miR164A- and miR164B-overexpressing lines (Fig. 3B and fig. S5B). Moreover, aging-induced cell death and senescence symptoms were more pronounced in the mORE1 overexpression lines (fig. S6), where the mORE1 transcript level was higher because of the escape from the miR164-guided cleavage (Fig. 2D). Therefore, miR164 negatively regulates aging-induced cell death and senescence through down-regulation of ORE1.

Fig. 3.

miR164 negatively regulates aging-induced cell death. (A and B) Photochemical efficiency (top), membrane ion leakage (middle), and transcript level of SAG12 (bottom) in mir164abc (A) and miR164A- and miR164B-overexpressing leaves (B). The values of photochemical efficiency and ion leakage are means ± 95 CI (n = 12 to 18).

Next, we asked how the age-dependent decline of miR164 expression occurs. Because miR164 negatively regulates leaf senescence, a genetic mutation in upstream regulatory elements that relieves age-dependent down-regulation of miR164 expression would lead to delayed senescence symptoms. We thus examined the age-dependent miR164 expression in the delayed senescence mutants ore1-1, ethylene insensitive 2-34 (ein2-34), ore12-1, and ore7-1D (Fig. 4A); ein2-34 was originally isolated as ore3-1 (4). Among the tested mutants, the ein2-34 mutant alone exhibited the least difference in miR164 expression between 8- and 20-day-old leaves. The age-dependent regulation of miR164 expression by EIN2 was examined in more detail at 4-day intervals (Fig. 4B). miR164 expression was barely altered with aging in the ein2-34 mutant. The results indicate that EIN2 mediates age-dependent down-regulation of miR164.

Fig. 4.

A trifurcate feed-forward pathway controls age-dependent leaf cell death. (A) Comparison of miR164 level between 8 and 20 days of leaf age in WT and delayed senescence mutant leaves. (B) Age-dependent expression of miR164 in ein2-34 leaves with aging. tRNA and 5S rRNA are loading controls. (C) Age-dependent expression level of ORE1 mRNA in WT and ein2-34 leaves. (D and E) Photochemical efficiency (D) and expression levels of SAG12 (E) of WT, ore1-1, ein2-34, and ore1-1 ein2-34 leaves. In (D), values are mean ± 95 CI (n = 12).

EIN2 is required for salt-induced expression of ORE1/AtNAC2 in seedlings (8). We found that age-dependent induction of ORE1 relies on EIN2, as the level of ORE1 transcript in the ein2-34 mutant was only 12% of that in the wild type in 28-day-old plants (Fig. 4C). Thus, up-regulation of ORE1 expression results from age-dependent induction through EIN2, as well as stabilization of ORE1 mRNA due to the age-dependent down-regulation of miR164 by EIN2. The functional relation between ORE1 and EIN2 in aging-induced leaf cell death was further examined using the ore1-1 ein2-34 double mutant. In the double mutant, the loss of photochemical efficiency and the induction of SAG12 expression exhibited a longer delay than did either of the single mutants (Fig. 4, D and E). Therefore, although age-dependent induction of ORE1 critically depends on EIN2, ore1-1 and ein2-34 did not show a simple epistatic interaction but rather a partially additive effect.

Our study leads to a trifurcate feed-forward pathway for regulation of age-dependent cell death and senescence in Arabidopsis leaves (fig. S7): ORE1 is a transcription factor that functions positively in cell death. ORE1 is induced in an age-dependent manner by EIN2. ORE1 is negatively regulated by miR164 at earlier stages, which is relieved at later stages because of the age-dependent down-regulation of miR164 expression by EIN2. However, EIN2 also uses another pathway to regulate aging-induced cell death that does not include ORE1. Additionally, miR164 may function in the age-dependent cell death pathway as a “brake” or “guard” against premature overexpression of ORE1 and may finely tune the timing of senescence and cell death. We propose that the trifurcate pathway exists to ensure senescence and the accompanying cell death when leaves are aged. This trifurcate pathway contains an embedded coherent feed-forward loop (16). Mathematical modeling (fig. S8) of this pathway showed that SAG12 is gradually induced upon a systematic, persistent action of EIN2, but it is not induced upon nonsystematic variations of EIN2 action. EIN2, a component of the trifurcate pathway, regulates other functions including ethylene signaling, cell growth control, and stress responses as well as leaf senescence (4, 17, 18). ORE1/AtNAC2 is also induced by salt as well as aging (8). Therefore, the age-dependent trifurcate pathway is probably interwoven with other developmental and environmental signals to tune leaf senescence and cell death processes.

miRNAs are widespread and are involved in a variety of biological processes, in both animals and plants (11, 19). Here, we place the function of the plant miR164 in the context of age-dependent developmental pathways. The members of the Arabidopsis miR164 target a group of NAC family genes (CUC1, CUC2, NAC1, ORE1, At5g07680, and At5g61430) and function in various developmental processes, including lateral root development and organ boundary formation in shoot meristem and flower development (1315). Our study showed that miR164 functions in regulation of age-dependent cell death in leaves through changes in its expression level over the life span of the leaves. Do the other miR164-binding NAC family genes also affect aging-induced cell death? Among the six NAC family genes, NAC1, ORE1, and At5g61430 were induced with leaf aging (fig. S9A) and were down-regulated in miR164-overexpression lines (fig. S9B), implying that they are targets of miR164 at the aged leaves. However, loss-of-function mutations of CUC1, CUC2, NAC1, and At5g07680 did not noticeably alter age-dependent leaf senescence (see fig. S9C for a representative example of NAC1). Thus, at least the four NAC genes we tested may not have a critical function in age-induced leaf senescence, or their effect on leaf senescence may be negligible to be detected in a single mutation. Expression of miR156 and miR172 in Arabidopsis is also regulated along the developmental time to control heteroblasty and flowering, respectively (20, 21). Thus, we note that miRNAs appear to participate in the regulation of a range of temporal events in plants. The mechanism presented here should provide insights into aging-induced cell death and senescence in plants as well as in other systems, including animals.

Supporting Online Material

Materials and Methods

Figs. S1 to S9

Tables S1 and S2


References and Notes

View Abstract

Navigate This Article