Simultaneous but spatially different regulation of non-photosynthetic callus formation and photosynthetic root development after shoot removal

ABSTRACT

Plants coordinate plastid differentiation and cellular differentiation during tissue development. Recently, we reported a mechanism of chloroplast development and photosynthetic improvement in Arabidopsis roots after shoot removal. Shoot removal induces the expression of WOUND INDUCED DEDIFFERENTIATION (WIND) transcription factors at the wound site, which activates type-B ARABIDOPSIS RESPONSE REGULATOR (ARR)-mediated cytokinin signaling in roots. The type-B ARR signaling upregulates chloroplast-related transcription factors along with photosynthesis-associated genes, thereby inducing chloroplast development in detached roots. However, at the wound site, WINDs promote the development of non-green callus cells in a type-B ARR-dependent manner. Thus, in shoot-removed roots, WIND-mediated cytokinin signaling has spatially distinct roles: it represses chloroplast development at the wound site while activating the development in adjacent regions. Because WINDs are strong inducers of cell dedifferentiation, spatial differences in their expression levels between the wound site and other areas may determine the fate of chloroplast development.

In the natural environment, the aboveground tissues of plants are sometimes damaged by animal trampling, wind lodging, attacks from herbivores and pathogens, and severe abiotic stresses such as heat and drought. When plants are physically damaged, they often produce a disorganized cell mass called callus to cover the wounded area for protection against pathogenic infection and water loss.¹ In some cases, new tissues are regenerated from calli formed at the wound site, which reflects the high pluripotency of the callus cells. Some plants can regenerate shoots from roots: Taraxacum and Cichorium species can easily regenerate shoot buds from calli developed at the cut surface of secondarily thickened roots.²

To be able to regenerate photosynthetic tissues after shoot removal, underground tissues need to supply energy and a carbon source to newly developing cells, at least until the regenerated tissues start photoautotrophic growth. Some plants may use a carbon source such as starch and lipids stored in roots or underground stems, and others may acquire carbon from nearby plants to which they are connected by stolons.

We recently found that shoot removal in Arabidopsis induced chloroplast development and photosynthetic improvement in roots, which may be a survival strategy for plants after shoot loss.^3,4 In this paper, we propose a model for how roots detached from the shoot induce callus formation at the wound site while undergoing chloroplast development in other regions.

Signaling pathway regulating callus formation at the wound site

A gene family of wound-induced AP2/ERF transcription factors, WOUND INDUCED DEDIFFERENTIATION (WIND), plays a central role in inducing cell dedifferentiation and callus formation at the wound site.¹ Among the 4 WIND homologs functioning redundantly in Arabidopsis, the roles of WIND1 have been well characterized.^5-7 WIND1 overexpression induces ectopic callus formation without exogenous phytohormone treatment, whereas the expression of a synthetic WIND1 chimeric repressor gene (WIND1-SRDX) under the WIND1 promoter strongly suppresses callus formation in a dominant-negative manner. Hence, WIND1 is a master regulator of callus formation. Downstream of WIND1, type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), which are positive regulators of the two-component cytokinin signaling pathway,⁸ are essential factors for callus formation and subsequent shoot regeneration.⁵ The activity of type-B ARRs, which is greatly enhanced by wounding in wild-type plants, is largely suppressed in WIND1-SRDX plants. Moreover, double knockout mutation of the major type-B ARR isoforms, ARR1 and ARR12 (arr1 arr12), strongly inhibits WIND1-induced callus formation.⁵ These findings suggest that wounding-induced WIND1 activates type-B ARR-mediated cytokinin signaling, thereby promoting callus formation (Fig. 1).

Figure 1.

A simplified model for the simultaneous but spatially separated regulation of callus formation and chloroplast development in shoot-removed roots. At the wound site, WIND1 expression, induced in response to wounding, upregulates ESR1 expression and cytokinin signaling mediated by ARR1 and ARR12. In addition, cytokinin and auxin signaling pathways positively affect local ESR1 expression to induce callus formation. Below the wound site, the ARR signaling pathway, activated by WIND1, induces GNL expression and directly upregulates some photosynthesis-associated genes. Shoot removal attenuates the inhibitory effect of shoot-derived auxin on GNL expression. In addition, GLK2 expression in roots, which is inducible in response to both enhanced cytokinin and inhibited auxin signaling, is slightly increased by shoot removal via ARR1 and ARR12. GNL promotes chloroplast development and increases photosynthetic efficiency in shoot-removed roots in concert with other chloroplast-related transcription factors including GLK2. Dashed line represents a minor involvement of a factor in the relevant pathway.

At the same time, WIND1 upregulates another AP2/ERF transcription factor, ENHANCER OF SHOOT REGENERATION1 (ESR1), by directly binding to its promoter region.⁹ The role of WIND1 in ESR1 expression is supported by exogenously expressed WIND1-SRDX strongly suppressing ESR1 level at the wound site. Because forced overexpression of ESR1 can rescue impaired callus formation in WIND1-SRDX plants,⁹ ESR1 likely functions in cellular reprogramming processes downstream of WIND1. In fact, loss-of-function mutations of ESR1 inhibit callus formation at the wound site, whereas gain-of-function modifications promote cellular reprogramming.⁹ The cytokinin signaling pathway mediated by type-B ARRs is also involved in wounding-induced ESR1, as represented by decreased ESR1 expression in arr1 arr12.⁹ Furthermore, ESR1 expression in response to wounding is strongly enhanced by exogenous supplementation with both auxin and cytokinin. Thus, ESR1 is subject to complex regulation by a direct WIND1 action and by auxin/cytokinin-mediated signaling (Fig. 1).

Chloroplast development in roots after shoot removal

Plant plastids plastically differentiate into various functional forms according to the host cell type.¹⁰ In roots of intact Arabidopsis seedlings, chloroplast development is suppressed via the shoot-derived auxin-dependent signaling pathway.³ However, in roots detached from the shoot, the auxin-mediated suppression of photosynthetic gene expression is attenuated, which leads to chloroplast development and photosynthetic improvement. We recently reported that in addition to negative auxin regulation, the WIND1-mediated cytokinin signaling pathway is deeply involved in this root greening response (Fig. 1).⁴ WIND1-SRDX expression driven by the WIND1 promoter decreased chlorophyll accumulation in roots after shoot removal. Moreover, in wild-type roots, shoot removal strongly activated type-B ARR signaling, which was inhibited in WIND1-SRDX plants. Multiple type-B ARR isoforms redundantly function in the root greening response downstream of WIND1, and in particular, ARR1 and ARR12 have a crucial role in this process.⁴ The arr1 arr12 double mutation strongly attenuates the upregulation of genes involved in chloroplast development and photosynthesis in detached roots, which results in perturbed root greening.⁴ Type-B ARRs may act on root greening via transcription factors regulating chloroplast development, in addition to directly upregulating some photosynthesis-related genes by binding to their promoter regions.¹¹

Class B GATA factors (B-GATA), a subfamily of transcription factors recognizing a G-A-T-A core cis-element, mediate various developmental processes, including chloroplast development.^12,13 Two B-GATAs — GATA, NITRATE-INDUCIBLE, CARBON METABOLISM INVOLVED (GNC) and GNC-LIKE/CYTOKININ-RESPONSIVE GATA TRANSCRIPTION FACTOR 1 (GNL/CGA1) — have been well characterized. Double knockout mutations (gnc gnl) impair chloroplast development in leaves, whereas overexpression of either gene induces ectopic development of chloroplasts with high photosynthetic activity in hypocotyls and roots.^4,14,15 Thus, B-GATAs are potent regulators of chloroplast development, although their direct targets related to chloroplast functions are unclear.¹⁴ The expression of B-GATAs, particularly GNL, is inducible by cytokinin via ARR1 and ARR12.¹⁵ Consistently, GNL expression in roots is strongly increased by shoot removal in the wild type but not the arr1 arr12 mutant.⁴ These data suggest that GNL upregulated via the ARR1 and ARR12 pathway promotes chloroplast development in detached roots. Because auxin treatment to detached roots strongly suppresses GNL expression,⁴ GNL may be a point of convergence between positive cytokinin and negative auxin signaling pathways (Fig. 1). In addition, the Golden2-like (GLK) transcription factors, GLK1 and GLK2, are involved in regulating chloroplast development in Arabidopsis roots,^3,16 mainly via the transcriptional activation of genes associated with chlorophyll biosynthesis and light harvesting.¹⁷ Overexpression of GLK1 or GLK2 strongly upregulates their target genes in roots and induces ectopic chloroplast development.¹⁶ Cytokinin treatment increases the expression of GLK2 in roots along with that of its target genes and induces root greening.³ Shoot removal also slightly increases GLK2 expression in roots via type-B ARR signaling,⁴ so in addition to GNL, this gene may contribute in part to the root greening response.

Suppression and promotion of chloroplast development by the same trigger

At wound sites, Arabidopsis generates transparent callus cells, with chloroplast development suppressed (Fig. 2A).¹ Indeed, we confirmed the formation of transparent calli at the cut surface of Arabidopsis roots when the shoot was removed at the root–hypocotyl junction (Fig. 2A).⁴ At the same time, chloroplast development occurred in the region adjacent to the cut site, so the shoot removal triggered the simultaneous chloroplast suppression at the wound site and promotion of chloroplast development in other areas in roots. The common signaling pathway via WIND1 and type-B ARRs was used for both responses (Fig. 1).

Figure 2.

Spatial patterns of callus formation, expression of WIND1 and ESR1, and activity of the type-B ARR-responsive promoter after wounding. (A) (Left panel) Arabidopsis wild-type leaf was cut by using sharp scissors and grown for 6 d.¹ (Middle panel) Arabidopsis root explant was cultured on the Murashige-Skoog (MS) medium for 16 d.⁴ In both cases, calli with transparent white cells (arrowheads) developed only at the wound site. (Right panel) XVE-WIND1 transgenic Arabidopsis was grown on the 17β-estradiol–containing MS medium for 8 d to induce ectopic WIND1 expression in the whole body.⁶ Undifferentiated callus cells extensively developed with WIND1 overexpression, with cotyledon greening completely impaired. (B) WIND1 expression in hypocotyl and root explants visualized by using the PRO_WIND1:WIND1-GFP reporter gene.⁵ Excised hypocotyls and roots were cultured on MS medium for 4 and 16 d, respectively. GFP, single GFP fluorescence (green); GFP + Chl, dual color fluorescence of GFP (green) and chlorophyll (red). (C) Cytokinin-responsive promoter activity mediated by type-B ARRs visualized by using the TCS:GFP reporter gene in shoot-removed roots.⁴ For the detached root sample, roots excised from 14-d-old seedlings were grown on MS medium for 7 d. The intact root control from 21-d-old seedlings was observed along with the detached roots. Single green GFP fluorescence is shown in the right panel. (D) ESR1 expression in root explants visualized by using the PRO_ESR1:GUS reporter gene.⁹ Excised roots were cultured on MS medium for 2 d. Blue staining represents ESR1 expression. (C and D) Vertical bar represents the wound site. Scale bars, 0.5 mm. All the images are different versions of published data as cited.

How does the plant simultaneously regulate these opposite processes, dedifferentiation and differentiation, using the same signaling components? Of note, WIND1 promoter activity is stronger at the wound site (Fig. 2B),^5,6 with type-B ARR-mediated activation of the cytokinin-responsive promoter increased at and around the wound site (Fig. 2C).^4,5 WIND1 may induce cellular dedifferentiation locally at its expression sites while more widely activating type-B ARR signaling. This assumption is supported by WIND1 inducing callus formation throughout the plant body when overexpressed in the whole plant.⁵ Particularly, in photosynthetic tissues, WIND1-induced cell dedifferentiation accompanies degreening of tissues (Fig. 2A),^5,6 presumably due to promoted plastid dedifferentiation into proplastids. The data imply that the action of WIND1 on chloroplast development is intrinsically negative. In fact, a transcriptome analysis revealed that overexpression of WIND1 strongly downregulated GLK1, GNC, and GNL along with photosynthesis-associated genes (Fig. 3).⁵ Thus, at the wound site, WIND1 may suppress key transcription factors involved in chloroplast development to coordinate plastid dedifferentiation along with cellular dedifferentiation. Meanwhile, WIND1 overexpression was also reported to increase endogenous levels of active cytokinin and its precursors.⁵ Thus, one attractive hypothesis is that, in shoot-removed roots, WIND1 induced at the wound site increases mobile cytokinin signals, thereby activates type-B ARR signaling remotely. Although there is no direct evidence showing increased cytokinin levels in shoot-removed roots, type-B ARRs activated by transportable cytokinin signals may induce chloroplast development at a distance from the wound site in the absence of negative WIND1 action.

Figure 3.

Relative mRNA levels of photosynthesis-associated nuclear genes in seedlings of the WIND1-overexpression line (35S:WIND1). Data are from microarray analysis by Iwase et al. (2011).⁵ The mRNA levels in 35S:WIND1 plants are presented as fold difference from wild-type seedlings (dotted line). Genes encoding transcription factors regulating chloroplast development and those associated with photosystem I and II functions are globally downregulated in 35S:WIND1.

Downstream of WIND1, ESR1 would contribute to the local dedifferentiation response, because ESR1 expression induced by WIND1 is also restricted to the wound site (Fig. 2D).⁹ Of note, the ESR1 expression in response to wounding can be further enhanced by exogenous auxin in addition to cytokinin.⁹ Therefore, auxin signaling, which has a negative effect on chloroplast development in roots,³ may also be involved in suppressing chloroplast development at the wound site. Because WIND1 likely acts on dedifferentiation processes independent of auxin signaling,⁵ two different pathways via WIND1 and auxin signaling might suppress chloroplast development at the wound site, but this point needs further investigation.

In summary, together with ESR1 and type-B ARRs, WIND1 promotes cellular dedifferentiation and callus formation at the wound site while simultaneously suppressing the expression of chloroplast-related genes and subsequent chloroplast development, at least during the early stage of callus development (Fig. 1). Although the type-B ARR pathway has potential to induce chloroplast development, this effect may be canceled or reversed via WIND1 at the wound site. In areas distant from the wound site, the type-B ARR pathway upregulates chloroplast-related genes and induces chloroplast development and photosynthetic improvement, presumably away from the negative effect of WIND1 and auxin on chloroplast biogenesis. How WIND1 locally suppresses chloroplast-related genes and widely activates the type-B ARR signaling pathway remains undetermined. One possibility for the remote action of WIND1 on type-B ARR signaling is that WIND1 induces mobile signals such as cytokinin molecules thereby activates type-B ARRs at a distance. This hypothesis awaits further evidence. Furthermore, how GATA transcription factors, particularly GNL, upregulate photosynthesis-associated genes and improve photosynthesis efficiency is unknown. Future studies are needed to elucidate the molecular mechanisms and physiologic roles of the greening and degreening of plant roots in response to loss of photosynthetic tissues.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by JSPS KAKENHI Grant Numbers 26711016 and 15KK0265.