Bonnie Bartel

Prediction of Plant MicroRNA Targets

We predict regulatory targets for 14 Arabidopsis microRNAs (miRNAs) by identifying mRNAs with near complementarity. Complementary sites within predicted targets are conserved in rice. Of the 49 predicted targets, 34 are members of transcription factor gene families involved in developmental patterning or cell differentiation. The near-perfect complementarity between plant miRNAs and their targets suggests that many plant miRNAs act similarly to small interfering RNAs and direct mRNA cleavage. The targeting of developmental transcription factors suggests that many plant miRNAs function during cellular differentiation to clear key regulatory transcripts from daughter cell lineages.

Criteria for Annotation of Plant MicroRNAs

MicroRNAs (miRNAs) are ∼21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs play critical roles in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interactions with specific target mRNAs. miRNAs are not the only Dicer-derived small RNAs produced by plants: A substantial amount of the total small RNA abundance and an overwhelming amount of small RNA sequence diversity is contributed by distinct classes of 21- to 24-nucleotide short interfering RNAs. This fact, coupled with the rapidly increasing rate of plant small RNA discovery, demands an increased rigor in miRNA annotations. Herein, we update the specific criteria required for the annotation of plant miRNAs, including experimental and computational data, as well as refinements to standard nomenclature.

MicroRNA-Directed Regulation of Arabidopsis AUXIN RESPONSE FACTOR17 Is Essential for Proper Development and Modulates Expression of Early Auxin Response Genes

The phytohormone auxin plays critical roles during plant growth, many of which are mediated by the auxin response transcription factor (ARF) family. MicroRNAs (miRNAs), endogenous 21-nucleotide riboregulators, target several mRNAs implicated in auxin responses. miR160 targets ARF10, ARF16, and ARF17, three of the 23 Arabidopsis thaliana ARF genes. Here, we describe roles of miR160-directed ARF17 posttranscriptional regulation. Plants expressing a miRNA-resistant version of ARF17 have increased ARF17 mRNA levels and altered accumulation of auxin-inducible GH3-like mRNAs, YDK1/GH3.2, GH3.3, GH3.5, and DFL1/GH3.6, which encode auxin-conjugating proteins. These expression changes correlate with dramatic developmental defects, including embryo and emerging leaf symmetry anomalies, leaf shape defects, premature inflorescence development, altered phyllotaxy along the stem, reduced petal size, abnormal stamens, sterility, and root growth defects. These defects demonstrate the importance of miR160-directed ARF17 regulation and implicate ARF17 as a regulator of GH3-like early auxin response genes. Many of these defects resemble phenotypes previously observed in plants expressing viral suppressors of RNA silencing and plants with mutations in genes important for miRNA biogenesis or function, providing a molecular rationale for phenotypes previously associated with more general disruptions of miRNA function.

MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs.

BACKGROUND MicroRNAs (miRNAs) are approximately 21 nucleotide (nt) RNAs that regulate gene expression in plants and animals. Most known plant miRNAs target transcription factors that influence cell fate determination, and biological functions of miRNA-directed regulation have been reported for four of 15 known microRNA gene families: miR172, miR159, miR165, and miR168. Here, we identify a developmental role for miR164-directed regulation of NAC-domain genes, which encode a family of transcription factors that includes CUP-SHAPED COTYLEDON1 (CUC1) and CUC2. RESULTS Expression of a miR164-resistant version of CUC1 mRNA from the CUC1 promoter causes alterations in Arabidopsis embryonic, vegetative, and floral development, including cotyledon orientation defects, reduction of rosette leaf petioles, dramatically misshapen rosette leaves, one to four extra petals, and one or two missing sepals. Reciprocally, constitutive overexpression of miR164 recapitulates cuc1 cuc2 double mutant phenotypes, including cotyledon and floral organ fusions. miR164 overexpression also leads to phenotypes not previously observed in cuc1 cuc2 mutants, including leaf and stem fusions. These likely reflect the misregulation of other NAC-domain mRNAs, including NAC1, At5g07680, and At5g61430, for which miR164-directed cleavage products were detected. CONCLUSIONS These results demonstrate that miR164-directed regulation of CUC1 is necessary for normal embryonic, vegetative, and floral development. They also show that proper miR164 dosage or localization is required for separation of adjacent embryonic, vegetative, and floral organs, thus implicating miR164 as a common regulatory component of the molecular circuitry that controls the separation of different developing organs and thereby exposes a posttranscriptional layer of NAC-domain gene regulation during plant development.

FKF1, a clock-controlled gene that regulates the transition to flowering in Arabidopsis.

Plant reproduction requires precise control of flowering in response to environmental cues. We isolated a late-flowering Arabidopsis mutant, fkf1, that is rescued by vemalization or gibberellin treatment. We positionally cloned FKF1, which encodes a novel protein with a PAS domain similar to the flavin-binding region of certain photoreceptors, an F box characteristic of proteins that direct ubiquitin-mediated degradation, and six kelch repeats predicted to fold into a beta propeller. FKF1 mRNA levels oscillate with a circadian rhythm, and deletion of FKF1 alters the waveform of rhythmic expression of two clock-controlled genes, implicating FKF1 in modulating the Arabidopsis circadian clock.

MicroRNAs: At the Root of Plant Development?

Although most genes use RNA in the form of mRNA as a coding intermediate for protein production, there are many genes whose final products are RNA. These noncoding RNAs range from the familiar transfer and ribosomal RNAs to the more recently discovered regulatory RNAs. One type of regulatory RNA was

A Gain-of-Function Mutation in IAA28 Suppresses Lateral Root Development

The phytohormone auxin is important in many aspects of plant development. We have isolated an auxin-resistant Arabidopsis mutant, iaa28-1, that is severely defective in lateral root formation and that has diminished adult size and decreased apical dominance. The iaa28-1 mutant is resistant to inhibition of root elongation by auxin, cytokinin, and ethylene, but it responds normally to other phytohormones. We identified the gene defective in the iaa28-1 mutant by using a map-based positional approach and found it to encode a previously uncharacterized member of the Aux/IAA gene family. IAA28 is preferentially expressed in roots and inflorescence stems, and in contrast to other Aux/IAA genes, IAA28 transcription is not induced by exogenous auxin. Studies of the gain-of-function iaa28-1 mutant suggest that IAA28 normally represses transcription, perhaps of genes that promote lateral root initiation in response to auxin signals.

The recognition component of the N-end rule pathway.

The N‐end rule‐based degradation signal, which targets a protein for ubiquitin‐dependent proteolysis, comprises a destabilizing amino‐terminal residue and a specific internal lysine residue. We report the isolation and functional analysis of a gene (UBR1) for the N‐end recognizing protein of the yeast Saccharomyces cerevisiae. UBR1 encodes a approximately 225 kd protein with no significant sequence similarities to other known proteins. Null ubr1 mutants are viable but are unable to degrade the substrates of the N‐end rule pathway. These mutants are partially defective in sporulation and grow slightly more slowly than their wild‐type counterparts. The UBR1 protein specifically binds in vitro to proteins bearing amino‐terminal residues that are destabilizing according to the N‐end rule, but does not bind to otherwise identical proteins bearing stabilizing amino‐terminal residues.

Plant Peroxisomes: Biogenesis and Function

Peroxisomes are eukaryotic organelles that are highly dynamic both in morphology and metabolism. Plant peroxisomes are involved in numerous processes, including primary and secondary metabolism, development, and responses to abiotic and biotic stresses. Considerable progress has been made in the identification of factors involved in peroxisomal biogenesis, revealing mechanisms that are both shared with and diverged from non-plant systems. Furthermore, recent advances have begun to reveal an unexpectedly large plant peroxisomal proteome and have increased our understanding of metabolic pathways in peroxisomes. Coordination of the biosynthesis, import, biochemical activity, and degradation of peroxisomal proteins allows for highly dynamic responses of peroxisomal metabolism to meet the needs of a plant. Knowledge gained from plant peroxisomal research will be instrumental to fully understanding the organelle’s dynamic behavior and defining peroxisomal metabolic networks, thus allowing the development of molecular strategies for rational engineering of plant metabolism, biomass production, stress tolerance, and pathogen defense.

Ubiquitin as a degradation signal.

For many short‐lived eukaryotic proteins, conjugation to ubiquitin, yielding a multiubiquitin chain, is an obligatory pre‐degradation step. The conjugated ubiquitin moieties function as a ‘secondary’ signal for degradation, in that their posttranslational coupling to a substrate protein is mediated by amino acid sequences of the substrate that act as a primary degradation signal. We report that the fusion protein ubiquitin‐‐proline‐‐beta‐galactosidase (Ub‐P‐beta gal) is short‐lived in the yeast Saccharomyces cerevisiae because its N‐terminal ubiquitin moiety functions as an autonomous, primary degradation signal. This signal mediates the formation of a multiubiquitin chain linked to Lys48 of the N‐terminal ubiquitin in Ub‐P‐beta gal. The degradation of Ub‐P‐beta gal is shown to require Ubc4, one of at least seven ubiquitin‐conjugating enzymes in S.cerevisiae. Our findings provide the first direct evidence that a monoubiquitin moiety can function as an autonomous degradation signal. This generally applicable, cis‐acting signal can be used to manipulate the in vivo half‐lives of specific intracellular proteins.