Stress and developmental responses in Arabidopsis thaliana: regulation through the transcription factor-interacting protein RCD1

Plants constantly face adverse environmental conditions, such as drought or extreme temperatures that threaten their survival. They demonstrate astonishing metabolic flexibility in overcoming these challenges and one of the key responses to stresses is changes in gene expression leading to alterations in cellular functions. This is brought about by an intricate network of transcription factors and associated regulatory proteins. Protein-protein interactions and post-translational modifications are important steps in this control system along with carefully regulated degradation of signaling proteins. This work concentrates on the RADICAL-INDUCED CELL DEATH1 (RCD1) protein which is an important regulator of abiotic stress-related and developmental responses in Arabidopsis thaliana. Plants lacking this protein function display pleiotropic phenotypes including sensitivity to apoplastic reactive oxygen species (ROS) and salt, ultraviolet B (UV-B) and paraquat tolerance, early flowering and senescence. Additionally, the mutant plants overproduce nitric oxide, have alterations in their responses to several plant hormones and perturbations in gene expression profiles. The RCD1 gene is transcriptionally unresponsive to environmental signals and the regulation of the protein function is likely to happen post-translationally. RCD1 belongs to a small protein family and, together with its closest homolog SRO1, contains three distinguishable domains: In the N-terminus, there is a WWE domain followed by a poly(ADP-ribose) polymerase-like domain which, despite sequence conservation, does not seem to be functional. The C-terminus of RCD1 contains a novel domain called RST. It is present in RCD1-like proteins throughout the plant kingdom and is able to mediate physical interactions with multiple transcription factors. In conclusion, RCD1 is a key point of signal integration that links ROS-mediated cues to transcriptional regulation by yet unidentified means, which are likely to include post-translational mechanisms. The identification of RCD1-interacting transcription factors, most of whose functions are still unknown, opens new avenues for studies on plant stress as well as developmental responses.

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.

Assessment of wheat cultivars for drought tolerance via osmotic stress imposed at early seedling growth stages

A study was conducted in the Department of Plant Breeding and Genetics,Sindh Agriculture University, Tandojam, Pakistan during the year 2009. Sixteen spring wheat cultivars (Triticum aestivum L.) were screened under osmotic stress with three treatments i.e. control-no PEG (polyethylene glycol), 15 percent and 25 percent PEG-6000 solution. The analysis of variance indicated significant differences among treatments for all seedling traits except seed germination percentage. Varieties also differed significantly in germination percentage, coleoptile length, shoot root length, shoot weight, root/shoot ratio and seed vigour index. However, shoot and root weights were non-significant. Significant interactions revealed that cultivars responded variably to osmotic stress treatments; hence provided better opportunity to select drought tolerant cultivars at seedling growth stages. The relative decrease over averages due to osmotic stress was 0.8 percent in seed germination, 53 percent in coleoptile length 62.9 percent in shoot length, 74.4 percent in root length, 50.6 percent in shoot weight, 45.1 percent in root weight, 30.2 percent in root/shoot ratio and 68.5 percent in seed vigour index. However, relative decrease of individual variety for various seedling traits could be more meaningful which indicated that cultivar TD-1 showed no reduction in coleoptile length, while minimum decline was noted in Anmol. For shoot length, cultivar Sarsabz expressed minimum reduction followed by Anmol. However, cultivars Anmol, Moomal, Inqalab-91, and Pavan gave almost equally lower reductions for root length suggesting their higher stress tolerance. In other words, cultivars Anmol, Moomal, Inqalab-91, Sarsabz, TD-1, ZA-77 and Pavan had relatively longer coleoptiles, shoots and roots, and were regarded as drought tolerant. Correlation coefficients among seedlings traits were significant and positive for all traits except germination percentage which had no significant correlation with any of other trait. The results indicated that increase in one trait may cause simultaneous increase in other traits; hence selection for any of these seedling attributes will lead to develop drought tolerant wheat cultivars.

Effect of Rht alleles on the tolerance of wheat grain set to high temperature and drought stress during booting and anthesis

Factorial pot experiments were conducted to compare the responses of GA-sensitive and GA-insensitive reduced height (Rht) alleles in wheat for susceptibility to heat and drought stress during booting and anthesis. Grain set (grains/spikelet) of near isogenic lines (NILs) was assessed following three day transfers to controlled environments imposing day temperatures (t) from 20 to 40°C. Transfers were during booting and/or anthesis and pots maintained at field capacity (FC) or had water withheld. Logistic responses (y = c/1+e-b(t -m)) described declining grain set with increasing t, and t5 was that fitted to give a 5% reduction in grain set. Averaged over NIL, t5 for anthesis at FC was 31.7±0.47°C (S.E.M, 26 d.f.). Drought at anthesis reduced t5 by <2°C. Maintaining FC at booting conferred considerable resistance to high temperatures (t5=33.9°C) but booting was particularly heat susceptible without water (t5 =26.5°C). In one background (cv. Mercia), for NILs varying at the Rht-D1 locus, there was progressive reduction in t5 with dwarfing and reduced gibberellic acid (GA) sensitivity (Rht-D1a, tall, 32.7±0.72; Rht-D1b, semi-dwarf, 29.5±0.85; Rht-D1c, severe dwarf, 24.2±0.72). This trend was not evident for the Rht-B1 locus, or for Rht-D1b in an alternative background (Maris Widgeon). The GA-sensitive severe dwarf Rht12 was more heat tolerant (t5=29.4±0.72) than the similarly statured GA-insensitive Rht-D1c. The GA-sensitive, semi-dwarfing Rht8 conferred greater drought tolerance in one experiment. Despite the effects of Rht-D1 alleles in Mercia on stress tolerance, the inconsistency of the effects over background and locus led to the conclusion that semi-dwarfing with GA-insensitivity did not necessarily increase sensitivity to stress at booting and flowering. In comparison to effects of semi-dwarfing alleles, responses to heat stress are much more dramatically affected by water availability and the precise growth stage at which the stress is experienced by the plants.

EFFECT OF TREATMENT WITH GROWTH-REGULATORS AND SCARIFICATION ON THE TOLERANCE TO WATER-STRESS IN SEEDS OF ORYZA-SATIVA LCVIAC-165

The effect of treatment of seeds of Oryza sativa L. cv. IAC 165 with gibberellic acid, 6-benzyladenine and ethrel on the tolerance to the water stress were analysed. Gibberellic acid had no effect and 6-benzyladenine and ethrel promoted slight increase in the tolerance to water stress, specially in darkness. After scarification, those growth regulator presented no effect. Our results suggest that 6-benzyladenine and ethrel promoted water stress tolerance due to the decrease in the resistance of the seed coat to embryo expansion.

Overexpression of UCP1 in tobacco induces mitochondrial biogenesis and amplifies a broad stress response

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of hormonal priming on seed germination of pigeon pea under cadmium stress

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mycorrhization alters foliar soluble amino acid composition and influences tolerance to Pb in Calopogonium mucunoides

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C (q)) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (beta-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.

Molecular basis of salt tolerance in Physcomitrella Patens model plant: potassium homeostasis and physiological roles of chx transporters

El suelo salino impone un estrés abiótico importante que causa graves problemas en la agricultura ya que la mayoría de los cultivos se ven afectados por la salinidad debido a efectos osmóticos y tóxicos. Por ello, la contaminación y la escasez de agua dulce, la salinización progresiva de tierras y el aumento exponencial de la población humana representan un grave problema que amenaza la seguridad alimentaria mundial para las generaciones futuras. Por lo tanto, aumentar la tolerancia a la salinidad de los cultivos es un objetivo estratégico e ineludible para garantizar el suministro de alimentos en el futuro. Mantener una óptima homeostasis de K+ en plantas que sufren estrés salino es un objetivo importante en el proceso de obtención de plantas tolerantes a la salinidad. Aunque el modelo de la homeostasis de K+ en las plantas está razonablemente bien descrito en términos de entrada de K+, muy poco se sabe acerca de los genes implicados en la salida de K+ o de su liberación desde la vacuola. En este trabajo se pretende aclarar algunos de los mecanismos implicados en la homeostasis de K+ en plantas. Para ello se eligió la briofita Physcomitrella patens, una planta no vascular de estructura simple y de fase haploide dominante que, entre muchas otras cualidades, hacen que sea un modelo ideal. Lo más importante es que no sólo P. patens es muy tolerante a altas concentraciones de Na+, sino que también su posición filogenética en la evolución de las plantas abre la posibilidad de estudiar los cambios claves que, durante el curso de la evolución, se produjeron en las diversas familias de los transportadores de K+. Se han propuesto varios transportadores de cationes como candidatos que podrían tener un papel en la salida de K+ o su liberación desde la vacuola, especialmente miembros de la familia CPA2 que contienen las familias de transportadores KEA y CHX. En este estudio se intenta aumentar nuestra comprensión de las funciones de los transportadores de CHX en las células de las plantas usando P. patens, como ya se ha dicho. En esta especie, se han identificado cuatro genes CHX, PpCHX1-4. Dos de estos genes, PpCHX1 y PpCHX2, se expresan aproximadamente al mismo nivel que el gen PpACT5, y los otros dos genes muestran una expresión muy baja. La expresión de PpCHX1 y PpCHX2 en mutantes de Escherichia coli defectivos en el transporte de K+ restauraron el crecimiento de esta cepa en medios con bajo contenido de K+, lo que viii sugiere que la entrada de K+ es energizada por un mecanismo de simporte con H+. Por otra parte, estos transportadores suprimieron el defecto asociado a la mutación kha1 en Saccharomyces cerevisiae, lo que sugiere que podrían mediar un antiporte en K+/H+. La proteína PpCHX1-GFP expresada transitoriamente en protoplastos de P. patens co-localizó con un marcador de Golgi. En experimentos similares, la proteína PpCHX2-GFP localizó aparentemente en la membrana plasmática y tonoplasto. Se construyeron las líneas mutantes simples de P. patens ΔPpchx1 y ΔPpchx2, y también el mutante doble ΔPpchx2 ΔPphak1. Los mutantes simples crecieron normalmente en todas las condiciones ensayadas y mostraron flujos de entrada normales de K+ y Rb+; la mutación ΔPpchx2 no aumentó el defecto de las plantas ΔPphak1. En experimentos a largo plazo, las plantas ΔPpchx2 mostraron una retención de Rb+ ligeramente superior que las plantas silvestres, lo que sugiere que PpCHX2 promueve la transferencia de Rb+ desde la vacuola al citosol o desde el citosol al medio externo, actuando en paralelo con otros transportadores. Sugerimos que transportadores de K+ de varias familias están involucrados en la homeostasis de pH de orgánulos ya sea mediante antiporte K+/H+ o simporte K+-H+.ix ABSTRACT Soil salinity is a major abiotic stress causing serious problems in agriculture as most crops are affected by it. Moreover, the contamination and shortage of freshwater, progressive land salinization and exponential increase of human population aggravates the problem implying that world food security may not be ensured for the next generations. Thus, a strategic and an unavoidable goal would be increasing salinity tolerance of plant crops to secure future food supply. Maintaining an optimum K+ homeostasis in plants under salinity stress is an important trait to pursue in the process of engineering salt tolerant plants. Although the model of K+ homeostasis in plants is reasonably well described in terms of K+ influx, very little is known about the genes implicated in K+ efflux or release from the vacuole. In this work, we aim to clarify some of the mechanisms involved in K+ homeostasis in plants. For this purpose, we chose the bryophyte plant Physcomitrella patens, a nonvascular plant of simple structure and dominant haploid phase that, among many other characteristics, makes it an ideal model. Most importantly, not only P. patens is very tolerant to high concentrations of Na+, but also its phylogenetic position in land plant evolution opens the possibility to study the key changes that occurred in K+ transporter families during the course of evolution. Several cation transporter candidates have been proposed to have a role in K+ efflux or release from the vacuole especially members of the CPA2 family which contains the KEA and CHX transporter families. We intended in this study to increase our understanding of the functions of CHX transporters in plant cells using P. patens, in which four CHX genes have been identified, PpCHX1-4. Two of these genes, PpCHX1 and PpCHX2, are expressed at approximately the same level as the PpACT5 gene, but the other two genes show an extremely low expression. PpCHX1 and PpCHX2 restored growth of Escherichia coli mutants on low K+-containing media, suggesting they mediated K+ uptake that may be energized by symport with H+. In contrast, these genes suppressed the defect associated to the kha1 mutation in Saccharomyces cerevisiae, which suggest that they might mediate K+/H+ antiport. PpCHX1-GFP protein transiently expressed in P. patens protoplasts co-localized with a Golgi marker. In similar experiments, the PpCHX2-GFP protein appeared to localize to tonoplast and plasma x membrane. We constructed the ΔPpchx1 and ΔPpchx2 single mutant lines, and the ΔPpchx2 ΔPphak1 double mutant. Single mutant plants grew normally under all the conditions tested and exhibited normal K+ and Rb+ influxes; the ΔPpchx2 mutation did not increase the defect of ΔPphak1 plants. In long-term experiments, ΔPpchx2 plants showed a slightly higher Rb+ retention than wild type plants, which suggests that PpCHX2 mediates the transfer of Rb+ from either the vacuole to the cytosol or from the cytosol to the external medium in parallel with other transporters. We suggest that K+ transporters of several families are involved in the pH homeostasis of organelles by mediating either K+/H+ antiport or K+-H+ symport.

Thermographic imaging: assessment of drought and heat tolerance in Spanish germplasm of Brachypodium distachyon.

The annual grass Brachypodium distachyon has been recently recognized as the model plant for functional genomics of temperate grasses, including cereals of economic relevance like wheat and barley. Sixty-two lines of B. distachyon were assessed for response to drought stress and heat tolerance. All these lines, except the reference genotype BD21, derive from specimens collected in 32 distinct locations of the Iberian Peninsula, covering a wide range of geo- climatic conditions. Sixteen lines of Brachypodium hybridum, an allotetraploid closely related to B. distachyon were used as reference of abiotic-stress well-adapted genotypes. Drought tolerance was assessed in a green-house trial. At the rosette-stage, no irrigation was applied to treated plants whereas their replicates at the control were maintained well watered during all the experiment. Thermographic images of treated and control plants were taken after 2 and 3 weeks of drought treatment, when stressed plants showed medium and extreme wilting symptoms. The mean leaf temperature of stressed (LTs) and control (LTc) plants was estimated based upon thermographic records from selected pixels (183 per image) that strictly correspond to leaf tissue. The response to drought was based on the analysis of two parameters: LTs and the thermal difference (TD) between stressed and control plants (LTs – LTc). The response to heat stress was based on LTc. Comparison of the mean values of these parameters showed that: 1) Genotypes better adapted to drought (B. hybridum lines) presented a higher LTs and TD than B. distachyon lines. 2) Under high temperature conditions, watered plants of B. hybridum lines maintained lower LTc than those of B. distachyon. Those results suggest that in these species adaptation to drought is linked to a more efficient stomata regulation: under water stress stomata are closed, increasing foliar temperature but also water use efficiency by reducing transpiration. With high temperature and water availability the results are less definite, but still seems that opening stomata allow plants to increase transpiration and therefore to diminish foliar temperature.

Transcriptome, carbohydrate, and phytohormone analysis of Petunia hybrida reveals a complex disturbance of plant functional integrity under mild chilling stress

Cultivation of chilling-tolerant ornamental crops at lower temperature could reduce the energy demands of heated greenhouses. To provide a better understanding of how sub-optimal temperatures (12 degrees C vs. 16 degrees C) affect growth of the sensitive Petunia hybrida cultivar 'SweetSunshine Williams', the transcriptome, carbohydrate metabolism, and phytohormone homeostasis were monitored in aerial plant parts over 4 weeks by use of a microarray, enzymatic assays and GC-MS/MS. The data revealed three consecutive phases of chilling response. The first days were marked by a strong accumulation of sugars, particularly in source leaves, preferential up-regulation of genes in the same tissue and down-regulation of several genes in the shoot apex, especially those involved in the abiotic stress response. The midterm phase featured a partial normalization of carbohydrate levels and gene expression. After 3 weeks of chilling exposure, a new stabilized balance was established. Reduced hexose levels in the shoot apex, reduced ratios of sugar levels between the apex and source leaves and a higher apical sucrose/hexose ratio, associated with decreased activity and expression of cell wall invertase, indicate that prolonged chilling induced sugar accumulation in source leaves at the expense of reduced sugar transport to and reduced sucrose utilization in the shoot. This was associated with reduced levels of indole-3-acetic acid and abscisic acid in the apex and high numbers of differentially, particularly up-regulated genes, especially in the source leaves, including those regulating histones, ethylene action, transcription factors, and a jasmonate-ZIM-domain protein. Transcripts of one Jumonji C domain containing protein and one expansin accumulated in source leaves throughout the chilling period. The results reveal a dynamic and complex disturbance of plant function in response to mild chilling, opening new perspectives for the comparative analysis of differently tolerant cultivars.

Alteration of cell-wall porosity is involved in osmotic stress-induced enhancement of aluminium resistance in common bean (Phaseolus vulgaris L.)

Aluminium (Al) toxicity and drought are the two major abiotic stress factors limiting common bean production in the tropics. Using hydroponics, the short-term effects of combined Al toxicity and drought stress on root growth and Al uptake into the root apex were investigated. In the presence of Al stress, PEG 6000 (polyethylene glycol)-induced osmotic (drought) stress led to the amelioration of Al-induced inhibition of root elongation in the Al-sensitive genotype VAX 1. PEG 6000 (>> PEG 1000) treatment greatly decreased Al accumulation in the 1 cm root apices even when the roots were physically separated from the PEG solution using dialysis membrane tubes. Upon removal of PEG from the treatment solution, the root tips recovered from osmotic stress and the Al accumulation capacity was quickly restored. The PEG-induced reduction of Al accumulation was not due to a lower phytotoxic Al concentration in the treatment solution, reduced negativity of the root apoplast, or to enhanced citrate exudation. Also cell-wall (CW) material isolated from PEG-treated roots showed a low Al-binding capacity which, however, was restored after destroying the physical structure of the CW. The comparison of the Al(3+), La(3+), Sr(2+), and Rb(+) binding capacity of the intact root tips and the isolated CW revealed the specificity of the PEG 6000 effect for Al. This could be due to the higher hydrated ionic radius of Al(3+) compared with other cations (Al(3+) >> La(3+) > Sr(2+) > Rb(+)). In conclusion, the results provide circumstantial evidence that the osmotic stress-inhibited Al accumulation in root apices and thus reduced Al-induced inhibition of root elongation in the Al-sensitive genotype VAX 1 is related to the alteration of CW porosity resulting from PEG 6000-induced dehydration of the root apoplast.

When supply does not meet demand-ER stress and plant programmed cell death

The endoplasmic reticulum (ER) is the central organelle in the eukaryotic secretory pathway. The ER functions in protein synthesis and maturation and is crucial for proper maintenance of cellular homeostasis and adaptation to adverse environments. Acting as a cellular sentinel, the ER is exquisitely sensitive to changing environments principally via the ER quality control machinery. When perturbed, ER-stress triggers a tightly regulated and highly conserved, signal transduction pathway known as the unfolded protein response (UPR) that prevents the dangerous accumulation of unfolded/misfolded proteins. In situations where excessive UPR activity surpasses threshold levels, cells deteriorate and eventually trigger programmed cell death (PCD) as a way for the organism to cope with dysfunctional or toxic signals. The programmed cell death that results from excessive ER stress in mammalian systems contributes to several important diseases including hypoxia, neurodegeneration, and diabetes. Importantly, hallmark features and markers of cell death that are associated with ER stress in mammals are also found in plants. In particular, there is a common, conserved set of chaperones that modulate ER cell death signaling. Here we review the elements of plant cell death responses to ER stress and note that an increasing number of plant-pathogen interactions are being identified in which the host ER is targeted by plant pathogens to establish compatibility.

Heat stress effects on grain sorghum productivity-biology and modelling

Heat stress can cause sterility in sorghum and the anticipated increased frequency of high temperature events implies increasing risk to sorghum productivity in Australia. Here we summarise our research on specific varietal attributes associated with heat stress tolerance in sorghum and evaluate how they might affect yield outcomes in production environments by a crop simulation analysis. We have recently conducted a range of controlled environment and field experiments to study the physiology and genetics of high temperature effects on growth and development of sorghum. Sorghum seed set was reduced by high temperature effects (>36-38oC) on pollen germination around flowering, but genotypes differed in their tolerance to high temperature stress. Effects were quantified in a manner that enabled their incorporation into the APSIM sorghum crop model. Simulation analysis indicated that risk of high temperature damage and yield loss depended on sowing date, and variety. While climate trends will exacerbate high temperature effects, avoidance by crop management and genetic tolerance seems possible.