LBD18/ASL20 Regulates Lateral Root Formation in Combination with LBD16/ASL18 Downstream of ARF7 and ARF19 in Arabidopsis

The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes encode proteins harboring a conserved amino acid domain, referred to as the LOB (for lateral organ boundaries) domain. While recent studies have revealed developmental functions of some LBD genes in Arabidopsis (Arabidopsis thaliana) and in crop plants, the biological functions of many other LBD genes remain to be determined. In this study, we have demonstrated that the lbd18 mutant evidenced a reduced number of lateral roots and that lbd16 lbd18 double mutants exhibited a dramatic reduction in the number of lateral roots compared with lbd16 or lbd18. Consistent with this observation, significant β-glucuronidase (GUS) expression in Pro_LBD18:GUS seedlings was detected in lateral root primordia as well as in the emerged lateral roots. Whereas the numbers of primordia of lbd16, lbd18, and lbd16 lbd18 mutants were similar to those observed in the wild type, the numbers of emerged lateral roots of lbd16 and lbd18 single mutants were reduced significantly. lbd16 lbd18 double mutants exhibited additively reduced numbers of emerged lateral roots compared with single mutants. This finding indicates that LBD16 and LBD18 may function in the initiation and emergence of lateral root formation via a different pathway. LBD18 was shown to be localized into the nucleus. We determined whether LBD18 functions in the nucleus using a steroid regulator-inducible system in which the nuclear translocation of LBD18 can be regulated by dexamethasone in the wild-type, lbd18, and lbd16 lbd18 backgrounds. Whereas LBD18 overexpression in the wild-type background induced lateral root formation to some degree, other lines manifested the growth-inhibition phenotype. However, LBD18 overexpression rescued lateral root formation in lbd18 and lbd16 lbd18 mutants without inducing any other phenotypes. Furthermore, we demonstrated that LBD18 overexpression can stimulate lateral root formation in auxin response factor7/19 (arf7 arf19) mutants with blocked lateral root formation. Taken together, our results suggest that LBD18 functions in the initiation and emergence of lateral roots, in conjunction with LBD16, downstream of ARF7 and ARF19.The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes (hereafter referred to as LBD) encode proteins harboring a LOB (for lateral organ boundaries) domain, which is a conserved amino acid domain that is detected only in plants, indicative of its function in plant-specific processes (Iwakawa et al., 2002; Shuai et al., 2002). There are 42 Arabidopsis (Arabidopsis thaliana) LBD genes, which have been assigned to two classes. Class I comprises 36 genes and class II comprises six genes (Iwakawa et al., 2002; Shuai et al., 2002). The class I proteins harbor LOB domains similar to those observed in the LOB protein, whereas the class II proteins are less similar to the class I proteins, which include the LOB domain as well as regions outside of the LOB domain. The LOB domain is approximately 100 amino acids in length and harbors a conserved 4-Cys motif with CX₂CX₆CX₃C spacing, a Gly-Ala-Ser block, and a predicted coiled-coil motif with LX₆LX₃LX₆L spacing, reminiscent of the Leu zipper found in the majority of class I proteins (Shuai et al., 2002). None of the class II proteins were predicted to form coiled-coil structures.Although we currently understand very little about the biological roles of the LBD genes, there have been some reports describing the developmental functions of LBD genes in Arabidopsis on the basis of gain-of-function studies. The gain-of-function mutants of LBD36/ASL1, designated downwards siliques1, showed shorter internodes and downward lateral organs such as flowers (Chalfun-Junior et al., 2005). Although the lbd36 loss-of-function mutants did not show morphological phenotypes, the analysis of lbd36 as2 double mutants showed that these two members act redundantly to control cell fate determination in the petals. Another Arabidopsis gain-of-function mutant, jagged lateral organs-D (jlo-D), generates strongly lobed leaves and the shoot apical meristem prematurely arrests organ initiation, terminating in a pin-like structure (Borghi et al., 2007). During embryogenesis, JLO (=LBD30/ASL19) is necessary for the initiation of cotyledons and development beyond the globular stage. The results of misexpression experiments indicate that during postembryonic development, JLO function is required for the initiation of plant lateral organs. A recent study showed that the LOB domain of AS2 cannot be functionally replaced by those of other members of the LOB family, indicating that dissimilar amino acid residues in the LOB domains are important for characteristic functions of the family members (Matsumura et al., 2009).Thirty-five LBD genes in rice (Oryza sativa) have been identified from the genome sequences of the two rice subspecies, a japonica rice (Nippobare) and an indica rice (9311; Yang et al., 2006). Analyses of rice mutants have provided evidence of the involvement of a variety of rice LBD genes in lateral organ development. CROWN ROOTLESS1 (CRL1), encoding a LBD protein, is crucial for crown root formation in rice (Inukai et al., 2005). The crl1 mutant showed auxin-related phenotypes, such as decreased lateral root number, auxin insensitivity in lateral root formation, and impaired root gravitropism. A rice AUXIN RESPONSE FACTOR (ARF) appears to directly regulate CRL1 expression in the auxin signaling pathway (Inukai et al., 2005). ADVENTITIOUS ROOTLESS1 encodes an auxin-responsive protein with a LOB domain that controls the initiation of adventitious root primordia in rice and turned out to be the same gene as CRL1 (Liu et al., 2005).Lateral roots of Arabidopsis are derived from a subset of the pericycle cells (pericycle founder cells), which are positioned at the xylem poles within the parent root tissues (Casimiro et al., 2003). The mature pericycle cells dedifferentiate to form lateral root primordium (LRP), which undergoes consistent anticlinal and periclinal cell divisions to generate a highly organized LRP (Malamy and Benfey, 1997). The LRP emerges from the parent root via cell expansion, and the activation of the lateral root meristem results in continued growth of the organized lateral root. A growing body of physiological and genetic evidence has been collected to suggest that auxin plays a profound role in lateral root formation. For example, many auxin-related mutants have been shown to affect lateral root formation (Casimiro et al., 2003). Lateral root formation in Arabidopsis was shown to be regulated by ARF7 and ARF19 via the direct activation of LBD16 and LBD29/ASL16 (Okushima et al., 2007). Overexpression of LBD16 and LBD29 induced lateral root formation in the absence of ARF7 and ARF19, and the dominant repression of LBD16 inhibited lateral root formation, thus suggesting that these LBDs function downstream of ARF7- and ARF19-mediated auxin signaling during lateral root formation. The results of selection and binding assays demonstrated that a truncated LOB protein harboring only the conserved LOB domain can preferentially bind to unique DNA sequences, which is indicative of a DNA-binding protein (Husbands et al., 2007). Recently, LBD18 was shown to regulate tracheary element differentiation (Soyano et al., 2008).In this study, we demonstrated that LBD18 is involved in the regulation of lateral root formation, based on the analysis of loss-of-function mutants and the complementation of lbd18 and lbd16 lbd18 mutants by dexamethasone (DEX)-inducible LBD18 expression. Double mutations in LBD16 and LBD18 resulted in a synergistic reduction in the number of lateral roots, particularly in initiation and emergence, compared with either the lbd16 or lbd18 single mutant. This finding is suggestive of a combinatorial interaction of LBD16 and LBD18 in the process of lateral root formation. LBD18 expression in arf7 and arf19 mutants by the DEX-inducible system increased the number of lateral roots, thus demonstrating that LBD18 functions downstream of ARF7 and ARF19 in lateral root formation.     

RLF,a cytochrome b5‐like heme/steroid binding domain protein,controls lateral root formation independently of ARF7/19‐mediated auxin signaling in Arabidopsis thaliana

Lateral root (LR) formation is important for the establishment of root architecture in higher plants. Recent studies have revealed that LR formation is regulated by an auxin signaling pathway that depends on auxin response factors ARF7 and ARF19, and auxin/indole‐3‐acetic acid (Aux/IAA) proteins including SOLITARY‐ROOT (SLR)/IAA14. To understand the molecular mechanisms of LR formation, we isolated a recessive mutant rlf (reduced lateral root formation) in Arabidopsis thaliana. The rlf‐1 mutant showed reduction of not only emerged LRs but also LR primordia. Analyses using cell‐cycle markers indicated that the rlf‐1 mutation inhibits the first pericycle cell divisions involved in LR initiation. The rlf‐1 mutation did not affect auxin‐induced root growth inhibition but did affect LR formation over a wide range of auxin concentrations. However, the rlf‐1 mutation had almost no effect on auxin‐inducible expression of LATERAL ORGAN BOUNDARIES‐DOMAIN16/ASYMMETRIC LEAVES2‐LIKE18 (LBD16/ASL18) and LBD29/ASL16 genes, which are downstream targets of ARF7/19 for LR formation. These results indicate that ARF7/19‐mediated auxin signaling is not blocked by the rlf‐1 mutation. We found that the RLF gene encodes At5g09680, a protein with a cytochrome b₅‐like heme/steroid binding domain. RLF is ubiquitously expressed in almost all organs, and the protein localizes in the cytosol. These results, together with analysis of the genetic interaction between the rlf‐1 and arf7/19 mutations, indicate that RLF is a cytosolic protein that positively controls the early cell divisions involved in LR initiation, independent of ARF7/19‐mediated auxin signaling.     

Effects of three auxin-inducible LBD members on lateral root formation in Arabidopsis thaliana

In Arabidopsis, two AUXIN RESPONSE FACTORs (ARF7 and ARF19) and several Aux/IAAs regulate auxin-induced lateral root (LR) formation. As direct targets of ARF7 and ARF19, LATERAL ORGAN BOUNDARIES DOMAIN 16 (LBD16), LBD29, and LBD18 have a biological function in the formation of lateral roots (LRs). However, the details of the functions of these three LBDs have remained unclear. Each single T-DNA insert mutant has been shown to have slightly fewer LRs than the wild type. We then created a triple mutant, which exhibited a dramatic defect in the LR formation. Our results show that the lbd mutations can lead to impairment in auxin-induced pericycle cell division and in the expression levels of some D-type cyclins (CYCDs). Simultaneously, PLETHORA (PLT) and PIN-FORMED (PIN), which have been well documented to promote cell mitotic activity and are required for auxin response effects, were down-regulated by these lbd mutations. Our results so far indicate that CYCDs, PLT, and PINs are the main targets of the LBDs. We believe that these three LBDs are involved in cell cycle progression of the pericycle in response to auxin. Overexpression of any of these three LBD genes in the triple mutant was found incapable of completely replacing the other two LBDs. The phenotypes of lbd29 mutants were not completely consistent with lbd16 or lbd18 mutants. This indicates that LBD29 may play a distinctive role compared with LBD16 or LBD18 and LBDs might play partially independent roles during the formation of LRs.     

WOX11 and 12 Are Involved in the First-Step Cell Fate Transition during de Novo Root Organogenesis in Arabidopsis

De novo organogenesis is a process through which wounded or detached plant tissues or organs regenerate adventitious roots and shoots. Plant hormones play key roles in de novo organogenesis, whereas the mechanism by which hormonal actions result in the first-step cell fate transition in the whole process is unknown. Using leaf explants of Arabidopsis thaliana, we show that the homeobox genes WUSCHEL RELATED HOMEOBOX11 (WOX11) and WOX12 are involved in de novo root organogenesis. WOX11 directly responds to a wounding-induced auxin maximum in and surrounding the procambium and acts redundantly with its homolog WOX12 to upregulate LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) and LBD29, resulting in the first-step cell fate transition from a leaf procambium or its nearby parenchyma cell to a root founder cell. In addition, our results suggest that de novo root organogenesis and callus formation share a similar mechanism at initiation.     

IN VITRO DEVELOPMENT OF THE ISOLATED SHOOT APICAL MERISTEM OF ANGIOSPERMS

Development of complete plants was achieved from isolated shoot apical meristems of Nicotiana tabacum L., Daucus carota L., Nicotiana glauca Grah., Tropaeolum majus L., and Coleus blumei Benth. The explants consisted of only meristematic dome tissue with no visible leaf primordia. A simple nutrient medium composed of the Murashige and Skoog salt mixture, 100 mg/liter myo-inositol, 0.4 mg/liter thiamin-HCl, 1-2 mg/liter IAA, 30 g/liter sucrose, and 1% agar was adequate. Histologically there occurred principally tissue enlargement during the first 3-6 days, followed by appearance of bipolar organization in 6-9 days and formation of a well-defined root apex and initiation of first leaf primordium by 12 days.