Bactericidal activity of pyrazinamide and clofazimine alone and in combinations with pretomanid and bedaquiline.

RATIONALE: New regimens to shorten tuberculosis treatment and manage patients with drug-resistant tuberculosis who are infected with HIV are urgently needed. Experimental and clinical evidence suggests that the new drugs bedaquiline (B) and pretomanid (Pa), combined with an existing drug, pyrazinamide (Z), and a repurposed drug, clofazimine (C), may assist treatment shortening of drug-susceptible and drug-resistant tuberculosis. OBJECTIVES: To evaluate the 14-day bactericidal activity of C and Z in monotherapy and in combinations with Pa and B. METHODS: Groups of 15 treatment-naive, sputum smear-positive patients with pulmonary tuberculosis were randomized to receive combinations of B with Z-C, Pa-Z, Pa-Z-C, and Pa-C, or C or Z alone, or standard combination treatment for 14 days. The primary endpoint was the mean daily fall in log10 Mycobacterium tuberculosis CFU per milliliter sputum estimated by joint nonlinear mixed-effects Bayesian regression modeling. MEASUREMENTS AND MAIN RESULTS: Estimated activities were 0.167 (95% confidence interval [CI], 0.075-0.257) for B-Pa-Z, 0.151 (95% CI, 0.071-0.232) for standard treatment, 0.124 (95% CI, 0.035-0.214) for B-Z-C, 0.115 (95% CI, 0.039-0.189) for B-Pa-Z-C, and 0.076 (95% CI, 0.005-0.145) for B-Pa-C. Z alone had modest activity (0.036; 95% CI, -0.026 to 0.099). C had no activity alone (-0.017; 95% CI, -0.085 to 0.053) or in combinations. Treatments were well tolerated and safe. CONCLUSIONS: B-Pa-Z, including two novel agents without resistance in prevalent M. tuberculosis strains, is a potential new tuberculosis treatment regimen. C had no measurable activity in the first 14 days of treatment. Clinical trial registered with www.clinicaltrials.gov (NCT 01691534).

Randomized dose-ranging study of the 14-day early bactericidal activity of bedaquiline (TMC207) in patients with sputum microscopy smear-positive pulmonary tuberculosis.

Bedaquiline is a new antituberculosis agent targeting ATP synthase. This randomized, double-blinded study enrolling 68 sputum smear-positive pulmonary tuberculosis patients evaluated the 14-day early bactericidal activity of daily doses of 100 mg, 200 mg, 300 mg, and 400 mg bedaquiline, preceded by loading doses of 200 mg, 400 mg, 500 mg, and 700 mg, respectively, on the first treatment day and 100 mg, 300 mg, 400 mg, and 500 mg on the second treatment day. All groups showed activity with a mean (standard deviation) daily fall in log10 CFU over 14 days of 0.040 (0.068), 0.056 (0.051), 0.077 (0.064), and 0.104 (0.077) in the 100-mg, 200-mg, 300-mg, and 400-mg groups, respectively. The linear trend for dose was significant (P = 0.001), and activity in the 400-mg dose group was greater than that in the 100-mg group (P = 0.014). All of the bedaquiline groups showed significant bactericidal activity that was continued to the end of the 14-day evaluation period. The finding of a linear trend for dose suggests that the highest dose compatible with safety considerations should be taken forward to longer-term clinical studies.

14-day bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin combinations: a randomised trial.

BACKGROUND: New drugs, but also shorter, better-tolerated regimens are needed to tackle the high global burden of tuberculosis complicated by drug resistance and retroviral disease. We investigated new multiple-agent combinations over the first 14 days of treatment to assess their suitability for future development. METHODS: In this prospective, randomised, early bactericidal activity (EBA) study, treatment-naive, drug-susceptible patients with uncomplicated pulmonary tuberculosis were admitted to hospitals in Cape Town, South Africa, between Oct 7, 2010, and Aug 19, 2011. Patients were randomised centrally by computer-generated randomisation sequence to receive bedaquiline, bedaquiline-pyrazinamide, PA-824-pyrazinamide, bedaquiline-PA-824, PA-824-moxifloxacin-pyrazinamide, or unmasked standard antituberculosis treatment as positive control. The primary outcome was the 14-day EBA assessed in a central laboratory from the daily fall in colony forming units (CFU) of M tuberculosis per mL of sputum in daily overnight sputum collections. Bilinear regression curves were fitted for each group separately and groups compared with ANOVA for ranks, followed by pair-wise comparisons adjusted for multiplicity. Clinical staff were partially masked but laboratory personnel were fully masked. This study is registered, NCT01215851. FINDINGS: The mean 14-day EBA of PA-824-moxifloxacin-pyrazinamide (n=13; 0·233 [SD 0·128]) was significantly higher than that of bedaquiline (14; 0·061 [0·068]), bedaquiline-pyrazinamide (15; 0·131 [0·102]), bedaquiline-PA-824 (14; 0·114 [0·050]), but not PA-824-pyrazinamide (14; 0·154 [0·040]), and comparable with that of standard treatment (ten; 0·140 [0·094]). Treatments were well tolerated and appeared safe. One patient on PA-824-moxifloxacin-pyrazinamide was withdrawn because of corrected QT interval changes exceeding criteria prespecified in the protocol. INTERPRETATION: PA-824-moxifloxacin-pyrazinamide is potentially suitable for treating drug-sensitive and multidrug-resistant tuberculosis. Multiagent EBA studies can contribute to reducing the time needed to develop new antituberculosis regimens. FUNDING: The Global Alliance for TB Drug Development (TB Alliance).

Accuracy and impact of Xpert MTB/RIF for the diagnosis of smear-negative or sputum-scarce tuberculosis using bronchoalveolar lavage fluid.

RATIONALE: The accuracy and impact of new tuberculosis (TB) tests, such as Xpert MTB/RIF, when performed on bronchoalveolar lavage fluid (BALF) obtained from patients with sputum-scarce or smear-negative TB is unclear. METHODS: South African patients with suspected pulmonary TB (n=160) who were sputum-scarce or smear-negative underwent bronchoscopy. MTB/RIF was performed on uncentrifuged BALF (1 ml) and/or a resuspended pellet of centrifuged BALF (∼10 ml). Time to TB detection and anti-TB treatment initiation were compared between phase one, when MTB/RIF was performed as a research tool, and phase two, when it was used for patient management. RESULTS: 27 of 154 patients with complete data had culture-confirmed TB. Of these, a significantly lower proportion were detected by smear microscopy compared with MTB/RIF (58%, 95% CI 39% to 75% versus 93%, 77% to 98%; p<0.001). Of the 127 patients who were culture negative, 96% (91% to 98%) were MTB/RIF negative. When phase two was compared with phase one, MTB/RIF reduced the median days to TB detection (29 (18-41) to 0 (0-0); p<0.001). However, more patients initiated empirical therapy (absence of a positive test in those commencing treatment) in phase one versus phase two (79% (11/14) versus 28% (10/25); p=0.026). Consequently, there was no detectable difference in the overall proportion of patients initiating treatment (26% (17/67; 17% to 37%) versus 36% (26/73; 26% to 47%); p=0.196) or the days to treatment initiation (10 (1-49) versus 7 (0-21); p=0.330). BALF centrifugation, HIV coinfection and a second MTB/RIF did not result in detectable changes in accuracy. CONCLUSIONS: MTB/RIF detected TB cases more accurately and more rapidly than smear microscopy and significantly reduced the rate of empirical treatment.

Changing character and waning impact of COVID-19 at a tertiary centre in Cape Town, South Africa.

Background: The emergence of genetic variants of SARS-CoV-2 was associated with changing epidemiological characteristics throughout coronavirus disease 2019 (COVID-19) pandemic in population-based studies. Individual-level data on the clinical characteristics of infection with different SARS-CoV-2 variants in African countries is less well documented. Objectives: To describe the evolving clinical differences observed with the various SARS-CoV-2 variants of concern and compare the Omicron-driven wave in infections to the previous Delta-driven wave. Method: We performed a retrospective observational cohort study among patients admitted to a South African referral hospital with COVID-19 pneumonia. Patients were stratified by epidemiological wave period, and in a subset, the variants associated with each wave were confirmed by genomic sequencing. Outcomes were analysed by Cox proportional hazard models. Results: We included 1689 patients were included, representing infection waves driven predominantly by ancestral, Beta, Delta and Omicron BA1/BA2 & BA4/BA5 variants. Crude 28-day mortality was 25.8% (34/133) in the Omicron wave period versus 37.1% (138/374) in the Delta wave period (hazard ratio [HR] 0.68 [95% CI 0.47-1.00] p = 0.049); this effect persisted after adjustment for age, gender, HIV status and presence of cardiovascular disease (adjusted HR [aHR] 0.43 [95% CI 0.28-0.67] p < 0.001). Hospital-wide SARS-CoV-2 admissions and deaths were highest during the Delta wave period, with a decoupling of SARS-CoV-2 deaths and overall deaths thereafter. Conclusion: There was lower in-hospital mortality during Omicron-driven waves compared with the prior Delta wave, despite patients admitted during the Omicron wave being at higher risk. Contribution: This study summarises clinical characteristics associated with SARS-CoV-2 variants during the COVID-19 pandemic at a South African tertiary hospital, demonstrating a waning impact of COVID-19 on healthcare services over time despite epidemic waves driven by new variants. Findings suggest the absence of increasing virulence from later variants and protection from population and individual-level immunity.

Brassinosteroid transport.

Brassinosteroids (BRs) are steroidal plant hormones that are important regulators of plant growth. These compounds are widely distributed throughout reproductive and vegetative plant tissues. This raises the question of whether or not BRs are transported over long distances between these tissues. Several lines of evidence indicate that this is not the case. Exogenous BRs move only slowly, if at all, after application to leaves; grafting BR-deficient mutants to wild-type plants has no phenotypic effect; removal of the apical bud or mature leaves does not reduce BR levels in the remaining internodes; and, in tomato, wild-type sectors do not substantially alter the growth of BR-deficient sectors when the two types are together in a variegated leaf. Although BRs do not undergo long-distance transport they may influence long-distance signalling by altering auxin transport. At the cellular level, BRs do appear to be transported. The enzymes for BR biosynthesis appear to be located within the cell, and to be associated with the endoplasmic reticulum, in particular. BR reception, on the other hand, is thought to occur on the exterior cell surface. Therefore, BRs must move from the interior of the cell to the exterior, where they are perceived by the same cell or by neighbouring cells. The existence of a feedback system, whereby bioactive BRs negatively regulate their own biosynthesis, provides further evidence that individual cells are able to both perceive and synthesize BRs.

Do brassinosteroids mediate the water stress response?

Brassinosteroids (BRs) have been suggested to increase the resistance of plants to a variety of stresses, including water stress. This is based on application studies, where exogenously applied bioactive BRs have been shown to improve various aspects of plant growth under water stress conditions. However, it is not known whether changes in endogenous BR levels are normally involved in mediating the plant's response to stress. We have utilized BR mutants in pea (Pisum sativum L.) to determine whether changes in endogenous BR levels are part of the plant's response to water stress and whether low endogenous BR levels alter the plant's ability to cope with water stress. In wild-type (WT) plants, we show that while water stress causes a significant increase in ABA levels, it does not result in altered BR levels in either apical, internode or leaf tissue. Furthermore, the plant's ability to increase ABA levels in response to water stress is not affected by BR deficiency, as there was no significant difference in ABA levels between WT, lkb (a BR-deficient mutant) and lka (a BR-perception mutant) plants before or 14 days after the cessation of watering. In addition, the effect of water stress on traits such as height, leaf size and water potential in lkb and lka was similar to that observed in WT plants. Therefore, it appears that, at least in pea, changes in endogenous BR levels are not normally part of the plant's response to water stress.

Uncoupling brassinosteroid levels and de-etiolation in pea.

The suggestion that brassinosteroids (BRs) have a negative regulatory role in de-etiolation is based largely on correlative evidence, which includes the de-etiolated phenotypes of, and increased expression of light-regulated genes in, dark-grown mutants defective in BR biosynthesis or response. However, we have obtained the first direct evidence which shows that endogenous BR levels in light-grown pea seedlings are increased, not decreased, in comparison with those grown in the dark. Similarly, we found no evidence of a decrease in castasterone (CS) levels in seedlings that were transferred from the dark to the light for 24 h. Furthermore, CS levels in the constitutively de-etiolated lip1 mutant are similar to those in wild-type plants, and are not reduced as is the case in the BR-deficient lkb plants. Unlike lip1, the pea BR-deficient mutants lk and lkb are not de-etiolated at the morphological or molecular level, as they exhibit neither a de-etiolated phenotype or altered expression of light-regulated genes when grown in the dark. Similarly, dark-grown WT plants treated with the BR biosynthesis inhibitor, Brz, do not exhibit a de-etiolated phenotype. In addition, analysis of the lip1lkb double mutant revealed an additive phenotype indicative of the two genes acting in independent pathways. Together these results strongly suggest that BR levels do not play a negative-regulatory role in de-etiolation in pea.

South African tobacco smoking cessation clinical practice guideline.

Tobacco smoking (i.e. cigarettes, rolled tobacco, pipes, etc.) is associated with significant health risks, reduced life expectancy and negative personal and societal economic impact. Smokers have an increased risk of cancer (i.e. lung, throat, bladder), chronic obstructive pulmonary disease (COPD), tuberculosis and cardiovascular disease (i.e. stroke, heart attack). Smoking affects unborn babies, children and others exposed to second hand smoke. Stopping or 'quitting' is not easy. Nicotine is highly addictive and smoking is frequently associated with social activities (e.g. drinking, eating) or psychological factors (e.g. work pressure, concerns about body weight, anxiety or depressed mood). The benefits of quitting, however, are almost immediate, with a rapid lowering of blood pressure and heart rate, improved taste and smell, and a longer-term reduction in risk of cancer, heart attack and COPD. Successful quitting requires attention to both the factors surrounding why an individual smokes (e.g. stress, depression, habit, etc.) and the symptoms associated with nicotine withdrawal. Many smokers are not ready or willing to quit and require frequent motivational input outlining the benefits that would accrue. In addition to an evaluation of nicotine dependence, co-existent medical or psychiatric conditions and barriers to quitting should be identified. A tailored approach encompassing psychological and social support, in addition to appropriate medication to reduce nicotine withdrawal, is likely to provide the best chance of success. Relapse is not uncommon and reasons for failure should be addressed in a positive manner and further attempts initiated when the individual is ready.Key steps in smoking cessation include: (i) identifying all smokers, alerting them to the harms of smoking and benefits of quitting; (ii) assessing readiness to initiate an attempt to quit; (iii) assessing the physical and psychological dependence to nicotine and smoking; (iv) determining the best combination of counselling/support and pharmacological therapy; (v) setting a quit date and provide suitable resources and support; (vi) frequent follow-up as often as possible via text/telephone or in person; (vii) monitoring for side-effects, relapse and on-going cessation; and (viii) if relapse occurs, providing the necessary support and encourage a further attempt when appropriate. 

A historical review of XDR tuberculosis in the Western Cape province of South Africa.

There are limited data on the temporal relationship between the regional introduction of multidrug-resistant tuberculosis (MDR-TB) treatment and the subsequent development of extensively drug-resistant TB (XDR-TB). The first XDR-TB case in the Western Cape province of South Africa was recorded in 1992, approximately 5 - 7 years after the regional introduction of MDR-TB-like treatment. Between 1990 and 2002 we identified 48 patients with XDR-TB in the Cape Metropole region of the Western Cape province. Patients were predominantly HIV-uninfected and median survival was 10.8 months. XDR-TB has therefore been present in the Western Cape at least since 1992. These data inform public health policy relevant to the introduction of new anti-TB drug regimens.

Clinical diagnostic utility of IP-10 and LAM antigen levels for the diagnosis of tuberculous pleural effusions in a high burden setting.

BACKGROUND: Current tools for the diagnosis of tuberculosis pleural effusions are sub-optimal. Data about the value of new diagnostic technologies are limited, particularly, in high burden settings. Preliminary case control studies have identified IFN-gamma-inducible-10 kDa protein (IP-10) as a promising diagnostic marker; however, its diagnostic utility in a day-to-day clinical setting is unclear. Detection of LAM antigen has not previously been evaluated in pleural fluid. METHODS: We investigated the comparative diagnostic utility of established (adenosine deaminase [ADA]), more recent (standardized nucleic-acid-amplification-test [NAAT]) and newer technologies (a standardized LAM mycobacterial antigen-detection assay and IP-10 levels) for the evaluation of pleural effusions in 78 consecutively recruited South African tuberculosis suspects. All consenting participants underwent pleural biopsy unless contra-indicated or refused. The reference standard comprised culture positivity for M. tuberculosis or histology suggestive of tuberculosis. PRINCIPAL FINDINGS: Of 74 evaluable subjects 48, 7 and 19 had definite, probable and non-TB, respectively. IP-10 levels were significantly higher in TB vs non-TB participants (p<0.0001). The respective outcomes [sensitivity, specificity, PPV, NPV %] for the different diagnostic modalities were: ADA at the 30 IU/L cut-point [96; 69; 90; 85], NAAT [6; 93; 67; 28], IP-10 at the 28,170 pg/ml ROC-derived cut-point [80; 82; 91; 64], and IP-10 at the 4035 pg/ml cut-point [100; 53; 83; 100]. Thus IP-10, using the ROC-derived cut-point, missed approximately 20% of TB cases and mis-diagnosed approximately 20% of non-TB cases. By contrast, when a lower cut-point was used a negative test excluded TB. The NAAT had a poor sensitivity but high specificity. LAM antigen-detection was not diagnostically useful. CONCLUSION: Although IP-10, like ADA, has sub-optimal specificity, it may be a clinically useful rule-out test for tuberculous pleural effusions. Larger multi-centric studies are now required to confirm our findings.

Brassinosteroids, de-etiolation and the re-emerging art of plant hormone quantification.

An increase in the use of molecular techniques has provided a significant insight into the function of genes, and how they are regulated and interact. However, in the field of plant hormone physiology, the increased use of these techniques has been accompanied by a reduction in the direct measurement of plant hormone levels by physiochemical methods. Instead, the transcript (mRNA) levels of genes involved in hormone metabolism are often used to predict endogenous hormone levels. The validity of this approach was recently tested by comparing the expression of a range of genes involved in BR synthesis, catabolism and perception, with the actual endogenous BR levels in pea seedlings grown under different light conditions.1,2 Based on this comparison, we now argue that gene expression analysis alone is not always a reliable indicator of endogenous hormone levels.

The hormonal regulation of de-etiolation.

De-etiolation involves a number of phenotypic changes as the plants shift from a dark-grown (etiolated) to a light-grown (de-etiolated) morphology. Whilst these light-induced, morphological changes are thought to be mediated by plant hormones, the precise mechanism/s are not yet fully understood. Here we provide further direct evidence that gibberellins (GAs) may play an important role in de-etiolation, because a similar light-induced reduction in bioactive GA levels was detected in barley (Hordeum vulgare L.), Arabidopsis (Arabidopsis thaliana L.), and pea (Pisum sativum L.). This is indicative of a highly conserved, negative-regulatory role for GAs in de-etiolation, in a range of taxonomically diverse species. In contrast, we found no direct evidence of a reduction in brassinosteroid (BR) levels during de-etiolation in any of these species.

Evidence that the mature leaves contribute auxin to the immature tissues of pea (Pisum sativum L.).

In plants such as the garden pea (Pisum sativum L.), it is widely thought that the auxin indole-3-acetic acid (IAA) is synthesised mainly in the immature tissues of the apical bud and then transported basipetally to other parts of the plant. Consistent with this belief are results showing that removal of the apical bud markedly reduces the IAA content in the stem. However, it has also been suggested that the mature leaves may synthesise substantial amounts of IAA, which enters the basipetal transport stream after being transported to the shoot apex in the phloem (Cambridge and Morris in Planta 99:583-588, 1996). To examine this theory, we defoliated pea plants and measured the effect on IAA content in the remaining shoot tissues. IAA levels were reduced in the internodes, and to a lesser extent in the apical bud, after defoliation, suggesting that mature leaves are indeed an important source of auxin for the shoot. Consistent with this idea, we have demonstrated that mature, fully expanded leaves are capable of de novo IAA synthesis. Furthermore, we report evidence for the presence of IAA in the phloem sap of pea. Together these results support those of Cambridge and Morris, suggesting that mature leaves are a source of the IAA in the basipetal transport stream.

A study of gibberellin homeostasis and cryptochrome-mediated blue light inhibition of hypocotyl elongation.

Cryptochromes mediate blue light-dependent photomorphogenic responses, such as inhibition of hypocotyl elongation. To investigate the underlying mechanism, we analyzed a genetic suppressor, scc7-D (suppressors of cry1cry2), which suppressed the long-hypocotyl phenotype of the cry1cry2 (cryptochrome1/cryptochrome2) mutant in a light-dependent but wavelength-independent manner. scc7-D is a gain-of-expression allele of the GA2ox8 gene encoding a gibberellin (GA)-inactivating enzyme, GA 2-oxidase. Although scc7-D is hypersensitive to light, transgenic seedlings expressing GA2ox at a level higher than scc7-D showed a constitutive photomorphogenic phenotype, confirming a general role of GA2ox and GA in the suppression of hypocotyl elongation. Prompted by this result, we investigated blue light regulation of mRNA expression of the GA metabolic and catabolic genes. We demonstrated that cryptochromes are required for the blue light regulation of GA2ox1, GA20ox1, and GA3ox1 expression in transient induction, continuous illumination, and photoperiodic conditions. The kinetics of cryptochrome induction of GA2ox1 expression and cryptochrome suppression of GA20ox1 or GA3ox1 expression correlate with the cryptochrome-dependent transient reduction of GA(4) in etiolated wild-type seedlings exposed to blue light. Therefore we propose that in deetiolating seedlings, cryptochromes mediate blue light regulation of GA catabolic/metabolic genes, which affect GA levels and hypocotyl elongation. Surprisingly, no significant change in the GA(4) content was detected in the whole shoot samples of the wild-type or cry1cry2 seedlings grown in the dark or continuous blue light, suggesting that cryptochromes may also regulate GA responsiveness and/or trigger cell- or tissue-specific changes of the level of bioactive GAs.

Characterization of two brassinosteroid C-6 oxidase genes in pea.

C-6 oxidation genes play a key role in the regulation of biologically active brassinosteroid (BR) levels in the plant. They control BR activation, which involves the C-6 oxidation of 6-deoxocastasterone (6-DeoxoCS) to castasterone (CS) and in some cases the further conversion of CS to brassinolide (BL). C-6 oxidation is controlled by the CYP85A family of cytochrome P450s, and to date, two CYP85As have been isolated in tomato (Solanum lycopersicum), two in Arabidopsis (Arabidopsis thaliana), one in rice (Oryza sativa), and one in grape (Vitis vinifera). We have now isolated two CYP85As (CYP85A1 and CYP85A6) from pea (Pisum sativum). However, unlike Arabidopsis and tomato, which both contain one BR C-6 oxidase that converts 6-DeoxoCS to CS and one BR C-6 Baeyer-Villiger oxidase that converts 6-DeoxoCS right through to BL, the two BR C-6 oxidases in pea both act principally to convert 6-DeoxoCS to CS. The isolation of these two BR C-6 oxidation genes in pea highlights the species-specific differences associated with C-6 oxidation. In addition, we have isolated a novel BR-deficient mutant, lke, which blocks the function of one of these two BR C-6 oxidases (CYP85A6). The lke mutant exhibits a phenotype intermediate between wild-type plants and previously characterized pea BR mutants (lk, lka, and lkb) and contains reduced levels of CS and increased levels of 6-DeoxoCS. To date, lke is the only mutant identified in pea that blocks the latter steps of BR biosynthesis and it will therefore provide an excellent tool to further examine the regulation of BR biosynthesis and the relative biological activities of CS and BL in pea.

Grapes on steroids. Brassinosteroids are involved in grape berry ripening.

Fruit ripening is a unique plant developmental process with direct implications for our food supply, nutrition, and health. In contrast to climacteric fruit, where ethylene is pivotal, the hormonal control of ripening in nonclimacteric fruit, such as grape (Vitis vinifera), is poorly understood. Brassinosteroids (BRs) are steroidal hormones, essential for normal plant growth and development but not previously implicated in the ripening of nonclimacteric fruit. Here we show that increases in endogenous BR levels, but not indole-3-acetic acid (IAA) or GA levels, are associated with ripening in grapes. Putative grape homologs of genes encoding BR biosynthesis enzymes (BRASSINOSTEROID-6-OXIDASE and DWARF1) and the BR receptor (BRASSINOSTEROID INSENSITIVE 1) were isolated, and the function of the grape BRASSINOSTEROID-6-OXIDASE gene was confirmed by transgenic complementation of the tomato (Lycopersicon esculentum) extreme dwarf (dx/dx) mutant. Expression analysis of these genes during berry development revealed transcript accumulation patterns that were consistent with a dramatic increase in endogenous BR levels observed at the onset of fruit ripening. Furthermore, we show that application of BRs to grape berries significantly promoted ripening, while brassinazole, an inhibitor of BR biosynthesis, significantly delayed fruit ripening. These results provide evidence that changes in endogenous BR levels influence this key developmental process. This may provide a significant insight into the mechanism controlling ripening in grapes, which has direct implications for the logistics of grape production and down-stream processing.

The brassinosteroid growth response in pea is not mediated by changes in gibberellin content.

The objective of this study was to increase our understanding of the relationship between brassinosteroids (BRs) and gibberellins (GAs) by examining the effects of BR deficiency on the GA biosynthesis pathway in several tissue types of pea (Pisum sativum L.). It was suggested recently that, in Arabidopsis, BRs act as positive regulators of GA 20-oxidation, a key step in GA biosynthesis [Bouquin et al. (2001) Plant Physiol 127:450-458]. However, this may not be the case in pea as GA20 levels were consistently higher in all shoot tissues of BR-deficient (lk and lkb) and BR-response (lka) mutants. The application of brassinolide (BL) to lkb plants reduced GA20 levels, and metabolism studies revealed a reduced conversion of GA19 to GA20 in epi-BL-treated lkb plants. These results indicate that BRs actually negatively regulate GA20 levels in pea. Although GA20 levels are affected by BR levels, this does not result in consistent changes in the level of the bioactive GA, GA1. Therefore, even though a clear interaction exists between endogenous BR levels and the level of GA20, this interaction may not be biologically significant. In addition to the effect of BRs on GA levels, the effect of altered GA1 levels on endogenous BR levels was examined. There was no significant difference in BR levels between the GA mutants and the wild type (wt), indicating that altered GA1 levels have no effect on BR levels in pea. It appears that the BR growth response is not mediated by changes in bioactive GA levels, thus providing further evidence that BRs are important regulators of stem elongation.

Brassinosteroids do not undergo long-distance transport in pea. Implications for the regulation of endogenous brassinosteroid levels.

It is widely accepted that brassinosteroids (BRs) are important regulators of plant growth and development. However, in comparison to the other classical plant hormones, such as auxin, relatively little is known about BR transport and its potential role in the regulation of endogenous BR levels in plants. Here, we show that end-pathway BRs in pea (Pisum sativum) occur in a wide range of plant tissues, with the greatest accumulation of these substances generally occurring in the young, actively growing tissues, such as the apical bud and young internodes. However, despite the widespread distribution of BRs throughout the plant, we found no evidence of long-distance transport of these substances between different plant tissues. For instance, we show that the maintenance of steady-state BR levels in the stem does not depend on their transport from the apical bud or mature leaves. Similarly, reciprocal grafting between the wild type and the BR-deficient lkb mutants demonstrates that the maintenance of steady-state BR levels in whole shoots and roots does not depend on either basipetal or acropetal transport of BRs between these tissues. Together, with results from (3)H-BR feeding studies, these results demonstrate that BRs do not undergo long-distance transport in pea. The widespread distribution of end-pathway BRs and the absence of long-distance BR transport between different plant tissues provide significant insight into the mechanisms that regulate BR homeostasis in plants.