Comparative expression analysis of the Arabidopsis karrikin (KAR)-receptor mutant, kai2-2, and wild type (WT) plants (Col-0) under dehydration conditions
ABSTRACT: To understand the role of KAR-signaling components in water stress response, we have carried out comparative expression analysis of the KAR-receptor kai2-2 mutant and WT plants under dehydration and well-watered (control) conditions. Aligent’s whole Arabidopsis Gene Expression Microarray (G2519F-021169, V4, 4x44K) was used. Overall design: Two-week-old WT and kai2-2 mutant plants were transferred from GM plates to soil and grown for 10 additional day. The aerial parts of 24-d-old plants were detached and exposed to dehydration on KimTowel papers for 0 (well-watered, control), 2 and 4 h. All rosette leaves of independent 24-d-old plants were collected. Total RNA was prepared and used for the microarray hybridization. Three independent biological replicates were used for each plant sample.
INSTRUMENT(S): Agilent-021169 Arabidopsis 4 Oligo Microarray (V4) (Probe Name version)
Project description:To understand the role of SL-signaling components in water stress response, we have carried out comparative expression analysis of the SL-response max2-3 mutant and WT plants under dehydration and well-watered (control) conditions. Aligent’s whole Arabidopsis Gene Expression Microarray (G2519F-021169, V4, 4x44K) was used. Two-week-old WT and max2-3 mutant plants were transferred from GM plates to soil and grown for 10 additional day. The aerial parts of 24-d-old plants were detached and exposed to dehydration on KimTowel papers for 0 (well-watered, control), 2 and 4 h. All rosette leaves of independent 24-d-old plants were collected. Total RNA was prepared and used for the microarray hybridization. Three independent biological replicates were used for each plant sample.
Project description:To understand the role of the Arabidopsis type-B Response Regulators ARR1, ARR10 and ARR12 in water stress response, we have carried out comparative expression analysis of the arr1,10,12 mutant and WT plants under dehydration and well-watered (control) conditions. Agilent’s whole Arabidopsis Gene Expression Microarray (G2519F-021169, V4, 4x44K) was used. Two-week-old WT and arr1,10,12 mutant plants were transferred from GM plates to soil and grown for 10 additional day. Aerial portions of 24-d-old plants were detached and exposed to dehydration on KimTowel papers for 0 (well-watered, control), 2 and 4 h. Rosette leaves collected in 3 biological repeats from arr1,10,12 and WT plants treated by dehydration for 0, 2 and 4 h were used for microarray and expression analyses. Total RNA was prepared and used for the microarray hybridization. Three independent biological replicates were used for each plant sample.
Project description:To understand the responses of plants to environmental stresses will help mitigate the problems via creating stress-tolerant crop cultivars. We have carried out comparative expression analysis of roots of two soybean varieties Williams 82 and DT2008 that have constrasting drought-responsive phenotype under dehydration and well-watered (control) conditions. Affymetrix’s whole Soybean Gene Expression Microarray (66K) was used. The Williams 82 and DT2008 soybean plants were grown for 14 days in the vermiculite soil under greenhouse conditions. The whole plants of 14-d-old plants were detached and exposed to dehydration on KimTowel papers for 0 (well-watered, control), 2 and 10 h. All roots of independent 14-d-old plants were collected. Total RNA was prepared and used for the microarray hybridization. Three independent biological replicates were used for each plant sample.
Project description:To understand the role of CK-signaling components, AHPs (His-containing phosphotransfer proteins) in drought stress response, we have employed transcriptional profiling of ahp2,3,5 and it's wild type plant, Col-0 under drought and well-water (control) conditions. Aligent’s Whole Arabidopsis Gene Expression Microarray (G2519F-021169, V4, 4x44K) was used. Two-week-old WT and ahp2,3,5 mutant plants were transferred from GM plates to soil and grown for an additional week. Three week-old plants were exposed to drought stress for 10 days or grown under well-watered condition in parallel. All rosette leaves of independent 31d-old plants were corrected. Total RNA was prepared and used for the microarray hybridization. Three replicative hybridization experiments were carried out for each independent biological sample.
Project description:Os02g31890 encodes a dehydration-responsive transcription factor (named ´ARID´) from rice (Oryza sativa, cv. Dongjin). Expression profiling was performed 90 min after the start of dehydration stress in roots of Oryza sativa wild-type plants (cv. Dongjin) and a knock-out (i.e. arid) mutant. Wild-type rice plants and a line carrying a T-DNA insertion in the third exon of the transcription factor gene (arid mutant) were subjected to dehydration stress for 90 min. Well-watered wild-type and T-DNA insertion plants were used as controls. Total RNA was extracted from roots and subjected to expression profiling using rice Affymetrix microarrays.
Project description:New shoot growth from underground adventitious buds of leafy spurge is critical for survival of this invasive perennial weed after episodes of severe abiotic stress. Because global climate change is expected to increase abiotic stress, such as dehydration, objectives of this study include examining the impact that dehydration stress has on molecular mechanisms associated with vegetative reproduction. Greenhouse plants were exposed to mild- (3-day), intermediate- (7-day), severe- (14-day) and extended- (21-day) dehydration treatments, prior to decapitation of aerial tissue and rehydration of soil to induce new vegetative shoot growth. Compared to well-watered control plants, mild-dehydration accelerated new vegetative shoot growth but intermediate- and severe-dehydration treatments both delayed and reduced shoot growth, and 21-day dehydration treatment inhibited initiation of new vegetative shoots and was considered a lethal treatment. Overall, transcriptome profiles revealed that 2109 genes were differentially-expressed (P<0.05) in crown buds in response to the various dehydration treatments. Sub-network enrichment analyses identified central hubs of over-represented genes involved in processes such as hormone responses and signaling (e.g., ABA, auxin, ethylene, GA, and JA), response to abiotic stress (DREB1A/2A) and light (PIF3), phosphorylation (CLV1, MPK3/4/6, SOS2), gene silencing (miRNA156/172a), circadian regulation (CRY2, LHY, PHYA/B), and flowering (AGL8/20, AP2, FLC). Further, results from this and previous studies highlight HY5, MAF3, MYB-like/RVE1 and RD22 as molecular markers for endodormancy in crown buds of leafy spurge. Early response to dehydration also highlighted involvement of upstream ethylene and jasmonate signaling, whereas longer-term dehydration impacted ABA signaling. The identification of conserved ABRE- and MYC-consensus, cis-acting elements in the promoter of a leafy spurge gene similar to Arabidopsis MYB-like/RVE1 (AT5G17300) implicates a potential role for ABA signaling in its dehydration-induced expression. Response of these molecular mechanisms to dehydration-stress provides insights on the ability of invasive perennial weeds to adapt and survive under harsh environments, which provide new insights for addressing future management practices. Changes in transcript abundance for underground adventitious buds of leafy spurge which were exposed various levels of dehydration stress (Day-3, -7, -14, -16, -21) are analysed relative to controls (Day-0).
Project description:Persistence of the invasive perennial weed leafy spurge is mainly attributed to seasonal production of underground adventitious buds (UABs), which undergo well-defined phases of dormancy (para-, endo- and eco-dormancy). These well-defined phases of dormancy also allow UABs of leafy spurge to survive extreme seasonal variations, including dehydration-stress. Consequently, objectives of this study include understanding the effects of dehydration-stress on vegetative reproduction, flowering competence, and transcript profiles at different phases of bud dormancy. The vegetative growth potential of UABs was monitored by removing the aerial portion of dehydration-stressed plants and re-watering the root system. Further, microarray analysis was used to follow transcriptome profiles to identify critical defense- and signaling-pathways at different phases of dormancy in UABs. Surprisingly, only 3 days of dehydration-stress is required to break the endodormant phase in UABs. In leafy spurge, vernalization of endodormant UABs has previously been shown to induce flower competence, while breaking of endodormancy via dehydration-stress did not induce floral induction. Thus, these two environmental treatments open a unique approach to independently dissect molecular mechanisms involved in endodormancy maintenance and floral competence. Bioinformatics analysis of transcriptome data helped to identify models and overlapping pathways. During endodormancy break, LEC1, PHOTOSYSTEM I RC, and brassinosteroids were identified as ooverlapping hubs of up-regulated genes, and DREB1A, CBF2, GPA1, MYC2, BHLH, BZIP, and flavonoids were identified as overlapping hubs of down-regulated genes. Additionally, key genes involved in metabolic activity, chromatin modification, and cross-talk between growth regulators were identified as playing a role during endodormancy maintenance. Overall design: Plant material, controlled environmental treatments, and vegetative growth: Three-month-old paradormant leafy spurge plants grown in a greenhouse were acclimated for one week, under growth chamber conditions, prior to being subjected to a ramp-down in temperature (27 °C → 10 °C) and photoperiod (16 h → 8 h light) for 12 weeks to induce endodormancy, as previously established (Doğramacı et al. 2010; Foley et al. 2009). Endodormancy status was confirmed by comparing vegetative growth rates of plants with and without a ramp-down treatment. To study the effects of dehydration-stress on vegetative growth from UABs, water was withheld from endodormant plants up to 21 days. After dehydration for 0, 1-, 3-, 7-, 14-, and 21-days, the aerial portion of the plants were decapitated at the soil surface and vegetative growth and floral competence of UABs were monitored under growth-conducive conditions by re-watering the root system. Vegetative shoot growth from six plants was recorded weekly for four weeks, and results of four replications were analyzed using SAS 9.2 (SAS Institute, Cary, NC, 2008) software as described by Doğramacı et al. (2010). Microarray analysis: At the end of each treatment, crown buds were collected, and RNA extraction, cDNA synthesis, fluorescent labeling, microarray hybridization using ~23K element arrays, and spot intensity analyses were performed as previously described by Doğramacı et al. (2010). Based on unexpected vegetative growth results obtained from this study, only microarray data obtained from 0-, 1-, and 3-day dehydration-stressed endodormant UABs, which were compared with microarray data for endodormant, and flowering competent ecodormant UABs from our previous study (Doğramacı et al. 2010; GSE19217), were used for bioinformatics analyses. However, to merge the datasets from these two separate experiments, T-tests were first performed on microarray data obtained from endodormant UABs from each experiment, and differentially-expressed genes (p<0.005) were removed from the dataset (~5% of the spots). After removing the outliers, the two endodormant transcriptome data sets were grouped together and used as the baseline control for further ANOVA, T-tests and other analyses. Array normalization, statistical analyses and clustering of the dataset and Venn diagram generation were done using GeneMaths XT 5.1 software as previously described by Doğramacı et al. (2010).
Project description:Arabidopsis thaliana plants that have experienced an initial exposure to dehydration stress (“trained plants”) have an increased ability to maintain leaf relative water content (RWC) during subsequent stresses than plants experiencing the stress for the first time and transcription of selected dehydration response genes is altered during successive exposures to dehydration stress. This physiological and transcriptional behavior of trained plants is consistent with a “memory “of an earlier stress. It is unknown whether such memory is present in other Angiosperm lineages and whether it is an evolutionarily conserved response to stress (see E-GEOD-48235). Here, we analyzed the behavior and transcriptomes of maize (Zea mays) plants experiencing multiple dehydration stresses and compare them with responses of the evolutionarily distant A. thaliana. We found structurally related genes in maize that displayed the same memory-type responses as in A. thaliana, providing evidence of the conservation of function and transcriptional memory in the evolution of plants’ dehydration stress response systems. Similar to A. thaliana, trained Z. mays plants retained higher RWC during dehydration stress than untrained plants, due in part to maintaining reduced stomatal conductance, despite full recovery of RWC, after the first stress. Divergent transcriptional memory responses were also expressed, suggesting diversification of function among stress memory genes. Some dehydration stress memory genes were also shared with other stress and hormone responding pathways, indicating complex and dynamic interactions between different plant signaling networks. The results provide new insight into how plants respond to multiple dehydration stresses and provide a platform for studies of the functions of memory genes in adaptive responses to water deficit in monocot and eudicot plants . For each condition (water, S1, and S3) the transcriptome was sequenced for two replicates. The watered condition is considered the control.
Project description:Arabidopsis plants were grown in plastic pots filled with peat moss for 3 weeks (principal growth stage 1.07-1.08) under a 16 h light/8 h dark regimen (40 ± 10 ?mol photons/m2/s) at 22 C.Dehydration treatment: The 3-week-old plants were grown for 2 or 3 days without watering. To obtain accurate results, we carefully raised single plants in Petri dishes, each containing an equal amount of soil. Soil moisture contents were calculated from soil dry weight. Untreated; the soil moisture content was 84.3%. Under dehydration, on the second day, the soil moisture content was 51.1%. Under dehydration, on the third day, the moisture content was 11.6%.
Project description:Wild-type rice plants (O. sativa L. cv. Nipponbare) were grown in plastic pots filled with nutrient soil for 2 weeks under flooded lowland conditions and a 12 h/12 h light/dark cycle (50 ± 10 ?mol photons/m2/s) at 28°C (day) and 25°C (night). For dehydration treatment, two-week-old plants were incubated for 3 days without watering. The soil moisture content was 15.6% on day 3 of dehydration.