Structure-Function Analysis of SMAX1 Reveals Domains That Mediate Its Karrikin-Induced Proteolysis and Interaction with the Receptor KAI2.

Karrikins (KARs) are butenolides found in smoke that can influence germination and seedling development of many plants. The KAR signaling mechanism is hypothesized to be very similar to that of the plant hormone strigolactone (SL). Both pathways require the F-box protein MORE AXILLARY GROWTH2 (MAX2), and other core signaling components have shared ancestry. Putatively, KAR activates the receptor KARRIKIN INSENSITIVE2 (KAI2), triggering its association with the E3 ubiquitin ligase complex SCFMAX2 and downstream targets SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE2 (SMXL2). Polyubiquitination and proteolysis of SMAX1 and SMXL2 then enable growth responses to KAR. However, many of the assumptions of this model have not been demonstrated. Therefore, we investigated the posttranslational regulation of SMAX1 from the model plant Arabidopsis (Arabidopsis thaliana). We find evidence that SMAX1 is degraded by KAI2-SCFMAX2 but is also subject to MAX2-independent turnover. We identify SMAX1 domains that are responsible for its nuclear localization, KAR-induced degradation, association with KAI2, and ability to interact with other SMXL proteins. KAI2 undergoes MAX2-independent degradation after KAR treatment, which we propose results from its association with SMAX1 and SMXL2. Finally, we discover an SMXL domain that mediates receptor-target interaction preferences in KAR and SL signaling, laying the foundation for understanding how these highly similar pathways evolved to fulfill different roles.

Functional redundancy in the control of seedling growth by the karrikin signaling pathway.

MAIN CONCLUSION: SMAX1 and SMXL2 control seedling growth, demonstrating functional redundancy within a gene family that mediates karrikin and strigolactone responses. Strigolactones (SLs) are plant hormones with butenolide moieties that control diverse aspects of plant growth, including shoot branching. Karrikins (KARs) are butenolide molecules found in smoke that enhance seed germination and seedling photomorphogenesis. In Arabidopsis thaliana, SLs and KARs signal through the α/ß hydrolases D14 and KAI2, respectively. The F-box protein MAX2 is essential for both signaling pathways. SUPPRESSOR OF MAX2 1 (SMAX1) plays a prominent role in KAR-regulated growth downstream of MAX2, and SMAX1-LIKE genes SMXL6, SMXL7, and SMXL8 mediate SL responses. We previously found that smax1 loss-of-function mutants display constitutive KAR response phenotypes, including reduced seed dormancy and hypersensitive growth responses to light in seedlings. However, smax1 seedlings remain slightly responsive to KARs, suggesting that there is functional redundancy in karrikin signaling. SMXL2 is a strong candidate for this redundancy because it is the closest paralog of SMAX1, and because its expression is regulated by KAR signaling. Here, we present evidence that SMXL2 controls hypocotyl growth and expression of the KAR/SL transcriptional markers KUF1, IAA1, and DLK2 redundantly with SMAX1. Hypocotyl growth in the smax1 smxl2 double mutant is insensitive to KAR and SL, and etiolated smax1 smxl2 seedlings have reduced hypocotyl elongation. However, smxl2 has little or no effect on seed germination, leaf shape, or petiole orientation, which appear to be predominantly controlled by SMAX1. Neither SMAX1 nor SMXL2 affect axillary branching or inflorescence height, traits that are under SL control. These data support the model that karrikin and strigolactone responses are mediated by distinct subclades of the SMXL family, and further the case for parallel butenolide signaling pathways that evolved through ancient KAI2 and SMXL duplications.

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.

SUPPRESSOR OF MORE AXILLARY GROWTH2 1 controls seed germination and seedling development in Arabidopsis.

Abiotic chemical signals discovered in smoke that are known as karrikins (KARs) and the endogenous hormone strigolactone (SL) control plant growth through a shared MORE AXILLARY GROWTH2 (MAX2)-dependent pathway. A SL biosynthetic pathway and candidate KAR/SL receptors have been characterized, but signaling downstream of MAX2 is poorly defined. A screen for genetic suppressors of the enhanced seed dormancy phenotype of max2 in Arabidopsis (Arabidopsis thaliana) led to identification of a suppressor of max2 1 (smax1) mutant. smax1 restores the seed germination and seedling photomorphogenesis phenotypes of max2 but does not affect the lateral root formation, axillary shoot growth, or senescence phenotypes of max2. Expression of three transcriptional markers of KAR/SL signaling, D14-LIKE2, KAR-UP F-BOX1, and INDOLE-3-ACETIC ACID INDUCIBLE1, is rescued in smax1 max2 seedlings. SMAX1 is a member of an eight-gene family in Arabidopsis that has weak similarity to HEAT SHOCK PROTEIN 101, which encodes a caseinolytic peptidase B chaperonin required for thermotolerance. SMAX1 and the SMAX1-like (SMXL) homologs are differentially expressed in Arabidopsis tissues. SMAX1 transcripts are most abundant in dry seed, consistent with its function in seed germination control. Several SMXL genes are up-regulated in seedlings treated with the synthetic SL GR24. SMAX1 and SMXL2 transcripts are reduced in max2 seedlings, which could indicate negative feedback regulation by KAR/SL signaling. smax1 seed and seedling growth mimics the wild type treated with KAR/SL, but smax1 seedlings are still responsive to 2H-furo[2,3-c]pyran-2-one (KAR2) or GR24. We conclude that SMAX1 is an important component of KAR/SL signaling during seed germination and seedling growth but is not necessary for all MAX2-dependent responses. We hypothesize that one or more SMXL proteins may also act downstream of MAX2 to control the diverse developmental responses to KARs and SLs.

An assessment of transgenomics as a tool for identifying genes involved in the evolutionary differentiation of closely related plant species.

• Transgenomics is the process of introducing genomic clones from a donor species into a recipient species and then screening the resultant transgenic lines for phenotypes of interest. This method might allow us to find genes involved in the evolution of phenotypic differences between species as well as genes that have the potential to contribute to reproductive isolation: potential speciation genes. • More than 1100 20-kbp genomic clones from Leavenworthia alabamica were moved into Arabidopsis thaliana by transformation. After screening a single primary transformant for each line, clones associated with mutant phenotypes were tested for repeatability and co-segregation. • We found 84 clones with possible phenotypic effects, of which eight were repeatedly associated with the same phenotype. One clone, 11_11B, co-segregated with a short fruit phenotype. Further study showed that 11_11B affects seed development, with as much as one-third of the seeds aborted in some fruit. • Transgenomics is a viable strategy for discovering genes of evolutionary interest. We identify methods to reduce false positives and false negatives in the future. 11_11B can be viewed as a potential speciation gene, illustrating the value of transgenomics for studying the molecular basis of reproductive isolation.

Studying starch content and sedimentation of amyloplast statoliths in Arabidopsis roots.

Amyloplasts, organelles responsible for the synthesis and storage of starch, are of critical importance to gravitropism in higher plants. We discuss two methods that are useful for describing the histology and behavior of amyloplasts. First, because mutants with little or no plastidic starch accumulation are defective in their gravitropic response, we review a method to observe starch accumulation quickly in plant tissue. Second, we discuss a method for measuring amyloplast sedimentation in the dynamic environment of Arabidopsis root columella cells, which is thought to provide a directional cue to a reoriented plant.

Joining forces: the interface of gravitropism and plastid protein import.

In flowering plants, gravity perception appears to involve the sedimentation of starch-filled plastids, called amyloplasts, within specialized cells (the statocytes) of shoots (endodermal cells) and roots (columella cells). Unfortunately, how the physical information derived from amyloplast sedimentation is converted into a biochemical signal that promotes organ gravitropic curvature remains largely unknown. Recent results suggest an involvement of the Translocon of the Outer Envelope of (Chloro)plastids (TOC) in early phases of gravity signal transduction within the statocytes. This review summarizes our current knowledge of the molecular mechanisms that govern gravity signal transduction in flowering plants and summarizes models that attempt to explain the contribution of TOC proteins in this important behavioral plant growth response to its mechanical environment.

A role for the TOC complex in Arabidopsis root gravitropism.

Arabidopsis (Arabidopsis thaliana) roots perceive gravity and reorient their growth accordingly. Starch-dense amyloplasts within the columella cells of the root cap are important for gravitropism, and starchless mutants such as pgm1 display an attenuated response to gravistimulation. The altered response to gravity1 (arg1) mutant is known to be involved with the early phases of gravity signal transduction. arg1 responds slowly to gravistimulation and is in a genetically distinct pathway from pgm1, as pgm1 mutants enhance the gravitropic defect of arg1. arg1 seeds were mutagenized with ethylmethane sulfonate to identify new mutants that enhance the gravitropic defect of arg1. Two modifier of arg1 mutants (mar1 and mar2) grow in random directions only when arg1 is present, do not affect phototropism, and respond like the wild type to application of phytohormones. Both have mutations affecting different components of the Translocon of Outer Membrane of Chloroplasts (TOC) complex. mar1 possesses a mutation in the TOC75-III gene; mar2 possesses a mutation in the TOC132 gene. Overexpression of TOC132 rescues the random growth phenotype of mar2 arg1 roots. Root cap amyloplasts in mar2 arg1 appear ultrastructurally normal. They saltate like the wild type and sediment at wild-type rates upon gravistimulation. These data point to a role for the plastidic TOC complex in gravity signal transduction within the statocytes.

Epsilon-tubulin is an essential component of the centriole.

Centrioles and basal bodies are cylinders composed of nine triplet microtubule blades that play essential roles in the centrosome and in flagellar assembly. Chlamydomonas cells with the bld2-1 mutation fail to assemble doublet and triplet microtubules and have defects in cleavage furrow placement and meiosis. Using positional cloning, we have walked 720 kb and identified a 13.2-kb fragment that contains epsilon-tubulin and rescues the Bld2 defects. The bld2-1 allele has a premature stop codon and intragenic revertants replace the stop codon with glutamine, glutamate, or lysine. Polyclonal antibodies to epsilon-tubulin show peripheral labeling of full-length basal bodies and centrioles. Thus, epsilon-tubulin is encoded by the BLD2 allele and epsilon-tubulin plays a role in basal body/centriole morphogenesis.