NODULE INCEPTION Directly Targets <em>NF-Y</em> Subunit Genes to Regulate Essential Processes of Root Nodule Development in <em>Lotus japonicus</em>

<div><p>The interactions of legumes with symbiotic nitrogen-fixing bacteria cause the formation of specialized lateral root organs called root nodules. It has been postulated that this root nodule symbiosis system has recruited factors that act in early signaling pathways (common <i>SYM</i> genes) partly from the ancestral mycorrhizal symbiosis. However, the origins of factors needed for root nodule organogenesis are largely unknown. <i>NODULE INCEPTION</i> (<i>NIN</i>) is a nodulation-specific gene that encodes a putative transcription factor and acts downstream of the common <i>SYM</i> genes. Here, we identified two Nuclear Factor-Y (NF-Y) subunit genes, <i>LjNF-YA1</i> and <i>LjNF-YB1</i>, as transcriptional targets of NIN in <i>Lotus japonicus</i>. These genes are expressed in root nodule primordia and their translational products interact in plant cells, indicating that they form an NF-Y complex in root nodule primordia. The knockdown of <i>LjNF-YA1</i> inhibited root nodule organogenesis, as did the loss of function of <i>NIN</i>. Furthermore, we found that <i>NIN</i> overexpression induced root nodule primordium-like structures that originated from cortical cells in the absence of bacterial symbionts. Thus, NIN is a crucial factor responsible for initiating nodulation-specific symbiotic processes. In addition, ectopic expression of either <i>NIN</i> or the <i>NF-Y</i> subunit genes caused abnormal cell division during lateral root development. This indicated that the <i>Lotus</i> NF-Y subunits can function to stimulate cell division. Thus, transcriptional regulation by NIN, including the activation of the <i>NF-Y</i> subunit genes, induces cortical cell division, which is an initial step in root nodule organogenesis. Unlike the legume-specific NIN protein, NF-Y is a major CCAAT box binding protein complex that is widespread among eukaryotes. We propose that the evolution of root nodules in legume plants was associated with changes in the function of NIN. NIN has acquired functions that allow it to divert pathways involved in the regulation of cell division to root nodule organogenesis.</p> </div

RT–PCR analysis of the expression of early nodulin genes and <i>LjNF-Y</i> subunit genes.

<p>Expression was analyzed for <i>ENOD40-1</i> (A), <i>ENOD40-2</i> (B), <i>ENOD2</i> (C), <i>LjNF-YA1</i> (D), <i>LjNF-YB1</i> (E), and <i>NIN</i> (F). Plants were cultured for 3 weeks without <i>M. loti</i> inoculation. Total RNA was isolated from roots transformed with either an empty vector, <i>ProLjUb-LjNF-YA1</i> plus <i>Pro35S-LjNF-YB1</i> (<i>NF-YA1 NF-YB1</i> OE), or <i>ProLjUb-NIN</i> (<i>NIN</i> OE). <i>ProLjUb-NIN</i> roots that exhibited altered structures (+) were harvested separately from roots with no morphological alterations (−). The means and SDs from 3 biological repeats are shown.</p

<i>NIN</i> overexpression induces cortical cell division.

<p>(A) A Gifu (wild-type) root was transformed with <i>ProLjUb-NIN</i> and then cultured for 6 weeks in the absence of <i>M. loti</i>. Bumps (arrowheads) and malformed lateral roots (arrows) are indicated. (B,C) Cleared roots that were transformed with either an empty vector (B) or <i>ProLjUb-NIN</i> (C). The fractions of plants with bumps are shown in parentheses. (D) A transverse section of a root nodule primordium (10 dai) formed on a MG-20 (wild-type) root that was transformed with the empty vector. (E,F) Transverse sections of bumps formed on uninoculated MG-20 roots that were transformed with <i>ProLjUb-NIN</i>. Blue and red lines in (F) represent the outer edges of the endodermis and the boundary of the region with dividing cortical cells, respectively. (G–I) <i>in situ</i> RNA hybridization of <i>ENOD40-1</i> in transverse sections of bumps caused by <i>NIN</i> overexpression, using either antisense (G,H) or sense probes (I). Asterisks indicate the central xylem. Arrows indicate the pericycle with <i>in situ</i> signals. Bars: 5 mm in (A); 0.2 mm in (B,C); 0.1 mm in (D–F); 50 µm in (G–I).</p

NIN directly targets <i>LjNF-YA1</i> and <i>LjNF-YB1</i>.

<p>(A) A diagram of the <i>LjNF-YA1</i> and <i>LjNF-YB1</i> promoter regions. These regions were used for the promoter-GUS reporters (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003352#pgen-1003352-g003" target="_blank">Figure 3</a>). Gray lines indicate 5′-UTRs. Regions analyzed by RT-PCR for the ChIP assays in (B) are shown as blue lines and probes used for EMSA in (C) and (D) are shown as green lines. The red numbers indicate regions and probes that gave positive results in the ChIP assays and EMSAs. Arrowheads indicate positions of the NIN-binding sites. (B) ChIP assays using either <i>ProLjUb-NIN-my</i>c roots or control (empty vector) roots. The means and SDs from 3 biological repeats are shown. (C,D) EMSAs for analyzing NIN binding to <i>LjNF-YA1</i> (C) and <i>LjNF-YB1</i> (D) promoter regions. NIN-myc (Full), NIN(520–878)-myc (C-ter), and <i>in vitro</i> translation products without templates (control) were incubated with ³²P-labeled probes shown in (A). Arrowheads indicate mobility-shifted bands specifically detected when incubated with NIN proteins. (E) An alignment of the partial nucleotide sequences of probes that were bound by NIN. yB1a, yB1b, yA1, E16a, are E16b correspond to NBSs found in the promoters of <i>LjNF-YB1</i>, <i>LjNF-YA1</i>, and the PLDP-encoding gene. Red and blue lines indicate nucleotides that are required for NIN-binding to NBS-yB1a, or that influence binding, respectively (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003352#pgen.1003352.s005" target="_blank">Figure S5</a>). NBS-yB1m is an NBS-yB1a derivative with nucleotide substitutions. Comparison of NIN-binding sequences illustrated by logo is shown at the bottom. (F) GUS expression in tobacco leaf disks transformed with the indicated constructs. Bar: 1 mm. (G) EMSA for analyzing NIN binding to NBS-yB1a and the NBS-yB1a-like sequences. Arrowheads indicate probes that were bound by NIN proteins.</p

Inhibition of root nodule development by the knockdown of <i>LjNF-YA1</i>.

<p>Roots that were transformed with either <i>ProLjUb-RNAi-LjNF-YA1</i> (A,C) or an empty vector (B,D) were inoculated with DsRed-labeled <i>M. loti</i> for 14 days. (A,B) Root nodule formation in transformed roots. Fluorescence is visible from GFP (the root transformation marker) and from DsRed expressed in <i>M. loti</i>. Arrowheads indicate root nodules. The fractions of plants that formed root nodules on GFP-positive roots are shown in parentheses. (C,D) Infection thread formation in transformed roots. Fluorescence from GFP and DsRed is shown in the upper panels and bright field images are shown in the bottom panels of the same roots. Arrowheads indicate infection threads visualized by DsRed-labeled <i>M. loti</i>. Bars: 5 mm in (A,B), 0.2 mm in (C,D). (E) RT-PCR analyses of gene expression in roots that were transformed with either the empty vector, <i>ProLjUb-RNAi-LjNF-YA1</i>, or <i>ProLjUb-RNAi-LjNF-YB1</i>. Roots were inoculated with (2 dai; days after inoculation) or without (−) <i>M. loti</i>. Expression was analyzed for <i>LjNF-YB1</i> and three <i>L. japonicus</i> NF-Y subunit A genes (<i>LjNF-YA1</i>, <i>LjNF-YA2</i>, and <i>CBF-A22 </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003352#pgen.1003352-Asamizu1" target="_blank">[57]</a>). The means and SDs from 3 biological repeats are shown.</p

Expression of candidate NIN-target genes in roots that were ectopically expressing the NIN protein.

<p>RT-PCR was used to analyze gene expression in <i>nin-2</i> roots that were transformed with <i>Pro35S-NIN-GR</i>. Roots were treated as indicated in the figure. For CHX plus DEX, the DEX was added after pre-incubation with CHX for 30 min. chr4.CM0179.190.r2.m encodes a plastocyanin-like domain-containing protein (PLDP) and chr6.CM1757.140.r2.m encodes an AP2/ERF family protein. These genes are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003352#pgen.1003352.s002" target="_blank">Figure S2</a>. The promoter of the former gene possesses NIN-binding nucleotide sequences (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003352#pgen-1003352-g002" target="_blank">Figure 2</a>). The latter gene was used as a negative control for DEX treatment. The means and SDs from 3 biological repeats are shown.</p

NIN and NF-Y subunits regulate cell division.

<p>(A–C) Lateral roots formed on MG-20 roots that were transformed with either an empty vector (A), <i>ProLjUb-LjNF-YA1</i> (B), or <i>ProLjUb-LjNF-YA1 Pro35S-LjNF-YB1</i> (C). Roots were cultured in the absence of <i>M. loti</i>. (D–G) Longitudinal sections of lateral roots formed on MG-20 roots that were transformed with either the empty vector (D), <i>ProLjUb-LjNF-YA1 Pro35S-LjNF-YB1</i> (E,F), or <i>ProLjUb-NIN</i> (G). An asterisk in (F) indicates an additional lateral root meristem-like structure. (H,I) Magnified images of the boxed regions in (D) and (E), respectively. (J) A longitudinal section of the root that was transformed with <i>ProLjUb-LjNF-YA1</i> and <i>Pro35S-LjNF-YB1</i>. Note the presence of small cells that were generated by cortical cell division. Bars: 1 mm in (A–C); 0.1 mm in (D–G,J); 0.2 mm in (H,I).</p

Tissue specificity of MIPS and seed storage proteins in developing seeds of as determined by immunoblot analysis with MIPS2, 2S albumin, and 12S globulin antibodies

Developing seeds (torpedo to mature stages) were separated into seed coat (Sc) and seed contents which were either left intact as endosperm and embryo (En+Emb), or washed to remove the endosperm (Emb). p2S and 2S are pro- and mature forms of 2S albumin, and p12S and 12S are pro- and mature forms of 12S globulin, respectively. The arrowhead at 60 kDa indicates MIPS.<p><b>Copyright information:</b></p><p>Taken from "Localization of -inositol-1-phosphate synthase to the endosperm in developing seeds of "</p><p></p><p>Journal of Experimental Botany 2008;59(11):3069-3076.</p><p>Published online 4 Jul 2008</p><p>PMCID:PMC2504351.</p><p></p

Investigating the Relationship between in Vitro–in Vivo Genotoxicity: Derivation of Mechanistic QSAR Models for in Vivo Liver Genotoxicity and in Vivo Bone Marrow Micronucleus Formation Which Encompass Metabolism

Strategic testing as part of an integrated testing strategy (ITS) to maximize information and avoid the use of animals where possible is fast becoming the norm with the advent of new legislation such as REACH. Genotoxicity is an area where regulatory testing is clearly defined as part of ITS schemes. Under REACH, the specific information requirements depend on the tonnage manufactured or imported. Two types of test systems exist to meet these information requirements, in vivo genotoxicity assays, which take into account the whole animal, and in vitro assays, which are conducted outside the living mammalian organism using microbial or mammalian cells under appropriate culturing conditions. Clearly, with these different broad experimental categories, results for a given chemical can often differ, which presents challenges in the interpretation as well as in attempting to model the results in silico. This study attempted to compare the differences between in vitro and in vivo genotoxicity results, to rationalize these differences with plausible hypothesis in concert with available data. Two proof of concept (Q)­SAR models were developed, one for in vivo genotoxicity effects in liver and a second for in vivo micronucleus formation in bone marrow. These “mechanistic models” will be of practical value in testing strategies, and both have been implemented into the TIMES software platform (http://oasis-lmc.org) to help predict the genotoxicity outcome of new untested chemicals

Investigating the Relationship between in Vitro–in Vivo Genotoxicity: Derivation of Mechanistic QSAR Models for in Vivo Liver Genotoxicity and in Vivo Bone Marrow Micronucleus Formation Which Encompass Metabolism

Strategic testing as part of an integrated testing strategy (ITS) to maximize information and avoid the use of animals where possible is fast becoming the norm with the advent of new legislation such as REACH. Genotoxicity is an area where regulatory testing is clearly defined as part of ITS schemes. Under REACH, the specific information requirements depend on the tonnage manufactured or imported. Two types of test systems exist to meet these information requirements, in vivo genotoxicity assays, which take into account the whole animal, and in vitro assays, which are conducted outside the living mammalian organism using microbial or mammalian cells under appropriate culturing conditions. Clearly, with these different broad experimental categories, results for a given chemical can often differ, which presents challenges in the interpretation as well as in attempting to model the results in silico. This study attempted to compare the differences between in vitro and in vivo genotoxicity results, to rationalize these differences with plausible hypothesis in concert with available data. Two proof of concept (Q)­SAR models were developed, one for in vivo genotoxicity effects in liver and a second for in vivo micronucleus formation in bone marrow. These “mechanistic models” will be of practical value in testing strategies, and both have been implemented into the TIMES software platform (http://oasis-lmc.org) to help predict the genotoxicity outcome of new untested chemicals