The Arabidopsis MAX pathway controls shoot branching by regulating auxin transport

BACKGROUND: Plants achieve remarkable plasticity in shoot system architecture by regulating the activity of secondary shoot meristems, laid down in the axil of each leaf. Axillary meristem activity, and hence shoot branching, is regulated by a network of interacting hormonal signals that move through the plant. Among these, auxin, moving down the plant in the main stem, indirectly inhibits axillary bud outgrowth, and an as yet undefined hormone, the synthesis of which in Arabidopsis requires MAX1, MAX3, and MAX4, moves up the plant and also inhibits shoot branching. Since the axillary buds of max4 mutants are resistant to the inhibitory effects of apically supplied auxin, auxin and the MAX-dependent hormone must interact to inhibit branching. RESULTS: Here we show that the resistance of max mutant buds to apically supplied auxin is largely independent of the known, AXR1-mediated, auxin signal transduction pathway. Instead, it is caused by increased capacity for auxin transport in max primary stems, which show increased expression of PIN auxin efflux facilitators. The max phenotype is dependent on PIN1 activity, but it is independent of flavonoids, which are known regulators of PIN-dependent auxin transport. CONCLUSIONS: The MAX-dependent hormone is a novel regulator of auxin transport. Modulation of auxin transport in the stem is sufficient to regulate bud outgrowth, independent of AXR1-mediated auxin signaling. We therefore propose an additional mechanism for long-range signaling by auxin in which bud growth is regulated by competition between auxin sources for auxin transport capacity in the primary stem.     

MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule

BACKGROUND: Plant development is exquisitely environmentally sensitive, with plant hormones acting as long-range signals that integrate developmental, genetic, and environmental inputs to regulate development. A good example of this is in the control of shoot branching, where wide variation in plant form can be generated in a single genotype in response to environmental and developmental cues. RESULTS: Here we present evidence for a novel plant signaling molecule involved in the regulation of shoot branching. We show that the MAX3 gene of Arabidopsis is required for the production of a graft-transmissible, highly active branch inhibitor that is distinct from any of the previously characterized branch-inhibiting hormones. Consistent with its proposed function in the synthesis of a novel signaling molecule, we show that MAX3 encodes a plastidic dioxygenase that can cleave multiple carotenoids. CONCLUSIONS: We conclude that MAX3 is required for the synthesis of a novel carotenoid-derived long-range signal that regulates shoot branching.     

拟南芥MAX2蛋白功能研究进展

独角金内酯(strigolactone, SLs)是一类新型植物激素,在植物生长发育的进程中发挥多种重要功能,包括调控植物的分枝,促进种子的萌发,以及影响根系建成等。MAX2 (more axillary growth 2)是SL信号传导途径的关键调控因子,位于合成途径基因MAX1、MAX3和MAX4的下游,几乎影响独脚金内酯所控制的所有表型。近年来,MAX2多样化的功能逐步得到揭示,大量数据表明MAX2不仅仅是SL信号的重要组分,同时也参与SL和多种激素信号间的交叉互作,在植物生长发育的各个环节,以及抵御生物和非生物胁迫的反应中都发挥至关重要的作用,但具体调控机制还有待更加深入的研究。对目前已知的MAX2功能进行了总结和阐述,以期为全面揭示MAX2功能及其调控多种激素信号的交叉机制提供理论参考。     

Characterization of MORE AXILLARY GROWTH Genes in Populus

Background

Strigolactones are a new class of plant hormones that play a key role in regulating shoot branching. Studies of branching mutants in Arabidopsis, pea, rice and petunia have identified several key genes involved in strigolactone biosynthesis or signaling pathway. In the model plant Arabidopsis, MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3 and MAX4 are four founding members of strigolactone pathway genes. However, little is known about the strigolactone pathway genes in the woody perennial plants.

Methodology/Principal Finding

Here we report the identification of MAX homologues in the woody model plant Populus trichocarpa. We identified the sequence homologues for each MAX protein in P. trichocarpa. Gene expression analysis revealed that Populus MAX paralogous genes are differentially expressed across various tissues and organs. Furthermore, we showed that Populus MAX genes could complement or partially complement the shoot branching phenotypes of the corresponding Arabidopsis max mutants.

Conclusion/Significance

This study provides genetic evidence that strigolactone pathway genes are likely conserved in the woody perennial plants and lays a foundation for further characterization of strigolactone pathway and its functions in the woody perennial plants.     

苹果MAX1基因的克隆和表达及启动子分析

MAX1(MORE AXILLARY GROWTH1)是独脚金内酯(Strigolactones,SLs)合成途径中的关键基因,为了研究MAX1在苹果分枝调控中的功能,以苹果‘长富2号’(Malus domestica‘Nagafu 2’)腋芽为材料,采用PCR方法,克隆MdMAX1基因,进行生物信息学和表达水平分析;采用瞬时表达转化烟草,进行GUS染色,分析MdMAX1启动子活性。结果表明:(1)成功克隆得到苹果MAX1,其开放阅读框(ORF)1620 bp,编码539个氨基酸;系统进化和基序分析表明,MdMAX1和已知的A1型MAX1相似。(2)qRT-PCR分析表明,MAX1基因在‘长富2号’嫁接苗茎中高表达,并在腋芽本身有表达;RNA-seq分析表明,细胞分裂素(6-BA)处理苹果腋芽96 h后MAX1基因的表达水平显著降低。(3)成功克隆获得‘长富2号’MAX1启动子序列片段(1500 bp),顺式作用元件预测显示MdMAX1启动子序列中存在光响应元件,GUS活性分析表明光照处理能够减弱MAX1启动子的活性。该研究为进一步研究苹果MAX1参与SLs合成、调控苹果分枝的功能奠定了基础。     

Modulation of hepatocyte nuclear factor-4alpha function by the peroxisome-proliferator-activated receptor-gamma co-activator-1alpha in the acute-phase response

Recent studies with the high-tillering mutants in rice (Oryza sativa), the max (more axillary growth) mutants in Arabidopsis thaliana and the rms (ramosus) mutants in pea (Pisum sativum) have indicated the presence of a novel plant hormone that inhibits branching in an auxin-dependent manner. The synthesis of this inhibitor is initiated by the two CCDs [carotenoid-cleaving (di)oxygenases] OsCCD7/OsCCD8b, MAX3/MAX4 and RMS5/RMS1 in rice, Arabidopsis and pea respectively. MAX3 and MAX4 are thought to catalyse the successive cleavage of a carotenoid substrate yielding an apocarotenoid that, possibly after further modification, inhibits the outgrowth of axillary buds. To elucidate the substrate specificity of OsCCD8b, MAX4 and RMS1, we investigated their activities in vitro using naturally accumulated carotenoids and synthetic apocarotenoid substrates, and in vivo using carotenoid-accumulating Escherichia coli strains. The results obtained suggest that these enzymes are highly specific, converting the C27 compounds beta-apo-10'-carotenal and its alcohol into beta-apo-13-carotenone in vitro. Our data suggest that the second cleavage step in the biosynthesis of the plant branching inhibitor is conserved in monocotyledonous and dicotyledonous species.     

MAX2 affects multiple hormones to promote photomorphogenesis

Ubiquitin-26S proteasome system (UPS) has been shown to play central roles in light and hormone-regulated plant growth and development. Previously, we have shown that MAX2, an F-box protein, positively regulates facets of photomorphogenic development in response to light. However, how MAX2 controls these responses is still unknown. Here, we show that MAX2 oppositely regulates GA and ABA biosynthesis to optimize seed germination in response to light. Dose-response curves showed that max2 seeds are hyposensitive to GA and hypersensitive to ABA in seed germination responses. RT-PCR assays demonstrated that the expression of GA biosynthetic genes is down-regulated, while the expression of GA catabolic genes is up-regulated in the max2 seeds compared to wild-type. Interestingly, expression of both ABA biosynthetic and catabolic genes is up-regulated in the max2 seeds compared to wild-type. Treatment with an auxin transport inhibitor, NPA, showed that increased auxin transport in max2 seedlings contributes to the long hypocotyl phenotype under light. Moreover, light-signaling phenotypes are restricted to max2, as the biosynthetic mutants in the strigolactone pathway, max1, max3, and max4, did not display any defects in seed germination and seedling de-etiolation compared to wild-type. Taken together, these data suggest that MAX2 modulates multiple hormone pathways to affect photomorphogenesis.     

Over-expression of the <Emphasis Type="Italic">IGI1</Emphasis> leading to altered shoot-branching development related to MAX pathway in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Shoot branching and growth are controlled by phytohormones such as auxin and other components in Arabidopsis. We identified a mutant (igi1) showing decreased height and bunchy branching patterns. The phenotypes reverted to the wild type in response to RNA interference with the IGI1 gene. Histochemical analysis by GUS assay revealed tissue-specific gene expression in the anther and showed that the expression levels of the IGI1 gene in apical parts, including flowers, were higher than in other parts of the plants. The auxin biosynthesis component gene, CYP79B2, was up-regulated in igi1 mutants and the IGI1 gene was down-regulated by IAA treatment. These results indicated that there is an interplay regulation between IGI1 and phytohormone auxin. Moreover, the expression of the auxin-related shoot branching regulation genes, MAX3 and MAX4, was down-regulated in igi1 mutants. Taken together, these results indicate that the overexpression of the IGI1 influenced MAX pathway in the shoot branching regulation.     

MAX1 and MAX2 control shoot lateral branching in Arabidopsis

Plant shoots elaborate their adult form by selective control over the growth of both their primary shoot apical meristem and their axillary shoot meristems. We describe recessive mutations at two loci in Arabidopsis, MAX1 and MAX2, that affect the selective repression of axillary shoots. All the first order (but not higher order) axillary shoots initiated by mutant plants remain active, resulting in bushier shoots than those of wild type. In vegetative plants where axillary shoots develop in a basal to apical sequence, the mutations do not clearly alter node distance, from the shoot apex, at which axillary shoot meristems initiate but shorten the distance at which the first axillary leaf primordium is produced by the axillary shoot meristem. A small number of mutant axillary shoot meristems is enlarged and, later in development, a low proportion of mutant lateral shoots is fasciated. Together, this suggests that MAX1 and MAX2 do not control the timing of axillary meristem initiation but repress primordia formation by the axillary meristem. In addition to shoot branching, mutations at both loci affect leaf shape. The mutations at MAX2 cause increased hypocotyl and petiole elongation in light-grown seedlings. Positional cloning identifies MAX2 as a member of the F-box leucine-rich repeat family of proteins. MAX2 is identical to ORE9, a proposed regulator of leaf senescence ( Woo, H. R., Chung, K. M., Park, J.-H., Oh, S. A., Ahn, T., Hong, S. H., Jang, S. K. and Nam, H. G. (2001) Plant Cell 13, 1779-1790). Our results suggest that selective repression of axillary shoots involves ubiquitin-mediated degradation of as yet unidentified proteins that activate axillary growth.     

The F-box protein MAX2 functions as a positive regulator of photomorphogenesis in Arabidopsis

Light is vital for plant growth and development. To respond to ambient light signals, plants are equipped with an array of photoreceptors, including phytochromes that sense red (R)/far-R (FR) regions and cryptochromes and phototropins that respond to the ultraviolet-A/blue (B) region of the light spectrum, respectively. Several positively and negatively acting components in light-signaling pathways have been identified using genetic approaches; however, the pathways are not saturated. Here, we characterize a new mutant named pleiotropic photosignaling (pps), isolated from a genetic screen under continuous R light. pps has longer hypocotyls and slightly smaller cotyledons under continuous R, FR, and B light compared to that of the wild type. pps is also hyposensitive to both R and FR light-induced seed germination. Although photosynthetic marker genes are constitutively expressed in pps in the dark at high levels, the expression of early light-regulated genes is reduced in the pps seedlings compared to wild-type seedlings under R light. PPS encodes MAX2/ORE9 (for MORE AXILLARY BRANCHES2/ORESARA9), an F-box protein involved in inflorescence architecture and senescence. MAX2 is expressed ubiquitously in the seedling stage. However, its expression is restricted to vascular tissues and meristems at adult stages. MAX2 is also localized to the nucleus. As an F-box protein, MAX2 is predicted to be a component of the SCF (for SKP, Cullin, and F-box protein) complex involved in regulated proteolysis. These results suggest that SCF(MAX2) plays critical roles in R, FR, and B light-signaling pathways. In addition, MAX2 might regulate multiple targets at different developmental stages to optimize plant growth and development.     

植物茎分枝的分子调控

植物茎分枝结构决定了不同植物的不同形态结构.本文从腋生分生组织的发生、腋芽的生长两个方面综述了近年来植物分枝发生发育相关的分子机理研究及其进展.发现在不同植物中腋分生组织形成的基本机制是相似的,LS（lateral suppressor）及其同源基因在不同植物中都参与腋生分生组织的形成,而BL(blind)及其同源基因也参与调控腋生分生组织的形成.腋生分生组织的形成可能也是受激素调控的.目前,对腋芽生长的分子调控机制的认识主要集中于生长素通过二级信使的作用调控腋芽的生长.而生长素调控腋芽生长的机制已经较为清楚的有两条途径：一是生长素通过抑制细胞分裂素合成来调控腋芽的生长;另一途径是一种类胡萝卜素衍生的信号物质参与生长素的运输调控(MAX途径)来调控腋芽的生长.最新研究表明,TB1的拟南芥同源基因在MAX途径的下游负调控腋芽的生长.此外,增强表达OsNAC2也促进腋芽的生长.     

The genetic architecture of shoot branching in Arabidopsis thaliana: a comparative assessment of candidate gene associations vs. quantitative trait locus mapping

Association mapping focused on 36 genes involved in branch development was used to identify candidate genes for variation in shoot branching in Arabidopsis thaliana. The associations between four branching traits and moderate-frequency haplogroups at the studied genes were tested in a panel of 96 accessions from a restricted geographic range in Central Europe. Using a mixed-model association-mapping method, we identified three loci--MORE AXILLARY GROWTH 2 (MAX2), MORE AXILLARY GROWTH 3 (MAX3), and SUPERSHOOT 1 (SPS1)--that were significantly associated with branching variation. On the basis of a more extensive examination of the MAX2 and MAX3 genomic regions, we find that linkage disequilibrium in these regions decays within approximately 10 kb and trait associations localize to the candidate genes in these regions. When the significant associations are compared to relevant quantitative trait loci (QTL) from previous Ler x Col and Cvi x Ler recombinant inbred line (RIL) mapping studies, no additive QTL overlapping these candidate genes are observed, although epistatic QTL for branching, including one that spans the SPS1, are found. These results suggest that epistasis is prevalent in determining branching variation in A. thaliana and may need to be considered in linkage disequilibrium mapping studies of genetically diverse accessions.     

A Role for MORE AXILLARY GROWTH1 (MAX1) in Evolutionary Diversity in Strigolactone Signaling Upstream of MAX2

Strigolactones (SLs) are carotenoid-derived phytohormones with diverse roles. They are secreted from roots as attractants for arbuscular mycorrhizal fungi and have a wide range of endogenous functions, such as regulation of root and shoot system architecture. To date, six genes associated with SL synthesis and signaling have been molecularly identified using the shoot-branching mutants more axillary growth (max) of Arabidopsis (Arabidopsis thaliana) and dwarf (d) of rice (Oryza sativa). Here, we present a phylogenetic analysis of the MAX/D genes to clarify the relationships of each gene with its wider family and to allow the correlation of events in the evolution of the genes with the evolution of SL function. Our analysis suggests that the notion of a distinct SL pathway is inappropriate. Instead, there may be a diversity of SL-like compounds, the response to which requires a D14/D14-like protein. This ancestral system could have been refined toward distinct ligand-specific pathways channeled through MAX2, the most downstream known component of SL signaling. MAX2 is tightly conserved among land plants and is more diverged from its nearest sister clade than any other SL-related gene, suggesting a pivotal role in the evolution of SL signaling. By contrast, the evidence suggests much greater flexibility upstream of MAX2. The MAX1 gene is a particularly strong candidate for contributing to diversification of inputs upstream of MAX2. Our functional analysis of the MAX1 family demonstrates the early origin of its catalytic function and both redundancy and functional diversification associated with its duplication in angiosperm lineages.Strigolactones (SLs) are carotenoid-derived terpenoid lactones, which have been identified as signaling molecules in several areas of plant biology. SLs were first identified as germination stimulants for seeds of plants in the genus Striga (Cook et al., 1966). Striga spp. and related Orobanchaceae are parasitic weeds that germinate in response to host plant root exudates and develop haustoria to penetrate the host tissue and draw nutrients. Striga spp. are major agricultural pests across much of tropical and subtropical Asia and are present in two-thirds of arable land in Africa, where they are the greatest biological cause of crop damage (Humphrey and Beale, 2006). The secretion of SLs by roots, despite its exploitation by Striga spp., has been preserved because it also serves to recruit arbuscular mycorrhizal (AM) fungi (Akiyama et al., 2005). AM fungi form symbiotic associations with most land plants, whereby the plant gains access to mineral nutrients, particularly phosphate, absorbed by the fungal hyphae, and in exchange the fungus gains fixed carbon from the plant. In several flowering plant species, SL production is correspondingly increased when phosphate availability is limiting, thereby presumably increasing fungal recruitment (Yoneyama et al., 2007, 2012).AM symbioses can be traced back to the origin of land plants, between 360 to 450 million years ago, and are thought to have facilitated plant colonization of the terrestrial environment (Simon et al., 1993). Although AM symbiosis has been lost from some lineages, such as Brassicaceae, it is still widespread, with 80% of land plants able to form associations with AM fungi (Schüssler et al., 2001). In support of a similarly ancient origin for SL secretion, the liverwort Marchantia polymorpha and the moss Physcomitrella patens, both basal land plant groups, have been shown to produce SLs (Proust et al., 2011; Delaux et al., 2012). Furthermore, the presence of SLs in charophyte algae indicates that SL production may predate the emergence of land plants (Delaux et al., 2012), and Chara corallina responds to SL treatment by producing longer rhizoids (Delaux et al., 2012). In P. patens, SLs appear to act as intercolony coordination signals, regulating colony growth and competition by controlling flexible developmental processes such as protonemal branching (Proust et al., 2011; Delaux et al., 2012). In flowering plants, SLs have also been implicated in development, including several processes regulated in response to phosphate limitation (Kohlen et al., 2011; Ruyter-Spira et al., 2011). In particular, SLs play important roles in the regulation of shoot branching in higher plants (Gomez-Roldan et al., 2008; Umehara et al., 2008). It is through work on their effects on shoot branching that some of the genes in the SL pathway were first identified.Arabidopsis (Arabidopsis thaliana) MORE AXILLARY GROWTH (MAX) mutants show increased branching and reduced stature relative to wild-type plants, and analogous phenotypes have been identified in pea (Pisum sativum; RAMOSUS [RMS]), petunia (Petunia hybrida; DECREASED APICAL DOMINANCE [DAD]), and rice (Oryza sativa; DWARF [D] or HIGH TILLERING DWARF) mutants. So far, six MAX/RMS/DAD/D genes have been identified, with roles in SL biosynthesis or signaling. MAX3/RMS5/HIGH TILLERING DWARF1/D17 (Booker et al., 2004; Johnson et al., 2006; Zou et al., 2006) and MAX4/RMS1/DAD1/D10 (Sorefan et al., 2003; Snowden et al., 2005; Arite et al., 2007) encode carotenoid cleavage dioxygenases (CCD7 and CCD8, respectively). These enzymes are capable of sequentially cleaving the carotenoid 9-cis-β-carotene to produce a novel compound, carlactone, a putative strigolactone intermediate (Alder et al., 2012). Another biosynthetic gene, D27, was originally mutationally defined in rice (Lin et al., 2009), and reverse genetic approaches in Arabidopsis indicate a similar function in this species (Waters et al., 2012a). D27 is an iron-containing protein with isomerase activity that can produce the 9-cis-β-carotene substrate for MAX3 from all-trans-β-carotene (Alder et al., 2012). The fourth gene known to be involved in SL biosynthesis, MAX1, encodes a cytochrome p450 monooxygenase belonging to the CYP711 clan (Booker et al., 2005). Mutant phenotypes associated with this gene have so far only been identified in one species, Arabidopsis, although the gene is present in all tracheophytes (Nelson et al., 2008). The excessive-branching phenotypes associated with mutations in all of these genes can be rescued by exogenous application of SL, while mutants in the two remaining genes in the pathway are SL insensitive. D14 encodes an α/β hydrolase, which is proposed to act in signaling or in the hydrolysis of SLs to an active compound and provides specificity to signaling via MAX2/RMS4/D3, an F-box protein that mediates both SL signaling and signaling of karrikins (Stirnberg et al., 2002, 2007; Ishikawa et al., 2005; Johnson et al., 2006; Arite et al., 2009; Hamiaux et al., 2012; Waters et al., 2012b). Karrikins are compounds structurally related to SLs that are found in smoke and act as germination stimulants for plants that colonize ground cleared by forest fires (Nelson et al., 2010; Waters et al., 2012b).Homology searches described in the original publications for each of the MAX/D genes revealed two general patterns. MAX1, MAX3, and MAX4 are members of widespread gene families and are more closely related to nonplant sequences than to other plant genes (Sorefan et al., 2003; Booker et al., 2005). By contrast, MAX2, D14, and D27 are members of plant-specific gene families (Stirnberg et al., 2002; Arite et al., 2009; Lin et al., 2009). These contrasting patterns of SL pathway gene ancestry and the diverse biological roles of SLs present interesting evolutionary questions. The identification of SLs and SL responses in charophyte algae demonstrate their early evolution, but these species lack many of the genes required for SL synthesis and signaling in angiosperms. In an attempt to trace the evolution of the angiosperm SL pathway, we conducted a phylogenetic analysis of the known SL biosynthesis and signaling genes, allowing the correlation of events in the evolution of the genes with the evolution of SL function. Our analysis suggests that the notion of a distinct SL pathway is inappropriate. Instead, the angiosperm pathway seems to have been defined by the rapid evolution of MAX2 in early land plants. Upstream of MAX2, there appears to be much greater flexibility, especially in the requirements for the synthesis of SLs. We present evidence for the contribution of MAX1 to this flexibility. Our functional analysis of MAX1 orthologs from phylogenetically diverse species demonstrates the early origin of its catalytic activity and both redundancy and functional diversification associated with its duplication in angiosperm lineages.     

SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis

The plant hormones strigolactones and smoke-derived karrikins are butenolide signals that control distinct aspects of plant development. Perception of both molecules in Arabidopsis thaliana requires the F-box protein MORE AXILLARY GROWTH2 (MAX2). Recent studies suggest that the homologous SUPPRESSOR OF MAX2 1 (SMAX1) in Arabidopsis and DWARF53 (D53) in rice (Oryza sativa) are downstream targets of MAX2. Through an extensive analysis of loss-of-function mutants, we demonstrate that the Arabidopsis SMAX1-LIKE genes SMXL6, SMXL7, and SMXL8 are co-orthologs of rice D53 that promote shoot branching. SMXL7 is degraded rapidly after treatment with the synthetic strigolactone mixture rac-GR24. Like D53, SMXL7 degradation is MAX2- and D14-dependent and can be prevented by deletion of a putative P-loop. Loss of SMXL6,7,8 suppresses several other strigolactone-related phenotypes in max2, including increased auxin transport and PIN1 accumulation, and increased lateral root density. Although only SMAX1 regulates germination and hypocotyl elongation, SMAX1 and SMXL6,7,8 have complementary roles in the control of leaf morphology. Our data indicate that SMAX1 and SMXL6,7,8 repress karrikin and strigolactone signaling, respectively, and suggest that all MAX2-dependent growth effects are mediated by degradation of SMAX1/SMXL proteins. We propose that functional diversification within the SMXL family enabled responses to different butenolide signals through a shared regulatory mechanism.