A novel regulatory circuit underlying plant response to canopy shade

Abstract

A plant growing in the field has the unique ability to sense the presence of other plants growing near by and adjust its growth rate accordingly. This ability to detect neighbors, which is referred to as shade avoidance response, is mediated by members of the phytochrome family which detect light in the red (R) and far-red (FR) region of the spectrum. Work done by several laboratories has shown that low R/FR provides the signal for shade avoidance response during which the elongation of stem-like organs occurs at the expense of leaf development. However, the mechanism by which the low R/FR signal is transduced to attenuate leaf development has remained largely unknown. In the August issue of Genes and Development, we have shown that low R/FR rapidly and transiently arrests the growth of the leaf primordium. By exploiting mutant analysis in combination with genome wide expression profiling, we have identified a novel regulatory circuit underlying plant response to canopy shade. Together, the data demonstrate that the growth arrest induced by low R/FR depends on auxin-induced cytokinin breakdown in pre-procambial cells of developing primordia. In this addendum, we discuss open questions to be addressed in the future.

To grow and develop optimally, all organisms need to perceive and process information from their environment. As sessile organisms, plants need to sense and respond to external stimuli more than most organisms, and have evolved sophisticated mechanisms to perceive environmental cues and adapt their developmental patterns to changes in the natural environment, thereby ensuring survival and reproduction. Being photosynthetic, plants are especially sensitive to their light environment and perceive the presence of neighbors as a warning of competition. The early perception of neighbor proximity depends on the detection of light quality changes. Within a vegetation, the ratio of red (R) to far-red (FR) light is lowered by the absorption of R light by photosynthetic pigments. This light quality change is perceived as an unambiguous signal of the proximity of neighbors through the phytochrome system, which triggers developmental responses that, when successful, result in the overgrowth of neighbors. Upon sensing a low R/FR ratio, shade avoiding plants react very rapidly and enhance elongation growth (e.g., hypocotyl and petioles) at the expenses of leaf development even before they are directly shaded. If the plants succeed in the attempt to overgrow their neighbors, the shade avoidance response is rapidly reverted through phytochrome photoconversion.^1–5

To maximize the capability of an organ to elongate, as in the shade avoidance response, plants must have evolved mechanisms tightly coupling distinct developmental processes: cell proliferation, cell differentiation, and direction of cell expansion. Consistently, previous studies have shown that the increase in extension growth of a seedling in low R/FR is the consequence of two events: a change in the orientation of cell expansion toward elongation in cells that do not divide, as the epidermal and cortical cells in the hypocotyl, and the inhibition of cambial cell proliferation that contributes to radial growth.⁶ It is known that these processes are dependent on the action of phytohormones, and it was earlier suggested that among them auxin is likely to act as a coordinator of growth across an organ, because it regulates many different aspects of plant development, including cell division, cell elongation, cell differentiation.⁷ Consistent with this suggestion, it was found that axr1, severely impaired in auxin response, does not elongate significantly in low R/FR. It was also shown that napthylphthalamic acid (NPA), an auxin transport inhibitor, significantly reduces hypocotyl elongation of wild-type seedlings in response to low R/FR.⁷ Furthermore, mutations in BIG, which is required in normal auxin efflux, result in attenuated shade avoidance responses.⁸

Despite considerable work in dissecting the elongation response induced by light quality changes, the effects of low R/FR on leaf development have remained largely unknown. However, among the plant organs, leaves are particularly interesting since they constitute most of the aboveground portion of the plant and are derived from determinate growth. Hence, we investigated the mechanism by which the low R/FR signal is transduced to attenuate leaf development.

Our study demonstrated that low R/FR rapidly and transiently arrests the growth of the leaf primordium affecting the frequency of cell division. Interestingly, we found that low R/FR does not affect cell proliferation in a cell-autonomous manner suggesting that this signal may affect an apical-basal gradient of a diffusible signal involved in the regulation of cell division, likely auxin. A simple analysis of wild type and seedlings lacking a functional TIR1 auxin receptor upon a brief exposure to low R/FR revealed that the growth arrest of leaf primordium is indeed an auxin response as it is absent from the tir1-1 mutant seedlings.^9–11 We then took a combinatorial approach of using genome wide expression profiling in combination with mutant analysis to identify the regulatory circuit involved in this response. Affymetrix Arabidopsis Genome GeneChip® array (ATH1) analyses on seedlings exposed to low R/FR for 1 h and 4 d identified 38 genes rapidly and transiently up-regulated by FR-rich light, previously described as auxin-inducible. Among them there is AtCKX6, a gene encoding a cytokinin oxidase involved in cytokinin breakdown.¹² To investigate whether AtCKX6 is functionally involved in the rapid arrest of leaf primordium growth provoked by low R/FR, we isolated and characterized a homozygous ckx6 T-DNA insertional line (Salk_070071, ckx6-1). The analysis of wild type and seedlings lacking a functional CKX6 enzyme upon a brief exposure to low R/FR revealed that the growth arrest of leaf primordium does indeed depend on auxin-induced cytokinin breakdown as it is absent from the ckx6-1 seedlings. Together, our data indicated that the induction of the auxin-regulated AtCKX6 gene by low R/FR promoting cytokinin breakdown diminishes cell proliferation in developing leaf primordia. This conclusion is further supported by the recent findings that the mature leaves of multiple cytokinin receptor mutants as well as those of plants overexpressing AtCKX1 contained significantly fewer cells compared with those of the wild type.^12–14

Interestingly, our data also showed that the induction of the synthetic DR5::GUS early-auxin responsive gene as well as that of the AtCKX6::GUS gene upon a brief exposure to low R/FR occurs in pre-provascular cells of young leaf primordia.^12,15 Hence, we speculated that induction of cytokinin degradation in the developing vasculature is by itself sufficient to arrest leaf primordium growth in low R/FR.

Further work is required to verify this hypothesis and decipher the mechanism through which the incipient vein cells might signal to all the other cells of the young primordium to arrest their division upon perception of neighbors. Cell-type specific cytokinin sensors will have to be developed to measure the changes in cytokinin content upon a brief exposure to low R/FR in all the cells of the young primordia. Furthermore, new methodologies will have to be set up to supply the ckx6-1 mutant with an intact version of the AtCKX6 gene in distinct cell types just before low R/FR treatment.

Further work is also required to understand how HFR1/SICS1, a master negative regulator attenuating virtually all plant responses to canopy shade, relates to the TIR1 pathway. Previous work has shown that leaf growth is significantly reduced in hfr1/sics1 mutants in low R/FR relative to wild type. Molecular data strongly suggested that HFR1/SICS1 may down-regulate key regulators of the shade avoidance response in low R/FR to prevent an exaggerated reaction to changes in R/FR. Among the genes up-regulated in hfr1/sics1, several genes encode hormone-related factors.¹⁶ Together, the data strongly suggest that the induction of the auxin signaling may persist longer in plant lacking HFR1/SICS1 and as a consequence the arrest in leaf cell proliferation may last longer resulting in smaller leaves. hfr1/sics1 tir1 and hfr1/sics1 ckx6 double mutants will have to be constructed and their leaf primordium phenotype upon a brief exposure to low R/FR will have to be examined.

Our work published in the August issue of Genes & Development demonstrated the existence of a previously unrecognized regulatory circuit underlying plant response to canopy shade and provided a basis for future work on cell-to-cell communication and pathway integration in the shade avoidance response.

Figure 1.

A schematic representation of the signaling pathway involved in the rapid arrest of leaf primordium growth upon low R/FR perception. Low R/FR light in a canopy is sensed through the phytochrome system, and the perception of a varying R/FR ratio is achieved through a change in the equilibrium of the Pr and Pfr forms of phytochromes. Phytochrome-mediated signals rapidly induce auxin changes perceived by TIR1. When auxin concentrations increase, auxin binds to the TIR1 receptor in the SCFTIR1 complex, leading to recruitment of the Aux/IAA repressors to TIR1. Once recruited to the SCFTIR1 complex, the repressors enter a pathway that leads to their destruction and the subsequent activation of ARF transcription factors. Among the ARF targets are the Aux/IAA genes themselves, which produce nascent Aux/IAA proteins that restore repression upon the pathway in a negative feedback loop. ARF activators bind to auxin-response elements in promoters of auxin-response genes. Among them is AtCKX6 a gene encoding a cytokinin oxidase involved in cytokinin breakdown. The activity of AtCKX6 resulting in a reduction of cytokinin levels diminishes cell proliferation in developing leaf primordia.

Addendum to: Canopy shade causes a rapid and transient arrest in leaf development through auxin-induced cytokinin oxidase activity. Carabelli M, Possenti M, Sessa G, Ciolfi A, Sassi M, Morelli G, Ruberti I. Genes Dev. 2007;21:1863–1868.