A conserved role for kinesin-5 in plant mitosis

The mitotic spindle of vascular plants is assembled and maintained by processes that remain poorly explored at a molecular level. Here, we report that AtKRP125c, one of four kinesin-5 motor proteins in arabidopsis, decorates microtubules throughout the cell cycle and appears to function in both interphase and mitosis. In a temperature-sensitive mutant, interphase cortical microtubules are disorganized at the restrictive temperature and mitotic spindles are massively disrupted, consistent with a defect in the stabilization of anti-parallel microtubules in the spindle midzone, as previously described in kinesin-5 mutants from animals and yeast. AtKRP125c introduced into mammalian epithelial cells by transfection decorates microtubules throughout the cell cycle but is unable to complement the loss of the endogenous kinesin-5 motor (Eg5). These results are among the first reports of any motor with a major role in anastral spindle structure in plants and demonstrate that the conservation of kinesin-5 motor function throughout eukaryotes extends to vascular plants

Nitrate regulates floral induction in Arabidopsis, acting independently of light, gibberellin and autonomous pathways

The transition from vegetative growth to reproduction is a major developmental event in plants. To maximise reproductive success, its timing is determined by complex interactions between environmental cues like the photoperiod, temperature and nutrient availability and internal genetic programs. While the photoperiod- and temperature- and gibberellic acid-signalling pathways have been subjected to extensive analysis, little is known about how nutrients regulate floral induction. This is partly because nutrient supply also has large effects on vegetative growth, making it difficult to distinguish primary and secondary influences on flowering. A growth system using glutamine supplementation was established to allow nitrate to be varied without a large effect on amino acid and protein levels, or the rate of growth. Under nitrate-limiting conditions, flowering was more rapid in neutral (12/12) or short (8/16) day conditions in C24, Col-0 and Laer. Low nitrate still accelerated flowering in late-flowering mutants impaired in the photoperiod, temperature, gibberellic acid and autonomous flowering pathways, in the fca co-2 ga1-3 triple mutant and in the ft-7 soc1-1 double mutant, showing that nitrate acts downstream of other known floral induction pathways. Several other abiotic stresses did not trigger flowering in fca co-2 ga1-3, suggesting that nitrate is not acting via general stress pathways. Low nitrate did not further accelerate flowering in long days (16/8) or in 35S::CO lines, and did override the late-flowering phenotype of 35S::FLC lines. We conclude that low nitrate induces flowering via a novel signalling pathway that acts downstream of, but interacts with, the known floral induction pathways

Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth

Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, total amino acids, photosynthetic pigments, starch and NAD(P)H. The plants showed little change in photosynthesis with the exception of a minor decrease in maximum photosynthetic efficiency (Fv/Fm), and a small decrease in growth compared to the wild type. These results reveal that even small changes in cytosolic NADP-dependent isocitrate dehydrogenase activity lead to noticeable alterations in the activities of enzymes involved in primary nitrate assimilation and in the synthesis of 2-oxoglutarate derived amino acids. These data are discussed within the context of current models for the role of the various isoforms of isocitrate dehydrogenase within plant amino acid metabolism

AXY3 encodes a α-xylosidase that impacts the structure and accessibility of the hemicellulose xyloglucan in Arabidopsis plant cell walls

Xyloglucan is the most abundant hemicellulose in the walls of dicots such as Arabidopsis. It is part of the load-bearing structure of a plant cell and its metabolism is thought to play a major role in cell elongation. However, the molecular mechanism by which xyloglucan carries out this and other functions in planta is not well understood. We performed a forward genetic screen utilizing xyloglucan oligosaccharide mass profiling on chemically mutagenized Arabidopsis seedlings to identify mutants with altered xyloglucan structures termed axy-mutants. One of the identified mutants, axy3.1, contains xyloglucan with a higher proportion of non-fucosylated xyloglucan subunits. Mapping revealed that axy3.1 contains a point mutation in XYLOSIDASE1 (XYL1) known to encode for an apoplastic glycoside hydrolase releasing xylosyl residues from xyloglucan oligosaccharides at the non-reducing end. The data support the hypothesis that AXY3/XYL1 is an essential component of the apoplastic xyloglucan degradation machinery and as a result of the lack of function in the various axy3-alleles leads not only to an altered xyloglucan structure but also a xyloglucan that is less tightly associated with other wall components. However, the plant can cope with the excess xyloglucan relatively well as the mutant does not display any visible growth or morphological phenotypes with the notable exception of shorter siliques and reduced fitness. Taken together, these results demonstrate that plant apoplastic hydrolases have a larger impact on wall polymer structure and function than previously thought

Oak root response to ectomycorrhizal symbiosis establishment: RNA-Seq derived transcript identification and expression profiling

Ectomycorrhizal symbiosis is essential for the life and health of trees in temperate and boreal forests where it plays a major role in nutrient cycling and in functioning of the forest ecosystem. Trees with ectomycorrhizal root tips are more tolerant to environmental stresses, such as drought, and biotic stresses such as root pathogens. Detailed information on these molecular processes is essential for the understanding of symbiotic tissue development in order to optimize the benefits of this natural phenomenon. Next generation sequencing tools allow the analysis of non model ectomycorrhizal plant-fungal interactions that can contribute to find the "symbiosis toolkits" and better define the role of each partner in the mutualistic interaction. By using 454 pyrosequencing we compared ectomycorrhizal cork oak roots with non-symbiotic roots. From the two cDNA libraries sequenced, over 2 million reads were obtained that generated 19,552 cork oak root unique transcripts. A total of 2238 transcripts were found to be differentially expressed when ECM roots were compared with non-symbiotic roots. Identification of up- and down-regulated gens in ectomycorrhizal roots lead to a number of insights into the molecular mechanisms governing this important symbiosis. In cork oak roots, ectomycorrhizal colonization resulted in extensive cell wall remodelling, activation of the secretory pathway, alterations in flavonoid biosynthesis, and expression of genes involved in the recognition of fungal effectors. In addition, we identified genes with putative roles in symbiotic processes such as nutrient exchange with the fungal partner, lateral root formation or root hair decay. These findings provide a global overview of the transcriptome of an ectomycorrhizal host root, and constitute a foundation for future studies on the molecular events controlling this important symbiosis.This work was funded by the Portuguese Foundation for Science and Technology (www.fct.pt) in the frame of the project Cork Oak EST Consortium SOBREIRO/0034/2009. Post-doc grant to MS was supported by the Portuguese Foundation for Science and Technology (SFRH/BPD/25661/2005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Cloning and Characterization of Maize miRNAs Involved in Responses to Nitrogen Deficiency

Although recent studies indicated that miRNAs regulate plant adaptive responses to nutrient deprivation, the functional significance of miRNAs in adaptive responses to nitrogen (N) limitation remains to be explored. To elucidate the molecular biology underlying N sensing/signaling in maize, we constructed four small RNA libraries and one degradome from maize seedlings exposed to N deficiency. We discovered a total of 99 absolutely new loci belonging to 47 miRNA families by small RNA deep sequencing and degradome sequencing, as well as 9 new loci were the paralogs of previously reported miR169, miR171, and miR398, significantly expanding the reported 150 high confidence genes within 26 miRNA families in maize. Bioinformatic and subsequent small RNA northern blot analysis identified eight miRNA families (five conserved and three newly identified) differentially expressed under the N-deficient condition. Predicted and degradome-validated targets of the newly identified miRNAs suggest their involvement in a broad range of cellular responses and metabolic processes. Because maize is not only an important crop but is also a genetic model for basic biological research, our research contributes to the understanding of the regulatory roles of miRNAs in plant adaption to N-deficiency stress

The OSU1/QUA2/TSD2-Encoded Putative Methyltransferase Is a Critical Modulator of Carbon and Nitrogen Nutrient Balance Response in Arabidopsis

The balance between carbon (C) and nitrogen (N) nutrients must be tightly coordinated so that cells can optimize their opportunity for metabolism, growth and development. However, the C and N nutrient balance perception and signaling mechanism remains poorly understood. Here, we report the isolation and characterization of two allelic oversensitive to sugar1 mutants (osu1-1, osu1-2) in Arabidopsis thaliana. Using the cotyledon anthocyanin accumulation and root growth inhibition assays, we show that the osu1 mutants are more sensitive than wild-type to both of the imbalanced C/N conditions, high C/low N and low C/high N. However, under the balanced C/N conditions (low C/low N or high C/high N), the osu1 mutants have similar anthocyanin levels and root lengths as wild-type. Consistently, the genes encoding two MYB transcription factors (MYB75 and MYB90) and an Asn synthetase isoform (ASN1) are strongly up-regulated by the OSU1 mutation in response to high C/low N and low C/high N, respectively. Furthermore, the enhanced sensitivity of osu1-1 to high C/low N with respect to anthocyanin accumulation but not root growth inhibition can be suppressed by co-suppression of MYB75, indicating that MYB75 acts downstream of OSU1 in the high C/low N imbalance response. Map-based cloning reveals that OSU1 encodes a member of a large family of putative methyltransferases and is allelic to the recently reported QUA2/TSD2 locus identified in genetic screens for cell-adhesion-defective mutants. Accumulation of OSU1/QUA2/TSD2 transcript was not regulated by C and N balance, but the OSU1 promoter was slightly more active in the vascular system. Taken together, our results show that the OSU1/QUA2/TSD2-encoded putative methyltransferase is required for normal C/N nutrient balance response in plants

Genome-Wide Identification of MicroRNAs in Response to Low Nitrate Availability in Maize Leaves and Roots

BACKGROUND: Nitrate is the major source of nitrogen available for many crop plants and is often the limiting factor for plant growth and agricultural productivity especially for maize. Many studies have been done identifying the transcriptome changes under low nitrate conditions. However, the microRNAs (miRNAs) varied under nitrate limiting conditions in maize has not been reported. MiRNAs play important roles in abiotic stress responses and nutrient deprivation. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used the SmartArray™ and GeneChip® microarray systems to perform a genome-wide search to detect miRNAs responding to the chronic and transient nitrate limiting conditions in maize. Nine miRNA families (miR164, miR169, miR172, miR397, miR398, miR399, miR408, miR528, and miR827) were identified in leaves, and nine miRNA families (miR160, miR167, miR168, miR169, miR319, miR395, miR399, miR408, and miR528) identified in roots. They were verified by real time stem loop RT-PCR, and some with additional time points of nitrate limitation. The miRNAs identified showed overlapping or unique responses to chronic and transient nitrate limitation, as well as tissue specificity. The potential target genes of these miRNAs in maize were identified. The expression of some of these was examined by qRT-PCR. The potential function of these miRNAs in responding to nitrate limitation is described. CONCLUSIONS/SIGNIFICANCE: Genome-wide miRNAs responding to nitrate limiting conditions in maize leaves and roots were identified. This provides an insight into the timing and tissue specificity of the transcriptional regulation to low nitrate availability in maize. The knowledge gained will help understand the important roles miRNAs play in maize responding to a nitrogen limiting environment and eventually develop strategies for the improvement of maize genetics

Immunolocalization of cell wall polymers in grapevine (Vitis vinifera) internodes under nitrogen, phosphorus or sulfur deficiency

Abstract The impact on cell wall (CW) of the deficiency in nitrogen (–N), phosphorus (–P) or sulphur (–S), known to impair essential metabolic pathways, was investigated in the economically important fruit species Vitis vinifera L. Using cuttings as an experimental model a reduction in total internode number and altered xylem shape was observed. Under –N an increased internode length was also seen. CW composition, visualised after staining with calcofluor white, Toluidine blue and ruthenium red, showed decreased cellulose in all stresses and increased pectin content in recently formed internodes under –N compared to the control. Using CW-epitope specific monoclonal antibodies (mAbs), lower amounts of extensins incorporated in the wall were also observed under –N and –P conditions. Conversely, increased pectins with a low degree of methyl-esterification and richer in long linear 1,5-arabinan rhamnogalacturonan-I (RG-I) side chains were observed under –N and –P in mature internodes which, in the former condition, were able to form dimeric association through calcium ions. –N was the only condition in which 1,5-arabinan branched RG- content was not altered, as –P and –S older internodes showed, respectively, lower and higher amounts of this polymer. Higher xyloglucan content in older internodes was also observed under –N. The results suggest that impairments of specific CW components led to changes in the deposition of other polymers to promote stiffening of the CW. The unchanged extensin amount observed under –S may contribute to attenuating the effects on the CW integrity caused by this stress. Our work showed that, in organized V. vinifera tissues, modifications in a given CW component can be compensated by synthesis of different polymers and/or alternative linking between polymers. The results also pinpoint different strategies at the CW level to overcome mineral stress depending on how essential they are to cell growth and plant development

Chemical PARP Inhibition Enhances Growth of Arabidopsis and Reduces Anthocyanin Accumulation and the Activation of Stress Protective Mechanisms

Poly-ADP-ribose polymerase (PARP) post-translationally modifies proteins through the addition of ADP-ribose polymers, yet its role in modulating plant development and stress responses is only poorly understood. The experiments presented here address some of the gaps in our understanding of its role in stress tolerance and thereby provide new insights into tolerance mechanisms and growth. Using a combination of chemical and genetic approaches, this study characterized phenotypes associated with PARP inhibition at the physiological level. Molecular analyses including gene expression analysis, measurement of primary metabolites and redox metabolites were used to understand the underlying processes. The analysis revealed that PARP inhibition represses anthocyanin and ascorbate accumulation under stress conditions. The reduction in defense is correlated with enhanced biomass production. Even in unstressed conditions protective genes and molecules are repressed by PARP inhibition. The reduced anthocyanin production was shown to be based on the repression of transcription of key regulatory and biosynthesis genes. PARP is a key factor for understanding growth and stress responses of plants. PARP inhibition allows plants to reduce protection such as anthocyanin, ascorbate or Non-Photochemical-Quenching whilst maintaining high energy levels likely enabling the observed enhancement of biomass production under stress, opening interesting perspectives for increasing crop productivity