Defective cytokinin signaling reprograms lipid and flavonoid gene-to-metabolite networks to mitigate high salinity in Arabidopsis.

Cytokinin (CK) in plants regulates both developmental processes and adaptation to environmental stresses. Arabidopsis histidine phosphotransfer ahp2,3,5 and type-B Arabidopsis response regulator arr1,10,12 triple mutants are almost completely defective in CK signaling, and the ahp2,3,5 mutant was reported to be salt tolerant. Here, we demonstrate that the arr1,10,12 mutant is also more tolerant to salt stress than wild-type (WT) plants. A comprehensive metabolite profiling coupled with transcriptome analysis of the ahp2,3,5 and arr1,10,12 mutants was conducted to elucidate the salt tolerance mechanisms mediated by CK signaling. Numerous primary (e.g., sugars, amino acids, and lipids) and secondary (e.g., flavonoids and sterols) metabolites accumulated in these mutants under nonsaline and saline conditions, suggesting that both prestress and poststress accumulations of stress-related metabolites contribute to improved salt tolerance in CK-signaling mutants. Specifically, the levels of sugars (e.g., trehalose and galactinol), amino acids (e.g., branched-chain amino acids and γ-aminobutyric acid), anthocyanins, sterols, and unsaturated triacylglycerols were higher in the mutant plants than in WT plants. Notably, the reprograming of flavonoid and lipid pools was highly coordinated and concomitant with the changes in transcriptional levels, indicating that these metabolic pathways are transcriptionally regulated by CK signaling. The discovery of the regulatory role of CK signaling on membrane lipid reprogramming provides a greater understanding of CK-mediated salt tolerance in plants. This knowledge will contribute to the development of salt-tolerant crops with the ability to withstand salinity as a key driver to ensure global food security in the era of climate crisis.

Effects of overproduced ethylene on the contents of other phytohormones and expression of their key biosynthetic genes.

Ethylene is involved in regulation of various aspects of plant growth and development. Physiological and genetic analyses have indicated the existence of crosstalk between ethylene and other phytohormones, including auxin, cytokinin (CK), abscisic acid (ABA), gibberellin (GA), salicylic acid (SA), jasmonic acid (JA), brassinosteroid (BR) and strigolactone (SL) in regulation of different developmental processes. However, the effects of ethylene on the biosynthesis and contents of these hormones are not fully understood. Here, we investigated how overproduction of ethylene may affect the contents of other plant hormones using the ethylene-overproducing mutant ethylene-overproducer 1 (eto1-1). The contents of various hormones and transcript levels of the associated biosynthetic genes in the 10-day-old Arabidopsis eto1-1 mutant and wild-type (WT) plants were determined and compared. Higher levels of CK and ABA, while lower levels of auxin, SA and GA were observed in eto1-1 plants in comparison with WT, which was supported by the up- or down-regulation of their biosynthetic genes. Although we could not quantify the BR and SL contents in Arabidopsis, we observed that the transcript levels of the potential rate-limiting BR and SL biosynthetic genes were increased in the eto1-1 versus WT plants, suggesting that BR and SL levels might be enhanced by ethylene overproduction. JA level was not affected by overproduction of ethylene, which might be explained by unaltered expression level of the proposed rate-limiting JA biosynthetic gene allene oxide synthase. Taken together, our results suggest that ET affects the levels of auxin, CK, ABA, SA and GA, and potentially BR and SL, by influencing the expression of genes involved in the rate-limiting steps of their biosynthesis.

Minimum values for midluteal plasma progesterone and estradiol concentrations in patients who achieved pregnancy with timed intercourse or intrauterine insemination without a human menopausal gonadotropin.

OBJECTIVE: The aim of the study was to assess the lower limits of midluteal plasma progesterone and estradiol concentrations in patients who achieved pregnancy with timed intercourse or intrauterine insemination without a human menopausal gonadotropin stimulation. RESULTS: We included 297 pregnant cycles of 297 women and assessed midluteal plasma progesterone and estradiol concentrations and pregnancy outcomes, retrospectively. These cycles were compared with the non-pregnant cycles (406 cycles) of the same women who became pregnant. Mean midluteal plasma P4 and E2 concentrations were significantly (P < 0.01) higher in pregnant cycles (14.5 and 188.5 pg/mL) than in non-pregnant cycles (10.7 and 162.6 pg/mL). The 5 percentiles of progesterone and estradiol in pregnant cycles were 5.6 and 70.2 pg/mL, respectively. The lowest progesterone and estradiol levels in pregnant cycles were 2.3 and 23.4 pg/mL, respectively. In non-pregnant cycles, many women had low P4 levels that were less than 5.6 ng/mL. Subgroup analyses showed slight differences among the four groups, which may have been due to the ovarian function of each group. Miscarriage was not related to progesterone and estradiol concentrations. These values may be useful for the evaluation of necessary values for pregnancy with timed intercourse or intrauterine insemination.

Is a high serum copper concentration a risk factor for implantation failure?

BACKGROUND: Copper-containing contraceptive devices may deposit copper ions in the endometrium, resulting in implantation failure. The deposition of copper ions in many organs has been reported in patients with untreated Wilson's disease. Since these patients sometimes exhibit subfertility and/or early pregnancy loss, copper ions were also considered to accumulate in the uterine endometrium. Wilson's disease patients treated with zinc successfully delivered babies because zinc interfered with the absorption of copper from the gastrointestinal tract. These findings led to the hypothesis that infertile patients with high serum copper concentrations may have implantation failure due to the excess accumulation of copper ions. The relationship between implantation (pregnancy) rates and serum copper concentrations has not yet been examined. The Japanese government recently stated that actual copper intake was higher among Japanese than needed. Therefore, the aim of the present study was to investigate whether serum copper concentrations are related to the implantation (pregnancy) rates of human embryos in vivo. METHODS: We included 269 patients (age <40 years old) who underwent vitrifying and warming single embryo transfer with a hormone replacement cycle using good blastocysts (3BB or more with Gardner's classification). Serum hCG, copper, and zinc concentrations were measured 16 days after the first date of progesterone replacement. We compared 96 women who were pregnant without miscarriage at 10 weeks of gestation (group P) and 173 women who were not pregnant (group NP). RESULTS: No significant differences were observed in age or BMI between the groups. Copper concentrations were significantly higher in group NP (average 193.2 µg/dL) than in group P (average 178.1 µg/dL). According to the area under the curve (AUC) on the receiver operating characteristic curve for the prediction of clinical pregnancy rates, the Cu/Zn ratio (AUC 0.64, 95% CI 0.54-0.71) was a better predictor than copper or zinc. When we set the cut-off as 1.59/1.60 for the Cu/Zn ratio, sensitivity, specificity, the positive predictive value, and negative predictive value were 0.98, 0.29, 0.71, and 0.88, respectively. CONCLUSIONS: Our single-center retrospective study suggests that high serum copper concentrations (high Cu/Zn ratio) are a risk factor for implantation failure.

Live birth rate following oral antibiotic treatment for chronic endometritis in infertile women with repeated implantation failure.

PROBLEM: The aim of this prospective study was to investigate the prevalence of chronic endometritis (CE) in infertile women with a history of repeated implantation failure (RIF) and to determine whether oral antibiotic treatment improves their live birth rate in the following embryo transfer (ET) cycles. METHOD OF STUDY: Endometrial biopsy samples obtained from infertile women with RIF were subjected to immunohistochemistrical/histopathologic diagnosis of CE. Following antibiotic administration to the RIF/CE group, their histopathologic cure rate, microbial detection rate, and reproductive outcome in the subsequent ET cycles were prospectively studied. RESULTS: 33.7% of infertile women with RIF were diagnosed with CE. Following the first-line doxycycline treatment, the histopathologic cure rate in the subsequent endometrial biopsy was 92.3%. Following the second-line metronidazole/ciprofloxacin treatment, the overall cure rate was 99.1%. The live birth rate in the first ET cycle (P=.031, RR 1.48, 95% CI 1.03-2.12) and cumulative three ET cycles (P=.037, RR 1.39, 95% CI 1.02-1.90) following antibiotic treatment in the cured RIF/CE group (32.8% and 38.8%, respectively) was significantly higher than in the RIF/non-CE group (22.1% and 27.9%, respectively). CONCLUSION: Chronic endometritis was found in one-third of infertile women with RIF. The oral antibiotic treatment against CE might be a promising therapeutic option for infertile women with RIF.

Clinical background affecting pregnancy outcome following local endometrial injury in infertile patients with repeated implantation failure.

Local endometrial injury (LEI) has been performed as a promising medical intervention to improve the pregnancy outcome in infertile women suffering from repeated implantation failure (RIF) in in vitro fertilization-embryo transfer cycles. The effect of LEI, however, remains controversial. The aim of this retrospective study was to identify the subgroups of patients with RIF who benefit from LEI. We compared the clinical parameters between the patients who had had a clinical pregnancy in the subsequent embryo transfer cycle following the LEI cycle (LEI-CP group, n = 94) and those who had resulted in negative pregnancy test (LEI-NP group, n = 114). The female age, basal follicle stimulating hormone concentration, number of past oocyte pickup cycles, and embryos/blastocysts transferred in the past three RIF cycles were significantly (p < 0.047) lower in the LEI-CP group than the LEI-NP group. The prevalence of polycystic ovarian syndrome was significantly (p = 0.0059) higher in the LEI-CP group than in the LEI-NP group. These findings suggest that LEI is most effective to improve the pregnancy outcome in patients undergoing RIF with uncompromised ovarian reserve, particularly in those with polycystic ovarian syndrome.

Arabidopsis type B cytokinin response regulators ARR1, ARR10, and ARR12 negatively regulate plant responses to drought.

In this study, we used a loss-of-function approach to elucidate the functions of three Arabidopsis type B response regulators (ARRs)--namely ARR1, ARR10, and ARR12--in regulating the Arabidopsis plant responses to drought. The arr1,10,12 triple mutant showed a significant increase in drought tolerance versus WT plants, as indicated by its higher relative water content and survival rate on drying soil. This enhanced drought tolerance of arr1,10,12 plants can be attributed to enhanced cell membrane integrity, increased anthocyanin biosynthesis, abscisic acid (ABA) hypersensitivity, and reduced stomatal aperture, but not to altered stomatal density. Further drought-tolerance tests of lower-order double and single mutants indicated that ARR1, ARR10, and ARR12 negatively and redundantly control plant responses to drought, with ARR1 appearing to bear the most critical function among the three proteins. In agreement with these findings, a comparative genome-wide analysis of the leaves of arr1,10,12 and WT plants under both normal and dehydration conditions suggested a cytokinin (CK) signaling-mediated network controlling plant adaptation to drought via many dehydration/drought- and/or ABA-responsive genes that can provide osmotic adjustment and protection to cellular and membrane structures. Expression of all three ARR genes was repressed by dehydration and ABA treatments, inferring that plants down-regulate these genes as an adaptive mechanism to survive drought. Collectively, our results demonstrate that repression of CK response, and thus CK signaling, is one of the strategies plants use to cope with water deficit, providing novel insight for the design of drought-tolerant plants by genetic engineering.

Chronic Endometritis: Potential Cause of Infertility and Obstetric and Neonatal Complications.

Chronic endometritis (CE) is a local inflammatory disease characterized by unusual plasmacyte infiltration in the endometrial stromal areas. CE has been neglected in gynecologic practice, as it is a less symptomatic benign disease that requires demanding and time-consuming histopathologic examinations for the definite diagnosis. Recent studies, however, suggest the association of CE with infertility and obstetric and neonatal complications. In this review article, we aimed to update the knowledge on epidemiology, etiology, and pathogenesis of CE as well as discuss its clinical management from diagnosis to treatment.

DT2008: a promising new genetic resource for improved drought tolerance in soybean when solely dependent on symbiotic N2 fixation.

Water deficit is one of the major constraints for soybean production in Vietnam. The soybean breeding research efforts conducted at the Agriculture Genetics Institute (AGI) of Vietnam resulted in the development of promising soybean genotypes, suitable for the drought-stressed areas in Vietnam and other countries. Such a variety, namely, DT2008, was recommended by AGI and widely used throughout the country. The aim of this work was to assess the growth of shoots, roots, and nodules of DT2008 versus Williams 82 (W82) in response to drought and subsequent rehydration in symbiotic association as a means to provide genetic resources for genomic research. Better shoot, root, and nodule growth and development were observed in the cultivar DT2008 under sufficient, water deficit, and recovery conditions. Our results represent a good foundation for further comparison of DT2008 and W82 at molecular levels using high throughput omic technologies, which will provide huge amounts of data, enabling us to understand the genetic network involved in regulation of soybean responses to water deficit and increasing the chances of developing drought-tolerant cultivars.

Spontaneous uterine rupture at 15 weeks' gestation in a patient with a history of cesarean delivery after removal of shirodkar cerclage.

A pregnant woman presented with acute upper abdominal pain and nausea at 15 weeks' gestation. She had a history of cesarean delivery for abruption after the removal of a Shirodkar cerclage that was placed because of cervical shortening caused by conization. She became pregnant again 14 months later. Ultrasonography revealed no significant findings, and a single intrauterine pregnancy with positive fetal heart activity was confirmed. An intestinal obstruction was suspected because abdominal radiography showed multiple air-fluid levels in the colon. Over the 3 hours following admission, her symptoms gradually worsened, and plain abdominal computed tomography (CT) showed a large hemorrhage in the abdominal cavity, but the uterine wall appeared intact at this time. Subsequently, dynamic CT revealed discontinuity of the uterine muscle layer. During laparotomy, uterine rupture with complete opening of the uterine wall at the site of the previous transverse scar was identified. A dead fetus was located within the amniotic sac in a blood-filled abdominal cavity. She received a total of 10 units of packed red blood cells and 6 units of fresh frozen plasma for the resuscitation. She was discharged on the eighth postoperative day without any complications.

Positive regulatory role of strigolactone in plant responses to drought and salt stress.

This report provides direct evidence that strigolactone (SL) positively regulates drought and high salinity responses in Arabidopsis. Both SL-deficient and SL-response [more axillary growth (max)] mutants exhibited hypersensitivity to drought and salt stress, which was associated with shoot- rather than root-related traits. Exogenous SL treatment rescued the drought-sensitive phenotype of the SL-deficient mutants but not of the SL-response mutant, and enhanced drought tolerance of WT plants, confirming the role of SL as a positive regulator in stress response. In agreement with the drought-sensitive phenotype, max mutants exhibited increased leaf stomatal density relative to WT and slower abscisic acid (ABA)-induced stomatal closure. Compared with WT, the max mutants exhibited increased leaf water loss rate during dehydration and decreased ABA responsiveness during germination and postgermination. Collectively, these results indicate that cross-talk between SL and ABA plays an important role in integrating stress signals to regulate stomatal development and function. Additionally, a comparative microarray analysis of the leaves of the SL-response max2 mutant and WT plants under normal and dehydrative conditions revealed an SL-mediated network controlling plant responses to stress via many stress- and/or ABA-responsive and cytokinin metabolism-related genes. Our results demonstrate that plants integrate multiple hormone-response pathways for adaptation to environmental stress. Based on our results, genetic modulation of SL content/response could be applied as a potential approach to reduce the negative impact of abiotic stress on crop productivity.

Rapid degeneration of noncoding DNA regions surrounding SlAP3X/Y after recombination suppression in the dioecious plant Silene latifolia.

Silene latifolia is a dioecious plant with heteromorphic XY sex chromosomes. Previous studies of sex chromosome-linked genes have suggested a gradual divergence between the X-linked and the Y-linked genes in proportion to the distance from the pseudoautosomal region. However, such a comparison has yet to be made for the noncoding regions. To better characterize the nonrecombining region of the X and Y chromosomes, we sequenced bacterial artificial chromosome clones containing the sex chromosome-linked paralogs SlAP3X and SlAP3Y, including 115 kb and 73 kb of sequences, respectively, flanking these genes. The synonymous nucleotide divergence between SlAP3X and SlAP3Y indicated that recombination stopped approximately 3.4 million years ago. Sequence homology analysis revealed the presence of six long terminal repeat retrotransposon-like elements. Using the nucleotide divergence calculated between left and right long terminal repeat sequences, insertion dates were estimated to be 0.083-1.6 million years ago, implying that all elements detected were inserted after recombination stopped. A reciprocal sequence homology search facilitated the identification of four homologous noncoding DNA regions between the X and Y chromosomes, spanning 6.7% and 10.6% of the X chromosome-derived and Y chromosome-derived sequences, respectively, investigated. Genomic Southern blotting and fluorescence in situ hybridization showed that the noncoding DNA flanking SlAP3X/Y has homology to many regions throughout the genome, regardless of whether they were homologous between the X and Y chromosomes. This finding suggests that most noncoding DNA regions rapidly lose their counterparts because of the introduction of transposable elements and indels (insertion-deletions) after recombination has stopped.

The auxin response factor transcription factor family in soybean: genome-wide identification and expression analyses during development and water stress.

In plants, the auxin response factor (ARF) transcription factors play important roles in regulating diverse biological processes, including development, growth, cell division and responses to environmental stimuli. An exhaustive search of soybean genome revealed 51 GmARFs, many of which were formed by genome duplications. The typical GmARFs (43 members) contain a DNA-binding domain, an ARF domain and an auxin/indole acetic acid (AUX/IAA) dimerization domain, whereas the remaining eight members lack the dimerization domain. Phylogenetic analysis of the ARFs from soybean and Arabidopsis revealed both similarity and divergence between the two ARF families, as well as enabled us to predict the functions of the GmARFs. Using quantitative real-time polymerase chain reaction (qRT-PCR) and available soybean Affymetrix array and Illumina transcriptome sequence data, a comprehensive expression atlas of GmARF genes was obtained in various organs and tissues, providing useful information about their involvement in defining the precise nature of individual tissues. Furthermore, expression profiling using qRT-PCR and microarray data revealed many water stress-responsive GmARFs in soybean, albeit with different patterns depending on types of tissues and/or developmental stages. Our systematic analysis has identified excellent tissue-specific and/or stress-responsive candidate GmARF genes for in-depth in planta functional analyses, which would lead to potential applications in the development of genetically modified soybean cultivars with enhanced drought tolerance.

Diversity of plant methionine sulfoxide reductases B and evolution of a form specific for free methionine sulfoxide.

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological conditions. Organisms evolved two distinct methionine sulfoxide reductase families (MSRA & MSRB) to repair oxidized methionine residues. We found that 5 MSRB genes exist in the soybean genome, including GmMSRB1 and two segmentally duplicated gene pairs (GmMSRB2 and GmMSRB5, GmMSRB3 and GmMSRB4). GmMSRB2 and GmMSRB4 proteins showed MSRB activity toward protein-based MetO with either DTT or thioredoxin (TRX) as reductants, whereas GmMSRB1 was active only with DTT. GmMSRB2 had a typical MSRB mechanism with Cys121 and Cys 68 as catalytic and resolving residues, respectively. Surprisingly, this enzyme also possessed the MSRB activity toward free Met-R-O with kinetic parameters similar to those reported for fRMSR from Escherichia coli, an enzyme specific for free Met-R-O. Overexpression of GmMSRB2 or GmMSRB4 in the yeast cytosol supported the growth of the triple MSRA/MSRB/fRMSR (Δ3MSRs) mutant on MetO and protected cells against H2O2-induced stress. Taken together, our data reveal an unexpected diversity of MSRBs in plants and indicate that, in contrast to mammals that cannot reduce free Met-R-O and microorganisms that use fRMSR for this purpose, plants evolved MSRBs for the reduction of both free and protein-based MetO.

Arabidopsis AHP2, AHP3, and AHP5 histidine phosphotransfer proteins function as redundant negative regulators of drought stress response.

Cytokinin is an essential phytohormone controlling various biological processes, including environmental stress responses. In Arabidopsis, although the cytokinin (CK)-related phosphorelay--consisting of three histidine kinases, five histidine phosphotransfer proteins (AHPs), and a number of response regulators--has been known to be important for stress responses, the AHPs required for CK signaling during drought stress remain elusive. Here, we report that three Arabidopsis AHPs, namely AHP2, AHP3, and AHP5, control responses to drought stress in negative and redundant manner. Loss of function of these three AHP genes resulted in a strong drought-tolerant phenotype that was associated with the stimulation of protective mechanisms. Specifically, cell membrane integrity was improved as well as an increased sensitivity to abscisic acid (ABA) was observed rather than an alteration in ABA-mediated stomatal closure and density. Consistent with their negative regulatory functions, all three AHP genes' expression was down-regulated by dehydration, which most likely resulted from a stress-induced reduction of endogenous CK levels. Furthermore, global transcriptional analysis of ahp2,3,5 leaves revealed down-regulation of many well-known stress- and/or ABA-responsive genes, suggesting that these three AHPs may control drought response in both ABA-dependent and ABA-independent manners. The discovery of mechanisms of activation and the targets of the downstream components of CK signaling involved in stress responses is an important and challenging goal for the study of plant stress regulatory network responses and plant growth. The knowledge gained from this study also has broad potential for biotechnological applications to increase abiotic stress tolerance in plants.

Characterization of the newly developed soybean cultivar DT2008 in relation to the model variety W82 reveals a new genetic resource for comparative and functional genomics for improved drought tolerance.

Soybean (Glycine max) productivity is adversely affected by drought stress worldwide, including Vietnam. In the last few years, we have made a great effort in the development of drought-tolerant soybean cultivars by breeding and/or radiation-induced mutagenesis. One of the newly developed cultivars, the DT2008, showed enhanced drought tolerance and stable yield in the field conditions. The purpose of this study was to compare the drought-tolerant phenotype of DT2008 and Williams 82 (W82) by assessing their water loss and growth rate under dehydration and/or drought stress conditions as a means to provide genetic resources for further comparative and functional genomics. We found that DT2008 had reduced water loss under both dehydration and drought stresses in comparison with W82. The examination of root and shoot growths of DT2008 and W82 under both normal and drought conditions indicated that DT2008 maintains a better shoot and root growth rates than W82 under both two growth conditions. These results together suggest that DT2008 has better drought tolerance degree than W82. Our results open the way for further comparison of DT2008 and W82 at molecular levels by advanced omic approaches to identify mutation(s) involved in the enhancement of drought tolerance of DT2008, contributing to our understanding of drought tolerance mechanisms in soybean. Mutation(s) identified are potential candidates for genetic engineering of elite soybean varieties to improve drought tolerance and biomass.

Differential gene expression in soybean leaf tissues at late developmental stages under drought stress revealed by genome-wide transcriptome analysis.

The availability of complete genome sequence of soybean has allowed research community to design the 66 K Affymetrix Soybean Array GeneChip for genome-wide expression profiling of soybean. In this study, we carried out microarray analysis of leaf tissues of soybean plants, which were subjected to drought stress from late vegetative V6 and from full bloom reproductive R2 stages. Our data analyses showed that out of 46,093 soybean genes, which were predicted with high confidence among approximately 66,000 putative genes, 41,059 genes could be assigned with a known function. Using the criteria of a ratio change > = 2 and a q-value<0.05, we identified 1458 and 1818 upregulated and 1582 and 1688 downregulated genes in drought-stressed V6 and R2 leaves, respectively. These datasets were classified into 19 most abundant biological categories with similar proportions. There were only 612 and 463 genes that were overlapped among the upregulated and downregulated genes, respectively, in both stages, suggesting that both conserved and unconserved pathways might be involved in regulation of drought response in different stages of plant development. A comparative expression analysis using our datasets and that of drought stressed Arabidopsis leaves revealed the existence of both conserved and species-specific mechanisms that regulate drought responses. Many upregulated genes encode either regulatory proteins, such as transcription factors, including those with high homology to Arabidopsis DREB, NAC, AREB and ZAT/STZ transcription factors, kinases and two-component system members, or functional proteins, e.g. late embryogenesis-abundant proteins, glycosyltransferases, glycoside hydrolases, defensins and glyoxalase I family proteins. A detailed analysis of the GmNAC family and the hormone-related gene category showed that expression of many GmNAC and hormone-related genes was altered by drought in V6 and/or R2 leaves. Additionally, the downregulation of many photosynthesis-related genes, which contribute to growth retardation under drought stress, may serve as an adaptive mechanism for plant survival. This study has identified excellent drought-responsive candidate genes for in-depth characterization and future development of improved drought-tolerant transgenic soybeans.

Regulatory roles of cytokinins and cytokinin signaling in response to potassium deficiency in Arabidopsis.

Potassium (K) is an important plant macronutrient that has various functions throughout the whole plant over its entire life span. Cytokinins (CKs) are known to regulate macronutrient homeostasis by controlling the expression of nitrate, phosphate and sulfate transporters. Although several studies have described how CKs signal deficiencies for some macronutrients, the roles of CKs in K signaling are poorly understood. CK content has been shown to decrease under K-starved conditions. Specifically, a CK-deficient mutant was more tolerant to low K than wild-type; however, a plant with an overaccumulation of CKs was more sensitive to low K. These results suggest that K deprivation alters CK metabolism, leading to a decrease in CK content. To investigate this phenomenon further, several Arabidopsis lines, including a CK-deficient mutant and CK receptor mutants, were analyzed in low K conditions using molecular, genetic and biochemical approaches. ROS accumulation and root hair growth in low K were also influenced by CKs. CK receptor mutants lost the responsiveness to K-deficient signaling, including ROS accumulation and root hair growth, but the CK-deficient mutant accumulated more ROS and exhibited up-regulated expression of HAK5, which is a high-affinity K uptake transporter gene that is rapidly induced by low K stress in ROS- and ethylene-dependent manner in response to low K. From these results, we conclude that a reduction in CK levels subsequently allows fast and effective stimulation of low K-induced ROS accumulation, root hair growth and HAK5 expression, leading to plant adaptation to low K conditions.

Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions.

Quantitative RT-PCR can be a very sensitive and powerful technique for measuring differential gene expression. Changes in gene expression induced by abiotic stresses are complex and multifaceted, which make determining stably expressed genes for data normalization difficult. To identify the most suitable reference genes for abiotic stress studies in soybean, 13 candidate genes collected from literature were evaluated for stability of expression under dehydration, high salinity, cold and ABA (abscisic acid) treatments using delta CT and geNorm approaches. Validation of reference genes indicated that the best reference genes are tissue- and stress-dependent. With respect to dehydration treatment, the Fbox/ABC, Fbox/60s gene pairs were found to have the highest expression stability in the root and shoot tissues of soybean seedlings, respectively. Fbox and 60s genes are the most suitable reference genes across dehydrated root and shoot tissues. Under salt stress the ELF1b/IDE and Fbox/ELF1b are the most stably expressed gene pairs in roots and shoots, respectively, while 60s/Fbox is the best gene pair in both tissues. For studying cold stress in roots or shoots, IDE/60s and Fbox/Act27 are good reference gene pairs, respectively. With regard to gene expression analysis under ABA treatment in either roots, shoots or across these tissues, 60s/ELF1b, ELF1b/Fbox and 60s/ELF1b are the most suitable reference genes, respectively. The expression of ELF1b/60s, 60s/Fbox and 60s/Fbox genes was most stable in roots, shoots and both tissues, respectively, under various stresses studied. Among the genes tested, 60s was found to be the best reference gene in different tissues and under various stress conditions. The highly ranked reference genes identified from this study were proved to be capable of detecting subtle differences in expression rates that otherwise would be missed if a less stable reference gene was used.

Identification and expression analysis of cytokinin metabolic genes in soybean under normal and drought conditions in relation to cytokinin levels.

Cytokinins (CKs) mediate cellular responses to drought stress and targeted control of CK metabolism can be used to develop drought-tolerant plants. Aiming to manipulate CK levels to improve drought tolerance of soybean cultivars through genetic engineering of CK metabolic genes, we surveyed the soybean genome and identified 14 CK biosynthetic (isopentenyltransferase, GmIPT) and 17 CK degradative (CK dehydrogenase, GmCKX) genes. Comparative analyses of GmIPTs and GmCKXs with Arabidopsis counterparts revealed their similar architecture. The average numbers of abiotic stress-inducible cis-elements per promoter were 0.4 and 1.2 for GmIPT and GmCKX genes, respectively, suggesting that upregulation of GmCKXs, thereby reduction of CK levels, maybe the major events under abiotic stresses. Indeed, the expression of 12 GmCKX genes was upregulated by dehydration in R2 roots. Overall, the expressions of soybean CK metabolic genes in various tissues at various stages were highly responsive to drought. CK contents in various organs at the reproductive (R2) stage were also determined under well-watered and drought stress conditions. Although tRNA-type GmIPT genes were highly expressed in soybean, cis-zeatin and its derivatives were found at low concentrations. Moreover, reduction of total CK content in R2 leaves under drought was attributable to the decrease in dihydrozeatin levels, suggesting a role of this molecule in regulating soybean's responses to drought stress. Our systematic analysis of the GmIPT and GmCKX families has provided an insight into CK metabolism in soybean under drought stress and a solid foundation for in-depth characterization and future development of improved drought-tolerant soybean cultivars by manipulation of CK levels via biotechnological approach.