BRI1 activity in the root meristem involves post-transcriptional regulation of PIN auxin efflux carriers

Abstract

Spatiotemporal coordination between multiple hormonal pathways is a key determinant of plant growth. This coordination can be mediated by distribution of the auxin network via the action of PIN auxin efflux carriers. We showed that brassinosteroids (BRs) promote cell proliferation and cell expansion of meristematic cells. Hence, roots with high epidermal expression of the BR receptor BRI1 have enlarged meristem whereas bri1 mutant has a reduced meristem size. Because the extent of mitotic activity and differentiation is tightly linked to auxin gradient we further asked how the BR pathway integrates with current proposed models for PIN regulation. We showed that the small meristem of BR deficient plants does not involve transcriptional modulation of PIN 1, 3 and 7 genes. Here, we found that PIN2 and PIN4 are under transcriptional regulation. However, their accumulation in the epidermis/cortex and columella respectively was also determined by BRs in a post-transcriptional manner. Thus, BRs impinge on auxin distribution through distinct regulatory modes and the self-organizing auxin system represents at least one mechanism that contributes to BR-mediated growth.

The BR receptor BRI1 signals from the epidermis to promote mitotic cell cycle activity and cell expansion, thus ensuring normal cell progression along the meristematic zones.¹ It is established that the extent of mitotic activity and differentiation is set by the distribution of the auxin network via the action of PIN auxin efflux carriers.^2,3 One immediate question is therefore how local BR activity integrates with the proposed role of PIN activity in regulating the size of the meristem.³ Enhanced differentiation is attributed to the cytokinin-mediated accumulation of the AUX/IAA transcriptional repressor gene SHY2 in the vascular tissue transition zone, where it inhibits the transcription of PIN1, 3 and 7.^4,5 We recently demonstrated that short BR treatment and low BR activity have no apparent effect on the transcript level of these PIN encoding genes. In agreement, we confirmed that PIN1 and PIN7 protein remained fairly unchanged when BR activity is impaired.¹ It should be noted however that our work does not exclude the effect of BRs on the aforementioned PIN transcript at specific developmental stages. For example, BRs were proposed to be rate limiting for BRX-mediated regulation of PIN3, at precise time after germination.⁶ Taken together, the small meristem size of BR deficient plants at one week after germination is not explained by lower transcription of PIN and increase of SHY2 in the vasculature (data not shown).

Notably, BRs controlled the accumulation of PIN2. PIN2 directs the intercellular auxin flow from the root tip shootward via the lateral root cap and epidermis, and recycles it back to the vasculature via the cortex. Using immunofluorescence analysis of endogenous PIN2 and live imaging of PIN2-GFP, we found that bri1 and plants treated with the BR biosynthesis inhibitor BRZ, had reduced PIN2 levels as compared with wild-type (Fig. 1B and C). Moreover, restricted BRI1 activity in the epidermis restored PIN2 expression while BRI1 activity in the inner cells had smaller effect (Fig. 1C). We also found that the transcript level of endogenous PIN2 and of the transgene PIN2-GFP slightly changed in response to short BR treatment and showed an opposing behavior as compared with the protein accumulation (Fig. 1A). Thus, PIN2 is regulated by BRs in a post-transcriptional manner. Interestingly, BRs had similar effect on PIN4 accumulation in the columella (Fig. 1A and B). That is, BRs induced a negative and a positive effect on PIN4 transcript and protein levels respectively. Such BR-mediated non-transcriptional control may also apply to other PIN proteins (i.e., PIN3).

Figure 1. BR signaling regulates the accumulation of PIN2 and PIN4 at multiple levels Note that BRs are required for PIN2 and PIN4 accumulation (B) but negatively affect the level of their corresponding transcript (A). (A) qRT-PCR analysis demonstrating the negative effect of BRs on PIN2, PIN2-GFP and PIN4 transcript level respectively (methods are as in ref. 1) (B) Confocal microscope image showing reduced PIN2-GFP and PIN4-GFP levels in bri1 and in the presence of BRZ. The images are false-colored to indicate GFP (green) and PI (red). (C) Immunofluorescence analysis with anti-PIN2 shows low accumulation of PIN2 in bri1 and BRZ treated plants. PIN2 accumulation is controlled by epidermal BRI1 activity (bri1;pGL2-BRI1-GFP) while BRI1 expression in the inner cells (bri1;pSCR-BRI1-GFP and bri1;pSHR-BRI1-GFP) have small effect. (D) A model proposing that epidermal BRI1 activity can control the size of the meristem, at least in part, by modulating auxin distribution. Arrow width represents the extent of auxin flow.

It seems therefore plausible that BRI1 activity in the epidermis promotes the mitotic cycle and cell size, at least in part, by modulating auxin distribution. In accordance, BRs have been shown to promote rootward and shootward auxin transport^7,8. Interestingly, BR effect on the quiescent center does not appear to involve alterations in auxin targets (i.e., PLT) or auxin efflux transport.⁹ Indeed, to what extent PIN-dependent auxin distribution mediates BR effect on growth is currently unknown. Difficulties in providing a direct answer to this question stems from the inherent self organizing property of the auxin network. In addition, as we further point here, the fate of PIN accumulation in bri1 goes beyond a simple modification of their transcription.

Interestingly, non-transcriptional control of PIN accumulation can be mediated by distinct hormonal and environmental signals and involves endocytosis and protein degradation mechanisms.^10-18 For example, decreased level of gibberellin and increased level of cytokinin has been recently shown to promote the vacuolar degradation of PIN2 and PIN1 respectively. Cytokinin also modulates the post-transcriptional accumulation of other PIN proteins via a yet unknown mechanism.¹⁹ How exactly these distinct modes of hormone-mediated regulation of PINs integrate to fine-tune auxin transport during growth and development is currently an open question.