Published On: Tue, Mar 12th, 2019

Reprogramming of the cambium regulators during adventitious root development upon wounding of storage tap roots in radish (Raphanus sativus L.) [RESEARCH ARTICLE]

PXY-WOX4 may be reprogramed to aid the adventitious root formation

Founder cells of ARs seemed to be derived from the cambium. During this reprogramming the cut radish taproots no longer grew in the radial direction as shown in the images of cut radishes pictured after decapitation and after the hydroponic incubation with or without auxin treatment for 7 days (Fig. S4). To find the relationships between changes in cambium activities and their regulators, we analyzed the expression patterns of RsPXY and RsWOX4. In the intact radish taproot undergoing active secondary growth, these genes showed expression specific to the cambium near emerging xylem vessels (Fig. 3A). When ARs developed from cut roots, we observed the significant reduction of RsWOX4 and RsPXY expression in qRT-PCR, which is consistent with the lack of the cambial activities in the cut roots generating ARs (Fig. 3B). PXY and WOX4 are known to drive the cambium cell proliferation in an auxin-dependent manner (Suer et al., 2011). We observed that auxin promotes AR development from the radish taproot cambium. If PXY and WOX4 are a part of AR development, expression of these genes is likely upregulated upon AR induction in response to auxin treatment (1 µM of IAA). Supporting this idea, when AR formation was induced by incubating cut roots with 1 µM of IAA, expression of both RsPXY and RsWOX4 significantly increased (Student’s t-test; P0.01) (Fig. 3C). To find how the expression of RsPXY and RsWOX4 was reprogrammed during AR organogenesis, we performed RNA in situ hybridization again. RsPXY was expressed in a gap between the vascular cambium and the AR primordia, which is where the vascularization of the AR happened (Fig. 3A; Fig. S5). Similarly, RsWOX4 was present in areas where vascularization was taking place between the AR primordia and the vascular cambium (Fig. 3A; Fig. S5).

Fig. 3.

Fig. 3.

WOX4 and PXY reprogramming for adventitious root formation. (A) RNA in situ hybridization for antisense RsWOX4 (right) and RsPXY (left) in a developing AR (top) and in a cambium with secondary growth (SG) (bottom). (B,C) Measurement of gene expression changes of RsWOX4 and RsPXY during AR development. (B) The graph shows the relative expression levels of the genes before and after the AR formation. qPCR experiment was performed twice using pooled biological samples with three technical replicates. Error bar indicates ±s.e. (n=8). Fold enrichments statistically significant are labeled with asterisks (Student’s t-test; *P0.05). (C) The graph shows the relative expression levels of the genes in the AR under auxin treatment in comparison to the non-treated control. qPCR experiment was performed three times using pooled biological samples with three technical replicates. The three bars show the genes’ tendency for upregulation in the biological replicates. Error bar indicates ±s.e. (n=3). (D) RNA in situ hybridization for antisense RsPXY and RsHB8 in mature AR and AR primordia. (E) The average number of ARs appearing in the wounded area in Col-0 (n=23) and pxy mutant (n=24). Data are presented as mean±s.e. (Mann–Whitney U-test; *P0.01). Scale bars: 200 µm.

Expression of RsPXY in a gap between the vascular cambium and the AR primordia was further examined by analyzing its expression in the root generating mature AR. During vascularization, auxin maximum is established along vascular precursors via PIN1. ARF5 activated by auxin then promotes HB8 transcription and HB8 in turn activates PIN1 expression. This positive feedback regulation supporting canalization model enables the formation of vascular strands along the auxin flow (Baima et al., 1995, 2001; Scarpella et al., 2006). We thus included RsHB8 as a vascularization marker and analyzed its expression together with RsPXY. RsHB8 showed an irregular expression pattern with high expression in the areas of vascular initiation during the AR initiation, as expected. Interestingly, RsPXY showed the expression pattern very similar to RsHB8 (Fig. 3D). In the mature AR, RsPXY and RsHB8 were expressed along the AR procambium which connects with the vascular cambium of the cut taproot (Santuari et al., 2011). These expression changes indicate that PXYWOX4 might function for the AR development when the secondary growth no longer happens in the cut taproot.

Our investigation in radish suggested the recruitment of PXY and WOX4 to the AR development. To find whether these regulators actually affect AR formation from the cut taproots, we counted the ARs in Arabidopsis pxy mutant. Col-0 (n=23) and pxy (n=24) plants were grown for 18 days in MS media, their primary roots were decapitated, and then the shoots without roots were grown in B5 media for 4 days (Fig. S7). The number of ARs from cut root surfaces decreased significantly from an average of 2.91 to 0.62 in the pxy (Fig. 3E).

In Arabidopsis, WOX11/12 promotes AR founder cells and subsequently activates LBD16 and WOX5, which coordinate the initiation of a root primordium and a root apical meristem (Hu and Xu, 2016). Our expression analyses indicate a similar program might operate in AR development from radish taproots and the cambium serve as foci for the AR initiation (Fig. 1). During AR development, we found the vascular connection being established between the cambium of a cut taproot and the AR primordium.

Auxin flow plays a key role in the formation of vascular connections during organogenesis, and HB8 and PIN1 promotes this process as parts of positive feedback regulation (Baima et al., 2001; Donner et al., 2010; Scarpella et al., 2006). Consistently, in radish we detected the expression of PIN1 and RsHB8 in the region where the vascular connection was made between cut taproot cambium and the AR primordium. Exogenous application of auxin to the cut taproot promoted the AR formation specifically from the cambium (Fig. 2). These collectively indicate that the stem cells in the cambium have a capacity to initiate AR formation in an auxin dependent manner. Our further investigation suggests that RsPXY and RsWOX4 that are known to be in charge of cambial cell division during secondary growth (Etchells et al., 2013; Suer et al., 2011) and regulation of xylem differentiation (Fisher and Turner, 2007; Kondo et al., 2014) are reprogramed to re-establish their expression in the junction between AR primordia and the cut taproot. In an RNA in situ hybridization, we observed RsPXY expression largely disappeared when the secondary growth of a taproot stopped upon the removal of the root base and then re-emerged intermittently where vascularization happened as AR primordia developed. At the point of AR growth, it showed homogeneous expression in the AR procambium that was connected to the vascular cambium (Fig. 3). RsHB8 also showed expression pattern similar to RsPXY, confirming the procambium-cambium connection (Baima et al., 1995; Santuari et al., 2011). Recruitment of RsPXY and RsWOX4 to AR development is also supported by their response to auxin. Exogenous auxin application to the cut taproot no longer promoted the secondary growth, however strongly induced the expression of RsPXY and RsWOX4 (Fig. 4). This process seems functionally important since pxy mutant showed significant reduction in the emergence of ARs from the cut taproots in Arabidopsis. We interpret this behavior as a plants’ survival strategy that allows them to initiate organ regeneration upon wounding in a time- and cost-efficient way. Unraveling molecular mechanisms underlying how auxin redirects the vascular stem cell function from organ growth to organ regeneration will advance our understanding of the remarkably dynamic nature of plant development.

Fig. 4.

Fig. 4.

Model for adventitious root development in radish. When the bottom part of the taproot is removed, localized auxin maxima are created. In this point, high levels of auxin activate the AR development program: RsWOX11/12 initiates the formation of AR founder cells from the cambium, and activates RsLBD16 that initiates the root primordium and RsWOX5 that establishes the new root apical meristem. Auxin also reprograms the cambium specific RsPXY-RsWOX4 pathway to induce vascularization in the areas for AR formation, thereby connecting the vascular tissue of the AR to the tap root.

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