Rice alcohol dehydrogenase 1 promotes survival and has a major impact on carbohydrate metabolism in the embryo and endosperm when seeds are germinated in partially oxygenated water

Background and Aims

Rice (Oryza sativa) has the rare ability to germinate and elongate a coleoptile under oxygen-deficient conditions, which include both hypoxia and anoxia. It has previously been shown that ALCOHOL DEHYDROGENASE 1 (ADH1) is required for cell division and cell elongation in the coleoptile of submerged rice seedlings by means of studies using a rice ADH1-deficient mutant, reduced adh activity (rad). The aim of this study was to understand how low ADH1 in rice affects carbohydrate metabolism in the embryo and endosperm, and lactate and alanine synthesis in the embryo during germination and subsequent coleoptile growth in submerged seedlings.

Methods

Wild-type and rad mutant rice seeds were germinated and grown under complete submergence. At 1, 3, 5 and 7 d after imbibition, the embryo and endosperm were separated and several of their metabolites were measured and compared.

Key results

In the rad embryo, the rate of ethanol fermentation was halved, while lactate and alanine concentrations were 2·4- and 5·7- fold higher in the mutant than in the wild type. Glucose and fructose concentrations in the embryos increased with time in the wild type, but not in the rad mutant. The rad mutant endosperm had lower amounts of the α-amylases RAMY1A and RAMY3D, resulting in less starch degradation and lower glucose concentrations.

Conclusions

These results suggest that ADH1 is essential for sugar metabolism via glycolysis to ethanol fermentation in both the embryo and endosperm. In the endosperm, energy is presumably needed for synthesis of the amylases and for sucrose synthesis in the endosperm, as well as for sugar transport to the embryo.     

Wait or escape? Contrasting submergence tolerance strategies of Rorippa amphibia,Rorippa sylvestris and their hybrid

Background and Aims

Differential responses of closely related species to submergence can provide insight into the evolution and mechanisms of submergence tolerance. Several traits of two wetland species from habitats with contrasting flooding regimes, Rorippa amphibia and Rorippa sylvestris, as well as F₁ hybrid Rorippa × anceps were analysed to unravel mechanisms underlying submergence tolerance.

Methods

In the first submergence experiment (lasting 20 d) we analysed biomass, stem elongation and carbohydrate content. In the second submergence experiment (lasting 3 months) we analysed survival and the effect of re-establishment of air contact on biomass and carbohydrate content. In a separate experiment we analysed expression of two carbohydrate catabolism genes, ADH1 and SUS1, upon re-establishment of air contact following submergence.

Key Results

All plants had low mortality even after 3 months of submergence. Rorippa sylvestris was characterized by 100 % survival and higher carbohydrate levels coupled with lower ADH1 gene expression as well as reduced growth compared with R. amphibia. Rorippa amphibia and the hybrid elongated their stems but this did not pay-off in higher survival when plants remained submerged. Only R. amphibia and the hybrid benefited in terms of increased biomass and carbohydrate accumulation upon re-establishing air contact.

Conclusions

Results demonstrate contrasting ‘escape’ and ‘quiescence’ strategies between Rorippa species. Being a close relative of arabidopsis, Rorippa is an excellent model for future studies on the molecular mechanism(s) controlling these strategies.     

Recovery dynamics of growth, photosynthesis and carbohydrate accumulation after de-submergence: a comparison between two wetland plants showing escape and quiescence strategies

Background and Aims

The capacity for fast-growth recovery after de-submergence is important for establishment of riparian species in a water-level-fluctuation zone. Recovery patterns of two wetland plants, Alternanthera philoxeroides and Hemarthria altissima, showing ‘escape’ and ‘quiescence’ responses, respectively, during submergence were investigated.

Methods

Leaf and root growth and photosynthesis were monitored continuously during 10 d of recovery following 20 d of complete submergence. Above- and below-ground dry weights, as well as carbohydrate concentrations, were measured several times during the experiment.

Key Results

Both species remobilized stored carbohydrate during submergence. Although enhanced internode elongation depleted the carbohydrate storage in A. philoxeroides during submergence, this species resumed leaf growth 3 d after de-submergence concomitant with restoration of the maximal photosynthetic capacity. In contrast, some sucrose was conserved in shoots of H. altissima during submergence, which promoted rapid re-growth of leaves 2 d after de-submergence and earlier than the full recovery of photosynthesis. The recovery of root growth was delayed by 1–2 d compared with leaves in both species.

Conclusions

Submergence tolerance of the escape and quiescence strategies entails not only the corresponding regulation of growth, carbohydrate catabolism and energy metabolism during submergence but also co-ordinated recovery of photosynthesis, growth and carbohydrate partitioning following de-submergence.     

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.     

A kinetic analysis of hyponastic growth and petiole elongation upon ethylene exposure in Rumex palustris

Background and Aims

Complete submergence is an important stress factor for many terrestrial plants, and a limited number of species have evolved mechanisms to deal with these conditions. Rumex palustris is one such species and manages to outgrow the water, and thus restore contact with the atmosphere, through upward leaf growth (hyponasty) followed by strongly enhanced petiole elongation. These responses are initiated by the gaseous plant hormone ethylene, which accumulates inside plants due to physical entrapment. This study aimed to investigate the kinetics of ethylene-induced leaf hyponasty and petiole elongation.

Methods

Leaf hyponasty and petiole elongation was studied using a computerized digital camera set-up followed by image analyses. Linear variable displacement transducers were used for fine resolution monitoring and measurement of petiole growth rates.

Key Results

We show that submergence-induced hyponastic growth and petiole elongation in R. palustris can be mimicked by exposing plants to ethylene. The petiole elongation response to ethylene is shown to depend on the initial angle of the petiole. When petiole angles were artificially kept at 0°, rather than the natural angle of 35°, ethylene could not induce enhanced petiole elongation. This is very similar to submergence studies and confirms the idea that there are endogenous, angle-dependent signals that influence the petiole elongation response to ethylene.

Conclusions

Our data suggest that submergence and ethylene-induced hyponastic growth and enhanced petiole elongation responses in R. palustris are largely similar. However, there are some differences that may relate to the complexity of the submergence treatment as compared with an ethylene treatment.     

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.     

The potential roles of strigolactones and brassinosteroids in the autoregulation of nodulation pathway

Background and Aims

The number of nodules formed on a legume root system is under the strict genetic control of the autoregulation of nodulation (AON) pathway. Plant hormones are thought to play a role in AON; however, the involvement of two hormones recently described as having a largely positive role in nodulation, strigolactones and brassinosteroids, has not been examined in the AON process.

Methods

A genetic approach was used to examine if strigolactones or brassinosteroids interact with the AON system in pea (Pisum sativum). Double mutants between shoot-acting (Psclv2, Psnark) and root-acting (Psrdn1) mutants of the AON pathway and strigolactone-deficient (Psccd8) or brassinosteroid-deficient (lk) mutants were generated and assessed for various aspects of nodulation. Strigolactone production by AON mutant roots was also investigated.

Key Results

Supernodulation of the roots was observed in both brassinosteroid- and strigolactone-deficient AON double-mutant plants. This is despite the fact that the shoots of these plants displayed classic strigolactone-deficient (increased shoot branching) or brassinosteroid-deficient (extreme dwarf) phenotypes. No consistent effect of disruption of the AON pathway on strigolactone production was found, but root-acting Psrdn1 mutants did produce significantly more strigolactones.

Conclusions

No evidence was found that strigolactones or brassinosteroids act downstream of the AON genes examined. While in pea the AON mutants are epistatic to brassinosteroid and strigolactone synthesis genes, we argue that these hormones are likely to act independently of the AON system, having a role in the promotion of nodule formation.     

AGO1 controls arabidopsis inflorescence architecture possibly by regulating TFL1 expression

Background and Aims

The TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators.

Methods

Mutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (β-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype.

Key Results

A mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants.

Conclusions

The results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression.     

Distinct mechanisms for aerenchyma formation in leaf sheaths of rice genotypes displaying a quiescence or escape strategy for flooding tolerance

Background and Aims

Rice is one of the few crops able to withstand periods of partial or even complete submergence. One of the adaptive traits of rice is the constitutive presence and further development of aerenchyma which enables oxygen to be transported to submerged organs. The development of lysigenous aerenchyma is promoted by ethylene accumulating within the submerged plant tissues, although other signalling mechanisms may also co-exist. In this study, aerenchyma development was analysed in two rice (Oryza sativa) varieties, ‘FR13A’ and ‘Arborio Precoce’, which show opposite traits in flooding response in terms of internode elongation and survival.

Methods

The growth and survival of rice varieties under submergence was investigated in the leaf sheath of ‘FR13A’ and ‘Arborio Precoce’. The possible involvement of ethylene and reactive oxygen species (ROS) was evaluated in relation to aerenchyma formation. Cell viability and DNA fragmentation were determined by FDA/FM4-64 staining and TUNEL assay, respectively. Ethylene production was monitored by gas chromatography and by analysing ACO gene expression. ROS production was measured by using Amplex Red assay kit and the fluorescent dye DCFH₂-DA. The expression of APX1 was also evaluated. AVG and DPI solutions were used to test the effect of inhibiting ethylene biosynthesis and ROS production, respectively.

Key Results

Both the varieties displayed constitutive lysigenous aerenchyma formation, which was further enhanced when submerged. ‘Arborio Precoce’, which is characterized by fast elongation when submerged, showed active ethylene biosynthetic machinery associated with increased aerenchymatous areas. ‘FR13A’, which harbours the Sub1A gene that limits growth during oxygen deprivation, did not show any increase in ethylene production after submersion but still displayed increased aerenchyma. Hydrogen peroxide levels increased in ‘FR13A’ but not in ‘Arborio Precoce’.

Conclusions

While ethylene controls aerenchyma formation in the fast-elongating ‘Arborio Precoce’ variety, in ‘FR13A’ ROS accumulation plays an important role.     

Escape from water or remain quiescent? Lotus tenuis changes its strategy depending on depth of submergence

Background and Aims

Two main strategies that allow plants to cope with soil waterlogging or deeper submergence are: (1) escaping by means of upward shoot elongation or (2) remaining quiescent underwater. This study investigates these strategies in Lotus tenuis, a forage legume of increasing importance in areas prone to soil waterlogging, shallow submergence or complete submergence.

Methods

Plants of L. tenuis were subjected for 30 d to well-drained (control), waterlogged (water-saturated soil), partially submerged (6 cm water depth) and completely submerged conditions. Plant responses assessed were tissue porosity, shoot number and length, biomass and utilization of water-soluble carbohydrates (WSCs) and starch in the crown.

Key Results

Lotus tenuis adjusted its strategy depending on the depth of submergence. Root growth of partially submerged plants ceased and carbon allocation prioritized shoot lengthening (32 cm vs. 24·5 cm under other treatments), without depleting carbohydrate reserves to sustain the faster growth. These plants also developed more shoot and root porosity. In contrast, completely submerged plants became quiescent, with no associated biomass accumulation, new shoot production or shoot elongation. In addition, tissue porosity was not enhanced. The survival of completely submerged plants is attributed to consumption of WSCs and starch reserves from crowns (concentrations 50–75 % less than in other treatments).

Conclusions

The forage legume L. tenuis has the flexibility either to escape from partial submergence by elongating its shoot more vigorously to avoid becoming totally submerged or to adopt a non-elongating quiescent strategy when completely immersed that is based on utilizing stored reserves. The possession of these alternative survival strategies helps to explain the success of L. tenuis in environments subjected to unpredictable flooding depths.     

Continuous,high-resolution biospeckle imaging reveals a discrete zone of activity at the root apex that responds to contact with obstacles

Background and Aims

Shining a laser onto biological material produces light speckles termed biospeckles. Patterns of biospeckle activity reflect changes in cell biochemistry, developmental processes and responses to the environment. The aim of this work was to develop methods to investigate the biospeckle activity in roots and to characterize the distribution of its intensity and response to thigmostimuli.

Methods

Biospeckle activity in roots of Zea mays, and also Jatropha curcas and Citrus limonia, was imaged live and in situ using a portable laser and a digital microscope with a spatial resolution of 10 μm per pixel and the ability to capture images every 0·080 s. A procedure incorporating a Fujii algorithm, image restoration using median and Gaussian filters, image segmentation using maximum-entropy threshold methods and the extraction of features using a tracing algorithm followed by spline fitting were developed to obtain quantitative information from images of biospeckle activity. A wavelet transform algorithm was used for spectral decomposition of biospeckle activity and generalized additive models were used to attribute statistical significance to changes in patterns of biospeckle activity.

Key Results

The intensity of biospeckle activity was greatest close to the root apex. Higher frequencies (3–6 Hz) contributed most to the total intensity of biospeckle activity. When a root encountered an obstacle, the intensity of biospeckle activity decreased abruptly throughout the root system. The response became attenuated with repeated thigmostimuli.

Conclusions

The data suggest that at least one component of root biospeckle activity resulted from a biological process, which is located in the zone of cell division and responds to thigmostimuli. However, neither individual cell division events nor root elongation is likely to be responsible for the patterns of biospeckle activity.     

A genetic approach of wine yeast fermentation capacity in nitrogen-starvation reveals the key role of nitrogen signaling

Background

In conditions of nitrogen limitation, Saccharomyces cerevisiae strains differ in their fermentation capacities, due to differences in their nitrogen requirements. The mechanisms ensuring the maintenance of glycolytic flux in these conditions are unknown. We investigated the genetic basis of these differences, by studying quantitative trait loci (QTL) in a population of 133 individuals from the F2 segregant population generated from a cross between two strains with different nitrogen requirements for efficient fermentation.

Results

By comparing two bulks of segregants with low and high nitrogen requirements, we detected four regions making a quantitative contribution to these traits. We identified four polymorphic genes, in three of these four regions, for which involvement in the phenotype was validated by hemizygote comparison. The functions of the four validated genes, GCN1, MDS3, ARG81 and BIO3, relate to key roles in nitrogen metabolism and signaling, helping to maintain fermentation performance.

Conclusions

This study reveals that differences in nitrogen requirement between yeast strains results from a complex allelic combination. The identification of three genes involved in sensing and signaling nitrogen and specially one from the TOR pathway as affecting nitrogen requirements suggests a role for this pathway in regulating the fermentation rate in starvation through unknown mechanisms linking nitrogen signaling to glycolytic flux.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-495) contains supplementary material, which is available to authorized users.     

A cell-type-specific defect in border cell formation in the Acacia mangium root cap developing an extraordinary sheath of sloughed-off cells

Background and Aims

Root caps release border cells, which play central roles in microbe interaction and root protection against soil stresses. However, the number and connectivity of border cells differ widely among plant species. Better understanding of key border-cell phenotype across species will help define the total function of border cells and associated genes.

Methods

The spatio-temporal detachment of border cells in the leguminous tree Acacia mangium was investigated by using light and fluorescent microscopy with fluorescein diacetate, and their number and structural connectivity compared with that in soybean (Glycine max).

Key Results

Border-like cells with a sheet structure peeled bilaterally from the lateral root cap of A. mangium. Hydroponic root elongation partially facilitated acropetal peeling of border-like cells, which accumulate as a sheath that covers the 0- to 4-mm tip within 1 week. Although root elongation under friction caused basipetal peeling, lateral root caps were minimally trimmed as compared with hydroponic roots. In the meantime, A. mangium columella caps simultaneously released single border cells with a number similar to those in soybean.

Conclusions

These results suggest that cell type-specific inhibitory factors induce a distinct defective phenotype in single border-cell formation in A. mangium lateral root caps.     

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.     

Phylogenetic utility of the nuclear genes AGAMOUS 1 and PHYTOCHROME B in palms (Arecaceae): an example within Bactridinae

Background and Aims

Molecular phylogenetic studies of palms (Arecaceae) have not yet provided a fully resolved phylogeny of the family. There is a need to increase the current set of markers to resolve difficult groups such as the Neotropical subtribe Bactridinae (Arecoideae: Cocoseae). We propose the use of two single-copy nuclear genes as valuable tools for palm phylogenetics.

Methods

New primers were developed for the amplification of the AGAMOUS 1 (AG1) and PHYTOCHROME B (PHYB) genes. For the AGAMOUS gene, the paralogue 1 of Elaeis guineensis (EgAG1) was targeted. The region amplified contained coding sequences between the MIKC K and C MADS-box domains. For the PHYB gene, exon 1 (partial sequence) was first amplified in palm species using published degenerate primers for Poaceae, and then specific palm primers were designed. The two gene portions were sequenced in 22 species of palms representing all genera of Bactridinae, with emphasis on Astrocaryum and Hexopetion, the status of the latter genus still being debated.

Key Results

The new primers designed allow consistent amplification and high-quality sequencing within the palm family. The two loci studied produced more variability than chloroplast loci and equally or less variability than PRK, RPBII and ITS nuclear markers. The phylogenetic structure obtained with AG1 and PHYB genes provides new insights into intergeneric relationships within the Bactridinae and the intrageneric structure of Astrocaryum. The Hexopetion clade was recovered as monophyletic with both markers and was weakly supported as sister to Astrocaryum sensu stricto in the combined analysis. The rare Astrocaryum minus formed a species complex with Astrocaryum gynacanthum. Moreover, both AG1 and PHYB contain a microsatellite that could have further uses in species delimitation and population genetics.

Conclusions

AG1 and PHYB provide additional phylogenetic information within the palm family, and should prove useful in combination with other genes to improve the resolution of palm phylogenies.