text:
Dual Effects of miR156-Targeted SPL Genes and CYP78A5/KLUH on Plastochron Length and Organ Size in Arabidopsis thaliana
Jia-Wei Wang, Rebecca Schwab, Benjamin Czech, Erica Mica, Detlef Weigel
Jia-Wei Wang
Department of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tübingen, Germany
Rebecca Schwab
Benjamin Czech
Erica Mica
Detlef Weigel
Published May 2008. DOI: https://doi.org/10.1105/tpc.108.058180
American Society of Plant Biologists
Leaves of flowering plants are produced from the shoot apical meristem at regular intervals, with the time that elapses between the formation of two successive leaf primordia defining the plastochron. We have identified two genetic axes affecting plastochron length in Arabidopsis thaliana. One involves microRNA156 (miR156), which targets a series of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. In situ hybridization studies and misexpression experiments demonstrate that miR156 is a quantitative, rather than spatial, modulator of SPL expression in leaf primordia and that SPL activity nonautonomously inhibits initiation of new leaves at the shoot apical meristem. The second axis is exemplified by a redundantly acting pair of cytochrome P450 genes, CYP78A5/KLUH and CYP78A7, which are likely orthologs of PLASTOCHRON1 of rice (Oryza sativa). Inactivation of CYP78A5, which is expressed at the periphery of the shoot apical meristem, accelerates the leaf initiation rate, whereas cyp78a5 cyp78a7 double mutants often die as embryos with supernumerary cotyledon primordia. The effects of both miR156-targeted SPL genes and CYP78A5 on organ size are correlated with changes in plastochron length, suggesting a potential compensatory mechanism that links the rate at which leaves are produced to final leaf size.
In the aerial part of flowering plants, all organs, including leaves, stems, and flowers, originate from a small population of stem cells. These cells are embedded in the shoot apical meristem, on the flanks of which new leaf and flower primordia are produced in a regular spatial and temporal pattern. The spatial pattern is known as phyllotaxis and can be, for example, spiral, alternating (distichous), or decussate (forming 180° angles). Most species show a single phyllotactic pattern, but in some cases, a plant can switch from one pattern to another in response to developmental or external cues. The temporal pattern is characterized by the time that elapses between the formation of primordia, called the plastochron, which is the inverse of the leaf initiation rate.
Classical surgical experiments have indicated that existing leaf primordia produce a diffusible substance that inhibits the formation of new primordia, with new primordia being formed at the position with a minimal inhibitor concentration (Snow, 1929). Recent analyses have suggested an alternative phyllotactic model that is based on positive effects of the growth hormone auxin (Reinhardt et al., 2000, 2003; Vernoux et al., 2000; Jönsson et al., 2006; Smith et al., 2006). According to this model, auxin is imported by the shoot apical meristem and redistributed primarily through the epidermal cell layer (L1), with existing primordia acting as auxin sinks. Additional auxin maxima can thus only form between existing primordia, once these are sufficiently spaced apart. New primordia are then initiated at these sites.
While there has been considerable progress in knowledge about how phyllotaxis is governed and what role auxin plays in this process, a coherent understanding of the regulation of plastochron length is lacking. Inactivation of several genes, including rice (Oryza sativa) PLASTOCHRON1 (PLA1), maize (Zea mays) TERMINAL EAR1 (TE1) and its rice ortholog PLA2, and ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis thaliana, causes a shortening of the plastochron (Conway and Poethig, 1997; Itoh et al., 1998; Veit et al., 1998; Helliwell et al., 2001; Miyoshi et al., 2004; Kawakatsu et al., 2006). By contrast, phytochrome b (phyb) and serrate (se) mutants in Arabidopsis suffer from delayed initiation of new leaves (Reed et al., 1993; Clarke et al., 1999; Prigge and Wagner, 2001). All of these genes have pleiotropic effects, and the diverse nature of the encoded products, including a photoreceptor (PHYB), a cytochrome P450 enzyme (PLA1), and an RNA binding protein (TE1/PLA2), suggests a complex network for plastochron regulation in many species.
Several lines of evidence point to cytokinin as a regulator of leaf initiation. For example, amp1 mutants contain elevated levels of cytokinin (Chaudhury et al., 1993), and overexpressing the CYTOKININ OXIDASE1 (CKX1) gene increases plastochron length in Arabidopsis (Werner et al., 2003). Furthermore, maize plants lacking the ABPHYL1 (ABPH1) gene have, like amp1 mutants in Arabidopsis, a larger shoot apical meristem and altered phyllotaxy (Jackson and Hake, 1999; Nogué et al., 2000; Giulini et al., 2004). The ABPH1 gene product belongs to a group of ARABIDOPSIS RESPONSE REGULATOR proteins, which are important modulators of the cytokinin response (Ferreira and Kieber, 2005). Related genes in Arabidopsis function directly downstream of the meristem regulator WUSCHEL (Leibfried et al., 2005). Nevertheless, how meristem size and cytokinin affect primordium formation or how this is regulated by crosstalk between auxin and cytokinin is unclear.
Here, we describe two genetic axes affecting plastochron length in Arabidopsis. One includes microRNA156 (miR156), which targets several SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor genes that are predominantly expressed at the shoot apex (Cardon et al., 1999; Rhoades et al., 2002; Schmid et al., 2003; Schwarz et al., 2008). By comparing endogenous expression domains of SPL genes with the results of misexpression studies, we demonstrate that miR156 limits plastochron length in developing leaf primordia. The effects of miR156/SPLs on leaf initiation rate appear to be independent of CYP78A5/KLUH, a likely ortholog of rice PLA1 (Itoh et al., 1998; Zondlo and Irish, 1999; Miyoshi et al., 2004; Anastasiou et al., 2007). Interestingly, organ size and plastochron length are coordinately affected by both miR156/SPL and CYP78A5, suggesting either that there are common regulators of organ size and plastochron length or that these two developmental parameters reciprocally influence each other.
Role of miR156-Targeted SPL Genes in Determining Plastochron Length
Among previously described mutants with increased plastochron lengths is se-1 (Clarke et al., 1999), which is defective in the processing of several miRNAs, including miR156 (Lobbes et al., 2006; Yang et al., 2006; Laubinger et al., 2008; see Supplemental Figures 1A and 1B online). We found that leaf initiation was similarly affected in ago1-27 plants, which carry a weak mutant allele of ARGONAUTE1 (AGO), the gene encoding the main slicer responsible for miRNA-directed transcript cleavage in Arabidopsis (Morel et al., 2002; Vaucheret et al., 2004; Baumberger and Baulcombe, 2005; see Supplemental Figure 1A online). Together, these observations indicate that an miRNA is limiting for the delay of the initiation of new leaf primordia. At least two lines of evidence suggest miR156 as the most promising candidate. First, overexpression of miR156 plants causes shortening of the plastochron (Schwab et al., 2005; see Supplemental Figure 1C online), a phenotype opposite to that of se-1 and ago1-27. Second, while Pro35S:MIR156f se-1 double mutants retained the serrated leaf phenotype typical of se-1 mutants, the plastochron defect caused by miR156 overexpression was suppressed in these plants (see Supplemental Figure 1C online).
In Arabidopsis, 11 of the 17 SPL genes are targeted by miR156 (Rhoades et al., 2002; Schwab et al., 2005; Gandikota et al., 2007). In microarray analyses, most miR156-targeted SPLs show similar patterns of RNA accumulation, with low levels in vegetative tissues and increased expression at the shoot apex upon flowering (Schmid et al., 2003; Schwab et al., 2005). However, the SPL proteins differ substantially in size. SPL3, SPL4, and SPL5 are much smaller than the other gene products, with the DNA binding domain making up most of the protein (Cardon et al., 1999). We identified several homozygous T-DNA insertion lines of miR156-targeted SPLs, including spl2, spl3, spl9, spl10, spl11, spl13, and spl15 (Figure 1A ; see Supplemental Figure 2A online). Similar to spl3 mutants (Wu and Poethig, 2006), the other single-insertion mutants were phenotypically normal in terms of leaf initiation rate and flowering time (data not shown), indicating a high degree of functional redundancy.
Modulation of Plastochron Length by SPL Genes.
(A) Diagram of SPL9 and SPL15 transcribed regions, with thin lines indicating introns. Arrowheads mark T-DNA insertion sites.
(B) Rosettes of 30-d-old short-day-grown plants.
(C) Appearance of visible leaves in wild-type, Pro35S:MIR156f, and spl9 spl15 plants grown in short days (n ≥ 10). Bars indicate sd.
Among the larger SPLs, two unlinked genes are particularly similar to each other, SPL9 and SPL15 (Cardon et al., 1999; Yang et al., 2008). We found that spl9 spl15 double mutants had short plastochrons (Figure 1; see Supplemental Table 1 online), in agreement with a recent independent report (Schwarz et al., 2008). Although the phenotype was milder than that of the miR156 overexpressers, in which expression of eight additional SPL genes is strongly reduced (Schwab et al., 2005), this finding indicated an important redundant effect of SPL9 and SPL15 on plastochron length.
Based on overexpression experiments, the SPL3 gene has been implicated primarily in the regulation of flowering time and phase change (Cardon et al., 1997; Wu and Poethig, 2006). Unfortunately, knockout alleles for the other two small SPL genes, SPL4 and SPL5, are not available, making it more difficult to test whether redundancy among the three small SPL genes masks their contribution to plastochron length. We therefore designed an artificial miRNA that targeted the mRNAs of both SPL4 and SPL5 (Schwab et al., 2006) (see Supplemental Figures 2B and 2C online) and introduced these into spl3 mutants. These plants did not show obvious differences in plastochron length (data not shown), suggesting that the three small SPL genes likely play minor roles, if any, in the regulation of leaf initiation rate.
Effects of miR156 Misexpression
To understand how miR156 and its SPL targets modulate leaf initiation rate, we analyzed the effects of expressing miR156 in different domains of the shoot apex (Figure 2 ). We chose the promoter of SHOOT MERISTEMLESS (STM) for the shoot apical meristem (Kim et al., 2003), that of FLOWERING LOCUS D (FD) for the shoot apical meristem and leaf anlagen (Abe et al., 2005; Wigge et al., 2005), that of LATERAL ORGAN BOUNDARIES (LOB) for the periphery of the shoot apical meristem (Shuai et al., 2002), those of AINTEGUMENTA (ANT) and ASYMMETRIC LEAVES1 (AS1) for young leaf primordia (Schoof et al., 2000; Eshed et al., 2001), that of the SCARECROW homolog BLS for late leaf primordia (Lifschitz et al., 2006), and that of the SUCROSE-PROTON SYMPORTER2 (SUC2) for vascular tissue (Truernit and Sauer, 1995; Imlau et al., 1999). Specific activity of the promoters, as reported in the literature, was confirmed by promoter β-glucuronidase (GUS) fusions (see Supplemental Figure 3 online).
Effects of MIR156f Misexpression.
(A) Thirty-day-old short-day-grown transgenic plants expressing MIR156f from different promoters, with expression domains indicated by blue color in the accompanying illustrations of shoot apices (see Supplemental Figure 3 online for expression data).
(B) Leaf initiation rates of transgenic plants, calculated from short-day-grown T1 individuals (n ≥ 10; see Supplemental Table 1 online). Bars indicate sd. Asterisks indicate a significant difference from the wild type (Student's t test with Bonferroni correction, P < 0.04).
Because one can imagine that the rate of primordium initiation is largely determined by the number of cells available in the periphery of the meristem, we suspected that miR156 targets would limit plastochron length in the meristem itself. To our surprise, transgenic miR156 expression in the meristem proper (ProFD:MIR156f and ProSTM:MIR156f) did not affect leaf initiation rate (Figure 2; see Supplemental Table 1 online). Similar to Pro35S:MIR156b plants (Schwab et al., 2005), directing miR156 expression to the periphery of the shoot apical meristem with the LOB promoter did not change plastochron length either (Figure 2; see Supplemental Table 1 online). By contrast, expression of MIR156f under control of the leaf primordium–specific ANT and AS1 promoters led to a similar plastochron phenotype to that seen in Pro35S:MIR156f plants. Thirty-day-old wild-type plants grown in short days had produced 20 leaves, whereas ProANT:MIR156f and ProAS1:MIR156f plants had produced 30 or half again as many leaves (Figure 2; see Supplemental Table 1 online). Finally, ProBLS:MIR156f and ProSUC2:MIR156f plants had an intermediate phenotype, indicating that misexpression of miR156 in older leaf primordia that already develop vasculature (or in the vasculature subtending the meristem proper) is less effective in altering plastochron length. Taken together, we conclude that miR156-targeted SPL genes contribute to nonautonomous effects of existing leaf primordia on the initiation of new leaf primordia at the shoot apical meristem.
Expression Pattern of SPL9
Consistent with other types of expression studies for SPL3 (Cardon et al., 1997; Wu and Poethig, 2006), microarray analyses have shown that most miR156-targeted SPL genes have a similar RNA expression pattern, with the highest levels in the shoot apex and an increase in expression during the transition to flowering (Schmid et al., 2003; Schwab et al., 2005). We used in situ hybridization to analyze in more detail the expression pattern of SPL9. During the vegetative phase, SPL9 mRNA was found in leaf anlagen and in developing leaf primordia (Figure 3A ). As young leaves grew, SPL9 expression became gradually restricted to vascular tissues. Importantly, we did not detect any SPL9 transcripts in the central part of the meristem. In the inflorescence apex, the expression pattern of SPL9 was similar to that in the vegetative shoot apex. SPL9 mRNA was found only transiently in the youngest floral primordia (Figure 3B). In Pro35S:MIR156f plants, SPL9 transcripts were strongly reduced (Figure 3C), confirming that miR156 can target SPL9 mRNAs for degradation by miRNA-guided cleavage.
In Situ Hybridizations Showing Expression Patterns of SPL9 and miR156.
(A) to (H) Expression of SPL9.
(A) and (B) Wild-type vegetative (A) and reproductive shoot apex (B).
(C) Pro35S:MIR156f vegetative shoot apex.
(D) to (F) Wild-type, se-1, and ago1-27 vegetative shoot apices. All three samples were stained for the same amount of time in the dark. The wild type was allowed to remain underdeveloped so that the stronger signals in the other two genotypes would not become saturated.
(G) and (H) Wild-type and Pro35S:MIM156 vegetative apices. Both samples were stained for the same amount of time in the dark. The wild type was allowed to remain underdeveloped so that the stronger signal in the Pro35S:MIM156 apex would not become saturated.
(I) Expression of miR156 in the wild type and se-1 (inset).
Shoot apices were dissected from 15-d-old plants grown in short days. Bars = 50 μm.
Since transgene-directed expression of miR156 in the meristem itself had little effect on plastochron length, one might conclude that this is the primary site of endogenous miR156 activity and that the main function of miR156 is to prevent accumulation of SPL transcripts in the meristem proper. We therefore analyzed SPL9 expression in two mutants with reduced miR156 levels, se-1 and ago1-27. Compared with the wild type, SPL9 RNA levels were substantially elevated in both mutants. However, the overall pattern of expression was similar to that seen in the wild type (Figures 3D to 3F). To exclude that this was an indirect effect of other miRNAs, we employed transgenic plants in which miR156 activity is specifically reduced by constitutive expression of a target mimic (Franco-Zorrilla et al., 2007). SPL9 mRNA expression was changed in these Pro35S:MIM156 plants in a similar manner as in se-1 and ago1-27 plants (Figures 3G and 3H).
The analysis of SPL9 expression in mutants indicates that miR156 primarily affects SPL9 expression in a quantitative, rather than spatial manner. That miR156 is not an important regulator of the domain of SPL9 expression was also supported by a direct analysis of miR156 expression by in situ hybridization, which revealed that miR156 accumulated to similar levels in both the shoot apical meristem proper and leaf primordia (Figure 3I). The specificity of the hybridization signal was confirmed using se-1 plants, in which the level of miR156 was greatly reduced (Figure 3I, inset).
In summary, the expression studies confirm that SPL9, and likely other SPL genes, either mediate or trigger nonautonomous effects of existing leaf primordia on the initiation of new leaf primordia by the meristem proper and that the primary role of miR156 is to dampen overall levels of SPL RNA (and probably also protein; Gandikota et al., 2007) in leaf primordia, rather than shaping its spatial expression pattern.
Increased Plastochron Length in Response to Elevated SPL9 and SPL10 Activity
To determine whether SPL genes are not only required to prevent shortening of plastochron length but are also sufficient to increase plastochron length, we prepared forms of SPL genes that can no longer be targeted by miR156 according to known rules for effective miRNA targeting in plants (Schwab et al., 2005). We refer to these mutants as nontargeted or resistant SPLs (rSPLs) (Figure 4A ).
Effects of a miR156-Insensitive Form of SPL9.
(A) Diagram of the miR156 target sites of the wild-type and modified version of SPL9. Capital letters below indicate encoded amino acids.
(B) Forty-day-old (ProSPL9:rSPL9 genotypes) and 20-d-old plants (all other genotypes) grown in short days. Misexpression of rSPL9 with different promoters causes a similar delay in leaf initiation. Numbers indicate the order of leaves, with 1 referring to the oldest leaf. Note that there is no apparent disruption of normal phyllotaxis in plants with decreased or increased SPL activity.
(C) Appearance of visible leaves in short-day-grown plants (n = 10). Bars indicate sd.
Overexpression of rSPL3 caused early flowering as reported (Wu and Poethig, 2006; Gandikota et al., 2007) but caused only a minor decrease in leaf initiation rate (see Supplemental Table 1 online), suggesting limited crosstalk between miR156 targets of the SPL3/4/5 and SPL9/15 groups.
It was difficult to recover Pro35S:rSPL9 plants, suggesting that very high levels of SPL9 protein cause embryonic lethality. Plants that expressed rSPL9 under the control of the native promoter had a very strong plastochron phenotype, with the leaf initiation rate reduced to one-third of that of the wild type (Figures 4B and 4C; see Supplemental Table 1 online). We observed a similar, but weaker, phenotype in a few plants expressing the nonmutated form of SPL9, in line with the hypothesis of a quantitative interaction between miR156 and its targets. We confirmed that rSPL9 is insensitive to miR156 action by introducing the Pro35S:MIR156f transgene into these plants. The doubly transgenic plants were indistinguishable from ProSPL9:rSPL9 plants (Figure 4B).
The results presented in the previous two sections had suggested that SPL9 functions nonautonomously in existing leaf primordia to time the emergence of new primordia from the shoot apical meristem. To assess whether SPL9 can also act directly in the meristem, we misexpressed rSPL9 from different promoters. Ectopic expression from the primordium-specific ANT and AS1 promoters reduced the number of leaves to less than half of that of the wild type after 30 d in short-day conditions. A similar phenotype was observed in ProFD:rSPL9 plants (Figure 4B; see Supplemental Table 1 online), in which SPL9 was ectopically expressed in the shoot meristem. Misexpression of rSPL10 had similar, though less dramatic, effects than rSPL9 (see Supplemental Table 1 online), consistent with overlapping roles of SPL9 and SPL10, which both belong to the group of large SPL genes.
In summary, elevated activity of SPL genes can increase plastochron length, indicating an instructive role of the miR156/SPL axis in timing the emergence of new leaf primordia.
Effects of CYP78A5/KLUH and CYP78A7 on Plastochron Length
A short plastochron phenotype has also been described for rice plants with mutations in PLA1, which is likely an ortholog of CYP78A5/KLUH in Arabidopsis. Both genes are expressed at the periphery of the meristem proper and at the base of new leaf primordia (Zondlo and Irish, 1999; Miyoshi et al., 2004; Anastasiou et al., 2007). We found that CYP78A5 loss-of-function mutants had shortened plastochrons in both long and short days. In addition, the first flowers formed a few days earlier than in the wild type (Figures 5A to 5C ; see Supplemental Table 2 online).
cyp78a5 and cyp78a7 Mutant Phenotypes.
(A) Diagram of CYP78A5 and CYP78A7 transcribed regions, with thin lines indicating introns. Arrowheads mark T-DNA insertion sites. The cyp78a5 allele has also been described as klu-4 (Anastasiou et al., 2007).
(B) Rosettes of 30-d-old plants grown in short days.
(D) and (E) Wild-type embryos at bent-cotyledon stage (D) and at maturity (E).
(F) and (G) cyp78a5 cyp78a7 embryos at bent-cotyledon stage (F) and at maturity (G). The latter was dissected from the seed coat. More than 10 siliques were examined, and representative embryos are shown. Note the enlarged shoot apical meristem in (F) and multiple cotyledons in (G).
Bars = 150 μm.
CYP78A5, located on chromosome 5, has a homolog on chromosome 1, CYP78A7. Unlike cyp78a5, the cyp78a7 single mutant appeared phenotypically normal (see Supplemental Figure 4 online). To reveal functional redundancy between these two members, we crossed the cyp78a5 and cyp78a7 mutant lines. Because we did not readily identify double mutants in the F2 generation, we examined developing embryos in siliques of cyp78a5/cyp78a5 cyp78a7/+ plants. Little phenotypic change was observed among embryos in cyp78a5/cyp78a5 cyp78a7/+ siliques until the torpedo stage. From that stage on, almost a quarter of embryos did not increase much in size. The shoot apical meristem, however, continued to enlarge and became much bigger than the arrested cotyledons. The abnormal shoot apical meristem initiated supernumerary cotyledons (Figures 5D to 5G). The double mutants could sometimes survive, and we recovered a few cyp78a5 cyp78a7 seedlings from a cyp78a5/cyp78a5 cyp78a7/+ parent (five double mutants among 87 progeny tested). These plants, which produced no seeds, were small with compacted rosette leaves and an increased leaf initiation rate. The seedlings had three or four cotyledons (see Supplemental Figure 4 online), similar to amp1 mutants, which also have an embryonic phenotype reminiscent of cyp78a5 cyp78a7 double mutants (Chaudhury et al., 1993; Conway and Poethig, 1997). In summary, these results indicate that CYP78A5 and CYP78A7 play redundant roles in regulating relative growth of the shoot apical meristem and the rest of the plant.
Genetic Interaction between miR156/SPL and CYP78A5
Our miR156 misexpression experiments indicated that miR156-targeted SPLs act predominantly in leaf primordia, while CYP78A5 is expressed in the periphery of the shoot apical meristem (Zondlo and Irish, 1999). Nevertheless, given the phenotypic similarity of Pro35S:MIR156f and cyp78a5 mutants, we wanted to test whether miR156/SPL and CYP78A5 operate in the same genetic pathway. Since SPL genes encode transcription factors, we first assessed RNA expression levels. There was no significant change of CYP78A5 transcript levels in either Pro35S:MIR156f or ProSPL9:rSPL9 plants (Figure 6A ). Similarly, SPL9 levels were unaffected by cyp78a5 (Figure 6A).
Genetic Interaction between miR156/SPL and CYP78A5.
(A) Expression of CYP78A5 and SPL9 in wild-type, mutant, and transgenic plants. Total RNA was extracted from 7-d-old long-day-grown plants and analyzed by real-time RT-PCR with three technical replicates. Expression was normalized relative to that of β-TUBULIN2. Two biological replicates were performed, both with similar results.
(B) Leaf initiation rate in wild-type, mutant, and transgenic plants, calculated from 10 short-day-grown individuals (see Supplemental Table 1 online).
Bars indicate sd.
To further clarify the relationship between CYP78A5 and the miR156/SPL pathway, we examined genetic interactions. The plastochron length of ProSPL9:rSPL9 cyp78a5 plants was intermediate between that of the parental lines, which have opposite phenotypes, suggesting parallel effects of the two genetic systems. This was supported by the Pro35S:MIR156f cyp78a5 combination, which had an even faster leaf initiation rate than either single mutant (Figure 6B).
CYP78A5, also known as KLUH, has recently been implicated in growth regulation, with small leaves and floral organs in cyp78a5 mutants because of prematurely arrested growth (Anastasiou et al., 2007), similar to what was shown for the apparent rice ortholog, PLA1 (Itoh et al., 1998). We found that Pro35S:MIR156f plants, which have a similar plastochron phenotype to cyp78a5 mutants, also exhibited smaller leaf size (Table 1 ; see Supplemental Figure 5 online). Similar effects on organ and cell size were observed in petals (see Supplemental Table 3 online). Thus, the effects of decreasing SPL activity on organ growth parallel the changes caused by inactivation of PLA1 or CYP78A5/KLUH (Itoh et al., 1998; Anastasiou et al., 2007). Together, these observations support a close relationship between leaf growth and plastochron length.
Leaf and Leaf Cell Sizes in Different Genotypes
Correlation between Plastochron Length, Cell Division Rate, and Meristem Size
From first principles, the leaf initiation rate could be affected either by the size of the meristem or by the rate of cell division in the meristem. Indeed, increased shoot apical meristem size in clv1 mutants is paralleled by shortened plastochron length (Kwon et al., 2005). We therefore examined vegetative shoot apices by scanning electron microscopy. Meristem shape and phyllotaxis of cyp78a5, and Pro35S:MIR156f and ProSPL9:rSPL9 plants were similar to those of the wild type, even though they had either shorter or longer plastochrons than the wild type, suggesting that the plastochron changes were not caused by defects in leaf positioning (Figures 7A to 7D ). The two lines with shortened plastochrons, cyp78a5 and Pro35S:MIR156f, had meristems of similar sizes to the wild type, while meristems were smaller in ProSPL9:rSPL9 plants (Figure 7, Table 2 ).
Morphology of Shoot Apical Meristem and Dividing Cells.
(A) to (D) Scanning electron micrographs of shoot apices from 30-d-old short-day-grown plants. At least 10 individuals were examined for each genotype, and representative images are shown.
(E) to (H) Histone H4 expression in shoot apices of 15-d-old short-day-grown plants.
The ProSPL9:rSPL9 plants were grown at a later time point, but the wild-type controls were similar as for the other two genotypes. Bars = 50 μm.
Shoot Apical Meristem Characteristics of Different Genotypes Grown for 30 d in Short Photoperiods
Histone H4 is highly transcribed in G1-S cells and is a useful marker for cell division (Krizek, 1999; Gaudin et al., 2000). We performed in situ hybridization with different genotypes and counted the number of Histone H4 positive cells per median longitudinal section (Table 2). We found that there were substantially fewer Histone H4–expressing cells in the ProSPL9:rSPL9 shoot apical meristem (Figure 7F), suggesting that the increased plastochron length in rSPL9 plants correlates with both fewer dividing cells and a smaller overall meristem. The picture was less clear for cyp78a5 and Pro35S:MIR156f plants, which have normal-sized shoot apical meristems. There were more Histone H4–expressing cells in cyp78a5 and Pro35S:MIR156f plants (Figure 7G), but the difference from the wild type was statistically significant only in cyp78a5 (Table 2, Figures 7E and 7G). Even so, together with the normal-sized shoot apical meristem, we conclude that more rapid cell division in the shoot apical meristems of both miR156-overexpressing plants and in cyp78a5 mutants compensates for an increase in leaf initiation rate.
miR156 and its SPL targets have previously been shown to affect flowering time and phase change (Cardon et al., 1997; Schwab et al., 2005; Wu and Poethig, 2006). Here, we have described a critical role of miR156/SPL in regulating plastochron length. Although the miR156/SPL axis appears to act largely in parallel with CYP78A5/KLUH, the likely ortholog of rice PLA1, the two genetic systems have similar effects on both organ size and plastochron length, suggesting a regulatory link between these traits, which are important determinants of overall plant growth.
Regulation of SPL Expression by miR156
It had been initially proposed that plant miRNAs mostly control the spatial expression pattern of their targets (Juarez et al., 2004; Parizotto et al., 2004). However, in other cases, miRNAs seem to be important for both spatially confining target mRNAs and for dampening their levels (McConnell et al., 2001; Kidner and Martienssen, 2004; Sieber et al., 2007). In se-1 and ago1-27 mutants as well as Pro35S:MIM156 plants, all of which have reduced miR156 activity, we observed a clear increase in the levels of the SPL9 target mRNA but no obvious expansion of its expression domain. Thus, miR156 appears to be at the other end of possibilities for miRNA target interactions, with its main role being quantitative control of target mRNA levels, similar to what has been reported for miR169 and its targets in snapdragon (Antirrhinum majus) and petunia (Petunia hybrida; Cartolano et al., 2007). This conclusion is supported by a comparison of SPL9 and miR156 expression patterns, which strongly overlap (Figure 3).
Nonautonomous Effects on Plastochron Length
Classic experiments have shown that removal of young leaf primordia accelerates the rate of leaf initiation at the shoot apex (Snow, 1929). Based on this finding, it has been proposed that young leaf primordia synthesize an inhibitory, mobile factor that acts in the meristem proper. Two lines of evidence indicate that the miR156/SPL axis fits the tenets of this hypothesis. First, overexpression of miR156 in leaves or leaf primordia shortens plastochron length, whereas increased SPL activity in the leaf primordium has an opposite effect. Second, at least one of the SPL genes, SPL9, is predominantly expressed in leaf primordia.
Auxin is required for the initiation of leaf primordia, and blocking auxin transport or signaling inhibits primordium formation (Reinhardt et al., 2000, 2003; Benkova et al., 2003). Since existing leaf primordia, as auxin sinks, have an important effect on the generation of new auxin maxima, which then direct the initiation of new primordia, it is formally possible that SPLs affect plastochron length by modulating auxin accumulation or sensitivity. A similar formal possibility is that SPL proteins themselves are the mobile signal, which would be consistent with our observation that overexpressing SPLs in either leaf primordia or the meristem proper had similar effects on plastochron length. The latter observations also conform to a model in which a secondary inhibitor is directly regulated by SPLs. Some of these alternatives could be resolved by identifying direct targets of SPL transcription factors.
Several other genes have similar effects on plastochron length to those of the miR156/SPL axis. Mutations in both TE1 and in its rice ortholog PLA2 cause shortened plastochrons (Itoh et al., 1998; Veit et al., 1998; Miyoshi et al., 2004). These genes encode RNA binding proteins related to yeast MEI2, a master regulator of meiosis (Ohno and Mattaj, 1999). Both are preferentially expressed in young leaf primordia, and they could potentially act in a pathway dependent on miR156 and its SPL targets, which are conserved in monocotyledonous plants (Xie et al., 2006; Chuck et al., 2007). Consistent with this hypothesis is the finding that PLA2 appears to function independently of PLA1 in rice (Kawakatsu et al., 2006), similar to the relationship between miR156/SPLs and CYP78A5 in Arabidopsis. Unfortunately, it is unclear whether TE1/PLA2 homologs fulfill related roles in Arabidopsis, especially since the most similar genes, TEL1 and TEL2, are expressed in the shoot apical meristem only, different from TE1 and PLA2 (Anderson et al., 2004).
In contrast with the case for PLA2, a clear candidate for a PLA1 ortholog exists in Arabidopsis, CYP78A5/KLUH, which delays the initiation of new leaves, just like PLA1 (Itoh et al., 1998; Miyoshi et al., 2004; this work). The phenotype of embryos that lack activity of both CYP78A5 and its most similar Arabidopsis homolog, CYP78A7, is reminiscent of amp1 mutants (Chaudhury et al., 1993; Conway and Poethig, 1997). The cyp78a double mutant phenotype appears to be stronger than that of amp1 mutants, being often embryonic lethal and always causing several cotyledons to develop in surviving seedlings. AMP1 encodes an enzyme with unknown substrate (Helliwell et al., 2001; Vidaurre et al., 2007), and it remains to be seen whether the CYP78A5/7 and AMP1 proteins operate in the same metabolic pathway.
Plastochron Length and Shoot Apical Meristem Size
Several reports have suggested that plastochron length and shoot apical meristem size are inversely correlated. Increased meristem size and shortened plastochron characterize clv1 mutants (Kwon et al., 2005), while the opposite is true of plants that overexpress CKX genes (Werner et al., 2003). A longer plastochron is also seen in plants with mutations in multiple cytokinin receptor genes (Riefler et al., 2006). These observations are consistent with our finding that ProSPL9:rSPL9 plants have both a reduced meristem and increased plastochron length.
There is, however, no simple correlation between shoot apical meristem size and plastochron length, as neither miR156-overexpressing plants nor cyp78a5 mutants have strongly enlarged shoot apical meristems, despite a substantial shortening of plastochron length. In these plants, it appears that the increase in plastochron length is mostly due to increased cell division rates, which is also the case in several rice mutants, including pla1 and pla2 (Itoh et al., 1998; Ikeda et al., 2005; Kawakatsu et al., 2006). A direct link between cell division and plastochron length has been made with plants that overexpress Cyclin D2, which leads to faster leaf initiation correlated with higher cell division rates. At least early on, size and organization of the shoot apical meristem are normal in these plants (Cockcroft et al., 2000; Boucheron et al., 2005). However, while cell division can under certain circumstances be limiting for shortening plastochron length, slowing down cell division with a dominant-negative version of CDC2a, a cyclin-dependent kinase that interacts with Cyclin D2, did not affect leaf initiation rate, apparently because increased cell size compensated for lower cell numbers (Hemerly et al., 1995).
Plastochron Length and Phase Change
Another defect of plants with reduced SPL activity due to miR156 overexpression is a delay in phase change, with the opposite effect caused by constitutive overexpression of an miRNA-insensitive version of SPL3 (Wu and Poethig, 2006). An even stronger phenotype is seen in ProSPL9:rSPL9 plants, in which the juvenile phase is lost (this work). Clonal analyses with maize have indicated that vegetative phase change is not conferred by the shoot apical meristem, but rather that phase identity is determined autonomously in each leaf primordium (Orkwiszewski and Poethig, 2000). Our results are consistent with this observation, as SPL9 is normally expressed in leaf primordia, and increasing SPL9 levels in leaf primordia by expression from the ANT promoter is sufficient to accelerate phase change.
A similar correlation between reduced plastochron length and delayed phase change is seen in rice pla1 and pla2 mutants (Itoh et al., 1998; Kawakatsu et al., 2006). A net outcome of this would be that phase change is more normal in these mutants when measured in absolute time. However, there does not seem to be an inevitable link between plastochron length and phase change. For example, Teopod mutants in maize suffer from delayed phase change, but leaf initiation rates are normal (Poethig, 1988). Conversely, inactivation of the TAS3 pathway causes an acceleration of phase change. However, leaf initiation rates are not altered in RNA-dependent RNA polymerase6, dicer-like4, or zippy/ago7 mutants (J.-W. Wang and D. Weigel, unpublished results), all of which are affected in TAS3-dependent phase change (Hunter et al., 2003, 2006; Xie et al., 2005; Adenot et al., 2006; Fahlgren et al., 2006; Garcia et al., 2006). As with meristem size, the relationship between phase change and plastochron length is complex.
A Compensatory Mechanism Linking Plastochron Length and Organ Size?
A final phenotype that is shared between several mutants is the reduced size of leaves and floral organs (Itoh et al., 1998; Veit et al., 1998; Kawakatsu et al., 2006; Anastasiou et al., 2007; this work). An increase in plastochron length, at least during inflorescence development, has also been suggested for the big brother mutant, which has enlarged organs as well (Disch et al., 2006). Similar to how coordinated changes in plastochron length and phase change would maintain the absolute timing of phase change, an effect of organ size on plastochron length or vice versa would lead to overall biomass changes being kept to a minimum. There is precedence for a related phenomenon during leaf growth, namely the compensation of changes in cell size by total cell number (Horiguchi et al., 2006). Alternatively, rather than organ size and plastochron length reciprocally affecting each other, one can envision that coordinated behavior of the two traits is due to a common regulator. Nevertheless, the connection between organ size and plastochron length is not static. For example, manipulating leaf size by altering activity of the STRUBBELIG-RECEPTOR FAMILY4 gene (Eyüboglu et al., 2007) does not have the same effects on plastochron length as altering miR156/SPL activity (K. Schneitz, personal communication).
In summary, we have described how and where the miR156/SPL axis affects plastochron length and organ size and found that it acts largely independently of CYP78A5/KLUH, despite similar mutant phenotypes. Apart from the nature of the leaf-derived signal that affects plastochron length, an important challenge for future studies is the question of how ubiquitous coordinated changes in plastochron length and leaf size are.
Oligonucleotide primers used in this work are given in Supplemental Table 4 online.
Arabidopsis thaliana plants, ecotype Columbia (Col-0), were grown at 23°C in long days (16 h light/8 h dark) or short days (8 h light/16 h dark). Individual spl and cyp78a T-DNA insertion lines (Tissier et al., 1999; Sessions et al., 2002; Alonso et al., 2003) were obtained from the European Arabidopsis Stock Centre (http://Arabidopsis.info). The cyp78a5 allele has also been described as klu-4 (Anastasiou et al., 2007). miR156 target mimic expressing Pro35S:MIM156 plants have been described (Franco-Zorrilla et al., 2007).
Transgenic Plants
For promoter fusions, AS1 and BLS promoters were amplified by PCR with AS1:LhG4 or BLS:LhG4 plasmid DNA as templates. All other promoters and SPL genes were amplified by PCR with genomic DNA from Col-0 as template, using Turbo Hotstart Pfu DNA polymerase (Stratagene). rSPL9 was made by two rounds of mutagenic PCR using Turbo Hot-start Pfu DNA polymerase (Stratagene). An artificial miRNA construct against SPL4 and SPL5 was generated as described (Schwab et al., 2006). The binary constructs were delivered into Agrobacterium tumefaciens strain GV3101 (pMP90RK) by the freeze-thaw method (Weigel and Glazebrook, 2002). Arabidopsis plants were transformed using the flower-dip method (Clough and Bent, 1998). Transgenic seedlings were selected with 50 μg/mL kanamycin on plates or 0.1% glufosinate (BASTA) on soil. At least 50 T1 seedlings were analyzed for each construct.
GUS Staining and Histology
One-week-old seedlings were fixed in 90% acetone for 20 min on ice. GUS staining was performed as described (Blázquez et al., 1997). Stained tissue was embedded, sectioned, and mounted in Clarion Mounting Medium (Sigma-Aldrich). Developing embryos were dissected from siliques and mounted in a mixture of chloralhydrate/glycerol/water (8:1:2) and photographed using differential interference contrast optics.
Expression Analysis
Total RNA was extracted from 1-week-old seedlings or vegetative shoot apices with the Plant RNeasy Mini kit (Qiagen). One microgram of total RNA was treated with DNase I and used for cDNA synthesis with oligo(dT) primer and Superscript reverse transcriptase (Invitrogen). Quantitative RT-PCR was performed with SYBR-Green PCR Mastermix (Invitrogen), and amplification was real-time monitored on an Opticon continuous fluorescence detection system (MJR). For small RNA gel blots, a mixed DNA/locked nucleic acid (LNA; Exiqon; Wahlestedt et al., 2000) oligonucleotide probe (gtgmCtcActmCtcTgtmCa, where uppercase letters indicate LNA bases and lowercase letters DNA bases) was used.
SPL9 and Histone H4 cDNAs were PCR amplified and cloned into pGEM-T easy (Promega). Digoxigenin-labeled sense and antisense probes were synthesized with T7 or SP6 RNA polymerase (Roche). For the miR156 probe, LNA oligonucleotides were end labeled with the DIG oligonucleotide 3′-end labeling kit (Roche). Shoot apices from 20-d-old short-day-grown plants were dissected and fixed in formalin/acetic acid/ethanol (1:1:18). Paraffin-embedded material was sectioned to 8 μm thickness. Hybridization and detection were performed as previously described (Palatnik et al., 2003).
Scanning Electron Microscopy and Shoot Apical Meristem and Organ Size Measurement
Vegetative shoot apices of 30-d-old short-day-grown plants were dissected, fixed in methanol, washed twice with 100% ethanol, critical point dried, and mounted. After gold coating, at least 10 apices per genotype were examined on a Hitachi S800 electron microscope. For size measurements, apices were embedded in paraffin and sectioned. The width and height of the meristem were scored for each section. To measure cell size, the leaves or petals were dissected, incubated in 80% ethanol for 2 h, mounted on slides with a drop of chloralhydrate/glycerol/water (8:1:2), and photographed using differential interference contrast optics. Cell size was calculated using AxioVision software (Zeiss).
Leaf Initiation Rate Measurement
The number of visible leaves (∼1 mm in width) was recorded daily. Average leaf initiation rate was calculated by dividing total leaf number by days after germination.
Accession Numbers
Arabidopsis Genome Initiative gene identifiers are as follows: SPL2 (At5g43270), SPL3 (At2g33810), SPL4 (At1g53160), SPL5 (At3g15270), SPL9 (At2g42200), SPL10 (At1g27370), SPL11 (At1g27360), SPL13 (At5g50670), SPL15 (At3g57920), CYP78A5 (At1g13710), CYP78A7 (At5g09970), LOB (At5g63090), SUC2 (At1g22710), FD (At4g35900), BLS (At3g49950), STM (At1g62360), ANT (At4g37750), AS1 (At2g37630), β-TUBULIN-2 (At5g62690), and Histone H4 (At5g59690).
The following materials are available in the online version of this article.
Supplemental Figure 1. miRNA Dependence of Plastochron Length.
Supplemental Figure 2. T-DNA Insertion Mutants and amiRNA Targeting SPL4/5.
Supplemental Figure 3. Activity of Promoter Fusions.
Supplemental Figure 4. Phenotype of the cyp78a5 cyp78a7 Double Mutant.
Supplemental Figure 5. Leaf and Cell Size in Wild-Type, Mutant, and Transgenic Plants.
Supplemental Table 1. Leaf Initiation Rates of Different Genotypes.
Supplemental Table 2. Flowering Time of cyp78a5 Mutants.
Supplemental Table 3. Petal and Petal Cell Size of Different Genotypes.
Supplemental Table 4. Oligonucleotide Primer Sequences.
We thank the European Arabidopsis Stock Centre for seeds, Yuval Eshed for the AS1 and BLS promoter constructs, Marco Todesco for Pro35S:MIM156 plants, Jürgen Berger for scanning electron microscopy, Kay Schneitz for communicating unpublished data, and members of Team MiRNA for discussion. This work was supported by an EMBO Long-Term Fellowship to J.-W.W. (ALTF 274-2006), by a DFG-SFB 446 grant, and by European Community FP6 IPs SIROCCO (Contract LSHG-CT-2006-037900) and AGRON-OMICS (Contract LSHG-CT-2006-037704).
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Detlef Weigel (weigel{at}weigelworld.org).
www.plantcell.org/cgi/doi/10.1105/tpc.108.058180
↵1 Current address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.
↵2 Current address: Department of Biomolecular Sciences and Biotechnology, University of Milan, 20133 Milan, Italy.
↵[OA] Open Access articles can be viewed online without a subscription.
↵[W] Online version contains Web-only data.
Received January 17, 2008.
Revised April 8, 2008.
Accepted May 1, 2008.
Published May 20, 2008.
Abe, M., Kobayashi, Y., Yamamoto, S., Daimon, Y., Yamaguchi, A., Ikeda, Y., Ichinoki, H., Notaguchi, M., Goto, K., and Araki, T. (2005). FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309: 1052–1056.
Adenot, X., Elmayan, T., Lauressergues, D., Boutet, S., Bouche, N., Gasciolli, V., and Vaucheret, H. (2006). DRB4-dependent TAS3 trans-acting siRNAs control leaf morphology through AGO7. Curr. Biol. 16: 927–932.
Alonso, J.M., et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657.
Anastasiou, E., Kenz, S., Gerstung, M., MacLean, D., Timmer, J., Fleck, C., and Lenhard, M. (2007). Control of plant organ size by KLUH/CYP78A5-dependent intercellular signaling. Dev. Cell 13: 843–856.
Anderson, G.H., Alvarez, N.D.G., Gilman, C., Jeffares, D.C., Trainor, V.C.W., Hanson, M.R., and Veit, B. (2004). Diversification of genes encoding Mei2-Like RNA binding proteins in plants. Plant Mol. Biol. 54: 653–670.
Baumberger, N., and Baulcombe, D.C. (2005). Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc. Natl. Acad. Sci. USA 102: 11928–11933.
Benkova, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertova, D., Jürgens, G., and Friml, J. (2003). Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115: 591–602.
Blázquez, M.A., Soowal, L., Lee, I., and Weigel, D. (1997). LEAFY expression and flower initiation in Arabidopsis. Development 124: 3835–3844.
Boucheron, E., Healy, J.H., Bajon, C., Sauvanet, A., Rembur, J., Noin, M., Sekine, M., Riou-Khamlichi, C., Murray, J.A., Van Onckelen, H., and Chriqui, D. (2005). Ectopic expression of Arabidopsis CYCD2 and CYCD3 in tobacco has distinct effects on the structural organization of the shoot apical meristem. J. Exp. Bot. 56: 123–134.
Cardon, G., Hohmann, S., Klein, J., Nettesheim, K., Saedler, H., and Huijser, P. (1999). Molecular characterisation of the Arabidopsis SBP-box genes. Gene 237: 91–104.
Cardon, G., Hohmann, S., Nettesheim, K., Saedler, H., and Huijser, P. (1997). Functional analysis of the Arabidopsis thaliana SBP-box gene SPL3: A novel gene involved in the floral transition. Plant J. 12: 367–377.
Cartolano, M., Castillo, R., Efremova, N., Kuckenberg, M., Zethof, J., Gerats, T., Schwarz-Sommer, Z., and Vandenbussche, M. (2007). A conserved microRNA module exerts homeotic control over Petunia hybrida and Antirrhinum majus floral organ identity. Nat. Genet. 39: 901–905.
Chaudhury, A.M., Letham, S., Craig, S., and Dennis, E.S. (1993). amp1 — A mutant with high cytokinin levels and altered embryonic pattern, faster vegetative growth, constitutive photomorphogenesis and precocious flowering. Plant J. 4: 907–916.
Chuck, G., Cigan, A.M., Saeteurn, K., and Hake, S. (2007). The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA. Nat. Genet. 39: 544–549.
Clarke, J.H., Tack, D., Findlay, K., Van Montagu, M., and Van Lijsebettens, M. (1999). The SERRATE locus controls the formation of the early juvenile leaves and phase length in Arabidopsis. Plant J. 20: 493–501.
Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743.
Cockcroft, C.E., den Boer, B.G.W., Healy, J.M.S., and Murray, J.A.H. (2000). Cyclin D control of growth rate in plants. Nature 405: 575–579.
Conway, L.J., and Poethig, R.S. (1997). Mutations of Arabidopsis thaliana that transform leaves into cotyledons. Proc. Natl. Acad. Sci. USA 94: 10209–10214.
Disch, S., Anastasiou, E., Sharma, V.K., Laux, T., Fletcher, J.C., and Lenhard, M. (2006). The E3 ubiquitin ligase BIG BROTHER controls Arabidopsis organ size in a dosage-dependent manner. Curr. Biol. 16: 272–279.
Eshed, Y., Baum, S.F., Perea, J.V., and Bowman, J.L. (2001). Establishment of polarity in lateral organs of plants. Curr. Biol. 11: 1251–1260.
Eyüboglu, B., Pfister, K., Haberer, G., Chevalier, D., Fuchs, A., Mayer, K.F.X., and Schneitz, K. (2007). Molecular characterisation of the STRUBBELIG-RECEPTOR FAMILY of genes encoding putative leucine-rich repeat receptor-like kinases in Arabidopsis thaliana. BMC Plant Biol. 7: 16.
Fahlgren, N., Montgomery, T.A., Howell, M.D., Allen, E., Dvorak, S.K., Alexander, A.L., and Carrington, J.C. (2006). Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Curr. Biol. 16: 939–944.
Ferreira, F.J., and Kieber, J.J. (2005). Cytokinin signaling. Curr. Opin. Plant Biol. 8: 518–525.
Franco-Zorrilla, J.M., Valli, A., Todesco, M., Mateos, I., Puga, M.I., Rubio-Somoza, I., Leyva, A., Weigel, D., García, J.A., and Paz-Ares, J. (2007). Target mimicry provides a new mechanism for regulation of microRNA activity. Nat. Genet. 39: 1033–1037.
Gandikota, M., Birkenbihl, R.P., Hohmann, S., Cardon, G.H., Saedler, H., and Huijser, P. (2007). The miRNA156/157 recognition element in the 3′ UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings. Plant J. 49: 683–693.
Garcia, D., Collier, S.A., Byrne, M.E., and Martienssen, R.A. (2006). Specification of leaf polarity in Arabidopsis via the trans-acting siRNA pathway. Curr. Biol. 16: 933–938.
Gaudin, V., Lunness, P.A., Fobert, P.R., Towers, M., Riou-Khamlichi, C., Murray, J.A., Coen, E., and Doonan, J.H. (2000). The expression of D-cyclin genes defines distinct developmental zones in snapdragon apical meristems and is locally regulated by the CYCLOIDEA gene. Plant Physiol. 122: 1137–1148.
Giulini, A., Wang, J., and Jackson, D. (2004). Control of phyllotaxy by the cytokinin-inducible response regulator homologue ABPHYL1. Nature 430: 1031–1034.
Helliwell, C.A., Chin-Atkins, A.N., Wilson, I.W., Chapple, R., Dennis, E.S., and Chaudhury, A. (2001). The Arabidopsis AMP1 gene encodes a putative glutamate carboxypeptidase. Plant Cell 13: 2115–2125.
Hemerly, A., Engler, J., Bergounioux, C., Van Montagu, M., Engler, G., Inzé, D., and Ferreira, P. (1995). Dominant negative mutants of the Cdc2 kinase uncouple cell division from iterative plant development. EMBO J. 14: 3925–3936.
Horiguchi, G., Ferjani, A., Fujikura, U., and Tsukaya, H. (2006). Coordination of cell proliferation and cell expansion in the control of leaf size in Arabidopsis thaliana. J. Plant Res. 119: 37–42.
Hunter, C., Sun, H., and Poethig, R.S. (2003). The Arabidopsis heterochronic gene ZIPPY is an ARGONAUTE family member. Curr. Biol. 13: 1734–1739.
Hunter, C., Willmann, M.R., Wu, G., Yoshikawa, M., de la Luz Gutiérrez-Nava, M., and Poethig, S.R. (2006). Trans-acting siRNA-mediated repression of ETTIN and ARF4 regulates heteroblasty in Arabidopsis. Development 133: 2973–2981.
Ikeda, K., Nagasawa, N., and Nagato, Y. (2005). ABERRANT PANICLE ORGANIZATION 1 temporally regulates meristem identity in rice. Dev. Biol. 282: 349–360.
Imlau, A., Truernit, E., and Sauer, N. (1999). Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues. Plant Cell 11: 309–322.
Itoh, J.I., Hasegawa, A., Kitano, H., and Nagato, Y. (1998). A recessive heterochronic mutation, plastochron1, shortens the plastochron and elongates the vegetative phase in rice. Plant Cell 10: 1511–1522.
Jackson, D., and Hake, S. (1999). Control of phyllotaxy in maize by the abphyl1 gene. Development 126: 315–323.
Jönsson, H., Heisler, M.G., Shapiro, B.E., Meyerowitz, E.M., and Mjolsness, E. (2006). An auxin-driven polarized transport model for phyllotaxis. Proc. Natl. Acad. Sci. USA 103: 1633–1638.
Juarez, M.T., Kui, J.S., Thomas, J., Heller, B.A., and Timmermans, M.C. (2004). microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity. Nature 428: 84–88.
Kawakatsu, T., Itoh, J.-I., Miyoshi, K., Kurata, N., Alvarez, N., Veit, B., and Nagato, Y. (2006). PLASTOCHRON2 regulates leaf initiation and maturation in rice. Plant Cell 18: 612–625.
Kidner, C.A., and Martienssen, R.A. (2004). Spatially restricted microRNA directs leaf polarity through ARGONAUTE1. Nature 428: 81–84.
Kim, J.Y., Yuan, Z., and Jackson, D. (2003). Developmental regulation and significance of KNOX protein trafficking in Arabidopsis. Development 130: 4351–4362.
Krizek, B.A. (1999). Ectopic expression of AINTEGUMENTA in Arabidopsis plants results in increased growth of floral organs. Dev. Genet. 25: 224–236.
Kwon, C.S., Chen, C., and Wagner, D. (2005). WUSCHEL is a primary target for transcriptional regulation by SPLAYED in dynamic control of stem cell fate in Arabidopsis. Genes Dev. 19: 992–1003.
Laubinger, S., Sachsenberg, T., Zeller, G., Busch, W., Lohmann, J.U., Rätsch, G., and Weigel, D. (2008). Dual roles of the nuclear cap binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA, in press.
Leibfried, A., To, J.P., Busch, W., Stehling, S., Kehle, A., Demar, M., Kieber, J.J., and Lohmann, J.U. (2005). WUSCHEL controls meristem function by direct regulation of cytokinin-inducible response regulators. Nature 438: 1172–1175.
Lifschitz, E., Eviatar, T., Rozman, A., Shalit, A., Goldshmidt, A., Amsellem, Z., Alvarez, J.P., and Eshed, Y. (2006). The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli. Proc. Natl. Acad. Sci. USA 103: 6398–6403.
Lobbes, D., Rallapalli, G., Schmidt, D.D., Martin, C., and Clarke, J. (2006). SERRATE: A new player on the plant microRNA scene. EMBO Rep. 7: 1052–1058.
McConnell, J.R., Emery, J., Eshed, Y., Bao, N., Bowman, J., and Barton, M.K. (2001). Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature 411: 709–713.
Miyoshi, K., Ahn, B.-O., Kawakatsu, T., Ito, Y., Itoh, J.-I., Nagato, Y., and Kurata, N. (2004). PLASTOCHRON1, a timekeeper of leaf initiation in rice, encodes cytochrome P450. Proc. Natl. Acad. Sci. USA 101: 875–880.
Morel, J.-B., Godon, C., Mourrain, P., Beclin, C., Boutet, S., Feuerbach, F., Proux, F., and Vaucheret, H. (2002). Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. Plant Cell 14: 629–639.
Nogué, F., Grandjean, O., Craig, S., Dennis, E., and Chaudhury, A. (2000). Higher levels of cell proliferation rate and cyclin CycD3 expression in the Arabidopsis amp1 mutant. Plant Growth Regul. 32: 275–283.
Ohno, M., and Mattaj, I.W. (1999). Meiosis: MeiRNA hits the spot. Curr. Biol. 9: R66–R69.
Orkwiszewski, J.A.J., and Poethig, R.S. (2000). Phase identity of the maize leaf is determined after leaf initiation. Proc. Natl. Acad. Sci. USA 97: 10631–10636.
Palatnik, J.F., Allen, E., Wu, X., Schommer, C., Schwab, R., Carrington, J.C., and Weigel, D. (2003). Control of leaf morphogenesis by microRNAs. Nature 425: 257–263.
Parizotto, E.A., Dunoyer, P., Rahm, N., Himber, C., and Voinnet, O. (2004). In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev. 18: 2237–2242.
Poethig, R.S. (1988). Heterochronic mutations affecting shoot development in maize. Genetics 119: 959–973.
Prigge, M.J., and Wagner, D.R. (2001). The Arabidopsis SERRATE gene encodes a zinc-finger protein required for normal shoot development. Plant Cell 13: 1263–1280.
Reed, J.W., Nagpal, P., Poole, D.S., Furuya, M., and Chory, J. (1993). Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development. Plant Cell 5: 147–157.
Reinhardt, D., Mandel, T., and Kuhlemeier, C. (2000). Auxin regulates the initiation and radial position of plant lateral organs. Plant Cell 12: 507–518.
Reinhardt, D., Pesce, E.-R., Stieger, P., Mandel, T., Baltensperger, K., Bennett, M., Traas, J., Friml, J., and Kuhlemeier, C. (2003). Regulation of phyllotaxis by polar auxin transport. Nature 426: 255–260.
Rhoades, M.W., Reinhart, B.J., Lim, L.P., Burge, C.B., Bartel, B., and Bartel, D.P. (2002). Prediction of plant microRNA targets. Cell 110: 513–520.
Riefler, M., Novak, O., Strnad, M., and Schmülling, T. (2006). Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism. Plant Cell 18: 40–54.
Schmid, M., Uhlenhaut, N.H., Godard, F., Demar, M., Bressan, R., Weigel, D., and Lohmann, J.U. (2003). Dissection of floral induction pathways using global expression analysis. Development 130: 6001–6012.
Schoof, H., Lenhard, M., Haecker, A., Mayer, K.F.X., Jürgens, G., and Laux, T. (2000). The stem cell population of Arabidopsis shoot meristems in maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100: 635–644.
Schwab, R., Ossowski, S., Riester, M., Warthmann, N., and Weigel, D. (2006). Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18: 1121–1133.
Schwab, R., Palatnik, J.F., Riester, M., Schommer, C., Schmid, M., and Weigel, D. (2005). Specific effects of microRNAs on the plant transcriptome. Dev. Cell 8: 517–527.
Schwarz, S., Grande, A.V., Bujdoso, N., Saedler, H., and Huijser, P. (2008). The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis. Plant Mol. Biol. 67: 183–195.
Sessions, A., et al. (2002). A high-throughput Arabidopsis reverse genetics system. Plant Cell 14: 2985–2994.
Shuai, B., Reynaga-Pena, C.G., and Springer, P.S. (2002). The LATERAL ORGAN BOUNDARIES gene defines a novel, plant-specific gene family. Plant Physiol. 129: 747–761.
Sieber, P., Wellmer, F., Gheyselinck, J., Riechmann, J.L., and Meyerowitz, E.M. (2007). Redundancy and specialization among plant microRNAs: role of the MIR164 family in developmental robustness. Development 134: 1051–1060.
Smith, R.S., Guyomarc'h, S., Mandel, T., Reinhardt, D., Kuhlemeier, C., and Prusinkiewicz, P. (2006). A plausible model of phyllotaxis. Proc. Natl. Acad. Sci. USA 103: 1301–1306.
Snow, R. (1929). The young leaf as the inhibiting organ. New Phytol. 28: 345–348.
Tissier, A.F., Marillonnet, S., Klimyuk, V., Patel, K., Torres, M.A., Murphy, G., and Jones, J.D.G. (1999). Multiple independent defective suppressor-mutator transposon Insertions in Arabidopsis: A tool for functional genomics. Plant Cell 11: 1841–1852.
Truernit, E., and Sauer, N. (1995). The promoter of the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene directs expression of β-glucuronidase to the phloem: Evidence for phloem loading and unloading by SUC2. Planta 196: 564–570.
Vaucheret, H., Vazquez, F., Crété, P., and Bartel, D.P. (2004). The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev. 18: 1187–1197.
Veit, B., Briggs, S.P., Schmidt, R.J., Yanofsky, M.F., and Hake, S. (1998). Regulation of leaf initiation by the terminal ear 1 gene of maize. Nature 393: 166–168.
Vernoux, T., Kronenberger, J., Grandjean, O., Laufs, P., and Traas, J. (2000). PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem. Development 127: 5157–5165.
Vidaurre, D.P., Ploense, S., Krogan, N.T., and Berleth, T. (2007). AMP1 and MP antagonistically regulate embryo and meristem development in Arabidopsis. Development 134: 2561–2567.
Wahlestedt, C., et al. (2000). Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc. Natl. Acad. Sci. USA 97: 5633–5638.
Weigel, D., and Glazebrook, J. (2002). Arabidopsis: A Laboratory Manual. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).
Werner, T., Motyka, V., Laucou, V., Smets, R., Van Onckelen, H., and Schmülling, T. (2003). Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15: 2532–2550.
Wigge, P.A., Kim, M.C., Jaeger, K.E., Busch, W., Schmid, M., Lohmann, J.U., and Weigel, D. (2005). Integration of spatial and temporal information during floral induction in Arabidopsis. Science 309: 1056–1059.
Wu, G., and Poethig, R.S. (2006). Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development 133: 3539–3547.
Xie, K., Wu, C., and Xiong, L. (2006). Genomic organization, differential expression, and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156 in rice. Plant Physiol. 142: 280–293.
Xie, Z., Allen, E., Wilken, A., and Carrington, J.C. (2005). DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 102: 12984–12989.
Yang, L., Liu, Z., Lu, F., Dong, A., and Huang, H. (2006). SERRATE is a novel nuclear regulator in primary microRNA processing in Arabidopsis. Plant J. 47: 841–850.
Yang, Z., Wang, X., Gu, S., Hu, Z., Xu, H., and Xu, C. (2008). Comparative study of SBP-box gene family in Arabidopsis and rice. Gene 407: 1–11.
Zondlo, S.C., and Irish, V.F. (1999). CYP78A5 encodes a cytochrome P450 that marks the shoot apical meristem boundary in Arabidopsis. Plant J. 19: 259–268.
You are going to email the following Dual Effects of miR156-Targeted SPL Genes and CYP78A5/KLUH on Plastochron Length and Organ Size in Arabidopsis thaliana
The Plant Cell May 2008, 20 (5) 1231-1243; DOI: 10.1105/tpc.108.058180


SUNNY WEDNESDAY: Sunshine for your Thanksgiving-Eve here in Cookeville. We had a few sprinkles overnight that resulted in .02" in the CoCoRaHS professional 4" rain gauge. Expect highs to reach around 56° today with the sunny skies. Some other sections of Tennessee, around Nashville, will climb over 60°. On Thanksgiving, you'll need to dress warmly as a cold front brings some sprinkles and strong west winds. Highs only in the upper 40's with lows tomorrow night in the lower 30's. We can't rule out a changeover to snow late Thursday/early Friday. Speaking of Friday, if you're shopping you'll need to dress warmly as we expect a chilly start with some flurries flying. Eventually, we'll clear out and high the upper 40's by Friday afternoon!
Interesting web site from NOAA allows you to look up weather records. Very handy!


Nephropathology is the subspeciality of surgical pathology which is focused on the diagnosis and characterization of medical diseases of the kidney. The diagnosis of renal diseases is usually made by percutaneous renal biopsy, to determine patient prognosis and therapy. Renal pathology interpretation is complex and challenging.
Nephropathologists use light microscopy, immunofluorescence, immunohistochemistry and electron microscopy to obtain a definitive diagnosis. Renal pathologists work closely with Nephrologists and the transplant team. Although we usually focus on diseases of the glomerulus the scope of renal pathology is broad and includes tubulointerstitial diseases and vascular diseases of the native kidneys and disorders of renal allografts.
The nephropatholgy unit at UMass Memorial Pathology processes approximately 130 renal biopsies annually. 50% are from native kidneys and the remaining 50% are from renal transplants. About 15% of our native kidney biopsies are from patients in the pediatric age group. In addition to acquired kidney diseases the latter group includes interesting and challenging hereditary and congenital kidney diseases.
In addition to diagnostic service the renal pathology unit is actively involved in clinicopathological conferences with clinicians from Nephrology, Rheumatology and Transplant medicine.
About 20% of renal biopsies are STAT cases requiring same day processing and preliminary critical value diagnoses. This service is available 24 hours a day, 7 days a week and is provided for biopsies of renal allografts and native kidneys.
For both STAT and routine services the clinician will typically call the general office of UMass Memorial Pathology.
In a typical case the resident or histotechnologist will attend the biopsy procedure and will inform the radiologist whether the tissue is adequate. The tissue obtained will be triaged into three parts. Each part should contain at least one glomerulus and should include a portion of the renal cortex. The majority of the specimen is placed in formalin fixative for light microscopy , special stains and immunohistochemical stains. Typically 10-15 glomeruli are needed for a definitive diagnosis. A smaller portion of the biopsy will be placed in Michel's medium available in the Pathology Department to be processed for immunofluorescence microscopy. Usually 1-3 glomeruli are needed. A third small portion of the biopsy will be placed in a special glutaraldehyde for electron microscopy. Usually one to two glomeruli are required.


text:
Q: libvirt: difference in exposing LVM logical volumes VS libvirt storage pools I started learning how to create a good virtualization stack with libvirt. I have created a LVM Volume Group that exposes a device block made of a Linux RAID. My idea is to give a Logical Volume to each virtual machine, but a person suggested me to use storage pools instead, like
<pool type='logical'>
  <name>VG_foo</name>
  <uuid>foo</uuid>
  <capacity unit='bytes'>0</capacity>
  <allocation unit='bytes'>0</allocation>
  <available unit='bytes'>0</available>
  <source>
    <device path='/dev/md2'/>
    <name>vg_foo</name>
    <format type='lvm2'/>
  </source>
  <target>
    <path>/dev/vg_foo</path>
    <permissions>
      <mode>0755</mode>
      <owner>-1</owner>
      <group>-1</group>
    </permissions>
  </target>
</pool>

I have read libvirt storage documentation but I did not understand the difference

A: The effect of both of these is the same, but in one case you are doing everything manually, and in the other case, libvirt is able to do everything for you.
With the storage pool defined, libvirt is able to create and manage volumes in that pool. Without the storage pool defined, you must manually create and manage the volumes outside of libvirt.
Note that a block device does not have to be part of a storage pool in order for a virtual machine managed by libvirt to use it. But if it is not, then you must manage it yourself.

A: As the above has said its very similar, a storage pool is basically a Volume group and libvirt manages the logical volumes created.
Whereas you would be manually creating a Volume group and manually creating the disks and then adding them to the VM.
Just add a storage pool and create the disk on VM creation.

redpajama_set_name: RedPajamaStackExchange


Description This 2017 Honda Civic EX is Honda Certified vehicle which includes 7 years/100,000 mile Warranty. CARFAX 1 owner and buyback guarantee... ATTENTION!!! . Do you want it all? Well, with this spacious Sedan, you are going to get it** . Reconditioning and Certification Fee included in our advertised prices. Call Us at (847) 831-4100 to Set an Appointment for yout Test Drive. This Vehicle is located at our Certified Pre-Owned Center on 150 Skokie Valley Road.


text:
Mystery solved: Volcanoes cooled Earth 13,000 years ago
August 3rd, 2020 Posted by Keith Randall-Texas A&M
The bottom line, is that "the chemical anomalies found in sediments dating to the beginning of the Younger Dryas are the result of volcanism and not an extraterrestrial impact," says Michael Walters. (Credit: Pixabay/Pexels)
early Earth
Ancient sediment found in a central Texas cave appears to solve the mystery of why the Earth cooled suddenly about 13,000 years ago, according to a new study.
Some researchers believed that an extraterrestrial impact with the Earth, such as a meteor collision, caused the event, which cooled the Earth about 3 degrees centigrade (about 5.4 degrees Fahrenheit), a huge amount.
But the researchers found that the evidence left in layers of sediment in Hall's Cave were almost certainly the result of volcanic eruptions.
The cave has a sediment record extending over 20,000 years, says Michael Walters, professor at Texas A&M University and director of The Center for The Study of the First Americans. Walters first began researching the cave in 2017.
"It is an exceptional record that offers a unique opportunity for interdisciplinary cooperation to investigate a number of important research questions," he says.
"One big question was, did an extraterrestrial impact occur near the end of the last ice age, about 13,000 years ago as the ice sheets covering Canada were melting, and cause an abrupt cooling that thrust the northern hemisphere back into the ice age for an extra 1,200 years?"
The researchers found layers of sediment within the cave, which Thomas Stafford of Stafford Research Laboratories in Colorado first found, dating back to the time of the proposed impact that could answer the question and perhaps even identify the trigger that started the ancient cold snap.
Missing: Geochemical fingerprints
The event also likely helped cause the extinction of large mammals such as mammoth, horse, and camel that once roamed North America.
"This work shows that the geochemical signature associated with the cooling event is not unique but occurred four times between 9,000 and 15,000 years ago," says Alan Brandon, professor of geosciences at the University of Houston and head of the research team.
"Thus, the trigger for this cooling event didn't come from space. Prior geochemical evidence for a large meteor exploding in the atmosphere instead reflects a period of major volcanic eruptions.
"I was skeptical," Brandon says. "We took every avenue we could to come up with an alternative explanation, or even avoid, this conclusion. A volcanic eruption had been considered one possible explanation but was generally dismissed because there was no associated geochemical fingerprint."
After a volcano erupts, the global spread of aerosols reflects incoming solar radiation away from Earth and may lead to global cooling post-eruption for one to five years, depending on the size and timescales of the eruption, the team says.
Goodbye horses, hello Clovis
"The Younger Dryas, which occurred about 13,000 years ago, disrupted distinct warming at the end of the last ice age," says coauthor Steven Forman, professor of geosciences at Baylor University.
The Earth's climate may have been at a tipping point at the end of Younger Dryas, possibly from the ice sheet discharge into the North Atlantic Ocean, enhanced snow cover, and powerful volcanic eruptions that may have in combination led to intense Northern Hemisphere cooling, Forman says.
"This period of rapid cooling coincides with the extinction of a number of species, including camels and horses, and the appearance of the Clovis archaeological tradition," Waters says.
Brandon and fellow University of Houston scientist Nan Sun completed the isotopic analysis of sediments collected from Hall's Cave. They found that elements such as iridium, ruthenium, platinum, palladium, and rhenium were not present in the correct proportions, meaning that a meteor or asteroid could not have caused the event.
"The isotope analysis and the relative proportion of the elements matched those that were found in previous volcanic gases," says Sun, lead author of the paper in Science Advances.
Volcanic eruptions cause their most severe cooling near the source, usually in the year of the eruption, with substantially less cooling in the years after the eruption, the team says.
The Younger Dryas cooling lasted about 1,200 years, "so a sole volcanic eruptive cause is an important initiating factor, but other Earth system changes, such as cooling of the oceans and more snow cover were needed to sustain this colder period, "Forman says.
The bottom line, Waters says, is that "the chemical anomalies found in sediments dating to the beginning of the Younger Dryas are the result of volcanism and not an extraterrestrial impact."
Source: Texas A&M University
Original Study DOI: 10.1126/sciadv.aax8587
'Polar wander' may be behind Earth's most recent ice age
Earth-changing asteroid impact theory gets new evidence
Volcanic eruptions might have given Earth its first oxygen

redpajama_set_name: RedPajamaCommonCrawl


0300042582 Item in acceptable condition including possible liquid damage. As well, answers may be filled in. Lastly, may be missing components, e.g. missing DVDs, CDs, Access Code, etc.
Book. 4to-over 9¾-12" tall. New in original slipcase box. Illustrated. First numberline printing. John Smybert (or Smibert) (1688-1751) was a Scottish American artist, who was born in Edinburgh, Scotland and died in Boston, Massachusetts. In 1728 he accompanied Bishop Berkeley to America, with the intention of becoming professor of fine arts in the college which Berkeley was planning to found in Bermuda. The college, however, was never established, and Smybert settled in Boston, where he married in 1730. He lived at the corner of Brattle Street and Queen-Street. In 1734, Smibert opened a shop where he sold paint, other artist's supplies, and prints. In his studio above the shop, he displayed casts and copies of Old Masters that he had painted in Europe. This collection, which Richard Saunders has termed "America's first art gallery", provided much of the early artistic education for Charles Willson Peale, Gilbert Stuart, and John Trumbull. Between 1740-42, he served as architect for the original Faneuil Hall, which he designed in the style of an English country market.
Large ex-library hardback w/dust jacket, mylar jacket, library stamps & labels, 280 pages, b&w and color photos, index, good condition.
Sewn binding. Cloth over boards. 294 p. Barra Foundation Book. Audience: General/trade.
Brown cloth. 280 pp. 26 color, over 200 bw plates. NO SLIPCASE. In addition to enlightening text, there is a catalogue raisonne organized as follows: Accepted Works; Copies after works by other artists; Disputed works; Works known only through prints, references in Smibert's notebook, or descriptions; Works wrongly attributed to Smibert. Extensively researched. NO SLIPCASE.
Book is new but slipcase has minor scuffs.
280 pages. Dark brown cloth covers, gilt titles to spine, glossy dust jacket illustrated in color, 26 full-color plates, profusely illustrated with b&w plates. Clean covers and dust jacket, pages crisp and unmarked, stiff binding; an extremely clean, tight copy in great condition. Record # 401493.


Anna needs to find the Lost Temple to save her grandfather from a cursed death.
He spent days exploring its mysteries. Upon his return home, Anna's grandfather was stricken ill, affected by its curse and is now dying from an unknown disease. Guide Anna to the Lost Temple in order to find a cure for her grandfather.


What is Difference in Conditions?
All over the country and around the world, natural disasters and man-made catastrophes seem to happen on a daily basis. Earthquakes, hurricanes, fires, and flooding have begun to take their toll in certain areas, causing ripple effects in the insurance industry.
After several years of hurricanes in the late 90s and early 2000s, Florida created Citizens Property Insurance Corporation in 2002. This was to be the insurer of last resort for homeowners, condominium associations, and co-ops that could no longer find property and/or wind insurance coverage through the open market.
Much like the FAIR Plan in California, Citizens offered coverage that was basic perils only. While it did cover windstorm, hail and hurricane coverage, it left many other important perils uninsured.
Situations like this are where a Difference in Conditions (DIC) policy comes in handy. Read on to find out more about DIC and if this coverage is right for you.
A Difference in Conditions policy is the property insurance equivalent of "gap" coverage. This coverage can be written to cover many different perils that may not be covered by a standard property insurance policy. If you want or need coverage not offered under your personal lines or commercial policy coverage, DIC is right for you.
Are All DIC Policies the Same?
There is no standard DIC policy. Every carrier writes Difference in Conditions policies on a manuscript format which allows the coverage to be tailored to the specific needs of the policyholder.
It is important when considering a DIC policy that you work with your broker to determine where your coverage gaps are and how best to cover them. You should also carefully review the policy language with your broker to be sure that you are getting both what you pay for and what you need from that coverage.
Do You Need DIC Coverage?
You have a FAIR Plan policy for your home or business. These are basic form coverage and do not offer the covered perils you need.
You have a basic or broad form property policy. Check your policy or review with your broker.
You don't have earthquake or earth movement coverage.
You don't have flood coverage.
Your special form policy excludes specific perils that you feel need coverage.
The best course of action is to pull out your property policy, dust it off and call your insurance broker to review what you have versus what you need to properly insure your property investments.
Hayes Brokers offers a free, no-obligation risk analysis to current and prospective clients. Bring us your current policies and we will review them to determine whether DIC coverage is right for you.


text:
Spectroscopy and Photochemistry of Planetary Atmospheres and Ionospheres
Mars, Venus, Titan, Triton and Pluto
Part of Cambridge Planetary Science
Author: Vladimir A. Krasnopolsky, Catholic University of America, Washington DC
Date Published: April 2019
$ 84.99 (P)
If you are interested in the title for your course we can consider offering an examination copy. To register your interest please contact collegesales@cambridge.org providing details of the course you are teaching.
The chemical composition of any planetary atmosphere is of fundamental importance in determining its photochemistry and dynamics in addition to its thermal balance, climate, origin and evolution. Divided into two parts, this book begins with a set of introductory chapters, starting with a concise review of the Solar System and fundamental atmospheric physics. Chapters then describe the basic principles and methods of spectroscopy, the main tool for studying the chemical composition of planetary atmospheres, and of photochemical modeling and its use in the theoretical interpretation of observational data on chemical composition. The second part of the book provides a detailed review of the carbon dioxide atmospheres and ionospheres of Mars and Venus, and the nitrogen-methane atmospheres of Titan, Triton and Pluto. Written by an expert author, this comprehensive text will make a valuable reference for graduate students, researchers and professional scientists specializing in planetary atmospheres.
Contains detailed chapters on the atmospheres and ionospheres of Mars, Venus, Titan, Triton and Pluto which allow readers to see the applications of spectroscopy and photochemistry, and to compare carbon dioxide atmospheres with those principally composed of nitrogen and methane
Presents concise introductory chapters which introduce readers to the fundamental principles of atmospheric physics, spectroscopy and photochemical modeling, making it a useful resource for those without prior knowledge of these topics
Written by an expert author who has worked on various space missions, and Earth-orbiting and ground-based observatories, drawing on his vast experience of spectroscopy and photochemical modeling to provide a comprehensive, up-to-date summary of these techniques
contains: 377 b/w illus. 8 tables
1. The Solar System
2. Atmospheric structure
3. Spectroscopy
4. Aerosol extinction and scattering
5. Quantitative spectroscopy
6. Spectrographs
7. Spectroscopic methods to study planetary atmospheres
8. Solar radiation, its absorption in the atmospheres, and airglow
9. Chemical kinetics
10. Photochemical modeling
11. Mars
13. Titan
14. Triton
15. Pluto and Charon
Index (75 KB)
Vladimir A. Krasnopolsky, Catholic University of America, Washington DC
Vladimir Krasnopolsky was previously a Research Professor in the Department of Physics at the Catholic University of America, Washington, DC. An expert on spectroscopy and photochemical modelling, he is the author of three books, four book chapters and 153 refereed publications. He is one of the most highly cited scientists working on planetary atmosphere research and was awarded the USSR State Prize in 1985 for his studies of Venus. He has worked on many space missions throughout his career and was the Principle Investigator of the airglow spectrometer on the Mars 5 spacecraft and the Venera 9 and 10 missions to Venus, the three-channel spectrometer on the Vega mission to Venus, and the infrared spectrometer on the Phobos 2 orbiter.
Exploring Planetary Climate
A History of Scientific Discovery on Earth, Mars, Venus and Titan
Ionospheres
Physics, Plasma Physics, and Chemistry
The Surface of Mars
Exploring Space, Exploring Earth
New Understanding of the Earth from Space Research
An Introduction to Space Plasma Complexity
International Journal of Astrobiology
The International Journal of Astrobiology is the peer-reviewed forum for practitioners in this exciting interdisciplinary…
The Journal of Navigation
The Journal of Navigation contains original papers on the science of navigation by man and animals over land and…


text:
Q: Why does this code print big values instead of the totals I expect? I'm trying to get 5 integer outputs and display odd count, even count, odd total and even total.  This code gives me bigger total values.  What's the problem?
#include <stdio.h>

int main(){
    int n,count,oc,ec;
    float ot,et,answer;

    for(count=0;count<=5;count++){
        printf("enter a odd or even number :");
        scanf("%d",&n);
    }
    answer=n%2;
    if (answer>0){
        oc=oc+1;
        ot=n+ot;
    }

    else {
        ec=ec+1;
        et=n+et;
    }
    printf("odd counter is %f",oc);
    printf("odd counter is %f",ot);
    printf("even counter is %f",ec);
    printf("even counter is %f",et);
}

A: Change your accumulators to integers and initialize them:
int oc, ot, ec, et;
oc = ot = ec = et = 0;

Move the closing brace as follows:
for(count=0;count<=5;count++){
    printf("enter a odd or even number :");
    scanf("%d",&n);

    answer=n%2;
    if (answer>0){
        oc=oc+1;
        ot=n+ot;
    }
    else {
        ec=ec+1;
        et=n+et;
    }
}

and change your printf's to use %d instead of %f
printf("odd counter is %d",oc);
printf("odd counter is %d",ot);
printf("even counter is %d",ec);
printf("even counter is %d",et);

A: the following proposed code:

*

*cleanly compiles

*properly checks for errors

*has no undefined behavior

*documents why each header file is being included

*performs the desired functionality

*uses no 'magic' numbers, 'magic' numbers are numbers with no basis.  I.E. 5.  See the #define and where the resulting (meaningful) name is being used.

*handles each input, as it is input

*uses text in the printf() statements that accurately says what the value being output actually means

*uses proper formatting of each output value.

*caveat: does not check for overflow of the summed values

*properly writes the for() loop.

*follows the axiom: only one statement per line and (at most) one variable declaration per statement.

*uses appropriate horizontal spacing (for readability) inside parens, after commas, after semicolons, around C operators.

*was compiled on linux using:
gcc -c main.c -Wall -Wextra -pedantic -Wconversion -std=gnu11 -o main.o

*was linked on linux using:
gcc main.o -o main

*was run using:
./main
And now the proposed coded:
#include <stdio.h>   // printf(), fprintf(), stderr, scanf()
#include <stdlib.h>  // exit(), EXIT_FAILURE

#define MAX_INPUTS 5

int main( void )
{
    int n;
    int oc = 0;
    int ec = 0;
    int ot = 0;
    int et = 0;

    for( int count = 0; count < MAX_INPUTS; count++ )
    {
        printf("enter a odd or even number :");
        if( 1 != scanf( "%d", &n ) )
        { // then input error
            fprintf( stderr, "scanf for input %d failed\n", count+1 );
            exit( EXIT_FAILURE );
        }

        // implied else, scanf successful

        if ( (n%2) > 0 )
        {  // input was ODD
            oc = oc + 1;
            ot = n + ot;
        }

        else
        {  // input was even
            ec = ec + 1;
            et = n + et;
        }
    }

    printf( "number of odd inputs is %d\n", oc );
    printf( "sum of odd inputs is %d\n", ot );
    printf( "number of even inputs is %d\n", ec );
    printf( "sum of even inputs is %d\n", et );
}

the output of a typical run of the code:
enter a odd or even number :1
enter a odd or even number :2
enter a odd or even number :3
enter a odd or even number :4
enter a odd or even number :5
number of odd inputs is 3
sum of odd inputs is 9
number of even inputs is 2
sum of even inputs is 6


Save $4.97 when you buy 2!
Full Description: Epiphyllum hybrid 'TROXEL'S DRACULA' Has a Large bloom with red, almost white throat with light purple and purple with a red midstripe on outer petals, strong flat basket growth and a great bloomer! Fast grower depending on conditions.


text:
CFDs are complex instruments and come with a high risk of losing money rapidly due to leverage. 62% of retail investor accounts lose money when trading CFDs with this provider.
CFDs are complex instruments and come with a high risk of losing money rapidly due to leverage. 62% of retail investor accounts lose money when trading CFDs with this provider. You should consider whether you understand how CFDs, FX or any of our other products work and whether you can afford to take the high risk of losing your money. Losses can exceed deposits on some products.
TradersInstitutional and Partners
Forex options
Listed options
SaxoTraderGO
SaxoTraderPRO
Connectivity and APIs
Personal relationship manager
Dedicated sales traders
In-house analysts
Quarterly Outlook
Outrageous Predictions
Recession Watch
SaxoStrats
Ole S. Hansen
John J. Hardy
Althea Spinozzi
Christopher Dembik
Kay Van-Petersen
Peter Garnry
Eleanor Creagh
Saxo account
ISA account
SIPP account
VIP offering
About Saxo
About Saxo Group
Account funding
Search on home.saxo
search.accessibility.search
General.Accessibility.Close
SaxoTraderGO Login
Pricing/Margin Overview
Funding Instructions
Refer a friend programme
Forex Trading Hours
Orders and Execution Statistics
Careers at Saxo
Video length: 1 minute
Q4 Outlook: US equity outperformance to end?
Head of Equity Strategy, Saxo Bank Group
Summary: Investors with pure equity exposure are at risk to a potential global recession.
The end of US equity outperformance
In April 2008, the USD weakness in real terms that started in February 2002 ended months before a historical credit crunch and tailspin that required astronomical policy effort to mitigate. Since then, the USD has strengthened by 22% in real terms. And in August 2019, it reached a high enough level for the US Treasury Secretary Steve Mnuchin to declare that the US government does not intend to intervene in the USD for now — although he also said that the Trump administration had weighed up intervention. In other words, the US is planning to intervene if the Fed does not manage to weaken the USD through monetary policy.
This quarterly outlook examines how this situation will impact equity markets.
Emerging markets will thrive on weaker USD
The Fed uses the trade-weighted broad USD index to measure how strong the USD is relative to its trade partners' currencies.
Over the period from July 1995 to August 2019, equity returns in the US, the world ex-US and emerging markets have had a negative correlation to the USD. In other words, whenever the USD weakens equity markets should strengthen. As the world's reserve currency, largest trading currency and the currency used in commodity markets, the USD is an important component in financial conditions. Since the 2008 crash, emerging market countries have seen a large increase in issuance of USD bonds which has added another growth constraint from the USD.
If we break the period into regimes of stronger or weaker real USD, then a clear pattern appears. Whenever the USD strengthens, the US equity market outperforms the world ex-US and emerging markets and vice versa. As explained in our Quarterly Outlook, some form of USD intervention is the next logical policy step. Monetary policy has lost its effectiveness and fiscal stimulus is coming to slowly to offset the weakness in the global economy (the OECD's leading indicators have been declining for 18 straight months). A great irony of any USD intervention is that it will partly help China, which is not exactly the strategy of the Trump administration but as with everything in life there are always trade-offs.
When the strong USD regime ends, investors should overweight European and especially emerging market equities. There is also good support for this strategic position from a valuation perspective as the valuation spread — measured on the dividend yield — has soared to historically attractive levels for the world ex-US and emerging markets.
US equities have had a historical run
As we have been writing over many quarters, US equities are expensive in both relative and absolute terms. They remain expensive due to multiple factors ranging from higher share of buybacks, safe-haven status, higher profit growth driven by technology monopolies and finally a healed financial sector. Valuation metrics are difficult to use as timing tools because the market can often remain insane for long periods and maybe we are going through such a period. But rich valuation premiums to other equity markets are typically not a good starting point for relative superior returns in the future.
In February 2019, equity markets celebrated the 10-year anniversary of the equity market bottom during the financial crisis. The celebration occurred with a historic equity outperformance over aggregate US government bonds of 339% or 16% annualised. This is one of the best 10-year periods for US equities relative to government bonds since 1973, only marginally topped by the dot-com peak. It is quite likely that the next 10 years will not offer attractive equity returns in terms of outperformance and especially not when factoring in the downside risk relative to upside risk.
Stronghold EUR is not in panic mode yet
Our team's tactical asset allocation strategy, Stronghold, went online during the summer on our trading platforms through Saxo Bank's managed portfolio service SaxoSelect. The model behind Stronghold is based on a pure mathematical framework aimed at reducing drawdowns while trying to maximise returns. The model has a strict risk budget and therefore cuts risk quickly if the market structure become highly correlated across asset classes.
The EUR version of the model is up 10.6% as of mid-September and increased exposure in equities slowly over the first half of the year. But in the last three months, its equity exposure has been reduced to around 40% with the main exposure in minimum volatility stocks. The portfolio is balanced as of September also reflecting our general stance to clients. Remain invested in equities but in high quality and minimum volatility stocks. Investors attempting to escape low yields with pure equity exposure are running portfolios that are at risk to policy mistakes and a potential global recession — which has a probability of around 25-40% within the next 6-12 months.
Source: Saxo Bank
Access both platforms from your single Saxo account.
Preview platform Open Account
Climate and inflation two new priorities for markets
For the first time since WWII we sense a shift in which climate and the environment - not growth - will become the priority of governments and their citizens.
It's time for a greener portfolio
We believe that these green stocks could, over time, become some of the world's most valuable companies - even eclipsing the current technology monopolies.
The US dollar is a tough one to turn
Many of the USD-positive drivers noted above look well entrenched as 2020 gets under way, but we still look for a low ceiling to what has effectively been a flat US dollar over the last 18 months.
The Saxo Bank Group entities each provide execution-only service and access to Analysis permitting a person to view and/or use content available on or via the website. This content is not intended to and does not change or expand on the execution-only service. Such access and use are at all times subject to (i) The Terms of Use; (ii) Full Disclaimer; (iii) The Risk Warning; (iv) the Rules of Engagement and (v) Notices applying to Saxo News & Research and/or its content in addition (where relevant) to the terms governing the use of hyperlinks on the website of a member of the Saxo Bank Group by which access to Saxo News & Research is gained. Such content is therefore provided as no more than information. In particular no advice is intended to be provided or to be relied on as provided nor endorsed by any Saxo Bank Group entity; nor is it to be construed as solicitation or an incentive provided to subscribe for or sell or purchase any financial instrument. All trading or investments you make must be pursuant to your own unprompted and informed self-directed decision. As such no Saxo Bank Group entity will have or be liable for any losses that you may sustain as a result of any investment decision made in reliance on information which is available on Saxo News & Research or as a result of the use of the Saxo News & Research. Orders given and trades effected are deemed intended to be given or effected for the account of the customer with the Saxo Bank Group entity operating in the jurisdiction in which the customer resides and/or with whom the customer opened and maintains his/her trading account. Saxo News & Research does not contain (and should not be construed as containing) financial, investment, tax or trading advice or advice of any sort offered, recommended or endorsed by Saxo Bank Group and should not be construed as a record of our trading prices, or as an offer, incentive or solicitation for the subscription, sale or purchase in any financial instrument. To the extent that any content is construed as investment research, you must note and accept that the content was not intended to and has not been prepared in accordance with legal requirements designed to promote the independence of investment research and as such, would be considered as a marketing communication under relevant laws.
Please read our disclaimers:
- Notification on Non-Independent Investment Research (https://www.home.saxo/legal/niird/notification)
- Full disclaimer (https://www.home.saxo/en-gb/legal/disclaimer/saxo-disclaimer)
Queries & Complaints
Saxo Markets
40 Bank Street, 26th floor
E14 5DA
International Australia 中文 Česká republika Danmark France 中國香港 Hong Kong S.A.R Italia UAE الشرق الأوسط Nederland Norge Polska По-русски Schweiz Singapore Slovenská republika Suisse Switzerland United Kingdom 日本
Saxo Markets is a registered Trading Name of Saxo Capital Markets Ltd ('SCML'). SCML is authorised and regulated by the Financial Conduct Authority, Firm Reference Number 551422. Registered address: 26th Floor, 40 Bank Street, Canary Wharf, London E14 5DA. Company number 7413871.
Your browser cannot display this website correctly.
Our website is optimised to be browsed by a system running iOS 9.X and on desktop IE 10 or newer. If you are using an older system or browser, the website may look strange. To improve your experience on our site, please update your browser or system.