Plant biology

Time for growth

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Analyses of growth kinetics in seedlings reveal exquisite connections between the signalling pathways controlled by the circadian clock and by light, and illuminate the molecular mechanisms involved.

Using infrared light imaging to observe cell elongation in the dark, Nozue and colleagues (page 358 of this issue)1 have made a fascinating discovery — that the growth rate of plant seedlings, specifically of a structure called the hypocotyl, is differentially regulated during a day–night cycle. Intriguingly, the maximal rates occur at dawn. To determine the molecular nature of this observation, the authors designed a series of experiments using several well-characterized mutants of Arabidopsis thaliana, a favoured subject in experimental plant biology. Their results allow them to separate the distinct contribution of light perception and the associated responses from that of the plant's circadian clock. Furthermore, they have identified two transcription factors — mediators of the production of messenger RNA from DNA — that regulate this cyclic mode of growth.

Genetic screens of Arabidopsis mutants have revealed the complex nature of growth regulation in the hypocotyl; this is a small region of about 20 epidermal cells in length that lies between the root and the embryonic leaves of young seedlings, and that grows mostly through longitudinal cell expansion2. Many mutations in genes involved in hormonal, light-perception and circadian pathways result in short or long hypocotyls2,3. Measurement of hypocotyl length in constant light or constant dark is commonly used to characterize light-signalling or clock mutants. Growth in constant darkness is thought to mimic the conditions experienced by seedlings that are emerging from the soil, and reaching for light at the surface. In the dark, seedlings enter a form of development termed etiolation, in which most of the plants' resources are channelled into elongation of the hypocotyl. In contrast, seedlings grown in the light follow a different path, including the inhibition of etiolation and initiation of the greening process that enables light capture through photosynthesis2,3.

Nozue and colleagues1 have integrated study of these two conditions, which are normally considered separately, by measuring hypocotyl growth rate under diurnal conditions — that is, cycles of light and dark. They first noticed that, following a few days of non-consolidated growth, seedlings seem to tune their maximum growth at dawn. They showed that seedlings with specific defects in light perception had weak or no growth rhythms, suggesting that light signalling is essential for rhythmic growth under diurnal cycles.

To distinguish the role of the circadian clock on hypocotyl growth from that of light, they performed similar experiments using plants with clock defects. The output of the clock creates a temporal matrix that is used to drive overt rhythms, such as photosynthesis and protective mechanisms against cold at night, and can also serve to anticipate changes between day and night. One of the hallmarks of plant circadian clocks is their capacity to confer cycling behaviour under constant light conditions, and mutants with a disrupted clock have been used4 to define a role for the clock in the timing of cell elongation.

Interestingly, Nozue et al. showed that, under conditions of diurnal cycles, maximal growth of mutants with an impaired clock occurred from dusk to dawn instead of being restricted to a few hours at dawn, suggesting that those seedlings were hyper-responsive to darkness. Additional experiments confirmed this observation, which implies that, under diurnal conditions, hypocotyl growth in wild-type (non-mutant) seedlings is partly controlled by light–dark transitions, whereas the circadian clock acts to suppress growth in the early part of the night.

What might be the molecular mechanism associated with growth control? To address this question, Nozue et al. carried out transcription profiling using whole-genome arrays. Taking advantage of the fact that clock mutants are hyper-responsive to darkness, they designed a strategy to find genes whose expression is associated with maximal growth rates. They identified two transcription factors — PIF4 and PIF5 (also known as PIL6), which belong to a family known as phytochrome-interacting factors (PIFs)5 — to be good candidates as light-regulated components of the mechanism. Overexpression of each factor alone in Arabidopsis led to seedlings that were hyper-responsive to the dark. Furthermore, double mutants had a very short hypocotyl that didn't display any growth rhythmicity. These striking results confirm the growth-promoting nature of the PIF4 and PIF5 proteins.

Finally, two different experiments helped to determine how both genes might be regulated by light and the circadian clock. Expression-profiling experiments revealed that both are under circadian regulation and are expressed at high levels in seedlings without a functional clock (Fig. 1a). Because in wild-type seedlings the expression maxima are at dawn, and the minima are at the beginning of the night, the authors propose that, during the early night, growth suppression by the circadian clock must in part occur through transcriptional repression of the PIF4 and PIF5 genes (Fig. 1b). Further experiments with transgenic plants overexpressing PIF4 and PIF5 showed that the abundance of both proteins decreased in the light and increased in the dark. These results complete an elegant model of events, in which one of the essential processes involved in the reduction of growth rate after dawn is the light-dependent degradation of PIF4 and PIF5.

Figure 1: From physiological observation to molecular mechanism.

Nozue and colleagues' comparison1 of hypocotyl growth rhythms in (a) clock mutants and (b) wild-type seedlings under light–dark cycles reveals the repressing effects of the circadian clock in the early night and of light during the day. a, Unregulated growth during the night; b, the normal growth pattern. Molecular studies identified two growth-promoting factors (PIF4 and PIF5), the messenger RNAs of which are normally clock-regulated (blue line) by a repressing action in the early night. In addition, the protein levels are reduced in a light-dependent manner (red line) possibly through a light-receptor-mediated mechanism. Thus, internal cues (clock) restrict transcriptional activation in the late night leading to hypocotyl elongation before dawn, whereas an external cue (sun) inhibits growth during the day by targeting the proteins for degradation. It is the coincidence of both cues that leads to the observed maximum growth at dawn.

Questions remain, of course. Which factor negatively regulates PIF4 and PIF5 mRNA in the early night? Which molecular complex controls their degradation? And which genes are targeted by PIF4 and PIF5? More generally, there is the issue of whether Nozue et al.1 have uncovered design principles that apply to growth regulation in other tissues, given that growth of the stem and leaf seem to be under similar control6,7. For the moment, however, publication of their discovery provides a considerable step forward in understanding the factors that shape the young seedling's quest for photons.


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    Nozue, K. et al. Nature 448, 358–361 (2007).

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    Nozue, K. & Maloof, J. N. Plant Cell Environ. 29, 396–408 (2006).

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    Wiese, A., Christ, M. M., Virnich, O., Schurr, U. & Walter, A. New Phytol. 174, 752–761 (2007).

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    Jouve, L., Gaspar, T., Kevers, C., Greppin, H. & Degli Agosti, R. Planta 209, 136–142 (1999).

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