Michael J. Prigge

Class III Homeodomain-Leucine Zipper Gene Family Members Have Overlapping, Antagonistic, and Distinct Roles in Arabidopsis Development

The Arabidopsis thaliana genome contains five class III homeodomain-leucine zipper genes. We have isolated loss-of-function alleles for each family member for use in genetic analysis. This gene family regulates apical embryo patterning, embryonic shoot meristem formation, organ polarity, vascular development, and meristem function. Genetic analyses revealed a complex pattern of overlapping functions, some of which are not readily inferred by phylogenetic relationships or by gene expression patterns. The PHABULOSA and PHAVOLUTA genes perform overlapping functions with REVOLUTA, whereas the PHABULOSA, PHAVOLUTA, and CORONA/ATHB15 genes perform overlapping functions distinct from REVOLUTA. Furthermore, ATHB8 and CORONA encode functions that are both antagonistic to those of REVOLUTA within certain tissues and overlapping with REVOLUTA in other tissues. Differences in expression patterns explain some of these genetic interactions, whereas other interactions are likely attributable to differences in protein function as indicated by cross-complementation studies.

Complex regulation of the TIR1/AFB family of auxin receptors.

Auxin regulates most aspects of plant growth and development. The hormone is perceived by the TIR1/AFB family of F-box proteins acting in concert with the Aux/IAA transcriptional repressors. Arabidopsis plants that lack members of the TIR1/AFB family are auxin resistant and display a variety of growth defects. However, little is known about the functional differences between individual members of the family. Phylogenetic studies reveal that the TIR1/AFB proteins are conserved across land plant lineages and fall into four clades. Three of these subgroups emerged before separation of angiosperms and gymnosperms whereas the last emerged before the monocot-eudicot split. This evolutionary history suggests that the members of each clade have distinct functions. To explore this possibility in Arabidopsis, we have analyzed a range of mutant genotypes, generated promoter swap transgenic lines, and performed in vitro binding assays between individual TIR1/AFB and Aux/IAA proteins. Our results indicate that the TIR1/AFB proteins have distinct biochemical activities and that TIR1 and AFB2 are the dominant auxin receptors in the seedling root. Further, we demonstrate that TIR1, AFB2, and AFB3, but not AFB1 exhibit significant posttranscriptional regulation. The microRNA miR393 is expressed in a pattern complementary to that of the auxin receptors and appears to regulate TIR1/AFB expression. However our data suggest that this regulation is complex. Our results suggest that differences between members of the auxin receptor family may contribute to the complexity of auxin response.

The Arabidopsis SERRATE Gene Encodes a Zinc-Finger Protein Required for Normal Shoot Development

Organogenesis in plants depends upon the proper regulation of many genes, but how such necessary changes in gene expression are coordinated is largely unknown. The serrate (se) mutant of Arabidopsis displays defects in the initiation and elaboration of cotyledons and post-embryonic lateral organs. Cloning the SE gene revealed that it encodes a protein with a single, C2H2-type, zinc finger related to genes in other eukaryotes. Consistent with a role in organogenesis, the SE gene is transcribed in shoot meristems and in emerging organ primordia throughout development. Expression of the SE cDNA under the control of a heterologous promoter caused both accelerated and arrested plant growth, and these phenotypes were due to overexpression and co-suppression of the SE gene, respectively. Our analysis of the se mutant and the SE gene suggests a role for the SE gene product in regulating changes in gene expression via chromatin modification. Consistent with this proposed function, a synergistic double mutant phenotype was seen for plants mutant at both the SE locus and the locus encoding the largest subunit of chromatin assembly factor I.

The TRANSPORT INHIBITOR RESPONSE2 gene is required for auxin synthesis and diverse aspects of plant development.

The plant hormone auxin plays an essential role in plant development. However, only a few auxin biosynthetic genes have been isolated and characterized. Here, we show that the TRANSPORT INHIBITOR RESPONSE2 (TIR2) gene is required for many growth processes. Our studies indicate that the tir2 mutant is hypersensitive to 5-methyl-tryptophan, an inhibitor of tryptophan synthesis. Further, treatment with the proposed auxin biosynthetic intermediate indole-3-pyruvic acid (IPA) and indole-3-acetic acid rescues the tir2 short hypocotyl phenotype, suggesting that tir2 may be affected in the IPA auxin biosynthetic pathway. Molecular characterization revealed that TIR2 is identical to the TAA1 gene encoding a tryptophan aminotransferase. We show that TIR2 is regulated by temperature and is required for temperature-dependent hypocotyl elongation. Further, we find that expression of TIR2 is induced on the lower side of a gravitropically responding root. We propose that TIR2 contributes to a positive regulatory loop required for root gravitropism.

CORONA, a Member of the Class III Homeodomain Leucine Zipper Gene Family in Arabidopsis, Regulates Stem Cell Specification and Organogenesis

Organogenesis at the shoot meristem requires a delicate balance between stem cell specification and differentiation. In Arabidopsis thaliana, WUSCHEL (WUS) is a key factor promoting stem cell identity, whereas the CLAVATA (CLV1, CLV2, and CLV3) loci appear to promote differentiation by repressing WUS expression. In a screen for mutations modifying clv1 mutants, we have identified a novel regulator of meristem development we term CORONA (CNA). Whereas cna single mutant plants exhibit subtle defects in meristem development, clv cna double mutants develop massively enlarged apices that display early loss of organogenesis, misexpression of WUS and CLV3, and eventual differentiation of the entire apex. The CNA gene was isolated by positional cloning and found to encode a class III homeodomain Leu zipper protein. A missense mutation resulting in the dominant-negative cna-1 allele was identified in a conserved domain of unknown function, and a likely null allele was shown to display a similar but weaker phenotype. CNA is expressed in developing vascular tissue, diffusely through shoot and flower meristems, and within developing stamens and carpels. Our analysis of WUS expression in wild-type, clv, and clv cna plants revealed that, contrary to current models, WUS is neither necessary nor sufficient for stem cell specification and that neither WUS nor CLV3 is a marker for stem cell identity. We propose that CNA functions in parallel to the CLV loci to promote organ formation.

Two Cap-Binding Proteins CBP20 and CBP80 are Involved in Processing Primary MicroRNAs

MicroRNAs (miRNAs) are 21 nt RNAs that regulate many biological processes in plants by mediating translational inhibition or cleavage of target transcripts. Arabidopsis mutants defective in miRNA biogenesis have overlapping and highly pleiotropic phenotypes including serrated leaves and ABA hypersensitivity. Recent evidence indicates that miRNA genes are transcribed by RNA polymerase II (Pol II). Since Pol II transcripts are capped, we hypothesized that CBP (cap-binding protein) 20 and 80 may bind to capped primary miRNA (pri-miRNA) transcripts and play a role in their processing. Here, we show that cbp20 and cbp80 mutants have reduced miRNA levels and increased pri-miRNA levels. Co-immunoprecipitation experiments revealed that pri-miRNAs 159, 166, 168 and 172 could be associated with CBP20 and CBP80. We found that CBP20 and CBP80 are stabilized by ABA by a post-translational mechanism, and these proteins are needed for ABA induction of miR159 during seed germination. The lack of miR159 accumulation in ABA-treated seeds of cbp20/80 mutants leads to increased MYB33 and MYB101 transcript levels, and presumably higher levels of these positive regulators result in ABA hypersensitivity. Genetic and molecular analyses show that CBP20 and 80 have overlapping function in the same developmental pathway as SE and HYL1. Our results identify new components in miRNA biogenesis.

Evolution of the class III HD-Zip gene family in land plants.

SUMMARY Arabidopsis class III homeodomain‐leucine zipper (HD‐Zip III) proteins play overlapping, distinct, and antagonistic roles in key aspects of development that have evolved during land plant evolution. To better understand this gene familys role in plant evolution and development as well as to address broader questions of how duplicated genes functionally diversify, we investigated the evolutionary history of this gene family. Phylogenetic analyses including homologs from diverse land plants indicate that a gene duplication event before the angiosperm‐‐gymnosperm split gave rise to two gene lineages that diversified during angiosperm plant radiation. Heterologous expression of an HD‐Zip III gene from the nonvascular plant moss within the Arabidopsis HD‐zip III revoluta mutant modified but did not complement the phenotype. Comparison of the expression domains of flowering and nonflowering plant homologs indicate an ancestral role in vascular development and organ initiation but not in specifying organ polarity, a prominent role for angiosperm homologs.

Physcomitrella patens Auxin-Resistant Mutants Affect Conserved Elements of an Auxin-Signaling Pathway

Auxin regulates most aspects of flowering-plant growth and development, including key developmental innovations that evolved within the vascular plant lineage after diverging from a bryophyte-like ancestor nearly 500 million years ago. Recent studies in Arabidopsis indicate that auxin acts by directly binding the TIR1 subunit of the SCF(TIR1) ubiquitin ligase; this binding results in degradation of the Aux/IAA transcriptional repressors and de-repression of auxin-responsive genes. Little is known, however, about the mechanism of auxin action in other plants. To characterize auxin signaling in a nonflowering plant, we utilized the genetically tractable moss Physcomitrella patens. We used a candidate-gene approach to show that previously identified auxin-resistant mutants of P. patens harbor mutations in Aux/IAA genes. Furthermore, we show that the moss Aux/IAA proteins interact with Arabidopsis TIR1 moss homologs called PpAFB and that a reduction in PpAFB levels results in a phenotype similar to that of the auxin-resistant mutants. Our results indicate that the molecular mechanism of auxin perception is conserved in land plants despite vast differences in the role auxin plays in different plant lineages.

Evolutionary crossroads in developmental biology: Physcomitrella patens

The moss Physcomitrella patens has recently emerged as a powerful genetically tractable model plant system. As a member of the bryophytes, P. patens provides a unique opportunity to study the evolution of a myriad of plant traits, such as polarized cell growth, gametophyte-to-sporophyte transitions, and sperm-to-pollen transition. The availability of a complete genome sequence, together with the ability to perform gene targeting efficiently in P. patens has spurred a flurry of elegant reverse genetic studies in this plant model that address a variety of key questions in plant developmental biology.

Untethering the TIR1 auxin receptor from the SCF complex increases its stability and inhibits auxin response.

Plant genomes encode large numbers of F-box proteins (FBPs), the substrate recognition subunit of SKP1–CULLIN–F-box (SCF) ubiquitin ligases. There are ∼700 FBPs in Arabidopsis, most of which are uncharacterized. TIR1 is among the best-studied plant FBPs and functions as a receptor for the plant hormone auxin. Here we use a yeast two-hybrid system to identify novel TIR1 mutants with altered properties. The analysis of these mutants reveals that TIR1 associates with the CULLIN1 (CUL1) subunit of the SCF through the N-terminal H1 helix of the F-box domain. Mutations that untether TIR1 from CUL1 stabilize the FBP and cause auxin resistance and associated growth defects, probably by protecting TIR1 substrates from degradation. Based on these results we propose that TIR1 is subject to autocatalytic degradation when assembled into an SCF. Further, our results suggest a general method for determining the physiological function of uncharacterized FBPs. Finally, we show that a key amino acid variation in the F-box domain of auxin signalling F-box (AFB1), a closely related FBP, reduces its ability to form an SCF, resulting in an increase in AFB1 levels.