Diversification,phylogeny and evolution of auxin response factor (ARF) family: insights gained from analyzing maize <Emphasis Type="Italic">ARF</Emphasis> genes

Auxin response factors (ARFs), member of the plant-specific B3 DNA binding superfamily, target specifically to auxin response elements (AuxREs) in promoters of primary auxin-responsive genes and heterodimerize with Aux/IAA proteins in auxin signaling transduction cascade. In previous research, we have isolated and characterized maize Aux/IAA genes in whole-genome scale. Here, we report the comprehensive analysis of ARF genes in maize. A total of 36 ARF genes were identified and validated from the B73 maize genome through an iterative strategy. Thirty-six maize ARF genes are distributed in all maize chromosomes except chromosome 7. Maize ARF genes expansion is mainly due to recent segmental duplications. Maize ARF proteins share one B3 DNA binding domain which consists of seven-stranded β sheets and two short α helixes. Twelve maize ARFs with glutamine-rich middle regions could be as activators in modulating expression of auxin-responsive genes. Eleven maize ARF proteins are lack of homo- and heterodimerization domains. Putative cis-elements involved in phytohormones and light signaling responses, biotic and abiotic stress adaption locate in promoters of maize ARF genes. Expression patterns vary greatly between clades and sister pairs of maize ARF genes. The B3 DNA binding and auxin response factor domains of maize ARF proteins are primarily subjected to negative selection during selective sweep. The mixed selective forces drive the diversification and evolution of genomic regions outside of B3 and ARF domains. Additionally, the dicot-specific proliferation of ARF genes was detected. Comparative genomics analysis indicated that maize, sorghum and rice duplicate chromosomal blocks containing ARF homologs are highly syntenic. This study provides insights into the distribution, phylogeny and evolution of ARF gene family.     

Auxin response factor family in rice

We isolated 11 rice genes homologous to the genes encoding auxin response factors (ARFs) in Arabidopsis. All of the genes encoded a well-conserved amino acid sequence in the N-terminal region, which is considered to be a DNA-binding domain (DBD). Phylogenetic analysis based on comparison of the DBDs indicated that rice has one or two closely related orthologs corresponding to a given respective ARF gene in Arabidopsis. We also analyzed the amino acid sequences of another conserved domain in the C-terminal conserved domain (CTD), which was shared by almost all the rice ARFs, with the exception of OsETTIN1 and OsETTIN2. These results agreed well with the evolutionary relationship deduced from the DBD comparison. In contrast to many ARFs, OsETTIN1 and OsETTIN2 do not contain the conserved C-terminal domain, but do share another consensus motif that is also found in Arabidopsis ETTIN. All of the above observations indicate that rice has functionally diversified ARF genes whose structures and functions correspond to those of various Arabidopsis ARFs, with one or two rice ARFs corresponding to a given Arabidopsis ARF. Thus, auxin signal transduction mechanisms may be well conserved between monocot and dicot plants.     

植物生长素反应因子研究进展

生长素反应因子（ARFs）是植物生长和发育的重要调节因子,在生长素早期反应蛋白（Aux／IAAs）的参与下,通过和生长素反应基因启动子区AuxRE元件的JTGTCTC序列结合,共同调控这些基因的表达。近年来关于生长素反应因子的分子结构和ARF与Aux／IAA的相互作用及其对植物生长和发育的影响、作用的靶基因以及分子机制受到人们的重视,并在这些方面做了大量的研究。     

生长素响应因子与植物的生长发育

生长素响应因子(Auxin response factor, ARF)作为一类调控生长素响应基因表达的转录因子, 是生长素研究的重要内容。它可与生长素响应基因启动子区域内的生长素响应元件结合, 促进或抑制基因的表达。文章介绍了植物体内ARF家族的分子生物学近年来的研究进展, 同时也讨论了ARF转录因子的结构、ARF基因的表达调控、ARF在植物生长发育及信号转导中的作用以及ARF对靶基因的调控机制等内容。植物ARF成员都有一定的同源性, 大多含有4个结构域, 在多种组织和器官中都有表达, 其表达受到转录及转录后调控, 并且在介导生长素与其它激素之间相互作用方面扮演重要角色。     

Genome-wide analysis of the auxin response factor (ARF) gene family in maize (<Emphasis Type="Italic">Zea mays</Emphasis>)

Auxin response factors (ARFs) are an important family involved in auxin-mediated response through specific binding to auxin response elements (AuxREs). A few members of the ARF family have been functionally characterized in Arabidopsis, rice (Oryza sativa), Poplar (Populous trichocarpa). However, little is known about ARF genes in maize (Zea mays). We performed a comprehensive bioinformatics analysis of the maize ARF gene family including analysis of the genome sequence, conserved domains, chromosomal locations, phylogenetic relationships, gene duplication, and expression profiles. 35 ZmARF genes were identified and categorized into four groups (Class I, II, III, and IV). In addition, a segmental ZmARF duplication event was shown to play an important role in maize ARF gene expansion. 7 ZmARF genes had no expression in specific tissues we obtained, but presented in mixed tissues according to the NCBI EST database, respectively. These studies have laid the theoretical foundation for further functional verification of these ZmARF genes.     

A role for auxin response factor 19 in auxin and ethylene signaling in Arabidopsis

Although auxin response factors (ARFs) are the first well-characterized proteins that bind to the auxin response elements, elucidation of the roles of each ARF gene in auxin responses and plant development has been challenging. Here we show that ARF19 and ARF7 not only participate in auxin signaling, but also play a critical role in ethylene responses in Arabidopsis (Arabidopsis thaliana) roots, indicating that the ARFs serve as a cross talk point between the two hormones. Both arf19 and arf7 mutants isolated from our forward genetic screens are auxin resistant and the arf19arf7 double mutant had stronger auxin resistance than the single mutants and displayed phenotypes not seen in the single mutants. Furthermore, we show that a genomic fragment of ARF19 not only complements arf19, but also rescues arf7. We conclude that ARF19 complements ARF7 at the protein level and that the ARF7 target sequences are also recognized by ARF19. Therefore, it is the differences in expression level/pattern and not the differences in protein sequences between the two ARFs that determines the relative contribution of the two ARFs in auxin signaling and plant development. In addition to being auxin resistant, arf19 has also ethylene-insensitive roots and ARF19 expression is induced by ethylene treatment. This work provides a sensitive genetic screen for uncovering auxin-resistant mutants including the described arf mutants. This study also provides a likely mechanism for coordination and integration of hormonal signals to regulate plant growth and development.     

Temporal and Spatial Distribution of Auxin Response Factor Genes During Tomato Flower Abscission

Abscission facilitates growth and reproduction and improves plant defenses against pathogens. This tightly regulated process is triggered by environmental cues and hormones such as ethylene and auxin. Because auxin is crucial for abscission, auxin response factors (ARFs) may play important roles in this process. Here, we examined changes in gene expression during abscission in tomato, focusing on regulation of genes encoding ARFs. Specifically, we analyzed the pattern of ARF gene expression in tomato flower pedicel explants treated with ethylene, the ethylene blocker 1-methylcyclopropene (1-MCP), or auxin to determine how auxin and ethylene affect ARF gene expression. In addition, we examined the spatial and temporal distribution of IAA during abscission by examining transgenic tomato plants expressing an IAA-inducible promoter fused to the GUS reporter gene (the P5::GUS ‘Chico III’ line). Flower removal from the explants quickly induced abscission by ethylene, which was inhibited by exogenous auxin or 1-MCP. During early abscission, auxin (or 1-MCP) regulated the expression of various ARFs, including ARF1, 2, 3, 4, 5, 7, 8-1, 9, 11, 12, 13, 13-1, 14, and 17, whereas ethylene had the opposite effect on most of these genes. Further analysis shows that during this stage, auxin may mediate the expression of ARF8-1, 9, 11, 12, 13, 13-1, and 14, whereas ethylene may mediate ARF13-1. During the later stage of abscission, ARF2, 8, 10, 11, and 19 were upregulated, and 8-1, 12, 13, and 13-1 were downregulated, compared with nonabscising parts of plants. Fluorometric GUS analysis indicated that GUS activity in the abscission zone remained stable at 4 h and sharply decreased after 8 h until abscission was complete (32 h).     

A common miRNA160‐based mechanism regulates ovary patterning,floral organ abscission and lamina outgrowth in tomato

Plant microRNAs play vital roles in auxin signaling via the negative regulation of auxin response factors (ARFs). Studies have shown that targeting of ARF10/16/17 by miR160 is indispensable for various aspects of development, but its functions in the model crop tomato (Solanum lycopersicum) are unknown. Here we knocked down miR160 (sly–miR160) using a short tandem target mimic (STTM160), and investigated its roles in tomato development. Northern blot analysis showed that miR160 is abundant in developing ovaries. In line with this, its down‐regulation perturbed ovary patterning as indicated by the excessive elongation of the proximal ends of mutant ovaries and thinning of the placenta. Following fertilization, these morphological changes led to formation of elongated, pear‐shaped fruits reminiscent of those of the tomato ovate mutant. In addition, STTM160‐expressing plants displayed abnormal floral organ abscission, and produced leaves, sepals and petals with diminished blades, indicating a requirement for sly–miR160 for these auxin‐mediated processes. We found that sly–miR160 depletion was always associated with the up‐regulation of SlARF10A, SlARF10B and SlARF17, of which the expression of SlARF10A increased the most. Despite the sly–miR160 legitimate site of SlARF16A, its mRNA levels did not change in response to sly–miR160 down‐regulation, suggesting that it may be regulated by a mechanism other than mRNA cleavage. SlARF10A and SlARF17 were previously suggested to function as inhibiting ARFs. We propose that by adjusting the expression of a group of ARF repressors, of which SlARF10A is a primary target, sly–miR160 regulates auxin‐mediated ovary patterning as well as floral organ abscission and lateral organ lamina outgrowth.