Pivotal role of LBD16 in root and root-like organ initiation

Abstract

In the post-embryonic stage of Arabidopsis thaliana, roots can be initiated from the vascular region of the existing roots or non-root organs; they are designated as lateral roots (LRs) and adventitious roots (ARs), respectively. Some root-like organs can also be initiated from the vasculature. In tissue culture, auxin-induced callus, which is a group of pluripotent root-primordium-like cells, is formed via the rooting pathway. The formation of feeding structures from the vasculature induced by root-knot nematodes also borrows the rooting pathway. In this review, we summarize and discuss recent progress on the role of LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16; also known as ASYMMETRIC LEAVES2-LIKE18, ASL18), a member of the LBD/ASL gene family encoding plant-specific transcription factors, in roots and root-like organ initiation. Different root and root-like organ initiation processes have distinct priming mechanisms to specify founder cells. All these priming mechanisms converge to activate LBD16 expression in the primed founder cells. The activation of LBD16 expression leads to organ initiation via promotion of cell division and establishment of root-primordium identity. Therefore, LBD16 might play a common and pivotal role in root and root-like organ initiation.

Introduction

In the post-embryonic stage of Arabidopsis thaliana, many types of roots can originate from the vasculature. Adventitious roots (ARs) can form from non-root organs, such as detached leaf or stem explants and the hypocotyl [1–8]. In a developing root, acropetal lateral roots (simply referred to LR in this review) form from the xylem-pole pericycle cells under the guidance of an oscillating auxin flux derived from the root cap [9–14]. An existing root can also produce adventitious lateral roots, mainly via the AR formation pathway [15–23].

Some root-like organs can also be initiated from the vasculature in A. thaliana. In tissue culture, callus can be induced on auxin-rich callus-inducing medium (CIM) [24–32]. Callus forms via the rooting pathway and the cellular nature of the newly formed callus is a group of root-primordium-like cells [33–38]. Callus cells are pluripotent, because they are competent for shoot regeneration on cytokinin-rich shoot-inducing medium (SIM) or root regeneration on root-inducing medium (RIM). In addition, root-knot nematodes can induce the formation of feeding structures from the vasculature of A. thaliana in either roots or detached leaf explants [39, 40]. The formation of feeding structures has been proposed to borrow the rooting pathway [40–48]. Therefore, it is possible that feeding structures induced by root-knot nematodes are also root-like organs.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD; also known as ASYMMETRIC LEAVES2-LIKE, ASL) transcription factor genes belong to a plant-specific family that is present in diverse taxa ranging from algae to seed plants. There are at least six classes of LBD genes in A. thaliana [49–54]. The class-IB, also known as the root-associated LBD/ASL clade, has ten members in A. thaliana [53, 54] (Fig. 1), and they are involved in many aspects of plant development. Here, we review recent progress in research on the role of A. thaliana LBD16 (Fig. 1), which belongs to class-IB LBD genes, in root and root-like organ initiation.

Fig. 1.

Phylogeny of class-IB LBD genes in land plants. Phylogenetic analysis of protein sequences of class-IB LBDs from a bryophyte Physcomitrella patens (Pp, in blue), a lycophytes Selaginella kraussiana (Sk, in green), a dicot Arabidopsis thaliana (At, in red), and monocot Oryza sativa (Os, in black) and Zea mays (Zm, in black). Sequence of SkLBDA was obtained by analyzing RNA-seq and DNA-seq data published previously [91] (GenBank, MH107252). Other sequences were obtained from the published data [53]. Phylogenetic tree of full length of the LBD proteins was constructed using the maximum-likelihood method (the Poisson model) with MEGA7.0 and default parameters [92]

LBD16 in LR initiation

When A. thaliana is grown vertically on the medium, the primary root usually produces LRs by an acropetal sequence. The formation of LR in A. thaliana involves successive steps of cell fate transitions [13, 14, 55–57] (Fig. 2a). Auxin is transported into and accumulates in the xylem-pole pericycle cells in the priming step for specification of a group of LR founder cells. After priming, the nuclei of the LR founder cells migrate to start the initiation step, and pairs of LR founder cells undergo asymmetric cell division to form the LR primordium. The LR primordium undergoes continuous cell division, and then, the LR primordium forms the LR apical meristem with functional domains in the patterning step [55].

Fig. 2.

Model of LBD16 in root and root-like organ formation. Model summarizing the common role of A. thaliana LBD16 in LR formation from primary root (a), AR formation from detached leaf explants (b), callus formation from detached leaf explant (c), and feeding structure formation from detached leaf explant (d). Cells and genes that are involved in the priming step are in blue, and those in the initiation step are in red

The expression of LBD16 is specifically induced in the primed LR founder cells before the nuclei migrate and asymmetric division occurs [58] (Fig. 2a). During the initiation step, LBD16 expression continues in the LR primordium, but its expression gradually decreases during the patterning step to form the LR apical meristem ([9, 20, 58] and our unpublished data) (Fig. 2a). The lbd16 mutant and LBD16-SRDX transgenic lines (in which the LBD16 pathway was blocked) formed fewer LRs from the primary root than did wild type [9, 20, 58–60]. Specifically, blocking of the LBD16 pathway resulted in defective polar nuclei migration in LR founder cells and defective asymmetrical cell division, but it did not affect the specification of LR founder cells with auxin accumulation in the priming step [58]. Therefore, LBD16 seems to be specifically involved in the initiation of the LR primordium.

In LR formation, LBD16 is activated by AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 (ARF7/19), two functionally redundant transcription factors [9, 58, 59, 61] (Fig. 2a). At a low auxin level, ARF7/19 form a protein complex with the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) protein IAA14 (also known as SOLITARY-ROOT, SLR) [62]. The interaction with IAA14 represses the transcriptional activation activity of ARF7/19. In the priming step of lateral rooting, auxin is transported into and highly accumulates in LR founder cells. This results in the degradation of the IAA14 protein and, therefore, the release of ARF7/19. The release of ARF7/19 derepresses their transcriptional activator role. In the primed founder cells, ARF7/19 can directly bind to the AUXIN RESPONSE ELEMENTs (AuxREs) on the promoter of LBD16 to activate transcription [9, 58, 59, 63]. The arf7 arf19 double mutant showed defective LR initiation and a loss of LBD16 expression when the plants were vertically cultured on growth medium. Overexpression of LBD16 in the arf7 arf19 double mutant background partially rescued the LR formation defect, suggesting that LBD16 acts downstream of ARF7/19 [9].

Overall, during LR organogenesis, ARF7/19 activate LBD16 expression in the primed LR founder cells, and then, LBD16 promotes cell division and fate transition during the initiation of LR primordium.

LBD16 in AR initiation

In A. thaliana, ARs can form from many tissues after wounding or in response to environmental signals [1–8]. By studying de novo root regeneration from detached A. thaliana leaf explants [64], the role of LBD16 in AR initiation was analyzed [8, 20, 36, 65] (Fig. 2b). After detachment of leaf explants, endogenous auxin is produced and polar-transported into regeneration-competent cells (i.e., procambium cells and some vascular parenchyma cells) near the wounded site. Accumulation of auxin in a regeneration-competent cell activates the expression of two partially redundant transcription factor genes, WUSCHEL-RELATED HOMEOBOX11 (WOX11) and WOX12 (WOX11/12), resulting in the priming step, i.e., cell fate transition from a regeneration-competent cell to an AR founder cell [36]. In the initiation step, the AR founder cell divides to form the AR primordium. To date, there is no solid evidence that nucleus migration and asymmetrical cell division of pairs of founder cells occur during the initiation of the AR primordium. Further analysis of the first round of cell division in AR formation from A. thaliana leaf explants will clarify how the AR primordium is initiated in the future.

LBD16 is specifically induced in the primed AR founder cell during the initiation step ([65] and our unpublished data) (Fig. 2b). LBD16 expression continues in the AR primordium and gradually decreases when the AR primordium enters the patterning step to differentiate into the AR apical meristem [20, 36, 65] (Fig. 2b). The lbd16 mutant showed defects in AR organogenesis from leaf explants [20], suggesting that LBD16 is required for AR initiation.

During AR formation from leaf explants, LBD16 is not activated by ARF7/19 [20, 36]. The arf7 arf19 double mutant was able to form ARs from detached leaf explants, suggesting that AR initiation from leaf explants is not strictly dependent on ARF7/19, in contrast to LR initiation [20]. Therefore, other upstream activators may induce LBD16 expression during AR formation. WOX11/12 were proposed to directly activate LBD16 [20]. In the WOX11–SRDX transgenic lines, LBD16 expression was not upregulated in the primed AR founder cells or the AR primordium. There are two WOX-binding elements on the LBD16 promoter, and WOX11 can directly bind to these elements. Mutations in the WOX-binding elements resulted in the loss of the LBD16 expression during AR initiation, suggesting that WOX11/12 are key upregulators of LBD16 in AR formation. Activation of LBD16 by WOX11/12 requires the presence of auxin, suggesting that the upregulation of LBD16 in the primed AR founder cells may require ARF proteins other than ARF7/19. Those ARFs may cooperate with WOX11/12 to upregulate LBD16 during AR primordium initiation from leaf explants [8] (Fig. 2b).

Although ARF7/19 do not regulate LBD16 expression during AR formation from leaf explants, the two ARFs have been shown to control AR formation from hypocotyls [66], indicating that different priming mechanisms might be involved in AR formations from different organs. It will be interesting to determine whether the auxin signaling pathways involved in AR formation differ between leaf explants and hypocotyls in the future.

WOX11/12 did not regulate LBD16 during LR formation when A. thaliana was vertically grown on the medium [20]. WOX11/12 are not expressed in LR founder cells; therefore, they cannot be responsible for LBD16 upregulation during LR initiation. Interestingly, ectopic WOX11-SRDX expression under the control of the 35S promoter in LR founder cells did not block LBD16 activation by ARF7/19, suggesting that there is some unknown mechanism to abolish the function of the WOX11 protein in the LR founder cells [20].

The A. thaliana primary root can produce not only LRs but also adventitious lateral roots [20–23]. The priming of founder cells during adventitious lateral rooting is more likely to follow the AR pathway than the LR pathway, i.e., involving WOX11/12 but not ARF7/19 [20]. It is possible that LBD16 could also be involved in the initiation step of adventitious lateral rooting, as it is in AR formation. Further research on the role of LBD16 in adventitious lateral root formation is required to test this hypothesis.

Overall, during AR organogenesis from leaf explants, WOX11/12 together with ARFs activate LBD16 expression in the primed AR founder cells. LBD16 then promotes the initiation of AR primordium.

LBD16 in callus initiation and pluripotency acquisition

De novo shoot regeneration (also known as de novo shoot organogenesis) in tissue culture usually experiences two phases: first, callus forms from detached explants on auxin-rich CIM; then, callus is moved to cytokinin-rich SIM for adventitious shoot induction [24–32]. The results of recent studies have suggested that the newly formed callus on CIM is a group of root-primordium-like cells and the process of callus initiation borrows the rooting pathway [33–38].

Using callus formation from A. thaliana leaf explants as an example, the priming and initiation steps of callus formation are similar to those that occur during AR formation [34, 36, 38, 67–70] (Fig. 2c). In the priming step, exogenous auxin in the medium activates WOX11/12 expression to trigger fate transition from a regeneration-competent cell to a callus founder cell [36, 38, 69]. Then, the callus founder cell undergoes division to become the newly formed callus cells, which have root-primordium-like features at the molecular level [36–38, 69]. When callus is cultured on CIM for a long time, partial differentiation into root apical meristem-like tissue can occur (i.e., the formation of partially differentiated callus) [69].

LBD16 expression is specifically induced in the primed callus founder cell in the initiation step on CIM ([34, 38] and our unpublished data) (Fig. 2c). LBD16 expression continues in the root-primordium-like newly formed callus [38]. The lbd16 mutant exhibited slowed cell division during callus initiation, indicating that LBD16 is required for proper cell division [34, 38]. When the callus is continuously cultured on the CIM for a long time, LBD16 expression level decreases in root apical meristem-like partially differentiated callus ([34, 69] and our unpublished data) (Fig. 2c).

When newly formed callus is moved to SIM, LBD16 expression sharply decreases and then disappears, suggesting that cytokinin might have a role in repressing LBD16 expression on SIM [38]. Interestingly, the callus of the lbd16 mutant showed defective shoot formation on SIM, suggesting that LBD16 is also required for the acquisition of pluripotency in the newly formed callus during its initiation [38]. The cellular basis of pluripotency in the newly formed callus was proposed to be the root-primordium identity [37, 38]. Therefore, LBD16 promotes establishment of the root-primordium cell fate identity in newly formed callus.

During callus formation from A. thaliana detached leaf explants on CIM, LBD16 expression is upregulated by auxin together with WOX11/12 [34, 38] (Fig. 2c), similar to the case during AR formation. The previous studies have indicated that ARF7/19 might be involved in activating LBD16 during callus initiation [34, 71]. However, LBD16 expression was not completely abolished in the arf7 arf19 double mutant background when callus was initiated from leaf explants (our unpublished data), suggesting that at least some other ARF proteins are also involved in the activation of LBD16 during this process (Fig. 2c). In addition, the chromatin factor ARABIDOPSIS TRITHORAX-RELATED 2 (ATXR2), a putative histone methyltransferase, acts as a co-activator with ARFs to upregulate LBD16 [71]. ATXR2 might regulate LBD16 expression via deposition of histone H3 lysine 36 trimethylation (H3K36me3) epigenetic markers on the LBD16 locus, thereby facilitating its transcription. Consistent with the molecular role of ATXR2, the atxr2 mutant showed partially defective callus formation from leaf explants, and the LBD16 expression level was lower in atxr2 leaf explants than in the wild-type leaf explants during callus initiation. Therefore, WOX11/12 and the auxin-mediated ARF pathway together with chromatin factors could act together to upregulate LBD16 during callus initiation on CIM. A recent study using enhanced yeast one-hybrid (eY1H) screening showed that LBD16 is involved in a complex gene regulatory network involving multiple key transcription factors related to regeneration [72].

The AtbZIP59 transcription factor interacts with LBD16 during callus formation [70]. Like LBD16 overexpression [34], AtbZIP59 overexpression induced autonomous callus formation in the absence of exogenous auxin [70]. The AtbZIP59–LBD16 complex was shown to directly regulate the expression of FAD-BINDING BERBERINE (FAD-BD), which encodes a BBE-like enzyme involved in cell wall metabolism during LR emergence [70, 73].

Therefore, callus initiation from leaf explants may borrow the AR formation pathway. Similarly, it is possible that callus formation from root explants may borrow the acropetal lateral rooting pathway or adventitious lateral rooting pathway. Regardless of the founder cells from different rooting pathways, LBD16 is the pivotal gene to be induced during callus initiation, and is required for the establishment of pluripotency (i.e., root-primordium identity) in the newly formed callus.

LBD16 in root-knot nematode feeding site formation

Root-knot nematodes, a type of plant endoparasitic nematode, can induce the formation of feeding structures from the vasculature in roots or in other organs (e.g., detached leaf explants) [39, 40]. Giant cells, the most commonly formed feeding cells in the feeding structures, are initiated from the vasculature and undergo repeated mitosis with aborted cytokinesis induced by nematode effectors [48].

Using A. thaliana roots and detached leaf explants as the model systems, a series of studies revealed the role of LBD16 in the formation of the feeding structures [40, 47, 48] (Fig. 2d). At the transcriptome level, the early stages of the formation of the feeding structure gall in A. thaliana roots resemble those of the formation of the root primordium [47]. In addition, in A. thaliana leaf explants, root-knot nematodes can feed on giant cells within a callus-like structure induced from the vasculature, similar to the gall induced in roots [40]. The formation of giant cells within these feeding structures (galls in roots or callus-like structures in leaf explants) is likely to borrow the root organogenesis pathways. The cells that are initiated to form the feeding structures have high auxin levels and express LBD16 [40, 47, 48] (Fig. 2d). Mutation of LBD16 led to partially defective feeding structure formation in roots and leaf explants [40, 47, 48]. The LBD16 expression level decreases when the feeding structures are partially differentiated [40, 47] (Fig. 2d). These observations suggested that the auxin signaling pathway and the LBD16-mediated molecular network are adopted for the formation of feeding structures induced by root-knot nematodes.

Interestingly, ARF7/19 are not strictly involved in the formation of giant cells and feeding structures. Primary roots of the arf7 arf19 double mutant were able to form feeding structures in response to root-knot nematodes [40]. Thus, upregulation of LBD16 and the formation of feeding structures either from the roots or leaf explants may occur mainly through the AR pathway instead of the LR pathway. At present, it is unclear which ARFs are involved in this process (Fig. 2d).

It is plausible to speculate that the initiation step of feeding structures is based on the AR formation mechanism involving auxin-induced LBD16 expression. In future research, it will be interesting to test whether WOX11/12 are also involved in this process.

Conclusion and perspectives

The common role of LBD16 in root and root-like organ formation is at the initiation step (see models in Fig. 2). Expression of LBD16 is induced in the specified founder cells after the priming step, and it continues to be expressed in the root primordium, the newly formed callus, or feeding structures. Its expression gradually decreases when the patterning step begins. The roles of LBD16 in root and root-like organ initiation may be to control cell division and to determine root-primordium identity.

Although LBD16 is upregulated in the initiation step during the formation of roots and root-like organs, the upstream mechanisms that activate LBD16 differ depending on the process. First, different ARFs are required to fulfill the auxin-mediated signaling pathway: ARF7/19 for LRs and other ARFs for ARs or AR-like organs (e.g., adventitious lateral roots from the primary root, callus from leaf explants, or feeding structures). Second, WOX11/12 are required for LBD16 upregulation in ARs or AR-like organs, but not LRs. The different molecular events’ upstream of LBD16 in the priming step might be due to the different status of founder cells in LR or AR organogenesis. While LR organogenesis requires several pairs of founder cells with nuclei migration and asymmetric founder cell division, there is no conclusive evidence that these factors are required for AR organogenesis or AR-like organ formation. Therefore, different molecular mechanisms operate in different LR and AR founder cells. One hypothesis was that WOX11/12-mediated AR organogenesis could be inherited from the ancient ability of intermediate-clade WOX (IC-WOX) genes in root founder cells [74]; because similar IC-WOX-mediated root organogenesis was also observed in a fern [75].

In summary, in the formation of root and root-like organs, diverse upstream priming mechanisms for the specification of founder cells may converge to activate LBD16 expression to achieve cell division and fate transition in organ initiation (see models in Fig. 2).

Many questions about LBD16 remain unanswered. First, it is largely unclear how other class-IB LBD genes interact with LBD16 during root and root-like organ formation. It will be interesting to test whether class-IB LBD genes have partially redundant or unique functions. For example, LBD29 is expressed in the root primordium during LR and AR formation [9, 36, 60, 76, 77], and its homologs CROWN ROTLESS1 (CRL1) in rice and ROOTLESS CONCERNING CROWN AND SEMINAL ROOTS (RTCS) in maize are involved in adventitious root (crown root) formation [78–82]. In addition, many class-IB LBD genes are upregulated during LR formation from the primary root or during callus formation on CIM [34, 36, 59, 73, 77, 83–87]. Furthermore, class-IB LBD proteins may form complexes to regulate downstream targets [88]. Further research is required to explore the shared and common roles of class-IB LBD genes. Second, it will be interesting to determine which ARF protein upregulates LBD16 expression in AR organogenesis and AR-like organ formation. Why different ARF proteins function in different types of founder cells is an interesting question. Third, the genome-wide target analysis of LBD16 will provide new insights into how it regulates organ initiation. Fourth, the mechanism that downregulates LBD16 expression in the patterning step is unclear. Fifth, further analysis of the protein complex involving LBD16 will provide new information about how LBD16 regulates gene expression in the specific context of organ initiation. Sixth, it will be important to determine how the LBD16 pathway acts synergistically with other pathways, for example the PLETHORAs (PLTs) and WOX5/7 pathways, in root and root-like organ formation. PLT3/5/7 and WOX5/7 are all expressed in the root primordium or newly formed callus [33, 36, 37]. Mutations in either PLT3/5/7 or WOX5/7 resulted in abnormal root-primordium development or the loss of pluripotency in callus cells ([37, 89, 90] and our unpublished data). It is unclear how LBD16, PLT3/5/7, and WOX5/7 form a regulatory network in this process [38]. Seventh, it will be interesting to explore how class-IB LBD genes have evolved and how and when were they recruited into root organogenesis, together with other rooting-related genes (e.g., ARFs and WOXs) [51–54, 74]. Answers to these questions will improve our understanding of root and root-like organ initiation and evolution.

Abbreviations

AR

Adventitious root

ARF

AUXIN RESPONSE FACTOR

ASL

ASYMMETRIC LEAVES2-LIKE

ATXR2

ARABIDOPSIS TRITHORAX-RELATED 2

Aux/IAA

AUXIN/INDOLE-3-ACETIC ACID

AuxRE

AUXIN RESPONSE ELEMENT

CIM

Callus-inducing medium

CRL1

CROWN ROTLESS1

eY1H

Enhanced yeast one-hybrid

FAD-BD

FAD-BINDING BERBERINE

LBD

LATERAL ORGAN BOUNDARIES DOMAIN

LR

Lateral root

PLT

PLETHORA

RIM

Root-inducing medium

RTCS

ROOTLESS CONCERNING CROWN AND SEMINAL ROOTS

SIM

Shoot-inducing medium

SLR

SOLITARY-ROOT

WOX

WUSCHEL-RELATED HOMEOBOX

Compliance with ethical standards

Conflict of interest

No conflicts of interest declared.