Analysis of Brachypodium distachyon UVR8 reveals conservation in UV-B receptors.

The Ultraviolet Resistance Locus 8 (UVR8) in plants recognizes ultraviolet-B (UV-B) light and plays a crucial role in regulating plant growth through a series of signal transduction events. However, the UVR8 in monocotyledon crops has not yet been systematically analysed. We identified BdUVR8 (BRADI_3g45740) from the genome of Brachypodium distachyon, a relative of wheat, by analysing the phylogenetic tree, the gene expression pattern, detecting accumulation of UV-B response metabolites, and checking for phenotype recovery. The BdUVR8 protein sequence is similar to the known UVR8 of other species. The phylogenetic tree of UVR8 shows clear divergence between dicotyledons and monocotyledons. Expression analysis revealed that UV-B downregulates BdUVR8 by 70% and upregulates the chalcone synthase (BdCHS) gene 3.4-fold in B. distachyon. The pCAMBIA1300::BdUVR8-mCherry construct introduced into Arabidopsis uvr8 mutants showed that the BdUVR8 protein is localized in the cytoplasm and translocates into the nucleus in response to UV-B irradiation. The introduction of BdUVR8 into uvr8 rescued hypocotyl elongation caused by UV-B and restored expression of HY5, Chalcone synthase, and Flavanone 3-hydroxylase, as well as accumulation of total flavonoids. Together, our results show that BdUVR8 is a photoreceptor that perceives UV-B in B. distachyon.

Da Vinci SP robotic approach to colorectal surgery: two specific indications and short-term results.

BACKGROUND: Da Vinci® Single Port (dvSP) was recently developed. Its application in colorectal surgery is under investigation. The aim of this study was to explore the safety and feasibility of dvSP for intersphincteric (dvSP-ISR), right colectomy (dvSP-RC), and transverse colectomy (dvSP-TC). Surgical indication and short-term results were analyzed. METHODS: All consecutive patients from a prospective database of patients who underwent dvSP-ISR, dvSP-RC, and dvSP-TC at Korea University Anam Hospital from November 2020 to December 2021, were analyzed. Perioperative, pathological, and oncological short-term outcomes were analyzed. RESULTS: A total of 7 dvSP-ISR, 5 dvSP-RC, and 1 dvSP-TC were performed. Median age was 56.0 (55.0-61.0) years for the dvSP-ISR and 54.0 (44.7-63.5) years for the dvSP-RC/TC. Median body mass index was 22.8 (17.1-24.8) kg/m2 for the dvSP-ISR and 23.6 (20.8-26.9) kg/m2 for the dvSP-RC/TC. All dvSP-ISR patients received neoadjuvant long-course chemoradiotherapy, including one patient with squamocellular carcinoma who was treated with 5-fluorouracil (5-FU)/mitomycin. All other patients, excluding one dvSP-RC patient with Crohn's disease, had an adenocarcinoma. Median operation time was 280 (240-370) minutes for the dvSP-ISR and 220 (201-270) minutes for the dvSP-RC/TC. Estimated blood loss was insignificant. No intraoperative complications or conversions to multiport/open surgery was reported. Median post-operative stay was 7.0 (6.0-10.0) days for the dvSP-ISR and 5.0 (4.0-6.7) days for the dvSP-RC/TC. Quality of mesorectum was complete for six patients, and nearly complete for one. Median number of retrieved lymph nodes were 21 (17-25) for the dvSP-ISR and 28 (24-49) for the dvSP-RC/TC. Proximal and distal resection margins were tumor free. Four patients experienced post-operative complications not related to the platform which were: ileus, voiding dysfunction, infected pelvic hematoma, and wound infection. Median follow-up was 9 (6-11) months and 11 (7-17) months for the dvSP-ISR and dvSP-RC/TC, respectively. Two patients had systemic recurrence; all others were tumor free. CONCLUSIONS: The dvSP platform is safe and feasible for intersphincteric resection with right lower quadrant access, and right/transverse colectomy with suprapubic access. Further studies are needed to evaluate benefit differences compared to multiport robotic platform.

Risk factors for local recurrence and long term survival after minimally invasive intersphincteric resection for very low rectal cancer: Multivariate analysis in 161 patients.

INTRODUCTION: Intersphincteric resection (ISR) is the ultimate anal-sparing technique as an alternative to abdominoperineal resection in selected patients. Oncological safety is still debated. This study analyses long-term oncological results and evaluates risk factors for local recurrence (LR) and overall survival (OS) after minimally-invasive ISR. MATERIALS AND METHODS: Retrospective single-center data were collected from a prospectively maintained colorectal database. A total of 161 patients underwent ISR between 2008 and 2018. OS and local recurrence-free survival (LRFS) were assessed using Kaplan-Meier analysis (log-rank test). Risk factors for OS and LRFS were assessed with Cox-regression analysis. RESULTS: Median follow-up was 55 months. LR occurred in 18 patients. OS and LRFS rates at 1, 3, and 5 years were 96%, 91%, and 80% and 96%, 89%, and 87%, respectively. Tumor size (p = 0.035) and clinical T-stage (p = 0.029) were risk factors for LRFS on univariate analysis. On multivariate analysis, tumor size (HR 2.546 (95% CI: 0.976-6.637); p = 0.056) and clinical T-stage (HR 3.296 (95% CI: 0.941-11.549); p = 0.062) were not significant. Preoperative CEA (p < 0.001), pathological T-stage (p = 0.033), pathological N-stage (p = 0.016) and adjuvant treatment (p = 0.008) were prognostic factors for OS on univariate analysis. Preoperative CEA (HR 4.453 (95% CI: 2.015-9.838); p < 0.001) was a prognostic factor on multivariate analysis. CONCLUSIONS: This study confirms the oncological safety of minimally-invasive ISR for locally advanced low-lying rectal tumors when performed in experienced centers. Despite not a risk factor for LR, tumor size and, locally advanced T-stage with anterior involvement should be carefully evaluated for optimal surgical strategy. Preoperative CEA is a prognostic factor for OS.

Robotic-assisted resection for beyond TME rectal cancer: a novel classification and analysis from a specialized center.

Locally advanced rectal cancer often requires an extended resection beyond the total mesorectal excision plane (bTME) to obtain clear resection margins. We classified three types of bTME rectal cancer following local disease diffusion: radial (adjacent pelvic organs), lateral (pelvic lateral lymph nodes) and longitudinal (below 3.5 cm from the anal verge, submitted to intersphincteric resection). The primary aim of this study was to evaluate the application of robotic surgery to the three types of bTME regarding the short and long-term oncological outcomes. Secondary aim was to identify survival prognostic factors for bTME rectal cancers. A total of 137 patients who underwent robotic-assisted bTME procedures between 2008 and 2018 were extracted from a prospectively collected database. Patient-related, operative and pathological factors were assessed. Morbidity was moderately high with 66% of patients reporting postoperative complications. Median follow up was 47 months (IQR, 31.5-66.5). Local recurrence rate was 15.3% with a statistical difference between the three types of bTME (p = 0.041). Disease progression/distant metastasis rate was 33.6%. Overall survival was significantly different (p = 0.023) with 1- and 3-years rates of: 77.8% and 55.0% (radial; n = 19); 96.6% and 84.8% (lateral; n = 30); 97.7% and 86.9% (longitudinal; n = 88). No statistical difference was observed for disease-free survival (p = 0.897). Local recurrence-free survival was significantly different between the groups (p = 0.031). Multivariate analysis showed that (y)pT (p = 0.028; HR (95% CI) 5.133 (1.192-22.097)), (y)pN (p = 0.014; HR (95% CI) 2.835 (1.240-6.482)) and type of bTME were associated to OS whilst (y)pT (p = 0.072) and type of bTME were not associated to LRFS.

A multicentre comparative study between laparoscopic and open surgery for intussusception in adults.

AIM: Intussusception in adults is rare and requires surgery in most cases. While abdominal laparoscopic surgery (LS) is becoming more popular, there are few reports on the outcomes of adult intussusception treated with LS. This study compared the feasibility of LS vs open surgery (OS) for adult intussusception. METHOD: We reviewed retrospectively the medical records of adult patients with intussusception from three tertiary hospitals between 2000 and 2016. The patients were divided into LS and OS groups, and their surgical outcomes were compared. RESULTS: Surgery was indicated in 71 patients with intussusception (41 LS and 30 OS). The median age of the patients was 49.0 and 51.5 years in the LS and OS groups, respectively (P = 0.930). Overall, nine (12.7%) patients had a negative laparotomy or laparoscopy with spontaneous reduction of the intussusception. Conversion to OS from LS was necessary in one patient (2.4%). The operative time and intra-operative and postoperative complication rates were not significantly different. However, there were more serious complications such as bowel perforation and major vessel injury in the LS group. The patients in the LS group had a shorter time to first food intake and hospital stay vs patients in the OS group (4.0 vs 6.0 days, P < 0.001, and 7.0 vs 10.5 days, P < 0.001, respectively). CONCLUSION: LS may be feasible for adult intussusception; there may be more severe intra-operative complications than in OS.

Intraobserver and interobserver reliability of the computed tomography-based radiographic classification of primary elbow osteoarthritis: comparison with plain radiograph-based classification and clinical assessment.

OBJECTIVES: To develop a staging system that could better reflect symptoms by the spurs quantification in the fossa and joint space narrowing using computed tomography (CT) for elbow arthritis and to evaluate its reproducibility with multiple readers. METHODS: This retrospective study evaluated 81 cases of primary elbow osteoarthritis using both plain radiography and CT. Qualitative and quantitative analyses were independently performed by four orthopedic surgeons using previous and newly proposed staging systems. The reproducibility of the new system was analyzed with intraclass correlation coefficients (ICC). Correlations between symptoms and radiologic classification were assessed using Pearson's correlation coefficient (PCC). RESULTS: The interobserver agreement (1) and intraobserver agreement (2) among the four evaluators was present by ICC. (1) The system of Hastings and Rettig [first observation, 0.544 (95% confidence interval (CI), 0.436-0.649); second observation, 0.582 (95% CI, 0.478-0.682)] and Broberg and Morrey's staging system [first observation, 0.620 (95% CI, 0.521-0.714); second observation, 0.656 (95% CI, 0.562-0.743)] showed substantial and moderate retrospective agreement, whereas the CT-based staging system showed almost perfect agreement [first observation, 0.867 (95% CI, 0.820-0.906); second observation, 0.909 (95% CI, 0.875-0.936)]. (2) The intraobserver agreement was almost perfect in the Brogerg and Morrey's and CT-based staging systems. CT-based staging showed high correlation with visual analogue scale (PCC 0.754, P < 0.001) and Mayo elbow performance score (PCC -0.614, P < 0.001) and moderate correlation with range of motion (PCC -0.458, P < 0.001). CONCLUSIONS: CT-based staging system was highly reproducible and clinically feasible than previous plain radiograph-based staging systems.

Hybrid transgastric approach for the treatment of gastroesophageal junction pathologies.

Flexible endoscopy has evolved to the point that it includes many endoluminal procedures that once required open or laparoscopic surgery, for instance, antireflux surgery, pyloromyotomy, mucosal and submucosal tumor resections, and even full-thickness resection. However, these procedures remain technically demanding due to flexible technology issues: difficult imaging, limited energy devices, lack of staplers, unsatisfactory suturing abilities, and so on. Transgastric laparoscopy or hybrid laparoscopy/flex endoscopy has been described for almost two decades as an alternative to a pure endoluminal approach, mainly for pancreatic pseudocyst drainage and full-thickness and mucosal resection of various lesions. The approach has never been widely adopted mostly due to cumbersome and difficult to maintain methods of gastric access. We propose to expand the indications of transgastric laparoscopy by using novel endoscopically placed ports to replicate endoscopic procedures particularly in the difficult to access proximal stomach such as endoluminal antireflux surgery. Under general anesthesia, five female pigs (mean weight: 27.6 kg) had endoscopic placement of 3, 5 mm intragastric ports (Endo-TAGSS, Leakwood KS, USA) using a technique similar to percutaneous endoscopic gastrostomy. A 5-mm laparoscope was used for visualization. Laparoendoscopic-assisted plication of the gastroesophageal junction (GEJ) was performed using 3-0 interrupted sutures (Polysorb®, Covidien, Mansfield, MA, USA). A functional lumen imagine probe (EndoFLIP®, Crospon, Inc., Galway, Ireland) was used to measure diameter, cross sectional area (CSA), distensibility, and compliance of GEJ before and after intervention. Once the TAGSS ports were removed, the gastrotomies were closed by using endoscopic over-the-scope clips. At the end of the procedure, animals were euthanized. Five laparoendoscopic-assisted endoluminal plications were performed. The mean operative time was 65.6 min (Endoscopic evaluation: 3.2 min, TAGSS Insertion: 11 min, EndoFLIP evaluation + GEJ Plication: 43.25 min, gastric wall closure: 15 minutes). In all cases, this technique was effective and allowed to achieve an adequate GEJ plication by endoscopic grading and EndoFLIP measurements. Median pre-plication GEJ diameter (D) and median pre-plication GEJ cross-sectional area (CSA) were 11.42 mm (8.6-13.6 mm) and 104.8 mm2 (58-146 mm2). After the procedure, these values were decreased to 6.14 mm (5.7-6.6 mm) and 29.8 mm2 (25-34 mm2) respectively (p = 0.0079). Median pre-plication distensibility (d) and compliance (C) were 7.87 mm2/mmHg (2.4-22.69 mm2/mmHg) and 190.56 mm3/mmHg (70.9-502.8 mm3/mmHg). After the procedure, these values decreased to 1.5 mm2/mmHg (0.7-2.2 mm2/mmHg) and 52.17 mm3/mmHg (21.9-98.7 mm3/mmHg) respectively (p = 0.0317). No intraoperative events were observed. Endoscopically, all valves were felt to be transitioned from a Hill grade 3 (normal state for the animal model) to a Hill grade 1 at the procedure completion. A hybrid laparoendoscopic approach is a feasible alternative for performing intragastric procedures with the assistance of conventional laparoscopic instruments; especially in cases where the intervention location limits the access to standard endoscopy or where endoscopic technology is inadequate. Further evaluation is planned in survival models and clinical trials.

Two guard cell mitogen-activated protein kinases, MPK9 and MPK12, function in methyl jasmonate-induced stomatal closure in Arabidopsis thaliana.

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9-1 and mpk12-1 single mutants as well as wild-type plants, but not in mpk9-1 mpk12-1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA-induced stomatal closure in wild-type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9-1, mpk12-1 and mpk9-1 mpk12-1 mutants, as well in wild-type plants. Furthermore, MeJA triggered elevation of cytosolic Ca(2+) concentration ([Ca(2+)]cyt ) in the mpk9-1 mpk12-1 double mutant as well as wild-type plants. Activation of S-type anion channels by MeJA was impaired in mpk9-1 mpk12-1. Together, these results indicate that MPK9 and MPK12 function upstream of S-type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca(2+)]cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.

Complete mesocolic excision with D3 lymph node dissection in laparoscopic colectomy for stages II and III colon cancer: long-term oncologic outcomes in 168 patients.

BACKGROUND: There is emerging evidence that complete mesocolic excision (CME) for colon cancer produces favorable oncologic outcomes. The applicability of CME technique in laparoscopic colectomy has not been fully explored. The aim of our retrospective study was to evaluate the feasibility of the CME technique with D3 lymphadenectomy in laparoscopic colectomy and its short- and long-term outcomes. METHODS: Between September 2006 and December 2009, 168 laparoscopic colectomies were performed for stages II and III colon cancer. Prospectively, collected data on demographics, tumor characteristics, complications, and outcomes were analyzed retrospectively. RESULTS: Eighty-seven patients (51.8 %) had stage II colon cancer, and 81 patients had stage III cancer. The mean operative time was 196.0 ± 61.2 min. The overall morbidity rate was 17.8 %, which included anastomotic leak in 10 patients (5.9 %). There was no operative mortality. The number of lymph nodes harvested was 27.8 ± 13.6. With a median follow-up of 57.3 months, locoregional recurrence and systemic metastasis developed in 6 (3.6 %) and 14 patients (8.3 %), respectively. Seven patients died of causes related to cancer, and all had stage III cancer. Disease-free survival at 5-years was 95.2 % for patients with stage II and 80.9 % for patients with stage III. CONCLUSIONS: Standardization of laparoscopic CME and D3 lymphadenectomy is expedient. The technique is associated with acceptable morbidity and provides excellent oncologic outcomes for stage II and stage III colon cancer. A longer follow-up is needed to validate the enhancement of oncological outcome related to this surgical concept.

First report: Robotic pelvic exenteration for locally advanced rectal cancer.

AIM: The aim of this study was to present the feasibility and surgical outcome of robotic en bloc resection of the rectum and with prostate and seminal vesicle invaded by rectal cancer. METHOD: The details of three consecutive cases involving male patients in their forties, with locally invasive low rectal cancers are presented. The da Vinci robotic system was used by experienced colorectal and urological surgeons to perform en bloc resection of the rectum, prostate and seminal vesicles. RESULTS: In the first case, coloanal and vesico-urethral anastomoses were performed, and the second included an end colostomy and vesico-urethral anastomosis. The bladder and bulbar urethra were also removed en bloc in the third case, with robotic intracorporeal ileal conduit formation and end colostomy. There was no major complication postoperatively. In the second patient there was a minor leakage at the vesico-urethral anastomosis. The third was readmitted the following week with a urinary infection which settled with intravenous antibiotics. In the first case, the circumferential resection margin was microscopically positive but the patient is currently free of recurrence after 14 months. In the second and third cases, all margins were clear. CONCLUSION: This the first report of the use of the da Vinci robotic system for pelvic exenteration in patients with locally advanced rectal cancer invading the prostate and seminal vesicles. The robot may have a potential role in selected patients requiring exenterative pelvic surgery particularly in men.

Robotic total mesorectal excision: operative technique and review of the literature.

In recent years, an increasing number of reports have been published on robotic colorectal surgery; this modality has also garnered an increasing amount of attention from the colorectal society. Most of the interest has been in robotic total mesorectal excision (TME) for rectal cancer. The purpose of this article is to briefly introduce our technique for total robotic TME and to review the recent literature regarding robotic TME for rectal cancer to summarize the current evidence on clinical and oncologic outcomes.

Two guard cell-preferential MAPKs, MPK9 and MPK12, regulate YEL signalling in Arabidopsis guard cells.

We report that two mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate abscisic acid (ABA)-induced stomatal closure in Arabidopsis thaliana. Yeast elicitor (YEL) induced stomatal closure accompanied by intracellular reactive oxygen species (ROS) accumulation and cytosolic free calcium concentration ([Ca(2+) ]cyt ) oscillation. In this study, we examined whether these two MAP kinases are involved in YEL-induced stomatal closure using MAPKK inhibitors, PD98059 and U0126, and MAPK mutants, mpk9, mpk12 and mpk9 mpk12. Both PD98059 and U0126 inhibited YEL-induced stomatal closure. YEL induced stomatal closure in the mpk9 and mpk12 mutants but not in the mpk9 mpk12 mutant, suggesting that a MAPK cascade involving MPK9 and MPK12 functions in guard cell YEL signalling. However, YEL induced extracellular ROS production, intracellular ROS accumulation and cytosolic alkalisation in the mpk9, mpk12 and mpk9 mpk12 mutants. YEL induced [Ca(2+) ]cyt oscillations in both wild type and mpk9 mpk12 mutant. These results suggest that MPK9 and MPK12 function redundantly downstream of extracellular ROS production, intracellular ROS accumulation, cytosolic alkalisation and [Ca(2+) ]cyt oscillation in YEL-induced stomatal closure in Arabidopsis guard cells and are shared with ABA signalling.

Sigmoid colon fistula following totally extraperitoneal hernioplasty: an improper treatment for mesh infection or iatrogenic injury?

BACKGROUND: There are only a few reports of severe mesh-related complications, and major bowel complications after totally extraperitoneal (TEP) hernioplasty are also rare. CASE REPORT: A 75-year-old male patient, who had undergone TEP hernioplasty for a left inguinal hernia 2 months previously, presented with a left inguinal swelling that was found to be due to sigmoid colon-related mesh complications following TEP hernioplasty. CONCLUSIONS: Infection is an accepted complication of hernia operation; however, it may be more serious following laparoscopic techniques. Successful TEP hernioplasty requires adequate dissection and complete exposure and coverage of all potential sites that cause inguinal hernia. If infection and suppuration are resistant to conservative methods or occur in the early postoperative period, aggressive imaging study and treatment provides definitive treatment and reduces the burden of complications.

Dominant negative guard cell K+ channel mutants reduce inward-rectifying K+ currents and light-induced stomatal opening in arabidopsis.

Inward-rectifying potassium (K+(in)) channels in guard cells have been suggested to provide a pathway for K+ uptake into guard cells during stomatal opening. To test the proposed role of guard cell K+(in) channels in light-induced stomatal opening, transgenic Arabidopsis plants were generated that expressed dominant negative point mutations in the K+(in) channel subunit KAT1. Patch-clamp analyses with transgenic guard cells from independent lines showed that K+(in) current magnitudes were reduced by approximately 75% compared with vector-transformed controls at -180 mV, which resulted in reduction in light-induced stomatal opening by 38% to 45% compared with vector-transformed controls. Analyses of intracellular K+ content using both sodium hexanitrocobaltate (III) and elemental x-ray microanalyses showed that light-induced K+ uptake was also significantly reduced in guard cells of K+(in) channel depressor lines. These findings support the model that K+(in) channels contribute to K+ uptake during light-induced stomatal opening. Furthermore, transpirational water loss from leaves was reduced in the K+(in) channel depressor lines. Comparisons of guard cell K+(in) current magnitudes among four different transgenic lines with different K+(in) current magnitudes show the range of activities of K+(in) channels required for guard cell K+ uptake during light-induced stomatal opening.

An mRNA cap binding protein, ABH1, modulates early abscisic acid signal transduction in Arabidopsis.

The plant hormone abscisic acid (ABA) regulates important stress and developmental responses. We have isolated a recessive ABA hypersensitive mutant, abh1, that shows hormone specificity to ABA. ABH1 encodes the Arabidopsis homolog of a nuclear mRNA cap binding protein and functions in a heterodimeric complex to bind the mRNA cap structure. DNA chip analyses show that only a few transcripts are down-regulated in abh1, several of which are implicated in ABA signaling. Consistent with these results, abh1 plants show ABA-hypersensitive stomatal closing and reduced wilting during drought. Interestingly, ABA-hypersensitive cytosolic calcium increases in abh1 guard cells demonstrate amplification of early ABA signaling. Thus, ABH1 represents a modulator of ABA signaling proposed to function by transcript alteration of early ABA signaling elements.

Guard cell abscisic acid signalling and engineering drought hardiness in plants.

Guard cells are located in the epidermis of plant leaves, and in pairs surround stomatal pores. These control both the influx of CO2 as a raw material for photosynthesis and water loss from plants through transpiration to the atmosphere. Guard cells have become a highly developed system for dissecting early signal transduction mechanisms in plants. In response to drought, plants synthesize the hormone abscisic acid, which triggers closing of stomata, thus reducing water loss. Recently, central regulators of guard cell abscisic acid signalling have been discovered. The molecular understanding of the guard cell signal transduction network opens possibilities for engineering stomatal responses to control CO2 intake and plant water loss.

Overexpression of the AtGluR2 gene encoding an Arabidopsis homolog of mammalian glutamate receptors impairs calcium utilization and sensitivity to ionic stress in transgenic plants.

We have identified a homolog of the mammalian ionotropic glutamate receptor genes in Arabidopsis thaliana (AtGluR2). This gene was found to alter Ca2+ utilization when overexpressed in A. thaliana. These transgenic plants displayed symptoms of Ca2+ deficiency, including browning and death of the shoot apex, necrosis of leaf tips, and deformation of leaves. Supplementation with Ca2+ alleviated these phenotypes. Overall levels of Ca2+ in tissues of control plants were not significantly different from those of transgenic plants, suggesting that overexpression of the AtGluR2 gene did not affect Ca2+ uptake. However, the relative growth yield as a function of Ca2+ levels revealed that the critical deficiency content of Ca2+ in transgenic plants was three times higher than that of control plants. The transgenic plants also exhibited hypersensitivity to Na+ and K+ ionic stresses. The ion hypersensitivity was ameliorated by supplementation with Ca2+. The results showed that overexpression of the AtGluR2 gene caused reduced efficiency of Ca2+ utilization in the transgenic plants. The promoter of the AtGluR2 gene was active in vascular tissues, particularly in cells adjacent to the conducting vessels. This suggests that AtGluR2 encodes a functional channel that unloads Ca2+ from the xylem vessels. The results together suggest that appropriate expression of the AtGluR2 protein may play critical roles in Ca2+ nutrition by controlling the ion allocation among different Ca2+ sinks both during normal development and during adaptation to ionic stresses.

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells.

Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca2+]cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.

A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis.

We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.