Jarosław Tyburski

Reactive oxygen species localization in roots of Arabidopsis thaliana seedlings grown under phosphate deficiency

Arabidopsis plants responding to phosphorus (P) deficiency increase lateral root formation and reduce primary root elongation. In addition the number and length of root hairs increases in response to P deficiency. Here we studied the patterns of radical oxygen species (ROS) in the roots of Arabidopsis seedlings cultured on media supplemented with high or low P concentration. We found that P availability affected ROS distribution in the apical part of roots. If plants were grown on high P medium, ROS were located in the root elongation zone and quiescent centre. At low P ROS were absent in the elongation zone, however, their synthesis was detected in the primary root meristem. The proximal part of roots was characterized by ROS production in the lateral root primordia and in elongation zones of young lateral roots irrespective of P concentration in the medium. On the other hand, plants grown at high or low P differed in the pattern of ROS distribution in older lateral roots. At high P, the elongation zone was the primary site of ROS production. At low P, ROS were not detected in the elongation zone. However, they were present in the proximal part of the lateral root meristem. These results suggest that P deficiency affects ROS distribution in distal parts of Arabidopsis roots. Under P-sufficiency ROS maximum was observed in the elongation zone, under low P, ROS were not synthesized in this segment of the root, however, they were detected in the apical root meristem.

Exogenous auxin regulates H2O2 metabolism in roots of tomato (Lycopersicon esculentum Mill.) seedlings affecting the expression and activity of CuZn-superoxide dismutase, catalase, and peroxidase

The aim of this study was to test the effect of auxin treatment on selected parameters of the redox metabolism in roots. We found that auxin application results in a reduction in the H2O2 level in roots. The hormone stimulated CuZn-superoxide dismutase, but simultaneously increased the activities of catalase, cell wall bound ferulic acid peroxidase, and soluble peroxidase izoenzymes. The analysis of the expression of genes coding for the cytosolic izoform of CuZn-superoxide dismutase, catalase, and cell wall associated peroxidase (TPX 1) involved in cell wall stiffening and lignification revealed the stimulatory effect of exogenous auxin on the expression of the aforementioned genes. The enzyme activity and gene expression in the roots of control and auxin-treated plants were studied in daily intervals, during a 3-day-long growth cycle. The stimulatory effect of auxin on the enzymatic activity was transient with the highest stimulation observed on the second day of treatment. On the third day, the activities of the enzymes decreased. The maximal enzyme activities were preceded by a rise in gene expression. The increase in the level of CuZn-superoxide dismutase and catalase transcripts were detected after 1 day of auxin treatment. Then the expression of the aforementioned genes decreased. The period of auxin-dependent stimulation of the TPX 1 gene expression encompassed the first and the second day of treatment. Auxin stimulated CuZn-superoxide dismutase and catalase activities only in the distal zone of the root while peroxidase activity was increased by auxin in the distal as well as in the proximal parts of the organ.

The role of light and polar auxin transport in root regeneration from hypocotyls of tomato seedling cuttings

A relationship between light conditions, auxin transport and adventitious root formation by hypocotyls of tomato seedling cuttings was demonstrated. Effective rooting of tomato seedling cuttings was observed under continuous white light (WL) irradiation. However, root formation was reduced in darkness or under red (RL) or blue light (BL). At least 3/4-day-long irradiation treatment with (WL) was necessary to increase the number of roots formed in comparison with control cuttings grown in darkness. Light was most effective if applied during the first half of the 13-day-long rooting period. The role of photoreceptor-dependent light perception in the light-regulation of rooting was tested using tomato photomorphogenic mutants: aurea (au) and high pigment (hp). When exposed to WL both mutants generated fewer roots then their isogenic wild type (WT). In darkness or under BL and RL less roots were formed on all plants and no difference was observed between mutants and WT plants. TIBA (2,3,5-triiodobenzoic acid) inhibited rooting in a dose-dependent manner both in darkness and under WL. However, although rooting was suppressed by 0.75 μM TIBA in the dark, 8 μM TIBA was necessary to block root formation in continuous WL. Inhibition of rooting by TIBA was most efficient when applied at the initial period of rooting, a 1-day-long treatment with TIBA being sufficient to suppress rooting if given during the first 2 days of culture. Later treatment had much less effect on the root formation.

Exogenous auxin affects ascorbate metabolism in roots of tomato seedlings

Ascorbate levels and redox states, as well as the activities of the enzymes of ascorbate metabolism, were analyzed in roots of tomato seedlings during the culture on a medium supplemented with auxin and compared to the control cultured on an auxin-free medium. Biochemical parameters were determined separately in the distal part of the root where the inhibitory effect of auxin on root elongation growth is observed and in the proximal half on the organ which reacts to auxin treatment with increased lateral root proliferation. ASC peroxidase activity was found to be stimulated by auxin treatment in the lateral-root forming part of the root. This effect was not observed in the distal part of the organ. On the other hand, ASC oxidase activity was raised by auxin exclusively in the distal part of the root. An inhibitory effect of auxin supplementation to the medium on ASC—reducing enzymes was observed. The dehydroascorbate reductase activity was found to be inhibited by auxin only in the proximal part, while the activity of monodehydroascorbate reductase in both, the proximal and distal parts of the root. Ascorbate content increased in roots during culture irrespective of the presence of auxin. However, auxin treatment resulted in higher DHA levels and more significant participation of DHA in the total ascorbate pool when compared to the control grown on the auxin-free medium. Similar to auxin, adding DHA to the culture medium stimulated lateral root formation and inhibited primary root elongation. In contrast to DHA, ASC treatment affected significantly neither lateral root formation nor primary root growth and partly reversed the stimulatory effect of IAA on root formation and the inhibitory effect on root elongation. These results suggest that auxin induced changes in ascorbate metabolism may be involved in developmental reactions in tomato roots.

A role for redox factors in shaping root architecture under phosphorus deficiency

The developmental response of the Arabidopsis root system to low phosphorus (P) availability involves the reduction in primary root elongation accompanied by the formation of numerous lateral roots. We studied the roles of selected redox metabolites, namely, radical oxygen species (ROS) and ascorbic acid (ASC) in the regulation of root system architecture by different P availability. Rapidly growing roots of plants grown on P-sufficient medium synthesize ROS in root elongation zone and quiescent centre. We have demonstrated that the arrest of root elongation at low P medium coincides with the disappearance of ROS from the elongation zone. P-starvation resulted in a decrease in ascorbic acid level in roots. This correlated with a decrease in cell division activity. On the other hand, feeding P-deficient plants with ASC, stimulated mitotic activity in the primary root meristem and partly reversed the inhibition of root growth imposed by low P conditions. In this paper, we discuss the idea of the involvement of redox agents in the regulation of root system architecture under low P availability.

Cloning and Expression Analysis of a Gene Encoding for Ascorbate Peroxidase and Responsive to Salt Stress in Beet (Beta vulgaris).

BvpAPX is a full-length cDNA-encoding peroxisomal ascorbate peroxidase isolated from leaves of salt-stressed beet (Beta vulgaris) plants. A high level of identity has been reported between the deduced amino acid sequence of BvpAPX and other known ascorbate peroxidases. The genomic sequence of BvpAPX revealed a gene composed of 5 exons and 4 introns. Several sequence motifs revealed in the 5’UTR region of the gene confer to BvpAPX a putative responsiveness to various abiotic stresses. We determined the effect of salt stress on BvpAPX expression in leaves of the cultivated beet varieties, Huzar and Janosik, and their wild salt-tolerant relative B. vulgaris ssp. maritima. Plants were subjected to salt stress during a 32-day culture period (long-term salt treatment). An alternative salinization protocol consisted of an 18-h incubation of detached beet leaves in media supplemented with toxic salt concentrations (short-term salt treatment). RT-Q-PCR analysis revealed that BvpAPX expression markedly increased in leaves of plants subjected to conditions of long-term treatment with salinity, whereas BvpAPX transcript levels remained unaffected in detached leaves during short-term salt treatment. In addition, several leaf redox system parameters, such as ascorbate peroxidase activity or ascorbic acid, hydrogen peroxide, and lipid hydroperoxide concentration, were determined in the leaves of beet plants subjected to salt stress conditions.

Role of Ascorbate in the Regulation of the Arabidopsis thaliana Root Growth by Phosphate Availability

Arabidopsis root system responds to phosphorus (P) deficiency by decreasing primary root elongation and developing abundant lateral roots. Feeding plants with ascorbic acid (ASC) stimulated primary root elongation in seedlings grown under limiting P concentration. However, at high P, ASC inhibited root growth. Seedlings of ascorbate-deficient mutant (vtc1) formed short roots irrespective of P availability. P-starved plants accumulated less ascorbate in primary root tips than those grown under high P. ASC-treatment stimulated cell divisions in root tips of seedlings grown at low P. At high P concentrations ASC decreased the number of mitotic cells in the root tips. The lateral root density in seedlings grown under P deficiency was decreased by ASC treatments. At high P, this parameter was not affected by ASC-supplementation. vtc1 mutant exhibited increased lateral root formation on either, P-deficient or P-sufficient medium. Irrespective of P availability, high ASC concentrations reduced density and growth of root hairs. These results suggest that ascorbate may participate in the regulation of primary root elongation at different phosphate availability via its effect on mitotic activity in the root tips.

Glutathione and glutathione disulfide affect adventitious root formation and growth in tomato seedling cuttings

To study the relationship between glutathione and rooting, tomato seedling cuttings, grown on basal- or on auxin-supplemented media, were treated with the reduced (GSH) or oxidized (GSSG) form of this antioxidant. In turn, the consequences of the depletion of GSH pool on rooting were tested using l-buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis. Effects of the aforementioned treatments on rooting response were assessed. GSH treatment promoted root formation on cuttings grown on both basal- and auxin-supplemented media. Whereas GSSG did not affect the number of roots formed by cuttings grown on basal medium, it strongly enhanced the rooting stimulatory effect of auxin treatment. GSH depletion resulting from BSO application did not change the number of roots formed. All the tested compounds, namely GSH, GSSG, BSO and auxin, had a strong inhibitory effect on the elongation of regenerated roots. Supplementing the rooting medium with glutathione efficiently increased the GSH level in the rooting zones, while addition of BSO led to a strong decrease in endogenous GSH level. Neither of the treatments affected the level of GSSG. Exogenous auxin affect neither GSH nor GSSG levels in rooting zones; however, in the regenerated roots, GSH level was significantly higher when the organs were formed on auxin-supplemented medium. Patterns of GSH distribution in the roots regenerated on basal- and auxin-enriched media were studied using the GSH-specific dye monochlorobimane and confocal laser scanning microscopy. GSH was found in the root apical meristem and in the elongation zone. Auxin did not change the GSH distribution; however, the number of fluorescent cells was higher when roots were regenerated on auxin-supplemented medium.

Transcriptomic profiling of the salt stress response in excised leaves of the halophyte Beta vulgaris ssp. maritima.

Beta vulgaris ssp. maritima is a halophytic relative of cultivated beets. In the present work a transcriptome response to acute salt stress imposed to excised leaves of sea beet was investigated. Salt treatments consisted of adding NaCl directly to the transpiration stream by immersing the petioles of excised leaves into the salt solutions. Sequencing libraries were generated from leaves subjected to either moderate or strong salt stress. Control libraries were constructed from untreated leaves. Sequencing was performed using the Illumina MiSeq platform. We obtained 32970 unigenes by assembling the pooled reads from all the libraries with Trinity software. Screening the nr database returned 18,362 sequences with functional annotation. Using the reference transcriptome we identified 1,246 genes that were differentially expressed after 48 h of NaCl stress. Genes related to several cellular functions such as membrane transport, osmoprotection, molecular chaperoning, redox metabolism or protein synthesis were differentially expressed in response to salt stress. The response of sea beet leaves to salt treatments was marked out by transcriptomic up-regulation of genes related to photosynthetic carbon fixation, ribosome biogenesis, cell wall-building and cell wall expansion. Furthermore, several novel and undescribed transcripts were responsive to salinity in leaves of sea beet.

Organogenetic Response of Photomorphogenic Mutants of Tomato

Summary The effect of white (WL) and red (RL) light on organogenesis in vitro was studied using explants isolated from seedlings of wild-type (WT) and two photomorphogenic mutants of tomato ( Lycopersicon esculentum Mill.) - aurea ( au ) and high pigment ( hp ). Explants excised from hypocotyls and cotyledons of green and etiolated seedlings were cultured on shoot or root inducing media. It was observed that both continuous white (CWL) and red light (CRL) stimulate shoot formation on hypocotyl explants isolated from green seedlings of WT and hp plants compared with control ones cultured in darkness. On the other hand, au mutant shows very low organogenetic response in spite of light conditions applied. Explants isolated from both green and etiolated seedlings were not able to form shoots when they were cultured in darkness. In contrast to green explants, etiolated ones formed roots in spite of being grown on a shoot inducing medium. Root regeneration from etiolated explants was stimulated by short, 5-min-long daily pulses of RL. This effect was reversed by subsequent far-red light (FRL) irradiation. Stimulation of shoot regeneration from etiolated explants was found when 2-h-long daily irradiation with WL or RL was applied. The highest and the lowest shoot regeneration response was obtained from hp and au explants, respectively, with an intermediate response from WE Under the same growth conditions shoot formation was accompanied by root formation, which also occurred in a light dependent manner. The highest number of roots regenerated from au -derived explants. The results that we have obtained may suggest that shoot formation is strongly dependent on the light sensitivity of plants and light conditions applied. It also seems that the pattern of organ (shoot and root) development in tomato is affected by the etiolated/deetiolated phenotype of explant. Therefore, we believe that the organogenetic response of tomato in vitro is at least partly regulated by phytochrome.