<Emphasis Type="Italic">ASYMMETRIC LEAVES2-LIKE38</Emphasis> gene,a Member of AS2/LOB family of <Emphasis Type="Italic">Arabidopsis</Emphasis>, causes leaf dorsoventral alternation in transgenic cockscomb plants

ASYMMETRIC LEAVES2-LIKE38/LBD41 gene of Arabidopsis is a member of the ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) domain gene family. To explore ASL38 function, we transformed 35S:ASL38 constructs into cockscomb (Celosia plumosus) plants via Agrobacterium tumefaciens and obtained T1 35S:ASL38 plants. The extremely folded or crinkly leaves were seen in these T1 cockscomb plants. The anatomical analysis of these malformed leaf blades indicated that adaxial cells revealed abaxialized traits, which were never seen in those of wild-type plants. These results suggested that ectopic expression of ASL38 might lead to alternations of dorsoventrality in folded or crinkly leaves of 35S:ASL38 cockscomb. In general, all data showed that ASL38 might be involved in dorsoventral determination in lateral organ development of plants.     

Expression of SHOOT MERISTEMLESS, WUSCHEL, and ASYMMETRIC LEAVES1 Homologs in the Shoots of Podostemaceae: Implications for the Evolution of Novel Shoot Organogenesis

Podostemaceae (the river weeds) are ecologically and morphologically unusual angiosperms. The subfamily Tristichoideae has typical shoot apical meristems (SAMs) that produce leaves, but Podostemoideae is devoid of SAMs and new leaves arise below the base of older leaves. To reveal the genetic basis for the evolution of novel shoot organogenesis in Podostemaceae, we examined the expression patterns of key regulatory genes for shoot development (i.e., SHOOT MERISTEMLESS (STM), WUSCHEL (WUS), and ASYMMETRIC LEAVES1/ROUGH SHEATH2/PHANTASTICA (ARP) orthologs) in Tristichoideae and Podostemoideae. In the SAM-mediated shoots of Tristichoideae, like in model plants, STM and WUS orthologs were expressed in the SAM. In the SAM-less shoots of Podostemoideae, STM and WUS orthologs were expressed in the initiating leaf/bract primordium. In older leaf/bract primordia, WUS expression disappeared and STM expression became restricted to the basal part, whereas ARP was expressed in the distal part in a complementary pattern to STM expression. In the reproductive shoots of Podostemoideae with a normal mode of flower development, STM and WUS were expressed in the floral meristem, but not in the floral organs, similar to the pattern in model plants. These results suggest that the leaf/bract of Podostemoideae is initiated as a SAM and differentiates into a single apical leaf/bract, resulting in the evolution of novel shoot-leaf mixed organs in Podostemaceae.     

<Emphasis Type="Italic">ASYMMETRIC LEAVES2-LIKE1</Emphasis>gene,a member of the AS2/LOB family,controls proximal–distal patterning in <Emphasis Type="Italic">Arabidopsis</Emphasis> petals

The formation and the development of the floral organs require an intercalate expression of organ-specific genes. At the same time, meristem-specific genes are repressed to complete the differentiation of the organs in the floral whorls. In an Arabidopsis activation tagging population, a mutant affected in inflorescence architecture was identified. This gain-of-function mutant, designateddownwards siliques1 (dsl1-D), has shorter internodes and the lateral organs such as flowers are bending downwards, similar to the loss-of-function brevipedicellus (bp) mutant. The affected gene in dsl1-D appeared to be ASYMMETRIC LEAVES2-LIKE1 (ASL1)/LATERAL ORGAN BOUNDARIESdomain gene 36 (LBD36), which is a member of the ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) domain gene family. Analysis of the loss-of-function mutant asl1/lbd36 did not show morphological aberration. Double mutant analysis of asl1/lbd36 together with as2, the ASL1/LBD36 closest homologue, demonstrates that these two members of the AS2/LOB family act partially redundant to control cell fate determination in Arabidopsis petals. Moreover, molecular analysis revealed that overexpression of ASL1/LBD36 leads to repression of the homeobox gene BP, which supports the model that an antagonistic relationship between ASL/LBD and homeobox members is required for the differentiation of lateral organs.     

A link between cytokinin and ASL9 (ASYMMETRIC LEAVES 2 LIKE 9) that belongs to the AS2/LOB (LATERAL ORGAN BOUNDARIES) family genes in Arabidopsis thaliana

In Arabidopsis thaliana, each member of a large family of AS2/LOB (ASYMMETRIC LEAVES 2/LATERAL ORGAN BOUNDARIES) genes encodes a plant specific protein. They are highly homologous to one other. A mutational lesion in the representative AS2 gene results in the development of anomalous asymmetric leaves, implying that these family members commonly play some roles in plant development. In this study, we found that ectopic overexpression of ASL9 (ASYMMETRIC LEAVES 2 LIKE 9) in transgenic plants displayed a markedly anomalous architecture during the development of adult plants. Then we found that among AS2/LOB family members, ASL9 is distinct from the others in that it is exclusively regulated by the plant hormone cytokinin in a manner dependent on His-Asp phosphorelay signal transduction. We further found that when supplied externally in a medium, cytokinin specifically affected the growth properties of ASL9-ox seedlings. Taken together, the results of this study suggest that the cytokinin-induced ASL9 gene is implicated in regulation of the development of Arabidopsis thaliana.     

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.     

拟南芥ASL25/LBD28基因过量表达对叶片形态建成的影响

为研究ASL25/LBD28基因在植物发育过程中的作用,该研究构建了拟南芥ASL25/LBD28的过量表达载体并将其转入野生型拟南芥中,结果发现,ASL25/LBD28基因的过量表达可导致转基因拟南芥的叶片变得狭长;在叶极性发育突变体as2中,ASL25/LBD28基因过量表达导致部分转基因植株在形成1～3片畸形叶后顶端分生组织的发育会终止;而许多转基因植株则会形成许多"针状"叶.扫描电镜观察表明,不正常的叶片近轴面或"针状"叶的表皮细胞具有远轴面化的长条形细胞,说明在as2突变体中过量表达ASL25/LBD28基因影响叶片的极性发育.     

Centering the Organizing Center in the Arabidopsis thaliana Shoot Apical Meristem by a Combination of Cytokinin Signaling and Self-Organization

Plants have the ability to continously generate new organs by maintaining populations of stem cells throught their lives. The shoot apical meristem (SAM) provides a stable environment for the maintenance of stem cells. All cells inside the SAM divide, yet boundaries and patterns are maintained. Experimental evidence indicates that patterning is independent of cell lineage, thus a dynamic self-regulatory mechanism is required. A pivotal role in the organization of the SAM is played by the WUSCHEL gene (WUS). An important question in this regard is that how WUS expression is positioned in the SAM via a cell-lineage independent signaling mechanism. In this study we demonstrate via mathematical modeling that a combination of an inhibitor of the Cytokinin (CK) receptor, Arabidopsis histidine kinase 4 (AHK4) and two morphogens originating from the top cell layer, can plausibly account for the cell lineage-independent centering of WUS expression within SAM. Furthermore, our laser ablation and microsurgical experiments support the hypothesis that patterning in SAM occurs at the level of CK reception and signaling. The model suggests that the interplay between CK signaling, WUS/CLV feedback loop and boundary signals can account for positioning of the WUS expression, and provides directions for further experimental investigation.     

The CLAVATA signaling pathway mediating stem cell fate in shoot meristems requires Ca2+ as a secondary cytosolic messenger

CLAVATA1 (CLV1) is a receptor protein expressed in the shoot apical meristem (SAM) that translates perception of a non‐cell‐autonomous CLAVATA3 (CLV3) peptide signal into altered stem cell fate. CLV3 reduces expression of WUSCHEL (WUS) and FANTASTIC FOUR 2 (FAF2) in the SAM. Expression of WUS and FAF2 leads to maintenance of undifferentiated stem cells in the SAM. CLV3 binding to CLV1 inhibits expression of these genes and controls stem cell fate in the SAM through an unidentified signaling pathway. Cytosolic Ca²⁺ elevations, cyclic nucleotide (cGMP)‐activated Ca²⁺ channels, and cGMP have been linked to signaling downstream of receptors similar to CLV1. Hence, we hypothesized that cytosolic Ca²⁺ elevation mediates the CLV3 ligand/CLV1 receptor signaling that controls meristem stem cell fate. CLV3 application to Arabidopsis seedlings results in elevation of cytosolic Ca²⁺ and cGMP. CLV3 control of WUS was prevented in a genotype lacking a functional cGMP‐activated Ca²⁺ channel. In wild‐type plants, CLV3 inhibition of WUS and FAF2 expression was impaired by treatment with either a Ca²⁺ channel blocker or a guanylyl cyclase inhibitor. When CLV3‐dependent repression of WUS is blocked, altered control of stem cell fate leads to an increase in SAM size; we observed a larger SAM size in seedlings treated with the Ca²⁺ channel blocker. These results suggest that the CLV3 ligand/CLV1 receptor system initiates a signaling cascade that elevates cytosolic Ca²⁺, and that this cytosolic secondary messenger is involved in the signal transduction cascade linking CLV3/CLV1 to control of gene expression and stem cell fate in the SAM.