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.     

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.     

Roles for Auxin, Cytokinin, and Strigolactone in Regulating Shoot Branching

Many processes have been described in the control of shoot branching. Apical dominance is defined as the control exerted by the shoot tip on the outgrowth of axillary buds, whereas correlative inhibition includes the suppression of growth by other growing buds or shoots. The level, signaling, and/or flow of the plant hormone auxin in stems and buds is thought to be involved in these processes. In addition, RAMOSUS (RMS) branching genes in pea (Pisum sativum) control the synthesis and perception of a long-distance inhibitory branching signal produced in the stem and roots, a strigolactone or product. Auxin treatment affects the expression of RMS genes, but it is unclear whether the RMS network can regulate branching independently of auxin. Here, we explore whether apical dominance and correlative inhibition show independent or additive effects in rms mutant plants. Bud outgrowth and branch lengths are enhanced in decapitated and stem-girdled rms mutants compared with intact control plants. This may relate to an RMS-independent induction of axillary bud outgrowth by these treatments. Correlative inhibition was also apparent in rms mutant plants, again indicating an RMS-independent component. Treatments giving reductions in RMS1 and RMS5 gene expression, auxin transport, and auxin level in the main stem were not always sufficient to promote bud outgrowth. We suggest that this may relate to a failure to induce the expression of cytokinin biosynthesis genes, which always correlated with bud outgrowth in our treatments. We present a new model that accounts for apical dominance, correlative inhibition, RMS gene action, and auxin and cytokinin and their interactions in controlling the progression of buds through different control points from dormancy to sustained growth.     

Branching in rice

Rice branching, including the formation of tillers and panicle branches, has been well investigated over the past several years because of its agronomic importance. A major breakthrough in elucidating rice tillering in the recent years was the discovery of strigolactones, a specific group of terpenoid lactones that can inhibit axillary bud outgrowth. Since that discovery, new tillering mutants, that is, dwarf 27 (d27) or dwarf14 (d14, also reported as d88 or htd2), have been identified with reduced strigolactone levels or strigolactone response. DWARF27 (D27) and DWARF14 (D14) probably act on strigolactone biosynthesis and signal transduction, respectively. Additionally, several genes controlling panicle branches have been identified recently. DEP1 and IPA1/WFP are essential dominant/semidominant regulators that determine rice panicle branches and thus affect the grain yields. More importantly, dep1 and ipa1 alleles have been shown to be applicable for the improvement of rice grain yields in molecular breeding.     

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.     

Strigolactones,karrikins and beyond

The plant hormones strigolactones are synthesized from carotenoids and signal via the α/β hydrolase DWARF 14 (D14) and the F‐box protein MORE AXILLARY GROWTH 2 (MAX2). Karrikins, molecules produced upon fire, share MAX2 for signalling, but depend on the D14 paralog KARRIKIN INSENSITIVE 2 (KAI2) for perception with strong evidence that the MAX2–KAI2 protein complex might also recognize so far unknown plant‐made karrikin‐like molecules. Thus, the phenotypes of the max2 mutants are the complex consequence of a loss of both D14‐dependent and KAI2‐dependent signalling, hence, the reason why some biological roles, attributed to strigolactones based on max2 phenotypes, could never be observed in d14 or in the strigolactone‐deficient max3 and max4 mutants. Moreover, the broadly used synthetic strigolactone analog rac‐GR24 has been shown to mimic strigolactone as well as karrikin(‐like) signals, providing an extra level of complexity in the distinction of the unique and common roles of both molecules in plant biology. Here, a critical overview is provided of the diverse biological processes regulated by strigolactones and/or karrikins. These two growth regulators are considered beyond their boundaries, and the importance of the yet unknown karrikin‐like molecules is discussed as well.     

Strigolactone Acts Downstream of Auxin to Regulate Bud Outgrowth in Pea and Arabidopsis

During the last century, two key hypotheses have been proposed to explain apical dominance in plants: auxin promotes the production of a second messenger that moves up into buds to repress their outgrowth, and auxin saturation in the stem inhibits auxin transport from buds, thereby inhibiting bud outgrowth. The recent discovery of strigolactone as the novel shoot-branching inhibitor allowed us to test its mode of action in relation to these hypotheses. We found that exogenously applied strigolactone inhibited bud outgrowth in pea (Pisum sativum) even when auxin was depleted after decapitation. We also found that strigolactone application reduced branching in Arabidopsis (Arabidopsis thaliana) auxin response mutants, suggesting that auxin may act through strigolactones to facilitate apical dominance. Moreover, strigolactone application to tiny buds of mutant or decapitated pea plants rapidly stopped outgrowth, in contrast to applying N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, which significantly slowed growth only after several days. Whereas strigolactone or NPA applied to growing buds reduced bud length, only NPA blocked auxin transport in the bud. Wild-type and strigolactone biosynthesis mutant pea and Arabidopsis shoots were capable of instantly transporting additional amounts of auxin in excess of endogenous levels, contrary to predictions of auxin transport models. These data suggest that strigolactone does not act primarily by affecting auxin transport from buds. Rather, the primary repressor of bud outgrowth appears to be the auxin-dependent production of strigolactones.     

Characterization of a new allelic mutant of DWARF3 in rice and analysing its function and stability in the presence of strigolactone

Strigolactones (SLs) are important intrinsic growth regulators that control plant architecture by coordinating shoot and root development. Recent studies demonstrate that SL signals act via targeting the degradation protein DWARF53 (D53) family of chaperonin-like proteins. This process requires DWARF14 (D14) as strigolactones signal receptor and DWARF3 (D3) forming Skp-Cullin-F-box (SCF) complex as ubiquitin E3 ligase. Although the interactions of these signal components can be expected, where and how the SLs signalling occur within cells in a tissue-specific manner is still uncertain. In this study, we characterize a rice high-tillering dwarf mutant, ext.-M1B, displaying resistance to synthetic strigolactone mixture rac-GR24. Through genetic analysis, we find that ext.-M1B is a new allelic mutant of D3 with a nucleotide mutation resulting in a truncated protein of wide-type D3. We demonstrate that the mutation affects neither gene expression level nor the protein sub-cellular localization, whereas it disrupts the perception of SLs signal in ext.-M1B mutant. Moreover, we find that overexpression of D3 in wild type background causes no significant phenotype, but suppression of D3 by RNA interfering results in a clear phenocopy of SL mutants. By expressing fluorescent D3 fusion protein in rice, we first show that D3 is stable consistently in the nucleus with or without strigolactone treatment. Taken together, our data indicates that D3 encoding an F-box protein in nucleus, as a stable signal component response to strigolactone regulating rice shoot architecture.     

Effects of strigolactone-biosynthesis inhibitor TIS108 on Arabidopsis

TIS108 is a triazole-type strigolactone (SL)-biosynthesis inhibitor that reduces the level of 2′-epi-5-deoxystrigol (epi-5DS) in rice. Here we report the effects of TIS108 on Arabidopsis. Treatment of TIS108 increased the number of branches and repressed root hair elongation as was observed in SL-deficient mutants, and co-application of GR24, a synthetic SL analog, recovered the TIS108-induced phenotype to that of wild-type. In addition, MAX3 and MAX4 genes in the SL-biosynthesis pathway were upregulated in TIS108-treated Arabidopsis, probably due to feedback regulation caused by SL deficiency. These results indicate that TIS108 is an effective tool for regulating SL production in Arabidopsis.     

The Interactions among DWARF10, Auxin and Cytokinin Underlie Lateral Bud Outgrowth in Rice

Previous studies have shown that DWARF10 (D10) is a rice ortholog of MAX4/RMS1/DAD1, encoding a carotenoid cleavage dioxygenase and functioning in strigolactones/strigolactone-derivatives (SL)biosynthesis. Here we use D10- RNA interference (RNAi) transgenic plants similar to d10 mutant in phenotypes to investigate the interactions among D10, auxin and cytokinin in regulating rice shoot branching. Auxin levels in node 1 of both decapitated D10-RNAi and wild type plants decreased significantly, showing that decapitation does reduce endogenous auxin concentration, but decapitation has no clear effects on auxin levels in node 2 of the same plants. This implies that node 1 may be the location where a possible interaction between auxin and D10 gene would be detected. D10 expression in node 1 is inhibited by decapitation, and this inhibition can be restored by exogenous auxin application,indicating that D10 may play an important role in auxin regulation of SL. The decreased expression of most OsPINs in shoot nodes of D10- RNAi plants may cause a reduced auxin transport capacity.Furthermore, effects of auxin treatment of decapitated plants on the expression of cytokinin biosynthetic genes suggest that D10 promotes cytokinin biosynthesis by reducing auxin levels. Besides, in D10- RNAi plants, decreased storage cytokinin levels in the shoot node may partly account for the increased active cytokinin contents, resulting in more tillering phenotypes.     

Identification and Functional Analysis of Two Cotton Orthologs of <Emphasis Type="Italic">MAX2</Emphasis> Which Control Shoot Lateral Branching

Cotton (Gossypium spp.), as the most important fiber and oilseed crop in the world, is extremely important for the industry. However, due to its indeterminate growth habit and complex branching system, massive labor costs are needed for shoot apex removal and branch pruning during cotton production. Therefore, it is very important to explore branch-controlling genes and genetically modify the branch architecture of cotton. Strigolactones (SLs) are a novel class of plant hormone that inhibit the outgrowth of lateral branches. To elucidate the role of SLs in branch development of cotton, we cloned and characterized GhMAX2a and GhMAX2b from tetraploid upland cotton (Gossypium hirsutum), the orthologs of Arabidopsis MAX2, rice D3, and petunia RMS4. GhMAX2a/2b was ubiquitously expressed in all tested tissues of cotton, with relatively higher expression levels in leaves and lateral buds. Subcellular localization assay showed that the GhMAX2-GFP fusion protein localized to the nucleus. Both GhMAX2a and GhMAX2b can fully rescue the dwarfed and highly branched phenotypes of the Arabidopsis max2-1 mutant, indicating that GhMAX2s have conserved functions with that of AtMAX2. The cotton GhMAX2b interacted with Arabidopsis Skp1-like 1 (ASK1) proteins in vitro which was further confirmed in the Arabidopsis protoplasts using the co-immunoprecipitation assay, indicating that GhMAX2b probably functions through forming an SCF E3 complex with Skp and other proteins in the Arabidopsis. These results suggest that the cotton GhMAX2s encode functional MAX2 that can inhibit the shoot lateral branching. Further functional analysis of GhMAX2s in determining cotton branch architecture and yield is underway.     

SlCCD7 controls strigolactone biosynthesis,shoot branching and mycorrhiza‐induced apocarotenoid formation in tomato

The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch‐inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C₁₃ cyclohexenone and C₁₄ mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source–sink interactions and production of arbuscular mycorrhiza‐induced apocarotenoids.