KLUH/CYP78A5 promotes organ growth without affecting the size of the early primordium

Abstract

Mobile signals play a key role in controlling the growth of organisms. In Arabidopsis, the cytochrome P450 CYP78A5/KLUH (KLU) non-cell autonomously stimulates cell proliferation in developing organs. In a recent study, we determined the range of KLU action, using a widely applicable system to predictably generate chimaeric plants. We showed that KLU acts not only within individual floral organs or flowers, but that its overall activity level is integrated across an inflorescence to determine organ size. Here, we address the question at which stage of petal development KLU acts to promote growth. We demonstrate that the size of the very young petal primordium in klu mutants is not altered, supporting the conclusion that KLU acts during later stages of organ outgrowth and a correspondingly longer range of the presumed KLU-dependent growth signal.

Non Cell-Autonomous Control of Arabidopsis Organ Growth by KLU

In plants, the flower and often the whole inflorescence function as integrated structures whose component organs need to grow to their correct relative sizes to ensure reproductive success. However, very little is known at present about how this coordination of growth is achieved. Plant organ size is influenced by several phytohormones,¹ yet their range of action in controlling growth has not been clearly established. A further growth signaling mechanism is defined by the KLUH (KLU)/CYP78A5 and the homologous CYP78A7 genes,^2,3 which appear to noncell autonomously promote plant organ growth via a novel mobile signal. To ask which developmental roles KLU signaling plays, in particular whether it may be involved in growth coordination in flowers, we aimed to determine the range of action of the KLU-dependent growth signal.

Long-range communication in plants has successfully been studied using mechanical grafting of scions onto genetically distinct stocks.⁴ However, mechanical grafting is limited in terms of spatial resolution and the accessible developmental stages. An alternative method to study intercellular signaling is the analysis of genetically distinct clones generated for example by Cre/loxP-mediated recombination.^5–7 However, the predictable generation of specific types of clones and chimaeras has been difficult, due to the nature of the Cre-source used. To circumvent these limitations and facilitate the analysis of intercellular communication in Arabidopsis, we have combined Cre/loxP-mediated recombination with temporally and spatially controlled expression of Cre in a widely applicable system that allows for the predictable generation of chimaeric plants.⁸

Using this system, we demonstrated that neither the final size of wild-type and klu mutant petals within the same flower, nor the final size of wild-type and mutant flowers within one inflorescence correlated with their genotypes. Instead, the petals within one flower or one inflorescence grew to sizes depending on the sum total of KLU activity in the respective inflorescence. By contrast, organs within an inflorescence that was chimaeric for another tested growth-control gene with largely autonomous behavior grew to sizes according to their genotype. Taken together these observations suggest that the overall activity level of KLU is integrated across an inflorescence to determine individual organ size and that growth is coordinated by a mobile KLU-dependent growth signal.

The Size of the Very Early Petal Primordium in klu Mutants is Unchanged

The comparatively long range of KLU activity beyond individual flowers that is suggested by the above results raises the question at which stage of petal development KLU acts to stimulate growth. The earlier this stage, the shorter would be the distance that the presumed growth signal has to cover to integrate growth in a flower or an inflorescence. From an analysis of dissected petals, it had been concluded that KLU acts towards the end of the proliferation phase in petals.² However, an effect on the size of the very early petal primordium at the time of its specification, called the petal anlage, could not be ruled out. The size of a floral organ anlage can be measured using the boundaries of marked sectors originating outside of the flower.⁹ The number of cells in the anlage is the inverse of the smallest fraction of the organ that can be occupied by a marked sector originating outside the flower.

To determine whether the petal anlage in klu mutants contains the same number of cells as in the wild type, we used the pCLV3-Alc-Cre and 35S:loxP-Stop-loxP:vYFPer transgenes to generate YFP-positive sectors originating in the stem cells of the shoot meristem (Fig. 1A and B)⁸ and searched for flowers and petals with a clonal boundary (Fig. 1C–E). Almost all of the identified sectors were located in the epidermis, limiting the following conclusions to this tissue layer. In a wild-type background, 84 split flowers contained 18 split petals, essentially all of which had the sector boundary running close to the midline of the organ (Fig. 1C and D), indicating two cells in the epidermis of the petal anlage in agreement with earlier studies.⁹ Similarly, in a klu-2 mutant background 86 split flowers contained 15 split petals, again with the sector boundary running close to the petal midline (Fig. 1E). Thus, at the time of its specification, the petal anlage in klu-2 mutants is not altered in size. Consistent with the rescue of petal size when KLU activity is provided only late in flower development in an otherwise klu mutant background,^2,8 this supports a role for KLU during later stages of organ outgrowth and a consequently longer range of the presumed KLU-dependent signal.

Figure 1.

Dissecting the size of the petal anlage by clonal analysis. (A) The 35S:loxP-Stop-loxP:vYFPer transgene for marking cells.² vYFPer is an endoplasmic reticulum-localized, cell-autonomous version of VENUS-YFP.¹⁰ (B) A mericlinal chimaera (genotype pCLV3-Alc-Cre; 35S:locP-Stop-loxPvYFPer) with a spontaneously arising YFP-positive stem-cell clone that has populated the epidermis of approximately one third of the shoot. Asterisk marks a rosette leaf with half of the epidermis expressing YFP. (C–E) Overlays of brightfield and YFP fluorescence micrographs (upper) and YFP fluorescence micrographs only (lower). (C) A mature flower from a pCLV3-Alc-Cre; 35S:loxP-Stop-loxP:vYFPer doubly transgenic plant in a KLU wild-type background with a sector boundary running through the middle of the flower. (D) A petal of the same genotype as the plant in (C) with a sector boundary running along the midline of the organ. (E) A petal from a pCLV3-Alc-Cre; 35S:loxP-Stop-loxP:vYFPer doubly transgenic plant in a klu-2 background with a sector boundary running along the midline of the organ. Scale bars are 1 mm in (B) and 500 µm in (C–E).

Identifying the presumed growth signal and further factors involved in its synthesis and perceptions are important future objectives for further defining the role of KLU in promoting and coordinating Arabidopsis organ growth.

Addendum to: Eriksson S, Stransfeld L, Adamski NM, Breuninger H, Lenhard M. KLUH/CYP78A5-Dependent growth signaling coordinates floral organ growth in Arabidopsis. Curr Biol. 2010;20:527–532. doi: 10.1016/j.cub.2010.01.039.