Auxin and cytokinin control formation of the quiescent centre in the adventitious root apex of arabidopsis

Background and Aims

Adventitious roots (ARs) are part of the root system in numerous plants, and are required for successful micropropagation. In the Arabidopsis thaliana primary root (PR) and lateral roots (LRs), the quiescent centre (QC) in the stem cell niche of the meristem controls apical growth with the involvement of auxin and cytokinin. In arabidopsis, ARs emerge in planta from the hypocotyl pericycle, and from different tissues in in vitro cultured explants, e.g. from the stem endodermis in thin cell layer (TCL) explants. The aim of this study was to investigate the establishment and maintenance of the QC in arabidopsis ARs, in planta and in TCL explants, because information about this process is still lacking, and it has potential use for biotechnological applications.

Methods

Expression of PR/LR QC markers and auxin influx (LAX3)/efflux (PIN1) genes was investigated in the presence/absence of exogenous auxin and cytokinin. Auxin was monitored by the DR5::GUS system and cytokinin by immunolocalization. The expression of the auxin-biosynthetic YUCCA6 gene was also investigated by in situ hybridization in planta and in AR-forming TCLs from the indole acetic acid (IAA)-overproducing superroot2-1 mutant and its wild type.

Key Results

The accumulation of auxin and the expression of the QC marker WOX5 characterized the early derivatives of the AR founder cells, in planta and in in vitro cultured TCLs. By determination of PIN1 auxin efflux carrier and LAX3 auxin influx carrier activities, an auxin maximum was determined to occur at the AR tip, to which WOX5 expression was restricted, establishing the positioning of the QC. Cytokinin caused a restriction of LAX3 and PIN1 expression domains, and concomitantly the auxin biosynthesis YUCCA6 gene was expressed in the apex.

Conclusions

In ARs formed in planta and TCLs, the QC is established in a similar way, and auxin transport and biosynthesis are involved through cytokinin tuning.     

Auxin transport in maize roots in response to localized nitrate supply

Background and Aims

Roots typically respond to localized nitrate by enhancing lateral-root growth. Polar auxin transport has important roles in lateral-root formation and growth; however, it is a matter of debate whether or how auxin plays a role in the localized response of lateral roots to nitrate.

Methods

Treating maize (Zea mays) in a split-root system, auxin levels were quantified directly and polar transport was assayed by the movement of [³H]IAA. The effects of exogenous auxin and polar auxin transport inhibitors were also examined.

Key Results

Auxin levels in roots decreased more in the nitrate-fed compartment than in the nitrate-free compartment and nitrate treatment appeared to inhibit shoot-to-root auxin transport. However, exogenous application of IAA only partially reduced the stimulatory effect of localized nitrate, and auxin level in the roots was similarly reduced by local applications of ammonium that did not stimulate lateral-root growth.

Conclusions

It is concluded that local applications of nitrate reduced shoot-to-root auxin transport and decreased auxin concentration in roots to a level more suitable for lateral-root growth. However, alteration of root auxin level alone is not sufficient to stimulate lateral-root growth.     

Nitric oxide modulates sensitivity to ABA

Nitric oxide (NO) is a gas with crucial signaling functions in plant defense and development. As demonstrated by generating a triple nia1nia2noa1-2 mutant with extremely low levels of NO (February 2010 issue of Plant Physiology), NO is synthesized in plants through mainly two different pathways involving nitrate reductase (NR/NIA) and NO Associated 1 (AtNOA1) proteins. Depletion of basal NO levels leads to a priming of ABA-triggered responses that causes hypersensitivity to this hormone and results in enhanced seed dormancy and decreased seed germination and seedling establishment in the triple mutant. NO produced under non-stressed conditions represses inhibition of seed developmental transitions by ABA. Moreover, NO plays a positive role in post-germinative vegetative development and also exerts a critical control of ABA-related functions on stomata closure. The triple nia1nia2noa1-2 mutant is hypersensitive to ABA in stomatal closure thus resulting in a extreme phenotype of resistance to drought. In the light of the recent discovery of PYR/PYL/RCAR as a family of potential ABA receptors, regulation of ABA sensitivity by NO may be exerted either directly on ABA receptors or on downstream signalling components; both two aspects that deserve our present and future attention.Key words: nitric oxide, abscisic acid, seed germination, stomata openingPlant development is the result of the succesfull execution of several programs that control the transition between different growth phases. Every developmental transition is regulated through coordinated mechanisms that involved exogenous environmental factors such as light and temperature as well as endogenous cues, including levels of primary and secondary metabolites. Among the latter, hormones such as gibberellins (GA), auxins, citokinins, ethylene and abscisic acid (ABA) participate in the control of most of the developmental transitions.^1,2 During the last years, nitric oxide (NO) has gained an increasing role as an essential player in plant defense responses³ as well as a co-regulator of many developmental processes.⁴ However, studies of NO function as a regulatory molecule in plants have been hampered by the scanty, limited and controversial knowledge on how this gas is synthesized in plants.^5,6 This situation has moved researchers in this area to adopt pharmacological approaches based on chemicals acting as artificial NO donors as well as inhibitors or scavengers of NO action. The lack of specificity and the inherent artificial effects of these chemicals can be overcome by genetic approaches based on the use of mutants with endogenous low levels of NO. In February 2010 issue of Plant Physiology, we report the generation and further characterization of a triple nia1nia2noa1-2 mutant that contains extremely low levels of NO due to the impairment of two NO biosynthetic pathways involving nitrate reductase (NIA/NR) or NO Associated 1 (AtNOA1) proteins.⁷ These findings support that NO is mainly produced through those pathways in Arabidopsis. However, the possible existence of a minor still uncharacterized pathways involved in the residual production of NO can not be ruled out at this time.Further functional characterization of nia1nia2noa1-2 mutant in terms of development has pointed to NO as an overall positive regulator of plant growth, affecting to almost every developmental stage from seed germination to reproductive development. Accordingly, triple mutant plants display a delayed growth resulting in small shoot and root size and they also produce low amounts of viable seeds.⁷Dormancy and seed germination are developmental programs largely regulated by the combined action of GA and ABA.¹ GA promote breaking of dormancy and promote germination whereas ABA acts as a brake in those processes, thus ensuring a timely seed germination. Our data from the characterization of dormancy and seed germination in the nia1nia2noa1-2 mutant suggest that NO’s role in the control of those processes may be exerted through modulation of the sensitivity to ABA (Fig. 1A). Seeds from NO deficient plants have increased dormancy and lower seed germination and seedling establishment rates than wild type seeds due to the enhanced ABA inhibitory action. These effects can be reversed by exogenous application of NO to nia1nia2noa1-2, suggesting that the sensitivity to ABA is actually controlled by the endogenous levels of NO. The recent identification of PYR/PYL/RCAR family of ABA receptors,^8,9 and the further characterization of the essential ABA regulatory module including receptor, protein phosphatases of the 2C class and kinases of the SnRK2 family¹⁰ point to these components as potential targets of NO in regulating sensitivity to ABA (Fig. 1B). This work is in progress in our lab but we already know that some of the PYR/PYL/RCAR receptors and SnRKs are actually regulated by NO and also that this regulation may be exerted at different levels (Lozano-Juste J and León J, unpublished data).Open in a separate windowFigure 1Interactions between NO and ABA results in modulated sensitivity to ABA throughout development. (A) NO synthesized through nitrate reductase (NR/NIA) and NO associatedI (AtNOA1) protein regulate germinative and post-germinative development as well as stomata movements through modulation of the sensitivity to ABA. Arrows and bars represent positive and negative effects, and the thickness of lines are proportional to the magnitud of regulatory effects. (B) Scheme of a minimal ABA signalling module and the potential targets of NO. Dashed lines represent effects still to be demonstrated. (C) ABA signalling in stomata guard cells through Ca²⁺-dependent and -independent pathways and the potential interactions with NO as represented by dashed lines.The enhanced sensitivity to ABA observed in germinative and post-germinative development of nia1nia2noa1-2, is extended throughout plant life cycle and it is actually the cause of the very strong resistance of nia1nia2noa1-2 plants to water deficit conditions.⁷ Stomatal aperture is a fine-tuned process controlled mainly through a balance between the light-promoted opening and the ABA-mediated promotion of closure and inhibition of opening¹¹ (Fig. 1A). It has been previously reported that ABA function on stomata movements involve the participation of NO as well as Ca²⁺ in such a way that Ca²⁺ chelators and NO scavengers block ABA action on stomata movements.¹² Stomata of nia1nia2noa1-2 leaves, despite of being depleted of NO, are not impaired for ABA inhibition of stomata opening but, in turn, they seem to be primed for a more efficient ABA response (Fig. 1A). Contrary to the Ca²⁺ requirements for ABA action on wild type stomata movements, this process is not affected by Ca²⁺ chelators in nia1nia2noa1-2 stomata, and it thus seems to be independent of Ca²⁺ in NO-deficient backgrounds (Fig. 1C). As mentioned above, NO might regulate sensitivity to ABA by acting on ABA receptors or on SnRKs, some of which are Ca²⁺-independent kinases. Both receptors and Ca^2+--independent kinases are likely targets of NO in the modulation of stomata sensitivity to ABA thus explaining the more efficient stomata closure in nia1nia2noa1-2 leaves, and the consequent low rates of evapotranspiration that leads to the extreme resistance of triple mutant plants to drought.The future characterization of the interactions between NO and key components of ABA signaling will be the basis for a better knowledge of the functional interactions between different hormones in plant development and defense, but it will also open up new possibilities of identifying new targets and strategies leading to improved drought resistance.     

Composite Cucurbita pepo plants with transgenic roots as a tool to study root development

Background and Aims

In most plant species, initiation of lateral root primordia occurs above the elongation zone. However, in cucurbits and some other species, lateral root primordia initiation and development takes place in the apical meristem of the parental root. Composite transgenic plants obtained by Agrobacterium rhizogenes-mediated transformation are known as a suitable model to study root development. The aim of the present study was to establish this transformation technique for squash.

Methods

The auxin-responsive promoter DR5 was cloned into the binary vectors pKGW-RR-MGW and pMDC162-GFP. Incorporation of 5-ethynyl-2′-deoxyuridine (EdU) was used to evaluate the presence of DNA-synthesizing cells in the hypocotyl of squash seedlings to find out whether they were suitable for infection. Two A. rhizogenes strains, R1000 and MSU440, were used. Roots containing the respective constructs were selected based on DsRED1 or green fluorescent protein (GFP) fluorescence, and DR5::Egfp-gusA or DR5::gusA insertion, respectively, was verified by PCR. Distribution of the response to auxin was visualized by GFP fluorescence or β-glucuronidase (GUS) activity staining and confirmed by immunolocalization of GFP and GUS proteins, respectively.

Key Results

Based on the distribution of EdU-labelled cells, it was determined that 6-day-old squash seedlings were suited for inoculation by A. rhizogenes since their root pericycle and the adjacent layers contain enough proliferating cells. Agrobacterium rhizogenes R1000 proved to be the most virulent strain on squash seedlings. Squash roots containing the respective constructs did not exhibit the hairy root phenotype and were morphologically and structurally similar to wild-type roots.

Conclusions

The auxin response pattern in the root apex of squash resembled that in arabidopsis roots. Composite squash plants obtained by A. rhizogenes-mediated transformation are a good tool for the investigation of root apical meristem development and root branching.     

Successive microsporogenesis affects pollen aperture pattern in the tam mutant of Arabidopsis thaliana

Background and Aims

The tam (tardy asynchronous meiosis) mutant of Arabidopsis thaliana, which exhibits a modified cytokinesis with a switch from simultaneous to successive cytokinesis, was used to perform a direct test of the implication of cytokinesis in aperture-pattern ontogeny of angiosperm pollen grains. The aperture pattern corresponds to the number and arrangement of apertures (areas of the pollen wall permitting pollen tube germination) on the surface of the pollen grain.

Methods

A comparative analysis of meiosis and aperture distribution was performed in two mutant strains of arabidopsis: quartet and quartet-tam.

Key Results

While the number of apertures is not affected in the quartet-tam mutant, the arrangement of the three apertures is modified compared with the quartet, resulting in a different aperture pattern.

Conclusions

These results directly demonstrate the relationship between the type of sporocytic cytokinesis and pollen aperture-pattern ontogeny.     

Combined in silico/in vivo analysis of mechanisms providing for root apical meristem self-organization and maintenance

Background and Aims

The root apical meristem (RAM) is the plant stem cell niche which provides for the formation and continuous development of the root. Auxin is the main regulator of RAM functioning, and auxin maxima coincide with the sites of RAM initiation and maintenance. Auxin gradients are formed due to local auxin biosynthesis and polar auxin transport. The PIN family of auxin transporters plays a critical role in polar auxin transport, and two mechanisms of auxin maximum formation in the RAM based on PIN-mediated auxin transport have been proposed to date: the reverse fountain and the reflected flow mechanisms.

Methods

The two mechanisms are combined here in in silico studies of auxin distribution in intact roots and roots cut into two pieces in the proximal meristem region. In parallel, corresponding experiments were performed in vivo using DR5::GFP Arabidopsis plants.

Key Results

The reverse fountain and the reflected flow mechanism naturally cooperate for RAM patterning and maintenance in intact root. Regeneration of the RAM in decapitated roots is provided by the reflected flow mechanism. In the excised root tips local auxin biosynthesis either alone or in cooperation with the reverse fountain enables RAM maintenance.

Conclusions

The efficiency of a dual-mechanism model in guiding biological experiments on RAM regeneration and maintenance is demonstrated. The model also allows estimation of the concentrations of auxin and PINs in root cells during development and under various treatments. The dual-mechanism model proposed here can be a powerful tool for the study of several different aspects of auxin function in root.     

Auxin distribution is differentially affected by nitrate in roots of two rice cultivars differing in responsiveness to nitrogen

Background and Aims

Although ammonium (NH₄⁺) is the preferred form of nitrogen over nitrate (NO₃⁻) for rice (Oryza sativa), lateral root (LR) growth in roots is enhanced by partial NO₃⁻ nutrition (PNN). The roles of auxin distribution and polar transport in LR formation in response to localized NO₃⁻ availability are not known.

Methods

Time-course studies in a split-root experimental system were used to investigate LR development patterns, auxin distribution, polar auxin transport and expression of auxin transporter genes in LR zones in response to localized PNN in ‘Nanguang’ and ‘Elio’ rice cultivars, which show high and low responsiveness to NO₃⁻, respectively. Patterns of auxin distribution and the effects of polar auxin transport inhibitors were also examined in DR5::GUS transgenic plants.

Key Results

Initiation of LRs was enhanced by PNN after 7 d cultivation in ‘Nanguang’ but not in ‘Elio’. Auxin concentration in the roots of ‘Nanguang’ increased by approx. 24 % after 5 d cultivation with PNN compared with NH₄⁺ as the sole nitrogen source, but no difference was observed in ‘Elio’. More auxin flux into the LR zone in ‘Nanguang’ roots was observed in response to NO₃⁻ compared with NH₄⁺ treatment. A greater number of auxin influx and efflux transporter genes showed increased expression in the LR zone in response to PNN in ‘Nanguang’ than in ‘Elio’.

Conclusions

The results indicate that higher NO₃⁻ responsiveness is associated with greater auxin accumulation in the LR zone and is strongly related to a higher rate of LR initiation in the cultivar ‘Nanguang’.     

Nitric oxide is the shared signalling molecule in phosphorus- and iron-deficiency-induced formation of cluster roots in white lupin (Lupinus albus)

Background and Aims

Formation of cluster roots is one of the most specific root adaptations to nutrient deficiency. In white lupin (Lupinus albus), cluster roots can be induced by phosphorus (P) or iron (Fe) deficiency. The aim of the present work was to investigate the potential shared signalling pathway in P- and Fe-deficiency-induced cluster root formation.

Methods

Measurements were made of the internal concentration of nutrients, levels of nitric oxide (NO), citrate exudation and expression of some specific genes under four P × Fe combinations, namely (1) 50 µm P and 10 µm Fe (+P + Fe); (2) 0 P and 10 µm Fe (–P + Fe); (3) 50 µm P and 0 Fe (+P–Fe); and (4) 0 P and 0 Fe (–P–Fe), and these were examined in relation to the formation of cluster roots.

Key Results

The deficiency of P, Fe or both increased the cluster root number and cluster zones. It also enhanced NO accumulation in pericycle cells and rootlet primordia at various stages of cluster root development. The formation of cluster roots and rootlet primordia, together with the expression of LaSCR1 and LaSCR2 which is crucial in cluster root formation, were induced by the exogenous NO donor S-nitrosoglutathione (GSNO) under the +P + Fe condition, but were inhibited by the NO-specific endogenous scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl- 3-oxide (cPTIO) under –P + Fe, +P–Fe and –P–Fe conditions. However, cluster roots induced by an exogenous supply of the NO donor did not secrete citrate, unlike those formed under –P or –Fe conditions.

Conclusions

NO plays an important role in the shared signalling pathway of the P- and Fe-deficiency-induced formation of cluster roots in white lupin.     

Auxin increases the hydrogen peroxide (H2O2) concentration in tomato (Solanum lycopersicum) root tips while inhibiting root growth

Background and Aims

The hormone auxin and reactive oxygen species (ROS) regulate root elongation, but the interactions between the two pathways are not well understood. The aim of this study was to investigate how auxin interacts with ROS in regulating root elongation in tomato, Solanum lycopersicum.

Methods

Wild-type and auxin-resistant mutant, diageotropica (dgt), of tomato (S. lycopersicum ‘Ailsa Craig’) were characterized in terms of root apical meristem and elongation zone histology, expression of the cell-cycle marker gene Sl-CycB1;1, accumulation of ROS, response to auxin and hydrogen peroxide (H₂O₂), and expression of ROS-related mRNAs.

Key Results

The dgt mutant exhibited histological defects in the root apical meristem and elongation zone and displayed a constitutively increased level of hydrogen peroxide (H₂O₂) in the root tip, part of which was detected in the apoplast. Treatments of wild-type with auxin increased the H₂O₂ concentration in the root tip in a dose-dependent manner. Auxin and H₂O₂ elicited similar inhibition of cell elongation while bringing forth differential responses in terms of meristem length and number of cells in the elongation zone. Auxin treatments affected the expression of mRNAs of ROS-scavenging enzymes and less significantly mRNAs related to antioxidant level. The dgt mutation resulted in resistance to both auxin and H₂O₂ and affected profoundly the expression of mRNAs related to antioxidant level.

Conclusions

The results indicate that auxin regulates the level of H₂O₂ in the root tip, so increasing the auxin level triggers accumulation of H₂O₂ leading to inhibition of root cell elongation and root growth. The dgt mutation affects this pathway by reducing the auxin responsiveness of tissues and by disrupting the H₂O₂ homeostasis in the root tip.     

Gene dosage effect of WEE1 on growth and morphogenesis from arabidopsis hypocotyl explants

Background and Aims

How plant cell-cycle genes interface with development is unclear. Preliminary evidence from our laboratory suggested that over-expression of the cell cycle checkpoint gene, WEE1, repressed growth and development. Here the hypothesis is tested that the level of WEE1 has a dosage effect on growth and development in Arabidospis thaliana. To do this, a comparison was made of the development of gain- and loss-of-function WEE1 arabidopsis lines both in vivo and in vitro.

Methods

Hypocotyl explants from an over-expressing Arath;WEE1 line (WEE1^oe), two T-DNA insertion lines (wee1-1 and wee1-4) and wild type (WT) were cultured on two-way combinations of kinetin and naphthyl acetic acid. Root growth and meristematic cell size were also examined.

Key Results

Quantitative data indicated a repressive effect in WEE1^oe and a significant increase in morphogenetic capacity in the two T-DNA insertion lines compared with WT. Compared with WT, WEE1^oe seedlings exhibited a slower cell-doubling time in the root apical meristem and a shortened primary root, with fewer laterals, whereas there were no consistent differences in the insertion lines compared with WT. However, significantly fewer adventitious roots were recorded for WEE1^oe and significantly more for the insertion mutant wee1-1. Compared with WT there was a significant increase in meristem cell size in WEE1^oe for all three ground tissues but for wee1-1 only cortical cell size was reduced.

Conclusions

There is a gene dosage effect of WEE1 on morphogenesis from hypocotyls both in vitro and in vivo.     

Phylogenetic analyses provide the first insights into the evolution of OVATE family proteins in land plants

Background and Aims

The OVATE gene encodes a nuclear-localized regulatory protein belonging to a distinct family of plant-specific proteins known as the OVATE family proteins (OFPs). OVATE was first identified as a key regulator of fruit shape in tomato, with nonsense mutants displaying pear-shaped fruits. However, the role of OFPs in plant development has been poorly characterized.

Methods

Public databases were searched and a total of 265 putative OVATE protein sequences were identified from 13 sequenced plant genomes that represent the major evolutionary lineages of land plants. A phylogenetic analysis was conducted based on the alignment of the conserved OVATE domain from these 13 selected plant genomes. The expression patterns of tomato SlOFP genes were analysed via quantitative real-time PCR. The pattern of OVATE gene duplication resulting in the expansion of the gene family was determined in arabidopsis, rice and tomato.

Key Results

Genes for OFPs were found to be present in all the sampled land plant genomes, including the early-diverged lineages, mosses and lycophytes. Phylogenetic analysis based on the amino acid sequences of the conserved OVATE domain defined 11 sub-groups of OFPs in angiosperms. Different evolutionary mechanisms are proposed for OVATE family evolution, namely conserved evolution and divergent expansion. Characterization of the AtOFP family in arabidopsis, the OsOFP family in rice and the SlOFP family in tomato provided further details regarding the evolutionary framework and revealed a major contribution of tandem and segmental duplications towards expansion of the OVATE gene family.

Conclusions

This first genome-wide survey on OFPs provides new insights into the evolution of the OVATE protein family and establishes a solid base for future functional genomics studies on this important but poorly characterized regulatory protein family in plants.     

The barley MATE gene,HvAACT1, increases citrate efflux and Al3+ tolerance when expressed in wheat and barley

Background and Aims

Aluminium is toxic in acid soils because the soluble Al³⁺ inhibits root growth. A mechanism of Al³⁺ tolerance discovered in many plant species involves the release of organic anions from root apices. The Al³⁺-activated release of citrate from the root apices of Al³⁺-tolerant genotypes of barley is controlled by a MATE gene named HvAACT1 that encodes a citrate transport protein located on the plasma membrane. The aim of this study was to investigate whether expressing HvAACT1 with a constitutive promoter in barley and wheat can increase citrate efflux and Al³⁺ tolerance of these important cereal species.

Methods HvAACT1

was over-expressed in wheat (Triticum aestivum) and barley (Hordeum vulgare) using the maize ubiquitin promoter. Root apices of transgenic and control lines were analysed for HvAACT1 expression and organic acid efflux. The Al³⁺ tolerance of transgenic and control lines was assessed in both hydroponic solution and acid soil.

Key Results and Conclusions

Increased HvAACT1 expression in both cereal species was associated with increased citrate efflux from root apices and enhanced Al³⁺ tolerance, thus demonstrating that biotechnology can complement traditional breeding practices to increase the Al³⁺ tolerance of important crop plants.     

Analyses of phylogeny, evolution, conserved sequences and genome-wide expression of the ICK/KRP family of plant CDK inhibitors

Background and Aims

The cell cycle is controlled by cyclin-dependent kinases (CDKs), and CDK inhibitors are major regulators of their activities. The ICK/KRP family of CDK inhibitors has been reported in several plants, with seven members in arabidopsis; however, the phylogenetic relationship among members in different species is unknown. Also, there is a need to understand how these genes and proteins are regulated. Furthermore, little information is available on the functional differences among ICK/KRP family members.

Methods

We searched publicly available databases and identified over 120 unique ICK/KRP protein sequences from more than 60 plant species. Phylogenetic analysis was performed using 101 full-length sequences from 40 species and intron–exon organization of ICK/KRP genes in model species. Conserved sequences and motifs were analysed using ICK/KRP protein sequences from arabidopsis (Arabidopsis thaliana), rice (Orysa sativa) and poplar (Populus trichocarpa). In addition, gene expression was examined using microarray data from arabidopsis, rice and poplar, and further analysed by RT-PCR for arabidopsis.

Key Results and Conclusions

Phylogenetic analysis showed that plant ICK/KRP proteins can be grouped into three major classes. Whereas the C-class contains sequences from dicotyledons, monocotyledons and gymnosperms, the A- and B-classes contain only sequences from dicotyledons or monocotyledons, respectively, suggesting that the A- and B-classes might have evolved from the C-class. This classification is also supported by exon–intron organization. Genes in the A- and B- classes have four exons, whereas genes in the C-class have only three exons. Analysis of sequences from arabidopsis, rice and poplar identified conserved sequence motifs, some of which had not been described previously, and putative functional sites. The presence of conserved motifs in different family members is consistent with the classification. In addition, gene expression analysis showed preferential expression of ICK/KRP genes in certain tissues. A model has been proposed for the evolution of this gene family in plants.     

Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

Background

Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca²⁺ concentration, which is a fundamental prerequisite for any Ca²⁺-based signalling system, is accomplished by complex mechanisms including Ca²⁺ binding proteins acting as Ca²⁺ buffers. In this work we investigated the occurrence of Ca²⁺ binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus.

Results

A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca²⁺-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca²⁺-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca²⁺ binding ability of the M. loti protein was demonstrated by ⁴⁵Ca²⁺-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca²⁺ adducts.

Conclusions

The present data indicate that ferredoxin II is a major Ca²⁺ binding protein in M. loti that may participate in Ca²⁺ homeostasis and suggest an evolutionarily ancient origin for protein-based Ca²⁺ regulatory systems.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0352-5) contains supplementary material, which is available to authorized users.     

The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein

Background and Aims

Rhamnogalacturonan-II (RG-II) is one of the pectin motifs found in the cell wall of all land plants. It contains sugars such as 2-keto-3-deoxy-d-lyxo-heptulosaric acid (Dha) and 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo), and within the wall RG-II is mostly found as a dimer via a borate diester cross-link. To date, little is known regarding the biosynthesis of this motif. Here, after a brief review of our current knowledge on RG-II structure, biosynthesis and function in plants, this study explores the implications of the presence of a Golgi-localized sialyltransferase-like 2 (SIA2) protein that is possibly involved in the transfer of Dha or Kdo in the RG-II of Arabidopsis thaliana pollen tubes, a fast-growing cell type used as a model for the study of cell elongation.

Methods

Two heterozygous mutant lines of arabidopsis (sia2-1+/– and qrt1 × sia2-2+/–) were investigated. sia2-2+/– was in a quartet1 background and the inserted T-DNA contained the reporter gene β-glucuronidase (GUS) under the pollen-specific promoter LAT52. Pollen germination and pollen tube phenotype and growth were analysed both in vitro and in vivo by microscopy.

Key Results

Self-pollination of heterozygous lines produced no homozygous plants in the progeny, which may suggest that the mutation could be lethal. Heterozygous mutants displayed a much lower germination rate overall and exhibited a substantial delay in germination (20 h of delay to reach 30 % of pollen grain germination compared with the wild type). In both lines, mutant pollen grains that were able to produce a tube had tubes that were either bursting, abnormal (swollen or dichotomous branching tip) or much shorter compared with wild-type pollen tubes. In vivo, mutant pollen tubes were restricted to the style, whereas the wild-type pollen tubes were detected at the base of the ovary.

Conclusions

This study highlights that the mutation in arabidopsis SIA2 encoding a sialyltransferase-like protein that may transfer Dha or Kdo on the RG-II motif has a dramatic effect on the stability of the pollen tube cell wall.     

GPT2: a glucose 6-phosphate/phosphate translocator with a novel role in the regulation of sugar signalling during seedling development

Background and Aims

GPT2, a glucose 6-phosphate/phosphate translocator, plays an important role in environmental sensing in mature leaves of Arabidopsis thaliana. Its expression has also been detected in arabidopsis seeds and seedlings. In order to examine the role of this protein early in development, germination and seedling growth were studied.

Methods

Germination, greening and establishment of seedlings were monitored in both wild-type Arabidopsis thaliana and in a gpt2 T-DNA insertion knockout line. Seeds were sown on agar plates in the presence or absence of glucose and abscisic acid. Relative expression of GPT2 in seedlings was measured using quantitative PCR.

Key Results

Plants lacking GPT2 expression were delayed (25–40 %) in seedling establishment, specifically in the process of cotyledon greening (rather than germination). This phenotype could not be rescued by glucose in the growth medium, with greening being hypersensitive to glucose. Germination itself was, however, hyposensitive to glucose in the gpt2 mutant.

Conclusions

The expression of GPT2 modulates seedling development and plays a crucial role in determining the response of seedlings to exogenous sugars during their establishment. This allows us to conclude that endogenous sugar signals function in controlling germination and the transition from heterotrophic to autotrophic growth, and that the partitioning of glucose 6-phosphate, or related metabolites, between the cytosol and the plastid modulates these developmental responses.     

Nitric oxide contributes to copper tolerance by influencing ROS metabolism in Arabidopsis

Key message

Nitric oxide improves copper tolerance via modulation of superoxide and hydrogen peroxide levels. This reflects the necessity of a well-coordinated interplay between NO and ROS during stress tolerance.

Abstract

Copper (Cu) excess causes toxicity and one probable consequence of this is the disturbance of cell redox state maintenance, inter alia, by reactive oxygen- (ROS) and nitrogen species (RNS). The objective of this paper was to examine the role of nitric oxide (NO) in Cu stress tolerance and its relationship with ROS in Arabidopsis. In agar-grown seedlings, concentration-dependent Cu accumulation was observed. The 5 μM Cu resulted in reduced cell viability in the NO overproducing nox1 and gsnor1-3 root tips compared to the wild-type (WT). In contrast, 25 and 50 μM Cu caused higher viability in these mutants, while in the NO-lacking nia1nia2 lower viability was detected than in the WT. The exogenous NO donor enhanced cell viability and scavenging endogenous NO decreased it in Cu-exposed WT seedlings. Besides, SNP in nia1nia2 roots led to the improvement of viability. The ascorbic acid-deficient mutants (vtc2-1, vtc2-3) possessing slightly elevated ROS levels proved to be Cu sensitive, while miox4 showing decreased ROS production was more tolerant to Cu than the WT. In nox1 and gsnor1-3, Cu did not induce superoxide formation, and H₂O₂ accumulation occurred only in the case of NO deficiency. Based on these, under mild stress NO intensifies cell injury, while in the case of severe Cu excess it contributes to better viability. ROS were found to be responsible for aggravation of Cu-induced damage. NO alleviates acute Cu stress via modulation of O ₂ ^·? and H₂O₂ levels reflecting the necessity of a well-coordinated interplay between NO and ROS during stress tolerance.     

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

Arabidopsis T-DNA insertional lines for CDC25 are hypersensitive to hydroxyurea but not to zeocin or salt stress

Background and Aims

In yeasts and animals, cyclin-dependent kinases are key regulators of cell cycle progression and are negatively and positively regulated by WEE1 kinase and CDC25 phosphatase, respectively. In higher plants a full-length orthologue of CDC25 has not been isolated but a shorter gene with homology only to the C-terminal catalytic domain is present. The Arabidopis thaliana;CDC25 can act as a phosphatase in vitro. Since in arabidopsis, WEE1 plays an important role in the DNA damage/DNA replication checkpoints, the role of Arath;CDC25 in conditions that induce these checkpoints or induce abiotic stress was tested.

Methods

arath;cdc25 T-DNA insertion lines, Arath;CDC25 over-expressing lines and wild type were challenged with hydroxyurea (HU) and zeocin, substances that stall DNA replication and damage DNA, respectively, together with an abiotic stressor, NaCl. A molecular and phenotypic assessment was made of all genotypes

Key Results

There was a null phenotypic response to perturbation of Arath;CDC25 expression under control conditions. However, compared with wild type, the arath;cdc25 T-DNA insertion lines were hypersensitive to HU, whereas the Arath;CDC25 over-expressing lines were relatively insensitive. In particular, the over-expressing lines consistently outgrew the T-DNA insertion lines and wild type when challenged with HU. All genotypes were equally sensitive to zeocin and NaCl.

Conclusions

Arath;CDC25 plays a role in overcoming stress imposed by HU, an agent know to induce the DNA replication checkpoint in arabidopsis. However, it could not enhance tolerance to either a zeocin treatment, known to induce DNA damage, or salinity stress.