Characterization of Arabidopsis sterol glycosyltransferase TTG15/UGT80B1 role during freeze and heat stress

Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of TTG15/UGT80B1 gene of Arabidopsis thaliana in freeze/thaw and heat shock stress using T-DNA insertional sgt knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, TTG15/UGT80B1 knockout mutant and p35S:TTG15/UGT80B1 restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at -1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, p35S:TTG15/UGT80B1 restored plants were more adapted to freeze stress than TTG15/UGT80B1 knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both β-sitosterol and sitosterol glycoside quantity was more in Col-0 and p35S:TTG15/UGT80B1 restored lines, whereas it was significantly less in TTG15/UGT80B1 knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress.     

Changes in homologous recombination frequency in Arabidopsis thaliana plants exposed to stress depend on time of exposure during development and on duration of stress exposure

In the past, we showed that exposure to abiotic and biotic stresses changes the homologous recombination frequency (HRF) in somatic tissue and in the progeny. In current work we planned to answer the following question: do stress intensity/duration and time during exposure influence changes in somatic HRF and transgenerational changes in HRF? Here, we tested the effects of exposure to UV-C, cold and heat on HRF at 7, 14, 21 and 28 days post germination (dpg). We found that exposure at 14 and 21 dpg resulted in a higher increase in HRF as compared to exposure at 7 dpg; longer exposure to UV-C resulted in a higher frequency of HR, whereas prolonged exposure to cold or heat, especially at later developmental stages, had almost no effect on somatic HRF. Exposure at 7 dpg had a positive effect on somatic growth of plants; plants exposed to stress at this age had larger leaves. The analysis of HRF in the progeny showed that the progeny of plants exposed to stress at 7 dpg had an increase in somatic HRF and showed larger sizes of recombination spots on leaves. The progeny of plants exposed to UV-C at 7 dpg and the progeny of plants exposed to cold or heat at 28 dpg had larger leaves as compared to control plants. To summarize, our experiments showed that changes in somatic and transgenerational HRF depend on the type of stress plants are exposed to, time of exposure during development and the duration of exposure.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-013-0197-z) contains supplementary material, which is available to authorized users.     

Phosphatidic acid is a major phospholipid class in reproductive organs of Arabidopsis thaliana

Phospholipids are the crucial components of biological membranes and signal transduction. Among different tissues, flower phospholipids are one of the least characterized features of plant lipidome. Here, we report that floral reproductive organs of Arabidopsis thaliana contain high levels of phosphatidic acid (PA), a known lipid second messenger. By using floral homeotic mutants enriched with specific floral organs, lipidomics study showed increased levels of PA species in ap3-3 mutant with enriched pistils. Accompanied gene expression study for 7 diacylglycerol kinases and 11 PA phosphatases revealed distinct floral organ specificity, suggesting an active phosphorylation/dephosphorylation between PA and diacylglycerol in flowers. Our results suggest that PA is a major phospholipid class in floral reproductive organs of A. thaliana.     

A protective role for the polyamine spermine against drought stress in Arabidopsis

Cellular polyamine content often changes in response to abiotic stresses. However, its physiological relevance is unknown. We found that an Arabidopsis mutant plant (acl5/spms), which cannot produce spermine, is hypersensitive to high salt. Examination of drought sensitivity of the mutant and comparison with wild type plants indicated hypersensitivity to drought. This phenotype was cured by spermine pretreatment but not by the other polyamines putrescine and spermidine, suggesting that drought-hypersensitivity exhibited by the mutant is due to spermine deficiency. The water loss rate of wild type and mutant plants were similar until 20 min after onset of dehydration stress, but after a longer exposure the rate in mutant plants was higher than in wild type plants. Consistent with this result, the stomata of the mutant leaves remained open while in wild type leaves they closed. Based on the collected data, we discuss a role for spermine in response to drought stress.     

Cortex proliferation in the root is a protective mechanism against abiotic stress

Although as an organ the root plays a pivotal role in nutrient and water uptake as well anchorage, individual cell types function distinctly. Cortex is regarded as the least differentiated cell type in the root, but little is known about its role in plant growth and physiology. In recent studies, we found that cortex proliferation can be induced by oxidative stress. Since all types of abiotic stress lead to oxidative stress, this finding suggests a role for cortex in coping with abiotic stress. This hypothesis was tested in this study using the spy mutant, which has an extra layer of cortex in the root. Interestingly, the spy mutant was shown to be hypersensitive to salt and oxidizing reagent applied to the leaves, but it was as tolerant as the wild type to these compounds in the soil. This result lends support to the notion that cortex has a protective role against abiotic stress arising from the soil.     

Nitric oxide accumulation is required to protect against iron-mediated oxidative stress in frataxin-deficient Arabidopsis plants

Frataxin is a mitochondrial protein that is conserved throughout evolution. In yeast and mammals, frataxin is essential for cellular iron (Fe) homeostasis and survival during oxidative stress. In plants, frataxin deficiency causes increased reactive oxygen species (ROS) production and high sensitivity to oxidative stress. In this work we show that a knock-down T-DNA frataxin-deficient mutant of Arabidopsis thaliana (atfh-1) contains increased total and organellar Fe levels. Frataxin deficiency leads also to nitric oxide (NO) accumulation in both, atfh-1 roots and frataxin null mutant yeast. Abnormally high NO production might be part of the defence mechanism against Fe-mediated oxidative stress.     

Mild ammonium stress increases chlorophyll content in Arabidopsis thaliana

Nitrate (NO₃⁻) and ammonium (NH₄⁺) are the main forms of nitrogen available in the soil for plants. Excessive NH₄⁺ accumulation in tissues is toxic for plants and exclusive NH₄⁺-based nutrition enhances this effect. Ammonium toxicity syndrome commonly includes growth impairment, ion imbalance and chlorosis among others. In this work, we observed high intraspecific variability in chlorophyll content in 47 Arabidopsis thaliana natural accessions grown under 1 mM NH₄⁺ or 1 mM NO₃⁻ as N-source. Interestingly, chlorophyll content increased in every accession upon ammonium nutrition. Moreover, this increase was independent of ammonium tolerance capacity. Thus, chlorosis seems to be an exclusive effect of severe ammonium toxicity while mild ammonium stress induces chlorophyll accumulation.     

Differential expression of two related,low-temperature-induced genes in Arabidopsis thaliana (L.) Heynh

Plant cold acclimation is correlated to expression of low-temperature-induced (lti) genes. By using a previously characterized lti cDNA clone as a probe we isolated a genomic fragment that carried two closely located lti genes of Arabidopsis thaliana. The genes were structurally related with the coding regions interrupted by three similarly located short introns and were transcribed in the same direction. The nucleotide sequences of the two genes, lti78 and lti65, predict novel hydrophilic polypeptides with molecular weights of 77856 and 64510, respectively, lti78 corresponding to the cDNA probe. Of the 710 amino acids of LTI78 and 600 amino acids of LTI65, 346 amino acids were identical between the polypeptides, which suggests that the genes may have a common origin.Both lti78 and lti65 were induced by low temperature, exogenous abscisic acid (ABA) and drought, but the responsiveness of the genes to these stimuli was markedly different. Both the levels and the temporal pattern of expression differed between the genes. Expression of lti78 was mainly responsive to low temperature, that of lti65 to drought and ABA. In contrast to the induction of lti78, which follows separate signal pathways during low-temperature, ABA and drought treatment, the drought induction of lti65 is ABA-dependent and the low-temperature induction appears to be coupled to the ABA biosynthetic pathway. This differential expression of two related genes may indicate that they have some-what different roles in the stress response.     

Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana

High-salinity, drought, and low temperature are three common environmental stress factors that seriously influence plant growth and development worldwide. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators that have also been linked to stress responses. However, the relationship between miRNA expression and stress responses is just beginning to be explored. Here, we identified 14 stress-inducible miRNAs using microarray data in which the effects of three abiotic stresses were surveyed in Arabidopsis thaliana. Among them, 10 high-salinity-, four drought-, and 10 cold-regulated miRNAs were detected, respectively. miR168, miR171, and miR396 responded to all of the stresses. Expression profiling by RT-PCR analysis showed great cross-talk among the high-salinity, drought, and cold stress signaling pathways. The existence of stress-related elements in miRNA promoter regions provided further evidence supporting our results. These findings extend the current view about miRNA as ubiquitous regulators under stress conditions.     

The Arabidopsis LECTIN RECEPTOR KINASE-VI.2 is a functional protein kinase and is dispensable for basal resistance to Botrytis cinerea

Sensing of microbial pathogens by pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) elicits a defense program known as PAMP-triggered immunity (PTI). Recently, we have shown that the Arabidopsis thaliana L-TYPE LECTIN RECEPTOR KINASE-VI.2 (LecRK-VI.2) positively regulates bacterial PTI. In this report, we suggest by in silico analysis that the kinase domain of LecRK-VI.2 is functional. LecRK-VI.2 also demonstrated auto-phosphorylation activity in vitro in the presence of divalent metal cations indicating that LecRK-VI.2 has the ability to auto-phosphorylate. We further investigate the role of LecRK-VI.2 in Arabidopsis resistance to the necrotrophic fungal pathogen Botrytis cinerea. Disruption of LecRK-VI.2 did not affect Arabidopsis resistance to B. cinerea. Accordingly, wild-type upregulation levels of PTI-responsive WRKY53, FRK1, NHL10, CYP81F2 and CBP60 g after treatment with the fungal PAMP chitin were observed in lecrk-VI.2-1. These data provide evidences that the kinase domain of LecRK-VI.2 is active and show that LecRK-VI.2 is not critical for resistance to the fungal pathogen B. cinerea.     

ATHB17 is a positive regulator of abscisic acid response during early seedling growth

We performed activation tagging screen to isolate abscisic acid (ABA) response mutants. One of the mutants, designated ahs10 (ABA-hypersensitive 10), exhibited ABA-hypersensitive phenotypes. TAIL-PCR analysis of the mutant revealed that T-DNA was inserted in the promoter region of the Arabidopsis gene, At2g01430, which encodes a homeodomain-leucine zipper protein ATHB17. Subsequent expression analysis indicated that ATHB17 was activated in ahs10. To recapitulate the mutant phenotypes, we prepared ATHB17 OX lines and investigated their phenotypes. The results showed that ATHB17 confers ABA-hypersensitivity and drought tolerance. On the contrary, ATHB17 knockout lines were ABA-insensitive and drought-sensitive, further demonstrating that ATHB17 is involved in ABA and water-stress responses. Interestingly, the ATHB17 effect on seedling growth in the presence of ABA was observed only during the postgermination seedling establishment stage, suggesting that it functions during a narrow developmental window of early seedling growth.     

RNA interference-mediated suppression of xanthine dehydrogenase reveals the role of purine metabolism in drought tolerance in Arabidopsis

We have previously demonstrated that RNA interference-mediated suppression of xanthine dehydrogenase (XDH), the rate-limiting enzyme in purine degradation, causes defects in the normal growth and development of Arabidopsis thaliana. Here, we investigated a possible role for XDH in drought tolerance, since this enzyme is also implicated in plant stress responses and acclimatization. When XDH-suppressed lines were subjected to drought stress, plant growth was markedly reduced in conjunction with significantly enhanced cell death and H₂O₂ accumulation. This drought-hypersensitive phenotype was reversed by pretreatment with exogenous uric acid, the catalytic product of XDH. These results suggest that fully functional purine metabolism plays a role in the Arabidopsis drought acclimatization.