Techniques for isolating and analysing leaf cell types have now been developed, leading to the discovery that circadian clocks in the plant vasculature communicate with and regulate clocks in neighbouring cells. See Letter p.419
The flowering plants in our gardens and in the countryside provide us with a colourful landscape, and are often thought of as nothing more than a dormant backdrop to our lives. But beneath their attractive exteriors, plants are capable of complex behaviour, such as measuring time. In this issue, Endo et al.1 (page 419) identify circadian clocks in leaf veins that signal to neighbouring cells — an indication that plant circadian clocks might be organized into a hierarchical system.
Plant leaves are sophisticated organs comprised of several cell types, each with a different function. Epidermal cells line the leaf surface, with the bulk of the leaf being composed of mesophyll cells, which are responsible for photosynthesis. In addition, the leaves and stem are infiltrated by the veins of the plant vasculature, which transports water and molecules such as sugars around the plant. Endo and colleagues developed a method for efficiently isolating epidermal, mesophyll and vasculature cells from Arabidopsis thaliana plants, allowing them to study spatiotemporal gene expression and circadian-clock regulation at high resolution.
Multicellular organisms ensure that cells are performing the correct processes at the right time of day through their circadian clocks, which have a period of approximately 24 hours, allowing anticipation of dawn and dusk. The timing of about 30% of gene activity in plants is modulated by circadian clocks. A clock's core consists of around 20 genes divided into two interlocking pathways — a morning loop of genes that are active during daylight hours and an evening loop active from dusk.
The researchers observed that morning-loop genes such as CCA1 were more active in the mesophyll than in the vasculature, whereas the opposite was true of evening-loop genes such as TOC1. Furthermore, when they measured genome-wide gene activity, the authors found differential gene expression in each tissue. Output genes (those regulated by circadian clocks) that were more active in the mesophyll than in the vasculature tended to be expressed in the morning, whereas output genes more active in the vasculature were likely to be expressed in the evening. This suggests that differences in the circadian clock of each tissue cause differential gene expression (Fig. 1).
Evidence of differences between the circadian clocks of leaves and roots2 suggests that cell-type-specific clocks regulate specialized plant-cell functions. The activation of mesophyll-specific genes in the morning might reflect the need for photosynthesis to begin around dawn3. Enhanced evening-loop activity in the vasculature might be required to ensure accurate measurement of the timing of dusk, and therefore of day length — a measurement that controls flower production in many species4. Indeed, Endo and colleagues demonstrated that disruption of the circadian clock in the vasculature, but not in the mesophyll, epidermis, stem or root affected the timing of flower production in Arabidopsis. The vascular clock might also regulate vascular-specific night-time activities, such as refilling vessels to remove air bubbles.
A common feature of multicellular organisms is that the circadian clocks of neighbouring cells can communicate with each other, forming synchronized groups of cells that either create a robust oscillating system or convey information about time to distant organs. In mammals, for example, a coupled clock in the hypothalamus region of the brain regulates clocks in other tissues. In plants, weak communication between individual circadian clocks has been observed5, and it has been proposed that circadian clocks in the leaves are masters over those in the roots2. Endo et al. now provide experimental evidence for local coupling of clock systems in plants. They stopped the vascular clock by overexpressing CCA1 in cells of the vasculature, and demonstrated that this also inhibited the clocks of the neighbouring mesophyll cells. This might be achieved through chemical signalling, perhaps involving sugars, because leaf-cell clocks are sensitive to changing sugar levels6.
The communication between the circadian clocks in the vasculature and mesophyll might be hierarchical. Overexpression of CCA1 in the mesophyll had little effect on the vascular circadian clock. Because the clocks of the two cell types are differentially enriched for morning and evening components, it will be interesting to determine whether signalling occurs from the vasculature if TOC1 is overexpressed in a cell-type-specific manner.
Is the plant vasculature an interconnected system, generating robust oscillations that regulate other cells, similar to the circadian pacemakers of mammalian brains? Or might it function as a pipeline that disseminates timing signals, analogous to the circadian clocks of red blood cells7? The vasculature is certainly more than just sophisticated plumbing; it acts as a conduit for rapid electrical8, oxidative9 and ionic10 signals, reminiscent of a nervous system. However, the analogies to mammalian systems break down under scrutiny. Plants do not require the rapid responses provided by a nervous system, because their movements — usually mediated by growth — are slower than those of animals.
Endo and colleagues' work will make it easier to study individual plant cell types. By optimizing protocols for dissection, sonication and enzyme treatments that degrade the cell wall, they have considerably shortened the time required to isolate cells for RNA measurement. Furthermore, the researchers have developed imaging techniques for studying spatiotemporal gene regulation in plants. They engineered two halves of the luminescent protein luciferase such that one half was produced only in a specific cell type and the other half only when the promoter that drives either CCA1 or TOC1 gene expression was active. Because both halves must be produced in a cell for luminescence to occur, light emission can be used as a measure of the activity of a circadian-clock gene in a given cell type. This approach can be extended to other cell types and responses, such as stress and developmental signals, simply by using different promoters to drive the two halves of luciferase.
The ability to study individual leaf cell types in detail will surely lead to a deeper understanding of circadian regulation of gene activity, development and photosynthesis. The first steps will be to determine why leaf circadian clocks communicate, and which signalling pathways convey information about time. Such knowledge is sorely needed if the challenge of improving crops to feed the growing human population is to be met.