Sequence and expression differences underlie functional specialization of Arabidopsis microRNAs miR159 and miR319

Many microRNAs (miRNAs) are encoded by small gene families. In a third of all conserved Arabidopsis miRNA families, members vary at two or more nucleotide positions. We have focused on the related miR159 and miR319 families, which share sequence identity at 17 of 21 nucleotides, yet affect different developmental processes through distinct targets. MiR159 regulates MYB mRNAs, while miR319 predominantly acts on TCP mRNAs. In the case of miR319, MYB targeting plays at most a minor role because miR319 expression levels and domain limit its ability to affect MYB mRNAs. In contrast, in the case of miR159, the miRNA sequence prevents effective TCP targeting. We complement these observations by identifying nucleotide positions relevant for miRNA activity with mutants recovered from a suppressor screen. Together, our findings reveal that functional specialization of miR159 and miR319 is achieved through both expression and sequence differences.     

Brassinosteroid negatively regulates jasmonate inhibition of root growth in Arabidopsis

Jasmonate (JA) inhibits root growth of Arabidopsis thaliana seedlings. The mutation in COI1, that plays a central role in JA signaling, displays insensitivity to JA inhibition of root growth. To dissect JA signaling pathway, we recently isolated one mutant named psc1, which partially suppresses coi1 insensitivity to JA inhibition of root growth. As we identified the PSC1 gene as an allele of DWF4 that encodes a key enzyme in brassinosteroid (BR) biosynthesis, we hypothesized and demonstrated that BR is involved in JA signaling and negatively regulates JA inhibition of root growth. In our Plant Physiology paper, we analyzed effects of psc1 or exogenous BR on the inhibition of root growth by JA. Here we show that treatment with brassinazole (Brz), a BR biosynthesis inhibitor, increased JA sensitivity in both coi1-2 and wild type, which further confirms that BR negatively regulates JA inhibition of root growth. Since effects of psc1, Brz and exogenous BR on JA inhibition of root growth were mild, we suggests that BR negatively finely regulates JA inhibition of root growth in Arabidopsis.Key words: jasmonate signaling, root growth, brassinosteroid, brassinazole, arabidopsisJasmonate (JA) regulates many plant developmental processes and stress responses.^1,2 COI1 plays a central role in JA signaling and is required for all JA responses in Arabidopsis.^3,4 coi1-1, a strong mutation in COI1, is male sterile and exhibits loss of all JA responses tested to date, such as JA inhibition of root growth, the expression of JA-induced genes, and susceptibility to insect attack and pathogen infection, and coi1-2, a weak mutant of COI1, shows similar JA responses to coi1-1 except for partially fertile that makes it able to produce a small quantity of seeds.⁵To investigate COI1-mediated JA responses and dissect JA signaling pathway, we conducted genetic screens for suppressors of coi1-2. Previously, we identified cos1 that completely suppresses coil-2 insensitive to JA.⁶ Recently, we isolated the psc1 mutant that partially suppresses coi1-2 insensitivity to JA, and found that PSC1 is an allele of DWF4.⁷Since the DWF4 gene encodes a key enzyme in brassinosteroid (BR) biosynthesis,⁸ we hypothesized that BR is involved in JA signaling. By physiological analysis, we showed that psc1 partially restored JA inhibition of root growth in coi1-2 background and displayed JA hypersensitivity in wild-type COI1 background, the effects of psc1 were eliminated by exogenous BR, and that exogenous BR could attenuated JA inhibition of root growth in wild type. These findings demonstrated that BR is involved in JA signaling and indicated that BR negatively regulates JA inhibition of root growth.BR is a family of polyhydroxylated steroid hormones involved in many aspects of plant growth and development. The BR-deficient mutants exhibited severely retarded growth that was able to be rescued by exogenous BR.⁹ Brassinazole (Brz) is a BR biosynthesis inhibitor. The Arabidopsis seedlings treated with Brz displayed a BR deficient-mutant-like phenotype, which could be elimilated by exogenous BR.¹⁰To determine wether treatment with Brz affects JA inhibition of root growth, the seedlings of wild type and coi1-2 were grown in MS medium supplemented with MeJA and/or Brz. As shown in Figure 1, the relative root length was obviously reduced in both coi1-2 and wild type when treated with Brz relative to without Brz, indicating that the repression of BR biosynthesis by Brz could increase JA sensitivity. These results further confirm BR negatively regulates JA inhibition of root growth.Open in a separate windowFigure 1Effect of Brz on JA inhibition of root growth. Brz increased JA inhibition of root growth in both coi1-2 and wild type (WT). Root length of 7-day-old seedlings grown in MS medium containing 0, 5 and 10 μM MeJA without (−) or with (+) 0.5 μM Brz was expressed as a percentage of root length in MS without (−) or with (+) 0.5 µM Brz. Error bars represent SE (n > 30).It has been demonstrated that JA connects with other plant hormones including auxin, ethylene, abscisic acid, salicylic acid and gibberellin to form complex regulatory networks modulating plant developmental and stress responses.^11–15 We found that BR negatively regulates JA inhibition of root growth, suggesting that a cross talk between JA and BR exists in planta, which extends our understandings on the JA signal transduction.COI1 is a JA receptor¹⁶ and DWF4 catalyzes the rate-limiting step in BR-biosynthesis pathway.⁸ We found that JA inhibits DWF4 expression, this inhibition was dependent on COI1,⁷ indicating that DWF4 is downregulated by JA and is located downstream of COI1 in the JA signaling pathway.Since the effects of psc1, Brz, and exogenous BR on JA inhibition of root growth were mild, and the DWF4 expression was partially repressed by JA (Ren et al. 2009, Fig. 1), we suggest that BR negatively finely regulates JA inhibition of root growth, and propose a model for these regulations. As shown in Figure 2A, JA signal passes COI1 repressing substrates, such as JAZs,^17,18 i.e., JA activates degradation of substrates via SCF^COI1-26S proteasome,^16–18 whereas substrates positively regulate root growth through other regulators. JA also partially inhibits DWF4 expression through COI1, reducing BR that is required for root growth.^7,9 Mutation in COI1 interrupts JA signaling for failing in degradation of substrates and repression of DWF4 as well, resulting in JA-insensitivity (Fig. 2B). However, mutation in DWF4 or treatment with Brz causes a reduction in BR, which affects root growth, leading to JA-hypersensitivity in wild-type COI1 background (Fig. 2C and E) and partial restoration of JA sensitivity in coi1-2 background (Fig. 2D and F). Whereas, an application of exogenous BR could eliminate the effect of BR reduction resulted from repression of DWF4 by JA on root growth, attenuating JA sensitivity in wild type (Fig. 2G). Because the inhibition of DWF4 expression by JA is dependent on COI1, the coi1 mutant treated with exogenous BR do not show alteration in JA sensitivity (Fig. 2H).Open in a separate windowFigure 2A model for that BR negatively finely regulates JA inhibition of root growth in Arabidopsis. (A–D) Treatment with JA in wild type (A), coi1-2 (B), psc1 (C) and psc1coi1 (D). (E and F) Treatments with JA and Brz in wild type (E) and coi1-2 (F). (G and H) Treatments with JA and exogenous BR in wild type (G) and coi1-2 (H). Arrows indicate positive regulation or enhancement, whereas blunted lines indicate repression or negative regulation. Crosses indicate interruption or impairment. The letter “S” indicates substrates of SCF^COI1. Thicker arrows and blunted lines represent the central JA signaling pathway regulating JA inhibition of root growth. Broken arrows represent JA signaling pathway in which other regulators are involved. The intensity of gray boxes represents the degree of JA inhibition on root growth.     

Potent induction of Arabidopsis thaliana flowering by elevated growth temperature

The transition to flowering is an important event in the plant life cycle and is modulated by several environmental factors including photoperiod, light quality, vernalization, and growth temperature, as well as biotic and abiotic stresses. In contrast to light and vernalization, little is known about the pathways that mediate the responses to other environmental variables. A mild increase in growth temperature, from 23 °C to 27 °C, is equally efficient in inducing flowering of Arabidopsis plants grown in 8-h short days as is transfer to 16-h long days. There is extensive natural variation in this response, and we identify strains with contrasting thermal reaction norms. Exploiting this natural variation, we show that FLOWERING LOCUS C potently suppresses thermal induction, and that the closely related floral repressor FLOWERING LOCUS M is a major-effect quantitative trait locus modulating thermosensitivity. Thermal induction does not require the photoperiod effector CONSTANS, acts upstream of the floral integrator FLOWERING LOCUS T, and depends on the hormone gibberellin. Analysis of mutants defective in salicylic acid biosynthesis suggests that thermal induction is independent of previously identified stress-signaling pathways. Microarray analyses confirm that the genomic responses to floral induction by photoperiod and temperature differ. Furthermore, we report that gene products that participate in RNA splicing are specifically affected by thermal induction. Above a critical threshold, even small changes in temperature can act as cues for the induction of flowering. This response has a genetic basis that is distinct from the known genetic pathways of floral transition, and appears to correlate with changes in RNA processing.     

Brassinosteroids promote root growth in Arabidopsis

Although brassinosteroids (BRs) are known to regulate shoot growth, their role in the regulation of root growth is less clear. We show that low concentrations of BRs such as 24-epicastasterone and 24-epibrassinolide promote root elongation in Arabidopsis wild-type plants up to 50% and in BR-deficient mutants such as dwf1-6 (cbb1) and cbb3 (which is allelic to cpd) up to 150%. The growth-stimulating effect of exogenous BRs is not reduced by the auxin transport inhibitor 2,3,5-triidobenzoic acid. BR-deficient mutants show normal gravitropism, and 2,3,5-triidobenzoic acid or higher concentrations of 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid inhibit root growth in the mutants to the same extent as in wild-type plants. Simultaneous administration of 24-epibrassinolide and 2,4-dichlorophenoxyacetic acid results in largely additive effects. Exogenous gibberellins do not promote root elongation in the BR-deficient mutants, and the sensitivity to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid is not altered. Thus, the root growth-stimulating effect of BRs appears to be largely independent of auxin and gibberellin action. Furthermore, we analyzed BR interactions with other phytohormones on the gene expression level. Only a limited set of auxin- and ethylene-related genes showed altered expression levels. Genes related to other phytohormones barely showed changes, providing further evidence for an autonomous stimulatory effect of BR on root growth.     

miR396-targeted AtGRF transcription factors are required for coordination of cell division and differentiation during leaf development in Arabidopsis

In plants, cell proliferation and polarized cell differentiation along the adaxial-abaxial axis in the primordium is critical for leaf morphogenesis, while the temporal-spatial relationships between these two processes remain largely unexplored. Here, it is reported that microRNA396 (miR396)-targeted Arabidopsis growth-regulating factors (AtGRFs) are required for leaf adaxial-abaxial polarity in Arabidopsis. Reduction of the expression of AtGRF genes by transgenic miR396 overexpression in leaf polarity mutants asymmetric leaves1 (as1) and as2 resulted in plants with enhanced leaf adaxial-abaxial defects, as a consequence of reduced cell proliferation. Moreover, transgenic miR396 overexpression markedly decreased the cell division activity and the expression of cell cycle-related genes, but resulted in an increased percentage of leaf cells with a higher ploidy level, indicating that miR396 negatively regulates cell proliferation by controlling entry into the mitotic cell cycle. miR396 is mainly expressed in the leaf cells arrested for cell division, coinciding with its roles in cell cycle regulation. These results together suggest that cell division activity mediated by miR396-targeted AtGRFs is important for polarized cell differentiation along the adaxial-abaxial axis during leaf morphogenesis in Arabidopsis.