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Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high-resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and various membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label-free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Diffuse alveolar damage (DAD) is the histological expression of acute respiratory distress syndrome and characterises lung pathology due to infection with SARS-CoV-2, and other respiratory pathogens of clinical significance. DAD reflects a time-dependent immunopathological process, progressing from an early/exudative stage through to an organising/fibrotic stage, yet within an individual these different stages of DAD may coexist. Understanding the progression of DAD is central to the development of new therapeutics to limit progressive lung damage. Here, we applied highly multiplexed spatial protein profiling to autopsy lung tissues derived from 27 patients who died from COVID-19 and identified a protein signature (ARG1, CD127, GZMB, IDO1, Ki67, phospho-PRAS40 (T246) and VISTA) that distinguishes early DAD from late DAD with good predictive accuracy. These proteins warrant further investigation as potential regulators of DAD progression., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both "early" and "recycling" endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved., (© 2022 Laidlaw et al.)

Introduction: Spatial analysis of lipids in inflammatory microenvironments is key to understand the pathogenesis of infectious disease. Granulomatous inflammation is a hallmark of leishmaniasis and changes in host and parasite lipid metabolism have been observed at the bulk tissue level in various infection models. Here, mass spectrometry imaging (MSI) is applied to spatially map hepatic lipid composition following infection with Leishmania donovani , an experimental mouse model of visceral leishmaniasis., Methods: Livers from naïve and L. donovani -infected C57BL/6 mice were harvested at 14- and 20-days post-infection (n=5 per time point). 12 µm transverse sections were cut and covered with norhamane, prior to lipid analysis using MALDI-MSI. MALDI-MSI was performed in negative mode on a Rapiflex (Bruker Daltonics) at 5 and 50 µm spatial resolution and data-dependent analysis (DDA) on an Orbitrap-Elite (Thermo-Scientific) at 50 µm spatial resolution for structural identification analysis of lipids., Results: Aberrant lipid abundances were observed in a heterogeneous distribution across infected mouse livers compared to naïve mouse liver. Distinctive localized correlated lipid masses were found in granulomas and surrounding parenchymal tissue. Structural identification revealed 40 different lipids common to naïve and d14/d20 infected mouse livers, whereas 15 identified lipids were only detected in infected mouse livers. For pathology-guided MSI imaging, we deduced lipids from manually annotated granulomatous and parenchyma regions of interests (ROIs), identifying 34 lipids that showed significantly different intensities between parenchyma and granulomas across all infected livers., Discussion: Our results identify specific lipids that spatially correlate to the major histopathological feature of Leishmania donovani infection in the liver, viz. hepatic granulomas. In addition, we identified a three-fold increase in the number of unique phosphatidylglycerols (PGs) in infected liver tissue and provide direct evidence that arachidonic acid-containing phospholipids are localized with hepatic granulomas. These phospholipids may serve as important precursors for downstream oxylipin generation with consequences for the regulation of the inflammatory cascade. This study provides the first description of the use of MSI to define spatial-temporal lipid changes at local sites of infection induced by Leishmania donovani in mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tans, Dey, Dey, Cao, Paul, Calder, O’Toole, Kaye and Heeren.)

This paper investigates the COVID-19 pandemic, and its impact on pro-environmental behaviour of individuals travelling internationally for leisure and recreational purposes. The aim of this manuscript is to investigate a conceptual framework created through the examination of current existing literature in the field of tourism science. The conceptual framework, consisting of certain constructs of the health belief model (HBM), and the theory of planned behaviour (TPB), is applied and tested using a partial least-squares-structural equation modelling (PLS-SEM). Data were collected from participants who have travelled internationally before and during the outbreak of the COVID-19 pandemic, and those who plan to travel post-COVID-19 pandemic. Results revealed that the conceptual framework tested positively against existing theory, highlighting the key influencing factors in which COVID-19 is likely to have on future pro-environmental behaviour of individuals travelling internationally for leisure and recreational purposes. Moreover, perceived safety threat and outcome expectations have a positive impact on attitude; attitude has a positive impact on behavioural intentions; subjective norm has a positive impact on behavioural intentions, and perceived behavioural control has a positive impact on behavioural intentions. The study results identify practical and theoretical implications for global and travel companies and organisations, presenting opportunities to adjust environmental policies and procedures accordingly, whilst identifying the most effective marketing and management strategies to rebuild a collapsed global travel industry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Calder, Radic, Ryu, Ariza-Montes and Han.)

Cell surface protein trafficking is regulated in response to nutrient availability, with multiple pathways directing surface membrane proteins to the lysosome for degradation in response to suboptimal extracellular nutrients. Internalized protein and lipid cargoes recycle back to the surface efficiently in glucose-replete conditions, but this trafficking is attenuated following glucose starvation. We find that cells with either reduced or hyperactive phosphatidylinositol 3-kinase (PI3K) activity are defective for endosome to surface recycling. Furthermore, we find that the yeast Gα subunit Gpa1, an endosomal PI3K effector, is required for surface recycling of cargoes. Following glucose starvation, mRNA and protein levels of a distinct Gα subunit Gpa2 are elevated following nuclear translocation of Mig1, which inhibits recycling of various cargoes. As Gpa1 and Gpa2 interact at the surface where Gpa2 concentrates during glucose starvation, we propose that this disrupts PI3K activity required for recycling, potentially diverting Gpa1 to the surface and interfering with its endosomal role in recycling. In support of this model, glucose starvation and overexpression of Gpa2 alter PI3K endosomal phosphoinositide production. Glucose deprivation therefore triggers a survival mechanism to increase retention of surface cargoes in endosomes and promote their lysosomal degradation.

Infectious diseases, including those of viral, bacterial, fungal, and parasitic origin are often characterized by focal inflammation occurring in one or more distinct tissues. Tissue-specific outcomes of infection are also evident in many infectious diseases, suggesting that the local microenvironment may instruct complex and diverse innate and adaptive cellular responses resulting in locally distinct molecular signatures. In turn, these molecular signatures may both drive and be responsive to local metabolic changes in immune as well as non-immune cells, ultimately shaping the outcome of infection. Given the spatial complexity of immune and inflammatory responses during infection, it is evident that understanding the spatial organization of transcripts, proteins, lipids, and metabolites is pivotal to delineating the underlying regulation of local immunity. Molecular imaging techniques like mass spectrometry imaging and spatially resolved, highly multiplexed immunohistochemistry and transcriptomics can define detailed metabolic signatures at the microenvironmental level. Moreover, a successful complementation of these two imaging techniques would allow multi- omics analyses of inflammatory microenvironments to facilitate understanding of disease pathogenesis and identify novel targets for therapeutic intervention. Here, we describe strategies for downstream data analysis of spatially resolved multi- omics data and, using leishmaniasis as an exemplar, describe how such analysis can be applied in a disease-specific context., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tans, Dey, Dey, Calder, O’Toole, Kaye and Heeren.)

Objective: To examine the feasibility of developing an in-reach parenteral iron infusion service to residents of residential aged care facilities (RACFs)., Methods: An audit comparing the use of iron infusions in RACFs prior to and following the introduction of an in-reach iron infusion service., Results: Of the 738 inpatient iron infusions administered to inpatients ≥65 years in the 12 months prior to the in-reach service, 52 (7%) lived in an RACF, with no significant adverse events reported. After implementation of an in-reach service, a total of 37 RACF residents received parenteral iron in the first 12 months of the service, with no significant adverse events reported., Conclusion: It is possible to safely provide parenteral iron through an in-reach service to residents in RACF. Further research is required to identify the person-level benefits achieved by this service., (© 2020 AJA Inc.)

Temperature is a key factor in the growth and development of all organisms 1,2 . Plants have to interpret temperature fluctuations, over hourly to monthly timescales, to align their growth and development with the seasons. Much is known about how plants respond to acute thermal stresses 3,4 , but the mechanisms that integrate long-term temperature exposure remain unknown. The slow, winter-long upregulation of VERNALIZATION INSENSITIVE 3 (VIN3) 5-7 , a PHD protein that functions with Polycomb repressive complex 2 to epigenetically silence FLOWERING LOCUS C (FLC) during vernalization, is central to plants interpreting winter progression 5,6,8-11 . Here, by a forward genetic screen, we identify two dominant mutations of the transcription factor NTL8 that constitutively activate VIN3 expression and alter the slow VIN3 cold induction profile. In the wild type, the NTL8 protein accumulates slowly in the cold, and directly upregulates VIN3 transcription. Through combining computational simulation and experimental validation, we show that a major contributor to this slow accumulation is reduced NTL8 dilution due to slow growth at low temperatures. Temperature-dependent growth is thus exploited through protein dilution to provide the long-term thermosensory information for VIN3 upregulation. Indirect mechanisms involving temperature-dependent growth, in addition to direct thermosensing, may be widely relevant in long-term biological sensing of naturally fluctuating temperatures.

The development of feeding systems that can individually measure and control feed intake in a group-housed environment would allow a greater understanding of sheep intake without compromising animal welfare and behavior through the removal of social interactions between sheep. This study validated an automated feeding system for measuring feed intake of individual sheep when housed in groups. Validation of the feeding system was conducted during three separate experiments. The validation sampling involved the activation of four individual "feed events," whereby four separate samples weighing approximately 50, 100, 200, and 400 g were removed from each feeder, with each feed event being linked to a specific radio frequency identification (RFID) tag. The feeder validation experiments evaluated the ability of the feeding system to 1) create a unique feed event every time a sample of pellets was collected from the feeder, 2) link the feed event to the correct RFID, and 3) accurately record the weight of feed that was manually removed. All feed events were initiated and logged in the feeding system with 100% of the events being linked to the correct test RFID. Concordance correlation coefficients between the feeding system-recorded feed weight and the manually removed weight were 0.99 within all three experiments. There was also no overall and little level-dependent bias between the weights measured by the feeding system and weights measured on the external scales. These results indicate the stability of the feeding system over time and consistency between the feeders within and across the three experiments. In conclusion, the automated feeding system developed for measuring individual animal feed intake was able to detect and record the unique electronic RFID associated with unique feed events and accurately capture the weight of feed removed. Furthermore, there was no change in the accuracy of the system from the start to the end of experimental periods, and the amount of feed removed in the feed event (or meal size) did not impact the accuracy of the results., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science.)

An important component of cellular biochemistry is the concentration of proteins and nucleic acids in non-membranous compartments 1,2 . These biomolecular condensates are formed from processes that include liquid-liquid phase separation. The multivalent interactions necessary for liquid-liquid phase separation have been extensively studied in vitro 1,3 . However, the regulation of this process in vivo is poorly understood. Here we identify an in vivo regulator of liquid-liquid phase separation through a genetic screen targeting factors required for Arabidopsis RNA-binding protein FCA function. FCA contains prion-like domains that phase-separate in vitro, and exhibits behaviour in vivo that is consistent with phase separation. The mutant screen identified a functional requirement for FLL2, a coiled-coil protein, in the formation of FCA nuclear bodies. FCA reduces transcriptional read-through by promoting proximal polyadenylation at many sites in the Arabidopsis genome 3,4 . FLL2 was required to promote this proximal polyadenylation, but not the binding of FCA to target RNA. Ectopic expression of FLL2 increased the size and number of FCA nuclear bodies. Crosslinking with formaldehyde captured in vivo interactions between FLL2, FCA and the polymerase and nuclease modules of the RNA 3'-end processing machinery. These 3' RNA-processing components colocalized with FCA in the nuclear bodies in vivo, which indicates that FCA nuclear bodies compartmentalize 3'-end processing factors to enhance polyadenylation at specific sites. Our findings show that coiled-coil proteins can promote liquid-liquid phase separation, which expands our understanding of the principles that govern the in vivo dynamics of liquid-like bodies.

Background: Recent developments in both microscopy and fluorescent protein technologies have made live imaging a powerful tool for the study of plant cells. However, the complications of keeping plant material alive during a long duration experiment while maintaining maximum resolution has limited the use of these methods., Results: Here, we describe an imaging chamber designed to overcome these limitations, which is flexible enough to support a range of sizes of plant materials. We were able use confocal microscopy to follow growth and development of plant cells and tissues over several days. The chamber design is based on a perfusion system, so that the addition of drugs and other experimental treatments are also possible., Conclusions: In this article we present a design of imaging chamber that makes it possible to image plant material with high resolution for extended periods of time.

Many studies have described benefits to patients from geriatric care in the emergency department (ED), yet few geriatric emergency departments exist nationally. As our nation ages and health care financing for these patients becomes more complex, it will be crucial for hospitals to develop ED services that address the needs of our sickest and frailest patients. In this article, we report on our experiences using advanced practice registered nurses (APRNs) embedded in an established ED. Our geriatric emergency medicine service (GEMS(SM)) model has improved patient satisfaction rates and decreased time spent in the ED. It has increased volume of geriatric patients in our hospital by 6%. Strong executive support for geriatric services has established our hospital as a local leader in geriatric emergency medicine. The program is fiscally neutral and serves a frail vulnerable population. We have improved healthcare for our seniors and believe this model of geriatric emergency care can easily be replicated nationally.

Background: Anorexia nervosa is a psychiatric disorder characterized by restrictive eating, low body weight, and severe bone loss. Recent data show a deleterious relationship between low circulating sodium levels and bone mass, and relative or absolute hyponatremia is a known complication of anorexia nervosa. Clinical studies of other medical conditions associated with hyponatremia suggest that detrimental effects of low sodium levels on health are seen even within the normal range. We hypothesized that women with anorexia nervosa and relatively low plasma sodium levels would have lower bone mineral density (BMD) than those with higher plasma sodium levels., Method: In a cross-sectional study (January 1, 1997-December 31, 2009) of 404 women aged 17 to 54 years (mean ± standard error of the mean [SEM] age = 25.6 ± 0.3 years) who met DSM-IV criteria for anorexia nervosa, we measured BMD using dual-energy x-ray absorptiometry. Bone mineral density was compared in women with plasma sodium levels < 140 mmol/L (midpoint of normal range) versus those with plasma sodium levels ≥ 140 mmol/L and in women with hyponatremia (plasma sodium < 135 mmol/L) versus those without. The study was conducted at the Neuroendocrine Unit of Massachusetts General Hospital, Boston., Results: Women with plasma sodium levels < 140 mmol/L had significantly lower BMD and t and z scores versus those with plasma sodium levels ≥ 140 mmol/L at the anterior-posterior (AP) spine (mean ± SEM z scores = -1.6 ± 0.1 vs -1.3 ± 0.1, P = .004) and total hip (mean ± SEM z scores = -1.2 ± 0.1 vs -0.9 ± 0.1, P = .029). In a model controlling for age, BMI, psychiatric drug use, and disease duration, differences in BMD and t and z scores remained significant at the AP spine. Women with hyponatremia had significantly lower BMD and t and z scores versus those without hyponatremia at the AP spine (mean ± SEM z scores = -2.2 ± 0.3 vs -1.3 ± 0.1, P = .009), lateral spine (mean ± SEM z scores = -2.4 ± 0.4 vs -1.5 ± 0.1, P = .031), and total hip (mean ± SEM z scores = -2.5 ± 0.5 vs -1.0 ± 0.1, P < .0001). In a model controlling for age, BMI, psychiatric drug use, and disease duration, differences in BMD and z and t scores remained significant at all sites., Conclusions: These data suggest that relative plasma sodium deficiency may contribute to anorexia nervosa-related osteopenia., (© Copyright 2012 Physicians Postgraduate Press, Inc.)

Context: Anorexia nervosa (AN) is associated with depletion of body fat, loss of bone mineral density (BMD), and impaired thermogenesis. Brown adipose tissue (BAT) is lower in obese individuals and decreases during aging. Recent studies have suggested a link between BAT and bone metabolism., Objective: Our objective was to investigate the presence and quantity of BAT in patients with AN, recovered AN (AN-R), and normal-weight controls and to study the relationship between BAT and BMD and body composition and investigate hormonal predictors of BAT., Design and Setting: This was a cross-sectional study at a clinical research center., Patients: Patients included 15 women: five with AN (mean age 30 ± 6.3 yr), five AN-R, and five healthy nonobese controls of comparable age., Main Outcome Measures: Cold-activated BAT was determined by fluorodeoxyglucose-positron emission tomography/computed tomography. BMD of total-body, spine, and hip, fat and lean mass was determined by dual-energy x-ray absorptiometry. Single-slice magnetic resonance imaging at L4 was done for abdominal fat compartments, and preadipocyte factor-1 (Pref-1), T₃, and T₄ were measured., Results: Within the AN group, one of five; in the AN-R group, two of five; and in the healthy nonobese control group, four of five subjects were BAT positive. Subjects were divided into groups based on the presence (n = 7) or absence (n = 8) of BAT. Both groups were of comparable age and body mass index. Women with BAT had higher total-body BMD, higher T₃, and lower Pref-1 compared with women without BAT. There was a positive correlation between BAT and BMD that remained significant after controlling for disease status and body mass index., Conclusion: Young women with AN have low cold-activated BAT, which may be due to impaired BAT thermogenesis. Young women with BAT have higher BMD and lower Pref-1 compared with women without BAT, suggesting that BAT may be involved in the regulation of stem cell differentiation into the bone lineage at the expense of adipogenesis.

The phytohormone gibberellin (GA) promotes plant growth by stimulating cellular expansion. Whilst it is known that GA acts by opposing the growth-repressing effects of DELLA proteins, it is not known how these events promote cellular expansion. Here we present a time-lapse analysis of the effects of a single pulse of GA on the growth of Arabidopsis hypocotyls. Our analyses permit kinetic resolution of the transient growth effects of GA on expanding cells. We show that pulsed application of GA to the relatively slowly growing cells of the unexpanded light-grown Arabidopsis hypocotyl results in a transient burst of anisotropic cellular growth. This burst, and the subsequent restoration of initial cellular elongation rates, occurred respectively following the degradation and subsequent reappearance of a GFP-tagged DELLA (GFP-RGA). In addition, we used a GFP-tagged α-tubulin 6 (GFP-TUA6) to visualise the behaviour of microtubules (MTs) on the outer tangential wall (OTW) of epidermal cells. In contrast to some current hypotheses concerning the effect of GA on MTs, we show that the GA-induced boost of hypocotyl cell elongation rate is not dependent upon the maintenance of transverse orientation of the OTW MTs. This confirms that transverse alignment of outer face MTs is not necessary to maintain rapid elongation rates of light-grown hypocotyls. Together with future studies on MT dynamics in other faces of epidermal cells and in cells deeper within the hypocotyl, our observations advance understanding of the mechanisms by which GA promotes plant cell and organ growth., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

RNA-binding proteins play a central role in post-transcriptional mechanisms that control gene expression. Identification of novel RNA-binding proteins in fungi is essential to unravel post-transcriptional networks and cellular processes that confer identity to the fungal kingdom. Here, we carried out the functional characterisation of the filamentous fungus-specific RNA-binding protein RBP35 required for full virulence and development in the rice blast fungus. RBP35 contains an N-terminal RNA recognition motif (RRM) and six Arg-Gly-Gly tripeptide repeats. Immunoblots identified two RBP35 protein isoforms that show a steady-state nuclear localisation and bind RNA in vitro. RBP35 coimmunoprecipitates in vivo with Cleavage Factor I (CFI) 25 kDa, a highly conserved protein involved in polyA site recognition and cleavage of pre-mRNAs. Several targets of RBP35 have been identified using transcriptomics including 14-3-3 pre-mRNA, an important integrator of environmental signals. In Magnaporthe oryzae, RBP35 is not essential for viability but regulates the length of 3'UTRs of transcripts with developmental and virulence-associated functions. The Δrbp35 mutant is affected in the TOR (target of rapamycin) signaling pathway showing significant changes in nitrogen metabolism and protein secretion. The lack of clear RBP35 orthologues in yeast, plants and animals indicates that RBP35 is a novel auxiliary protein of the polyadenylation machinery of filamentous fungi. Our data demonstrate that RBP35 is the fungal equivalent of metazoan CFI 68 kDa and suggest the existence of 3'end processing mechanisms exclusive to the fungal kingdom.

Xylem vessels are cells that develop a specifically ornamented secondary cell wall to ensure their vascular function, conferring both structural strength and impermeability. Further plasticity is given to these vascular cells by a range of different patterns described by their secondary cell walls that-as for the growth of all plant organs-are developmentally regulated. Microtubules and their associated proteins, named MAPs, are essential to define the shape, the orientation, the position and the overall pattern of these secondary cell walls. Key actors in this process are the land-plant specific MAP70 proteins which not only allow the secondary cell wall to be positioned at the cell cortex but also determine the overall pattern described by xylem vessel secondary cell walls.

Microtubules are classically described as being transverse, which is perpendicular to the direction of cell elongation. However, fixation studies have indicated that microtubules can be variably aligned across the epidermis of elongating shoots. In addition, microtubules are reported to have different orientations on inner and outer epidermal surfaces, undermining the idea of hoop-reinforcement. Here, long-term movies of Arabidopsis seedlings expressing GFP-TUA6 allowed microtubule alignment to be directly correlated with the rate of elongation within individual growing cells. We also investigated whether microtubule alignment at the inner or the outer epidermal wall better reflected the growth rate. Movies confirmed that transverse microtubules form on the inner wall throughout elongation, but orientation of microtubules is variable at the outer wall, where they tend to become transverse only during episodes of accelerated growth. Because this appears to contradict the concept that circumferential arrays of transverse microtubules or microfibrils are essential for cell elongation, we checked the organisation of cellulose synthase tracks using GFP-CESA3 and found a similar mismatch between trajectories on inner and outer epidermal surfaces. We conclude that microtubule alignment on the inner wall appears to be a more stable predictor of growth anisotropy, whereas outer-wall alignment is more sensitive to the elongation rate.

One isoform of callose synthase, Glucan Synthase-Like7 (GSL7), is tightly coexpressed with two isoforms of sucrose synthase (SUS5 and SUS6) known to be confined to phloem sieve elements in Arabidopsis (Arabidopsis thaliana). Investigation of the phenotype of gsl7 mutants of Arabidopsis revealed that the sieve plate pores of stems and roots lack the callose lining seen in wild-type plants. Callose synthesis in other tissues of the plant appears to be unaffected. Although gsl7 plants show only minor phenotypic alterations during vegetative growth, flowering stems are reduced in height and all floral parts are smaller than those of wild-type plants. Several lines of evidence suggest that the reduced growth of the inflorescence is a result of carbohydrate starvation. Levels of sucrose, hexoses, and starch are lower in the terminal bud clusters of gsl7 than in those of wild-type plants. Transcript levels of "starvation" genes expressed in response to low sugars are elevated in the terminal bud clusters of gsl7 plants, at the end of the night, and during an extended night. Pulse-chase experiments with (14)CO(2) show that transport of assimilate in the flowering stem is much slower in gsl7 mutants than in wild-type plants. We suggest that the callose lining of sieve plate pores is essential for normal phloem transport because it confers favorable flow characteristics on the pores.

Splicing of plant organellar transcripts is facilitated by members of a large protein family, the pentatricopeptide repeat proteins. We have identified a pentatricopeptide repeat protein in a genetic screen for mutants resistant to inhibition of root growth by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis and consequently named BIR6 (BSO-insensitive roots 6). BIR6 is involved in splicing of intron 1 of the mitochondrial nad7 transcript. Loss-of-function mutations in BIR6 result in a strongly reduced accumulation of fully processed nad7 transcript. This affects assembly of Complex I and results in moderate growth retardation. In agreement with disruption of Complex I function, the genes encoding alternative NADH oxidizing enzymes are induced in the mutant, and the mutant plants are less sensitive to mannitol and salt stress. Mutation in the BIR6 gene allowed normal root growth in presence of BSO and strongly attenuated depletion of glutathione content at these conditions. The same phenotype was observed with other mutants affected in function of Complex I, thus reinforcing the importance of Complex I function for cellular redox homeostasis.