Javier F. Palatnik

A loop-to-base processing mechanism underlies the biogenesis of plant microRNAs miR319 and miR159

The first step in microRNA (miRNA) biogenesis usually involves cleavage at the base of its fold‐back precursor. Here, we describe a non‐canonical processing mechanism for miRNAs miR319 and miR159 in Arabidopsis thaliana. We found that their biogenesis begins with the cleavage of the loop, instead of the usual cut at the base of the stem–loop structure. DICER‐LIKE 1 (DCL1) proceeds then with three additional cuts until the mature miRNA is released. We further show that the conserved upper stem of the miR319 precursor is essential to organize its biogenesis, whereas sequences below the miRNA/miRNA* region are dispensable. In addition, the bulges present in the fold‐back structure reduce the accumulation of small RNAs other than the miRNA. The biogenesis of miR319 is conserved in the moss Physcomitrella patens, showing that this processing mechanism is ancient. These results provide new insights into the plasticity of small‐RNA pathways.

Functional specialization of the plant miR396 regulatory network through distinct microRNA-target interactions.

MicroRNAs (miRNAs) are ∼21 nt small RNAs that regulate gene expression in animals and plants. They can be grouped into families comprising different genes encoding similar or identical mature miRNAs. Several miRNA families are deeply conserved in plant lineages and regulate key aspects of plant development, hormone signaling, and stress response. The ancient miRNA miR396 regulates conserved targets belonging to the GROWTH-REGULATING FACTOR (GRF) family of transcription factors, which are known to control cell proliferation in Arabidopsis leaves. In this work, we characterized the regulation of an additional target for miR396, the transcription factor bHLH74, that is necessary for Arabidopsis normal development. bHLH74 homologs with a miR396 target site could only be detected in the sister families Brassicaceae and Cleomaceae. Still, bHLH74 repression by miR396 is required for margin and vein pattern formation of Arabidopsis leaves. MiR396 contributes to the spatio-temporal regulation of GRF and bHLH74 expression during leaf development. Furthermore, a survey of miR396 sequences in different species showed variations in the 5′ portion of the miRNA, a region known to be important for miRNA activity. Analysis of different miR396 variants in Arabidopsis thaliana revealed that they have an enhanced activity toward GRF transcription factors. The interaction between the GRF target site and miR396 has a bulge between positions 7 and 8 of the miRNA. Our data indicate that such bulge modulates the strength of the miR396-mediated repression and that this modulation is essential to shape the precise spatio-temporal pattern of GRF2 expression. The results show that ancient miRNAs can regulate conserved targets with varied efficiency in different species, and we further propose that they could acquire new targets whose control might also be biologically relevant.

Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development

BackgroundDevelopment of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood.ResultsWe characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes.ConclusionsOur findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility.

Identification of MicroRNA Processing Determinants by Random Mutagenesis of Arabidopsis MIR172a Precursor

MicroRNAs (miRNAs) are widespread posttranscriptional regulators of gene expression. They are processed from longer primary transcripts that contain foldback structures (reviewed in). In animals, a complex formed by Drosha and DGCR8/Pasha recognizes the transition between the single-stranded RNA sequences and the stem loop to produce the first cleavage step in miRNA biogenesis. Whereas animal precursors are of uniform size and shape, their plant counterparts comprise a collection of variable stem loops, and little is known about the structural clues recognized during their processing. Here, we designed an unbiased approach based on the random mutagenesis of the MIR172a precursor to study miRNA processing in plants. Randomly mutated precursors were overexpressed in Arabidopsis, and their activity was determined in vivo. We gathered sequence data from these transgenes and used it to build a MIR172a precursor map highlighting relevant and neutral positions for its processing. A 15 nucleotide stem segment below the miRNA/miRNA(*) duplex was essential for MIR172a processing. In contrast, mutations in the terminal-loop region were mostly neutral, yet a loop was required for miR172 biogenesis. The results could be extended to other precursors, suggesting the existence of common features in at least part of the plant precursors.

Functional replacement of ferredoxin by a cyanobacterial flavodoxin in tobacco confers broad-range stress tolerance.

Chloroplast ferredoxin (Fd) plays a pivotal role in plant cell metabolism by delivering reducing equivalents to various essential oxidoreductive pathways. Fd levels decrease under adverse environmental conditions in many microorganisms, including cyanobacteria, which share a common ancestor with chloroplasts. Conversely, stress situations induce the synthesis of flavodoxin (Fld), an electron carrier flavoprotein not found in plants, which can efficiently replace Fd in most electron transfer processes. We report here that chloroplast Fd also declined in plants exposed to oxidants or stress conditions. A purified cyanobacterial Fld was able to mediate plant Fd-dependent reactions in vitro, including NADP+ and thioredoxin reduction. Tobacco (Nicotiana tabacum) plants expressing Fld in chloroplasts displayed increased tolerance to multiple sources of stress, including redox-cycling herbicides, extreme temperatures, high irradiation, water deficit, and UV radiation. Oxidant buildup and oxidative inactivation of thioredoxin-dependent plastidic enzymes were decreased in stressed plants expressing plastid-targeted Fld, suggesting that development of the tolerant phenotype relied on productive interaction of this flavoprotein with Fd-dependent oxidoreductive pathways of the host, most remarkably, thioredoxin reduction. The use of Fld provides new tools to investigate the requirements of photosynthesis in planta and to increase plant stress tolerance based on the introduction of a cyanobacterial product that is free from endogenous regulation in higher plants.

Changes in amino acid composition and nitrogen metabolizing enzymes in ripening fruits of Lycopersicon esculentum Mill

The free amino acid content of tomato (Lycopersicon esculentum Mill.) fruits from cultivars Platense, Vollendung and Cherry were determined during ripening. It was found that glutamate markedly increased in red fruits of the three cultivars under study. At this stage, the cv Cherry had the highest relative glutamate molar content (52%) of all the analyzed tomato fruit cultivars. Measurements of nitrogen-assimilating enzyme activities of these fruits showed a decrease in glutamine synthetase (GS, EC 6.3.1.2) during fruit ripening and a concomitant increase in NADH-glutamate dehydrogenase (GDH, EC 1.4.1.3) and aspartate aminotransferase (EC 2.6.1.1) activities. Western blot analysis of protein extracts revealed that while GS was principally present in green fruit extracts, GDH was almost exclusively observed in the extracts of red fruits. These results suggest a reciprocal pattern of induction between GS and GDH during tomato fruit ripening.

The Flavoenzyme Ferredoxin (Flavodoxin)-NADP(H) Reductase Modulates NADP(H) Homeostasis during the soxRS Response of Escherichia coli

Escherichia coli cells from strain fpr, deficient in the soxRS-induced ferredoxin (flavodoxin)-NADP(H) reductase (FPR), display abnormal sensitivity to the bactericidal effects of the superoxide-generating reagent methyl viologen (MV). Neither bacteriostatic effects nor inactivation of oxidant-sensitive hydrolyases could be detected in fpr cells exposed to MV. FPR inactivation did not affect the MV-driven soxRS response, whereas FPR overexpression led to enhanced stimulation of the regulon, with concomitant oxidation of the NADPH pool. Accumulation of a site-directed FPR mutant that uses NAD(H) instead of NADP(H) had no effect on soxRS induction and failed to protect fpr cells from MV toxicity, suggesting that FPR contributes to NADP(H) homeostasis in stressed bacteria.

Repression of Cell Proliferation by miR319-Regulated TCP4

Leaf development has been extensively studied on a genetic level. However, little is known about the interplay between the developmental regulators and the cell cycle machinery--a link that ultimately affects leaf form and size. miR319 is a conserved microRNA that regulates TCP transcription factors involved in multiple developmental pathways, including leaf development and senescence, organ curvature, and hormone biosynthesis and signaling. Here, we analyze the participation of TCP4 in the control of cell proliferation. A small increase in TCP4 activity has an immediate impact on leaf cell number, by significantly reducing cell proliferation. Plants with high TCP4 levels have a strong reduction in the expression of genes known to be active in G2-M phase of the cell cycle. Part of these effects is mediated by induction of miR396, which represses Growth-Regulating Factor (GRF) transcription factors. Detailed analysis revealed TCP4 to be a direct regulator of MIR396b. However, we found that TCP4 can control cell proliferation through additional pathways, and we identified a direct connection between TCP4 and ICK1/KRP1, a gene involved in the progression of the cell cycle. Our results show that TCP4 can activate different pathways that repress cell proliferation.

Oxidative Stress Causes Ferredoxin-NADP+ Reductase Solubilization from the Thylakoid Membranes in Methyl Viologen-Treated Plants

The flavoenzyme ferredoxin-NADP+ reductase (FNR) is a member of the cellular defense barrier against oxidative damage in Escherichia coli. We evaluated the responses of chloroplast FNR to methyl viologen, a superoxide radical propagator, in wheat (Triticum aestivum L.) plants and chloroplasts. Treatments with the herbicide showed little effect on the levels of FNR protein or transcripts, indicating that expression of this reductase is not up-regulated by oxidants in plants. Viologens and peroxides caused solubilization of active FNR from the thylakoids into the stroma, converting the enzyme from a membrane-bound NADPH producer to a soluble NADPH consumer. This response appeared specific for FNR, since other thylakoid proteins were unaffected by the treatments. The reductase-binding protein was released together with FNR, suggesting that it might be the target of oxidative modification. Stromal accumulation of a functional NADPH reductase in response to oxidative stress is formally analogous to the induction of FNR synthesis observed in E. coli under similar conditions. FNR solubilization may be playing a crucial role in maintaining the NADPH/NADP+ homeostasis of the stressed plastid. The unchecked accumulation of NADPH might otherwise increase the risks of oxidative damage through a rise in the Mehler reaction rates and/or the production of hydroxyl radicals.

miR396 affects mycorrhization and root meristem activity in the legume Medicago truncatula

The root system is crucial for acquisition of resources from the soil. In legumes, the efficiency of mineral and water uptake by the roots may be reinforced due to establishment of symbiotic relationships with mycorrhizal fungi and interactions with soil rhizobia. Here, we investigated the role of miR396 in regulating the architecture of the root system and in symbiotic interactions in the model legume Medicago truncatula. Analyses with promoter-GUS fusions suggested that the mtr-miR396a and miR396b genes are highly expressed in root tips, preferentially in the transition zone, and display distinct expression profiles during lateral root and nodule development. Transgenic roots of composite plants that over-express the miR396b precursor showed lower expression of six growth-regulating factor genes (MtGRF) and two bHLH79-like target genes, as well as reduced growth and mycorrhizal associations. miR396 inactivation by mimicry caused contrasting tendencies, with increased target expression, higher root biomass and more efficient colonization by arbuscular mycorrhizal fungi. In contrast to MtbHLH79, repression of three GRF targets by RNA interference severely impaired root growth. Early activation of mtr-miR396b, concomitant with post-transcriptional repression of MtGRF5 expression, was also observed in response to exogenous brassinosteroids. Growth limitation in miR396 over-expressing roots correlated with a reduction in cell-cycle gene expression and the number of dividing cells in the root apical meristem. These results link the miR396 network to the regulation of root growth and mycorrhizal associations in plants.