Functional divergence in the Arabidopsis LOB-domain gene family

The Arabidopsis LOB-domain (LBD) gene family is composed by 43 members divided in two classes based on amino acid conservation within the LOB-domain. The LOB domain is known to be responsible for DNA binding and protein-protein interactions. There is very little functional information available for most genes in the LBD family and many lbd single mutants do not exhibit conspicuous phenotypes. One plausible explanation for the limited loss-of-function phenotypes observed in this family is that LBD genes exhibit significant functional redundancy. Here we discuss an example of one phylogenetic subgroup of the LBD family, in which genes that are closely related based on phylogeny exhibit distinctly different expression patterns and do not have overlapping functions. We discuss the challenges of using phylogenetic analyses to predict redundancy in gene families.     

蒺藜苜蓿LBD转录因子基因家族全基因组分析

LBD是植物中所特有的转录因子基因家族,在调控植物侧生组织发育、营养代谢以及响应逆境胁迫等方面具有重要作用。该研究利用生物信息学手段,从全基因组水平筛选和鉴定了蒺藜苜蓿LBD基因家族,并对基因结构、系统进化、进化压力、保守域、染色体定位以及基因表达模式等进行了分析。研究结果共鉴定出2类5亚类共计56个蒺藜苜蓿LBD家族基因,在8条染色体上均有分布,但分布不均匀。该家族成员外显子数目都不超过2个,结构简单,基因间在进化时存在负向选择作用。基因表达模式分析发现,该家族成员的表达具有一定的时空特异性,并受干旱和氮素调控。该研究结果对蒺藜苜蓿LBD基因功能研究及进化分析具有重要的意义。     

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.     

沙地云杉LBD基因家族鉴定及盐胁迫响应分析北大核心CSCD

沙地云杉(Picea mongolica)是我国稀有珍贵树种,具有耐旱、耐寒、耐沙埋的优良抗性。LBD(lateral organ boundaries domain)是植物侧生器官中重要的转录因子,在植物生长发育和胁迫应答进程中起关键作用,但目前尚未报道有关沙地云杉中LBD基因家族的研究。本研究参考挪威云杉全基因组数据及沙地云杉转录组数据鉴定沙地云杉LBD基因,进行生物信息学分析并利用qRT-PCR检测LBD基因在不同组织(茎尖、主根、侧根、茎和叶)及盐胁迫胁迫条件下的表达水平。结果表明,在沙地云杉转录组中共鉴定出30个LBD基因(PmLBD1-30),蛋白序列长度在119~309 aa之间,分子量为10.5~33.4 kD,等电点介于5.15~9.26之间,Cell-PLoc亚细胞定位显示均位于细胞核中;所有的LBD蛋白结构域、基因结构高度保守,并由相似的基序组成;根据系统发育树可将其分为5个亚家族(Class I a~e),沙地云杉在各类中的成员依次为4,11,5,1,9个;qRT-PCR试验结果显示,PmLBDs在不同组织中均有表达,如PmLBD2/5/18/19在茎中高表达,ClassⅠb中的PmLBD9/20/23基因在侧根中强烈表达;大多数PmLBDs的表达强烈响应盐胁迫,17个PmLBD基因在盐处理后上调表达,而6个PmLBD基因在盐处理后下调表达,且同一亚族基因表达情况呈现相似趋势。